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1.
Determination of levels of glycolytic intermediates and nucleotides in platelets by pulse-labeling with [32P]orthophosphate 总被引:1,自引:0,他引:1
Holm Holmsen Carol A. Dangelmaier Jan-Willem N. Akkerman 《Analytical biochemistry》1983,131(1):266-272
A method is presented for the separation and quantification of 32P-labeled carbohydrates and nucleotides in blood platelets which have been pulse-labeled with [32P]orthophosphate. The procedure is based on two-dimensional paper chromatography, identification of the spots by radioautography and enzymatic methods, and quantitation of 32P radioactivity by liquid scintillation counting. The data show that 32P is homogeneously distributed among the compounds studied so that the total radioactivity is proportional to the levels of these compounds in the metabolic compartment of the cells. Thus, this method provides a sensitive and accurate means to evaluate phosphorylated intermediates in glycolysis and nucleotide metabolism and to assess the transfer of energy-rich phosphate groups between these pathways in particular. 相似文献
2.
Identification of the phosphorylated intermediate of the Neurospora plasma membrane H+-ATPase as beta-aspartyl phosphate 总被引:4,自引:0,他引:4
The chemical nature of the phosphoryl enzyme linkage of the electrogenic proton-translocating ATPase (ATP phosphohydrolase, EC 3.6.1.3) in the plasma membrane of Neurospora has been identified as a mixed anhydride between phosphate and the beta-carboxyl group of an aspartic acid residue in the polypeptide chain. Incubation of isolated Neurospora plasma membrane vesicles containing 32P-labeled ATPase in buffers of increasing pH followed by analysis of the hydrolysis products yielded a pH versus hydrolysis profile characteristic of an acyl phosphate linkage. Reaction of labeled membranes with hydroxylamine at pH 5.3 also released [32P]i from the ATPase. Amino acid analyses of the Na[3H]BH4 reduction products obtained from membranes containing phosphorylated and dephosphorylated ATPase identified [3H]homoserine, the expected reduction product of beta-aspartyl phosphate, as the only additional tritiated reduction product in the samples from phosphorylated membranes. Tritium was not found in alpha-amino-delta-hydroxyvaleric acid, the reduction product of gamma-glutamyl phosphate, nor in proline, the degradation product of alpha-amino-delta-hydroxyvaleric acid. These results indicate that the phosphorylated intermediate of the Neurospora plasma membrane ATPase is a beta-aspartyl phosphate identical with that already known to exist in the Na+:K+- and Ca2+-translocating ATPases of animal cell origin. A common model for the mechanisms of all 3 ion-translocating ATPases is presented. 相似文献
3.
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
4.
Marit Nilsen-Hamilton Richard T. hamilton 《Biochimica et Biophysica Acta (BBA)/General Subjects》1979,588(3):322-331
Cyclic AMP-dependent protein kinases from several mammalian sources inhibit Na+-dependent α-aminoisobutyric acid transport by membrane vesicles isolated from 3T3 cells. Evidence is provided that phosphorylation of membrane proteins by the enzyme is responsible for the inhibition. Lysis of the vesicles, or a reduction in the intravesicular volume is not the cause of reduced transport.The cyclic AMP-dependent protein kinase and its catalytic subunit phosphorylate a number of membrane proteins. Most of these proteins are phosphorylated, but to a lesser extent in the absence of protein kinase or cyclic AMP. The phosphorylated proteins remain associated with the membranes during hypotonic lysis treatments, which would be expected to release intra-vesicular contents and loosely associated membrane proteins. 32P-labeled bands detected on sodium dodecyl sulfate polyacrylamide gels after phosphorylation of membranes by the catalytic subunit of the cyclic AMP-dependent kinase are eliminated by treatment with either pronase or 1 N NaOH, but not by ribonuclease nor by phospholipase C. The stability of the incorporated radioactivity to hot acid and hydroxylamine relative to hot base suggests that most of the 32P from [γ-32P]ATP is incorporated into protein phosphomonoester linkages. 相似文献
5.
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
6.
