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1.
The specific radioactivity of the γ-phosphorus of ATP has been determined by an indirect method. Galactokinase is employed to transfer the terminal phosphate group of [γ-32P] ATP to [1-3H] galactose. The doubly labeled galactose-1-phosphate is purified by ion exchange chromatography on QAE Sephadex. The specific radioactivity of the phosphorus is calculated from the 32P3H ratio. The method is extremely sensitive, requiring only 0.005 μmoles of ATP with a specific radioactivity of 1 μCi/μmole, and the chromatographic isolation of galactose-1-phosphate is simple and reproducible. The method is directly applicable to the determination of the specific radioactivity of [γ-32P] ATP in biological samples.  相似文献   

2.
A rapid method for the measurement of [γ-32P]ATP specific radioactivity in tissue extracts containing other 32P-labeled compounds is described. The neutralized acid extract is incubated with cyclic AMP-dependent protein kinase, cyclic AMP and casein. The incorporation of 32P into casein from [γ-32P]ATP is measured by perchloric acid precipitation of the protein on filter paper. 32P-Casein formation is linearly related to the specific radioactivity of the [γ-32P]ATP. Separation of ATP from other 32P-labeled compounds is not required for the assay. Application of this method in the evaluation of [γ-32P]ATP specific radioactivity in two rat cardiac muscle preparations exposed to 32Pi is demonstrated.  相似文献   

3.
The specific radioactivity of a choline phospholipid has been determined by a double-isotope method. Purified phospholipid was hydrolyzed to release labeled choline, and choline kinase was employed to label the choline with 32P from [γ-32P]ATP. The double-labeled phosphorylcholine was purified by ion-exchange chromatography on QAE-Sephadex, and the specific radioactivity of the choline was calculated from the isotope ratio. The method is sensitive, requiring only 5 nmol of choline with a specific radioactivity of 1 μCl/μmol, and the chromatographic isolation of phosphorylcholine is simple and reproducible.  相似文献   

4.
1. The specific activity of the γ-32P position of ATP was measured in various tissue preparations by two methods. One employed HPLC and the enzymatic conversion of ATP to glucose 6-phosphate and ADP. The other was based on the phosphorylation of histone by catalytic subunit of cAMP-dependent protein kinase (Hawkins, P.T., Michell, R.H. and Kirk, C.J. (1983) Biochem. J. 210, 717–720). The HPLC method also allowed the incorporation of 32P into the (α + β)-positions of ATP to be determined. 2. In rat epididymal fat-pad pieces and fat-cell preparations the specific activity of [γ-32P]ATP attained a steady-state value after 1–2 h incubation in medium containing 0.2 mM [32P]phosphate. Addition of insulin or the β-agonist isoprenaline increased this value by 5–10% within 15 min. 3. Under these conditions the steady-state specific activity of [γ-32P]ATP was 30–40% of the initial specific activity of the medium [32P]phosphate. However, if allowance was made for the change in medium phosphate specific activity during incubations the equilibration of the γ-phosphate position of ATP with medium phosphate was greater than 80% in both preparations. The change in medium phosphate specific activity was a combination of the expected equilibration of [32P]phosphate with exchangeable intracellular phosphate pools plus the net release of substantial amounts of tissue phosphate. At external phosphate concentrations of less than 0.6 mM the loss of tissue phosphate to the medium was the major factor in the change in medium phosphate specific activity. 4. It is concluded that little advantage is gained in employing external phosphate concentrations of less than 0.6 mM in experiments concerned with the incorporation of phosphate into proteins and other intracellular constituents. Indeed, a low external phosphate concentration may cause depletion of important intracellular phosphorus-containing components.  相似文献   

