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A new method for assaying the enzymes uridine diphosphoglucose (UDPglucose):thiohydroximate glucosyltransferase and 3'-phosphoadenosine-5'-phosphosulfate:desulfoglucosinolate sulfotransferase has been designed. The assay system is based on the separation of nonionic [14C]desulfobenzylglucosinolate from anionic [14C]UDPglucose and anionic [14C]benzylglucosinolate, respectively, by differential adsorption to DEAE-ion-exchange disks. The procedure eliminates elaborate chromatographic techniques. The method was used to measure both enzymes in several Brassica spp. In addition, sulfotransferase activity was monitored during partial purification from seedlings of Brassica napus (cv Westar).

Human skin fibroblasts from patients with Type IV glycogen storage disease, in which there is a demonstrable deficiency of glycogen branching enzyme, were shown to be able to synthesize [14C]glycogen containing [14C]glucose at branch points when sonicates containing endogenous glycogen synthase a were incubated with UDP[14C]glucose. The branch point content of the glycogen synthesized by the Type IV cells was essentially the same as that formed by normal cells, but the total synthetic capacity of the Type IV cells was lower. A new assay for the branching enzyme using glycogen synthase as the indicator enzyme has been developed. Using this assay it has been shown that the residual branching enzyme of affected children and of their heterozygote parents is less easily inhibited by an IgG antibody raised in rabbits against the normal human liver enzyme than is the branching enzyme of normal fibroblasts.

Debranching enzyme was purified 150-fold from rabbit skeletal muscle by a three-step procedure which utilised ammonium sulphate precipitation, ion-exchange chromatography on DEAE-cellulose and "hydrophobic" chromatography on Sepharose-NH(CH2)4NH2. The preparation was completed within three days, and 200 mg enzyme was isolated from 1000 g muscle, which represented an overall yield of 60%. The preparation was homogeneous by the criteria of polyacrylamide gel electrophoresis and ultracentrifugal analysis. The sedimentation coefficient, s20,w, was 8.1 S. The amino acid composition was determined, and the absorption coefficient, A280 1%, measured refractometrically was 17.5. The subunit molecular weight determined by gel electrophoresis in the presence of sodium dodecyl sulphate was 166000 and this value was supported by sedimentation equilibrium in the presence of 6 M guanidinium chloride (1550oo). The molecular weight of the native enzyme measured by high-speed sedimentation equilibrium was 164000, showing that the debranching enzyme is a monomeric protein at the concentrations which exist in muscle (0.7 mg/ml). The results indicate that the two different enzyme activities which are associated with debranching enzyme, 1,4-glucan-4-glycosyltransferase and amylo-1,6-glucosidase, reside on the same polypeptide chain. Protein-glycogen particles isolated from skeletal muscle showed seven major protein-staining components by polyacrylamide gel electrophoresis, one of which was identified as debranching enzyme. ,our of the other components were the alpha and beta subunits of phosphorylase kinase, glycogen phosphorylase and glycogen synthetase. A new titrimetric assay for debranching enzyme was developed; it was used to demonstrate that the maximum potential activity of debranching enzyme is only 5--10% that of phosphorylase at the concentrations of the two enzymes in skeletal muscle. Since the activity of debranching enzyme is unaffected by every mechanism which leads to the activation of glycogen phosphorylase and phosphorylase kinase, the evidence suggests that the hormonal control of muscle glycogenolysis by adrenalin might be confined to a stimulation of rate of degradation of the outermost branches of glycogen.

An N-acetylglucosaminyltransferase III which catalyzes the addition of N-acetylglucosamine through a beta 1-4 linkage (bisecting N-acetylglucosamine) to the beta-linked mannose of the trimannosyl core structure of N-linked oligosaccharides of glycoproteins was measured in human serum, and liver and hepatoma tissues. The enzyme activity in serum was significantly elevated in patients with hepatomas and liver cirrhosis, and the activity markedly decreased on the transcatheter arterial embolization treatment. High activities were also found in the hepatoma and cirrhotic liver tissues, indicating that the serum activity reflected the activity in the tissues. The assaying of the enzyme activity in serum appears to be useful for the detection and monitoring of primary hepatomas.

