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PubMed1978

Acid beta-glucosidase and the diagnosis of Gaucher's disease in liver and spleen.

Variable amounts of residual 4-methylumbelliferyl-beta-glucosidase activity gave rise to difficulties in the diagnosis of Gaucher's disease using spleen and particularly liver. Soluble and particulate components of beta-glucosidase, which interfere with the diagnosis, may be eliminated by preincubation of homogenates at pH 3.0 or with 100 mM sodium chloride at pH 4.0. After either of these treatments the optimum of acid beta-glucosidase was pH 4.5 and diagnosis of Gaucher's disease could be more easily made using homogenates of spleen and liver. There was residual acid beta-glucosidase activity in one of the Gaucher livers.

Gaucher Disease↗

PubMed1978

alpha-glucosidase activity in human leucocytes: choice of lymphocytes for the diagnosis of Pompe's disease and the carrier state.

alpha-Glucosidase activity was assayed in polymorphonuclear cells and lymphocytes from human peripheral blood with 4-methylumbelliferyl-alpha-D-glucopyranoside as substrate in the presence of sodium taurocholate. The pH vs. activity curve of the alpha-glucosidase indicated that differential estimation between acid and neutral alpha-glucosidases was difficult to perform with polymorphonuclear cells, but easily accessible with lymphocytes. The use of peripheral blood lymphocytes for the enzymatic diagnosis of Pompe's disease seemed to be more reliable than the use of whole leucocytes; this also the case with a classical Pompe's patient. The lymphocytes from the parents had normal or low normal activity of acid alpha-glucosidase in the freshly isolated state, but when cultured with phytohaemagglutinin for 72 h, the stimulated lymphocytes of both parents showed about half the enzyme activity of the cultured controls. It was deemed possible in all probability to identify the carrier state by assay of the enzyme activity in phytohaemagglutinin-stimulated lymphocytes.

Adult↗

PubMed1981

Human amniotic fluid alpha-glucosidase.

Amniotic fluid in midpregnancy contains significant alpha-glucosidase activity. This enzyme is distinguishable from the lysosomal acid alpha-glucosidase, deficiency of which is associated with Pompe's disease. The two enzymes differ in optimum pH, thermal stability, electrophoretic migration, isoelectric point, molecular mass, and immunological response. Amniotic alpha-glucosidase is also different from the classical neutral form. Immuno-cross reactions suggest that the amniotic fluid enzyme has a double fetal origin: renal and intestinal. It seems that alpha-glucosidase in amniotic fluid is linked to lipids.

Amniotic Fluid↗

PubMed1981

The characterization of a thermostable activator of beta-D-glucosidase in normal human saliva.

The existence of thermostable activators of beta-D-glucosidase (EC 3.2.1.21) in Gaucher tissues is known. We demonstrate the presence of a thermostable factor in normal human saliva which activates the beta-D-glucosidase of saliva. The activator is specific for beta-glucosidase. It is dialysable and susceptible to pronase digestion. Sephadex G-25 gel filtration indicates that the activator is of low molecular weight, and polyacrylamide gel electrophoresis shows the low molecular weight fraction to migrate as two closely moving protein bands. A major proportion of the activator does not bind to concanavalin A-Sepharose. The activator appears to act by binding to or modifying the salivary beta-glucosidase in a time-dependent manner.

Chromatography, Affinity↗

PubMed1983

Zonal differences of alpha-glucosidases in human kidney: studies in controls and in patients with glycogenosis type II.

In the kidneys of two infants who died from glycogenosis type II (deficiency of lysosomal acid alpha-glucosidase) the activity of alpha-glucosidase at acid pH was absent only from the medulla but not the cortex. This prompted an investigation of acid and neutral alpha-glucosidases of the two zones of normal human kidney and of liver. In the kidney medulla, a single, fast migrating electrophoretic band was active at neutral but not at acid pH. In the cortex, three additional bands were detected at neutral pH and two in the liver. One slowly migrating band was unique to the cortex and active over a broad pH range. It had comparatively high sensitivity to the inhibitor turanose and high heat stability. Its properties suggested a close relationship or identity to glucoamylase of the intestinal brush border membrane and to alpha-glucosidase in leukocytes and amniotic fluid and cells.

