Macular cherry-red spots and myoclonus with dementia: coexistent neuraminidase and beta-galactosidase deficiencies.

A patient was previously characterized as having a variant form of G_M1 gangliosidosis based on severe deficiencies in β-galactosidase activity in both leukocytes and fibroblasts using 4-methylumbelliferyl-β-D-galactoside and G_M1 ganglioside. Reexamination of her cultured fibroblasts revealed a severe deficiency in neuraminidase activity using neuramin lactose, fetuin and 2-(3′-methoxyphenyl)-N-acetyl-D-neuraminic acid as substrates, but normal neuraminidase activity using G_M3 ganglioside as a substrate. The presence of normal levels of β-galactosidase activity in leukocytes from the mother of the patient indicates that the β-galactosidase deficiency is not the primary enzyme defect in this type of patient.     

Correction of combined β-galactosidase/neuraminidase deficiency in human fibroblasts

The combined deficiency of β-galactosidase and neuraminidase in human fibroblasts can be corrected to nearly normal values. This can be accomplished by addition of concentrated culture medium obtained after NH₄Cl stimulation of different types of human fibroblasts, including those with an isolated β-galactosidase or neuraminidase deficiency. The corrective factor is a macromolecular glycoprotein, which is labile at 60°C. Its uptake by human fibroblasts is competitively inhibited by mannose-6-phosphate and its corrective action within β-gal^?/neur^? fibroblasts continues during a “chase” of 72 hours.     

Purification and properties of a neuraminidase from Streptococcus K 6646

A neuraminidase was purified from the culture filtrate of Streptococcus 6646 (group K) by means of ammonium sulfate fractionation and successive column chromatographies on N-(p-aminophenyl)oxamic acid-substituted Sepharose derivative and p-aminophenyl-2-acetamido-2-deoxy-1-thio-β-d-glucopyranoside-substituted Sepharose derivative. The former adsorbent was found to bind a β-galactosidase and a β-N-acetylhexosaminidase in addition to the neuraminidase, and the latter adsorbent bound the β-galactosidase in addition to the β-d-N-acetylhexosaminidase. These adsorbents effectively eliminated the contaminating glycosidase activities and a 1,500-fold purification of the neuraminidase was achieved by this procedure.The neuraminidase thus purified was homogeneous by electrophoresis on polyacrylamide gel, and its molecular weight was estimated to be 110,000 by gel filtration on Biogel P-200. The activity of the purified neuraminidase was slightly stimulated by Ca²⁺, Mg²⁺, Mn²⁺, and Co²⁺, and strongly inhibited by heavy metals. The specificity of the purified neuraminidase was almost the same with Vibrio cholerae or Clostridium perfringens neuraminidase. It completely hydrolyzes sialic acid residues in neuraminyl lactose and porcine thyroglobulin, but it liberates only 50% of sialic acid residues from porcine submaxillary mucin and ganglioside GD_1a.     

Analysis of oligomannosyl cores of cellular glycopeptides by digestion with endo-beta-N-acetylglucosaminidases.

Most of mannose-labeled glycopeptides from SV-40 transformed fibroblasts were hydrolyzed either by endo-β-N-acetylglucosaminidase D in the presence of β-galactosidase, β-N-acetylglucosaminidase and neuraminidase or by endo-β-N-acetylglucosaminidase H. The products were oligosaccharides with the probable structure of Man_nGlcNAc (n=3,5,6,….). The D enzyme preferentially released smaller oligosaccharides, while the H enzyme released larger oligosaccharides. The results indicate the structural homology between oligomannosyl cores of the cellular glycopeptides and those of non-membrane glycopeptides.     

