Acid glycohydrolase in Chinese hamster with spontaneous diabetes II. N-acetyl-beta-D-hexosaminidase in plasma and tissues

Excessively high activity of N-acetyl-beta-D-hexosaminidase (2-acetamido-2-deoxy-beta-D-glucoside acetamidodeoxy-glucohydrolase, EC 3.2.1.30) was found in the plasma of hereditary diabetic XA line animals, which however showed similar activity of this enzyme in both 12 00 X g supernatant and precipitate fractions of kidney homogenates as the nondiabetic M line animals. 0.1% Triton X-100 extracts of kidney, spleen, hind leg muscle, cheek pouch and spinal cord of XA and M line animals also showed similar activities of this enzyme whereas the XA animal liver extracts showed significantly higher activity than the M extracts. On a Sepharose CL-6B column, plasma N-acetyl-beta-D-hexosaminidase was eluted as two major peaks at 0 and 0.05 M NaCl (isozyme B1 and B2). Both isozymes showed pH optima between 3.5 and 4.0 and the same Michaelis constants for p-nitrophenyl-N-acetyl-beta-D-glucosaminide at pH 4.5, i.e. 0.18 mM. Isozymes from XA and M animals showed identical properties. N-acetyl-beta-D-hexosaminidase in the liver extracts was separated into 3 isozymes, A, B1 and B2, by successive column chromatography runs on Sepharose 6B and DEAE-Sepharose CL-6B. At 49 degrees C, isozyme B1 showed thermostability whereas B2 and A lost 20% and 76% of their activities after 30 min incubation, pH optima for A, B1 and B2 were 4.0--4.5, 3.5 and 3.5--4.0 respectively. The Km values for p-nitrophenyl-N-acetyl-beta-D-glucosaminide were 0.48 mM for A and 0.19 for B1 and B2. The XA animal liver extracts showed higher activities in all three isozymes than the M animal livers. Identical results, however, were obtained for liver isozymes from M and XA animals with regard to thermostability, pH vs. activity, elution profile on ion exchange column and affinity to p-nitrophenyl-N-acetyl-beta-D-glucosaminide.     

Acid glycohydrolase in Chinese hamster (Cricetulus griseus) with spontaneous diabetes--V. Subcellular distribution in the kidney.

1. Seven renal glycohydrolases were measured in four subcellular fractions prepared from highly inbred aglycosuric (AV-line) and glycosuric (XA-line) Chinese hamsters. 2. alpha-D-galactosidase and beta-D-galactosidase were highest in the nuclear (N) and supernatant (S) fractions; both fractions showed reduced activities in the XA animals. 3. alpha-D-mannosidase was chiefly a particulate enzyme and its decrease in XA animals was evident in N, lysosomal-mitochondrial (LM) and mitochondrial-microsomal (MM) fractions. 4. No significant difference in N-acetyl-beta-D-glucosaminidase was found in any of these subcellular fractions between AV and XA animals. 5. Although total alpha-L-fucosidase and beta-D-fucosidase levels were similar in AV and XA kidneys, a difference was observed in the S fraction. 6. beta-D-glucuronidase was virtually absent in N and LM fractions and the S fraction of AV kidneys showed higher activity than the XAs.     

Comparative studies of three exo-beta-glycosidases of Aspergillus oryzae.

beta-Glucosidase [beta-D-glucoside glucohydrolase EC 3.2.1.21] and beta-galactosidase [beta-D-galactoside galactohydrolase, EC 3.2.1.23] of Takadiastase were purified by acetone fractionation, DEAE-cellulose, and hydroxylapatite chromatography. Purity was confirmed by disc electrophoresis, ultracentrifugation and measurement of other glycosidase activities which coexisted in Takadiastase. Molecular weight of the beta-glucosidase was 218,000 by sedimentation equilibrium and 110,000-116,000 by SDS-disc electrophoresis. Molecular weight of the beta-galactosidase was 112,000 by sedimentation and 56,000-59,000 by SDS-disc electrophoresis. These values showed that both enzymes consisted of two subunits. Taka-beta-N-acetylglucosaminidase also consisted of two subunits. Both enzymes were glycoproteins containing glucosamine and neutral sugar. Stability, pH optima, isoelectric points, and some specificities were observed.     

