Search

Galactosidases, ubiquitous in nature, are complex carbohydrate-active enzymes and find extensive applications in food, pharma, and biotechnology industries. The present study deals with the production of galactosidases from fungi by solid-state fermentation. Fifteen fungi were screened and Aspergillus awamori (MTCC 548), exhibited the highest α and β-galactosidase activities of 75.11±0.29 U/g and 155.34±1.26 U/g, respectively. 30 g of wheat bran substituted with 6% defatted soy flour, at 28°C, pH 5.0 for 120 h, was established as the optimum production conditions by one-factor approach. The enzyme was purified to homogeneity with an apparent mass of 118 ± 2 kDa by ammonium sulfate precipitation (50-80%), ion exchange and hydrophobic interaction chromatography. Specific activities for α and β-galactosidase were 22 and 74 U/mg, respectively. Optimum temperature and pH ranges for enzyme activities were 55-60 °C, 5.0-5.5, respectively. The thermal inactivation mid-point was 65 °C. The purified enzyme not only exhibited α and β-galactosidase activities, but also exhibited β-xylosidase and β-glucosidase activities, indicating the enzyme has broad substrate specificity. Sequence analysis by in-gel digestion and tandem mass spectrometry (MS/MS) revealed that the enzyme was a probable β-galactosidase A, belonging to glycoside hydrolase 35 family, and is being reported for the first time., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Background: It has been demonstrated recently that α1,3-galactosidase from Bacteroides fragilis can efficiently convert human group B red blood cells (RBC) to group O cells. In addition, in vitro data indicated that the enzymatic conversion process did not affect the physiological or metabolic parameters of the RBC. The aim of this study was to investigate the lifespan of enzyme- treated RBC in vivo in the circulation., Materials and Methods: This was an experimental, randomised study. The rat was selected as the experimental subject because it expresses α-1,3galactosyl on its RBC. The efficiency of Galα1,3Gal epitope removal from RBC treated with α1,3-galactosidase was tested before the transfusion experiment to track the survival of RBC in the circulation. The animals were divided into three groups and injected via the tail vein with native, mock-treated or enzyme-treated RBC labelled with fluorescein isothiocyanate. The survival rates of the fluorescently labelled RBC were monitored by flow cytometry., Results: Flow cytometry showed that α-galactosidase (0.02 mg/mL for RBC with a haematocrit of 30%) efficiently removed Galα1,3Gal epitopes from rat erythrocytes, although small amounts of remaining Galα1,3Gal epitopes were still detected. The in vivo data demonstrated that the half-life of enzyme-treated RBC was a little shorter than that of native RBC. However, the 24-hour survival fractions of native, mock-treated and enzyme-treated RBC were virtually identical. Most importantly, the enzyme-treated RBC, like the native RBC, were still detectable 35 days after transfusion., Discussion: Our results indicate that α-glycosidase treatment had little effect on the in vivo survival kinetics of RBC. These data add further support to the feasibility of translating enzymatic conversion technology into clinical practice.

The genomic data on heterotrophic marine bacteria suggest the crucial role that microbes play in the global carbon cycle. However, the massive presence of hypothetical proteins hampers our understanding of the mechanisms by which this carbon cycle is carried out. Moreover, genomic data from marine microorganisms are essentially annotated in the light of the biochemical knowledge accumulated on bacteria and fungi which decompose terrestrial plants. However marine algal polysaccharides clearly differ from their terrestrial counterparts, and their associated enzymes usually constitute novel protein families. In this study, we have applied a combination of bioinformatics, targeted activity screening and structural biology to characterize a hypothetical protein from the marine bacterium Zobellia galactanivorans, which is distantly related to GH43 family. This protein is in fact a 1,3-α-3,6-anhydro-l-galactosidase (AhgA) which catalyses the last step in the degradation pathway of agars, a family of polysaccharides unique to red macroalgae. AhgA adopts a β-propeller fold and displays a zinc-dependent catalytic machinery. This enzyme is the first representative of a new family of glycoside hydrolases, especially abundant in coastal waters. Such genes of marine origin have been transferred to symbiotic microbes associated with marine fishes, but also with some specific human populations., (© 2011 Society for Applied Microbiology and Blackwell Publishing Ltd.)

