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1.
A radiometric assay for 5′-nucleotidese (EC 3.1.3.5) has been developed, which is applicable for all 5′-nucleotide substrates. Various column materials and eluants were evaluated for their suitability in the separation of purine and pyrimidine bases and nucleosides produced in the reaction. Neutral alumina columns were found to be the best. The unadsorbed nucleosides and their bases could be quantitatively eluted with 0.1 m Tris-HCl, pH 7.4; subsequent elution of the 5′-nucleotide was then accomplished with 0.2 m sodium phosphate, pH 7.4. Differential measurement of 5′-nucleotidase can be accomplished in the presence of acid or alkaline phosphatases by inclusion of concanavalin A into the reaction mixture. It completely inhibits 5′-nucleotidase without effecting the phosphatases. The applicability of this assay has been demonstrated by studying the properties of 5′-nucleotidase present in a purified plasma membrane preparation from a rat tumor which is enriched with both 5′-nucleotidase and alkaline phosphatase. 相似文献
2.
Bacteriophage T4 induced polynucleotide kinase was found to be ineffective in transferring 32P from [γ-32P]ATP to the 5′-terminus of 5′-phosphorylated tRNAHis using the ADP mediated exchange reaction. However, prior dephosphorylation with alkaline phosphatase allowed polynucleotide kinase catalyzed phosphorylation of tRNAHis. Contrary to reports for other tRNA species, alkaline phosphatase catalyzed 5′-terminus dephosphorylation destroys the amino acid accepting ability of tRNAHis. Aminoacylation competency of the tRNAHis is restored after phosphorylation with polynucleotide kinase. 相似文献
3.
John I. Toohey 《Biochemical and biophysical research communications》1982,109(2):313-319
An ether-extractable product formed from 5′-methylthioadenosine by extracts of malignant murine lymphocytic cells is shown to be 2-keto-4-methylthiobutyric acid. When 5′-methylthio [U-14C]adenosine was used as substrate, the product was labelled, confirming earlier reports that carbons of the keto acid are derived from carbons of the ribose. When hydroxylamine was added to the reaction mixture, the ketomethylthiobutyric acid was trapped as the oxime. When glutamine was added, the main product was methionine. 相似文献
4.
The level of 5′-AMP deaminase in homogenates of human term placenta has been measured by means of a simple radiometric assay. The assay uses 14C-labeled AMP as substrate and incorporates conditions of pH and K+ concentration, which optimize the 5′-AMP deaminase activity, and inhibitors of 5′-nucleotidase and adenosine deaminase to reduce interference from these enzymes. Assay products are separated by descending paper chromatography and quantitated by liquid scintillation counting. The activity of 5′-AMP deaminase in human term placenta determined by this assay was 474 ± 37 nmol min?1 g?1 at 30°C and was less than the 5′-AMP phosphatase activity evident under the same assay conditions. The assay is suitable for measurement of 5′-AMP deaminase in extracts of other tissues in which high levels of phosphatases and adenosine deaminase preclude assay of 5′-AMP deaminase by such techniques as ultraviolet absorption changes or ammonia estimation. 相似文献
5.
The properties of an assay for the 5-phosphoribosyl-1-pyrophosphate (PRPP) content of cultured mammalian fibroblasts are described. The assay is based upon the PRPP-dependent release of 14CO2 from [carboxyl-14C]orotic acid by a commercially available preparation of yeast orotidine-5′-monophosphate pyrophosphorylase and orotidine-5′-monophosphate decarboxylase. The advantages of the assay include the fact that it is based on the enzymatic recognition of PRPP, employs an irreversible reaction, and does not involve either the chromatographic separation of substrate and product or the purification of a phosphoribosyltransferase. The disadvantage of the assay is that the efficiency of PRPP measurement varies somewhat, in part because the yeast enzyme preparation contains 5′-nucleotidase activity. A calibration procedure is described which corrects for variation in efficiency both between and within experiments. This procedure seems to yield highly reliable estimates of PRPP content. The assay will readily detect 0.6 nmol, and the cell strain studied contained . 相似文献
6.
SYNOPSIS Deamination at pH 7.5 of adenosine, deoxyadenosine, cytidine and deoxycytidine by cell-free preparations of Tetrahymena pyriformis W was observed both in the presence and absence of fluoride. Deamination of 5′-AMP, 5′-dAMP, 5′-CMP, and 5′-dCMP was found only in the absence of fluoride. Dephosphorylation of the above nucleotides by acid phosphatases occurred at pH 4.5; reduced activity was noted at pH 7.5. Fluoride effectively blocked acid phosphatase activity at both pH values. This correlation of phosphatase and deaminase activities suggests a catabolic pathway for 5′-AMP and 5′-CMP whereby dephosphorylation precedes deamination. Radiolabelled substrates were used to test this hypothesis. The experiments were designed so that conversion of as little at 1.0% of the radiolabelled substrate to the deaminated product could be detected. No 5′-IMP or 5′-UMP, the expected deamination products of 5′-AMP and 5′-CMP, respectively, was recovered after incubation of the radiolabelled substrates with cell-free enzyme preparations. Thus, it appears that Tetrahymena has no 5′-AMP or 5′-CMP deaminases and that these compounds are deaminated only after conversion to nucleosides. Acid phosphatase activity toward 5′-GMP, 5′-dGMP, 5′-TMP, 5′-UMP, and 5′-XMP was also found. 相似文献
7.