Sexual differences in galactose metabolism: galactosyl ceramide galactosidase and other galactosidases in mouse kidney (Short Communication)

Male and female mice were compared at two ages, 15 and 50 days, with respect to the activities of three galactosidases in kidney. No sex difference in enzyme activity was seen in the young mice, but appreciable differences were found in the older animals. The male kidneys had about one-third higher specific activities of cerebroside beta-galactosidase and nitrophenyl beta-galactosidase, but there was no difference with nitrophenyl alpha-galactosidase. A listing and discussion of other galactose-metabolizing enzymes influenced by sex differences is presented.     

Studies on the kinetics of glycosidases from chemically-induced rat colonic tumours and normal rat colon.

K-m values of beta-N-acetylglucosaminidase (2-acetamido-2-deoxy-beta-D-glucoside acetamidodeoxyglucohydrolase EC 3.2.1.30), beta-N-acetylgalactosaminidase (EC 3.2.1.53), beta-galactosidase (beta-D-galactoside galactohydrolase EC 3.2.1.23) and alpha-L-fucosidase (alpha-L-fucoside fucohydrolase EC 3.2.1.51) of distal colonic tumours, induced in rats by 1,2-dimethylhydrazine, were found to be significantly different compared with the values for the enzymes of the colonic mucosa of the control and tumour-bearing animals and of the proximal colonic tumours. The inhibition kinetics data also showed a significant difference between the enzymes of the distal colon tumours and of other experimental tissues. The data on the effect of pH on enzyme kinetics (pK values) showed no significant difference in the catalytic groups of the active centres of enzymes from tumours and from the control colonic mucosa. Tumour beta-N-acetylglucosaminidase and beta-N-acetylgalactosaminidase compared with the enzymes from other experimental tissues were found to be different in their thermal inactivation kinetics. K-m values of 14 days old foetal intestinal beta-N-acetylglucosaminidase and beta-N-acetylgalactosaminidase were significantly different from the values obtained for the adult mucosal enzymes but were similar to those of the distal colonic tumour enzymes.     

Stimulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase in mouse uterine epithelial cells by oestradiol-17 beta.

Two alpha-D-galactosidases (alpha-D-galactoside galactohydrolase, EC 3.2.1.22) produced by Aspergillus tamarii were purified from the mycelial extract by a procedure including chromatography on hydroxyapatite, DEAE-cellulose and ECTEOLA-cellulose. Each of these enzymes showed a single protein band corresponding to the alpha-D-galactosidase activity when examined by polyacrylamide-gel electrophoresis. They catalysed the hydrolysis of o-nitrophenyl alpha-D-galactoside, melibiose, raffinose and stachyose, but did not attack the galactomannans. Their Mr values were respectively 265000 +/- 5000 and 254000 +/- 5000 by the method of Hedrick & Smith [(1968) Arch. Biochem. Biophys. 126, 155-164]. Polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate in each case showed a single protein band, with Mr 88000 and 77500 respectively. The purified enzymes contained carbohydrate, consisting of N-acetylglucosamine, mannose, glucose and galactose in the estimated molar proportions of 1:9:5:8 in alpha-galactosidase I.     

Plasma alpha-galactosidase A:properties and comparisons with tissue alpha-galactosidases.

The human plasma form of alpha-galactosidase A (alpha-D-galactoside galactohydrolase, EC 3.2.1.22) was highly purified and exhibited apparent Km values of 1.9 mM with 4-methylumbelliferyl-alpha-D-galactopyranoside and 0.23 mM with globotriglycosylceramide. Its inhibition with myo-inositol (Ki = 0.29 M) was similar to that observed with alpha-galactosidase A from various tissues. The plasma form of this lysosomal enzyme has a lower molecular weight of 96 600, a lower pI of 3.7 and faster electrophoretic mobility in polyacrylamide gels than the enzyme obtained from human liver. These data and the increased pI obtained after neuraminidase treatment suggest that the plasma form is an isoenzyme with a more highly sialylated carbohydrate moiety than the tissue isoenzymes.     

