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A 75 kDa protein was purified to homogeneity from granule extracts of normal human granulocytes using Sephadex G-75 chromatography, Mono-S cation exchange chromatography and chromatofocusing. The protein consisted of one chain with a molecular mass of 75 kDa, as determined by SDS/PAGE. Tryptic peptide analysis by MALDI-TOF (matrix-assisted laser-desorption ionization-time-of-flight) MS and sequence analysis by MS/MS identified the protein to be N-acetylglucosamine-6-sulphatase (EC 3.1.6.14). The identity of the protein was confirmed by demostrating enzymatic activity towards the substrate N-acetylglucosamine 6-sulphate. The enzyme was active over a broad pH range with an optimum of pH 7.0, and showed a K(m) value of 13.0 mM and a V(max) value of approximately 1.8 microM/min per mg. The enzyme also showed O-desulphation activity towards heparan sulphate-derived saccharides. Subcellular fractionation of neutrophil organelles showed the presence of enzymatic activity mainly in the same fractions as primary granules. Furthermore, PMA treatment of the neutrophils induced release of the enzyme, indicating its matrix protein nature. The presence of N-acetylglucosamine-6-sulphatase in human neutrophils implies that neutrophils may play a role in the modulation of cell surface molecules and extracellular matrix by O-desulphation.

Ecto-5′-nucleotidase (ecto-5′-NT) is a glycosylphosphatidylinositol-anchored membrane-bound protein that is ubiquitous in mammalian tissues. It is a target for a number of therapeutic drugs since increased levels of the enzyme correlate with various disease states. In this investigation, we describe the properties of a soluble ecto-5′-NT derived from bull seminal plasma. The protein was highly heterogeneous as demonstrated by chromatofocusing and two-dimensional PAGE. Sequencing analyses revealed a truncated polypeptide lacking the glycosylphospatidylinositol attachment site, suggesting that it is produced post-translationally by cleavage at Gln547 and/or Phe548. Heterogeneity was largely due to differential glycosylation, especially in the oligosaccharides linked to Asn403. Significant differences in substrate specificity were observed between isoforms and, on the basis of molecular-modelling studies, were interpreted in terms of variable glycosylation causing steric hindrance of the substrate-binding site. Thus the soluble forms of ecto-5′-NT found in bull seminal plasma are unique both biochemically and structurally, and have a putative role in signalling interactions with spermatozoa following ejaculation and capacitation in the female reproductive tract.

Since glutathione transferases (GSTs) are suggested to be involved in the prevention of tissue damage by oxidative stress, quantitative and qualitative alterations of GST forms were examined in rat skin after induction of inflammation by 0.6 and 1% 1-chloro-2,4-dinitrobenzene (CDNB) treatment. With 0.6% CDNB, the GST activity in supernatant preparations was 1.8-fold higher than that for control skin, with most GSTs in both cases being bound to S-hexyl-GSH–Sepharose. Major GST subunits of control skin were identified as subunits 7, 4 and 2 by HPLC and chromatofocusing at pH 11–7. These subunits were increased in inflamed skin by 0.6% CDNB and, in addition, the subunit 1 of the Alpha class and subunit 6, both hardly detectable in control skin, were expressed. The specific activity value for GST 7-7 from the inflamed skin by 0.6% CDNB was 2.4-fold lower than that from control skin. However, in the case of inflamed skin after application of 1% CDNB, GST activity was decreased to 69% of the control value and most activity was recovered in fractions binding to a GSH–Sepharose but not a S-hexyl-GSH–Sepharose column. GSTs eluted from the former column demonstrated a restored capacity to bind to the latter, suggesting the GSTs in inflamed skin to be partly inactivated and that they regained activity on exposure to GSH. The Km and Vmax values for GSH of GST 4-4 from inflamed skin after 1% CDNB treatment were 6-fold and 2-fold higher, respectively, than those for the enzyme from control skin, suggesting partial enzyme modification. These results suggest that not only quantitative but also qualitative alterations of GST subunits occur with CDNB-induced inflammation in vivo.

We previously showed that human liver hydroxymethylglutaryl-CoA (HMG-CoA) lyase (HL; EC 4.1.3.4) is found in both mitochondria and peroxisomes. HL contains a 27-residue N-terminal mitochondrial targeting sequence which is cleaved on mitochondrial entry, as well as a C-terminal Cys-Lys-Leu peroxisomal targeting motif. Because peroxisomal HL has a greater molecular mass and more basic pI value than mitochondrial HL, we predicted that peroxisomal HL retains the mitochondrial leader. To test this hypothesis, we expressed both the precursor (pHL) and mature (mHL) peptides in Escherichia coli and studied their properties. pHL purified by ion-exchange and hydrophobic chromatography had a pI of 7.6 on FPLC chromatofocusing and a molecular mass of 34.5 kDa on SDS/PAGE, similar to our findings for peroxisomal HL. For purified mHL, pI (6.2) and molecular mass (32 kDa) values resemble those of mitochondrial HL. Purified pHL is similar to mHL in Km for HMG-CoA (44.8 μM), kcat (6.3 min-1) and pH optimum (9.0–9.5). However, the quaternary structures of pHL and mHL differ. On Superose 12 FPLC gel filtration and also on ultrafiltration, both in the presence and in the absence of HMG-CoA, pHL behaves as a monomer whereas mHL migrates as a dimer. We conclude that the HL precursor is probably identical to peroxisomal HL, that its catalytic properties resemble those of mature mitochondrial HL, and that the mitochondrial leader peptide prevents dimerization of pHL.

A metalloprotease has been identified in ovine chromaffin granules which cleaves the proenkephalin fragment BAM12P to produce adrenorphin-Gly. This cleavage occurs at a single arginine residue and is an intermediate step in the production of the opiate adrenorphin in vivo. The identity of the product was confirmed by reverse-phase and ion-exchange chromatography. The adrenorphin-Gly-generating enzyme (AGE) was determined by chromatofocusing to have a pI value of 5.2 and bound strongly to a metal-chelate affinity column. After purification by gel-filtration and ion-exchange chromatography AGE was free of contaminating activities, as cleavage of radiolabelled BAM12P generated a single product as judged by reverse-phase and ion-exchange chromatography. The enzyme has a molecular mass of approx. 45 kDa and a pH optimum of 8.6 in Mops, Taps and Hepes buffers, but was inhibited by phosphate buffers. It was inhibited by micromolar concentrations of copper and zinc ions, but not by millimolar concentrations of calcium or manganese ions. The addition of BAM22P, dynorphin 1-13 or dynorphin 1-8 to the incubation mixture inhibited the cleavage of radiolabelled BAM12P. The cleavage was also inhibited by the presence of catecholamines at concentrations similar to those found within the chromaffin granule. This may explain the known effect of reserpine on chromaffin cells of reducing catecholamine levels and simultaneously increasing adrenorphin levels. It may also indicate a function for AGE and adrenorphin as reporters of intragranular conditions.

1. We have purified membrane-associated Ins(1,4,5)P3/Ins(1,3,4,5)P4 5-phosphatases from bovine testis and human erythrocytes by chromatography on several media, including a novel 2,3-bisphosphoglycerate affinity column. 2. The enzymes have apparent molecular masses of 42 kDa (testis) and 70 kDa (erythrocyte), as determined by SDS/PAGE, and affinities for Ins(1,4,5)P3 of 14 microM and 22 microM respectively. 3. The two enzymes hydrolyse both Ins(1,4,5)P3 and Ins(1,3,4,5)P4 and are therefore type I Ins(1,4,5)P3 5-phosphatases [nomenclature of Hansen, Johanson, Williamson and Williamson (1987) J. Biol. Chem. 262, 17319-17326]. 4. On chromatofocusing, the partially purified testicular enzyme migrates as two peaks of activity, with pI values of about 5.8 and 5.5. The erythrocyte enzyme exhibits only the latter peak. 5. The testis 5-phosphatase is labile at 37 degrees C, but its activity can be maintained in the presence of 50 mM phorbol dibutyrate (PdBu). After PdBu treatment, a third form of the enzyme, with pI about 6.2, appears on chromatofocusing, but without change in its Km or Vmax. 6. Consideration of the properties of these enzymes and of the 5-phosphatases from other tissues suggests that type I Ins(1,4,5)P3 5-phosphatases are of two well-defined subtypes. We propose that these be termed type Ia [typified by the testis enzyme: approximately 40 kDa, higher affinity for Ins(1,4,5)P3] and Type Ib [typified by the erythrocyte enzyme: approximately 70 kDa, lower affinity for Ins(1,4,5)P3].