5.
In order to meet a need for a cAMP assay which is not subject to interference by compounds in plant extracts, and which is suitable for use on occasions separated by many 32P half-lives, an assay based on cAMP-dependent protein kinase has been developed which does not require the use of [γ-32P]ATP. Instead of measuring the cAMP-stimulated increase in the rate of transfer of [γ-32P] phosphate from [γ-32P]ATP to protein, the rate of loss of ATP from the reaction mixture is determined. The ATP remaining after the protein kinase reaction is assayed by ATP-dependent chemiluminescence of the firefly luciferin-luciferase system. Under conditions of the protein kinase reaction in which a readily measurable decrease in ATP concentration occurs, the logarithm of the concentration of ATP decreases in proportion to the cAMP concentration, i.e., the reaction can be described by the equation: [ATP] = [ATP]0 e?[cAMP]kt. The assay based on this relationship can detect less than 1 pmol of cAMP. The levels of cAMP found with this assay after partial purification of the cAMP from rat tissue, algal cells, and the media in which the cells were grown agreed with measurements made by the cAMP binding-competition assay of Gilman, and the protein kinase stimulation assay based on transfer of [32P] phosphate from [γ-32P]ATP to protein. All of the enzymes and chemicals required for the assay of cAMP by protein kinase catalyzed loss of ATP can be stored frozen for months, making the assay suitable for occasional use.  相似文献   

6.
After conversion of unlabeled DNA and RNA to 3′-mononucleotides accurate base compositional analysis can be performed on as little as 10 ng of the hydrolysate. The 3′-mononucleotides are first quantitatively postlabeled with [γ-32P]ATP by T4 polynucleotide kinase and are then separated as mononucleoside diphosphates on Whatman DE-81 ion-exchange paper at pH 3.5 after hydrolysis of surplus [γ-32P]ATP to 32P1. The locations of the four labeled nucleoside diphosphates are determined by autoradiography and the ratio of radioactivity in the four spots gives the base ratio of the sample.  相似文献   

7.
A rapid method for the measurement of [γ-32P]ATP specific radioactivity in tissue extracts containing other 32P-labeled compounds is described. The neutralized acid extract is incubated with cyclic AMP-dependent protein kinase, cyclic AMP and casein. The incorporation of 32P into casein from [γ-32P]ATP is measured by perchloric acid precipitation of the protein on filter paper. 32P-Casein formation is linearly related to the specific radioactivity of the [γ-32P]ATP. Separation of ATP from other 32P-labeled compounds is not required for the assay. Application of this method in the evaluation of [γ-32P]ATP specific radioactivity in two rat cardiac muscle preparations exposed to 32Pi is demonstrated.  相似文献   

8.
(i) A new, rapid method for the measurement of [γ-32P]ATP specific radioactivity in tissue extracts in the presence of other 32P-containing compounds is described. The deproteinized extract is incubated with phosphorylase b and phosphorylase kinase, and the incorporation of 32P into protein from [γ-32P]ATP is measured by precipitation on filter paper in trichloroacetic acid. No separation of ATP or other treatment of the extracts is required for the assay. (ii) 32Pi uptake in perfused rat heart was found to be a relatively slow process, with a Km of 0.084 mm, whereas equilibration between intracellular 32Pi and [γ-32P]ATP occurred rapidly.  相似文献   

9.
A soluble fraction, obtained by extracting E. coli cytoplasmic membrane vesicles with water, transfers radioactivity from [γ-32P]ATP to a protein present in this soluble fraction. The formation of the [32P]phosphoprotein appears to be reversible. Thus the protein can transfer its 32P to ADP to form [32P]ATP, and the phosphate on the protein can exchange with the phosphate of ATP. Preliminary evidence indicates that the phosphate moiety is linked to a histidine residue of the protein. The Mn2+ and ATP dependencies of [32P]phosphoprotein formation are almost identical to the diglyceride kinase reaction previously reported in intact membrane vesicles. Although indirect evidence supports the involvement of the phosphoprotein in the diglyceride kinase reaction, the soluble fraction catalyzes only a slow formation of [32P]phosphatidie acid from [γ-32P]ATP and α,β-diglyceride.  相似文献   

10.
The straightforward, rapid synthesis and purification of radiochemically pure [1-32P]fructose-1,6-bisphosphate starting from radioactively labeled inorganic phosphate is described. The product has a relatively high specific radioactivity (2 × 105 Ci/mol). Using this method, carrier-free [1-32P]fructose-1,6-bisphosphate could be obtained. The use of [1-32P]fructose-1,6-bisphosphate in the assay of fructose bisphosphatase is illustrated.  相似文献   