The gene encoding homodimeric β-galactosidase (lacA) from Bacillus licheniformis DSM 13 was cloned and overexpressed in Escherichia coli, and the resulting recombinant enzyme was characterized in detail. The optimum temperature and pH of the enzyme, for both o-nitrophenyl-β-D: -galactoside (oNPG) and lactose hydrolysis, were 50°C and 6.5, respectively. The recombinant enzyme is stable in the range of pH 5 to 9 at 37°C and over a wide range of temperatures (4-42°C) at pH 6.5 for up to 1 month. The K(m) values of LacA for lactose and oNPG are 169 and 13.7 mM, respectively, and it is strongly inhibited by the hydrolysis products, i.e., glucose and galactose. The monovalent ions Na(+) and K(+) in the concentration range of 1-100 mM as well as the divalent metal cations Mg²(+), Mn²(+), and Ca²(+) at a concentration of 1 mM slightly activate enzyme activity. This enzyme can be beneficial for application in lactose hydrolysis especially at elevated temperatures due to its pronounced temperature stability; however, the transgalactosylation potential of this enzyme for the production of galacto-oligosaccharides (GOS) from lactose was low, with only 12% GOS (w/w) of total sugars obtained when the initial lactose concentration was 200 g/L.

The cyanobacterium Nostoc commune is adapted to the terrestrial environment and forms a visible colony in which the cells are embedded in extracellular polysaccharides (EPSs), which play a crucial role in the extreme desiccation tolerance of this organism. When natural colonies were immersed in water, degradation of the colonies occurred within 2 days and N. commune cells were released into the water. The activities that hydrolyze glycoside bonds in various N. commune fractions were examined using artificial nitrophenyl-linked sugars as substrates. A beta-D-glucosidase purified from the water-soluble fraction was resistant to 20 min of boiling. The beta-D-glucosidase, with a molecular mass of 20 kDa, was identified as a cyanobacterial fasciclin protein based on its N-terminal amino-acid sequence. The 36-kDa major protein in the water-soluble fraction was purified, and the N-terminal amino-acid sequence of the protein was found to be identical to that of the water-stress protein (WspA) of N. commune. This WspA protein also showed heat-resistant beta-D-galactosidase activity. The fasciclin protein and WspA in the extracellular matrix may play a role in the hydrolysis of the EPSs surrounding the cells, possibly as an aid in the dispersal of cells, thus expanding the colonies of this cyanobacterium.

A beta-fructosidase (EC 3.2.1.26) was isolated from the midgut of larval sugar cane stalk borer Diatraea saccharalis by mild-denaturing electrophoresis and further purified to near homogeneity by gel filtration. beta-Fructosidase hydrolysed sucrose, raffinose and the fructosyl-trisaccharide isokestose, but it had no activity against maltose, melibiose and synthetic substrates for alpha-glucosidases. Two other sucrose hydrolases, one resembling a alpha-glucosidase (EC 3.2.1.20) and the other one active specifically against sucrose (sucrase) were detected in the larval midgut of D. saccharalis. All three sucrose hydrolases were associated with the midgut epithelium of larval D. saccharalis. Relative molecular mass (M(r)) of the beta-fructosidase was estimated around 45,000 (by gel filtration). The other two sucrose hydrolases had M(r) of 54,000 (alpha-glucosidase) and 59,000 (sucrase). The pH optima of the sucrose hydrolases were 5-10 for both alpha-glucosidase and sucrase and 7-8 for beta-fructosidase. Considering V(max)/K(m) ratios, beta-fructosidase preferentially cleaves isokestose rather than raffinose and sucrose. In order to evaluate the possible contribution of microorganisms isolated from the midgut to the pool of sucrose hydrolases, washed midgut epithelia were homogenised and plated onto appropriate media. Seven bacterial and one yeast species were isolated. None of the sucrose hydrolases extracted from the microorganisms corresponded to the enzymes isolated from midgut tissue homogenates. This result suggests that the major sucrose hydrolases found in the midgut of larval D. saccharalis were probably produced by the insect themselves not by the gut microflora.

Two proteins with alpha-galactosidase activity, alpha-galactosidase A (alpha-GalA) and alpha-galactosidase B (alpha-GalB, or alpha-N-acetylgalactosaminidase; alpha-NAGA) have a high homology of amino-acid sequence. Point mutations of the alpha-GalA gene have been reported only in the exons 1, 2 or 6. In this study, the exon 1-2 and/or 6 sequences of alpha-GalA cDNA were partly substituted by the corresponding regions of alpha-GalB cDNA, and three chimeric proteins were prepared by the baculovirus expression system: CMB12 with substitution at the exon 1-2 region, CMB6 at the exon 6 region, and CMB126 at both regions. They all preserved alpha-GalA antigenicity. Their kinetic properties toward 4-methylumbelliferyl alpha-galactopyranoside were compared with those of alpha-GalA. The catalytic activity was slightly low in CMB12, decreased to 1/10 in CMB6, and restored to a significant degree in CMB126. Km was more than 4-fold higher for CMB6 and CMB126 than for alpha-GalA. The pH optimum was 4.0 for both CMB12 and alpha-GalA, 4.8 for CMB6, and 4.6 for CMB126 and alpha-GalB. The catalytic activity was inhibited most by galactosamine in CMB6, and less in alpha-GalB, CMB126, alpha-GalA and CMB12 in decreasing order. The 50% inhibition concentrations of melibiose (Gal alpha 1-6Glc) and methyl alpha-galactopyranoside were 2.5- to 3-fold higher for CMB126 than for alpha-GalA. These results indicate that the low affinity of CMB126 to the substrate was caused by a reduced affinity to terminal alpha-linked galactose. We conclude that (1) the two regions encoded by exons 1-2 and 6 contribute to the alpha-galactosidic cleavage, and (2) an increase in Km of CMB6 or CMB126, with chimeric substitutions at the exon 6 region, was caused by a loss of affinity toward terminal alpha-linked galactose.