Intracellular distribution of beta-galactosidases in mucosal cells from hog small intestine.

1. Intracellular distribution of three types of beta-galactosidases (beta-D-galactoside galactohydrolase EC 3.2.1.12) i.e. hetero beta-galactosidase, lactase and acid beta-galactosidase, was studied by examining the properties of subcellular fractions isolated by a systematic fractionation of mucosal cells of the small intestine of the hog. Localization of hetero beta-galactosidase in cytosol could be shown. 2. Localization of lactase in the brush borders was shown by analyzing the purified brush borders prepared separately. 3. To domonstrate the lysosomal localization of acid belta-galactosidase, lysosomes were purified separately and their extract was chromatographed on a hydroxylapatite column. The activities of various enzymes in the purified lysosomes as well as in the intermediary fractions obtained during lysosome purification and the pattern of the hydroxylapatite chromatography led us to conclude that acid beta-galactosidase is a lysosomal enzyme.     

Purification and characterization of extremely thermostable beta-mannanase, beta-mannosidase, and alpha-galactosidase from the hyperthermophilic eubacterium Thermotoga neapolitana 5068.

Thermostable and thermoactive beta-mannanase (1,4-beta-D-mannan mannanohydrolase [EC 3.2.1.78]), beta-mannosidase (beta-D-mannopyranoside hydrolase [EC 3.2.1.25]) and alpha-galactosidase (alpha-D-galactoside galactohydrolase [EC 3.2.1.22]) were purified to homogeneity from cell extracts and extracellular culture supernatants of the hyperthermophilic eubacterium Thermotoga neapolitana 5068 grown on guar gum-based media. The beta-mannanase was an extracellular monomeric enzyme with a molecular mass of 65 kDa. The optimal temperature for activity was 90 to 92 degrees C, with half-lives (t1/2) of 34 h at 85 degrees C, 13 h at 90 degrees C, and 35 min at 100 degrees C. The beta-mannosidase and alpha-galactosidase were found primarily in cell extracts. The beta-mannosidase was a homodimer consisting of approximately 100-kDa molecular mass subunits. The optimal temperature for activity was 87 degrees C, with t1/2 of 18 h at 85 degrees C, 42 min at 90 degrees C, and 2 min at 98 degrees C. The alpha-galactosidase was a 61-kDa monomeric enzyme with a temperature optimum of 100 to 103 degrees C and t1/2 of 9 h at 85 degrees C, 2 h at 90 degrees C, and 3 min at 100 degrees C. These enzymes represent the most thermostable and thermoactive versions of these types yet reported and probably act synergistically to hydrolyze extracellular galactomannans to monosaccharides by T. neapolitana for nutritional purposes. The significance of such substrates in geothermal environments remains to be seen.     

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.     

Enzymological properties and immunological characterization of alpha-galactosidase isoenzymes from normal and Fabry human liver.

1. A method is described for the rapid isolation of alpha-galactosidases A and B (alpha-D-galactoside galactohydrolase, EC 3.2.1.22) from normal human liver. 2. When the same method is applied to Fabry liver, most of the alpha-galactosidase activity is recovered in the fraction corresponding to normal alpha-galactosidase B. In agreement with Romeo, G., D'Urso, M., Pisacane, A., Blum, E., De Falco, A. and Ruffilli, A. (1975) Biochem. Genet. 13, 615-628) [18], a small amount of alpha-galactosidase activity is found in the fraction corresponding to normal alpha-galactosidase A. 3. The kinetic properties of the B-like activity from Fabry liver are similar to those of normal alpha-galactosidase B. In agreement with Romeo et al. [18], it was found that the kinetic properties of the A-like activity from Fabry liver are similar to those of normal alpha-galactosidase A. 4. Using antisera raised against normal alpha-galactosidase A and normal alpha-galactosidase B, it is shown that the normal alpha-galactosidase isoenzymes are immunologically distinct and that the B-like activity from Fabry liver is immunologically related to normal alpha-galactosidase B. Furthermore, the A-like activity from Fabry liver is immunologically related to normal alpha-galactosidase B and not to normal alpha-galactosidase A. 5. Normal alpha-galactosidase B is converted into an A-like form during storage. 6. It is concluded that the B-like alpha-galactosidase in Fabry tissues is identical to normal alpha-galactosidase B, and that the small amount of A-like activity found in Fabry material is due to a modified form of alpha-galactosidase B.     

