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 author reports a modification of the UV method UltraZyme Plus alpha-Amyl Harleco and the adaptation to the Eppendorf Enzymautomat 5010. alpha-amylase acts on an oligosaccharide mixture yielding maltose, which is hydrolysed by alpha-glucosidase. The liberated glucose is determined specifically by the hexokinase/glucose-6-phosphate dehydrogenase (NAD+-dependent) method+ by addition of pyruvate, lactate dehydrogenase and ATP. Thereafter the lactate dehydrogenase reaction is stopped by addition of oxamate and the alpha-amylase activity is measured.
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PMID:[Kinetic determination of alpha-amylase in serum and urine with an oligosaccharide as substrate--modification for a fully mechanized enzyme measuring device (author's transl)]. 9 28

I describe a new kinetic enzymatic saccharogenic method for assaying alpha-amylase in human serum and urine. alpha-Amylase liberates maltose from starch. This is successively acted on by alpha-glucosidase, mutarotase, and glucose dehydrogenase. The resulting conversion of NAD+ to NADH, measured at 340 nm, during a 20-min incubation reflects amylase activity. Endogenous glucose is destroyed before measurement of amylase activity is begun.
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PMID:New saccharogenic determination of alpha-amylase in serum and urine. 21 41

We explored whether glucocorticoid administration, a known stimulus of renal gluconeogenesis (GNG), could decrease avid inorganic phosphate (Pi) reabsorption in rats stabilized on low-phosphorus diet (LPD). Rats adapted to LPD were injected with the glucocorticoid (GCD) triamcinolone acetonide (1.25 or 2.5 mg.100 g body wt-1.day-1 ip) for 2 days; they showed a profound increase in urinary excretion of Pi during the injection period. In clearance studies GCD increased the clearance and fractional excretion of Pi but did not change the filtered load of Pi. Initial "uphill" Na+-gradient (Nao+ greater than Nai+)-dependent uptake of 32Pi by luminal brush-border membrane (BBM) vesicles prepared from renal cortex of rats treated with GCD was markedly (greater than 40%) decreased compared with control rats; Na+-gradient-dependent uptake of D-[3H]glucose was not diminished. At the "equilibrium" time interval, measured at 120 min, BBM vesicles from control and GCD-treated rats did not differ in the uptake of 32Pi or D-[3H]glucose. With kinetic analysis, BBM from GCD-treated rats showed a marked decrease (-40%) in the maximum velocity (Vmax) of initial Na+-dependent 32Pi uptake, but the apparent affinity of the BBM transport system for Pi (apparent Km = 0.078 mM Pi) was not different from that of controls. Alkaline phosphatase specific activity was much lower (-40%) in BBM from GCD-treated rats compared with controls, but the activities of three other BBM enzymes (maltase, leucine aminopeptidase, and gamma-glutamyl transferase) were not different. The addition of triamcinolone to BBM in vitro had no effect on either Na+-dependent uptake of 32Pi or alkaline phosphatase activity. The rate of GNG from alpha-ketoglutarate was significantly increased in cortical slices from GCD-treated rats adapted to LPD. Also, the NAD+-to-NADH ratio was higher in the renal cortex of GCD-treated rats, although the total content of NAD [NAD+ + NADH] was not different from controls. Renal excretory, BBM, and metabolic changes elicited controls. Renal excretory, BBM, and metabolic changes elicited by GCD treatment were similar in intact and thyroparathyroidectomized rats. Phosphaturia elicited in rats fed LPD by GCD administration in vivo appears to be at least in part due to a decreased capacity of luminal BBM of proximal tubules for decreased capacity of luminal BBM of proximal tubules for Na+-dependent uptake of Pi. Although the causal relationship between observed parameters is not established, our results are compatible with the interpretation that an increase in the rate of renal GNG, perhaps via action of NAD+ on BBM (J. Clin. Invest. 67: 1347-1360, 1981), decreases luminal uptake and reabsorption of Pi in proximal tubules.
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PMID:Mechanism of glucocorticoid effect on renal transport of phosphate. 675 2

