Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.2.1.20 (alpha-glucosidase)
 4,237 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The polybasic macromolecules DEAE-dextran (diethylaminoethyl-dextran, molecular weight 500000) and poly-DL-lysine (molecular weight 30000-70000) were absorbed with a high affinity by spheroplasts of Candida utilis and subsequently, induced lysis. The extent of lysis of spheroplasts and of the liberated vacuoles was studied under various conditions using alpha-glucosidase activity and soluble arginine as cytoplasmic and vacuolar markers, respectively. Adsorption of polybases was rapidly completed even at 0 degrees C; however, with small doses, lysis was poor at 0-12 degrees C and extensive at temperatures above 12 degrees C. This permitted the completion of adsorption before initiating lysis. The purified vacuoles were also sensitive to polybases though less so than the spheroplasts; however, after lysis of spheroplasts the liberated vacuoles were well protected against the action of polybases. A treatment with polybases which disrupted more than 99% of the spheroplasts left at least 70% of the vacuoles intact. Potassium chloride in high concentrations and calcium chloride in low concentrations inhibited polybase induced lysis of spheroplasts by preventing or even reversing the polybase adsorption. A polyacidic macromolecule, dextran sulfate, could prevent but not reverse the adsorption of polybase and subsequent lysis. Metabolic inhibitors reduced the susceptibility of spheroplasts to polybase induced lysis. Vacuoles isolated from polybase lysed spheroplasts still contained large pools of soluble amino acids, and their ability to transport arginine specifically is a further indication of their functional integrity.
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PMID:Polybase induced lysis of yeast spheroplasts. A new gentle method for preparation of vacuoles. 24 1

Bacteroides forsythus is a fastidious anaerobic gram-negative organism associated with various forms of periodontal disease. It is dependent on N-acetylmuramic acid for growth. A method for rapid presumptive identification of human-derived strains of B. forsythus is presented, based on the following eight criteria: (i) positive activity for alpha-glucosidase, (ii) positive activity for beta-glucosidase, (iii) positive activity for sialidase, (iv) positive activity for trypsinlike enzyme, (v) negative indole production, (vi) requirement for N-acetylmuramic acid, (vii) colonial morphology, and (viii) gram stain morphology from blood agar medium deficient in N-acetylmuramic acid. Enzymes were assayed with rapid filter paper spot tests based on fluorogenic substrates (4-methylumbelliferone derivatives and N alpha-carbobenzoxy-L-arginine-7-amino-4-methylcoumarin hydrochloride). Gas-liquid chromatography analysis of the metabolic products of B. forsythus grown in peptone yeast extract broth supplemented with N-acetylmuramic acid and heat-inactivated horse serum revealed predominant amounts of acetate, propionate, butyrate, isovalerate, and phenyl acetate, with minor amounts of isobutyrate and succinate. The described presumptive identification scheme facilitated recognition of four strains of B. forsythus which were isolated from subgingival plaque samples from monkeys (Macaca fascicularis). With the exception of indole production, these organisms were essentially identical to the human strains of B. forsythus for all phenotypic and genotypic characteristics examined.
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PMID:Rapid presumptive identification and further characterization of Bacteroides forsythus. 155 81