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.     

alpha-Galactosidases II, III and IV from seeds of Trifolium repens. Purification, physicochemical properties and mode of galactomannan hydrolysis in vitro.

Five alpha-galactosidases (alpha-D-galactoside galactohydrolase, EC 3.2.1.22) were identified by chromatography and by their different electrophoretic mobilities, in the germinated seeds of Trifolium repens (white clover). alpha-Galactosidases II, III and IV were purified to homogeneity, with increases in specific activity of approx. 4600-, 4900- and 2800-fold respectively. The enzymes were purified by a procedure that included (NH4)2SO4 precipitation, hydroxyapatite, Sephadex G-75 and DEAE-cellulose chromatography, and preparative polyacrylamide-gel disc electrophoresis. The purified enzymes showed a single protein band, corresponding to the alpha-galactosidase activity, when examined by polyacrylamide-gel electrophoresis. The pH optimum was determined with o-nitrophenyl alpha-D-galactoside and the galactomannan of T. repens To as substrate. All three enzymes are highly thermolabile. Hydrolysis of oligosaccharides and galactomannans was examined, including two galactomannans from the germinated seed of T. repens (T24 and T36). By sodium dodecyl sulphate/polyacrylamide-gel electrophoresis the mol.wts. of the multiple forms of enzyme were found to be identical (41 000).     

Concentration of phosphoribosyl pyrophosphate in renal hypertrophy. Contrasting effects of early diabetes and unilateral nephrectomy.

Studies were made of the renal phosphoribosyl pyrophosphate (PPRibP) content and PPRibP synthetase (EC 2.7.6.1) activity in rats diabetic for 5, 14 or 20 days, or unilaterally nephrectomized (UN) for 5 days, and in doubly lesioned animals. Approximately equal degrees of renal enlargement were found after 5 days diabetes or 5 days UN. In the doubly lesioned animals the increment of growth was additive. Unilateral nephrectomy of 5 days duration, in contrast with diabetes, had no effect on the PPRibP content of the contralateral kidney, nor did it modify the renal PPRibP content when performed on animals diabetic for 5, 14 or 20 days. The activity of PPRibP synthetase was unaffected by diabetes, UN or diabetes +UN. The results pinpoint a stage of nucleotide synthesis which is differentially affected by the two stimuli, in line with evidence for differences in regulation of nucleic acid turnover in the two conditions.     

Characterization of two new glycosyl hydrolases from the lactic acid bacterium Carnobacterium piscicola strain BA.

Three genes with homology to glycosyl hydrolases were detected on a DNA fragment cloned from a psychrophilic lactic acid bacterium isolate, Carnobacterium piscicola strain BA. A 2.2-kb region corresponding to an alpha-galactosidase gene, agaA, was followed by two genes in the same orientation, bgaB, encoding a 2-kb beta-galactosidase, and bgaC, encoding a structurally distinct 1.76-kb beta-galactosidase. This gene arrangement had not been observed in other lactic acid bacteria, including Lactococcus lactis, for which the genome sequence is known. To determine if these sequences encoded enzymes with alpha- and beta-galactosidase activities, we subcloned the genes and examined the enzyme properties. The alpha-galactosidase, AgaA, hydrolyzes para-nitrophenyl-alpha-D-galactopyranoside and has optimal activity at 32 to 37 degrees C. The beta-galactosidase, BgaC, has an optimal activity at 40 degrees C and a half-life of 15 min at 45 degrees C. The regulation of these enzymes was tested in C. piscicola strain BA and activity on both alpha- and beta-galactoside substrates decreased for cells grown with added glucose or lactose. Instead, an increase in activity on a phosphorylated beta-galactoside substrate was found for the cells supplemented with lactose, suggesting that a phospho-galactosidase functions during lactose utilization. Thus, the two beta-galactosidases may act synergistically with the alpha-galactosidase to degrade other polysaccharides available in the environment.     