Purification and properties of phosphorylated isocitrate dehydrogenase of Escherichia coli. 总被引:5,自引:0,他引:5
Phosphorylated NADP+-isocitrate dehydrogenase (EC 1.1.1.42) has been purified to electrophoretic homogeneity from in vivo 32P-labeled Escherichia coli. The cells used as the source of phosphorylated enzyme were harvested 1 h after the addition of 5 mCi of [32P]orthophosphoric acid and 25 mM sodium acetate to cultures grown to early stationary phase on a low phosphate medium with limiting glucose. Double immunodiffusion and autoradiography demonstrated immunological identity between the 32P-labeled NADP+-isocitrate dehydrogenase and the enzyme isolated from glucose-grown E. coli. The phosphoenzyme had an apparent subunit molecular weight of 51,000 as determined by denaturing acrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, and the radioactivity co-electrophoresed with NADP+-isocitrate dehydrogenase activity when purified enzyme was subjected to nondenaturing gel electrophoresis. [32P]Phosphoserine was identified following partial acid hydrolysis of the purified phosphoenzyme. 相似文献
7.
Burst titration experiments conducted on a highly purified isoenzyme of wheat germ acid phosphatase under conditions where [S]o > Km indicate that there is one titratable active site per molecule of enzyme of molecular weight 59,000. The enzyme is labeled to only a small extent with inorganic [32P]phosphate ion. Incubation of wheat germ acid phosphatase with 32P-labeled substrates such as p-nitrophenyl phosphate or inorganic pyrophosphate followed by quenching in alkali results in the stoichiometric trapping of a base-stable, acid-labile phosphorylated protein. The extent of 32P incorporation parallels the degree of purity of the enzyme and corresponds to the incorporation of 1 mol of phosphate per mole of enzyme. The incorporation is eliminated by the simultaneous presence of excess unlabeled phosphate ion (a competitive inhibitor) and is not observed when a noncatalytic protein (such as bovine serum albumin) is substituted for the enzyme. Complete alkaline hydrolysis of the labeled protein results in the recovery of an 85% yield of τ-phosphohistidine, identified by ion-exchange chromatography, high-voltage paper electrophoresis, and comparison with a synthetic sample. A 32P-labeled tryptic tetradecapeptide was isolated following hydrolysis of the labeled, reduced, and carboxymethylated protein with trypsin at pH 8.3, separation of the labeled peptide, and purification by two methods including a novel variant of a diagonal electrophoresis technique. The end groups and composition of the peptide are reported. The data are consistent with the interpretation that a phosphohistidine-enzyme intermediate is formed as an obligatory intermediate in the catalytic reaction involving this enzyme. 相似文献
8.
S.M. Thacher 《生物化学与生物物理学报:生物膜》1981,648(2):199-205
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
9.
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
10.
Kate Bárány David L. Vander Meulen Ronald F. Ledvora Michael Bárány 《Archives of biochemistry and biophysics》1982,217(1):392-396
The phosphorylation of myosin light chain was quantitated in fast and slow chicken skeletal muscles and in frog sartorius and semitendinosus muscles. The phosphate content of light chain was determined either as moles [32P]phosphate per mole of light chain in 32P-labeled muscles or as percentage phosphorylated light chain of the total P-light chain, measured by densitometry after separating the phospho and dephospho forms of P-light chain with two-dimensional gel electrophoresis. Both methods revealed that the percentage of total P-light chain which was phosphorylated did not exceed 50% either in maximally tetanized or caffeine-contracted skeletal muscle. This suggests that one of the two P-light chains is selectively phosphorylated in skeletal muscle. 相似文献
11.
32P-labeled peptides obtained by pronase digestion of unfractionated nonhistone nuclear proteins were resolved on columns of Dowex 50, DEAE-Sephadex, Bio-Gel P2, and paper electrophoresis at pH 1.8. Each of 30 peptides analyzed contained predominantly glycine, glutamic acid and proline. The chain length ranged from 7 to 19 residues, including 1 to 4 phosphorylated residues per peptide. These results suggest phosphorylation sites in nonhistones involve a high negative charge density in non-helical regions of these proteins. 相似文献
12.
