Purification and partial characterization of the carbohydrate structure of lysosomal N-acetyl-beta-D-hexosaminidases from bovine brain

1. The lysosomal forms A and B, and an intermediate form I of N-acetyl-beta-D-hexosaminidase (EC 3.2.1.30) were isolated from bovine brain, resulting in the following purification factors and specific activities: hexosaminidase A 20255, 103 U mg-1; hexosaminidase B 34715, 134 U mg-1; hexosaminidase I 15241, 78 U mg-1. 2. The molecular weights of the polypeptide chains were identical for each isoenzyme: two bands of 50 and 53 k daltons were found. 3. Carbohydrate analysis showed the presence of mannose, galactose, N-acetylglucosamine and sialic acid. This composition, and the absence of N-acetylgalactosamine, indicated that only N-glycosidically linked oligosaccharide chains are present. 4. The amino-acid composition showed no substantial differences for the three isoenzymes.     

Synergy between immunotoxins prepared with native ricin A chains and chemically-modified ricin B chains

Ricin B chains treated with chloramine-T in the presence or absence of NaI show a 100-fold to 200-fold reduction in their ability to bind to the galactose-containing protein asialofetuin. Such treated B chains do not form covalently associated homodimers with treated B chains or heterodimers with native ricin A chains. Furthermore, they cannot enhance the toxicity of a ricin A chain-containing rabbit anti-human immunoglobulin (RAHIg-A) for Daudi cells. However, when such B chains are coupled to goat anti-rabbit Ig (GARIg), they potentiate the killing of RAHIg-A-treated Daudi cells only slightly less effectively than GARIg coupled to native B chains. Furthermore, if GARIg-B chain conjugates are treated with chloramine-T after coupling, they fail to bind to asialofetuin but enhance the killing of Daudi cells treated with RAHIg-A. These results demonstrate that the ability of ricin B chains to bind to galactose and to enhance the toxicity of ricin A chains (in the form of an antibody-A chain) can be operationally separated. Thus, the two functions of the B chain may reside on separate domains of the molecule.     

The preparation and chemical composition of the multiple forms of beta-glucuronidase from the female rat preputial gland.

Beta-Glucuronidase isolated from the preputial gland of the female rat has previously been shown to be a tetrameric glycoprotein. We have now separated the enzyme into several molecular forms by chromatography on hydroxylapatite columns. The three major forms (A, B, and C) have a very similar or identical amino acid composition, and kinetic and stability studies on forms B and C disclosed no differences between these two forms. However, from C contained much more carbohydrate than forms A and B, which were very similar in carbohydrate composition. The sugars in forms A and B are mannose (2.8%), glucosamine (1.9%), fucose (0.2%), galactose (0.16%), and glucose (0.17%). Form C is a little higher in mannose content, but, more distinctively, is much richer in fucose (0.6%), galactose (1.1%), and glucose (1.5%). The presence of glucose was established by paper chromatography as well as by gas-liquid chromatography, and several special experiments were performed to rule out the possibility that this hexose was present in a persistent contaminant. Direct chemical analysis for sialic acid consistently showed the absence of this sugar in the enzyme. The fact that the carbohydrate-protein linkage is alkali-stable suggests that the linkage involves an asparaginyl-N-acetylglucosamine residue. The NH2-terminal amino acid in the polypeptide chain is leucine.     

The carbohydrate prosthetic groups of rat fibrinogen plasmic fragment E.

Rat fibrinogen plasmic fragment E was found to contain one oligosaccharide chain per gamma-chain attached by a glycosylamine linkage. The oligosaccharide was composed of 1 sialic acid, 1 galactose, 2 mannose and 2 glucosamine residues. The probable sequence from the nonreducing end was sialic acid leads to galactose beta leads to mannose alpha leads to mannose alpha leads to glucosamine leads to glucosamine. No difference in the rate of clearance from the rat circulation could be detected between native and desialated fragment E. A non-denaturing method for the purification of fragment E is described.     