Child↗

PubMed1984

Sodium taurocholate effect on beta-glucosidase activity: a new approach for identification of Gaucher disease using the synthetic substrate and leucocytes.

In this work we have studied the leucocytes and sera of 3 Gaucher patients, 4 obligate heterozygotes, 11 brothers and sisters of patients and 11 controls. Beta-glucosidase activity with 4-M-U-beta-glucopyranoside has been assayed at different pH's, in the presence of pure sodium taurocholate. At pH 4.5 and 5.0 sodium taurocholate activates the beta-glucosidase of control leucocytes, but inhibits the residual enzyme present in Gaucher leucocytes. The ratio of beta-glucosidase activity in the presence and absence of this effector seems to be a good approach to the diagnosis of Gaucher disease and it has proved indispensible in one patient's diagnosis. The apparent Km of beta-glucosidase determined for the same substrate, at pH 4.5 and 5.5 in the presence of sodium taurocholate showed markedly lower values in the patients than in the controls. An increased serum acid phosphatase activity, previously described as a secondary alteration in Gaucher disease, has also been studied and seems to be a useful complementary test, particularly when its age dependence is taken into account.

Acid Phosphatase↗

PubMed1982

Adult forms of glycogenosis type II. A defect in an early stage of acid alpha-glucosidase realization.

The activity of acid alpha-glucosidase in cultured fibroblasts from adult patients with the lysosomal storage disease glycogenosis type II is only 10% of normal. A normal activity per molecule is found for the mature as well as for the precursor form of acid alpha-glucosidase in adult mutant fibroblasts. Excessive lysosomal breakdown of mature enzyme purified from mutant fibroblasts and taken up by acceptor cells does not occur. However, the NH4Cl-stimulated secretion of a precursor form of acid alpha-glucosidase by adult mutant fibroblasts is markedly reduced. The results are indicative of a defect during the production of acid alpha-glucosidase.

Age Factors↗

PubMed1982

Biosynthesis of acid alpha-glucosidase in late-onset forms of glycogenosis type II (Pompe's disease).

Cultured human skin fibroblasts from control persons and from patients with the generalized and late-onset forms of Pompe's disease were labelled with radioactive leucine and the incorporation of radioactivity into acid alpha-glucosidase and cathepsin D was analysed by immunoprecipitation, gel electrophoresis and fluorography. When the labelling was carried out for 6-12 h in the presence of NH4Cl, the labelling of secreted alpha-glucosidase relative to that of secreted cathepsin D in fibroblasts from patients with the late-onset form of Pompe's disease was less than 15% of that in fibroblasts from control persons. However, when the fibroblasts were labelled for less than 1 h, the relative rate of incorporation of radioactivity into acid alpha-glucosidase was rather similar in the two types of fibroblasts. In fibroblasts from patients with the generalized form of Pompe's disease no incorporation of radioactivity into acid alpha-glucosidase could be detected.

Cathepsin D↗

PubMed1985

The molecular basis for charge heterogeneity in human acid alpha-glucosidase.

The molecular basis for charge heterogeneity in human hepatic alpha-glucosidase (alpha-D-glucoside glucohydrolase, EC 3.2.1.20) was determined by analysis of the carbohydrate and polypeptide components of the enzyme. Only small remnants of high mannose chains that contained neither sialic acid nor mannose 6-phosphate were detected in the carbohydrate structure. Four enzymatically active forms of alpha-glucosidase separated by chromatofocusing were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and were found to contain different polypeptides. The absence of charged residues in oligosaccharide chains and variability in the polypeptide subunits of the charge forms of hepatic alpha-glucosidase suggest that charge heterogeneity results from differences in the protein structure of the charge forms. The pattern of differences in the polypeptide subunits suggests that the charge forms for hepatic alpha-glucosidase may be the product of proteolysis.

Electrochemistry↗

PubMed1989

Inhibition of beta-glucosidase by imidazoles.

