A rapid method for assay of glycosidases involved in glycoprotein biosynthesis

A rapid procedure to measure processing glycosidases with labeled oligosaccharide as substrate is described, using assay of the specific processing alpha-mannosidase from Saccharomyces cerevisiae as an example. After incubation of [3H]mannose-labeled Man9GlcNAc with the mannosidase, a solution of concanavalin A is added, followed by polyethylene glycol to precipitate the oligosaccharide-lectin complex. The radioactivity present in the supernatant after centrifugation is then measured to determine the amount of labeled mannose released. It is shown that the results of this procedure are similar to those obtained previously using small columns of concanavalin A-Sepharose (B. Saunier, R. D. Kilker, Jr., J. S. Tkacz, A. Quaroni, and A. Herscovics (1982) J. Biol. Chem. 257, 14155-14161). The precipitation procedure, which can be applied to the assays of other processing enzymes, is much more convenient when a large number of samples must be analyzed.     

Fractionation of glycopeptides by affinity column chromatography on concanavalin A-sepharose.

Using [3H]-labeled oligosaccharides, we found that the presence of at least two alpha-mannosyl residues with free hydroxyl groups at C-3, 4, and 6 is required for oligosaccharides to be related by a concanavalin A-Sepharose column. This finding is also applicable to N-[14C]acetylated glycopeptides. Thus, the concanavalin A-Sepharose column might become a useful tool for structural studies of glycopeptides and oligosaccharides and for their fractionation. Glycopeptides prepared from the trypsinate of rat fibroblasts, which has been purified by paper electrophoresis, were further separated into two fractions by chromatography on a concanavalin A-Sepharose column.     

A direct continuous spectrophotometric assay for glycosidases with 3-nitro-2-pyridyl glycosides by tautomerization of 2-hydroxy-3-nitropyridine

Two kinds of 3-nitro-2-pyridyl glycosides were synthesized and evaluated as substrates for continuous spectrophotometric assay for glycosidases. The liberated aglycon, 2-hydroxy-3-nitropyridine, immediately tautomerized to 3-nitro-2(1H)-pyridone, causing an absorption shift of ca. 60 nm even under acidic conditions (pH 3-6). Consequently, the enzymatic hydrolysis of these glycosides was monitored continuously in the acidic to neutral pH range (pH 4-7), the optimum pH for most glycosidases. The absorbance of liberated aglycon increased linearly at 390 nm until 10% consumption of the substrate to enable the initial rate to be determined at once without terminating the reaction. The kinetic parameters for the hydrolysis of 3-nitro-2-pyridyl glycosides were obtained from the slopes of the progress curves and were compared with those obtained from the conventional discontinuous assay using p- and o-nitrophenyl glycosides as substrates. The kinetic parameters indicated that 3-nitro-2-pyridyl glycosides were more activated and specific substrates, but with less affinity to the enzymes than the corresponding nitrophenyl glycosides. Moreover, the absorbance shift by tautomerization should promise further applications to continuous spectrophotometric assays for other enzymes acting under acidic conditions, such as acid proteases and acid phosphatases.     

Postnatal changes in N-linked oligosaccharides of glycoproteins in rat liver.

[3H]Mannose-labelled glycopeptides in the slices of livers from neonatal and 1-, 2-, 3- and 5-week-old rats were characterized by column chromatographies on Sephadex G-50 and concanavalin A-Sepharose and by endo-beta-N-acetylglucosaminidase H digestion. The proportion of complex-type glycopeptides was increased with time until 2 weeks post partum and then returned to the neonatal level. This was mainly due to the increased proportion of concanavalin A-bound (biantennary) species. These changes were accompanied by consistent changes in the activities of processing enzymes in liver microsomal fraction, especially of N-acetylglucosaminyltransferase I. Complex-type glycopeptides from neonatal and 2- and 5-week-old rat livers were further characterized by column chromatographies on Bio-Gel P-6 and DE 52 DEAE-cellulose in combination with neuraminidase digestion. No significant difference was found between concanavalin A-bound species from neonatal liver and those from liver 5 weeks post partum, most of which were sialylated. Concanavalin A-bound species 2 weeks post partum were comparatively smaller in size and less sialylated. On the other hand, there was no significant difference among concanavalin A-unbound species from the three different sources, most of which were sialylated. Since glycoproteins from regenerating rat liver also contain a higher proportion of complex-type oligosaccharides, as previously reported, such changes in N-linked oligosaccharides of glycoproteins may be related to control of the growth of liver cells.     