Quantitative isolation of bovine prolactin was accomplished by immunoaffinity chromatography using clonal antibody as the stationary ligand. The phosphorylated and non-phosphorylated (native) prolactins contained in the immunopurified preparations were separated by chromatofocusing. Isolates from individual pituitaries revealed that phosphorylated prolactin represented between 20 and 80% of the total prolactin. The stoichiometry of phosphate in phosphorylated prolactin was 1.4:1 when determined by amino acid analysis after preparation of the S-ethylcysteine derivative. One major phosphorylation site, serine-90, and two minor sites, serine-26 and -34, were determined by mapping and sequencing studies. Serine-90 was conserved in prolactins, growth hormones and placental lactogens. Serine-26 and -34 were conserved in prolactins, but were not found in growth hormones or placental lactogens. Absorption spectroscopy of the aromatic amino acid residues indicated that phosphorylation of prolactin was associated with a unique structural conformation.

Major isoenzymes of androsterone-sulphating sulphotransferase (AD-ST) were isolated from liver cytosols of weanling and young adult female rats and their isoelectric properties were compared. On chromatofocusing the enzyme activity of young adults was eluted over a wider range of pH than was that of weanling rats. The activity at pH 7.8-7.2 (fraction I) is obvious at both ages, whereas the activity eluted over the pH 6.6-5.5 range (fraction II) is much lower in weanlings than in young adults. The AD-ST activities eluted in fractions I and II were separately purified by 3′-phosphoadenosine 5′-phosphate-agarose affinity chromatography at both ages. Two-dimensional gel electrophoresis of the isolated enzyme revealed several subunits with distinct pI values, but with the same molecular mass, namely 30 kDa. The relative levels of the pI 6.7 and pI 7.2 subunits are high and the relative level of the pI 6.1 is low in fraction I. In fraction II, the levels of pI 6.1 and pI 6.7 subunits are high and the level of the pI 7.2 subunit is low. There is no significant difference in the relative levels of the pI variants in each fraction between weanlings and young adults. The N-terminal amino acid sequences of the pI variants are identical within the area determined, irrespective of animal age or pI values. These results demonstrate that the pI variants of AD-ST are derived from the same precursor by post-translational modification or that they are products of closely related, but distinct, genes. The pI 6.1 and 6.7 subunits presumably increased during the development from the weanling stage to adulthood, resulting in the increase in acidic form(s) of AD-ST (fraction II) in adult females.

Three (1->3)-beta-D-glucan glucanohydrolase (EC 3.2.1.39) isoenzymes GI, GII and GIII were purified from young leaves of barley (Hordeum vulgare) using (NH4)2SO4 fractional precipitation, ion-exchange chromatography, chromatofocusing and gel-filtration chromatography. The three (1->3)-beta-D-glucanases are monomeric proteins of apparent M(r)32,000 with pI values in the range 8.8-10.3. N-terminal amino-acid-sequence analyses confirmed that the three isoenzymes represent the products of separate genes. Isoenzymes GI and GII are less stable at elevated temperatures and are active over a narrower pH range than is isoenzyme GIII, which is a glycoprotein containing 20-30 mol of hexose equivalents/mol of enzyme. The preferred substrate for the enzymes is laminarin from the brown alga Laminaria digitata, an essentially linear (1->3)-beta-D-glucan with a low degree of glucosyl substitution at 0-6 and a degree of polymerization of approx. 25. The three enzymes are classified as endohydrolases, because they yield (1->3)-beta-D-oligoglucosides with degrees of polymerization of 3-8 in the initial stages of hydrolysis of laminarin. Kinetic analyses indicate apparent Km values in the range 172-208 microM, kcat. constants of 36-155 s-1 and pH optima of 4.8. Substrate specificity studies show that the three isoenzymes hydrolyse substituted (1->3)-beta-D-glucans with degrees of polymerization of 25-31 and various high-M(r), substituted and side-branched fungal (1->3;1->6)-beta-D-glucans. However, the isoenzymes differ in their rates of hydrolysis of a (1->3;1->6)-beta-D-glucan from baker's yeast and their specific activities against laminarin vary significantly. The enzymes do not hydrolyse (1->3;1->4)-beta-D-glucans, (1->6)-beta-D-glucan, CM-cellulose, insoluble (1->3)-beta-D-glucans or aryl beta-D-glycosides.

Eight dimeric isoenzymes of glutathione S-transferase (GST) were purified from liver, kidney and testis of the Syrian golden hamster, using S-hexylglutathione affinity chromatography and chromatofocusing. The isoenzymes were characterized according to their substrate selectivity, physical properties and amino acid sequence analysis. Thus a classification into Alpha, Mu and Pi classes was made in analogy with GSTs of other species. Two Alpha-class GSTs were purified, termed A1A1 (pI 8.9) and A1A2 (pI 8.6). Four Mu-class subunits were detected (M1-M4), all forming homodimers, with M2 and M3 also forming a heterodimer. The isoelectric points ranged from 5.9 to 8.6. One Pi-class isoenzyme was purified and termed P1P1 (pI 6.8). Using h.p.l.c. analysis, the subunit composition was determined in a number of organs. The major subunits in liver were A1 and M1. Subunit A1 was also the major subunit in the kidney. Subunit M1 was not detected in kidney, while subunit P1 was not found in the liver. Pancreas and trachea contained predominantly the Pi-class subunit, P1. GST in the testis was mostly of the Mu class. The major subunit was M4, and subunits M2 and M3 were exclusively detected in the testis.

Six forms of glutathione transferase (GST) were resolved from the cytosolic fraction of Bufo bufo embryos at developmental stage 4 by GSH-Sepharose affinity chromatography followed by f.p.l.c. chromatofocusing in the 9-6 pH range. They have apparent isoelectric points at pH 8.37 (GST I), 8.22 (GST II), 8.10 (GST III), 7.84 (GST IV), 7.37 (GST V) and 7.12 (GST VI), and each displayed an apparent subunit molecular mass of 23 kDa by SDS/PAGE. The Bufo bufo embryo enzymes showed very similar structural, catalytic and immunological properties, as indicated by their substrate-specificities, inhibition characteristics, c.d. spectra, h.p.l.c. elution profiles and immunological reactivities, as well as by their N-terminal amino acid sequences. Although Bufo bufo embryo GSTs do not correspond to any other known GSTs, the results of our experiments indicate that amphibian GSTs could be included in the Pi family of GSTs. This conclusion is supported by the analysis of c.d. spectra, and by the fact that mammalian Pi class GSTs and amphibian GSTs showed about 80% identity in their N-terminal amino acid sequences. Furthermore, antisera prepared against Bufo bufo GST III cross-reacted in immunoblotting analysis with Pi class GSTs, and vice versa.

Glucosamine-6-sulphatase (6S) activity towards a series of radiolabelled heparin-derived trisaccharide substrates was determined in cultured human skin fibroblast and leucocyte homogenates, and in urine supernatants of normal individuals and patients affected with 6S deficiency [Sanfilippo D syndrome; mucopolysaccharidosis (MPS) type IIID]. The N-sulphated and N-acetylated derivatives of the trisaccharide substrate O-(alpha-glucosamine 6-sulphate)-(1----4)-L-O-(alpha-iduronic acid 2-sulphate)-(1----4)-D-O-2,5-anhydro[1-3H]mannitol 6-sulphate (GlcNH6S-IdoA2S-anM6S) were prepared by enzymic digestion of a pentasulphated tetrasaccharide isolated following the HNO2 deamination of heparin. Purified lysosomal enzymes and MPS-patient skin fibroblasts were used along with chemical degradation to confirm the structure of each of the substrates that were utilized to study the interaction of the enzyme activities required to degrade the highly sulphated regions of heparan sulphate. Human liver, skin fibroblast and urine 6S activities were separated by chromatofocusing into at least four and possibly up to six individual activities. 6S activities present in each of the tissues generally had similar catalytic properties, including Km values, pH optima and inhibition with NaCl, Na2SO4 and NaH2PO4. Leucocyte and skin fibroblast 6S activities towards GlcNAc6S-IdoA2S-anM6S were maximal at pH 4.1 and 3.9 respectively, with Km values of 2.8 microM and 0.9-1.7 microM respectively. Urine 6S activity towards GlcNAc6S-IdoA2S-anM6S was stimulated 30-fold by BSA at pH 3.9, which shifted the pH optimum from 5.1 to 4.2 and decreased the Km value at pH 4.2 from 4.0 microM to 0.5 microM. Residual 6S activity present in the skin fibroblast homogenates from MPS IIID patients was characterized for activity towards GlcNAc6S-IdoA2S-anM6S and observed to have similar pH optima and Km values to normal skin fibroblast 6S activities, although the residual 6S activity was less than 1% of the normal control range.