Oligosaccharide structure and amino acid sequence of the major glycopeptides of mature human beta-hexosaminidase

Human beta-hexosaminidase (EC 3.2.1.52) is a lysosomal enzyme that hydrolyzes terminal N-acetylhexosamines from GM2 ganglioside, oligosaccharides, and other carbohydrate-containing macromolecules. There are two major forms of hexosaminidase: hexosaminidase A, with the structure alpha(beta a beta b), and hexosaminidase B, 2(beta a beta b). Like other lysosomal proteins, hexosaminidase is targeted to its destination via glycosylation and processing in the rough endoplasmic reticulum and Golgi apparatus. Phosphorylation of specific mannose residues allows binding of the protein to the phosphomannosyl receptor and transfer to the lysosome. In order to define the structure and placement of the oligosaccharides in mature hexosaminidase and thus identify candidate mannose 6-phosphate recipient sites, the major tryptic/chymotryptic glycopeptides from each isozyme were purified by reverse-phase high-performance liquid chromatography. Two major concanavalin A binding glycopeptides, localized to the beta b chain, and one non concanavalin A binding glycopeptide, localized to the beta a chain, were found associated with the beta-subunit in both hexosaminidase A and hexosaminidase B. A single major concanavalin A binding glycopeptide was found to be associated with the alpha subunit of hexosaminidase A. The oligosaccharide structures were determined by nuclear magnetic resonance spectrometry. Two of them, the alpha and one of the beta b glycans, contained a Man3-GlcNAc2 structure, while the remaining one on the beta b chain was composed of a mixture of Man5-7-GlcNAc2 glycans. The unique glycopeptide associated with the beta a chain contained a single GlcNAc residue. Thus, all three mature polypeptides comprising the alpha and beta subunits of hexosaminidase contain carbohydrate, the structures of which have the appearance of being partially degraded in the lysosome. In the alpha chain we found only one possible site for in vivo phosphorylation. In the beta it is unclear if only one or all three of the sites could have contained phosphate. However, mature placental hexosaminidase A and B can be rephosphorylated in vitro. This requires the presence of an oligosaccharide containing an alpha 1,2-linked mannose residue. Only the single Man6-7 (of the Man5-7-GlcNAc2 glycans) containing site on the beta b chain retains this type of residue. Therefore, this site may act as the sole in vitro substrate in both of the mature isozymes for the phosphotransferase.     

Isolation and characterization of the two glycosylation isoforms of low molecular weight mannose 6-phosphate receptor from bovine testis. Effect of carbohydrate components on ligand binding.

Low molecular weight mannose 6-phosphate receptor from bovine testis exhibits two isoforms on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with Mr values of 45,000 (MPR-2A) and 41,000 (MPR-2B), respectively. Each isoform was purified to near homogeneity by the sequential application of differential centrifugation and affinity chromatography. The isoforms contain a common polypeptide core, but differ in their carbohydrate content. Treatment with specific endoglycosidases demonstrated that each isoform contains two high mannose and/or hybrid and two complex N-linked oligosaccharide chains. The results obtained from treatment of each isoform with endo-beta-galactosidase and neuraminidases and from lectin affinity chromatography reveal that MPR-2A contains a linear polylactosamine sequence(s) comprised of approximately 5 lactosamine units. A majority of the outer branches of the complex chains associated with MPR-2A are terminated with sialic acid residues. In contrast, MPR-2B lacks a polylactosamine sequence and a majority of the outer branches of the complex chains are terminated with galactose residues. MPR-2A exhibited a lower affinity than MPR-2B for mannose 6-phosphate-containing ligands. Treatment of MPR-2A with endo-beta-galactosidase and/or neuraminidases followed by affinity chromatography revealed that polylactosamine and sialic acid residues impair the ability of MPR-2A to bind ligands.     

Cell surface and intracellular functions for ricin galactose binding.

The role of the two galactose binding sites of ricin B chain in ricin toxicity was evaluated by studying a series of ricin point mutants. Wild-type (WT) ricin and three ricin B chain point mutants having mutations in either 1) the first galactose binding domain (site 1 mutant, Met in place of Lys-40 and Gly in place of Asn-46), 2) the second galactose binding domain (site 2 mutant, Gly in place of Asn-255), or 3) both galactose binding domains (double site mutant containing all three amino acid replacements formerly stated) were expressed in Xenopus oocytes and then reassociated with recombinant ricin A chain. The different ricin B chains were mannosylated to the same extent. Cytotoxicity of these toxins was evaluated when cell entry was mediated either by galactose-containing receptors or through an alternate receptor, the mannose receptor of macrophages. WT ricin and each of the single domain mutants was able to kill Vero cells following uptake by galactose containing receptors. Lactose blocked the toxicity of each of these ricins. Site 1 and 2 mutants were 20-40 times less potent than WT ricin, and the double site mutant had no detectable cytotoxicity. WT ricin, the site 1 mutant, and the site 2 mutant also inhibited protein synthesis of mannose receptor-containing cells. Ricin can enter these cells through either a cell-surface galactose-containing receptor or through the mannose receptor. By including lactose in the cell medium, galactose-containing receptor-mediated uptake is blocked and cytotoxicity occurs solely via the mannose receptor. WT ricin, site 1, and site 2 mutants were cytotoxic to macrophages in the presence of lactose with the relative potency, WT greater than site 2 mutant greater than site 1 mutant. The double site mutant lacked cytotoxicity either in the absence or presence of lactose. Thus, even for mannose receptor-mediated toxicity of ricin, at least one galactose binding site remains necessary for cytotoxicity and two galactose binding sites further increases potency. These results are consistent with the model that the ricin B chain galactose binding activity plays a role not only in cell surface binding but also intracellularly for ricin cytotoxicity.     