An exo-beta-(1----3)-D-galactanase from Driselase, a commercial enzyme preparation from Irpex lacteus (Polyporus tulipiferae) has been purified 166-fold. Apparent molecular weights of the purified enzyme, estimated by denaturing gel electrophoresis and gel filtration, were found to be 51,000 and 42,000, respectively. It hydrolyzed specifically oligosaccharides and polymers of (1----3)-linked beta-D-galactopyranosyl residues, and exhibited a maximal activity toward these substrates at pH 4.6. Based on the mode of the liberation of D-galactose from beta-(1----3)-D-galactan and the methyl beta-glycoside of beta-(1----3)-D-galactopentaose, the enzyme can be classified as an exo-glycanase capable of catalyzing the sequential hydrolytic release of single D-galactosyl residues from the nonreducing termini. The extent of the hydrolysis of the carbohydrate portion of acacia gum and radish arabinogalactan-proteins increased with their decreasing branching. Isolation and characterization of the major products formed from the proteoglycans indicated the action pattern of the enzyme to include the capability of bypassing the branching points. Consequently, the side chains carrying an additional D-galactosyl group at the reducing termini are released as neutral (1----6)-linked beta-D-galactooligosaccharides and their acidic derivatives having a 4-O-methyl-beta-D-glucuronosyl residue as the nonreducing end-group. The specificity and the mode of action showed the enzyme to be a useful tool for analyzing the fine structure of type II arabinogalactans and arabinogalactan-protein conjugates.

Lysosomal neuraminidase (sialidase; EC 3.2.1.18) and beta-galactosidase (EC 3.2.1.23), together with a carboxypeptidase, the so-called 'protective protein', were co-purified from the human placenta by affinity chromatography on a concanavalin A-Sepharose column followed by a thiogalactoside-agarose affinity column for beta-galactosidase. Analysis of the purified material by gel-filtration h.p.l.c. revealed three distinct molecular forms, all with high beta-galactosidase specific activity, but only the largest one expressed neuraminidase activity. Rechromatography of each individual species separately indicated that all three are in fact part of an equilibrium system (the neuraminidase-beta-galactosidase-carboxypeptidase complex or NGC-complex) and that these species undergo slow conversion into one another through dissociation and association of protomeric components. Each species was sufficiently stable for the determination of their hydrodynamic properties by gel-filtration h.p.l.c. and sedimentation velocity. The largest species had an apparent sedimentation coefficient S20.w, of 18.8 S and a Stokes' radius of 8.5 nm, giving a molecular mass of 679 kDa and a fractional ratio, f/f min, of 1.47. The latter value indicates that the macromolecule is asymmetric or highly hydrated. This large species is composed of four types of polypeptide chains of molecular mass 66 kDa (neuraminidase), 63 kDa (beta-galactosidase), 32 kDa and 20 kDa (carboxypeptidase heterodimer). The 32 kDa and 20 kDa protomers are linked together by a disulphide bridge. Glycopeptidase F digestion of the NGC-complex transformed the diffuse 66-63 kDa band on the SDS gel into two close but sharp bands at 58 and 56 kDa. The two smaller species which were separated on the h.p.l.c. column correspond to tetrameric and dimeric forms of the 66-63 kDa protomers and express exclusively beta-galactosidase activity. Treatment of the NGC-complex with increasing concentrations of guanidinium hydrochloride up to 1.5 M also resulted in dissociation of the complex into the same smaller species mentioned above plus two protomers of molecular mass around 60 and 50 kDa. A model of the largest molecular species as a hexamer of the 66-63 kDa protomers associated to five carboxypeptidase heterodimers (32 kDa and 20 kDa) is proposed

A plant has been constructed for the free-flow electrophoresis. It permits carrying out electrophoretic separation both in a free flow and with the use of neutral grained fillers. Beta-galactosidase has been purified from the culture liquid of the Escherichia coli cell phage lysate. Its content in the purified material amounted to 81.2