Over 25 nitrogen-containing heterocycles were tested as inhibitors of sweet almond beta-glucosidase (EC 3.2.1.21). Among the most potent of these are some imidazole derivatives. The pH dependence indicates that the unprotonated inhibitor binds most tightly to the catalytically active species of the enzyme. This is analogous to the situation with 1-deoxynojirimycin where the permanently cationic species, N,N-dimethyl-1-deoxynojirimycin, binds at least two orders of magnitude less tightly to the enzyme than does the unprotonated 1-deoxynojirimycin. The binding of imidazole derivatives show a general tendency of increasing affinity with increasing basicity (beta approximately 0.4). One derivative which shows a significant positive deviation from this correlation (- log Ki vs. pKa) is 4-phenylimidazole. 4-Phenylimidazole is one of the most potent reversible inhibitors of beta-glucosidase with a pH-independent Ki = 0.8 microM. It is also fairly specific for beta-glucosidase, binding at least three orders of magnitude less tightly to any of the other exoglycosidases tested. This inhibitor combines, in a mono-molecular species, the binding affinities of benzene, which binds at the hydrophobic aglycone binding site, and imidazole, which binds at the sugar binding site of beta-glucosidase. The binding energy of 4-phenylimidazole can be attributed to the sum of the intrinsic binding energies of the phenyl and imidazole moieties. Thus, there is no significant entropic advantage of combining the component parts of phenylimidazole in a single species. This indicates that there is no significant uncompensated entropy loss upon binding of either benzene or imidazole to the enzyme. Nevertheless, the additivity of binding energy, even in the absence of an entropic advantage, results in the most powerful known inhibitor of the enzyme.

1-Deoxynojirimycin↗

PubMed1979

Destruction and resynthesis of mouse beta-glucosidases.

1. Injection of a single dose of conduritol B epoxide into mice produced almost complete destruction of glucocerebrosidase (D-glucosyl-N-acylsphingosine glucohydrolase, EC 3.2.1.45) in liver, spleen, brain, and kidney within 5 h. Restoration of activity became noticeable within 1 day (2 days in the case of brain) and was about 80% of normal within 16 days. 2. The same injection produced less destruction of aryl beta-glucosidase (EC 3.2.1.21), measured at pH 5.4 with methylumbelliferyl glucoside in the absence of taurocholate. Brain showed the least amount of destruction, about 50%, but measurements of activity at lower pH values revealed complete loss of activity. This suggests that brain contains two different aryl glucosidases with differing sensitivity to the inhibitor. Liver, on the other hand, did not show differential destruction when assayed at different pH values. Resynthesis of the enzyme activities was almost complete by 16 days. 3. Injection of phenylhydrazine produced hemolysis and spleen enlargement, with concomitant increases in specific activities of glucocerebrosidase and aryl glucosidase in liver and spleen (but not in kidney). When this experiment was done in mice previously treated with conduritol B expoxide, the reappearance of cerebrosidase was found to be accelerated. This is interpreted to mean that the increased load of glucolipids from the erythrocytes had induced an enhanced synthesis of the glucohydrolase. A similar explanation may apply to aryl glucosidase and glucopeptides in the cells.

Animals↗

PubMed1979

Enzymic effects of beta-glucosidase destruction in mice. Changes in glucuronidase levels.

1. The injection into mice of a single dose of conduritol B epoxide, a covalent inhibitor of glucosidases, quickly produced changes in tissue levels of beta-D-glucuronidase (EC 3.2.1.31). The specific activity of the enzyme decreased in liver, spleen and kidney while brain showed little change. The inhibitor did not act on glucuronidase in vitro, so the effect of the inhibitor is complex, possibly a result of the loss of glucosidase activity. Since glucuronidase contains glucose, we suggest that the transport of the enzyme between subcellular regions and tissues involves loss of part of the glucose moieties. 2. Levels of glucocerebrosidase (D-glucosyl-N-acylsphingosine glucohydrolase, EC 3.2.1.45) dropped very rapidly after epoxide injection, reaching a minimum at 1 h in liver. There was a noticeable restoration of activity within the next 1--2 h. Aryl beta-glucosidase (EC 3.2.1.21) decrease somewhat less than cerebrosidase, reaching a minimum within 2 h. It too showed some recovery of activity within 3 h. 3. Acid phosphatase rose slightly in liver but not in brain. alpha-L-Fucosidase and angiotensin-converting enzyme were not affected by the epoxide injection. The latter two enzymes are known to contain glucose. 4. Injection of a hemolyzing agent, phenylhydrazine, produced an increased level of glucuronidase in liver and spleen within 6 days, but not in kidney. This enhancement was a little less in mice previously injected with the glucosidase inhibitor. 5. Mice injected with the epoxide once a day eight times showed a distinct rise in brain glucuronidase level, as well as a rise in brain weight. However, the other organs showed only the same decrease in glucuronidase specific activity noted with the single injection protocol. It is suggested that the difference is due to the blood-brain barrier, which could slow the loss of brain glucuronidase from the extracellular fluid.