Characterization of the N- and O-linked oligosaccharides in glycoproteins synthesized by Schistosoma mansoni schistosomula

This report describes the structural analyses of the O- and N-linked oligosaccharides contained in glycoproteins synthesized by 48-hr-old Schistosoma mansoni schistosomula. Schistosomula were prepared by mechanical transformation of cercariae and were then incubated in media containing either [2-3H] mannose, [6-3H]glucosamine, or [6-3H]galactose to metabolically radiolabel the oligosaccharide moieties of newly synthesized glycoproteins. Analysis by SDS-polyacrylamide gel electrophoresis and fluorography demonstrated that many glycoproteins were metabolically radiolabeled with the radioactive mannose and glucosamine precursors, whereas few glycoproteins were labeled by the radioactive galactose precursor. Glycopeptide were prepared from the radiolabeled glycoproteins by digestion with pronase and fractionated by chromatography on columns of concanavalin A-Sepharose and pea lectin-agarose. The structures of the oligosaccharide chains in the glycopeptides were analyzed by a variety of techniques. The major O-linked sugars were not bound by concanavalin A-Sepharose and consisted of simple O-linked monosaccharides that were terminal O-linked N-acetylgalactosamine, the minor type, and terminal O-linked N-acetylglucosamine, the major type. The N-linked oligosaccharides were found to consist of high mannose- and complex-type chains. The high mannose-type N-linked chains, which were bound with high affinity by concanavalin A-Sepharose, ranged in size from Man6GlcNAc2 to Man9GlcNAc2. The complex-type chains contained mannose, fucose, N-acetylglucosamine, and N-acetylgalactosamine. No sialic acid was present in any metabolically radiolabeled glycoproteins from schistosomula.     

Isolation of a concanavalin A receptor from mouse L cells.

A cell surface glycoprotein receptor for concanavalin A (Con A) has been isolated from mouse L cells. The isolation procedure involved dissolving whole L cells in 0.3 M lithium diiodosalicylate and extracting with aqueous phenol. The Con A receptor, which was found in the aqueous phase of this extract, was further purified by affinity chromatography on a column of Con A-Sepharose; the receptor was adsorbed to Con A-Sepharose and eluted with 0.1 M methyl alpha-D-glucopyranoside or with 0.1 M methyl alpha-D-mannopyranoside, but not with other monosaccharides. The cell surface location of the Con A receptor purified in this way was confirmed by showing that it can be isolated from purified L cell plasma membranes and by demonstrating that it can be labeled from the exterior surface of intact L cells by the nonpenetrating galactose oxidase-KB3H4 system. Biochemical studies of the Con A receptor have shown that it migrates on sodium dodecyl sulfate-polyacrylamide gels as a single component having an apparent molecular weight of approximately 100,000. Its N-terminal amino acid is valine and it has carbohydrate attached at several (at least five) different sites along the polypeptide chain.     

Assay for phosphatidylserine decarboxylase utilizing DEAE-cellulose column chromatography

A simple assay for phosphatidylserine decarboxylase is described. Following incubation of a mitochondrial fraction from Saccharomyces cerevisiae with purified, exogenous phosphatidyl[3H]serine, the lipid extract is applied to a small DEAE-cellulose column equilibrated in CHCI3-CH3OH (1:1). The unreacted substrate, phosphatidyl[3H]serine, is quantitatively bound by the ion-exchange column while the product, phosphatidyl[3H]ethanolamine, is eluted by sequential washing with CHCI3-CH3OH (1:1) and CH3OH. The organic solvents are evaporated, and the amount of radiolabeled phosphatidyl[3H]ethanolamine formed by enzymatic decarboxylation is determined by liquid scintillation spectrometry. The reliability of this assay was established by showing that several enzymatic properties of the yeast enzyme, defined by the new assay, were essentially identical to the properties characterized by a more tedious paper chromatographic assay described previously. Virtually identical rates of enzymatic decarboxylation of phosphatidyl[3H]serine were also obtained for mitochondrial fractions from pig brain and rat liver when the activities were compared by the column and paper chromatographic methods.     

