Purification and characterization of two alpha-galactosidases associated with catabolism of guar gum and other alpha-galactosides by Bacteroides ovatus.

When Bacteroides ovatus is grown on guar gum, a galactomannan, it produces alpha-galactosidase I which is different from alpha-galactosidase II which it produces when grown on galactose, melibiose, raffinose, or stachyose. We have purified both of these enzymes to apparent homogeneity. Both enzymes appear to be trimers and have similar pH optima (5.9 to 6.4 for alpha-galactosidase I, 6.3 to 6.5 for alpha-galactosidase II). However, alpha-galactosidase I has a pI of 5.6 and a monomeric molecular weight of 85,000, whereas alpha-galactosidase II has a pI of 6.9 and a monomeric molecular weight of 80,500. alpha-Galactosidase I has a lower affinity for melibiose, raffinose, and stachyose (Km values of 20.8, 98.1, and 8.5 mM, respectively) than does alpha-galactosidase II (Km values of 2.3, 5.9, and 0.3 mM, respectively). Neither enzyme was able to remove galactose residues from intact guar gum, but both were capable of removing galactose residues from guar gum which had been degraded into large fragments by mannanase. The increase in specific activity of alpha-galactosidase which was associated with growth on guar gum was due to an increase in the specific activity of enzyme I. Low, constitutive levels of enzyme II also were produced. By contrast, enzyme II was the only alpha-galactosidase that was detectable in bacteria which had been grown on galactose, melibiose, raffinose, or stachyose.     

Affinity purification of alpha-galactosidase A from human spleen, placenta, and plasma with elimination of pyrogen contamination. Properties of the purified splenic enzyme compared to other forms

The substrate analog alpha-D-galactosylamine was synthesized, linked to 6-aminohexanoic acid, and coupled to carboxyhexyl-Sepharose. This affinity support permitted the purification of human alpha-galactosidase A (alpha-D-galactoside galactohydrolase, EC 3.2.1.22) from spleen, placenta, and plasma. When used in conjunction with conventional procedures, affinity chromatography enabled the rapid and specific purification of alpha-galactosidase A from each source. Significantly, pyrogenic endotoxins were eliminated from enzyme preparations by the use of the affinity column. Splenic alpha-galactosidase A was purified in high yield (38%) with a specific activity of 1.9 X 10(6) units/mg. The purified enzyme was a homodimer with a native molecular weight of 101,000 and a subunit weight of 49,800. The UV absorption coefficient was E280 1% = 18 and the lambda max was 282 nm. The plasma form was purified with a markedly improved yield to a specific activity (229,000 units/mg) which was 3 times greater than that achieved previously. The enzymes from plasma, spleen, and placenta were immunologically identical. The physical and kinetic properties of the purified enzymes were consistent with and confirmed previous findings.     

Properties of acid beta-N-acetyl-D-glucosaminidase from cock semen.

Two forms (I and II) of beta-N-acetyl-D-glucosaminidase from cock seminal plasma and one form (III) from spermatozoa were separated by chromatofocusing. The active enzyme forms I and II had pI values of 6.6 and 6.3, respectively, while form III had two subforms with pI values of 6.3 and 6.1, as determined by polyacrylamide gel electrofocusing. The molecular weights were 76,000 for forms I and III and 32,000 for form II. The optimum pH of enzyme forms I and III ranged from 3.6 to 4.0. In contrast, form II showed one distinct maximum at pH 3.7. The Km values obtained with p-nitrophenyl-beta-N-acetyl-D-glucosaminide as substrate were 0.35, 0.28, and 0.39 mM for forms I, II, and III, respectively. It is assumed that both cock spermatozoa and cock seminal plasma contain a common, enzymatically active beta-N-acetyl-D-glucosaminidase subunit with M(r) about 32,000 and pI 6.3.     