A thermophilic and thermostable beta-galactosidase activity was purified to homogeneity from crude extracts of the archaebacterium Sulfolobus solfataricus, by a procedure including ion-exchange and affinity chromatography. The homogeneous enzyme had a specific activity of 116.4 units/mg at 75 degrees C with o-nitrophenyl beta-galactopyranoside as substrate. Molecular mass studies demonstrated that the S. solfataricus beta-galactosidase was a tetramer of 240 +/- 8 kDa composed of similar or identical subunits. Comparison of the amino acid composition of beta-galactosidase from S. solfataricus with that from Escherichia coli revealed a lower cysteine content and a lower Arg/Lys ratio in the thermophilic enzyme. A rabbit serum, raised against the homogeneous enzyme did not cross-react with beta-galactosidase from E. coli. The enzyme, characterized for its reaction requirements and kinetic properties, showed a thermostability and thermophilicity notably greater than those reported for beta-galactosidases from other mesophilic and thermophilic sources.

The amino acid and sugar composition of the enzyme protein, the effect of urea, sodium dodecyl sulphate and Concanavalin A on the purified alpha-galactosidase (EC 3.2.1.22) from the mold Cephalosporium acremonium has been studied. The results obtained by gas liquid chromatography indicated the presence of N-acetylglucosamine, mannose, galactose and N-acetylneuramic acid in the molar proportions 2:7:3:11. The presence of two types of Asn-linked oligosaccharide structures in the enzyme molecule is assumed. The alpha-galactosidase liberates alpha(1-3), alpha(1-4) and alpha(1-6)-linked D-galactose units from various synthetic and natural substrates which have been tested. The effects of pH, substrate concentration and temperature on the catalytic activity of the enzyme are described. The purified alpha-galactosidase also exhibited a lectin activity with an affinity towards glucose, and to some extent mannose.

Extracellular alpha-galactosidase, a glycoprotein from the extracellular culture fluid of Aspergillus ficuum grown on glucose and raffinose in a batch culture system, was purified to homogeneity in five steps by ion exchange and hydrophobic interaction chromatography. The molecular mass of the enzyme was 70.8 Kd by SDS polyacrylamide gel electrophoresis and 74.1 Kd by gel permeation HPLC. On the basis of a molecular mass of 70.7 Kd, the molar extinction coefficient of the enzyme at 279 nm was estimated to be 6.1 X10(4) M-1 cm-1. The purified enzyme was remarkably stable at 0 degrees C. It had a broad temperature optimum and maximum catalytic activity was at 60 degrees C. It retained 33% of its activity after 10 min. at 65 degrees C. It had a pH optimum of 6.0. It retained 62% of its activity after 12 hours at pH 2.3. The Kms for p-nitrophenyl-alpha-D-galactopyranoside, o-nitrophenyl-alpha-D-galactopyranoside and m-nitrophenyl-alpha-D-galactopyranoside are: 1462, 839 and 718 microM. The enzyme was competitively inhibited by mercury (19.8 microM), silver (21.5 microM), copper (0.48 mM), zinc (0.11 mM), galactose (64.0 mM) and fructose (60.3 mM). It was inhibited non-competitively by glucose (83.2 mM) and uncompetitively by mannose (6.7 mM).

alpha-Galactosidase A (alpha-D-galactoside galactohydrolase, EC 3.2.1.22) was purified from human placenta. The purified enzyme showed one major band on polyacrylamide gel electrophoresis and a single precipitin line on double immunodiffusion. Electrophoresis of the purified, S-carboxymethylated enzyme on sodium dodecyl sulfate polyacrylamide gel showed one component with a molecular weight of about 65 000, but electrophoresis of the non-S-carboxymethylated enzyme showed two components, a major band with a molecular weight of 67 500 and a diffuse band with a molecular weight of 47 000. We suggest that the smaller diffuse component is a degradation product and that the enzyme is a dimer with a molecular weight of approximately 150 000 and a subunit of molecular weight of about 67 500. Antibody raised against the purified enzyme quantitatively precipitated alpha-galactosidase A, but not alpha-galactosidase in Fabry's disease fibroblasts. The alpha-galactosidase A is very heat labile and pH sensitive. It is most stable in concentrated solution at low temperature and at a pH of 5.0 to 6.0. When added to plasma at 37 degrees C, it has a half-life of only 17 min. This imposes a serious obstacle to its use in the treatment of Fabry's disease.