A rat liver mitochondrial-matrix fraction was prepared and shown to have 1-chloro-2,4-dinitrobenzene(CDNB)-metabolizing glutathione transferase (GST) activity. Further fractionation by sequential gel filtration, isoelectric focusing or chromatofocusing and hydroxyapatite chromatography yielded three GSTs of pI 9.3, 8.9 and 7.5, none of which bound to a GSH-agarose affinity matrix. Most of the activity was associated with the pI-9.3 form, which was selected for further study. Its activity was tested with the following potential substrates in addition to CDNB: 1,2-dichloro-4-nitrobenzene, p-nitrobenzyl chloride, trans-4-phenylbut-3-en-2-one, 1,2-epoxy-3-(p-nitrophenoxy)propane, ethacrynic acid, menaphthyl sulphate, cumene hydroperoxide, linoleic acid hydroperoxide and 4-hydroxynon-2-enal. Appreciable activity was obtained only with CDNB and ethacrynic acid (82 and 26 mumol/min per mg of protein respectively). The apparent Km for GSH, using 1 mM-CDNB, was 1.9 mM. The enzyme is a dimer of subunit Mr 26,500. It has a free N-terminus, which has enabled the first 33 amino acids to be sequenced. This portion of primary structure has a sequence in common with members of the Theta class of GSTs (eg. 36% identity with subunit 12) and also a sequence which might function as a mitochondrial import signal. It is novel and has been named ‘GST 13-13’.

Human leucocyte aspartylglucosaminidase (AGA: 1-aspartamido-beta-N-acetylglucosamine amidohydrolase, EC 3.5.1.26) was purified to homogeneity by using affinity chromatography, gel filtration, chromatofocusing and reverse-phase h.p.l.c. As shown by SDS/PAGE, the homogeneous purified enzyme preparation consists of four polypeptide chains with molecular masses of 25, 24, 18 and 17 kDa. In the native polyacrylamide gel these polypeptides migrate as one active enzyme complex, and by gel filtration the peak of enzyme activity can be detected in a position of about 65 kDa. Digestion with endoproteinase Lys-C or endoproteinase Asp-N, followed by peptide analysis with reverse-phase h.p.l.c., reveals an identical peptide pattern for the 24 and 25 kDa bands as well as for the 17 and 18 kDa bands. This treatment further demonstrated a totally different peptide pattern for the 24/25 kDa versus the 17/18 kDa subunit. The N-terminal sequences of the 17 kDa and the 18 kDa peptides were identical, as determined by Edman degradation. The N-termini of the 24 kDa and the 25 kDa peptides were blocked. The enzyme was partly resistant to endoglycosidases H and F, but N-glycosidase F transformed the 24/25 kDa band into one 23 kDa band and the 17/18 kDa band into one 16 kDa band. Also, immunological data obtained with antisera produced against these subunits showed that AGA consists of two non-identical polypeptides.

Carboxylesterase (EC 3.1.1.1) has played an important part in our understanding of the toxicokinetic behaviour of the organophosphorus cholinesterase inhibitors. Carboxylesterases are a heterogeneous group of enzymes that can be separated on the basis of their isoelectric points and by their substrate-specificity. We have purified the isoenzyme (pI 5.8) present in greatest activity in rat lung to near homogeneity. The enzyme was purified by (NH4)2SO4 precipitation, gel filtration, chromatofocusing, separation on anion- and cation-exchangers and hydrophobic-interaction chromatography. The purified enzyme has a molecular mass of approx. 180 kDa with subunits of 60 kDa. The substrate and inhibitor specificities of the enzyme have been characterized. Edman degradation revealed the first 19 amino acid residues of the enzyme. The N-terminus was found to be tyrosine. Inhibition of the enzyme by organophosphorus compounds differed greatly from that of cholinesterases, despite the partial analogy at the N-terminal region.

The cytosolic glutathione S-transferases (GST) from human skeletal muscle were purified by a combination of affinity chromatography and anion-exchange chromatography followed by either chromatofocusing or hydroxyapatite chromatography. Pi-class and Mu-class GST, but not Alpha-class GST, were isolated from muscle. In addition to a Pi-class GST subunit, which exists as a homodimer, this tissue also contains a total of three distinct neutral-type Mu-class GST subunits, which hybridize to form homodimers or heterodimers. The neutral-type subunits are referred to as N1-N3 and are defined by the decreasing isoelectric points of the homodimers; GST N1N1, N2N2 and N3N3 have estimated pI values of 6.1, 5.3 and less than 5.0 respectively. SDS/PAGE showed that N1, N2 and N3 have Mr values of 26,700, 26,000 and 26,300 respectively. The N1, N2 and N3 subunits are catalytically distinct, with N1 possessing a high activity for trans-4-phenylbut-3-en-2-one and N2 having high activity with 1,2-dichloro-4-nitrobenzene. In skeletal muscle the expression of the N1 subunit, but not of N2 and N3 subunits, was found to differ from specimen to specimen. The N1 subunit was absent from about 50% of samples examined, and the purification results from two different specimens are presented to illustrate this inter-individual variation. Skeletal muscle from one individual (M1), which did not express N1, contained only GST N2N2, N2N3 and pi, whereas the second sample examined (M2) contained GST N1N2, N2N2 and N2N3 as well as GST pi. N-Terminal amino acid sequence analysis supported the electrophoretic evidence that the N2 subunit in GST N1N2, N2N2 and N2N3 represents the same polypeptide. The peptides obtained from CNBr digests of N2 were subjected separately to automated amino acid sequencing, and the results indicate that N2 is distinct but closely related to the protein encoded by the human Mu-class cDNA clone GTH4 [DeJong, Chang, Whang-Peng, Knutsen & Tu (1988) Nucleic Acids Res. 16, 8541-8554]. GST N2N2 is probably identical with GST 4 [Board, Suzuki & Shaw (1988) Biochim. Biophys. Acta 953, 214-217], as over the 24 N-terminal residues of GST 4 there is complete identity between the two enzymes. Our data suggest that the GST 1 and GST 4 loci are part of the same multi-gene family.

The cistron encoding the B subunit of Escherichia coli Shiga-like toxin I (SLT-I) was cloned under control of the tac promoter in the expression vector pKK223-3 and the SLT-I B subunit was expressed constitutively in a wild-type background and inducibly in a lacIq background. The B subunit was located in the periplasmic space, and less than 10% was found in the culture medium after 24 h incubation. Polymyxin B extracts contained as much as 160 micrograms of B subunit/ml of culture. B subunit was purified to homogeneity by ion-exchange chromatography followed by chromatofocusing. Cross-linking analysis of purified native B subunit showed that it exists as a pentamer. In gels containing 0.1% SDS the native protein dissociated into monomers. B subunit was found to have the same glycolipid-receptor-specificity as SLT-I holotoxin. Competitive binding studies showed that B subunit and holotoxin had the same affinity for the globotriosylceramide receptor. We conclude that this recombinant plasmid is a convenient source of large amounts of purified SLT-I B subunit, which could be used for biophysical and structural studies or as a natural toxoid.

Glutathione S-transferase (GST) subunits in rat liver cytosol were separated by reverse-phase h.p.l.c.; five major proteins were isolated and identified as subunits 1, 2, 3, 4 and 8. F.p.l.c. chromatofocusing resolved the affinity-purified GST pool into nine different isoenzymes. The five basic (Alpha class) dimeric peaks of GST activity were 1-1, 1-2a, 1-2b, 2-2a and 2-2b. Reverse-phase h.p.l.c. analysis revealed that subunit 8 was also present in the protein peaks designated 1-1, 1-2a and 1-2b. The four neutral (Mu class) isoenzymes were 3-3, 3-4, 3-6 and 4-4. The GST pool was methylated in vitro before reverse-phase h.p.l.c. or f.p.l.c. chromatofocusing. Chromatofocusing indicated that the Mu class isoforms (3-3, 3-4 and 4-4) were the primary GSTs methylated, and h.p.l.c. analysis confirmed that subunits 3 and 4 were the major methyl-accepting GST subunits. The addition of calmodulin stimulated the methylation in vitro of GST isoenzymes 3-3, 3-4 and 4-4 by 3.0-, 7.5- and 9.9-fold respectively. Reverse-phase h.p.l.c. also indicated that only the methylation of GST subunits 3 and 4 was stimulated by calmodulin. Basic GST isoenzymes were minimally methylated and the methylation was not enhanced by calmodulin. Investigation of the time course of methylation of GST subunits 3 and 4 indicated that at incubation times less than 4 h the methylation of both Mu class subunits was stimulated by calmodulin, and that under such conditions subunit 4 was the preferred substrate. In contrast, there was essentially no calmodulin-stimulated methylation at incubation times of 4 or 6 h, and the methylation of subunit 3 was predominant. Kinetic parameters at 2 h of incubation were determined in the presence and in the absence of calmodulin. The addition of calmodulin doubled the Vmax. for methylation of both subunits 3 and 4 and decreased the Km of subunit 4 for S-adenosyl-L-methionine 3.6-fold. Finally, methylation was substoichiometric and after 6 h of incubation ranged from 2.8 to 7.6% on a mole-to-mole basis for subunits 4 and 3 respectively.