Acid Phosphatase↗

PubMed2026

Design, synthesis and biological evaluation of hydroxybenzothiazole-linked benzothiazole/benzoxazole conjugates as potent dual α-amylase and α-glucosidase inhibitors.

The current study focuses on the synthesis and evaluation of novel Hydroxybenzothiazole-Linked Benzothiazole/Benzoxazole Conjugates to target Diabetes Mellitus (DM) by inhibiting α-amylase and α-glucosidase. Spectroscopic methods, including 1H and 13C NMR spectroscopy, were employed to confirm the structures of newly synthesized conjugates. The findings of in-vitro analysis displayed that the synthesized derivatives inhibited α-amylase and α-glucosidase enzymes with IC50 values ranging from 3.65 ± 0.20 μM to 32.15 ± 3.20 μM on α-amylase and 5.92 ± 0.80 μM to 35.60 ± 3.40 μM on α-glucosidase, in contrast to the reference drug Acarbose (α-amylase IC50 = 8.25 ± 0.80 μM; α-glucosidase IC50 = 10.75 ± 1.10 μM). Among the series 9a-9f and 10a-10f, analogs 10 f, 10b, 9b, and 9e displayed superior anti-diabetic activity compared to the reference drug Acarbose. The inhibitory activity of these conjugates can be attributed to their favorable and stable interactions with critical amino acid residues of targeted enzymes, as revealed through molecular docking analysis. ADMET predictions and drug-likeness evaluations showed favorable pharmacokinetic features, while DFT investigations revealed electronic insights related to bioactivity. Experimental outcomes and in silico support display that these potent Hydroxybenzothiazole-Linked Benzothiazole/Benzoxazole Conjugates were comparable to an existing diabetic mellitus inhibitor while conserving an acceptable safety profile, specifying potential for further therapeutic development and optimization against diabetic Mellitus.

Benzothiazoles↗

PubMed2000

Investigation of the slow inhibition of almond beta-glucosidase and yeast isomaltase by 1-azasugar inhibitors: evidence for the 'direct binding' model.

(-)-1-Azafagomine [(3R,4R,5R)-4,5-dihydroxy-3-hydroxymethylhexahydropyridazine; inhibitor 1] is a potent glycosidase inhibitor designed to mimic the transition state of a substrate undergoing glycoside cleavage. The inhibition of glycosidases by inhbitor 1 and analogues has been found to be a relatively slow process. This 'slow inhibition' process was investigated in the inhibition of almond beta-glucosidase and yeast isomaltase by inhibitor 1 and analogues. Progress-curve experiments established that the time-dependent inhibition of both enzymes by inhibitor 1 was a consequence of relatively slow dissociation and association of the inhibitor from and to the enzyme, and not a result of slow interchanges between protein conformations. A number of hydrazine-containing analogues of inhibitor 1 also inhibited beta-glucosidase and isomaltase slowly, while the amine isofagomine [(3R,4R,5R)-3,4-dihydroxy-5-hydroxymethylpiperidine; inhibitor 5] only inhibited beta-glucosidase slowly. Inhibitor 1 and related inhibitors were found to leave almond beta-glucosidase with almost identical rate constants, so that the difference in K(i) values depended almost entirely on changes in the binding rate constant, k(on). The same trend was observed for the inhibition of yeast isomaltase by inhibitor 1 and a related inhibitor. The values of the rate constants were obtained at 25 degrees C and at pH 6.8.

Enzyme Inhibitors↗

PubMed1986

Hydrolysis of a naturally occurring beta-glucoside by a broad-specificity beta-glucosidase from liver.