Papaya beta-galactosidase/galactanase isoforms in differential cell wall hydrolysis and fruit softening during ripening.

The potential significance of the previously reported papaya (Carica papaya L.) beta-galactosidase/galactanase (beta-d-galactoside galactohydrolase; EC 3.2.1.23) isoforms, beta-gal I, II and III, as softening enzymes during ripening was evaluated for hydrolysis of pectins while still structurally attached to unripe fruit cell wall, and hemicelluloses that were already solubilized in 4 M alkali. The enzymes were capable of differentially hydrolyzing the cell wall as evidenced by increased pectin solubility, pectin depolymerization, and degradation of the alkali-soluble hemicelluloses (ASH). This enzyme catalyzed in vitro changes to the cell walls reflecting in part the changes that occur in situ during ripening. beta-Galactosidase II was most effective in hydrolyzing pectin, followed by beta-gal III and I. The reverse appeared to be true with respect to the hemicelluloses. Hemicellulose, which was already released from any architectural constraints, seemed to be hydrolyzed more extensively than the pectins. The ability of the beta-galactanases to markedly hydrolyze pectin and hemicellulose suggests that galactans provide a structural cross-linkage between the cell wall components. Collectively, the results support the case for a functional relevance of the papaya enzymes in softening related changes during ripening.     

Microbial alpha-galactosidase (a review)

The review discusses properties, distribution and potential use of microbial alpha-galactosidase (alpha-D-galactoside galactohydrolase, EC 3.2.1.22), the enzyme catalyzing degradation of alpha-D-galactoside bonds. Recent years have witnessed many publications describing microbial alpha-galactosidase which, in contrast to the similar enzyme from higher plants, has been poorly studied. The microbial enzyme has certain specificities: a smaller substrate specificity, existence in one molecular form, etc. The present communication is an attempt to systematize the data about microbial alpha-galactosidase and to outline the most important investigations for the future.     

Glycoprotein enzymes secreted by Aspergillus niger: purification and properties of alpha-glaactosidase.

An alpha-galactosidase (alpha-D-galactoside galactohydrolase [EC 3.2.1.22]) was purified to homogeneity from the culture filtrate of Aspergillus niger. The enzyme had an apparent molecular weight of 45,000 and was a glycoprotein. Radioactive enzyme was prepared by growing cells in [14C]fructose and this enzyme was used to prepare 14C-labeled glycopeptides. The glycopeptides emerged from Sephadex G-50 between stachyose and the glycopeptide from ovalbumin. Based on calibration of the column with various-sized dextran oligosaccharides, the glycopeptides appeared to have a molecular weight of 1,200 to 1,400. Analysis of the glycopeptide(s) indicated that it contained mannose and N-acetylglucosamine (GlcNAc) in an approximate ratio of 3 or 4 to 1. Assuming that there are two GlcNAc residues in the oligosaccharide and based on the molecular weight of the glycopeptide, the oligosaccharide probably contains eight to nine sugar residues. Alks probably attached to the protein by a GlcNAc leads to asparagine linkage. The purified alpha-galactosidase was most active on raffinose (Km = 5 x 10--4 M, Vmax = 3 mumol/min per mg of protein), but also showed good activity on p-nitrophenyl-alpha-D-galactoside ans somewhat less activity on stachyose and melibitol. The enzyme also hydrolyzed guar flour and locust bean gum, but did not attack the p-nitrophenyl glycosides of beta-galactose, alpha- or beta-glucose, or alpha- or beta-mannose.