Cell specificity in the developmental regulation of acid hydrolases by temporal genes

The cell specificity of expression of three distinct trans acting temporal gene systems determining the developmental control of α-galactosidase, β-galactosidase and β-glucuronidase was tested in mouse liver. For α-galactosidase and β-galactosidase, expression was limited to hepatocytes; no effect was seen in nonhepatocytes. For β-glucuronidase the data suggest that expression of the Gus-t temporal locus is also limited to hepatocytes, and that the smaller enzyme reduction seen in nonhepatocytes of some strains is due to a separate systemic regulatory locus that is also present in the [Gus] gene complex. We conclude that the temporal gene-determined timing mechanisms initiating switches in rates of enzyme synthesis are intrinsic to the cells themselves and are not communicated to adjacent cells. This conclusion applies to the temporal locus for β-glucuronidase that is proximate to its structural gene as well as those for α-galactosidase and β-galactosidase that are distant from the structural genes that they regulate.     

Sialic acid: A specific role in hematopoietic spleen colony formation

Vibrio cholerae neuraminidase (VCN) treatment of donor bone marrow cells results in a reduction in the number of hematopoietic colonies (CFUs) formed in the spleens of lethally irradiated mice. Treatment of marrow cells with sodium periodate under mild conditions, known to preferentially oxidze sialic acid, also reduced CFUs while subsequent potassium borohydride reduction restored CFUs to 80% of control levels. Innoculum viability as measured by in vitro incorporation of tritiated precursors into proteins, nucleic acids, and oligosaccharides was unaffected by VCN treatment. The ability of bone marrow cells in culture to respond to the hormone erythropoietin, as measured by the incorporation of ⁵⁹Fe into cyclohexanone-extractable heme, was also not affected by neuraminidase, making a cytotoxic effect of the VCN preparation unlikely. Incubation of VCN-treated marrow with either β-galactosidase or trypsin had no effect on the VCN-induced reduction in CFUs. These results are consistent with the idea that membrane sialic acid plays a direct and specific role in the implantation and development of CFUs.     

The effect of nalidixic acid on the expression of some genes in Escherichia coli K-12.

The synthesis of maltodextrin phosphorylase and the phage λ receptor of Escherichia coli K-12 is substantially inhibited by the presence of 50 μg nalidixic acid/ml in the culture medium. β-galactosidase synthesis is inhibited to a lesser extent and no inhibition of L-tryptophanase synthesis is observed. The inhibition of enzyme synthesis is apparently not due to the effect of nalidixic acid on deoxyribonucleic acid synthesis.     

Ganglioside GM1 beta-galactosidase: studies in human liver and brain

A microcolumn assay for ganglioside GM₁ β-galactosidase (EC 3.2.1.23) has been developed using GM₁ tritiated exclusively in the terminal galactose residue. The reaction is stimulated up to 100-fold by anionic and cationic detergents; this stimulation is inhibited by neutral detergents. 4-Methylumbelliferyl β-d-galactopyranoside is hydrolyzed about seven times more rapidly than GM₁ in human brain (gray matter) and liver. Agarose gel filtration separated two forms of GM₁ β-galactosidase in both brain and liver. The major form (ganglioside GM₁ β-galactosidase A) had a molecular weight of 60–70 × 10³ and the minor form (ganglioside GM₁ β-galactosidase B) 600–800 × 10³. The liver and brain GM₁ β-galactosidases and 4-methylumbelliferyl β-galactosidase A cochromatographed on fractionation. The two forms of the enzyme in liver isolated by gel filtration corresponded to the two major forms found on starch gel electrophoresis and were converted to electrophoretically slower-moving forms after treatment with neuraminidase (EC 3.2.1.8, Cl. perfringens) suggesting that both are sialylated glycoproteins. The activity of GM₁ β-galactosidase in the brain and liver tissue of patients with GM₁ gangliosidosis Types I and II was less than 2% of control values. The mutation in each GM₁ gangliosidosis appears to result in a severe reduction of activity of two ganglioside GM₁ β-galactosidases.     

Neurochemical abnormality in I-cell disease: chemical analysis and a possible importance of beta-galactosidase deficiency.