We have isolated from guinea-pig liver a broad-specificity beta-glucosidase of unknown function that utilizes as its substrate non-physiological aryl glycosides (e.g. 4-methylumbelliferyl beta-D-glucopyranoside, p-nitrophenyl beta-D-glucopyranoside). The present paper documents that this enzyme can be inhibited by various naturally occurring glycosides, including L-picein, dhurrin and glucocheirolin. In addition, L-picein, which acts as a competitive inhibitor of the broad-specificity beta-glucosidase (Ki 0.65 mM), is also a substrate for this enzyme (Km 0.63 mM; Vmax. 277,000 units/mg). Heat-denaturation, kinetic competition studies, chromatographic properties and pH optima all argue strongly that the broad-specificity beta-glucosidase is responsible for the hydrolysis of both the non-physiological aryl glycosides and L-picein. This paper demonstrates that beta-glucosidase can catalyse the hydrolysis of a natural glycoside, and may provide a key to understanding the function of this enigmatic enzyme. A possible role in the metabolism of xenobiotic compounds is discussed.

Animals↗

PubMed1985

Major contribution of epididymis to alpha-glucosidase content of ram seminal plasma.

Acid alpha-glucosidase and L-carnitine (a well-known epididymal marker) were measured in rete testis and epididymal fluids of adult male rams. These fluids were collected by selective catheterization or by a micropuncture technique, respectively. Both parameters remained at a low and constant level in rete testis and all along caput and corpus epididymidis. Then they increased at equivalent rates in cauda epididymidis to much higher levels than those in seminal plasma (5 mU/mg protein and 10 mM, respectively). An optimum pH study of alpha-glucosidase activity in these fluids showed two well-separated peaks in rete testis and caput epididymal fluids around pH 4 and 7, respectively, but only a single peak at pH 4 in cauda epididymidis that was comparable to the one in seminal plasma. Sucrose density gradient fractions analyzed for their enzyme content in the absence or presence of sodium dodecyl sulfate (1% w/v), a selective inhibitor of acid alpha-glucosidase activity, allowed the demonstration of two enzyme forms at pH 6.8 in rete testis fluid sedimenting in the 7S and 4S regions of the gradient, while a unique 4S form was encountered in cauda epididymidis and in seminal plasma. Although the fate of the minor 7S component of the rete testis fluid in its epididymal transit is presently unknown, similarities between the enzyme in cauda epididymidis and seminal plasma are strong enough to support the hypothesis that epididymis contributes primarily to the acid alpha-glucosidase content of ram seminal plasma.

Animals↗

PubMed1997

Thermotoga neapolitana bglB gene, upstream of lamA, encodes a highly thermostable beta-glucosidase that is a laminaribiase.

The gene for thermostable 1,3-beta-glucosidase BglB was cloned from the chromosome of Thermotoga neapolitana and its primary sequence was determined. The purified recombinant beta-glucosidase B had a monomer molecular mass of 81 kDa in accordance with the amino acid sequence predicted from the nucleotide sequence of clone pTT51. It was a member of glycosylhydrolase family 3 and belonged to enzyme class EC 3.2.1.21. beta-Glucosidase B had a specific activity of 255 U mg-1 on 4-nitrophenyl(PNP)-beta-glucoside at the optima of pH (5.5) and temperature (90 degrees C), and K(m) values of 0.1, 10 and 50 mM for PNP-beta-glucoside, laminaribiose and cellobiose, respectively. The gene bglB was located immediately upstream of the laminarinase gene lamA. Both genes were transcribed from the same DNA strand and were not separated by a palindromic transcription terminator. The two purified enzymes 1,3-beta-glucosidase BglB (laminaribiase) and 1,3-beta-glucanase LamA (laminarinase) were together capable of completely degrading laminarin to glucose.

Amino Acid Sequence↗

PubMed1981

Multiple forms of membrane-bound beta-glucosidase in Gaucher's disease.

Two forms of membrane-bound beta-glucosidase in the spleen of normal individuals were distinguished by their thermostability properties. The heat-labile form A predominates; it catalyzes the hydrolysis of the natural substrate, glucosylceramide, and is activated by the detergent, sodium taurocholate. The minor heat-stable form B is inactive against glucosylceramide and is inhibited by taurocholate. The activity of form A increases from childhood to adult life, as does the activity of the soluble beta-glucosidase and of glucosylceramide beta-glucosidase. In the spleen of nine patients with different types of Gaucher's disease the residual membrane-bound beta-glucosidase was predominantly heat-stable and inhibited by taurocholate. There was no clear correlation between the properties of the residual enzyme in the different types of the disorder and their respective clinical severity. The results are discussed in relation to the biochemical pathogenesis and the enzymatic diagnosis of Gaucher's disease.

Adult↗