A highly sensitive microassay method and a microscale purification system were developed to isolate the residual acid beta-galactosidase in GM1-gangliosidosis fibroblasts. The sensitivity of the microassay system, composed of a 96-well microplate and a microplate fluorometer, was 100-fold higher than that of the conventional system and the response was linear in the pmole range. Acid beta-galactosidase was characterized as a thiol enzyme which was inactivated by a mercuric compound. This enzyme was completely adsorbed on an Hg-agarose column and was easily eluted from the column by 10 mM 2-mercaptoethanol. The microscale purification system using Con A-Sepharose, PAT-Sepharose, and Hg-agarose column chromatography achieved 565- and 7,970-fold purifications of acid beta-galactosidase with an overall yields of 44% and 45% from normal and GM1-gangliosidosis fibroblasts, respectively. The purified enzyme fractions did not contain any other lysosomal enzyme activities except for a small amount of beta-N-acetylhexosaminidase activity.

Forty-one strains of Kluyveromyces fragilis (Jörgensen) van der Walt 1909 varied 60-fold in ability to produce lactase (beta-galactosidase). The four best strains were UCD No. 55-31 (Northern Regional Research Center NRRL Y-1196), UCD No. C21(-), UCD No. 72-297(-), and UCD No. 55-61 (NRRL Y-1109). Biosynthesis of lactase during the growth of K. fragilis strain UCD No. 55-61 was followed on both lactose and sweet whey media. Maximum enzyme yield was obtained at the beginning of the stationary phase of growth. Bets lactase yields from K. fragilis UCD No. 55-61 were obtained with 15% lactose and an aeration rate of at least .2 mmol oxygen/liter per min. Supplementary growth factors were unneccessary for good lactase yeilds when yeast was grown on whey media. Best extraction of lactase from fresh yeast cells was obtained by toluene autolysis (2% vol/vol) at 37 C in .1 M potassium phosphate buffer, pH 7.0, containing .1 mM manganese chloride and .5 mM magnesium sulfate. The enzyme was concentrated and purified partially by acetone precipitation. At least 95% of the enzyme activity of the concentrated solution was retained after storage for 7 days at 22 C, for 3 wk at 4 C, and for 6 wk at -20 C.

An improved method is described for the preparation of bovine testicular beta-galactosidase that allows the isolation of enzyme fractions that bind avidly to phosphomannosyl receptors. The procedure permits removal of a contaminating beta-hexosaminidase and yields nearly homogeneous beta-galactosidase. Enzyme eluted from DEAE-Sephacel was arbitrarily divided into pools that exhibited differing ability to bind phosphomannosyl receptors. A high binding fraction was rapidly assimilated by cultured cells and bound to both low and high molecular weight phosphomannosyl receptors. Carbohydrate analysis of the high binding fraction indicates an average content of one complex and one high mannose oligosaccharide chain per molecule and an average mannose 6-phosphate content of two residues per molecule. However, electrofocusing studies indicated that all the fractions were heterogeneous with respect to sialic acid and phosphate content. The purification procedure also provides highly purified beta-galactosidase suitable for removing beta-galactosidase residues from a variety of complex carbohydrates.

A method is described for the preparation of a monomeric Fab'-beta-D-galactosidase conjugate, which is required for the development of a sensitive immunoenzymometric assay. Anti-human IgG F(ab')2 was labeled with 2,4-dinitrophenyl groups, split into Fab' by reduction and reacted with excess maleimide groups which had been introduced into beta-D-galactosidase through thiol groups using N,N'-o-phenylenedimaleimide. The monomeric 2,4-dinitrophenyl Fab'-beta-D-galactosidase conjugate was subsequently separated from unconjugated beta-D-galactosidase by affinity chromatography on a column of (anti-2,4-dinitrophenyl) IgG-Sepharose 4B. In the monomeric conjugate preparation, 98% of beta-D-galactosidase activity was associated with Fab' and 90% was associated with specific (anti-human IgG) Fab'. This conjugate allowed the measurement of 0.1 fmol of human IgG by an immunoenzymometric assay technique.

beta-D-Galactosidase activity has been detected previously in the cotyledons of germinated nasturtium (Tropaeolum majus L.) seeds and has been linked to the hydrolysis in vivo of storage xyloglucan (amyloid) (Edwards, M., Dea, I. C. M., Bulpin, P. V., and Reid, J. S. G. (1985) Planta (Berl.) 163, 133-140). The major beta-D-galactosidase present in extracts from the cotyledons of 9-day seedlings has now been purified to apparent homogeneity. The enzyme (Mr 97,000, no subunits) comprised a range of closely related molecular species ranging in isoelectric point from pH 6.6 to 7.1. Further purification to give a single protein band on isoelectric focusing (pI = 7.1) was achieved by chromatofocusing. The pH optimum of the enzyme (mixed molecular species) was 4.0-5.0 (stable from pH 3-10), and the temperature optimum was 50 degrees C (stable to 50 degrees C). It hydrolyzed lactose and beta-D-galactopyranosides but not melibiose and alpha-D-galactopyranosides. It did not release the terminal nonreducing alpha-D-galactopyranosyl residues from seed galactomannans, but catalyzed the rapid removal of terminal nonreducing beta-D-galactopyranosyl residues from xyloglucans. On the basis of the ability of the enzyme to hydrolyze xyloglucans, the kinetics of xyloglucan hydrolysis, and an experimental demonstration of a clear correlation between xyloglucan depletion and the activity in vitro of this enzyme, it is argued that the cell-wall storage xyloglucan of the nasturtium seed is its natural substrate.