Dephosphorylation of cardiac myofibril C-protein by protein phosphatase 1 and protein phosphatase 2A 总被引:1,自引:0,他引:1
C-protein purified from chicken cardiac myofibrils was phosphorylated with the catalytic subunit of cAMP-dependent protein kinase to nearly 3 mol [32P]phosphate/mol C protein. Digestion of 32P-labeled C-protein with trypsin revealed that the radioactivity was nearly equally distributed in three tryptic peptides which were separated by reversed-phase HPLC. Fragmentation of 32P-labeled C-protein with CNBr showed that the isotope was incorporated at different ratios in three CNBr fragments which were separated on polyacrylamide gels in the presence of sodium dodecyl sulfate. Phosphorylation was present in both serine and threonine residues. Incubation of 32P-labeled C-protein with the catalytic subunit of protein phosphatase 1 or 2A rapidly removed 30-40% of the [32P]phosphate. The major site(s) dephosphorylated by either one of the phosphatases was a phosphothreonine residue(s) apparently located on the same tryptic peptide and on the same CNBr fragment. CNBr fragmentation also revealed a minor phosphorylation site which was dephosphorylated by either of the phosphatases. Increasing the incubation period or the phosphatase concentration did not result in any further dephosphorylation of C-protein by phosphatase 1, but phosphatase 2A at high concentrations could completely dephosphorylate C-protein. These results demonstrate that C-protein phosphorylated with cAMP-dependent protein kinase can be dephosphorylated by protein phosphatases 1 and 2A. It is suggested that the enzyme responsible for dephosphorylation of C-protein in vivo is phosphatase 2A. 相似文献
13.
J C Lawrence J F Hiken A A DePaoli-Roach P J Roach 《The Journal of biological chemistry》1983,258(17):10710-10719
The effects of insulin and epinephrine on the phosphorylation of glycogen synthase were investigated using rat hemidiaphragms incubated with [32P]phosphate. Antibodies against rabbit skeletal muscle glycogen synthase were used for the rapid purification of the 32P-labeled enzyme under conditions that prevented changes in its state of phosphorylation. The purified material migrated as a single radioactive species (Mapp = 90,000) when subjected to electrophoresis in sodium dodecyl sulfate. Insulin decreased the [32P]phosphate content of glycogen synthase. This effect occurred rapidly (within 15 min) and was observed with physiological concentrations of insulin (25 microunits/ml). The amount of [32P]phosphate removed from glycogen synthase by either different concentrations of insulin or times of incubation with the hormone was well correlated to the extent to which the enzyme was activated. Epinephrine (10 microM) inactivated glycogen synthase and increased its content of [32P]phosphate by about 50%. Cleavage of the immunoprecipitated enzyme with cyanogen bromide yielded two major 32P-labeled fragments of apparent molecular weights equal to approximately 28,000 and 15,000. The larger fragment (Fragment II) displayed electrophoretic heterogeneity similar to that observed with the corresponding CNBr fragment (CB-2) from purified rabbit skeletal muscle glycogen synthase phosphorylated by different protein kinases. Epinephrine increased [32P]phosphate content of both fragments; however, the increase in the radioactivity of the smaller fragment (Fragment I) was more pronounced. Insulin decreased the amount of [32P] phosphate present in Fragments I and II by about 40%. The results presented provide direct evidence that both insulin and epinephrine control glycogen synthase activity by regulating the phosphate present at multiple sites on the enzyme. 相似文献
14.
Chinese hamster ovary cells in monolayer culture were incubated with [3 2P] phosphate. Ribosome-associated proteins, including both structural proteins and those tightly bound to washed, centrifuged ribosomes, were isolated and separated by electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. The electrophoretic pattern showed five major regions or peaks of 3 2P radioactivity which represented phosphorylated ribosome-associated proteins with molecular weights of 17 500, 23 000, 30 000, 38 000, and 57 000. When asynchronous cells were pulse-labeled with [3 2P] phosphate, the predominant peak of 3 2P radioactivity was associated with the protein of 38 000 daltons. Similar results were obtained with cells synchronized in the G1, S, or G2 phase of the mammalian cell cycle. Conversely, proteins isolated from the ribosomes of mitotic cells, collected and labeled with [3 2P] phosphate in the presence of colcemid, showed a new and predominant peak of 3 2P radioactivity migrating with a protein of 45 000 daltons. When cells labeled in mitosis were allowed to progress into G1 phase, this peak of 3 2P radioactivity rapidly disappeared from the electrophoretic pattern. These results suggest that a specific protein associated with the ribosomes was phosphorylated uniquely during the mitotic phase of the cell cycle. 相似文献
15.