Identification of the 6-sulfate binding site unique to alpha-subunit-containing isozymes of human beta-hexosaminidase

In humans, beta-hexosaminidase A (alphabeta) is required to hydrolyze GM2 ganglioside. A deficiency of either the alpha- or beta-subunit leads to a severe neurological disease, Tay-Sachs or Sandhoff disease, respectively. In mammals beta-hexosaminidase B (betabeta) and S (alphaalpha) are other major and minor isozymes. The primary structures of the alpha- and beta-subunits are 60% identical, but only the alpha-containing isozymes can efficiently hydrolyze beta-linked GlcNAc-6-SO(4) from natural or artificial substrates. Hexosaminidase has been grouped with glycosidases in family 20. A molecular model of the active site of the human hexosaminidase has been generated from the crystal structure of a family 20 bacterial chitobiase. We now use the chitobiase structure to identify residues close to the carbon-6 oxygen of NAG-A, the nonreducing beta-GlcNAc residue of its bound substrate. The chitobiase side chains in the best interactive positions align with alpha-Asn(423)Arg(424) and beta-Asp(453)Leu(454). The change in charge from positive in alpha to negative in beta is consistent with the lower K(m) of hexosaminidase S, and the much higher K(m) and lower pH optimum of hexosaminidase B, toward sulfated versus unsulfated substrates. In vitro mutagenesis, CHO cell expression, and kinetic analyses of an alphaArg(424)Lys hexosaminidase S detected little change in V(max) but a 2-fold increase in K(m) for the sulfated substrate. Its K(m) for the nonsulfated substrate was unaffected. When alphaAsn(423) was converted to Asp, again only the K(m) for the sulfated substrate was changed, increasing by 6-fold. Neutralization of the charge on alphaArg(424) by substituting Gln produced a hexosaminidase S with a K(m) decrease of 3-fold and a V(max) increased by 6-fold for the unsulfated substrate, parameters nearly identical to those of hexosaminidase B at pH 4.2. As well, for the sulfated substrate at pH 4.2 its K(m) was increased 9-fold and its V(max) decreased 1.5-fold, values very similar to those of hexosaminidase B obtained at pH 3.0, where its betaAsp(453) becomes protonated.     

Sargassan: A sulphated heteropolysaccharide from Sargassum linifolium

A new sulphated heteropolysaccharide containing glucuronic acid, mannose, galactose, xylose, fucose and a protein moiety has been extracted from Sargassum linifolium. The polysaccharide extracted with HCl was richer in its carbohydrate and protein contents and contained lower amounts of ash than that extracted with oxalic acid.     

Characterization and comparison of the major glycoprotein from three strains of Rous sarcoma virus.

The major glycoprotein g2 was purified from three strains of Rous sarcoma virus, representing subgroups A, B, and C. Carbohydrate analysis showed that glucosamine, mannose, galactose, fucose and sialic acid constitute 40% of the weight of the subgroup A glycoprotein and 15% of the subgroup B and C glycoproteins. The molar ratios of sugars were very similar and amino acid compositions were similar but not identical for the three glycoproteins. Glycosidase digestions of subgroup A and C glycoproteins suggested the presence of two types of oligosaccharide chains, the complex serum type, with terminal sequences sialic acidα-Galβ-GlcNAcβ- and the high mannose type with terminal α-linked mannosyl residues. After removal of 70% of the carbohydrate by glycosidases, subgroup A glycoprotein contained only glucosamine and mannose, in the molar ratio 2.0:1.3. The sequence of sugar release was consistent with oligosaccharide structures such as those which have been described for other glycoproteins. The plant lectins concanavalin A and wheat germ agglutinin were shown to interact strongly with the g2 glycoprotein from viruses of all three subgroups.     