An oestrogen sulphotransferase, active towards both oestrone and oestradiol, and of high specific activity, is present in cytosol prepared from adrenal glands of both sexes of English Shorthair and Hartley guinea pigs. The ovarian and testicular cytosolic activities of this enzyme are markedly low in comparison with the adrenal activity. The adrenal enzyme is distinct from an accompanying pregnenolone sulphotransferase as judged by f.p.l.c. gel filtration, chromatofocusing, and differences in activation brought about by the addition of thiol groups. The oestrogen sulphotransferase behaved as a 67 kDa protein on a Sephadex G100 column and as a 48 kDa protein on f.p.l.c. gel-filtration columns. Two forms of the enzyme with apparent pI values of 6.1 and 5.5 were eluted during f.p.l.c. chromatofocusing. Sequential salt fractionation, f.p.l.c. gel filtration and elution from an agarose-hexane-adenosine-3′,5′-diphosphate affinity gel has resulted in a preparation which, when resubmitted to f.p.l.c. gel filtration, yields a considerably purified oestrogen sulphotransferase. When submitted to SDS/polyacrylamide-gel electrophoresis under reducing conditions, a main protein band of 34-36 kDa is observed. It is suggested that the enzyme may exist as a dimer in the cytosol.

By using octyl glucoside in the presence of glycerol, it is possible to obtain a solubilized malonyl-CoA-sensitive carnitine palmitoyltransferase (CPTo) from the outer membranes of rat liver mitochondria. H.p.l.c. on hydroxyapatite column has now allowed a clear separation of the CPTo from the malonyl-CoA-insensitive CPT activity of the inner membranes (CPTi). The separated CPTo activity showed inhibition by low micromolar concentrations of malonyl-CoA, 2-tetradecylglycidyl-CoA and etomoxir-CoA. On solubilization and fractionation, the CPTo rapidly lost activity, unlike the relatively stable CPTi activity. Reconstitution into asolectin liposomes enhanced the activity and the malonyl-CoA-sensitivity of the CPTo fractions, whereas it had no such effect on the activity or malonyl-CoA insensitivity of the CPTi fractions. A polyclonal antibody raised against the malonyl-CoA-insensitive enzyme, purified from the inner membranes, precipitated the CPTi activity, but showed no reactivity with the CPTo fractions. In Western blots, the above antibody did not react with any polypeptide of the CPTo fractions. Incubation of the outer-membrane preparations with [3H]etomoxir, in the presence of ATP and CoA, led to labelling of a 90 kDa polypeptide that in the above hydroxyapatite chromatography was eluted in the same region as the CPTo. No such polypeptide labelling was seen in the CPTi fractions. With heart and skeletal-muscle mitochondria, the correspondingly labelled polypeptide was of about 86 kDa. These results show that the CPTo and CPTi are distinct proteins, that a subunit of 90 kDa for liver and 86 kDa for muscle constitutes a component of their respective CPTo systems, and that the 66 kDa subunit of the CPTi does not constitute a part of the CPTo system.

Cytosolic glutathione transferases (GSTs) were purified from the rat spleen by S-hexyl-GSH-Sepharose chromatography, and two major forms were identified as GSTs 2-2 and 7-7 (GST P). Besides these forms an acidic form (pI 5.8) was purified by chromatofocusing at pH 7-4 and it accounted for about 1% of the total GST activity bound to S-hexyl-GSH-Sepharose. Two-dimensional gel electrophoresis revealed that it is a homodimer (subunit Mr 26,000 with pI 5.8). Immunoblot analysis demonstrated that it was immunologically related to GSTs 2-2 and 1-1, and its N-terminal amino acid was apparently blocked, similarly to other forms of the class Alpha. This form had a low activity towards cumene hydroperoxide or 4-hydroxynon-2-enal, indicating that this form differed from GSTs 10-10 and 8-8 as well as from GSTs 1-1 and 2-2. These results suggest that it is a new form of GST belonging to the class Alpha.

Organophosphate-resistant and -susceptible strains of Culex quinquefasciatus (mosquito) have been compared on the basis of their esterase activities. The homozygous resistant strain (Dar) shows two highly active esterases after starch-gel electrophoresis, of Rm 0.2 and 0.4, which are absent from susceptible strains (Apo, Mon), and which previous selection studies have shown to be inseparable from organophosphate resistance. After SDS/polyacrylamide-gel electrophoresis and silver staining of total C. quinquefasciatus proteins, a 62 kDa band is observed in strain Dar at high concentrations, and in susceptible strains in trace amounts. After Western blotting, this 62 kDa protein is recognized by antisera raised against the two esterases eluted from starch gels. After chromatofocusing of Dar proteins, the 62 kDa protein is seen to be associated with esterase activity, and of a similar pI to that observed for esterases after isoelectric focusing. Post-translational modification is not required for recognition of the 62 kDa putative esterase, since the protein is immunoprecipitated by the anti-esterase serum from products of translation of Dar mRNA in vitro.

Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase (EC 4.1.3.5) was purified to homogeneity from ox liver and obtained essentially free from acetoacetyl-CoA thiolase activity. The purification procedure included substrate elution from cellulose phosphate and chromatofocusing. The relative molecular mas was about 100 000 and S20,w0 was 6.36S. The enzyme appears to be a dimer of identical subunits (Mr 47 900). The Km for acetoacetyl-CoA is extremely low (less than 0.5 microM), and acetoacetyl-CoA (Acac-CoA) gives marked substrate inhibition (KiAcac-CoA = 3.5 microM) that is competitive with respect to acetyl-CoA. Both CoA and DL-3-hydroxy-3-methylglutaryl-CoA give mixed product inhibition with respect to acetyl-CoA, which is compatible with a Ping Pong mechanism in which both products can form kinetically significant complexes with two forms of the enzyme. The two forms are most likely to be free enzyme and an acetyl-enzyme intermediate.

We have examined phospholipid-transfer activities in cytosols from rat and mouse whole lung, isolated rat alveolar type II cells and alveolar type II cell-derived mouse pulmonary adenomas. We report an enrichment in phosphatidylcholine and phosphatidylglycerol (but not phosphatidylinositol) protein-catalysed transfer in the type II cell and adenoma cytosols compared with the whole-lung cytosols. The activities from these cytosols were resolved using column chromatofocusing, which clearly demonstrated the presence of a phosphatidylcholine-specific transfer protein in each of the four tissues. In addition, two proteins (rat) or three proteins (mouse) catalysing both phosphatidylcholine and phosphatidylglycerol transfer were resolved from whole lung, whereas in both the rat isolated alveolar type II cells and the mouse type II cell-derived adenomas one of these less specific proteins is not present.

Human glucuronate 2-sulphatase (GAS), which is involved in the degradation of the glycosaminoglycans heparan sulphate and chondroitin 6-sulphate, was purified almost 2,000,000-fold to homogeneity in 8% yield from liver with a four-step six-column procedure, which consists of a concanavalin A-Sepharose/Blue A-agarose coupled step, a DEAE-Sephacel/octyl-Sepharose coupled step, CM-Sepharose chromatography and gel-permeation chromatography. Although more than 90% of GAS activity had a pI of greater than 7.5, other forms with pI values of 5.8, 5.3, 4.7 and less than 4.0 were also present. The pI greater than 7.5 form of GAS had a native molecular mass of 63 kDa. SDS/polyacrylamide-gel-electrophoretic analysis resulted in two polypeptide subunits of molecular mass 47 and 19.5 kDa. GAS was active towards disaccharide substrates derived from heparin [O-(beta-glucuronic acid 2-sulphate)-(1----4)-O-(2,5)-anhydro[1-3H]mannitol 6-sulphate (GSMS)] and chondroitin 6-sulphate [O-(beta-glucuronic acid 2-sulphate-(1----3)-O-(2,5)-anhydro[1-3H]talitol 6-sulphate (GSTS)]. GAS activity towards GSMS and GSTS was at pH optima of 3.2 and 3.0 respectively with apparent Km values of 0.3 and 0.6 microM respectively and corresponding Vmax values of 12.8 and 13.7 mumol/min per mg of protein respectively. Sulphate and phosphate ions are potent inhibitors of enzyme activity. Cu2+ ions stimulated, whereas EDTA inhibited enzyme activity. It was concluded that GAS is required together with a series of other exoenzyme activities in the lysosomal degradation of glycosaminoglycans containing glucuronic acid 2-sulphate residues.