A radiometric assay for phenylethanolamine N-methyltransferase and catechol O-methyltransferase in a single tissue sample: application to rat hypothalamic nuclei, pineal gland, and heart

A simple and highly sensitive method for simultaneous assay of phenylethanolamine N-methyltransferase (PNMT) and catechol O-methyltransferase (COMT) is described. These enzymes are determined in a single tissue homogenate using S-[methyl-3H] adenosyl-L-methionine as methyl donor and sequentially incubating with the substrates phenylethanolamine and epinephrine. The radioactive products of the enzymatic reactions, N-methylphenylethanolamine and metanephrine, are extracted and then separated by thin-layer chromatography. The identity of the reaction products has been established chromatographically and the conditions for both enzymatic reactions in the assay procedure have been defined. Measurement of PNMT activity in the rat pineal gland or in minute fragments of other tissues (e.g., brain nuclei) has not been possible using previously described methods. Activities of PNMT and COMT in the rat pineal gland, various hypothalamic nuclei, and the auricular and ventricular myocardia are herein reported.     

Studies on the carbohydrate-protein linkage region in bovine corneal keratan sulfate. III. Evidence for a biantennary structure

Peptidokeratan sulfates were retained by a concanavalin A-Sepharose column and readily eluted with 20 mM methyl alpha-D-mannoside from the column. Linkage region-enriched glycopeptides, prepared by endo-beta-galactosidase digestion of the peptidokeratan sulfates, behaved similarly to the peptidokeratan sulfates on the same affinity chromatography. Further, behaviors of both the peptidokeratan sulfates and the linkage region-enriched glycopeptides on pea lectin-Agarose columns were consistent with the results of the chromatography on concanavalin A-Sepharose. These findings confirmed the typical biantennary structure previously suggested for the linkage region on the basis of structural studies of the linkage region-enriched glycopeptides.     

Identification of latent procathepsin H in microsomal lumen: characterization of proteolytic processing and enzyme activation

Procathepsin H in kidney and liver microsomal lumen was identified to have a molecular mass of 41 kDa by immunoblot analysis. The proenzyme was then concentrated by applying the microsomal contents to a concanavalin A-Sepharose column. When the concanavalin A-adsorbed fraction was incubated at pH 4.0 at 20 degrees C, the activity measured with synthetic substrate increased 3.5 times over that of the control after 24 h incubation. Immunoblot analysis showed that acidic treatment caused the disappearance of procathepsin H. Thus the proenzyme might be processed to the mature enzyme under acidic conditions. The marked increase of enzymatic activity and the conversion of proenzyme were completely blocked with pepstatin which is a potent inhibitor of aspartic proteases. These results suggested that a protease for processing procathepsin H might be cathepsin D, a major lysosomal aspartic protease. Therefore, procathepsin H seems to be synthesized first in the enzymatically inactive form in endoplasmic reticulum and successively converted into the active form in lysosomes during biosynthesis.     

A Sensitive and Reliable Assay for Dopamine (β-Hydroxylase in Tissue

A new assay procedure for dopamine β-hydroxylase (DBH) in tissue extracts is described. Solubilized DBH was adsorbed from crude extracts on Concanavalin A-Sepharose (Con A-Sepharose), resulting in enrichment of the enzyme as well as removal of endogenous catecholamines and inhibitory substances. The enzymatic assay was carried out with DBH still adsorbed to Con A-Sepharose. The adsorption of the DBH to Con A-Sepharose offers three advantages over previous assay procedures. (1) Because of removal of the endogenous inhibitory substances, a single Cu²⁺ concentration can be used for the determination of DBH activity, regardless of the tissue dilution or inhibitor content of the analysed sample. Using this procedure, the optimal Cu²⁺ concentration for DBH of bovine adrenal gland extracts was 3 μM and for rat brain 10 μM. (2) Because of removal of endogenous catecholamines, dopamine, the main physiological substrate of DBH in noradrenergic neurons, can be used for the assay. The enzymatic reaction product, noradrenaline, was determined by high performance liquid chromatography and electrochemical detection (hplc-ec). This procedure resulted in an approx. 10-fold increase in sensitivity of the assay compared with other procedures, e.g., the radioenzymatic assay. (3) Direct determination of the immediate product of the enzymatic reaction (noradrenaline) permits kinetic analysis. It was found that the Michaelis constants for the substrate (dopamine) and co-factor (ascorbic acid) (2 mM and 0.65 mM, respectively) determined in bovine adrenal tissue extracts by the described procedure were identical with the values for the purified DBH preparation.     