Human placental N-acetyl-beta-D-hexosaminidase isozymes. Activity toward native hyaluronic acid.

The activity of purified human hexosaminidases A and B toward hyaluronic acid (HA) isolated from cultured human skin fibroblasts was investigated. The cleavage of N-acetylglucosaminyl residues to monosaccharide N-acetylglucosamines by hexosaminidase isozymes was determined in the presence and absence of purified human β-glucuronidase. The pH optima of this reaction, with and without β-glucuronidase, were 4.5 for hexosaminidase A and 4.0 for hexosaminidase B. The hydrolysis of HA by both hexosaminidase isozymes proceeds linearily for at least 18 h in the presence of β-glucuronidase. Concentrations of 0.5–5 units of either isozyme showed a linear relationship with rate of hydrolysis. Without β-glucuronidase, hexosaminidase only cleaved the terminal N-acetylglucosamine residue. However, under optimal conditions, with β-glucuronidase, the hydrolytic activity of hexosaminidase B was about 30% as efficient as that of hexosaminidase A. Approximately 70% of the HA could be degraded by 5 units of hexosaminidase A in the presence of 0.5 unit of β-glucuronidase, as opposed to 25% degraded by hexosaminidase B. These results probably reflect intrinsic differences in the activities of the two isozymes. Since the substrate (HA) did not inhibit the hydrolysis of a synthetic substrate (4-methylumbelliferyl-β-glucosaminide) by hexosaminidase B, the linear kinetics of HA hydrolysis implies no product inhibition. These data indicate that native HA can be hydrolyzed by the combined activities of β-glucuronidase with hexosaminidase A or hexoaminidase B.     

Proteolytic processing of pro-alpha and pro-beta precursors from human beta-hexosaminidase. Generation of the mature alpha and beta a beta b subunits

There are two major isozymes of human lysosomal beta-hexosaminidase (beta-N-acetylhexosaminidase, EC 3.2.1.52), hexosaminidase A, alpha(beta a beta b), and hexosaminidase B, 2(beta a beta b). The alpha subunit contains a single polypeptide chain, while the beta subunit is composed of two nonidentical chains (beta a and beta b) derived from a common pro-beta precursor. The mature subunits, like those of most lysosomal enzymes, are produced through the proteolytic processing of propolypeptides once they enter the lysosome. In order to define the structure of the alpha and beta subunits generated in the lysosome, the alpha, beta a, and beta b polypeptides of hexosaminidase A and B were separated by a combination of molecular sieve and ion exchange high performance liquid chromatography, and amino-terminal sequences were determined. These were localized to the deduced amino acid sequences of previously isolated cDNAs coding for the prepro-alpha and beta polypeptides. From this analysis, the sites of hydrolysis generating the mature alpha, beta a, and beta b chains from hexosaminidase A and B could be determined. First, the signal peptide, required for processing of the pre-propolypeptides through the rough endoplasmic reticulum was predicted from the first in-frame Met residue on the cDNA. Second, amino acid sequencing defined the amino termini of the mature polypeptide chains and identified the pro-sequences removed from both the pro-alpha and pro-beta polypeptides. Third, an internal cleavage resulted in the removal of a tetrapeptide, Arg-Gln-Asn-Lys, and tripeptide, Arg-Gln-Asn, from the pro-beta chain of hexosaminidase A and B, respectively , to generate the beta b and beta a chains. This result localized the beta b and beta a chains to the amino-terminal and carboxyl-terminal halves of the pro-beta sequence, respectively. Finally, we previously reported minimal or no carboxyl-terminal processing of the pro-beta chain in the lysosome. On the other hand, we suggest that there is trimming at the carboxyl terminus of the pro-alpha chain based on comparison of molecular weights of deglycosylated alpha with the isolated beta b and beta a chains comprising the mature beta subunit with those predicted from the cDNA. Thus, in the lysosome the pro forms of hexosaminidase A and B undergo extensive proteolytic processing which, while specific in nature, has the appearance of removing easily accessible, nonessential domains, rather than contributing to biosynthetic maturation of function.     