Administration of thyrotropin to porcine thyroid follicles, obtained in a serum-free chemically defined medium, provoked marked increases in the activities of several glycosyltransferases involved in protein N-glycosylation. The coincidence of these effects with a previously demonstrated enhancement of thyroglobulin production renders a relationship between these events likely. The most important stimulation was for peptide oligosaccharyltransferase (3-fold). Among the enzymes involved in the synthesis of the lipid oligosaccharide donor, Dol-P glycosyl- and mannosyltransferases were increased 1.5-fold, and Dol-P N-acetylglucosaminylphosphotransferase only 1.15-fold. As regards terminal glycosyltransferases, asialofetuin sialyltransferase was increased 2-fold and ovomucoid galactosyltransferase only 1.2-fold. There was a continuous release of the latter two enzymes into the culture medium.

GTF activity was separated into water-insoluble (GTF-I) and water-soluble (GTF-S) polyglucan-synthesizing enzymes. Each preparation demonstrated a single band on 6% SDS PAGE. Only water-insoluble or water-soluble polyglucan was synthesized by the respective enzyme preparation. Rats were immunized, on Days 1 and 14, with either GTF-I or GTF-S in adjuvant. Animals were bled 13, 35 and 54 days after the initial immunization. Individual antisera were tested against either the GTF-I or the GTF-S for inhibition of radioactive glucose incorporation into glucan, and in gel diffusion, and by Western transfer analyses. The respective antisera reacted with the homologous, but not the heterologous enzyme in gel diffusion and Western transfer. GTF-I activity was not inhibited by antibody to GTF-S, but antibody to GTF-I inhibited GTF-I by 68%. GTF-S was inhibited by more than 60% by each of 3 anti-GTF-S sera. Only one anti-GTF-I serum inhibited GTF-S at as much as a modest 30% level. These data support the antigenic and functional distinctiveness of the GTF enzymes of S. sobrinus 6715.

The localization of chitin synthase in the cells of Mucor rouxii was studied by a method which combined permeabilization of the cells with toluene/ethanol and incubation with the radioactive substrate UDP-[3H]GlcNAc followed by high resolution autoradiography. By this technique it was demonstrated that most of the chitin synthesized by these cells was located within the cytoplasm, and only a small amount of the enzyme product appeared at the cell surface. It was concluded that most of the chitin synthase of M. rouxii is located in the cytoplasm of the cells.

Multiple forms of purified dextransucrase have been observed in the presence of low detergent concentrations ( Luzio , G.A., Grahame , D. A. and Mayer, R.M. (1982) Arch. Biochem. Biophys. 216, 751-757). We now show these forms to arise partly as a result of proteolysis, and partly due to incomplete dissociation of the enzyme. Upon 25 degrees C incubation of the crude enzyme, several new bands appeared with little or no change in total activity. The electrophoretic pattern of aged, crude enzyme was similar to that of partially purified enzyme. Specific detection of dextransucrase on SDS gels revealed a single polypeptide of 174 kDa, which is converted to a 156 kDa protein during the aging process. The observation indicates the occurrence of proteolysis. The polypeptide composition of several of the enzyme forms was determined by two-dimensional electrophoresis. Forms Ia and IIa are composed exclusively of 174 kDa polypeptides. Forms III and IVa consist of 156 kDa units, as does the newly observed form Ic. It is likely that form Ib contains both 174 and 156 kDa polypeptides. The results indicate that incomplete dissociation of aggregates of the 174 kDa unit accounts for all of the bands observed on native gels run on fresh culture extracts. Additional enzyme forms result from aggregation of the 156 kDa proteolysis product alone, and from aggregation with unproteolyzed units to form hybrid aggregates.

A simplified assay procedure has been developed for the determination of collagen glucosyltransferase activity in tissue extracts. Using degraded gelatine as acceptor it was possible to isolate the reaction product by precipitation on to a glass fibre disc. Under our conditions degraded gelatine is glucosylated with a reaction rate which is 3--4 times lower compared with the glucosylation of basement membrane derived glycopeptides. Good reproducibility is demonstrated by the coefficient of variation of 4% in the same assay and an interassay variation coefficient below 8%. As the assay allows the testing of large numbers of samples in a few hours, it should prove a useful tool to determine the enzyme level in the tissue of diabetic animals. In humans the activity of the glucosyltransferase could provide a biochemical parameter related to diabetic microangiopathy.