John I. Toohey 《Archives of biochemistry and biophysics》1983,223(2):533-542
Typical enzyme kinetics were observed when 5′-methylthioadenosine was used as substrate with extracts of malignant murine cells in a diffusion assay. The volatile product was measured after diffusion into a solution of the sulfhydryl reagent, 5,5′-dithiobis(2-nitrobenzoic acid), which it reduced to a yellow chromophore. Cysteine was required in the system. The volatile product was identified as H2S derived from the cysteine. The yield of H2S was similar to the amount of 2-keto-4-methylthiobutyric acid (KMTB) formed from methylthioadenosine when the KMTB was measured simultaneously in an ether extraction assay. KMTB could replace methylthioadenosine as a substrate capable of causing the formation of the diffusible product from cysteine. It is concluded that the following sequence of reactions takes place in the diffusion assay system: (1) 5′-methylthioadenosine + Pi → adenine + 5-methylthioribose-1-P, (2) 5-methylthioribose-1-P → KMTB, (3) KMTB + cysteine → methionine + 3-mercaptopyruvate, (4) 3-mercaptopyruvate + excess R-SH → pyruvate + H2S, (5) H2S + 5,5′-dithiobis(2-nitrobenzoic acid) → 5-mercapto-2-nitrobenzoic acid. Thus, the diffusion assay measures the amount of KMTB formed. The key enzyme, cysteine aminotransferase, EC 2.6.1.3, was partially purified from malignant cells and from liver and several of its characteristics are described. The diffusion assay using this enzyme is useful in measuring de novo synthesis of α-keto acids and it is applicable to crude enzyme preparations. The sensitivity is about 5 nmol of keto acid and the accurate range is 5 to 100 nmol. 相似文献
8.
The primary acid product of DPNH 总被引:1,自引:0,他引:1
N J Oppenheimer 《Biochemical and biophysical research communications》1973,50(3):683-690
Analysis of the proton magnetic resonance spectra obtained at 220 MHz confirms the axial conformation of the C-6 hydroxyl in the model primary acid product 1-n-(2,6-dichlorobenzyl)-6-hydroxy-1,4,5,6-tetrahydronicotinamide. In the primary acid product of DPNH however the reaction occurs stereospecifically with the substitution at the C-6 position equatorial and on the B-side of the pyridine ring and the C-4A proton axial. A cyclic structure α,O2′-6B cyclotetrahydronicotinamide is proposed for the primary acid product of DPNH, formed by epimerization of βDPNH to the α configuration followed by protonation at C-5 and subsequent attack of the ribose C-2′-OH on the C-6 position forming a new five membered ring. 相似文献
9.
Improved rapidity and precision in the determination of brain 2',3'-cyclic nucleotide 3'-phosphohydrolase 总被引:33,自引:0,他引:33
A rapid and precise method for the determination of brain 2′,3′-cyclic nucleotide 3′-phosphohydrolase (CNP) activity has been developed. Total brain homogenates were treated with deoxycholate, and CNP activity was measured as inorganic phosphate (phosphomolybdic acid, 410 nm) released from the product, 2′-AMP, by alkaline phosphatase. Measurements were carried out under optimal conditions of temperature (30°C) and pH (6.2) using the whole brain of the rat, chicken, and quaking mouse. The entire assay was applicable to multiple samples and could be completed in less than 1 hr. 相似文献
10.
The anionic detergents sodium dodecyl sulfate (SDS) and Alipal CO-433 and the non-ionic detergent Trition X-100 at concentrations of 0.02–0.10% cause a more rapid solubilization of phospholipid than proteins in isolated rat liver plasma membranes. All three detergents cause an increase in membrane turbidity at low detergent concentration (0.01–0.04%) but then decrease the turbidity at higher detergent concentration (0.04–0.10%). Each detergent gives a characteristic turbidity-detergent concentration profile which is pH dependent.The activities of the membrane-bound enzymes Mg2+ ATPase, 5′-nucleotidase and acid and aklaline phosphatase were influenced by each detergent to a different extent. Each enzyme gave a characteristic activity-detergent concentration profile. Mg2+ ATPase was inhibited by all detergents. 5′-Nucleotidase was stimulated by Triton and Alipal but inhibited by SDS. Alkaline phosphatase was stimulated by Alipal and SDS and not influenced by Triton. Acid phosphatase was stimulated by Triton and inhibited by Alipal and SDS. 56% of the total membrane-bound alkaline phosphatase and 23% of the total membrane-bound 5′-nucleotidase was solubilized in an active form by 0.06% and 0.05% SDS respectively. 相似文献
11.