UDP-glucuronosyltransferase (EC 2.4.1.17) activity was solubilized from male Wistar rat liver microsomal fraction in Emulgen 911, and six fractions with the transferase activity were separated by chromatofocusing on PBE 94 (pH 9.4 to 6.0). Fraction I was further separated into Isoforms Ia, Ib and Ic by affinity chromatography on UDP-hexanolamine-Sepharose 4B. UDP-glucuronosyltransferase in Fraction III was further purified by rechromatofocusing (pH 8.7 to 7.5). UDP-glucuronosyltransferases in Fractions IV and V were purified by UDP-hexanolamine-Sepharose chromatography. The transferase isoforms in Fractions II, III, IV and V were finally purified by h.p.l.c. on a TSK G 3000 SW column. Purified UDP-glucuronosyltransferase Isoforms Ia (Mr 51,000), Ib (Mr 52,000), Ic (Mr 56,000), II (Mr 52,000), IV (Mr 53,000) and V (Mr 53,000) revealed single Coomassie Blue-stained bands on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Isoform III enzyme showed two bands of Mr 52,000 and 53,000. Comparison of the amino acid compositions by the method of Cornish-Bowden [(1980) Anal. Biochem. 105, 233-238] suggested that all UDP-glucuronosyltransferase isoforms are structurally related. Reverse-phase h.p.l.c. of tryptic peptides of individual isoforms revealed distinct ‘maps’, indicating differences in primary protein structure. The two bands of Isoform III revealed distinct electrophoretic peptide maps after limited enzymic proteolysis. After reconstitution with phosphatidylcholine liposomes, the purified isoforms exhibited distinct but overlapping substrate specificities. Isoform V was specific for bilirubin glucuronidation, which was not inhibited by other aglycone substrates. Each isoform, except Ia, was identified as a glycoprotein by periodic acid/Schiff staining.

Cysteine-conjugate beta-lyase (EC 4.4.1.13) was purified about 880-fold from human liver obtained post mortem. The purification procedure included (NH4)2SO4 precipitation, chromatography on DEAE-cellulose and hydroxyapatite, gel filtration on Sephadex G-200, and chromatofocusing. The purified enzyme cleaves the C-S bond of several S-aryl-L-cysteines to yield equimolar amounts of thiols, pyruvic acid and ammonia via an alpha beta-elimination reaction. The Mr of the enzyme was estimated to be 88,000 by gel filtration. The enzyme is thermolabile, has a pH optimum of 8.5, and an apparent Km of 0.7 mM towards S-(p-bromophenyl)-L-cysteine. The enzyme requires pyridoxal 5′-phosphate as a cofactor, and hence the enzyme activity was completely abolished by hydroxylamine. No effect of EDTA or thiol-blocking reagents was observed on the activity of the enzyme.

Cathepsin S was detected in bovine kidney, spleen, lymph nodes and lung by immunochemical methods. The immunostaining of cathepsin S in kidney was concentrated to the cells of the proximal tubule, where the enzyme was present in cytoplasmic granules. The purification method for cathepsin S from bovine spleen involved (NH4)2SO4 fractionation, chromatography on CM-Sephadex C-50, gel filtration on Sephacryl S-200 and chromatofocusing (pH 8.0-6.0). The enzyme was partially destroyed by autolysis of the homogenate at pH 4.2. The isoelectric point of cathepsin S was 7.0. Cathepsin S was found to hydrolyse proteins at a similar rate to cathepsin L below pH 7.0. At pH values of 7.0-7.5 cathepsin S retained most of its activity, whereas cathepsin L was completely inactive.

beta-D-Mannosidase activity in selected normal adult, neonatal and foetal goat tissues and in tissues from animals affected with caprine beta-mannosidosis was examined with the use of 4-methylumbelliferyl beta-D-mannopyranoside as substrate. The enzyme in normal adult thyroid, kidney and brain exhibited a sharp unimodal pH optimum at pH 5.0, whereas the enzyme in both normal adult and mutant liver exhibited broad pH ranges of activity (pH 4.5-8.0). No residual enzyme was detectable in mutant kidney or brain; in contrast, residual activity in mutant liver was 52% of that in a neonatal control. Concanavalin A-Sepharose 4B (Con A-Sepharose) fractionation of normal adult liver beta-D-mannosidase resolved the enzyme into an unbound (non-lysosomal) from (52%) with a broad pH range of activity (pH 4.5-8.0) and a bound (lysosomal) form (48%) with a sharp pH optimum of 5.5. The enzyme in mutant liver consisted entirely of the unbound (non-lysosomal) form. Beta-D-Mannosidase activity in normal adult thyroid, kidney and brain was resolved by chromatofocusing into two major isoenzymes, with pI 5.5 and 5.9, and traces of a minor isoenzyme, with pI 5.0. In normal adult liver the enzyme was also resolved into three isoenzymes with similar pI values; however, that with pI 5.0 predominated. The predominant form of the enzyme in 60-day-foetal liver was bound by Con A, exhibited a unimodal pH optimum (5.0) and was resolved into two isoenzymes, with pI 5.4 and 5.8; only traces of an isoenzyme with pI 5.0 were detectable. Total hepatic beta-D-mannosidase activity increased progressively towards adult values during the last 90 days of gestation as a result of increasing non-lysosomal isoenzyme activity (pI 5.0). Lysosomal beta-D-mannosidase was shown to occur in all normal goat tissues studied as multiple isoenzymes, which are genetically and developmentally distinct from the non-lysosomal isoenzyme occurring predominantly, if not exclusively, in liver.

1. The alpha-L-arabinofuranosidase isoenzyme designated AFIII [Laborda, Archer, Fielding & Byrde (1974) J. Gen. Microbiol. 81, 151-163] was purified by sequential isoelectric focusing, hydrophobic chromatography, gel filtration and chromatofocusing. 2. The enzyme is a monomer of Mr 40,000. 3. On inactivation of the enzyme with 3H-labelled 1-alpha-L-arabinofuranosylmethyl-3-p-nitrophenyltriazene, 0.64 mol of alpha-L-arabinofuranosylmethyl residues/mol of enzyme is estimated to become attached to protein. 5. Neither first-order nor second-order rate constants for hydrolyses of aryl alpha-L-arabinofuranosides are dependent upon leaving-group acidity [beta lg(V) = −0.16 +/− 0.11; Beta lg(V/K) = −0.11 +/- 0.07; n = 7; delta pKa = 4.5] 6. Bond-breaking is nonetheless rate-limiting, as is shown by a value of 18(V) of 1.030 +/− 0.007 for the hydrolysis of p-nitrophenyl arabinoside. 7. Proton-donation to the leaving group is thus far advanced at the rate-limiting transition state for this enzyme. 8. Four alpha-L-arabinofuranosyl pyridinium salts are substrates, and an approximate beta lg(V) value of −0.9 can be estimated. 9. The absolute rate enhancement with the 4-bromoisoquinolinium salt, 2.5 × 10(9), is comparable with that observed with pyranosidases. 10. Ring-opening mechanisms can therefore be dismissed, even though they are known in the acid-catalysed hydrolysis of arabinofuranosides.

Hepatic microsomal UDP-glucuronosyltransferases towards androsterone and testosterone were purified by chromatofocusing and UDP-hexanolamine affinity chromatography in Wistar rats which had genetic deficiency of androsterone UDP-glucuronosyltransferase activity. In rats with the high-activity phenotype, androsterone (the 3-hydroxy androgen) UDP-glucuronosyltransferase was eluted at about pH 7.4 and had a subunit Mr of 52 000, whereas testosterone (the 17-hydroxy steroid) UDP-glucuronosyltransferase was eluted at about pH 8.4 and had a subunit Mr of 50 000. The transferase that conjugates both androsterone and testosterone was eluted at about pH 8.0, had subunit Mr values of 50 000 and 52 000, and appeared to be an aggregate or hybrid of androsterone and testosterone UDP-glucuronosyltransferases. In rats with the low-activity phenotype, androsterone UDP-glucuronosyltransferase was absent, whereas testosterone UDP-glucuronosyltransferase was eluted at around pH 8.5, with a subunit Mr of 50 000.