Mannose receptor-mediated uptake of ricin toxin and ricin A chain by macrophages. Multiple intracellular pathways for a chain translocation

The role of the high mannose carbohydrate chains in the mechanism of action of ricin toxin was investigated. Ricin is taken up by two routes in macrophages, by binding to cell surface mannose receptors, or by binding of the ricin galactose receptor to cell surface glycoproteins. Removal of carbohydrate from ricin by periodate oxidation led to a large loss in toxicity via both routes of uptake by an effect on the B chain not due to a loss of galactose binding affinity. These data suggest that the carbohydrate chains of ricin B chain may be required for full toxicity. The pathway of uptake of ricin by the macrophage mannose receptor was found to differ in several respects from uptake via the galactose-specific pathway. Analysis of intoxication of macrophages by ricin in the presence of ammonium chloride suggested that mannose receptor bound ligand passes through acidic vesicles prior to translocation, unlike galactose bound ligand. Intoxication by ricin via galactose-specific uptake was potentiated by swainsonine but not by castanospermine, suggesting that ricin may be attacked by an endogenous mannosidase within the cell, and that ricin passes through either a lysosomal or a Golgi compartment prior to translocation.     

Study of the glycosylation of apolipoprotein H

Apolipoprotein H is a single chain polypeptide composed of 326 amino acids highly glycosylated. Its carbohydrate content is approximately 19% of the molecular weight. We show that it is rich in sialic acid linked alpha (2-6) to galactose or N-acetylgalactosamine. Sialic acid is not alpha (2-3) linked to galactose. Galactose is beta (1-4) linked to N-acetylglucosamine and beta (1-3) linked to N-acetylgalactosamine. Carbohydrate O-linked chains (mainly sialic acid) are alpha (2-6) linked to galactose or N-acetylgalactosamine. Galactose is also organised in O-linked chains and beta (1-4) linked to N-acetylglucosamine and beta (1-3) linked to acetylgalactosamine. Concanavalin A lectin was used to isolate two groups of apolipoprotein H molecules bearing biantennary and truncated hybrids and high mannose and hybrid oligosaccharides. Apolipoprotein H fails to bind lysine-Sepharose. Our results thus show that it presents truncated hybrid or hybrid-type carbohydrate chains which bear few unmasked mannose residues as a terminal sugar. Biochemical analysis of carbohydrate structures conducted on single isoforms separated through IEF revealed that no specific carbohydrate complex is bound to a single isoform.     

Carbohydrate content of human erythrocyte membrane. Variations with ABO-blood group.

The study of the carbohydrates of human erythrocyte membranes has been mainly focused on their glycopeptidic and glycolipidic complexes. Modifications of these carbohydrates have been described in subjects with various pathological states. In order to characterize possible changes of the glycopeptides, or glycolipids obtained from erythrocyte membrane in various pathological situations, the determination of the carbohydrate content of the whole membrane appeared a necessary preliminary. This study concerns the determination of the normal values of the main carbohydrates of whole human erythrocyte membranes, with respect to their blood group. Erythrocyte membranes were prepared from donors of the four ABO blood groups. After acidic hydrolysis, the contents of fucose, mannose, galactose, glucose, glucosamine, galactosamine and N-acetylneuraminic acid in each blood group were determined and compared with one another. The galactosamine content of A, B and AB erythrocyte membranes is significantly higher than that of the O-erythrocyte. For galactose, the differences are significant for the following pairs: A/O; B/O; AB/O; A/B; A/AB. Significant differences in the mannose contents of O-erythrocytes and A, B and AB erythrocytes have also been found. This result suggests that a basic difference, in the core of the oligosaccharide chains, may exist between O and A, B, AB erythrocyte membranes.     

Studies on pig serum lipoproteins. IV. Isolation and characterization of glycopeptides from pig serum low density lipoprotein.

Three glycopeptides were isolated from the pronase digest of the protein moiety of pig serum low density lipoprotein. The isolation procedure consisted of pronase digestion, gel filtration on Sephadex G-25 and G-50 columns, paper chromatography and DEAE-Sephadex A-50 column chromatography. Based on the carbohydrate analysis, the isolated glycopeptides were classified into two types. One type (GDI) consisted of mannose and N-acetylglucosamine residues in the molar ratio of 6:2 and had a molecular weight of about 2,300. The other type (GDII and GDIII) consisted of sialic acid, mannose, galactose, fucose, and N-acetylglucosamine residues in the molar ratio of 1:4:2:1:3 and 2:4:3:1:3, respectively. The molecular weights of GDII and GDIII were about 2,100 and 3,100, respectively. The results on the strong alkaline treatment of these glycopeptides suggested that all carbohydrate chains were linked to the peptide chains through N-acetylglucosaminyl-asparagine linkages. Of these glycopeptides and pig serum lipoproteins, only glycopeptide GDI and native LDL strongly interacted with concanavalin A.     