Expression and characterization of glycosylated and catalytically active recombinant human alpha-galactosidase A produced in Pichia pastoris

Fabry disease is an X-linked inborn error of glycolipid metabolism caused by deficiency of the lysosomal enzyme alpha-galactosidase A. This enzyme is responsible for the hydrolysis of terminal alpha-galactoside linkages in various glycolipids. An improved method of production of recombinant alpha-galactosidase A for use in humans is needed in order to develop new approaches for enzyme therapy. Human alpha-galactosidase A for use in enzyme therapy has previously been obtained from human sources and from recombinant clones derived from human cells, CHO cells, and insect cells. In this report we describe the construction of clones of the methylotrophic yeast Pichia pastoris that produce recombinant human alpha-galactosidase A. Recombinant human alpha-galactosidase A is secreted by these Pichia clones and the level of production is more than 30-fold greater than that of previously used methods. Production was optimized using variations in temperature, pH, cDNA copy number, and other variables using shake flasks and a bioreactor. Expression of the human enzyme increased with increasing cDNA copy number at 25 degrees C, but not at the standard growth temperature of 30 degrees C. The recombinant alpha-galactosidase A was purified to homogeneity using ion exchange (POROS 20 CM, POROS 20 HQ) and hydrophobic (Toso-ether, Toso-butyl) chromatography with a BioCAD HPLC Workstation. Purified recombinant alpha-galactosidase A was taken up by fibroblasts derived from Fabry disease patients and normal enzyme levels could be restored under these conditions. Analysis of the carbohydrate present on the recombinant enzyme indicated the predominant presence of N-linked high-mannose structures rather than complex carbohydrates.     

A method for isolating human plasma lecithin:cholesterol acyltransferase without using anti-apolipoprotein D, and its characterization

A method for isolating human plasma lecithin:cholesterol acyltransferase (EC 2.3.1.43) purified more than 50 000-fold is described. The crude enzyme obtained by initial ammonium sulfate and citric acid treatment of 21 of human plasma is subjected to repeated DEAE-cellulose chromatography to yield a preparation purified more than 600-fold. Hydroxyapatite chromatography of concentrates from this fraction using 0.5 mM phosphate buffer, pH 6.8, yields enzyme preparations purified more than 50 000-fold. The enzyme isolated by this procedure was free of apolipoprotein D, as shown by the absence of an arc in immunodiffusion with anti-apolipoprotein D. The enzyme showed a single band by polyacrylamide gel electrophoresis in the presence and absence of SDS. Upon analytical isoelectrofocusing the enzyme separated into three iso forms with isoelectric points below that of egg albumin (pI 4.6). The enzyme was characterized by a high content of glutamic acid, leucine and glycine, and a lower content of tyrosine. The enzyme possessed both transferase and phospholipase A2 activities and both activities show absolute requirement for apolipoprotein A-I. The purified enzyme was injected into Balb/c mice and the antiserum reacted both with the purified enzyme and normal human serum in immunodiffusion, giving lines of complete identity. The antiserum gave no precipitation lines with albumin or apolipoprotein D, providing additional evidence for the absence of apolipoprotein D in the purified enzyme. The gamma-globulin isolated from the antiserum inhibited human lecithin:cholesterol acyltransferase activity.     

Soluble and membrane-bound neutral alpha-glucosidase from the human kidney

In human kidney cortex neutral alpha-glucosidases 1 and 2 are represented by two forms, soluble (cytosolic) and membrane-bound (brush border) ones. It has been shown that the soluble enzyme preexists in human kidney but does not derive from the membrane-bound form. Similar to the membrane-bound enzyme the soluble form is a glycoprotein. Both enzyme forms possess identical electrophoretic mobility, pH-optimum, heat sensibility and Km values for maltose (0.7 mM) and 4-methylumbelliferyl-alpha-D-glucopyranoside (0.57 mM), but differ by molecular weights as determined by gel filtration chromatography. The molecular weights of the soluble neutral alpha-glucosidases 1 and 2 are lower than those of the comparable brush border enzymes (470 000, 360 000, 520 000 and 440 000, correspondingly). Neutral membrane-bound alpha-glucosidase 1 is a sialylated enzyme with a pI of 4.10 +/- 0.02. The soluble enzyme contains no or only traces of neuraminic acid and has a pI 4.40 +/- 0.03. The soluble and membrane-bound neutral alpha-glucosidases are apparently independent forms of the enzyme, differing by the degree of sialylation and by the presence of an "anchor" in the membrane-bound enzyme. The synthesis of both forms is presumably coded by the same structural gene.     

Endocytosis of lysosomal alpha-galactosidase A by cultured fibroblasts from patients with Fabry disease.