Sphingolipid composition in both gray and white matter of a patient with I-cell disease was normal except for the higher proportion.of G_MI-ganglioside in gray and white matter. In the patient's liver and kidney there was a significant accumulation of ceramide dihexoside and ceramide trihexoside and of sulphatide in kidney. Non-lipid hexosamine and sialic acid concentration in brain was increased 1.2-1.5 times above normal. Recovery of myelin from I-cell's white matter was 80-100%, suggesting that demyelination, if present, is minimal. Myelin lipid and myelin specific glycoprotein patterns were normal. Except for β-galactosidase activity the activity of other brain lysosomal enzymes were within the normal range. This finding was similar to that of Hurler's syndrome. Only β-galactosidase activity was reduced to less than 10% of normal in the patient's brain. To examine the possible metabolic significance of β-galactosidase deficiency in I-cell disease the physical characteristics of this enzyme, isolated from tissues from I-cell, Hurler and control patients, were compared using isoelectric focusing, Con A-Sepharose and Sephadex G-150 chromatography. The isoelectric point and the binding affinity of I-cell β-galactosidase with Con A-Sepharose was comparable to normal. However, the isoenzyme patterns of brain and liver I-cell β-galactosidase with Sephadex G-150 gel filtration revealed decreased acid β-galactosidase. Effects of the addition of sodium chloride on each fraction of β-galactosidase isoenzymes isolated from I-cell tissues were markedly different from controls, whereas the pH optimum of these enzymes were similar to normal. These enzyme characteristics in I-cell tissues were different from normal and Hurler's syndrome. These findings suggest that β-galactosidase deficiency in I-cell disease is a more specific phenomenon rather than secondary inhibition as found in the mucopolysaccharidoses and thus may have an important role for the pathogenesis of brain damage and disease occurrence.     

Mediator-induced macrophage activation, as shown by enhanced cytotoxicity for tumor, requires macrophage surface fucose and sialic acid

Macrophage-activating factor (MAF) activates macrophages so that their cytotoxic capacity is enhanced. This effect of MAF is inhibited by removing fucose from the macrophage cell surface by incubation with fucosidase, or by removing sialic acid by treatment with neuraminidase. After incubation with fucosidase or neuraminidase the average inhibition of cytotoxicity was 92 and 73%, respectively. β-Galactosidase had no effect. Addition of the specific products, fucose or sialic acid, to the incubation mixture of macrophages and enzyme blocked the effect of the enzymes. Taken together these observations indicate that macrophage surface fucose and sialic acid are essential for the interaction of MAF with macrophages which results in enhanced cytotoxicity for tumor cells.     

云南传统发酵豆豉中产β-半乳糖苷酶乳酸菌的筛选及其产酶条件的研究

目的从云南豆豉样品中筛选产β-半乳糖苷酶的乳酸菌,并对其产酶条件进行研究。方法从云南省元阳、红河、建水、石屏等地采集豆豉样品,并从中分离得到355株微生物。结果经明胶诱导、脱脂乳平板实验,复筛得到87株蛋白酶产生菌,从中筛选产β-半乳糖苷酶的乳酸菌。通过X-Gal平板实验,共获得34株产β-半乳糖苷酶菌株,通过酶活测定,最终筛选得到1株高产β-半乳糖苷酶菌株GJ-1-3L,经16S rDNA序列分析鉴定为短乳杆菌;GJ-1-3L在以葡萄糖为碳源、多聚蛋白胨为氮源、起始pH 6.5的MRS培养基中,接种量为4%,35℃发酵培养12 h,其β-半乳糖苷酶活性高达6.73 U/mL,Cu2＋、Ba2＋对酶活有抑制作用,而K2HPO4、MgSO4则能促进酶活。结论 GJ-1-3L菌株来源于豆豉,能够产生β-半乳糖苷酶发酵乳糖,同时产生乳酸,其在食品与乳品加工等方面具有很好的应用前景。     

The specific inhibition of the expression of the lactose operon by D,L-threo-phenylserine inEscherichia coli

Phenylserine, one of the phenylalanine analogues, is incorporated into proteins ofEscherichia coli and replaces the natural amino acid. The incorporation results in the inhibition of the synthesis of both inducible and constitutive β-galactosidase. The rate of the synthesis of β-galactosidase specific m-RNA is only slightly influenced by phenylserine, the steady-state level being decreased by about 40%. The m-RNA formed in the present of the analogue functions normally and its translation after the removal of the inhibitor results in the formation of normal β-galactosidase. The character of the inhibition of the enzyme synthesis by phenylserine is similar to that caused by chloramphenicol. However, phenylserine specifically inhibits only the synthesis of β-galactosidase, whereas other cell proteins are synthesized. No protein immunologically cross-reacting with the antiserum against normal β-galactosidase is formed by inducible ánd constitutiveEscherichia coli strains. The active transport is completely inhibited as the cells induced in the presence of phenylserine do not accumulate¹⁴C-TMG. It follows from the results that phenylserine inhibits both the formation of TMG-specific permease and the synthesis of the active molecule of β-galactosidase inEscherichia coli.     