We measured UDP-GlcNAc:Gal (beta 1-4) Glc (or GlcNAc) (beta 1-3) N-acetylglucosaminyltransferase activities in bovine (Holstein and Jersey cow) and human colostrums, and found in human colostrums sufficient activity to study the enzyme properties while not in bovine colostrums. The properties (requirements, pH optimum, acceptor specificity and Km values for lactose and N-acetyllactosamine) of the enzyme from human colostrum were very similar to those from human serum and urine. The reaction product was hydrolyzed by beta-N-acetylhexosaminidase, indicating that the N-acetylglucosaminyl residue was beta-linked to lactose. Methylation and hydrolysis of the reaction product from lactose [3H] labeled at the terminal galactose yielded 2, 4, 6-tri-O-methyl [3H] galactose. Thus the structure of the product was demonstrated to be GlcNAc (beta 1-3) Gal (beta 1-4) Glc (lacto-N-triose II). On the other hand, bovine sera contained N-acetylglucosaminyltransferase catalyzing the transfer of N-acetylglucosamine from UDP-GlcNAc to lactose. The enzyme activities were approximately 1/6-1/4 of that contained in human serum. The presence of (beta 1-3) N-acetylglucosaminyltransferase in human colostrum and its absence in bovine colostrums, apparently corresponds with the presence and absence of oligosaccharides containing lacto-N-triose II structure in colostrum.

The complete nucleotide sequence was determined for the Streptococcus sobrinus MFe28 gtfI gene, which encodes a glucosyltransferase that produces an insoluble glucan product. A single open reading frame encodes a mature glucosyltransferase protein of 1,559 amino acids (Mr, 172,983) and a signal peptide of 38 amino acids. In the C-terminal one-third of the protein there are six repeating units containing 35 amino acids of partial homology and two repeating units containing 48 amino acids of complete homology. The functional role of these repeating units remains to be determined, although truncated forms of glucosyltransferase containing only the first two repeating units of partial homology maintained glucosyltransferase activity and the ability to bind glucan. Regions of homology with alpha-amylase and glycogen phosphorylase were identified in the glucosyltransferase protein and may represent regions involved in functionally similar domains.

Implantation of cobalt powder in the cerebral cortex of rat determines an epileptogenic focus where two types of reactive astrocytes are observed. The first type is mostly represented in the subcortical white matter but it does exist in the cortex around the implant. Phosphorylase and branching enzyme are both very active in these cells which are filled with glycogen. The second type is limited to the cortex and phosphorylase activity leads to an unbranched polysaccharid. These cells correspond to the "activated astrocytes" described by the authors in a previous paper and observed round irritative lesions which, in the cerebral cortex, produce epileptogenic foci.

The isolation and characterization of cellulase-containing membranes of Achlya ambisexualis Raper was attempted by differential and density gradient centrifugations. Maximum cellulase activity was found at an isopycnic density of 1.19 g/cm3, although some activity was found at other densities. A similar distribution of activity was shown by IDPase, ATPase, UDPG transferase, and by sedimentable carbohydrate. The coequilibration and steady enrichment of these activities during purification suggests their presence in a single type of subcellular particle. It was not possible to identify clearly the particle(s) in question from isolated fractions by electron microscopy, but when compared with the cytochemical localization of carbohydrate and IDPase in intact hypha, cytoplasmic vesicles with 150-mm diameters seem to be likely candidates.

Metabolic activities of S mutans were selectively affected by Procion Blue, known to cause covalent bonds and stable cross links in relation to carbohydrate and protein. In this study, despite the reduction in extra polysaccharide, the level of activity of glucosyltransferase was not significantly changed from the control level and colonial morphology was not transformed from smooth to rough. The results of the investigation imply at least interference with formation of extra cellular polysaccharide, possibly in the bacterial cell wall.

The activities of prolyl 4-hydroxylase (PH) and galactosylhydroxylysyl glucosyltransferase (GGT), both enzymes of collagen biosynthesis, and the concentration of hydroxyproline (HYP) were measured in the gastrocnemius, soleus, and tibialis anterior muscles of rats after sciatic nerve neurectomy combined with cast immobilization of the denervated limb for 1 and 3 wk. PH and GGT were also observed in Achilles and tibialis anterior tendons after cast immobilization without neurectomy. After neurectomy the specific PH activity in the denervated gastrocnemius muscle increased by 215% (P less than 0.001). The specific GGT activity increased by 92-110% (P less than 0.01) in the denervated gastrocnemius, soleus, and tibialis anterior muscles. Elevation of the muscular HYP concentration by 118-170% (P less than 0.001) in the denervated muscles was observed. The PH, GGT, and HYP responses of the denervated muscles immobilized at a lengthened or shortened position during denervation atrophy did not generally differ significantly from those of the unfixed denervated ones. The specific PH and GGT activities of the disused tendons decreased by 62 (P less than 0.01) and 25% (P less than 0.001), respectively, in tendons immobilized in a chronically shortened position. The results suggest that denervation atrophy of skeletal muscle is associated with both an increased level of muscular collagen biosynthesis and with an increased muscular collagen concentration. The PH and GGT responses of the cast-immobilized tendons suggest adaptive changes in collagen biosynthesis of the disused tendon.