A study of adenosine 3':5'-cyclic monophosphate, sodium butyrate and cortisol as inducers of HeLa alkaline phosphatase 总被引:7,自引:0,他引:7
The role of adenosine 3′:5′-cyclic monophosphate in the cortisol-mediated induction of HeLa 65 alkaline phosphatase was investigated. Although growth of these cells with 0.5–1.0 mmN6,O2′-dibutyryl adenosine 3′:5′-cyclic monophosphate induces a 5- to 8-fold increase in cellular phosphatase activity after 72 hr, neither cAMP nor theophylline induce at concentrations up to 1 mm. Sodium butyrate induces the enzyme as well as dibutyryl cAMP. Moreover, induction kinetics show sodium butyrate to be a more efficient inducer than dibutyryl cAMP, inducing activity as quickly as cortisol. This suggests that the butyric acid cleaved from dibutyryl cAMP by HeLa cells is the mediator of induction when the cyclic nucleotide derivative is used. 相似文献
12.
LuAnn Langham Barbara M. Garber Daphne A. Roe Michael N. Kazarinoff 《Analytical biochemistry》1982,125(2):329-334
A radiometric assay for pyridoxamine 5′-phosphate oxidase (pyridoxamine (pyridoxine) 5′-phosphate:O2 oxidoreductase (deaminating), EC 1.4.3.5) has been developed utilizing N-(5′-phosphopyridoxyl)[3H]tryptamine. This assay is more sensitive than previously used colorimetric and fluorescent assays for this oxidase and furthermore is applicable to erythrocytes. Tritiated substrate is incubated with an enzyme sample in the presence of excess unlabeled truptamine and the radiolabeled tryptamine product is extracted into toluene and quantitated by liquid scintillation counting. 相似文献
13.
Ochromonas danica cell homogenate can be fractionated by differential centrifugation into chloroplast, mitochondrial, ribosome, lysosomal, plasma membrane and soluble fractions. The plasma membrane fraction was further purified by discontinuous sucrose density gradient centrifugation and was found to be enriched 4–16-fold in the following enzymes: β-galactosidase, acid phosphatase, alkaline phosphatase, 5′-nucleotidase, and (Na+, K+)-ATPase. The role of plasma membrane phosphatase in the phosphate metabolism of plants is discussed. 相似文献
14.
An ultraviolet absorbance method for quantitation of acetylsalicylic acid esterase (hydrolase) activity has been developed and validated. The sensitivity of the method was found to be 2.8 nmol/ml-min in the assay cuvette. Linearity of the reaction with enzyme concentration and time has been demonstrated. The product of the enzymatic reaction, salicylic acid, has been identified by thin-layer chromatography using acetyl-[14C]salicylic acid. The quantities of salicylic acid produced in 5, 10, and 15 min of incubation were equal when assayed by the spectrophotometric method and by the acetyl-[14C]salicylic acid thin-layer chromatographic method. The time required for assay by ultraviolet absorbance is approximately 3 min/sample. 相似文献
15.
Abstract The 4-Thiothymidine-5′-triphosphate 1 (S4TTP) was known to be a substrate for polymerase, however a commercial sample of this compound failed to be incorporated into DNA. Mass spectrometry combined to alkaline phosphatase digestion and 31P-NMR showed that this sample was in fact 5′-chloro-5′-deoxy-4-thiothymidine-3′-triphosphate 2 . The desired S4TTP was synthesized by two alternate routes, was fully characterized and was shown to be incorporated in a DNA polymerase assay. 相似文献
16.
Two methods are described for the assay of sulfotransferases which are active with sulfate acceptors bearing the hydroxyl functional group. Assays were developed for enzymes which transfer sulfate from 3′-phosphoadenosine–5′-phosphosulfate (PAPS) to sterols, phenols, and simple alcohols thereby forming the corresponding sulfate esters. With a filter binding assay, useful with crude and purified enzyme preparations, a radioactive sterol substrate is used and subsequently separated from labeled product, allowing the determination of between 50 and 400 pmol of product. In a second method, [35S]PAPS is used and the labeled product is separated from PAPS and inorganic sulfate by a thin-layer technique in which product migrates close to the solvent front; the assay is useful with a broad array of substrates and is more sensitive than the filter binding assay. 相似文献
17.