11.
Pyridoxal [32P] phosphate was prepared using [γ-32P]ATP, pyridoxal, and pyridoxine kinase purified from Escherichia coli B. The pyridoxal [32P] phosphate obtained had a specific activity of at least 1 Ci/mmol. This reagent was used to label intact influenza virus, red blood cells, and both normal and transformed chick embryo fibroblasts. The cell or virus to be labeled was incubated with pyridoxal [32P] phosphate. The Schiff base formed between pyridoxal [32P] phosphate and protein amino groups was reduced with NaBH4. The distribution of pyridoxal [32P] phosphate in cell membrane or virus envelope proteins was visualized by autoradiography of the proteins separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis.The labeling of the proteins of both influenza and chick cells appeared to be limited exclusively to those on the external surface of the virus or plasma membrane. With intact red blood cells the major portion of the probe was bound by external proteins, but a small amount of label was found associated with the internal proteins spectrin and hemoglobin.  相似文献   

12.
Upon subcellular fractionation of (murine) Friend erythroleukaemic cells (FELCs), purified plasma membranes were identified by their high enrichment in specific marker enzymes and typical plasma membrane lipids. When FELCs were incubated for short periods with 32Pi before cell fractionation, the lipid-bound radioactivity was almost exclusively present in phosphatidylinositol-4-phosphate (DPI) and phosphatidylinositol-4,5-bisphosphate (TPI), and its distribution closely matched that of the plasma membrane markers. In addition, purified plasma membranes actively incorporated 32P from [γ-32P]ATP into polyphosphoinositides, and the specific activities of the involved kinases were again mostly enriched in the plasma membrane fraction.  相似文献   

13.
Radioactive phosphate was incorporated from [γ-32P]ATP into a 160 000 dalton protein from preparations of highly purified toad retinal rod outer segment membranes. Maximal incorporation occurred at 1μM ATP, and turnover in the presence of nonradioactive substrate was rapid, showing that the 160 kdalton protein catalyzes ATP hydrolysis. The 160 kdalton intermediate was sensitive to hydroxylamine, suggesting an acyl linkage between the protein and phosphate. Ionic requirements for phosphorylation showed the ATPase is different from other membrane-bound ionic pumps. The phosphorylated intermediate was almost completely suppressed by 20 μM vanadate, and partial suppression occurred at lower concentrations. About one 160 kdalton protein was labelled per 30 000 molecules of rhodopsin. Although [γ-32P]GTP labeled the protein, the ATPase was far more specific for adenine than guanine nucleotides. The specificity for ATP and sensitivity to vanadate of the intermediate suggest a relation to an ATP-dependent structural change which occurs in stacks of outer segment discs (Thacher, S.M.; (1980) Fed. Proc. 39, 2066).  相似文献   

14.
The sodium-potassium adenosinetriphosphatase (NaK ATPase), partially purified from beef brain, has been phosphorylated with [γ-32P]ATP in the presence of Na and Mg and digested with pronase. A single 32P-labeled peptide spot has been identified on paper electrophoresis, accounting for 60% of the radioactivity in the 32P-labeled enzyme, the remainder of the radioactivity being [32P]-orthophosphate resulting from breakdown of the highly labile acyl phosphate during pronase digestion. The 32P in the pronase peptide was released as [32P]-orthophosphate by N-propylhydroxylamine—as to be expected of an acyl phosphate compound. The pH stability of the acyl phosphate in the denatured phosphorylated NaK ATPase, in the pronase peptide and in acetyl phosphate were quite different. The phosphorylated protein had the lowest stability of higher pHs, acetyl phosphate had the highest stability, and the pronase peptide had an intermediate stability. These results indicate that the neighboring groups in the polypeptide chain containing the acyl phosphate residue influence the stability of the acyl phosphate bond.  相似文献   

15.
Protein kinase (EC 2.7.1.37) catalyzes the phosphorylation of serine and threonine residues of a number of proteins. Histone is widely used as an acceptor substrate in measuring the activity of this enzyme isolated from a variety of sources. We have devised a rapid procedure for resolving phosphohistone from ATP and its metabolites based on the specific absorption of phosphorylated histone onto phosphocellulose paper. Using [γ-32P]ATP as the phosphoryl donor, aliquots of the protein kinase assay mixture are applied to phosphocellulose-paper disks that are then immersed in water which elutes [γ-32P]ATP and metabolites. After brief organic solvent extraction and drying, bound radioactivity is measured by liquid scintillation spectrometry.  相似文献   

