Catalytic properties of alkaline phosphatase from pig kidney

Hiwada, Kunio; Wachsmuth, E. D.

doi:10.1042/bj1410283

Cited by 53 publications

(17 citation statements)

References 30 publications

(39 reference statements)

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“…Therefore, the experimental data are consistent with the equimolar Zn2+/dimeric triptophanyltRNA synthetase ratio. Similar results have been obtained for alkaline phosphatase from pig kidney [21]; in this case, the 100% activity correlates with two zinc atoms per tetramer.…”

Section: Discussionsupporting

confidence: 85%

Bovine Tryptophanyl‐tRNA Synthetase

Kisselev¹,

Фаворова²,

Nurbekov³

et al. 1981

European Journal of Biochemistry

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As is found by atomic absorption spectroscopy, the highly purified bovine tryptophanyl-tRNA synthetase contains up to 0.9 mol Zn2+/mol enzyme while some other bivalent metal ions are absent. The enzyme is inactivated either upon treatment with 1 ,lO-phenanthroline (a zinc-chelating agent) or upon prolonged dialysis (which eliminates bound Zn2+ ions); addition of zinc reactivates the enzyme. Exposed histidine residue(s) and carboxylic group(s) of the enzyme are involved in the Zn2+ binding, as is shown using chemical modification. Circular dichroism spectra suggest that elimination of Zn2 + ions affects the tertiary rather than the secondary structure of the tryptophanyl-tRNA synthetase. The kinetics of inhibition with 1,lO-phenanthroline toward ATP, tryptophan and tRNATrp indicates that removal of zinc prevents the ATP binding to the enzyme.Many enzymes of nucleic acid metabolism, viz. D N A polymerase, RNA polymerase, terminal nudeotidyltransferase, poly(A) polymerase, etc. (see [1,2]), contain zinc ions. These enzymes are inactivated in the presence of 1 ,lo-phenanthroline, a chelating agent with a high affinity for ZnZt [3].It has long been known that the activity of certain Eschcvichiu coli aminoacyl-tRNA synthetases is inhibited with phenanthroline [4]. Hence, bivalent metal (iron, cadmium, zinc, or manganese) ions may be involved in the functioning of these enzymes. Zinc essential for the activity and manganese have recently been found in E. coli methionyl-tRNA synthetase [5]. Zinc ions are involved in the tRNA-independent ATP hydrolysis catalyzed by yeast phenylalanyl-tRNA synthetase [6]. The Zn2+ ion (0.94 mol/mol enzyme) unessential for the aminoacylation activity has been found in E. coli isoleucyl-tRNA synthetase [7].The purpose of this work is to clarify whether tryptophanyl-tRNA synthetase ( M , 120000, ~2 ) from beef pancreas belongs to the group of metalloenzymes and what is the role of zinc in the enzymatic functioning. This enzyme is the best studied aminoacyl-tRNA synthetase from multicellular organisms [8]. MATERIALS AND METHODSThe following reagents were used in the experiments : ATP, Tris (Reanal) ; L-tryptophan (Sigma) ; sodium pyrophosphate (Merck) ; dithiothreitol, diethylpyrocarbonate and 4-niorpholinepropanesulfonic acid (Mops, Serva) ; 2-mercaptoethanol (Calbiochem); cetyltrimethylammonium bromide (cetavlon) and nitrocellulose filters Synpor 3 and 6 (Chemapol); chelating resin Chelex-100 (Bio-Rad); Sephadex (3-100 and (3-50, fine grade (Pharmacia Fine Chemicals); diphenylthiocarbazone (ditizone), 1,lO-phenanthroline and Tryptophanyl-tRNA synthetase was isolated from beef pancreas as described elsewhere [8]. The preparation was homogeneous after gel electrophoresis under denaturing conditions. The protein concentration was determined spectrophotometrically at 280 nm using the coefficient A@,,, = 0.90 [8]. The activity of tryptophanyl-tRNA synthetase in the ATP-[32P]pyrophosphate exchange and the tRNA aminoacylation reactions was assayed as in [8].Solutions were prepared with deionized...

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Section: Discussionsupporting

confidence: 85%

Bovine Tryptophanyl‐tRNA Synthetase

Kisselev¹,

Фаворова²,

Nurbekov³

et al. 1981

European Journal of Biochemistry

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“…A higher concentration of Zn2+ (4mM) inhibited these activities (data not shown). These results on inhibition agree with those ob tained in previous studies with purified al-ease from other sources (14,15). Table 8 shows the effects of various inhibitors on the TDPase and p-NPPase activities in the purified preparation.…”

Section: Purification Of Intestinal Tdpase and P-nppasesupporting

confidence: 82%

Characterization of thiamine diphosphatase in rat small intestine.

Matsuda

Maeda

Baba

et al. 1978

Journal of Nutritional Science and Vitaminology, J Nutr Sci Vit

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SummaryThe properties of thiamine diphosphatase (TDPase) and p-nitrophenylphosphatase (p-NPPase) in rat small intestine were investi gated. TDPase activity, like p-NPPase activity, was high in the mucosa and in the proximal region. Both activities were high in the membrane associated fractions of the duodenal mucosa. Furthermore, TDPase had the same properties as intestinal alkaline phosphatase (al-Pase). These results suggest that thiamine diphosphate (TDP) and p-nitrophenyl phosphate (p-NPP) are hydrolyzed by a single enzyme, al-Pase, in the intestine.Recently, we (1) found that TDPase from rat intestine was purified with p -NPPase by n-butanol extraction, ethanol precipitation, cellulose ion-exchange chromatography and gel filtration and that the TDPase activity in the purified material was competitively inhibited by p-NPP, a substrate of p-NPPase. From these results it seemed likely that the enzymatic hydrolysis of TDP might be catalyzed by al-Pase in the intestine. This paper describes the distributions of TDPase and p-NPPase in rat small intestine and the characterizations of the two enzymes. The results support our previous idea that TDPase is identical with p-NPPase in the intestine. EXPERIMENTALMale Sprague-Dawley rats, weighing 150-300g, were used throughout. The rats were killed and the duodenal segment (the proximal 12cm), jejunal segment (the middle 15cm) and ileal segment (the distal 20cm) of the intestine were re moved. The mucosa, obtained by gently scraping it off the underlying tissue with a glass slide, was homogenized with 30 volumes of ice-cold 0.25M sucrose-5mM EDTA (pH 7.4).123

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“…Хотя, как известно, ЩФ -это цинксодержащий энзим, для полного проявления его активно-сти необходимо присутствие ионов Mg 2+ [17]. Стимулирующий эффект Mg 2+ был установлен для препаратов ЩФ из различных источников [28][29][30][31]. В каталитическом механизме ЩФ E. coli роль катионов Mg 2+ состоит в координации мо-лекулы воды, обеспечивающей протонирование/ депротонирование остатка Ser-102 [32].…”

Section: рис 3 влияние ионов Mgunclassified

Identification of thiamine monophosphate hydrolyzing enzymes in chicken liver

Kolas¹,

Makarchikov²

2014

Ukr.Biochem.J.

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Catalytic properties of alkaline phosphatase from pig kidney

Cited by 53 publications

References 30 publications

Bovine Tryptophanyl‐tRNA Synthetase

Bovine Tryptophanyl‐tRNA Synthetase

Characterization of thiamine diphosphatase in rat small intestine.

Identification of thiamine monophosphate hydrolyzing enzymes in chicken liver

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