Two GM1 ganglioside beta-galactosidases, multimeric form (enzyme I) and monomeric form (enzyme IV), have been purified from bovine liver by the procedures comprising Sephadex G-100 gel filtration, affinity chromatographies on Concanavalin A (Con A)-Sepharose and p-aminophenyl thio-beta-galactoside-CH-Sepharose (PATG-Sepharose) and Sephadex G-200 gel filtration. The multimeric form of the enzyme was purified 13,000-fold and monomeric form was 68,700-fold. On sodium dodecyl sulfate poly acrylamide gel electrophoresis, the monomeric form of the enzyme gave a single protein band with a molecular weight of 65,000, while the multimeric form gave two minor protein bands with molecular weights of 32,000 and 20,000 in addition to the major band at 65,000. Both enzymes liberated the terminal galactose from GM1 ganglioside and lactosylceramide. Enzyme I showed a broad pH optimum between pH 4.3 and 5.0, while enzyme IV was most active at pH 4.75. The pI values of beta-galactosidases I and IV were 4.6 and 5.8, respectively. Both enzymes were quite stable upon preincubation at 45 degrees C under acidic condition (pH 4.5), but rapidly lost their activities under neutral condition (pH 7.0). The apparent Km values for GM1 ganglioside of beta-galactosidases I and IV were calculated to be 2.0 x 10(-4) M and 3.3 x 10(-4) M, respectively.

Although lysosomal enzyme activities are known to vary in response to numerous physiological and pharmacological stimuli, the relationship between lysosomal enzyme activity and enzyme concentration has not been systematically studied. Therefore we developed radioimmunoassays for two lysosomal glycosidases in order to determine lysosomal enzyme concentration. beta-Galactosidase and beta-glucuronidase were purified from rat liver 2780-fold and 1280-fold respectively, by using differential centrifugation, affinity chromatography, ion-exchange chromatography and molecular-sieve chromatography. Polyclonal antibodies to these enzymes were raised in rabbits, and two radioimmunoassays were established. Antibody specificity was shown by: (i) selective immunoprecipitation of enzyme activity; (ii) identical bands of purified enzyme on SDS/polyacrylamide-gel electrophoresis and immunoelectrophoresis; (iii) single immunoreactive peaks in molecular-sieve chromatography experiments. Sensitivities of the assays were such that 15 ng of beta-galactosidase and 45 ng of beta-glucuronidase decreased the ratio of bound to free radiolabel by 50%; minimal detectable amounts of immunoreactive enzymes were 2 ng and 10 ng respectively. The assays were initially used to assess the effects of physiological perturbations (i.e. fasting and age) on enzyme concentrations in rat liver; these experiments showed that changes in enzyme concentrations do not always correlate with changes in enzyme activities. This represents the first report of radioimmunoassays for lysosomal glycosidases. The results suggest that these radioimmunoassays provide useful technology for the study of regulatory control mechanisms of the concentrations of lysosomal glycosidases in mammalian tissues.

The technique of affinity chromatography has been used in the partial purification of complementable fractions and complemented enzyme of beta-galactosidase from Escherichia coli mutant M15. The crude extract of mutant M15 was incubated with fragment CM-B. The complemented enzyme and complementable fractions were passed through a small column of p-aminophenyl-beta-D-thiogalactoside to which inhibitors had been covalently attached. A high percentage of the nonspecific protein passed directly through the affinity column while the specific enzymatic protein remained bound to the gel. Phosphate buffer with NaCl was used to elute the complementable fractions from the column. Sodium borate buffer was used to elute the bound complemented enzyme from the affinity support. The results of this study show that 100% of the complemented enzyme was bound to the column. The partially purified enzyme had the same position in disc gel electrophoresis as beta-galactosidase from E. coli.

alpha-Galactosidase was isolated from germinating guar. The extract also contained small amounts of alpha-mannosidase and beta-mannosidase activities. The fractionation of the enzyme extract with ammonium sulphate (75% saturation) resulted in the appearance of all the three enzymes in a floating lipid complex. The inclusion of detergents such as Triton X-100 and sodium deoxycholate in the extraction medium failed to prevent the appearance of these enzymes in the floating lipid complex. However, by using acetone powder of the seedlings, alpha-galactosidase could be sedimented with ammonium sulphate. The presence of detergents in the extraction medium affected the molecular properties of the enzyme. Using a set of carefully selected conditions alpha-galactosidase was purified to apparent homogeneity. Analytical ultracentrifugation and gel filtration studies of the purified enzyme showed association-dissociation phenomenon as a function of pH and temperature. The effect of pH on the association-dissociation indicates the predominance of electrostatic interactions in the association of subunits.