16.
An existing enzymic method for preparing [γ-32P]ATP from 32Pi has been modified toyield [γ-32P]ATP free of salt and buffer. 32P is incorporated into the γ-position of ATP by isotopic exchange in the presence of glyceraldehyde 3-phosphate dehydrogenase and 3-phosphoglycerate kinase. Unreacted 32Pi is separated from [γ-32P]ATP by column chromatography on Dowex 1 bicarbonate. [γ-32P]ATP is eluted with 2 m triethylammonium bicarbonate, which is then completely removed by freeze-drying.  相似文献   

17.
A sensitive method for measuring phosphorylase kinase activity by the incorporation of 32P from [γ-32]ATP into phosphorylase in the presence of other phosphorylation reactions is described. The kinase reaction is carried out in a crude homogenate. After stopping the reaction, a portion of the reaction mixture is withdrawn for assay of phosphorylase conversion and the rest is applied on a 5′-AMP Sepharose column. Phosphorylase in both forms is retained on the column while other phosphorylated proteins and [γ-32P]ATP are washed out. The phosphorylase is then eluted by 10 mm AMP and the radioactivity incorporated is counted.  相似文献   

18.
The regulation of the pyruvate dehydrogenase multienzyme complex of isolated beef heart mitochondria by a phosphorylation-dephosphorylation mechanism was investigated. From mitochondria incubated under conditions favoring either a protein kinasemediated inactivation or a phosphatase-mediated reactivation, the pyruvate dehydrogenase complex was extracted and partially purified. Incorporation of 32P from [γ-32P]ATP into the pyruvate dehydrogenase complex corresponded to the loss of enzymatic activity. Upon incubation of the mitochondria that were preincubated with [γ-32P]ATP under metabolic conditions favoring the phosphatase reaction, the amount of radioactivity in the 32P-labeled fraction decreased significantly with a concomitant increase in the pyruvate dehydrogenase activity. The estimated molecular weight of the 32P-labeled fraction derived from the mitochondrial incubation was 41,000, corresponding to the reported molecular weight of the α-subunit of the pyruvate dehydrogenase portion of the multienzyme complex.  相似文献   

19.
  • 1.1. Adenylylation, a posttranslational modification of proteins, was investigated in saponin-permeabilized acinar cells of the rat parotid gland.
  • 2.2. When cells were incubated with [2,8-3H]ATP, several proteins, including a 26 kDa protein in the particulate fraction, were labeled.
  • 3.3. Upon incubation of cells with [α-32P]ATP in the presence of cAMP and 3-isobutyl-lmethylxanthine, 32P-labeling of the 26 kDa protein was observed.
  • 4.4. After treatment with snake venom phosphodiesterase, [32P]AMP was released from the 26kDa protein. Such release was not observed when cells were labeled with [γ-32P]ATP.
  • 5.5. The 32P-labeling pattern of proteins with [α-32P]ATP was clearly different from that with [adenylate-32P]NAD+.
  • 6.6. The results suggest that the 26 kDa protein is one of the adenylylation substrates in rat parotid acinar cells.
  相似文献   

20.
Incorporation of 32P from [γ-32P]ATP into a homogeneous preparation of rat hepatic 6-phosphofructo 2-kinase/fructose 2,6-bisphosphatase was catalyzed by a homogeneous preparation of the catalytic subunit of the cyclic AMP dependent protein kinase from rat liver. Approximately 2 mol of phosphate were incorporated per mol of the dimeric enzyme and this was associated with inhibition of the phosphotransferase activity and activation of the phosphohydrolase activity. Acid hydrolysis of the enzyme that was phosphorylated in,vitro revealed that only seryl residues were labeled. Fructose 2,6-bisphosphate inhibited the initial rate of phosphorylation of the enzyme. It is concluded that both activities of this bifunctional enzyme are regulated in a reciprocal manner by cyclic AMP-dependent phosphorylation and that this phosphorylation can be modulated by fructose 2,6-bisphosphate.  相似文献   

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