Microsomal 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase kinase has been purified to apparent homogeneity by a process involving the following steps: solubilization from microsomes and chromatography on Affi-Gel Blue, phosphocellulose, Bio-Gel A 1.5m, and agarose-hexane-ATP. The apparent Mr of the purified enzyme as judged by gel-filtration chromatography is 205,000 and by sodium dodecyl sulfate-gel electrophoresis is 105,000. Immunoprecipitation of homogeneous reductase phosphorylated by reductase kinase and [γ-32P]ATP produces a unique band containing 32P bound to protein which migrates at the same Rf as the reductase subunit. Incubation of 32P-labeled HMG-CoA reductase with reductase phosphatase results in a time-dependent loss of protein-bound 32P radioactivity, as well as an increase in enzymic activity. Reductase kinase, when incubated with ATP, undergoes autophosphorylation, and a simultaneous increase in its enzymatic activity is observed. Tryptic treatment of immunoprecipitated, 32P-labeled HMG-CoA reductase phosphorylated with reductase kinase produces only one 32P-labeled phosphopeptide with the same Rf as one of the two tryptic phosphopeptides that have been reported in a previous paper. The possible existence of a second microsomal reductase kinase is discussed. 相似文献
16.
Cyclic AMP-dependent protein kinases from several mammalian sources inhibit Na+-dependent alpha-aminoisobutyric acid transport by membrane vesicles isolated from 3T3 cells. Evidence is provided that phosphorylation of membrane proteins by the enzyme is responsible for the inhibition. Lysis of the vesicles, or a reduction in the intravesicular volume is not the cause of reduced transport. The cyclic AMP-dependent protein kinase and its catalytic subunit phosphorylate a number of membrane proteins. Most of these proteins are phosphorylated, but to a lesser extent in the absence of protein kinase or cyclic AMP. The phosphorylated proteins remain associated with the membranes during hypotonic lysis treatments, which would be expected to release intravesicular contents and loosely associated membrane proteins. 32P-labeled bands detected on sodium dodecyl sulfate polyacrylamide gels after phosphorylation of membranes by the catalytic subunit of the cyclic AMP-dependent kinase are eliminated by treatment with either pronase or 1 N NaOH, but not by ribonuclease nor by phospholipase C. The stability of the incorporated radioactivity to hot acid and hydroxylamine relative to hot base suggests that most of the 32P from [gamma-32P]ATP is incorporated into protein phosphomonoester linkages. 相似文献
17.
《Insect Biochemistry》1987,17(3):401-415
Phosphorylation of vitellogenin (yolk protein precursor) and vitellin (major yolk protein) polypeptides of Leucophaea maderae was studied by [32P]ortho phosphate labeling and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) autoradiography. The vitellogenin molecule was isolated from the hemolymph and fat body by antibody precipitation and high-performance liquid chromatography (HPLC), and shown to consist of at least five polypeptides (“subunits”) which had apparent molecular masses of 155, 112, 95, 92 and 54 kD. Labeling studies with 32P showed that the covalently attached phosphorus was distributed in an uneven fashion among the five polypeptides. The two heavily-phosphorylated polypeptides, 112 and 54 kD, corresponded to the large and small, mature vitellin subunits. Quantitative SDS-PAGE analysis of long-term 32P-labeled vitellin showed that these large and small “subunits” contained 55 and 30%, respectively, of the total radioactivity.When fat body was pulse-labeled with 32P we found a heavily-phosphorylated intracellular 215 kD polypeptide which was precipitable with anti-vitellogenin. The synthesis of this intracellular precursorform of vitellogenin (pre-Vg) was under absolute juvenile hormone control. In vitro32P pulse-chase experiments showed that pre-Vg was proteolytically processed within the fat body into some (or possibly all) of the mature vitellogenin subnits. Furthermore, peptide mapping confirmed that all of the phosphorylated vitellogenin subunits were derived from pre-Vg. Since previous studies have shown that phosphoserine residues account for essentially all of the covalently-attached phosphorus of the native vitellogenin molecule, we speculate that the asymmetric pattern of vitellogenin and vitellin subunit-phosphorylation is due to an uneven distribution of phosphoserine residues along the initial pre-Vg polypeptide chain. Finally, we conclude that phosphorylation of vitellogenin occurred post-translationally in the fat body endoplasmic reticulum because we could identify 32P-labeled pre-Vg in purified microsomal vesicles but not in nascent vitellogenin polypeptide chains attached to vitellogenin polyribosomes. 相似文献
18.