Canine fibrinogen glycopeptides

Canine fibrinogen was digested by a complex of proteases from Streptomyces griseus. The degradation products were purified by gel-filtration, DEAE-cellulose chromatography and electrophoresis, resulting in nine glycopeptides, eight of which contained aspartic acid and one--serine. The other amino acids were found only in trace amounts. The glycopeptides were shown to contain hexoamines, mannose, galactose and sialic acid. The oligosaccharide chains form a sequence of structurally similar variants. The individual microheterogeneity of canine fibrinogen with respect to carbohydrate chains was detected. A comparison of the carbohydrate composition of fibrinogen and glycopeptides suggests the presence of four carbohydrate chains in the protein molecule.     

The relationship of structure of glucoamylase and glucose oxidase to antigenicity

Glucoamylase and glucose oxidase fromAspergillus niger have been purified to homogeneity by chromatography on DEAE-cellulose and the purified enzymes have been used to investigate structural and antigenicity relationships. In structure, glucoamylase and glucose oxidase are glycoproteins containing 14% and 16% carbohydrate. Earlier methylation and reductive -elimination results have shown that glucoamylase has an unusual arrangement of carbohydrate residues, with 20 single mannose units and 25 di-, tri-, or tetrasaccharide chains of mannose, glucose, and galactose, all attached O-glycosidically to serine and threonine residues of the protein moiety. The antigenicity of the glucoamylase has now been found to reside predominantly in the types and arrangement of the carbohydrate chains. Glucose oxidase contains mannose, galactose, and glucosamine in the N-acetyl form in the native enzyme, but the complete structure of the carbohydrate chains has not yet been determined. The antigenicity of this enzyme does not reside in the carbohydrate units, but rather in the polypeptide chains of the two subunits of the enzyme. Glucose oxidase can be dissociated into subunits by mercaptoethanol and sodium dodecyl sulfate treatment, while glucoamylase cannot be dissociated, but undergoes only an unfolding of the polypeptide chain under these conditions. The subunits of glucose oxidase do not react with the anti-glucose oxidase antibodies, but the unfolded molecule and peptide fragments produced from glucoamylase by cyanogen bromide cleavage do react with antiglucoamylase antibodies.     

Epithelial basement membrane of bovine renal tubuli. Isolation and analysis of the carbohydrate chains.

The carbohydrate chains present in the tubular basement membrane of bovine kidney were studied. Digestion with collagenase followed with pronase resulted in a complete solubilization of the basement membrane. The different glycopeptides were purified by gel filtration and ion-exchange chromatography. Two kinds of carbohydrate chains could be characterized: oligosaccharides composed of glucosamine, mannose, galactose, fucose and sialic acid, and glucosylgalactose disaccharides. A very small portion of the oligosaccharide chains (ca. 4%) appeared to be free of sialic acid. The bulk of these chains contained sialic acid and fucose, although in small amounts. Only traces of galactosamine were found.     

Purification and characterization of beta-N-acetylhexosaminidase I from human placenta

beta-N-Acetylhexosaminidase (hexosaminidase) I, which has an intermediate charge character between those of hexosaminidases A(alpha beta 2) and B[beta beta)2), was purified 1,500-fold from human placenta by procedures including chromatographies on concanavalin A (Con A)-Sepharose and an immunoadsorbent column. The isolated hexosaminidase I was heat-stable, and antigenically cross-reactive to anti-beta chain-IgG but not to anti-alpha chain-IgG. The results of substrate specificity experiments using 3H-labeled natural substrates indicated that the hexosaminidase I hydrolyzed Gb4Cer to Gb3Cer but not GM2 to GM3. The tryptic peptide map of the hexosaminidase I was similar to that of hexosaminidase B, though some differences were observed. The hexosaminidase I after treatment with neuraminidase or endo-beta-N-acetylglucosaminidase H was partly converted to less acidic forms. Treatment of the hexosaminidase I with acid phosphatase did not change the charge character. Therefore hexosaminidase I is an acidic variant form of hexosaminidase B, possibly resulting from sialylation and the presence of phosphodiester bonds at the carbohydrate moiety.