Glycoprotein as a Constituent of Purified γ-Aminobutyric Acid/Benzodiazepine Receptor Complex: Structures and Physiological Roles of Its Carbohydrate Chain

The effect of treatments with various enzymes and chemically modifying agents on [3H]muscimol binding to a purified gamma-aminobutyric acid (GABA)/benzodiazepine receptor complex from the bovine cerebral cortex was examined. Treatments with pronase, trypsin, guanidine hydrochloride, and urea significantly decreased the binding of [3H]muscimol, but dithiothreitol, N-ethylmaleimide, reduced glutathione, oxidized glutathione, cysteine, and cystine had no significant effect. These results indicate that the GABA receptor indeed consists of protein, but -SH and -S-S- groups in the protein are not involved in the exhibition of the binding activity. On the other hand, column chromatography using concanavalin A-Sepharose eluted protein having [3H]muscimol binding activity and staining of glycoprotein using an electrophoresed slab gel indicated the existence of two bands originating from the subunits of the GABA/benzodiazepine receptor complex. Furthermore, treatments with various glycosidases such as glycopeptidase A, beta-galactosidase, and alpha-mannosidase significantly increased the binding of [3H]muscimol. These results strongly suggest that GABA/benzodiazepine receptor complex is a glycoprotein and that its carbohydrate chain may be a hybrid type. Treatment with beta-galactosidase resulted in the disappearance of the low-affinity site for [3H]muscimol binding and in an increase of Bmax of the high-affinity site, without changing the KD value. These results suggest that the carbohydrate chain in the receptor complex may have a role in exhibiting the low-affinity binding site for GABA. The observation that the enhancement of [3H]muscimol binding by treatments with beta-galactosidase and glycopeptidase A were much higher than that with alpha-mannosidase may also indicate a special importance of the beta-galactosyl residue in the inhibition of GABA receptor binding activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Occurrence of glucose polymer in undifferentiated PC12 cells

A large glucose polymer was found, following pronase digestion, in PC12 pheochromocytoma cells metabolically labeled with [1-3H]galactose. The polymer was excluded from a Bio-Gel A-0.5 m column and adsorbed by immobilized concanavalin A-Sepharose from which it was eluted with 10 mM alpha-methylmannoside. Glucose was found to be the sole component monosaccharide. Except for those capable of degrading glycogen, no exo- or endo-glycosidases cleaved the polymer. This is the first report on the occurrence of a glucose polymer in undifferentiated PC12 cells.     

Temporal aspects of O-glycosylation of glycoprotein C from herpes simplex virus type-1.

Herpes simplex virus type-1 glycoprotein C (gC1) contains several O-linked oligosaccharides clustered near N-linked chains, and Pronase digestion produces glycopeptides carrying both oligosaccharide types. We have taken advantage of this fact to investigate the temporal relationship between the initiation of O-linked chains and the processing of N-linked oligosaccharides. gC1 was isolated from herpes-simplex-virus-infected BHK (baby-hamster kidney) cells after short labelling periods with [3H]glucosamine, and the labelled Pronase-cleaved glycopeptides fractionated on concanavalin A-Sepharose. N-[3H]Acetylgalactosamine, mostly convertible into free N-[3H]acetylgalactosaminitol on mild alkaline-borohydride treatment, was found in glycopeptides with an affinity to concanavalin A-Sepharose corresponding to that of glycopeptides carrying Man8GlcNAc2 or larger N-linked chains. Since there is evidence that the processing of N-linked chains up to Man8GlcNAc2 involves enzymes located in the rough endoplasmic reticulum, current results strongly suggest that gC1 acquires O-linked N-acetylgalactosamine before the glycoprotein routing to the Golgi apparatus. The addition of the second sugar to the nascent O-linked chain appeared to occur after a relatively long lag time.     