The role of glycosidically bound mannose in the assimilation of β-galactosidase by generalized gangliosidosis fibroblasts

Bovine testicular β-galactosidase is rapidly assimilated by generalized gangliosidosis skin fibroblasts. The enzyme contains equimolar amounts of mannose and glucosamine and strongly binds to concanavalin A-Sepharose. Pretreatment of β-galactosidase with a mannosidase preparation from $\text{Aspergillus}\text{niger}$ reduced the rate of assimilation of the enzyme 97%. These data indicate that mannosyl residues play a role in assimilation of the enzyme. This conclusion is supported by observed inhibition of β-galactosidase assimilation by mannose, methyl α- and β-mannopyranosides, and mannose-containing testicular glycoproteins.     

I-Cell disease: Activities of lysosomal enzymes toward natural and synthetic substrates

Cultured skin fibroblasts from a patient with I-Cell disease (mucolipidosis II) were assayed for a number of lysosomal enzymes using both natural and synthetic substrates. The cells from this patient were found to have very low activity for galactosylceramide β-galactosidase, lactosylceramide β-galactosidases (using two assay methods that measure different enzymes), G_M1 ganglioside β-galactosidase and sphingomyelinase. Glucosylceramide β-glucosidase activity was found to be normal. Acid hydrolase activities toward many synthetic substrate were measured and all except β-glucosidase and acid phosphatase were found to be extremely low (as has been reported by others). Acid phosphatase and β-glucosidase were in the low normal range. These studies expand on previously published reports on I-Cell disease that only present data from synthetic substrates, and also report the fibroblast culture deficiencies of galactosyl-ceramide β-galactosidase (the Krabbe disease enzyme) and sphingomyelinase (the Niemann-Pick disease enzyme) activities for the first time. Those two enzymes do not have a readily available synthetic analog to assay. Acid β-galactosidase activity measured with both the 4-methylumbelliferyl derivative and G_M1 ganglioside was partially deficient in leukocytes prepared from this patient. New methods for measuring 4-methylumbelliferyl-β-D-glucoside and glucosylceramide β-glucosidase activities are also presented.     

Glucoheptonic acid derivative as a new transgalactosylation product

Transgalactosylation is increasingly used for modification of selected compounds because it may introduce new bioactive properties or improve existing ones. This paper presents the application of transgalactosylation activity of Kluyveromyces lactis β-galactosidase (EC 3.2.1.23) for a new derivative of glucoheptonic acid synthesis. The study concerned the impact of following factors on the course of reaction: content and ratio of substrates, enzyme dose, pH of the solution and the presence of salts. In the most favourable conditions (without salt), the final product concentration reached 54.5?g/L, which corresponded to 10.9% of dry matter. Research has shown that higher initial dry matter content results in higher product content (as % dm). The addition of 0.5–0.75?M MgCl₂ or 1?M NaCl led to significantly increased yield. In contrast, the presence of MnCl₂ or the lowest enzyme dose seemed to slow down the synthesis process. Increasing the pH over the optimal value for hydrolytic activity of β-galactosidase caused inhibition of transgalactosylation reaction. The molar ratio of 1.9:1 (sodium glucoheptonate:lactose) was the best among tested options. The described method allowed to successfully obtain a new compound with satisfactory yield in comparison to other transgalactosylation products.     

Intercellular exchange of lysosomal enzymes: enzyme assays in single human fibroblasts after co-cultivation.

