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At least 613 records · Page 34Linked to original sources
PubMed1986

Three diadenosine 5',5''-P1,P4-tetraphosphate hydrolytic enzymes from Physarum polycephalum with differential effects by calcium: a specific dinucleoside polyphosphate pyrophosphohydrolase, a nucleotide pyrophosphatase, and a phosphodiesterase.

Two new enzymes that hydrolyze diadenosine tetraphosphate (Ap4A) have been isolated from the acellular slime mold Physarum polycephalum. Both enzymes are different from the Physarum Ap4A symmetrical pyrophosphohydrolase previously described on the basis of their substrate specificities, reaction products, molecular weights, and divalent cation requirements. One enzyme is a nucleotide pyrophosphatase that asymmetrically hydrolyzes Ap4A to AMP and ATP. This enzyme hydrolyzes several mono- and dinucleotides with the corresponding nucleotide monophosphate as one of the products. The percentage hydrolysis of NAD+, Ap4A, and Ap4G, each at 10 microM, was 100, 56, and 51, respectively. A divalent cation is required for activity, with Ca2+ yielding 20-30 times greater activity than Mg2+ or Mn2+. Values of Km for Ap4A and Vmax are similar to the corresponding values for Ap4A symmetrical pyrophosphohydrolase. The second enzyme is a phosphodiesterase I with broad substrate reactivity. This enzyme also asymmetrically hydrolyzes Ap4A, but it does not hydrolyze NAD+. Activity of the phosphodiesterase I is stimulated by divalent cations, with Ca2+ being 50-60 times more stimulatory than Mg2+ or Mn2+. The apparent molecular weights of the nucleotide pyrophosphatase and phosphodiesterase are 184,000 and 45,000, respectively. In contrast, the Ap4A pyrophosphohydrolase hydrolyzes Ap4A to ADP, is inhibited by Ca2+ and other divalent cations, and has an apparent molecular weight of 26,000 as previously reported.

Adenine Nucleotides↗
PubMed1996

Reactivity of a paramagnetic enzyme--CO adduct in acetyl-CoA synthesis and cleavage.

Partial reactions of acetyl-CoA cleavage by the Methanosarcina barkeri acetyl-CoA decarbonylase synthase enzyme complex were investigated by UV--visible and electron paramagnetic resonance (EPR) spectroscopy. Reaction of the enzyme complex with carbon monoxide generated an EPR-detectable adduct with principal g values of 2.089, 2.076, and 2.028, and line widths of 13.76, 16.65, and 5.41 G, respectively. The EPR signal intensity was dependent upon both enzyme and carbon monoxide concentration. A second signal with gav = 2.050 was generated by storage of the CO-exposed enzyme for 17 months at -70 degrees C. Reaction of the enzyme complex with low levels of CO caused reduction of the enzyme complex, but did not result in immediate formation of the NiFeC signal (designated NiFeC based on isotopic substitution studies carried out by others in analogous systems from Clostridium thermoaceticum and Methanosarcina thermophila). Further addition of CO generated the NiFeC signal, and the signal amplitude then increased progressively with increasing CO concentration. UV-visible spectra showed that enzyme Fe-S and corrinoid centers were already fully reduced at levels of CO significantly lower than needed for maximal EPR signal intensity. This result indicated that the EPR signal is formed by reaction of the reduced enzyme with CO (or a reduced one-carbon species), rather than with a one-carbon unit at the oxidation level of CO2. Addition of coenzyme A, acetyl-CoA, or tetrahydrosarcinapterin had no effect on the EPR signal. In contrast, addition of N5-methyltetrahydrosarcinapterin (CH3-H4SPt) abolished the EPR signal. EPR spectra recorded at 20-21 K revealed that reaction with CH3-H4SPt affects only the enzyme NiFeC signal, and does not influence other EPR-detectable Fe-S center(s). The results suggest that the enzyme--CO adduct reacts with CH3-H4SPt to form an EPR-silent enzyme-acetyl species. Preincubation of the enzyme complex with CO and CH3-H4SPt, both of which were required, produced an approximately 44-fold increase in the turnover rate of acetyl-CoA synthesis. The relevance of these findings to mechanisms involving possible reductive methylation of the enzyme and/or acetyl-enzyme formation is discussed.

Acetyl Coenzyme A↗
PubMed1990

The unfolding and attempted refolding of mitochondrial aspartate aminotransferase from pig heart.