Glutathione transferases from rat kidney cytosol were purified about 40-fold by chromatography on S-hexylglutathione linked to epoxy-activated Sepharose 6B. Further purification by fast protein liquid chromatography with chromatofocusing in the pH interval 10.6-7.6 resolved five major peaks of activity with 1-chloro-2,4-dinitrobenzene as the second substrate. Four of the peaks were identified with rat liver transferases 1-1, 1-2, 2-2 and 4-4 respectively. The criteria used for identification included physical properties, reactions with specific antibodies, substrate specificities and sensitivities to several inhibitors. The fourth major peak is a ‘new’ form of transferase, which has not been found in rat liver. This isoenzyme, glutathione transferase 7-7, has a lower apparent subunit Mr than any of the transferases isolated from rat liver cytosol, and does not react with antibodies raised against the liver enzymes. Glutathione transferases 3-3 and 3-4, which are abundant in liver, were only present in very small amounts. In a separate chromatofocusing separation in a lower pH interval, an additional peak was eluted at pH 6.3. This isoenzyme is characterized by its high activity with ethacrynic acid.

We compared immunochemical and biochemical properties of the vitamin D-dependent Ca2+-binding protein (CaBP) from rat and mouse intestine. The two intestinal CaBP species were extensively purified by gel filtration and successive anion-exchange chromatographies. Both had a similar mol.wt. of 9000. Their pI values differed markedly, being 8.0 and 4.9 in rat and mouse CaBP respectively. Accordingly, mouse CaBP displayed more anodal migration in electrophoresis under non-denaturing conditions. Both mouse and rat CaBP only exhibited partial immunochemical similarities, but their amino acid compositions were very similar. Chromatofocusing was also found to be a good method of detecting calcium-dependent changes in their pI. We developed a sensitive radioimmunoassay for mouse CaBP enabling us to detect substantial amounts of CaBP in uterus, yolk sac and chorio-allantoic placenta. During normal mouse gestation, CaBP appeared on day 12 in the chorio-allantoic placenta but was already present on day 9 in the yolk sac, where its level rose sharply between days 9.5 and 10. CaBP may therefore be considered as a new marker for mouse yolk-sac differentiation.

Two extracellular proteinases that probably play a central role in the metabolism and pathogenesis of the most common dermatophyte of man, Trichophyton rubrum, were purified to homogeneity. Size-exclusion chromatography and Chromatofocusing were used to purify the major proteinases 42-fold from crude fungal culture filtrate. The major enzyme has pI 7.8 and subunit Mr 44 000, but forms a dimer of Mr approx. 90 000 in the absence of reducing agents. A second enzyme with pI 6.5 and subunit Mr 36 000, was also purified. It is very similar in substrate specificity to the major enzyme but has lower specific activity, and may be an autoproteolysis product. The major proteinase has pH optimum 8, a Ca2+-dependence maximum of 1 mM, and was inhibited by serine-proteinase inhibitors, especially tetrapeptidyl chloromethane derivatives with hydrophobic residues at the P-1 site. Kinetic studies also showed that tetrapeptides containing aromatic or hydrophobic residues at P-1 were the best substrates. A kcat./Km of 27 000 M-1 X S-1 was calculated for the peptide 3-carboxypropionyl-Ala-Ala-Pro-Phe-p-nitroanilide. The enzyme has significant activity against keratin, elastin and denatured type I collagen (Azocoll).

A 1,3-1,4-beta-D-glucanase (lichenase, 1,3-1,4-beta-D-glucan 4-glucanohydrolase, EC 3.2.1.73) from Bacteroides succinogenes cloned in Escherichia coli was purified 600-fold by chromatography on Q-Sepharose and hydroxyapatite. The cloned enzyme hydrolysed lichenin and oat beta-D-glucan but not starch, CM(carboxymethyl)-cellulose, CM-pachyman, laminarin or xylan. The enzyme had a broad pH optimum with maximum activity at approx. pH 6.0 and a temperature optimum of 50 degrees C. The pH of elution from a chromatofocusing column for the cloned enzyme was 4.7 (purified) and 4.9 (crude) compared with 4.8 for the mixed-linkage beta-D-glucanase activity in B. succinogenes. The Mr of the cloned enzyme was estimated to be 37,200 by gel filtration and 35,200 by electrophoresis. The Km values estimated for lichenin and oat beta-D-glucan were 0.35 and 0.71 mg/ml respectively. The major hydrolytic products with lichenin as substrate were a trisaccharide (82%) and a pentasaccharide (9.5%). Hydrolysis of oat beta-D-glucan yielded a trisaccharide (63.5%) and a tetrasaccharide (29.6%) as the major products. The chromatographic patterns of the products from the cloned enzyme appear to be similar to those reported for the mixed-linkage beta-D-glucanase isolated from Bacillus subtilis. The data presented illustrate the similarity in properties of the cloned mixed-linkage enzyme and the 1,3-1,4-beta-D-glucanase from B. subtilis and the similarity with the 1,4-beta-glucanase in B. succinogenes.

gamma-Butyrobetaine hydroxylase [4-trimethylaminobutyrate, 2-oxoglutarate:oxygen oxidoreductase (3-hydroxylating), EC 1.14.11.1] from human kidney was resolved into three forms by chromatofocusing. After further chromatography on an anion-exchanger, each form appeared as a single band on electrophoresis in polyacrylamide gel containing sodium dodecyl sulphate. The isoelectric points of isoenzymes 1, 2 and 3 were 5.6, 5.7 and 5.8 respectively, as estimated by isoelectric focusing. Their specific activities were 17-29 mu kat/g of protein. The concentrations of the three isoenzymes were about equal, possibly slightly lower for isoenzyme 1. The requirement for Fe2+ and the Km values for gamma-butyrobetaine and 2-oxoglutarate were about the same for the different enzyme forms. L- and D-Carnitine caused decarboxylation of 2-oxoglutarate to the same extent (8 and 29%) with the three forms. The enzyme forms had the same mass, 64 kDa, as determined by gel filtration in nondenaturing media. The same subunit mass, 42 kDa, was obtained for the multiple forms by electrophoresis in polyacrylamide gels containing sodium dodecyl sulphate. Isoenzyme 2 was resolved into two protein bands by isoelectric focusing in polyacrylamide gels containing urea. Isoenzyme 1 contained only one of these bands and isoenzyme 3 the other. The three enzyme forms of gamma-butyrobetaine hydroxylase thus appear to be dimeric combinations of two subunits differing in charge but not in size. gamma-Butyrobetaine hydroxylase from crude extracts of human, rat and calf liver was also separated into multiple forms by a chromatofocusing technique. The isoenzyme pattern was the same in human liver and kidney. The technique used to resolve the mammalian enzymes gave no evidence for the presence of multiple forms of the bacterial enzyme from Pseudomonas sp. AK 1.

Qualitative and quantitative differences of purified hepatic 3 alpha-hydroxysteroid UDP-glucuronosyltransferase were investigated in Wistar and Sprague-Dawley rats. Individual differences in the glucuronidation rate of androsterone and chenodeoxycholic acid were observed in hepatic microsomal fractions from Wistar but not Sprague-Dawley rats. No individual variation was observed in the glucuronidation of testosterone, p-nitrophenol or oestrone. The 3 alpha-hydroxysteroid UDP-glucuronosyltransferases from livers of Wistar and Sprague-Dawley rats were isolated and highly purified by using Chromatofocusing and affinity chromatography. The amount of 3 alpha-hydroxysteroid UDP-glucuronosyltransferase in the liver of Wistar rats exhibiting low rates for androsterone glucuronidation is about 10% or less than that found in hepatic microsomal fractions obtained from Wistar rats having high rates for androsterone glucuronidation. The apparent Km for androsterone with purified 3 alpha-hydroxysteroid UDP-glucuronosyltransferase from Wistar rats with high glucuronidation activity (6 microM) was not different from that observed for the enzyme purified from Sprague-Dawley animals, whereas that for the enzyme purified from Wistar rats with low glucuronidation activity was substantially higher (120 microM). Despite the differences in apparent Km values for androsterone, the apparent Km for UDP-glucuronic acid (0.3 mM) was not different in the different populations of rats.