Intercellular exchange of N-acetyl-β-D-glucosaminidase (EC 3.2.1.30) β-galactosidase (EC 3.2.1.23) and acid α-glucosidase (EC 3.2.1.20) was studied after cocultivation of normal and enzyme deficient human fibroblasts in confluent cultures. Enzyme activities were measured in single cells using microchemical procedures. After co-cultivation of normal control fibroblasts and those from a patient with Sandhoff's disease an increase of activity of N-acetyl-β-D-glucosaminidase was found in Sandhoff cells, together with a decrease of activity in normal control cells. After co-cultivation of normal fibroblasts and those from patients with glycogenosis II and GM1-gangliosidosis, no indication was found for intercellular transfer of acid α-glucosidase and β-galactosidase respectively. The significance of the results is discussed in respect of the hypothesis of Hickman and Neufeld about secretion and uptake of lysosomal enzymes.     

蚕豆β－半乳糖苷酶活性必需氨基酸残基分析

 蚕豆β－半乳糖苷酶活性必需氨基酸残基分析龚笑海,孙册（中国科学院上海生物化学研究所,上海２０００３１）化学修饰作为一种研究酶活性基团的方法,对研究糖苷酶的催化机理有其独到的优点,并已有这方面的报道．从蚕豆纯化的β－半乳糖苷酶,分子量为７０ｋＤ,由两个...     

Preparation of immobilized β-galactosidase by poly(vinylpyrrolidone) and the continuous hydrolysis of lactose in acid whey

Immobilized β-galactosidase gel was prepared using poly(vinylpyrrolidone) (PVP) under β-ray irradiation. In contrast to the gelation of N-vinylpyrrolidone monomer–enzyme solution, the gelation of PVP-β-galactosidase solution (PVP content: 10%) was almost completely uneffected by the dose rate and amount of phosphate present. PVP-enzyme solution was gelled by irradiation with 3.0 Mrad. The expressed activity of the PVP-enzyme gel was about 30% of the initial activity and added activity was almost totally entrapped. No leakage of enzyme from these gels could be detected. Leakage was, however, detected in the case of the gelation of PVP-enzyme solution containing more than 1% of enzyme protein. When the general properties of the gel were compared with those of the native enzyme, the gel proved to be slightly inferior to the native enzyme with respect to optimum temperature, heat stability, pH activity, and pH stability. Continuous hydrolysis of lactose in acid whey could be carried out at 50°C using a column packed with the gel and sawdust and the degree of hydrolysis was found to be almost, constant for 12 days. The merits of using PVP in the immobilization of enzymes include the simplicity of the procedure and the fact that the PVP-enzyme gel can be used in the food industry without anxiety because of its high degree of compatibility with living organisms.     

Characterization of Lactose Utilization and β-Galactosidase in Lactobacillus brevis KB290, the Hetero-Fermentative Lactic Acid Bacterium

Unlike dairy lactic acid bacteria, Lactobacillus brevis cannot ferment milk. We characterized the lactose utilization by L. brevis KB290. In a carbohydrate fermentation assay using API 50 CHL, we showed during 7?days L. brevis did not ferment lactose. L. brevis grew to the stationary phase in 2?weeks in MRS broth containing lactose as the carbon source. L. brevis slowly consumed the lactose in the medium. L. brevis hydrolyzed lactose and a lactose analog, o-nitrophenyl-β-d-galactopyranoside (ONPGal). This β-galactosidase activity for ONPGal was not repressed by glucose, galactose, fructose, xylose, or maltose showing the microorganism may not have carbon catabolite repression. We purified the L. brevis β-galactosidase using ammonium sulfate precipitation and several chromatographies. The enzyme’s molecular weight is estimated at 72 and 37?kDa using SDS-PAGE analysis. The N-terminal amino acid sequence of the larger protein was 90?% similar to the sequence of the putative β-galactosidase (YP_796339) and the smaller protein was identical to the sequence of the putative β-galactosidase (YP_796338) in L. brevis ATCC367. This suggests the enzyme is a heterodimeric β-galactosidase. The specific activity of the purified enzyme for lactose is 55?U/mg. We speculate inhibition of lactose transport delays the lactose utilization in L. brevis KB290.