The endocytosis of alpha-galactosidase A was studied in cultured fibroblasts from patients with Fabry disease. Alpha-galactosidase A was purified from human placenta by chromatography on concanavalin A-Sepharose, DEAE-cellulose, and N-epsilon-aminocaproyl-alpha-D-galactosylamine-Sepharose. Separation of the high-uptake form of the enzyme from the low-uptake form was accomplished by chromatography on ECTEOLA-cellulose. With the high-uptake form of the enzyme, the uptake was linear at low concentrations of enzyme and had a Kuptake of 0.01 U/ml of medium that corresponds to a Km of 5.0 x 10(-9) M. At high concentrations of enzyme, it became saturated. The high-uptake form could be converted to the low-uptake form by treatment with acid phosphatase. Mannose-6-P strongly inhibited the active uptake of the enzyme. Once taken up into the lysosomes of Fabry disease fibroblasts, alpha-galactosidase A activity was rapidly lost in the first 2 days of incubation at 37 degrees C, but was fairly stable for the next 6 days. The half-life of internalized alpha-galactosidase A activity was calculated to be 4 days. Crosslinking of the enzyme with hexamethylene diisocyanate did not increase the intracellular stability of alpha-galactosidase A activity.     

Difference in renal alpha-galactosidase levels in male and female Chinese hamsters (Cricetulus griseus).

1. The activity of alpha-galactosidase was found to be significantly higher in the kidney of female than that of male Chinese hamsters in a highly inbred colony but its activity in liver, heart and spleen remained similar between female and male animals. 2. Partially purified renal alpha-galactosidase by sequential column chromatography on Sepharose 6B and DEAE-Sepharose CL-6B showed identical elution profiles, pH optima (4.5), KmS (4.4 mM) and heat-inactivation curves between enzymes of male and female animals. 3. Thus, the observed higher activity of renal alpha-galactosidase in the females was due to elevated enzyme concentration, not a result of enzyme polymorphism.     

Purification to homogeneity of human placental acid sphingomyelinase

Acid sphingomyelinase was purified to homogeneity from human placenta in the presence of a dialyzable detergent, n-octyl-beta-D-glucopyranoside. The major steps in the procedure included column chromatographies with Con A-Sepharose, sphingosylphosphorylcholine-Sepharose 4B, hexyl-agarose, and Mono P. The purified enzyme with pI 7.4 had a specific activity of approx 170,000 units/mg protein with a yield of 3.6%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single protein band of Mr 62,000. Gel filtration with a Superose 12 column gave a single peak, and the enzyme in the presence 50 mM n-octyl-beta-D-glucopyranoside was of Mr 123,000, indicating that the native enzyme occurs in a dimeric form. The optimal pH was 5.5 with both sphingomyelin and an artificial substrate, 2-N-hexadecanoylamino-4-nitrophenylphosphorylcholine. The Km values were 55 microM with sphingomyelin and 340 microM with the artificial substrate. The enzyme activity was not affected by Mg2+ (1-5 mM), confirming that the enzyme is acid sphingomyelinase. The enzyme was stable at -80 degrees C for more than 4 months. In addition to the enzyme with pI 7.4, the Mono P chromatofocusing gave two peaks (pI 7.0 and 6.7) possessing the enzymatic activity.     

Cloning and characterization of a novel alpha-galactosidase from Bifidobacterium breve 203 capable of synthesizing Gal-alpha-1,4 linkage.

A novel alpha-galactosidase gene (aga2) was cloned from Bifidobacterium breve 203. It contained an ORF of 2226-bp nucleotides encoding 741 amino acids with a calculated molecular mass of 81.5 kDa. The recombinant enzyme Aga2 was heterogeneously expressed, purified and characterized. Regarding substrate specificity for hydrolysis, Aga2 was highly active towards p-nitrophenyl-alpha-d-galactopyranoside (pNPG). The Km value for pNPG was estimated to be 0.27 mM and for melibiose it was estimated to be 4.3 mM. Aga2 was capable of catalyzing transglycosylation as well as hydrolysis. The enzyme synthesized a trisaccharide (Gal-alpha-1, 4-Gal-alpha-1, 6-Glc) using melibiose as a substrate. It was a new oligosaccharide produced by glycosidase and contained Gal-alpha-1,4 linkage, a novel galactosidic link formed by microbial alpha-galactosidase. In the presence of pNPG as a donor, Aga2 was able to catalyze glycosyl transfer to various acceptors including monosaccharides, disaccharides and sugar alcohols.     