Human kidney prolinase, assayed with Pro-Ala, and non-specific dipeptidase, assayed with Gly-Leu, were purified by using DEAE-cellulose, gel-filtration, metal-ion-chelate, hydrophobic and adsorption chromatography and chromatofocusing. Both enzymes gave single peaks of activity that were congruent and the ratio of their activities was constant throughout the purification. Gel filtration indicated an Mr of 100 000 and chromatofocusing a pI of 5.4. Ni2+-chelate chromatography demonstrated the presence of exposed histidine residues on the enzyme and was an effective separative procedure. Polyacrylamide-gel electrophoresis of the final preparation showed the two enzyme activities to be coincident. Both enzyme activities decayed at the same rate at 53 degrees C and were inhibited to the same extent by p-hydroxymercuribenzoate. Of six non-specific dipeptidase substrates tested Gly-Leu gave the highest activity, and of six prolinase substrates Pro-Leu had the highest activity. Gly-Leu was hydrolysed at double the rate of Pro-Leu. Pro-Ala was a competitive inhibitor of activity towards Gly-Leu, and Gly-Leu was a competitive inhibitor of activity towards Pro-Ala. Mixed-substrate studies strongly suggested that Gly-Leu and Pro-Ala were hydrolysed at a common active site. The data are consistent with prolinase and non-specific dipeptidase activity in human kidney being due to a single enzyme.

A fraction has been isolated from human urine which exhibits antiproliferative activity against human tumour cell lines without affecting the growth of several normal diploid cell lines or tumour cells of mouse or hamster origin. The major protein present in this fraction has been characterized and tentatively designated antineoplastic urinary protein (ANUP). An S020,W value of 3.69 S was obtained by sedimentation velocity analysis, and a subunit molecular mass of 16 300 Da was obtained by sedimentation equilibrium and by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Centrifugation data also indicated that the protein self-associates. The amino acid analysis of ANUP was consistent with its low pI (4.2) as determined by chromatofocusing analysis. Furthermore, the amino acid composition exhibited some features similar to collagen, as shown by high levels of proline and glycine, the absence of cysteine, and the presence of low levels of hydroxyproline.

The distribution of glucocorticoids incubated with rat liver cytosol preparations or administered in vivo to adrenalectomized rats was analysed by chromatographic procedures. Corticosterone or dexamethasone was co-eluted with Yb-type GSH S-transferases in anion-exchange and gel-permeation chromatography systems, and these glucocorticoids also were bound to Yb forms in analyses by immunoadsorbent and lysyl-GSH affinity matrices. Pretreatment of cytosol with lysyl-GSH to extract GSH S-transferases or incubation with excess bilirubin, which is expected to compete with steroids for binding to the protein, yielded preparations that were devoid of this major steroid-binding component. In mixtures of the multiple rat GSH S-transferases, corticosterone preferentially interacted with Yb forms rather than Ya and Yc subgroups. All of the multiple Yb forms resolved by chromatofocusing procedures retained the steroid-binding capacity. It is suggested that these abundant proteins can account for a considerable share of intracellular glucocorticoid binding and represent a high-affinity non-saturable binding component with potential to function in steroid-hormone metabolism and action.

The major glutathione transferases in the rat small-intestine cytosol were isolated and characterized. The enzymes active with 1-chloro-2,4-dinitrobenzene as second substrate were almost quantitatively recovered after affinity chromatography on immobilized S-hexylglutathione. The different basic forms of glutathione transferase, which account for 90% of the activity, were resolved by chromatofocusing. Fractions containing enzymes with lower isoelectric points were not further resolved. The isolated fractions were characterized by their elution position in chromatofocusing, apparent subunit Mr, reactions with specific antibodies, substrate specificities and inhibition characteristics. The major basic forms identified were glutathione transferases 1-1, 4-4 and 7-7. In addition, evidence for the presence of a variant form of subunit 1, as well as trace amounts of subunits 2 and 3, was obtained. A significant amount of transferase 8-8 in the fraction of acidic enzyme forms was demonstrated by immunoblot and Ouchterlony double-diffusion analysis. In the comparison of the occurrence of the different forms of glutathione transferase in liver, lung, kidney and small intestine, it was found that the small intestine is the richest source of glutathione transferase 7-7.

The glutathione S-transferases are dimeric proteins and comprise subunits of Mr 25 500 (Ya), 26 500 (Yn), 27 000 (Yb1 and Yb2) and 28 500 (Yc). Enzymes containing Ya and/or Yc subunits have been isolated as have forms containing binary combinations of Yn, Yb1 and Yb2 subunits. To date only one enzyme, transferase S, has been described that is a YbYn heterodimer [Hayes & Chalmers (1983) Biochem. J. 215, 581-588]; the identity of the Yb monomer found in transferase S has not been reported previously. The identification and isolation of a YnYn dimer (transferase N) from rat testis is now described. This has enabled structural and functional comparisons to be made between Yb1, Yb2 and Yn monomers. Reversible dissociation experiments between the YnYn and Yb1Yb1 homodimers and between the YnYn and Yb2Yb2 homodimers demonstrated that Yn monomers can hybridize with both Yb1 and Yb2 monomers. Reversible dissociation of transferases N and C (Yb1Yb2) showed that both Yb1 and Yb2 monomers can hybridize with Yn monomers under competitive conditions. The hydridization data suggest that transferase S represents the Yb2Yn subunit combination. A knowledge of the elution position from chromatofocusing columns of the Yb1Yn hybrid that was formed in vitro enabled a purification scheme to be devised for an enzyme from rat liver (transferase P) believed to consist of Yb1Yn subunits. A comparison of the chromatographic behaviour of the YnYn, Yb1Yb1 and Yb2Yb2 dimers on chromatofocusing and hydroxyapatite columns with the behaviour of transferases P and S on the same matrices suggests these two enzymes may be identified as the Yb1Yn and Yb2Yn dimers respectively. The catalytic activities and the inhibitory effects of non-substrate ligands on transferases P and S are significantly different and again suggest they comprise Yb1 and Yn subunits and Yb2 and Yn subunits respectively; transferase P exhibits a 6-fold higher specific activity for 1,2-dichloro-4-nitrobenzene than does transferase S, whereas, conversely, transferase S possesses a 9-fold higher specific activity for trans-4-phenylbut-3-en-2-one than does transferase P. The quaternary structure of transferases P and S was verified by using peptide mapping and ‘Western blotting’ techniques.

We have characterized the properties of human aspartylglucosaminidase (EC 3.5.1.26), the lysosomal enzyme which is deficient in the human inherited disease aspartylglucosaminuria. The purification procedure from human liver included affinity chromatography, gel filtration, strong-anion- and strong-cation-exchange h.p.l.c., chromatofocusing and reverse-phase h.p.l.c. In a denaturing SDS/polyacrylamide-gel electrophoresis, the 6600-fold purified enzyme was shown to be composed of three non-identical inactive polypeptide chains of molecular masses 24, 18 and 17 kDa. In a native polyacrylamide-gel electrophoresis, these polypeptide chains ran as one active enzyme complex. As judged from the elution position of the native enzyme in a Biogel P-100 gel filtration, the approximate molecular mass of this complex was 60 kDa. The enzyme had a pI of 5.7, a pH optimum at 6, of 0.48 mM and a specific activity of 200,000 nkat for the substrate 2-acetamido-1-beta-(L-aspartamido)-1,2-dideoxy-D-glucose. The enzyme showed a 57% loss of activity at 60 degrees C after 45 h but was practically inactive after incubation at 72 degrees C for a few minutes. The molecular structure, Km and specific activity as well as the thermostability of the enzyme described here are different from those reported previously for human aspartylglucosaminidase.

A nucleoside antibiotic, ascamycin (9-beta-[5′-0-(N-L-alanyl) sulphamoyl-D-ribofuranosyl]-2-chloroadenine), has a selective antibacterial activity against Xanthomonas species. When ascamycin was dealanylated, dealanylascamycin showed a broad antibacterial activity against various Gram-negative and Gram-positive bacteria. Xanthomonas citri is susceptible to ascamycin by virtue of the ascamycin-dealanylating enzyme on the cell surface [Osada & Isono (1985) Antimicrob. Agents Chemother. 27, 230-233]. The enzyme (Xc aminopeptidase) was purified from X. citri cells by successive DEAE-cellulose, chromatofocusing and Sephadex G-100 column chromatography to a homogeneous state. The purified enzyme exhibited a single band with an Mr of 38 000 in SDS/polyacrylamide-gel electrophoresis. Gel filtration on a calibrated column indicated a similar Mr value. The isoelectric point of the enzyme was 5.7. The enzyme catalysed the hydrolysis of the alanyl group of ascamycin and liberated alanine from the sulphamoyl nucleoside. The enzyme also catalysed the hydrolysis of L-proline beta-naphthylamide and L-alanine beta-naphthylamide. The optimal pH and temperature for enzyme activity were pH 7.5-8.0 and 35-40 degrees C respectively. The enzyme was inhibited by thiol-enzyme inhibitors (i.e. rho-chloromercuribenzoate and N-ethylmaleimide), but was not affected by various naturally occurring aminopeptidase inhibitors (i.e. amastatin, bestatin, pepstatin and leupeptin). Mn2+ and Mg2+ activated the enzyme, whereas Cu2+, Zn2+ and Cd2+ were inhibitory.