The gene for the alpha-glucosidase AglA of the hyperthermophilic bacterium Thermotoga maritima MSB8, which was identified by phenotypic screening of a T. maritima gene library, is located within a cluster of genes involved in the hydrolysis of starch and maltodextrins and the uptake of maltooligosaccharides. According to its primary structure as deduced from the nucleotide sequence of the gene, AglA belongs to family 4 of glycosyl hydrolases. The enzyme was recombinantly expressed in Escherichia coli, purified, and characterized. The T. maritima alpha-glucosidase has the unusual property of requiring NAD+ and Mn2+ for activity. Co2+ and Ni2+ also activated AglA, albeit less efficiently than Mn2+. T. maritima AglA represents the first example of a maltodextrin-degrading alpha-glucosidase with NAD+ and Mn2+ requirement. In addition, AglA activity depended on reducing conditions. This third requirement was met by the addition of dithiothreitol (DTT) or beta-mercaptoethanol to the assay. Using gel permeation chromatography, T. maritima AglA behaved as a dimer (two identical 55-kDa subunits), irrespective of metal depletion or metal addition, and irrespective of the presence or absence of NAD+ or DTT. The enzyme hydrolyzes maltose and other small maltooligosaccharides but is inactive against the polymeric substrate starch. AglA is not specific with respect to the configuration at the C-4 position of its substrates because glycosidic derivatives of D-galactose are also hydrolyzed. In the presence of all cofactors, maximum activity was recorded at pH 7.5 and 90 degrees C (4-min assay). AglA is the most thermoactive and the most thermostable member of glycosyl hydrolase family 4. When incubated at 50 degrees C and 70 degrees C, the recombinant enzyme suffered partial inactivation during the first hours of incubation, but thereafter the residual activity did not drop below about 50% and 20% of the initial value, respectively, within a period of 48 h.
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PMID:Thermotoga maritima AglA, an extremely thermostable NAD+-, Mn2+-, and thiol-dependent alpha-glucosidase. 1097 87

The NAD+-requiring enzymes of glycoside hydrolase family 4 (GHF4) contain a region with a conserved Gly-XXX-Gly-Ser (GXGS) motif near their N-termini that is reminiscent of the fingerprint region of the Rossmann fold, a conserved structural motif of classical nicotinamide nucleotide-binding proteins. The function of this putative NAD+-binding motif in the alpha-glucosidase AglA of Thermotoga maritima was probed by directed mutagenesis. The K(d) for NAD+ of the AglA mutants G10A, G12A and S13A was increased by about 300-, 5-, and 9-fold, respectively, while their K(m) for p-nitrophenyl-alpha-glucopyranoside was not seriously affected. The results indicate that the GXGS motif is indeed important for NAD+ binding by the glycosidases of GHF4.
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PMID:Identification of residues important for NAD+ binding by the Thermotoga maritima alpha-glucosidase AglA, a member of glycoside hydrolase family 4. 1206 50

Glycoside hydrolase family 4 represents an unusual group of glucosidases with a requirement for NAD+, divalent metal cations, and reducing conditions. The family is also unique in its inclusion of both alpha- and beta-specific enzymes. The alpha-glucosidase A, AglA, from Thermotoga maritima is a typical glycoside hydrolase family 4 enzyme, requiring NAD+ and Mn2+ as well as strongly reducing conditions for activity. Here we present the crystal structure of the protein complexed with NAD+ and maltose, refined at a resolution of 1.9 A. The NAD+ is bound to a typical Rossman fold NAD+-binding site, and the nicotinamide moiety is localized close to the maltose substrate. Within the active site the conserved Cys-174 and surrounding histidines are positioned to play a role in the hydrolysis reaction. The electron density maps indicate that Cys-174 is oxidized to a sulfinic acid. Most likely, the strongly reducing conditions are necessary to reduce the oxidized cysteine side chain. Notably, the canonical set of catalytic acidic residues common to other glucosidases is not present in the active site. This, combined with a high structural homology to NAD-dependent dehydrogenases, suggests an unusual and possibly unique mechanism of action for a glycoside-hydrolyzing enzyme.
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PMID:Crystal structure of Thermotoga maritima alpha-glucosidase AglA defines a new clan of NAD+-dependent glycosidases. 1258 67

The aglB and aglA genes from the starch/maltodextrin utilization gene cluster of Thermotoga neapolitana were subcloned into pQE vectors for expression in Escherichia coli. The recombinant proteins AglB and AglA were purified to homogeneity and characterized. Both enzymes are hyperthermostable, the highest activity was observed at 85 degrees C. AglB is an oligomer of identical 55-kDa subunits capable of aggregation. This protein hydrolyses cyclodextrins and linear maltodextrins to glucose and maltose by liberating glucose from the reducing end of the molecules, and it is a cyclodextrinase with alpha-glucosidase activity. The pseudo-tetrasaccharide acarbose, a potent alpha-amylase and alpha-glucosidase inhibitor, does not inhibit AglB but, on the contrary, acarbose is degraded quantitatively by AglB. Recombinant AglB is activated in the presence of CaCl2, KCl, and EDTA, as well as after heating of the enzyme. AglA is a dimer of two identical 54-kDa subunits, and it hydrolyses the alpha-glycoside bonds of disaccharides and short maltooligosaccharides, acting on the substrate from the non-reducing end of the chain. It is a cofactor-dependent alpha-glucosidase with a wide action range, hydrolysing both oligoglucosides and galactosides with alpha-link. Thereby, the enzyme is not specific with respect to the configuration at the C4 position of its substrate. For the enzyme to be active, the presence of NAD+, DTT, and Mn2+ is required. Enzymes AglB and AglA supplement one another in substrate specificity and ensure complete hydrolysis to glucose for the intermediate products of starch degradation.
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PMID:[Thermotoga neopolitina gene cluster, participating in degradation of starch and maltodextrins: expression of aglB and aglA gene in Escherichia coli, properties of recombinant enzymes]. 1459 17