Furostanol glycoside 26-O-beta-glucosidase from the leaves of Solanum torvum

A beta-glucosidase (torvosidase) was purified to homogeneity from the young leaves of Solanum torvum. The enzyme was highly specific for cleavage of the glucose unit attached to the C-26 hydroxyl of furostanol glycosides from the same plant, namely torvosides A and H. Purified torvosidase is a monomeric glycoprotein, with a native molecular weight of 87 kDa by gel filtration and a pI of 8.8 by native agarose IEF. Optimum pH of the enzyme for p-nitrophenyl-beta-glucoside and torvoside H was 5.0. Kinetic studies showed that Km values for torvoside A (0.06 3mM) and torvoside H (0.068 mM) were much lower than those for synthetic substrates, pNP-beta-glucoside (1.03 mM) and 4-methylumbelliferyl-beta-glucoside (0.78 mM). The enzyme showed strict specificity for the beta-d-glucosyl bond when tested for glycone specificity. Torvosidase hydrolyses only torvosides and dalcochinin-8'-beta-glucoside, which is the natural substrate of Thai rosewood beta-glucosidase, but does not hydrolyse other natural substrates of the GH1 beta-glucosidases or of the GH3 beta-glucosidase families. Torvosidase also hydrolyses C5-C10 alkyl-beta-glucosides, with a rate of hydrolysis increasing with longer alkyl chain length. The internal peptide sequence of Solanum beta-glucosidase shows high similarity to the sequences of family GH3 glycosyl hydrolases.     

A novel thermostable alpha-galactosidase from the thermophilic fungus Thermomyces lanuginosus CBS 395.62/b: purification and characterization

High levels of an extracellular alpha-galactosidase are produced by the thermophilic fungus Thermomyces lanuginosus CBS 395.62/b when grown in submerse culture and induced by sucrose. The enzyme was purified 114-fold from the culture supernatant by (NH(4))(2)SO(4) fractionation, and by chromatographical steps including Sepharose CL-6B gel filtration, DEAE-Sepharose FF anion-exchange, Q-Sepharose FF anion-exchange and Superose 12 gel filtration. The purified enzyme exhibits apparent homogeneity as judged by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and iso-electric focusing (IEF). The native molecular weight of the monomeric alpha-galactosidase is 93 kDa with an isoelectric point of 3.9. The enzyme displays a pH and temperature optimum of 5-5.5 and 65 degrees C, respectively. The purified enzyme retains more than 90% of its activity at 45 degrees C in a pH range from 5.5 to 9.0. The enzyme proves to be a glycoprotein and its carbohydrate content is 5.3%. Kinetic parameters were determined for the substrates p-nitrophenyl-alpha-galactopyranoside, raffinose and stachyose and very similar K(m) values of 1.13 mM, 1.61 mM and 1.17 mM were found. Mn(++) ions activates enzyme activity, whereas inhibitory effects can be observed with Ca(++), Zn(++) and Hg(++). Five min incubation at 65 degrees with 10 mM Ag(+) results in complete inactivation of the purified alpha-galactosidase. Amino acid sequence alignment of N-terminal sequence data allows the alpha-galactosidase from Thermomyces lanuginosus to be classified in glycosyl hydrolase family 36.     

Expression of the human alpha-galactosidase A in Escherichia coli K-12

We used the prokaryotic expression vector, ptrpL1, for the expression in Escherichia coli K-12 of a cDNA clone specific for the human lysosomal hydrolase, alpha-galactosidase A. The 5' terminus of the cDNA clone was engineered so that an ATG codon precedes the first codon of the mature form of the enzyme. A clone with elevated expression of this human enzyme was constructed by increasing the distance between the Shine-Dalgarno site and the ATG start codon from 6 to 8 bp. Clones with alpha-galactosidase A specific cDNA encoding the proenzyme produce a protein of 45 kDa, the size expected for the intact proenzyme. The 45-kDa protein is specifically precipitated by antibody to alpha-galactosidase A, and its expression is repressed by tryptophan and induced by 3-beta-indoleacrylic acid as expected for this expression vector. The human enzyme is produced in E. coli in a catalytically active form at levels sufficient to support the growth of cells using alpha-galactosides as sole sources of carbon and energy. In addition, bacterial colonies that produce the human enzyme turn blue in the presence of 5-bromo-4-chloro-3-indolyl-alpha-D-galactopyranoside.     