The unfolding of the mitochondrial isoenzyme of aspartate aminotransferase from pig heart in solutions of guanidinium chloride (GdnHCl) has been studied. By a number of criteria (enzyme activity, protein fluorescence, c.d., thiol-group reactivity), the enzyme was judged to be almost completely unfolded in 2 M-GdnHCl. On dilution of the GdnHCl, no re-activation of the enzyme could be observed, whether or not pyridoxal 5'-phosphate and dithiothreitol were present. The behaviour of the mitochondrial isoenzyme is in marked contrast with that of the cytoplasmic isoenzyme [West & Price (1989) Biochem. J. 261, 189-196], despite the similarities in the amino acid sequences and tertiary structures of the two isoenzymes. The implications of these findings for the process of folding and assembly of the mitochondrial isoenzyme in vivo are discussed.

Animals↗
PubMed2001

Chaperone-like activity of tubulin. binding and reactivation of unfolded substrate enzymes.

The eukaryotic cytoskeletal protein tubulin is a heterodimer of two subunits, alpha and beta, and is a building block unit of microtubules. In a previous communication we demonstrated that tubulin possesses chaperone-like activities by preventing the stress-induced aggregation of various proteins (Guha, S., Manna, T. K., Das, K. P., and Bhattacharyya, B. (1998) J. Biol. Chem. 273, 30077-30080). As an extension of this observation, we explored whether tubulin, like other known chaperones, also protected biological activity of proteins against thermal stress or increased the yields of active proteins during refolding from a denatured state. We show here that tubulin not only prevents the thermal aggregation of alcohol dehydrogenase and malic dehydrogenase but also protects them from loss of activity. We also show that tubulin prevents the aggregation of substrates during their refolding from a denatured state and forms a stable complex with denatured substrate. The activity of malic dehydrogenase, alpha-glucosidase, and lactate dehydrogenase during their refolding from urea or guanidium hydrochloride denatured states increased significantly in presence of tubulin compared with that without tubulin. These results suggest that tubulin, in addition to its role in mitosis, cell motility, and other cellular events, might be implicated in protein folding and protection from stress.

Alcohol Dehydrogenase↗
PubMed1991

A membrane-bound metallo-endopeptidase from rat kidney hydrolyzing parathyroid hormone. Purification and characterization.

A metallo-endopeptidase, which appears to be an integral membrane protein of rat kidney, was purified to homogeneity by a series of standard chromatographic procedures. This enzyme significantly hydrolyzed human parathyroid hormone [hPTH(1-84)] and a synthetic substrate Suc-Leu-Leu-Val-Tyr-Mec (Suc = succinyl, Mec = 4-methyl-coumarinyl-7-amide). The purified enzyme had apparent molecular masses of 250 kDa on gel filtration, and 88 kDa and 245 kDa on sodium dodecyl sulfate/polyacrylamide gel electrophoresis under reducing and non-reducing conditions, respectively. Its pH optimum for activity was 8.0-8.5 and its isoelectric point was pH 4.9. Its activity was inhibited by EDTA, EGTA and o-phenanthroline, but not by phosphoramidon. The metal-depleted enzyme was reactivated by the addition of metal ions. The enzyme was also inhibited by chymostatin and eglin C, and by thiol compounds. Of the synthetic substrates examined, the enzyme hydrolyzed only Suc-Leu-Leu-Val-Tyr-Mec, one of the synthetic substrates for alpha-chymotrypsin. It did not hydrolyze synthetic substrates with less than four amino acid residues with tyrosine in the P1 position. The enzyme hydrolyzed hPTH and reduced hen egg lysozyme but did not hydrolyze azocasein or [3H]methyl-casein. NH2-terminal amino acid sequence analyses of the degradation products of hPTH(1-84) and reduced hen egg lysozyme by the purified enzyme revealed that the enzyme preferentially cleaved these peptides at peptide bonds flanked by hydrophilic amino acid residues. Amino acid analyses showed that the main degradation products of PTH were hPTH(17-29), hPTH(30-38) and hPTH(74-84). The ability of the enzyme to hydrolyze peptide bonds flanked by hydrophilic amino acid residues and its inability to degrade azocasein distinguish it from several other kidney endopeptidases reported, such as endopeptidase 24.11 and meprin.

Amino Acid Sequence↗
PubMed1994

Effects of histoplasmin M antigen chemical and enzymatic deglycosylation on cross-reactivity in the enzyme-linked immunoelectrotransfer blot method.