To examine the function of islet lysosomal enzymes in islet hormone secretory mechanisms, we investigated the effects of the lysosomotropic drug chloroquine on islet lysosomal enzyme activities and basal as well as stimulated insulin and glucagon secretion. Chloroquine, added to islet homogenates, did not affect the activities of the lysosomal enzymes acid amyloglucosidase, acid alpha-glucosidase, or N-acetyl-beta-D-glucosaminidase. The activity of acid phosphatase, however, was inhibited at a high concentration of chloroquine (10(-3) M). When injected together with glucose, chloroquine (2 or 10 mumol/kg) inhibited the peak plasma insulin response. Similarly, at 24 hrs after chloroquine injection (100 mumol/kg), the plasma insulin response to glucose was reduced. In contrast, islets isolated from mice pretreated 24 hrs before with chloroquine, displayed glucose-stimulated insulin secretion in vitro that was not different from controls. Such islets showed, furthermore, enhanced activities of the enzymes acid phosphatase and neutral alpha-glucosidase but not of acid amyloglucosidase, acid alpha-glucosidase or N-acetyl-beta-D-glucosaminidase. Arginine-stimulated insulin response in vivo displayed a complex pattern; it was increased when arginine was injected together with chloroquine but decreased at 24 hrs after chloroquine administration. Arginine-stimulated glucagon secretion was not affected by chloroquine. We conclude that chloroquine pretreatment 24 hrs prior to glucose injection decreases glucose-stimulated insulin secretion in vivo by mechanisms that are not correlated to an inhibitory action on islet activities of glycogenolytic lysosomal enzymes.
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PMID:Islet hormone secretion and islet lysosomal enzyme activities in the mouse: effects of chloroquine. 307 44

The glucose-producing amylolytic activity in pancreatic islet tissue was characterized with regard to its properties with glycogen (amyloglucosidase) and maltose (maltase) as substrate, its optimum activity in islets from different strains of mice (NMRI, CBA and C-57BL) and after fasting, and its relation to the insulin secretory response after different secretagogues in vivo. Additionally the effects and fate of injected fungal acid amyloglucosidase were assessed. In the pancreatic islets of NMRI mice both the glycogen-splitting activity and the maltose-splitting activity displayed latency and an acid pH-optimum of about 5.0. After differential centrifugation a significant part of amyloglucosidase activity was found to be confined to the mitochondrial-lysosomal fraction. In crude islet homogenate the apparent Km for maltose at pH 5.0 was 2.1 mM. No Km for glycogen could be given because of complex kinetics in the presence of this substrate. The maltase activity was about 30% lower than the amyloglucosidase activity in islet tissue from NMRI mice. The reverse pattern was observed in the liver. Moreover, the liver amyloglucosidase activity was only one fourth of that of the islet tissue. The amyloglucosidase but not the maltase activity in islet tissue from CBA mice was lower than in islets from NMRI mice. Both activities were very low in islets from C-57 mice. A 24 hr fasting period reduced the amyloglucosidase but not the maltase activity in islets from NMRI mice. The insulin secretory response in vivo to an i.v. arginine load in the different strains and after fasting displayed the same pattern as the islet amyloglucosidase activity, whereas the insulin response following a glucagon injection was largest in the C-57 strain and unaffected by the fasting state. Pretreatment of mice with 0.05 mumol/kg of highly purified fungal amyloglucosidase, moderately (about 35%) enhanced the insulin secretory response to arginine, did not affect the response to glucagon, and greatly (about 100%) enhanced the response to glucose and tolbutamide. Moreover, treatment of mice with lysosomal stabilizers (glucocorticoids) reduced the insulin response to sulphonylureas and glucose, had no effect on the insulin response to beta-adrenergic and cholinergic stimulation, and increased ACTH-induced insulin release. A lysosomal labilizer (progesterone) enhanced the insulin response induced by glucose and tolbutamide.(ABSTRACT TRUNCATED AT 400 WORDS)
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PMID:Islet amyloglucosidase activity: some characteristics, and its relation to insulin secretion stimulated by various secretagogues. 308 68