Purification and characterization of rat plasma kallikrein

Kallikrein enzyme initially was isolated from rat plasma by passage of citrated plasma through a DEAE-Sephadex column at pH 7.2. The active fraction was purified to electrophoretic apparent homogeneity by precipitation to 60% ammonium sulfate saturation, sequential passage through DE-52 cellulose, Sephadex G-200 and SP-Sephadex columns and finally by chromatofocusing on a PBE-94 column. The kallikrein content of each fraction during purification was monitored on the synthetic substrate N-alpha-tosyl-L-arginine methyl ester (TAME) and by its ability to form kinin from heat-treated rat plasma. The molecular weight was estimated by gel filtration to be 50,000 and by SDS-gel electrophoresis 41,000. Multiple isozymic forms were obtained with pI values ranging from 4.2 to 5.0. The enzyme has a pH optimum of 8.3. The Km and Vmax values for TAME, Bz-pro-phe-arg-pNA and H-D-val-leu-lys-pNA were 1.6, 0.16 and 1.7 mM and 3.09, 0.96 and 0.25 microM/mg/min respectively. The enzyme was inhibited by soybean trypsin inhibitor but not by lima bean trypsin inhibitor.     

Synthesis and processing of alpha-galactosidase A in human fibroblasts. Evidence for different mutations in Fabry disease

The synthesis and processing of the human lysosomal enzyme alpha-galactosidase A was examined in normal and Fabry fibroblasts. In normal cells, alpha-galactosidase A was synthesized as an Mr = 50,500 precursor, which contained phosphate groups in oligosaccharide chains cleavable by endoglucosaminidase H. The precursor was processed via ill-defined intermediates to a mature Mr 46,000 form. Processing was complete within 3-7 days after synthesis. In the presence of NH4Cl and in I-cell fibroblasts, the majority of newly synthesized alpha-galactosidase A was secreted as an Mr = 52,000 form. For comparison, the processing and stability of alpha-galactosidase A were examined in fibroblasts from five unrelated patients with Fabry disease, which is caused by deficient alpha-galactosidase A activity. In one cell line, synthesis of immunologically cross-reacting polypeptides was not detectable. In another, the synthesis, processing, and stability of alpha-galactosidase A was indistinguishable from that in normal fibroblasts. In a third Fabry cell line, the mutation retarded the maturation of alpha-galactosidase A. Finally, in two cell lines, alpha-galactosidase A polypeptides were synthesized that were rapidly degraded following delivery to lysosomes. These results clearly indicate that Fabry disease comprises a heterogeneous group of mutations affecting synthesis, processing, and stability of alpha-galactosidase A.     

Purification and characterization of human seminal plasma aminopeptidase

A 50.4-fold purification of aminopeptidase is achieved by alcohol precipitation, DEAE-cellulose, CM-cellulose and finally Sephadex G-200 chromatography. On polyacrylamide gel electrophoresis of the purified enzyme after molecular sieving on Sephadex G-200, only one band was obtained, suggesting that the enzyme preparation was obtained almost homogeneous by three steps of column chromatography. Aminopeptidase showed highest activity at pH 7.0, using a buffer system, of 70 mM Na-phosphate. The enzyme was found to be active at 40 degrees C, even at 60 degrees C (80% activity), suggesting that the human seminal plasma enzyme is fairly thermostable. Amongst the various aminoacyl derivatives evaluated as substrates in the present study, L-alanine beta-naphthylamide hydrochloride was found to have the highest rate of hydrolysis. Ovalbumin showed effective cleavage in comparison to that of other natural substrates. The Km value for the purified seminal plasma aminopeptidase towards L-alanine beta-naphthylamide hydrochloride was 4 x 10(-4) M. Hg+2 showed highest inhibitory effect than other metal ions tested in the present study. Concentration causing 50% inhibition of the enzyme (I50) by Hg2+ was 4.7 x 10(-6) M. Inhibition by EDTA at 1 mM concentration in the incubation system was higher than by EGTA and sodium azide, suggesting that the enzyme contains a metallo group at the active site. A 50% inhibition of the enzyme by EDTA was obtained at 5.11 x 10(-3) M. The Ackerman and Potter plot for EDTA inhibition suggests that EDTA is a reversible inhibitor of seminal plasma aminopeptidase. A single molecular form of aminopeptidase was found to be present in human seminal plasma as shown by polyacrylamide activity gel electrophoresis.     