Jean A. Boutin Anne-Pascale Ernould Gilles Ferry Annie Genton 《Journal of chromatography. B, Analytical technologies in the biomedical and life sciences》1992,583(2)
A new HPLC method has been developed to assay tyrosine protein kinase activity. Using hydrophilic interaction chromatography, it is possible to resolve the four components of the incubation medium: substrate peptide, [32P]phosphorylated peptide, unreacted [γ-32P]ATP, and 32P-labelled inorganic phosphate. ATP interacts so strongly with the stationary phase material that it can be removed selectively from the incubation medium with solid-phase extraction cartridges packed with the same type of material. The three remaining components of interest can then be resolved by reversed-phase or hydrophilic interaction HPLC. This procedure permits the evaluation of almost every type of peptide as a substrate of tyrosine protein kinase. 相似文献
19.
A satisfactory method for the determination of the specific activity of highly labeled [γ-32P]ATP has not been reported previously. Yields of high specific activity 32P labeled material usually are too small to be detected by ultraviolet spectrophotometry or phosphate analysis. Recent reports describing the assay of ATP by enzyme catalyzed phosphate transfer to 3H labeled glucose (1) or galactose (2) are not suitable for use with highly labeled 32P material since the crossover into the 3H channel will greatly exceed the radioactivity of the 3H labeled phosphate acceptor. Recently Schendel and Wells reported the preparation of essentially carrier free [γ-32P]ATP. They indicated, however, that the specific activity of the labeled product could not be determined by conventional methods (3). We have developed and now routinely use an expedient method for the determination of the specific activity of picomole quantities of highly labeled [γ-32P]ATP. This procedure measures the phosphate transfer from [γ-32P]ATP to oligothymidylic acid [dT(pT)10] catalyzed by bacteriophage T4 induced polynucleotide kinase. The specific activity is determined by measuring the radioactivity present in d-32pT(pT)10, and can be verified by an isotope dilution method employing the same assay. Specific activities as high as 240 Ci/mmole have been determined. 相似文献
20.
Mechanism of action of clostridial glycine reductase: isolation and characterization of a covalent acetyl enzyme intermediate 总被引:1,自引:0,他引:1
Clostridial glycine reductase consists of proteins A, B, and C and catalyzes the reaction glycine + Pi + 2e(-)----acetyl phosphate + NH4+. Evidence was previously obtained that is consistent with the involvement of an acyl enzyme intermediate in this reaction. We now demonstrate that protein C catalyzes exchange of [32P]Pi into acetyl phosphate, providing additional support for an acetyl enzyme intermediate on protein C. Furthermore, we have isolated acetyl protein C and shown that it is qualitatively catalytically competent. Acetyl protein C can be obtained through the forward reaction from protein C and Se-(carboxymethyl)selenocysteine-protein A, which is generated by the reaction of glycine with proteins A and B [Arkowitz, R. A., & Abeles, R. H. (1990) J. Am. Chem. Soc. 112, 870-872]. Acetyl protein C can also be generated through the reverse reaction by the addition of acetyl phosphate to protein C. Both procedures lead to the same acetyl enzyme. The acetyl enzyme reacts with Pi to give acetyl phosphate. When [14C]acetyl protein C is denaturated with TCA and redissolved with urea, radioactivity remained associated with the protein. At pH 11.5 radioactivity was released with t1/2 = 57 min, comparable to the hydrolysis rate of thioesters. Exposure of 4 N neutralized NH2OH resulted in the complete release of radioactivity. Treatment with KBH4 removes all the radioactivity associated with protein C, resulting in the formation of [14C]ethanol. We conclude that a thiol group on protein C is acetylated. Proteins A and C together catalyze the exchange of tritium atoms from [3H]H2O into acetyl phosphate.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献