Prolyl hydroxylase activity, immunoreactive prolyl hydroxylase protein and galactosylhydroxylysyl glucosyltransferase activity were determined in primary human breast cancer tissues and sera of the same patients, and compared to the level of enzyme activities and protein in normal breast tissues and control sera. The mean prolyl hydroxylase (PH) activity, B-IRPH protein, and B-GGT values in primary breast cancer tissues were 13.86, 5.28 and 5.00 times the mean levels of PH activity, B-IRPH protein and B-GGT in normal breast tissues, respectively. There was no difference in the levels of serum immunoreactive prolyl hydroxylase protein and serum galactosylhydroxylysyl glucosyltransferase activity in breast cancer patients and the controls. These results suggest that the prolyl hydroxylase activity, immunoreactive prolyl hydroxylase protein and galactosylhydroxylysyl glucosyltransferase activity may be elevated in primary breast cancer tissues to compensate for an increase rate of collagen synthesis.

A cDNA sequence encoding a nodule-specific protein, nodulin-100, was identified among the abundant transcripts of poly(A)+ RNA from soybean nodules. Purification of nodulin-100 from the soluble fraction of nodule extract yielded an abundant protein with a subunit of approximately 90 kDa having properties of sucrose synthase (UDP-glucose:D-fructose 2-alpha-D-glucosyltransferase, EC 2.4.1.13). Nodule sucrose synthase of soybean is a tetrameric enzyme. Antibodies raised against this protein cross-reacted with the hybrid-released translation product of nodulin-100 cDNA, suggesting that nodulin-100 is the subunit of this enzyme. This was confirmed by partial DNA sequence analysis which showed 73% sequence homology at the amino acid level with maize sucrose synthase. Nodule sucrose synthase was found to dissociate rapidly into monomers in the presence of heme, suggesting that the availability of free heme may regulate the activity of this enzyme. Sucrose synthase activity increases rapidly during nodule development and declines during senescence. A model is presented which suggests that this enzyme plays a key role in maintaining the carbon economy of the nodules and the free heme may be involved in the flow of carbon to the bacteroids. As the degradation of leghemoglobin occurs during senescence, a concomitant decrease in sucrose synthase activity is observed.

Extracellular proteins from continuous cultures of serotype c and g Streptococcus mutans strains were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. Gels stained with raffinose after electrophoresis revealed that although serotype c strains secrete two fructosyltransferases of molecular mass 68 kDa and 79 kDa, no fructosyltransferase was secreted by the serotype g strain K1. A sucrose activity stain was used to detect two glucosyltransferases (GTF) of molecular mass 162 kDa (bifunctional 1,6-alpha-D-glucan 3-alpha- and 6-alpha GTF or 'dextransucrase') and 153 kDa (a 1,3-alpha-D-glucan 3-alpha-GTF) in samples from cariogenic serotype c strains. Neither the 153 kDa protein nor the corresponding GTF activity was secreted by the non-cariogenic mutant C 67-25. The molecular masses of the corresponding 1,3-alpha and 1,6-alpha-GTF proteins from the serotype g strain K1 were 164 kDa and 158 kDa, respectively. All of the GTF proteins were degraded to discrete bands of lower molecular mass on storage at 4 degrees C even after extensive purification. The results provide an explanation for several outstanding controversies in the GTF literature.

Multiple forms of the extracellular dextransucrase [EC 2.4.1.5] from Leuconostoc mesenteroides NRRL B-512F strain were characterized by polyacrylamide gel electrophoresis. Based on the Rm (Relative mobility) values, a newly devised simple plot of log (Rm X 10/(1-Rm)) vs. degree of association of the enzyme showed a good correlation with the results obtained by the Hedrick-Smith method. Both results indicated that the B-512F dextransucrase aggregates were a mixture of two types of forms, i.e., oligomers of a 65 kDa protomer and their charge isomers. Boiling and treatment of the enzyme at pH 10.5 suggested that enzyme aggregates contained dextran or its fragments bound to the enzyme and the enzyme-dextran complex showed the charge isomerism. Since the highly aggregated forms showed higher activity for dextran synthesis than the dissociated forms, the endogenous dextran may serve as a source of primer and may stabilize the enzyme molecule. Besides allosteric regulation of the activity, the occurrence of oligomeric forms of the enzyme may play an important role in the control of dextran synthesis in vivo.