The enzyme-linked immunoelectrotransfer blot (EITB) method was evaluated as a suitable method for detecting antibodies against M antigen of Histoplasma capsulatum by use of both glycosylated and deglycosylated M protein of histoplasmin (HMIN). Sera from patients with histoplasmosis, paracoccidioidomycosis, blastomycosis, coccidioidomycosis, and aspergillosis were tested by the EITB with glycosylated M protein of HMIN. This assay demonstrated 100% sensitivity with histoplasmosis serum samples, all of which reacted with the 94-kDa glycoprotein (M antigen). Although the EITB is highly sensitive, it is not specific for histoplasmosis when glycosylated M protein is used as an antigen. A total of 81% of paracoccidioidomycosis, 25% of blastomycosis, 33% of coccidioidomycosis, 73% of aspergillosis, and 16% of tuberculosis serum samples cross-reacted with M protein of HMIN and yielded patterns indistinguishable from those obtained with histoplasmosis serum samples. The EITB reactions with both untreated M antigen and M antigen altered by periodate oxidation or by deglycosylation with endoglycosidases were compared. Cross-reactions with heterologous sera in the EITB could be attributed to periodate-sensitive carbohydrate epitopes, as reflected by the increase in the test specificity from 46.1 to 91.2% after periodate treatment of M protein. The EITB for the detection of antibodies to M antigen is a potential diagnostic test for histoplasmosis, provided that periodate-treated M protein is used as an antigen.

Antibody Specificity↗
PubMed2006

Thiol-dependent serine alkaline proteases from Bacillus sp. HR-08 and KR-8102: isolation, production, and characterization.

Two Bacillus sp. strains, HR-08 and KR-8102, isolated from soil of the west and north parts of Iran were screened on gelatin agar medium for their ability to produce alkaline protease. The enzymes were active in a wide pH range (6.0-11.0) and stable in the alkaline range (7.0-12.0). The optimum temperatures for the protease from HR-08 and KR-8102 were 65 and 50 degrees C, respectively. The irreversible thermoinactivation of HR-08 and KR-8102 proteases showed that the stability of HR-08 enzyme was higher than that of KR-8102 and the half-lives of these enzymes were 95 and 32 min at 50 degrees C, respectively. In the presence of 10 mM Ca(2+), HR-08 retained 100, 90, and 20% of its initial activity after heating for 30 min at 50, 60, and 70 degrees C, respectively. Enzymes were inhibited by phenylmethylsulfonyl fluoride and iodoacetate. After inhibition by iodoacetate, both enzymes were reactivated by dithiothreitol. These data show that the enzymes seem to be thiol-dependent serine alkaline proteases. The enzymes especially from HR-08 were stable in the presence of H(2)O(2), surfactants, and local detergents; their activities were enhanced in the presence of 5 mM Fe(2+); and the presence of 5 mM metal ions such as Mg(2+), Cu(2+), and Mn(2+) produced almost no effect.

Bacillus↗
PubMed1986

Reactivity of old yellow enzyme with alpha-NADPH and other pyridine nucleotide derivatives.

The reaction of Old Yellow Enzyme (OYE) with pyridine nucleotides has been examined using steady state kinetics, rapid reaction kinetics, and equilibrium binding. alpha-NADPH, beta-NADPH, and the acid breakdown products of NADPH all bind to oxidized OYE with dissociation constants below 1 microM. These complexes produce characteristic red shifts in the absorption spectrum of OYE. A similar red shift which occurs after multiple turnovers of OYE with NADPH has been found to be due to an impurity in the NADPH preparation, possibly an acid breakdown product. Anions such as chloride, acetate, azide, and phenolates compete with the pyridine nucleotides for binding to a common site in oxidized OYE. Anaerobic reduction of OYE by NADPH proceeds via two intermediates to establish a readily reversible equilibrium. In contrast to most other NADPH-dependent enzymes, both alpha- and beta-NADPH are capable of reducing OYE, and alpha-NADPH is more effective. Using beta-[4(R)-2H]NADPH, a primary deuterium isotope effect was observed in the reduction reaction. Results from rapid reaction and steady state studies showed that reduction of OYE was rate limiting in turnover. Consistent with this, the turnover number with alpha-NADPH was significantly higher than that with beta-NADPH.

Chromatography, High Pressure Liquid↗