This paper describes a new, sensitive, and reproducible method for the determination of 2′,3′-cyclic-nucleotide 3′-phosphodiesterase (EC 3.1.4.37) activity in both central and peripheral nervous system tissue, as well as in extraneural tissue. Radioactive [8-3H]adenosine 2′,3′-cyclic monophosphate was used as the substrate. The [8-3H]2′-AMP product formed was isolated by thin-layer chromatography, or, alternatively, the reaction was coupled with an excess of Escherichia coli alkaline phosphatase, and the [8-3H]adenosine formed was isolated by column chromatography on AG 1-X2 resin. The values obtained by the two methods were compared with those obtained using a spectrophotometric method. Hydrolysis rates of 0.50 nmol/min could be reproducibly measured in 18-day fetal rat spinal cord. 相似文献
18.
A Stevens 《Biochemical and biophysical research communications》1978,81(2):656-661
Using poly(A) as a substrate, an exoribonuclease has been purified from the high-salt wash of ribosomes of . The product of the reaction of the exoribonuclease is 5′ AMP. Hydrolysis of [3H](pA)3[14C](pA)n shows that both labels are released at the same rate, suggesting that the enzyme acts in a processive manner. Removal of the terminal phosphate of poly(A) with alkaline phosphatase reduces the rate of hydrolysis by 80%. Treatment of the terminally dephosphorylated poly(A) with polynucleotide kinase restores the activity. Two 5′ capped mRNA's have been tested and they are hydrolyzed slowly, if at all, by the enzyme. In contrast, phage T4 mRNA, ribosomal RNA, and encephalomyocarditis viral RNA are hydrolyzed at greater than 50% of the rate of poly(A). 相似文献
19.
Takemitsu Mizunaga 《Bioscience, biotechnology, and biochemistry》2013,77(9):2021-2027
The regulation of acid phosphatase synthesis by various phosphate compounds was examined in Baker’s yeast protoplasts. Synthesis was repressed by inorganic phosphate and phosphomonoesters. Phosphomonoesters were hydrolysed by a small amount of non-specific acid phosphatase present in the protoplast membrane. The inorganic phosphate that was liberated and incorporated into protoplasts probably repressed acid phosphatase synthesis. Phosphodiesters, such as 3′, 5′-cyclic AMP, 3′, 5′-cyclic CMP and 3′, 5′-cyclic GMP, promoted acid phosphatase synthesis. The effect of 3′, 5′-cyclic AMP was not to overcome hexose repression, because high hexose did not repress acid phosphatase synthesis. 3′, 5′-cyclic AMP did not overcome repression of the enzyme synthesis by inorganic phosphate. From these observations 3′, 5′-cyclic nucleotides probably had some effect on the yeast acid phosphatase-synthesizing system but the exact role of the nucleotides is obscure. 相似文献
20.
Eric G. Brown Malcolm J. Edwards Russell P. Newton Christopher J. Smith 《Phytochemistry》1980,19(1):23-30
Cyclic nucleotide phosphodiesterase was extracted from intact chloroplasts and partially purified. Peak 1c activity from Sephadex G-200 was resolved by electrophoresis into two major bands (MWs 1.87 × 105 and 3.7 × 105). Both also possessed acid phosphatase, ribonuclease, nucleotidase and ATPase. The chloroplast peak 1c cyclic nueleotide phosphodiesterase was located in the envelope. Peak 1m cyclic nucleotide phosphodiesterase obtained from the microsomal fraction had a MW of 2.63 × 105. Electrophoresis separated 1m into two bands of cyclic nucleotide phosphodiesterase activity (MWs 2.63 × 105 and 1.28 × 105). Both contain ATPase, ribonuclease, nucleotidase, but not acid phosphatase. Peak 1c has high activity towards 3′:5′-cyclic AMP and 3′:5′-cyclic GMP but little towards 2′:3′-cyclic nucleotides. Peak 1m showed most activity towards 2′:3′-cyclic AMP, 2′:3′-cyclic GMP and 2′:3′-cyclic CMP with little activity towards 3′:5′-cyclic nucleotides. With 1c, 3′:5′-cyclic AMP and 3′:5′-cyclic GMP exhibit mixed-type inhibition towards one another. The 2′:3′-cyclic AMP phosphodiesterase 1m was competitively inhibited by 2′:3′-cyclic GMP. p-Chloromercuribenzoate inhibits 1c but not 1m. Electrophoresis after dissociation indicates that 1c and 1m are both enzyme complexes. After dissociation, the 1c complex but not that of 1m could be reassociated. The ribonuclease of the 1m complex hydrolyses RNA to yield 2′:3′-cyclic nucleotides as the main products. These results are compatible with the 1c cyclic nucleotide phosphodiesterase complex being involved in the metabolism of 3′:5′-cyclic AMP, and the 1m complex being concerned with RNA catabolism. 相似文献