Proteins from rabbit kidney brush border membranes were solubilized with 1% Nonidet P-40 (crude membrane proteins) and fractionated according to their isoelectric points (pI) by chromatofocusing. The eluate was pooled into three fractions according to the pI of the samples (1, greater than 6.8; 2, 6.8-5.4; 3, 5.4-4.0). The crude membrane proteins as well as the three fractions were reconstituted into liposomes and transport of Pi was measured by a rapid filtration technique in the presence of an inwardly directed K+ or Na+ gradient. Arsenate-inhibitable Na+-dependent transport of Pi was reconstituted into an osmotically active intravesicular space from both the crude membrane proteins and Fraction 1. In contrast, Fractions 2 and 3 were inactive. Treatment of the crude membrane proteins and the three fractions with the method for extracting phosphorin (a Pi-binding proteolipid found in brush border membranes) yielded Mn2+-dependent binding of Pi characteristic of phosphorin only in the extracts from crude membrane proteins and Fraction 1, the same fractions in which Na+-dependent transport of Pi was found in the reconstituted system. When reconstituted into liposomes, phosphorin was, however, unable to yield Na+-dependent transport of Pi. Moreover, we cannot eliminate the possibility that Na+-Pi transport can occur in the absence of phosphorin, since complete recovery of Na+-Pi transport was not achieved. However, the present data showing localization of the recovered binding and transport systems for Pi in the same protein fraction lend support to the hypothesis that phosphorin might be a constituent of the renal Pi transport system. Whether the presence of phosphorin is necessary or accessory for Na+-dependent Pi transport in intact brush border membrane vesicles or in liposomes reconstituted with crude or purified membrane proteins requires further investigation.

Histidine decarboxylase was purified 800-fold from the kidneys of thyroxine-treated mice. The purification procedure included precipitation of protein from a crude supernatant after heating it to 55 degrees C at pH 5.5, fractionation with (NH4)2SO4, phosphocellulose column chromatography, chromatofocusing, DEAE-Sepharose column chromatography, gel filtration on Sephacryl S-300 and preparative polyacrylamide-gel electrophoresis. The native enzyme had an estimated Mr of 113 000. The protein was analysed in SDS/10%-polyacrylamide gels and formed a single band corresponding to a subunit Mr of 55 000, indicating that it is a dimer. Three forms of the enzyme were resolved on isoelectrofocusing gels, with pI 5.3, 5.5 and 5.7.

A Ca2+-binding protein (CaBP) from human kidney was purified by two different procedures. The first involved heat-precipitation of a kidney cytosol fraction followed by gel filtration and chromatofocusing. This resulted in a 200-fold increase in the specific Ca2+-binding activity with a yield of 10%. A specific antibody was raised against the purified CaBP, as demonstrated by one precipitate in crossed immunoelectrophoresis of a kidney cytosol fraction. The antibody was coupled to Sepharose 4B and CaBP was then purified by immunoadsorbent chromatography. Applying this technique, a 500-fold purification of CaBP with a yield of 50% was obtained. Both preparations appeared homogeneous in crossed immunoelectrophoresis against a polyvalent antiserum and migrated as a single band corresponding to a mol.wt. of 26000 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. In gel filtration under non-denaturing conditions CaBP was eluted corresponding to a mol.wt. of 28000. The association constant for the high-affinity Ca2+-binding sites of CaBP was estimated by gel filtration to be 0.1 × 10(6)M-1, and the protein displayed Ca2+-dependent electrophoretic mobility, with more rapid anodic migration in the presence of EDTA. The protein eluted at a position corresponding to a pI of 4.5 in chromatofocusing. Immunochemical experiments with the specific antibody showed no cross-reaction between renal and intestinal CaBP.

Human N-acetylglucosamine-6-sulphate sulphatase was purified at least 50,000-fold to homogeneity in 78% yield from liver with a simple three-step four-column procedure, which consists of a concanavalin A-Sepharose/Blue A-agarose coupled step, chromatofocusing and Cu2+-chelating Sepharose chromatography. In all, four forms were isolated and partially characterized. Forms A and B, both with a pI greater than 9.5 and representing 30% and 60% respectively of the recovered enzyme activity, were separated by hydroxyapatite chromatography of the enzyme preparation obtained from the Cu2+-chelating Sepharose step. Both forms A and B had native molecular masses of 75 kDa. When analysed by SDS/polyacrylamide-gel electrophoresis, form A consists of a single polypeptide of molecular mass 78 kDa, whereas form B contained 48 kDa and 32 kDa polypeptide subunits. Neither form A nor form B was taken up from the culture medium into cultured human skin fibroblasts. The two other forms (C and D), with pI values of 5.8 and 5.4 respectively, represented approx. 7% and 3% of the total recovered enzyme activity. The native molecular masses of forms C and D were 94 kDa and approx. 75 kDa respectively. Form C contained three polypeptides with molecular masses of 48, 45 and 32 kDa. N-Acetylglucosamine-6-sulphate sulphatase activity was measured with a radiolabelled disaccharide substrate derived from heparin. The development of this substrate enabled the isolation and characterization of N-acetylglucosamine-6-sulphate sulphatase to proceed efficiently. Forms A, B and C had pH optima of 5.0, Km values of 11.7, 14.2 and 11.1 microM respectively and Vmax. values of 105, 60 and 53 nmol/min per mg of protein respectively. The molecular basis of the multiple forms of this sulphatase is not known. It is postulated that the differences in structure and properties of the four enzyme forms are due to differences in the state of processing of a large subunit.

The FAD-containing short-chain acyl-CoA dehydrogenase was purified from ox liver mitochondria by using (NH4)2SO4 fractionation, DEAE-Sephadex A-50 and chromatofocusing on PBE 94 resin. The enzyme is a tetramer, with a native Mr of approx. 162 000 and a subunit Mr of 41 000. Short-chain acyl-CoA dehydrogenases are usually isolated in a green form. The chromatofocusing step in the purification presented here partially resolved the enzyme into a green form and a yellow form. In the dye-mediated assay system, the enzyme exhibited optimal activity towards 50 microM-butyryl-CoA at pH 7.1. Kinetic parameters were also determined for a number of other straight-chain acyl-CoA substrates. The u.v.- and visible-absorption characteristics of the native forms of the enzyme are described, together with complexes formed by addition of butyryl-CoA, acetoacetyl-CoA and CoA persulphide.

The properties of 3 alpha-hydroxysteroid/dihydrodiol dehydrogenase from Sprague-Dawley rat liver cytosol have been re-examined in light of several reports which suggest that multiple forms of the enzyme may exist in this tissue. During enzyme purification, chromatography on DE-52 cellulose and chromatofocusing columns indicated the existence of only one form of the protein. Re-chromatography of the purified enzyme by either of these techniques failed to resolve the protein into additional forms. When the purified enzyme was subjected to SDS/polyacrylamide-gel electrophoresis a single band corresponding to Mr 34,000 was detected. Two-dimensional gels showed one predominant protein with a pI of 5.9. Using the homogeneous enzyme as antigen, high-titre polyclonal antibody was raised in rabbits. Western-blot analysis of cytosolic proteins prepared from male and female Sprague-Dawley rat liver indicated the presence of a single immunoreactive band with an Mr of 34,000 in both sexes. All of the 3 alpha-hydroxysteroid dehydrogenase activity present in rat liver cytosol could be immunotitrated with the antibody and the resulting titration curve was superimposable on the titration curve obtained with the purified enzyme. Western-blot analysis of cytosolic proteins prepared from livers of male Wistar and Fischer rats also revealed the presence of a single immunoreactive protein with an Mr of 34,000. These data indicate that, contrary to previous reports, only one form of the dehydrogenase may exist in liver cytosols prepared from a variety of rat strains. Although 3 alpha-hydroxysteroid dehydrogenase activity is known to be widely distributed in male Sprague-Dawley rat tissues, Western blots indicate that only the liver, lung, testis and small intestine contain immunoreactive protein with an Mr of 34,000. The levels of immunoreactive protein in these tissues follow the distribution of dihydrodiol dehydrogenase.