Characterization of the O2-induced manganese-containing superoxide dismutase from Bacteroides fragilis

A manganese-containing superoxide dismutase (MnSOD) has been isolated from extracts of O2-induced Bacteroides fragilis. The enzyme, Mr 43,000, was a dimer composed of noncovalently associated subunits of equal size. A preparation whose specific activity was 1760 U/mg had 1.1 g-atoms Mn, 0.3 g-atoms Fe, and 0.2 g-atoms Zn per mol dimer. Exposing the enzyme to 5 M guanidinium chloride, 20 mM 8-hydroxyquinoline abolished enzymatic activity. Dialysis of the denatured apoprotein in buffer containing either Fe (NH4)2(SO4)2 or MnCl2 restored O2-. scavenging activity. The iron-reconstituted enzyme was inhibited 89% by 2 mM NaN3, similar to other Fe-containing superoxide dismutases. The Mn-reconstituted and native MnSOD were inhibited approximately 50% by 20 mM NaN3. Addition of ZnSO4 to dialysis buffer containing either the iron or manganese salt inhibited restoration of enzymatic activity to the denatured apoprotein. MnSOD migrated as a single protein band coincident with a single superoxide dismutase activity band in 7.5 or 10% acrylamide gels. Isoelectric focusing resulted in a major isozymic form with pI 5.3 and a minor form at pI 5.0. Mixtures of the MnSOD and the iron-containing superoxide (FeSOD), isolated from anaerobically maintained B. fragilis [E. M. Gregory and C. H. Dapper (1983) Arch. Biochem. Biophys. 220, 293-300], migrated as a single band on acrylamide gels and isoelectrically focused to a major protein band (pI 5.3) and a minor band at pI 5.0. The amino acid composition of MnSOD was virtually identical to that of the FeSOD. The data are consistent with synthesis of a single superoxide dismutase apoprotein capable of accepting either Mn or Fe to form the holoenzyme.     

Purification and properties of arylsulphatase A from rabbit testis.

Rabbit testis arylsulphatase A was purified 140-fold with a recovery of 20% from detergent extracts of an acetone-dried powder by using DE-52 cellulose column chromatography, gel filtration on Sephadex G-200 and preparative isoelectric focusing. The purified enzyme showed one major band with one minor contaminant on electrophoresis in a 7.5% (w/v) polyacrylamide gel at pH8.3. On sodiumdodecyl sulphate/polyacrylamidegel electrophoresis, a single major band was observed with minor contaminants. The final preparation of enzyme was free from general proteolytic, esterase, hyaluronidase, beta-glucuronidase and beta-galactosidase activities. Rabbit testicular arylsulphatase A exists as a dimer of mol.wt. 110000 at pH7.1. At pH5.0 the enzyme is a tetramer of mol.wt. 220000. Arylsulphatase A appears to consist of two identical subunits of mol.wt. 55000 each. The highly purified enzyme has pI4.6. The enzyme hydrolyses p-nitrocatechol sulphate with Km and Vmax, of 4.1 mM and 80nmol/min respectively, but has no activity toward p-nitrophenyl sulphate. The pH optimum of the enzyme varies with the incubation time. By applying Sephacex G-200 chromatography and preparative isoelectric focusing, one form of enzyme was obtained. The enzyme has properites common to arylsulphatase A of other sources with respect to the anomalous time-activity relationship, pI, inhibition by PO42-, SO32- and Ag+ ions and substrate affinity to p-nitrocatechol sulphate. However, the enzyme shows the temperature optimum of arylsulphatase B of other species.     

alpha-galactosidase A from human placenta. Stability and subunit size.

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.