Human intestinal lactase/phlorizin hydrolase (EC 3.2.1.23/62) was purified in its amphiphilic form by immunoadsorbent chromatography. The purification factor was approximately 600 and the recovery 14%. The enzyme was essentially free from other known brush-border peptidases and disaccharidases and appeared homogeneous in crossed immunoelectrophoresis and polyacrylamide gel electrophoresis in sodium dodecylsulphate. The purified enzyme hydrolyzed lactose (pH optimum 5.8--6.0, Km 21 mM), phlorizin (Km 0.44mM) and other beta-galactosides and beta-glucosides. Tris inhibited the hydrolysis of lactose whereas phlorizin hydrolysis was almost unaffected. The activity against these two substrates also showed different thermal stability. It is suggested that the human enzyme has two different sites: one for lactose hydrolysis, inhibited by phlorizin and one for phlorizin hydrolysis. By gel filtration on Ultrogel AcA 34 the amphiphilic form of the enzyme had a molecular weight of 320000 while the hydrophilic form (papain-treated) had a molecular weight of 280000. This indicates that the anchoring segment(s) plus the bound detergent has a molecular weight of approximately 40000. In polyacrylamide gel electrophoresis in sodium dodecylsulphate the fully denatured enzyme had an apparent molecular weight of 160000. It is therefore suggested that the human lactase/phlorizin hydrolase is composed of two monomers each with a molecular weight of 160000. The electromicroscopic picture gives further evidence for this suggestion. In addition the possibility of a high molecular weight, one polypeptide chain is discussed.

BETA-Galactosidase (EC 3.2.1.23), prepared from strains ML 308 and K12 3300 of Escherichia coli, dissociated into an inactive monomer in the presence of Ag+. When such a monomer preparation is treated with excess of thiol an enzymically active dimer is formed in addition to an active tetramer. It is suggested that Ag+ may be of value in studies on other multimeric proteins as a mild dissociating agent.

The effects of overloading of the sample zone in density gradient centrifugation have been studied by use of a three-component shelf-lavered sample in which the total protein concentration was increased by addition of different amounts of albumin. It is found that overloading of the gradient gives rise to particle movements which are not predictable from the Svedberg equation. The two typical effects of overloading are dislocation of the zone mass centres and changes in the zone shapes. It is found that the magnitude of the calculated sedimentation coefficients increases nearly linearly with increasing sample load. The changes in zone shapes are found to depend on the specific load and two different patterns may be distinguished. The zone of the sample component which causes the overloading is defined as primarily overloaded and the others as secondarily overloaded. In primarily overloaded zones the original Gaussian shape is lost, while in secondarily overloaded zones the Gaussian zone shape is maintained, although a zone broadening is seen. Extreme high loads are found to be able to divide single zones. As a whole these experiments show that evidence for a non-overloaded set of experimental conditions must be provided, when density gradient centrifugation is used for determination of sedimentation coefficients. For preparative gradient centrifugations the power of resolution will decrease with increasing sample load. A simple method to detect overloading in density gradient centrifugations is described.

Pig intestinal lactase/phlorizin hydrolase (EC 3.2.1.23/62) was purified in its amphiphilic form by immunoadsorbent chromatography. The purified enzyme was free of other known brush border enzymes and appeared homogeneous in immunoelectrophoresis and polyacrylamide gel electrophoresis in the presence of SDS. Pig lactase/phlorizin hydrolase was shown to have the same quaternary structure as the human enzyme, i.e., built up of two polypeptides of the same molecular weight (160000). In addition to hydrolyzing lactose, phlorizin and a number of synthetic substrates, both the human and the pig enzyme were shown to have a considerable activity against cellotriose and cellotetraose, and a low but significant activity against cellulose. The lactase/phlorizin hydrolase isolated from pigs in which the pancreatic ducts had been disconnected 3 days before death and from Ca2+-precipitated enterocyte membranes (basolateral and intracellular membranes) exhibited in SDS-polyacrylamide gel electrophoresis the same size of constituent polypeptides and the same catalytic and immunological properties as a normal brush border lactase/phlorizin hydrolase.