Sequential deglycosylation and utilization of the N-linked, complex-type glycans of human alpha1-acid glycoprotein mediates growth of Streptococcus oralis

Streptococcus oralis is the agent of a large number of infections in immunocompromised patients, but little is known regarding the mechanisms by which this fermentative organism proliferates in vivo. Glycoproteins are widespread within the circulation and host tissues, and could provide a source of fermentable carbohydrate for the growth of those pathogenic organisms with the capacity to release monosaccharides from glycans via the production of specific glycosidases. The ability of acute phase serum alpha1-acid glycoprotein to support growth of S.oralis in vitro has been examined as a model for growth of this organism on N-linked glycoproteins. Growth was accompanied by the production of a range of glycosidases (sialidase, N-acetyl-beta-D-glucosaminidase, and beta-D-galactosidase) as measured using the 4-methylumbelliferone-linked substrates. The residual glycoprotein glycans remaining during growth of this organism were released by treatment with hydrazine and their analysis by HPAEC-PAD and MALDI demonstrated extensive degradation of all glycan chains with only terminal N-acetylglucosamine residues attached to asparagines of the protein backbone remaining when growth was complete. Monosaccharides were released sequentially from the glycans by S.oralis glycosidases in the order sialic acid, galactose, fucose, nonterminal N-acetylglucosamine, and mannose due to the actions of exo-glycosidic activities, including mannosidases which have not previously been reported for S.oralis. All released monosaccharides were metabolized during growth with the exception of fucose which remained free in culture supernatants. Direct release of oligosaccharides was not observed, indicating the absence of endo-glycosidases in S.oralis. We propose that this mechanism of deglycosylation of host glycoproteins and the subsequent utilization of released monosaccharides is important in the survival and persistence of this and other pathogenic bacteria in vivo.     

Radiolabeled heparan sulfate immobilized on microplate as substrate for the detection of heparanase activity

We developed a quantitative assay to monitor the enzymatic activity of heparanase, a protein responsible for the degradation of heparan sulfate (HS) present on cell surface and extracellular matrix. Our assay is based on a new procedure to immobilize radiolabeled HS to a solid support by a single end which is adaptable to a microplate format, thus allowing the rapid analysis of numerous samples. First, HS was radiolabeled by partial de-N-acetylation and re-N-acetylation with [3H] acetic anhydride, second, after reductive amination at the reducing terminus, it was covalently linked to an amino-reactive biotin analog, and third it was immobilized on a streptavidin-coated plate. The degradation of our solid-phase tritiated HS by heparanase was monitored by measuring the soluble radioactivity released in the well. The heparanase-induced release of radioactivity was linear with respect either to time or to the amount of enzyme and was inhibited by heparin or high ionic strength. The linearity of this assay for time and enzyme concentrations could be useful to determine the potency of heparanase inhibitors. Moreover, this assay was shown to be suitable for monitoring HS-degrading activity of either heparanase endogenously expressed by the HCT 116 tumor cell line or recombinant forms of this protein.     

Preparation of homogeneous human prostatic acid phosphatase using concanavalin A-sepharose 4-B.

A simple, rapid and efficient procedure is presented for the purification of human prostatic acid phosphatase (orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2) to homogeneity. The method employs two steps suitable for use with large quantities of material, followed by chromatography on concanavalin A-Sepharose as its sole column step. The procedure also permits the recovery of purified enzyme in higher yields than earlier methods.     

Characterization of functional domains of the lymphocyte plasma membrane

Highly purified plasma membranes of calf thymocytes were fractionated by means of affinity chromatography on concanavalin A-Sepharose into two subfractions; one (fraction 1) eluted freely from the affinity column, the second (fraction 2) adhered specifically to concanavalin A-Sepharose. Previous analysis showed that both subfractions were right-side-out (Resch, K., Schneider, S. and Szamel, M. (1981) Anal. Biochem. 117, 282-292). The ratio of cholesterol to phospholipid was nearly identical in plasma membrane and both subfractions. When isolated plasma membranes were labelled with tritiated NaBH4, both subfractions exhibited identical specific radioactivities. After enzymatic radioiodination of thymocytes, the relative distribution of labelled proteins and externally exposed phospholipids was very similar in isolated plasma membranes and in both membrane subfractions, indicating the plasma membrane nature of the subfractions separated by affinity chromatography on concanavalin A-Sepharose. This finding was further substantiated by the nearly identical specific activities of some membrane-bound enzymes, Mg2+-ATPase, alkaline phosphatase and gamma-glutamyl transpeptidase. The specific activities of (Na+ + K+)-ATPase and of lysolecithin acyltransferase were several-fold enriched in fraction 2 compared to fraction 1, especially after rechromatography of fraction 1 on concanavalin A-Sepharose. Unseparated membrane vesicles contained two types of binding site for concanavalin A. In contrast, isolated subfractions showed a linear Scatchard plot; fraction 2 exhibited fewer binding sites for concanavalin A: the association constant was, however, 3.5-times higher than that measured in fraction 1. When plasma membranes isolated from concanavalin A-stimulated lymphocytes were separated by affinity chromatography, the yield of the two subfractions was similar to that of membranes from unstimulated lymphocytes. Upon stimulation with concanavalin A, Mg2+-ATPase, gamma-glutamyl transpeptidase and alkaline phosphatase were suppressed in their activities in both membrane subfractions. In contrast, the specific activities of (Na+ + K+)-ATPase and lysolecithin acyltransferase were enhanced preferentially in the adherent fraction (fraction 2). The data suggest the existence of domains in the plasma membrane of lymphocytes which are formed by a spatial and functional coupling of receptors with high affinity for concanavalin A, and certain membrane-bound enzymes, implicated in the initiation of lymphocyte activation.     