Homogenates of Giardia lamblia trophozoites exhibited the following hydrolase activities: acid phosphatase (EC 3.1.3.2), proteinase (EC 3.1.4) with urea-denatured hemoglobin and N-benzoyl-DL-arginine-2-naphthylamide as substrates, deoxyribonuclease (EC 3.1.4.5), and ribonuclease (EC 2.7.7.16). beta-N-Acetylglucosaminidase (EC 3.2.1.30), beta-galactosidase (EC 3.2.1.23), beta-glucuronidase (EC 3.2.1.31), alpha-D-glucosidase (EC 3.2.1.20), beta-D-glucosidase (EC 3.2.1.21), and beta-D-xylosidase (EC 3.2.1.37) activities were below the level of detection. Differential and isopycnic centrifugation of homogenates demonstrated that giardial hydrolases were localized in a single-particle population sedimenting at 7200g for 30 min. The particles had a buoyant density in sucrose of 1.15 and exhibited latency. Latency was completely destroyed by Triton X-100 or 15 cycles of freezing and thawing. After centrifugation of Triton- or freeze-thaw-treated particle fractions, the hydrolase activities, though no longer latent, were still sedimentable suggesting tight binding to the organelle membrane. Latency was destroyed simultaneously for all hydrolases, in direct proportion to the amount of Triton added to a particle preparation or to the number of times a particle preparation was subjected to freezing and thawing. These results support the suggestion that the hydrolases of G. lamblia trophozoites are localized in a single-particle population of lysosome-like organelles.
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PMID:Giardia lamblia: localization of hydrolase activities in lysosome-like organelles of trophozoites. 327 50

Sixteen isolates from the vaginal discharge of women with bacterial vaginosis (non-specific vaginitis) and six provided by other investigators were divided into two groups on the basis of morphology and biochemical tests. Only one organism type was isolated from any one patient. The two groups differed in size, number of flagella, enzyme production, ability to ferment carbohydrates, hydrolyse hippurate and arginine, reduce nitrate, and produce ONPG, and in sensitivity to metronidazole. Both groups fermented glucose and maltose, produced succinate from glucose and alpha-glucosidase and leucine arylamidase.
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PMID:Two curved rods in non-specific vaginitis. 659 27

Maltase/glucoamylase from the rat intestinal brush-border membrane was solubilized by homogenization of the intestinal mucosa in buffer containing 0.5% Triton X-100. After removal of the detergent with butan-1-ol, the enzyme was purified by chromatography on Sepharose 4B and DEAE-cellulose. The final specific activity was 70.3 units/mg of protein in six preparations, comparing favourably with the specific activity of 65.0 units/mg of protein of a pure papain-solubilized maltase/glucoamylase previously isolated and characterized by us [Flanagan & Forstner (1978) Biochem. J.173, 553-563]. The two enzymes were compared. Both migrated as single bands with the same mobility on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, were eluted at the same volume from Sepharose 4B, and had the same sedimentation pattern in mannitol gradients. The amino acid composition was similar; content of total apolar residues differed by 1.0mol%. Antibodies prepared against either enzyme gave identical precipitin lines with each. Neither enzyme bound tritiated Triton X-100. The only difference noted was the tendency of the detergent-solubilized enzyme to aggregate on storage, whereas the papain-solubilized enzyme remained unchanged. Both enzymes had two N-termini, glycine and arginine. When the two enzymes were dissociated by boiling in sodium dodecyl sulphate, each exhibited the same five species on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Single N-termini were found in the two smaller species, 1 (glycine) and 2 (arginine), whereas larger species (3-5) had both N-terminal amino acids. Both the Triton- and papain-solubilized enzymes appear to be oligomers of species 1 and 2, indicating that the native enzyme contains two subunit types. Aggregation in aqueous solutions does not depend on a proteolytically susceptible peptide fragment at the N-terminus of either subunit.
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PMID:Isolation of a detergent-solubilized maltase/glucoamylase from rat intestine and its comparison with a maltase/glucoamylase solubilized by papain. 677 61