A beta-D-galactosidase from bovine liver was purified to apparent homogeneity. The major purification step was affinity chromatography on a column of D-galactose attached to a Sepharose support activated with divinyl sulfone. Affinity media prepared by binding ligands to Sepharose activated with cyanogen bromide were unsuitable for purification of the enzyme, even though such media have been used to purify beta-D-galactosidases from other sources. The molecular weight of the denatured enzyme was 67,000. The molecular weight of the native enzyme at pH 7.0 was 68,000, and at pH 4.5 or 5.0, was 141,000. These data suggest that the enzyme has a single, fundamental subunit with a molecular weight of 67,000, and that the enzyme exists as a monomer at pH 7.0, and a dimer at pH 4.5 or 5.0. The Vmax values of the enzyme with p-nitrophenyl beta-D-galactoside, p-nitrophenyl beta-D-fucoside, lactose, and beta-Gal-(1----4)-beta-GlcNAc-1---- OC6H4NO2 -p were 10,204, 11,550, 9,479, and 8,859 nmol/min/mg of protein, respectively, and the Km values for these substrates were 0.08, 14.9, 14.2, and 1.6mM, respectively. D-Galactose, beta-D- galactosylamine , p-aminophenyl 1-thio-beta-D-galactoside, and D- galactono -1,4-lactone were competitive inhibitors of the enzyme, with Ki values of 0.9, 0.6, 0.6, and 0.8mM, respectively. The enzyme catalyzed the transfer of the D-galactosyl group from p-nitrophenyl beta-D-galactoside to D-glucose. The pH optimum of the enzyme was 4.5, and the pI was 4.7.

Human intestinal lactase-phlorizin hydrolase (lactase) was selectively isolated with monospecific polyclonal antibodies to rat lactase. In addition to their immunologic similarities indicated by this isolation, human and rat lactase have similar kinetic characteristics but different subunit structure when analyzed by gel electrophoresis under reducing conditions. Rabbits immunized by injecting human lactase complexed with anti-rat lactase produced specific antibodies to human lactase that exhibited little cross-reactivity to the rat enzyme. The simple single-step procedure allows isolation of human lactase in high purity from small biologic samples and preparation of specific antisera to the human enzyme.

Two forms, I and II, of an acid beta-galactosidase from rabbit spleen were separated by DEAE-cellulose chromatography and then characterized. Both forms of the enzyme showed different heat-stability (form I being heat-labile and form II heat-stable), and different pI (6.7 for form I and 5.3 and 6.7 for form II). Their gel filtration patterns were also different: form I was resolved in a single peak of mol. wt. 75,000, whereas form II was resolved in one or two peaks of mol. wt. 120,000 and greater than 200,000, depending on the pH of elution. However, both forms had similar pH stability and behavior toward alpha-methyl-beta-D-galactopyranoside, alpha-methyl-beta-D-glucopyranoside, urea and KCl. Differences in pH optima, optimal temperature and Km values were not marked.

The common identity of human acidic beta-D-glucosidase (beta-D-glucoside glucohydrolase, EC 3.2.1.21) and beta-D-xylosidase (1,4-beta-D-xylan xylohydrolase, EC 3.2.1.37) as one enzyme and that of acidic beta-D-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23), beta-D-fucosidase (no allotted EC number) and alpha-L-arabinosidase (alpha-L-arabinofuranoside arabinohydrolase, EC 3.2.1.55) as another enzyme is indicated by similar binding patterns of glycosidase activities of each enzyme to various lectins. by similar ratios between their intra- and extracellular levels in normal and I-cell fibroblasts and by their deficiencies in liver tissues from patients with Gaucher disease and GM1 gangliosidosis, respectively. A third enzyme, neutral beta-D-galactosidase, purified to homogeneity from human liver has been shown to possess all these five glycosidase activities at neutral pH. These neutral enzymic activities were not bound by any of the lectins examined and found to be reduced in liver and spleen of a patient with neutral beta-D-galactosidase deficiency. An additional form of beta-D-xylosidase with optimal activity at pH 7.4 was bound by the fucose-binding lectin from Ulex eurpaeus while no binding was observed for the acidic (pH 4.8) and neutral (pH 7.0) beta-D-xylosidase activities of the multiple glycosidase enzymes.

Extra- and intracellular glycanohydrolases were isolated from Aspergillus flavus and partially characterized. Both preparations exhibited beta-galactosidase activity. Gel chromatography of the extracellular enzyme preparation on Sephadex revealed one protein fraction containing beta-galactosidase activity and a second one exhibiting mainly beta-xylosidase activity. Electrophoresis in starch gel and disc electrophoresis in polyacrylamide gel showed that the preparation obtained from the cultivation broth contained five protein fractions, whereas two protein fractions could be detected in the intracellular preparation. Hydrolysis of a partially degraded polysaccharide of peach gum by the above preparations yielded D-galactose as the main product and traces of D-mannose, L-arabinose, D-xylose and a number of oligosaccharides.