Purification and chemical characterization of human hexosaminidases A and B.

N-Acetyl-beta-hexosaminidases A and B were purified to homogeneity from human placenta. In the initial step of purification, the enzymes were adsorbed on concanavalin A-Sepharose 4B and eluted from the column with alpha-methyl D-mannosides. Subsequent purification steps included DEAE-cellulose column chromatography, QAE-Sephadex [diethyl-(2-hydroxypropyl)aminoethyl-Sephadex] column chromatography, Sephadex G-200 gel filtration and preparative disc polyacrylamide-gel electrophoresis, followed by another QAE-Sephadex chromatography for the hexosaminidase A preparation, and DEAE-cellulose column chromatography, calcium phosphate gel chromatography, Sephadex G-200 gel filtration, QAE-Sephadex chromatography and CM-cellulose chromatography for the hexosaminidase B preparation. The purified preparations, particularly hexosaminidase A, had significantly higher specific enzyme activities than previously reported. The preparations moved on polyacrylamide-gel electrophoresis as single protein bands, which also stained for enzyme activity. Sedimentation-equilibrium centrifugation indicated homogenous dispersion of the enzymes, and the molecular weight was estimated as about 110000 for both enzymes. Complete amino acid and carbohydrate compositions of the two isoenzymes were determined, and, in contrast with previous suggestions, no sialic acid was found in the enzymes.     

I-Cell disease: isoelectric focusing, concanavalin A-Sepharose 4B binding and kinetic properties of human liver acid beta-D-galactosidases.

Isoelectric focusing of the acid beta-D-galactosidases (beta-D-galactoside galactohydrolase, EC 3.2.1.23) in normal crude liver supernatant fluids demonstrated multiple isoelectric forms in the pH range 4.58-5.15, while corresponding I-cell disease samples showed an absence of isoelectric forms in the pH range 4.99-5.15. Concanavalin A-Sepharose 4B chromatography of the I-cell disease mutant C.A. demonstrated a 31% and 37% decrease in the binding of 4-methyl-umbelliferyl-beta-D-galactosidase and GM1 beta-D-galactosidase activities, respectively, when compared to normal samples. Isoelectric focusing profiles of the concanavalin A-Sepharose 4B alpha-methyl-D-mannoside effluents containing normal and I-cell disease acid beta-D-galactosidase were generally similar, but the unadsorbed I-cell disease enzyme from concanavalin A-Sepharose 4B demonstrated more activity in the pH range 4.21-4.49 than normals. Normal and I-cell disease acid beta-D-galactosidase "A" and "B", separated by gel column chromatography were found to have similar properties with respect to apparent molecular weights pH vs. activity profiles and apparent Km values for the 4 methylumbelliferyl-beta-D-galactopyranoside, GM1-ganglioside and asialofetuin (ASF) substrates. However, the apparent V values for the ICD samples were consistently reduced when compared to the results obtained with the corresponding normal fractions. The greatest decreases in apparent V were obtained for acid beta-D-galactosidase activities in I-cell disease crude supernatant fluids, and for the separated I-cell disease "B" enzyme. The differences in the isoelectric focusing profiles, the altered binding to concanavalin A-Sepharose 4B, and the reduced V values with natural and synthetic substrates may be related to changes in carbohydrate composition of I-cell disease acid beta-D-galactosidase.