Glycogen-storage disease type II (GSDII) is caused by the deficiency of lysosomal alpha-glucosidase (acid maltase). This paper reports on the analysis of the mutant alleles in an American black patient with an adult form of GSDII (GM1935). The lysosomal alpha-glucosidase precursor of this patient has abnormal molecular features: (i) the molecular mass is decreased, (ii) the phosphorylation is deficient and (iii) the proteolytic processing is impaired. Sequence analysis revealed four mutations leading to amino acid alterations: Asp-645-->Glu, Val-816-->Ile, Arg-854-->Stop and Thr-927-->Ile. By using allele-specific oligonucleotide hybridization on PCR-amplified cDNA we have demonstrated that the Arg-854-->Stop mutation is located in one allele that is not expressed, and that the other allele contains the remaining three mutations. Each of the mutations was introduced in wild-type cDNA and expressed in COS cells to analyse the effect on biosynthesis, transport and phosphorylation of lysosomal alpha-glucosidase. The Val-816-->Ile substitution appeared to have no significant effect in contrast with results [Martiniuk, Mehler, Bodkin, Tzall, Hirshhorn, Zhong and Hirschhorn (1991) DNA Cell Biol. 10, 681-687] and was therefore defined as a polymorphism. The Thr-927-->Ile substitution deleting one of the seven glycosylation sites was found to be responsible for the decrease in molecular-mass, but not for the deficient proteolytic processing and phosphorylation. It did not cause the enzyme deficiency either. The third mutation leading to the Asp-645-->Glu substitution was proven to account in full for the observed defects in transport, phosphorylation and proteolytic processing of the newly synthesized alpha-glucosidase precursor of the patient.
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PMID:The conservative substitution Asp-645-->Glu in lysosomal alpha-glucosidase affects transport and phosphorylation of the enzyme in an adult patient with glycogen-storage disease type II. 809 13

To evaluate the role of dietary polyamines in maturation of the rat small intestine, spermine was given orally twice daily to suckling pups from day 10 to day 14 postpartum at different doses: 0, 0.2, 0.5, 1, 2.5, and 5 mumol/dose. Compared to saline treated controls, spermine (5 mumol) produced significant increases in mucosal mass parameters (+12 to +57%, P < 0.05), induced prematurely an adult pattern of microvillous enzymes, and enhanced, respectively, by 19- and 3.5-fold (P < 0.01 vs controls) the concentration of the secretory component of p-immunoglobulins in villous and crypt cells. The response of microvillous enzymes (lactase, sucrase, maltase, and aminopeptidase) to spermine was dose-dependent and -specific since oral administration of arginine (5 mumol) or ornithine (5 mumol) was without effect. Intestinal changes were found to be significant (P < 0.05) for doses of spermine exceeding 1 mumol/day, which is in the range of the amount of polyamines provided by solid pellets at weaning (0.4 mumol/g). However, intestinal changes were undetectable at the physiological amounts of polyamines consumed by pups from rat milk during the suckling period (less than 0.3 mumol/day). Consistent with a direct effect of spermine on the intestinal cell, the cytosolic activity of ornithine decarboxylase was depressed by 27-fold (P < 0.005 vs controls) in the jejunum, while inhibition of ornithine decarboxylase by alpha-difluoromethylornithine did markedly decrease but did not suppress the cell response to spermine. Alternately, plasma corticosteronemia, which was virtually absent by day 14 in controls, ranged between 1.4 and 4.6 micrograms/dl in 60% (N = 9) of the spermine-treated rats.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Maturation of villus and crypt cell functions in rat small intestine. Role of dietary polyamines. 850 5

We have designed a new method for enzyme immobilization using a fusion protein of yeast alpha-glucosidase containing at its C-terminus a polycationic hexa-arginine fusion peptide. This fusion protein can be directly adsorbed from crude cell extracts on polyanionic matrices in a specific, oriented fashion. Upon noncovalent immobilization by polyionic interactions, the stability of the fusion protein is not affected by pH-, urea-, or thermal-denaturation. Furthermore, the enzymatic properties (specific activity at increasing enzyme concentration, Michaelis constant, or activation energy of the enzymatic reaction) are not influenced by this noncovalent coupling. The operational stability of the coupled enzyme under conditions of continuous substrate conversion is, however, increased significantly compared to the soluble form. Fusion proteins containing polyionic peptide sequences are proposed as versatile tools for the production of immobilized enzyme catalysts.
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PMID:A fusion protein designed for noncovalent immobilization: stability, enzymatic activity, and use in an enzyme reactor. 963 Sep 15


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