An easy and rapid method for the measurement of [γ-32P]ATP specific radioactivity in tissue extracts obtained from in vitro rat heart preparations labeled with 32Pi

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 ³²P_i is demonstrated.     

An easy and rapid method for the measurement of [γ-P]ATP specific radioactivity in tissue extracts obtained from in vitro rat heart preparations labeled with Pi

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 ³²P_i is demonstrated.     

A novel method for the synthesis of nucleoside triphosphates labelled with inorganic [32P]phosphate specifically in the beta-position.

A method was developed for the introduction of [32p]Pi specifically into the beta-position of ATP and GTP. The method is based on two separate reactions involving (a) phosphorolysis of poly(A) or poly(G) [Soreq, Nudel, Salomon, Revel & Littauer (1974) J. Mol Biol. 88, 233-245] in the presence of [32P]Pi and (b) conversion of the labelled diphosphate into the corresponding triphosphate by transferring the active phosphate group from 1,3-diphosphoglycerate in a coupled reaction as decribed by Glynn & Chappell [(1964) Biochem. J. 90, 147-149]. Radioactivity in the beta- and gamma-phosphate groups of the labelled triphosphate was measured by using polynucleotide kinase. No detectable radioactivity was found in the gamma-phosphate group. The suitability of this method for the synthesis of other nucleoside triphosphates specifically labelled in the beta-position is discussed.     

Rates of production and consumption of phosphatidic acid upon thrombin stimulation of human platelets

Human platelets were labelled with [32P]Pi and [3H]glycerol before gel filtration. In unstimulated cells, the specific 32P radioactivity in phosphatidic acid (PtdOH) was similar to that of phosphatidylinositol (PtdIns) but only 4% of that of the gamma-phosphate of ATP. Upon 3 min of stimulation with 0.5 U/ml of thrombin, there was a 20-fold increase in specific 32P radioactivity of PtdOH which approached that of the ATP gamma-phosphate. Based on constant rates of synthesis and removal, this thrombin-induced increase in specific 32P radioactivity in PtdOH allowed us to calculate the flux of phosphate through PtdOH upon stimulation. Synthesis and removal occurred at rates of 107 and 52 nmol min-1/10(11) cells, respectively. The specific [3H]glycerol radioactivity was similar in PtdIns, phosphatidylinositol 4-phosphate and phosphatidylinositol 4,5-bisphosphate in unstimulated platelets. In PtdOH, it was 50% of that of the inositol phospholipids. Thrombin stimulation induced no changes in the specific 3H radioactivity of the inositol phospholipids whereas specific [3H]PtdOH increased to the level of these lipids. It is concluded that PtdIns, PtdInsP and PtdInsP2 exist in a metabolic homogenous pool in human platelets.     

Turnover of the phosphomonoester groups of polyphosphoinositol lipids in unstimulated human platelets

The metabolic activity of the polyphosphoinositol lipids in unstimulated human platelets was studied by short-term labelling with [32P]Pi, by replacement of [32P]Pi from pre-labelled platelets with unlabelled phosphate, and by depriving the cells of metabolic ATP. Under short-term labelling conditions, the 4- and 5-phosphate groups of phosphatidylinositol 4-phosphate (PtdIns4P) and phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] had the same specific 32P radioactivity as the gamma-phosphate of metabolic ATP. The specific 32P radioactivity of the 1-phosphates of phosphatidylinositol, PtdIns4P and PtdIns(4,5)P2 was similar, but only 4-13% compared to that of the ATP-gamma-phosphate. When [32P]Pi pre-labelled platelets were incubated with up to 25 mM of unlabelled phosphate, the displacement of the 32P label from PtdIns4P, PtdIns(4,5)P2 and metabolic ATP followed similar kinetics. Inhibition of ATP regeneration in platelets pre-labelled with [32P]Pi resulted in a rapid fall in metabolic ATP with a much slower fall in [32P]PtdIns(4,5)P2, whereas [32P]PtdIns4P increased initially. However, ATP turnover was not abolished, as indicated by the marked (25% of the control) incorporation of extracellular [32P]Pi into PtdIns4P and PtdIns(4,5)P2 in metabolically inhibited platelets. This low phosphate turnover may explain the relative resistance of PtdIns4P and PtdIns(4,5)P2 to metabolic inhibition. We conclude that PtdIns4P and PtdIns(4,5)P2 are present as a single metabolic pool in human platelets. Turnover of the 4- and 5-phosphates of PtdIns4P and PtdIns(4,5)P2 in unstimulated platelets is as rapid as that of the gamma-phosphate of metabolic ATP, and accounts for about 7% of basal ATP consumption.     

Exchange of the actin-bound nucleotide in intact arterial smooth muscle

The actin-bound ADP was separated from cytoplasmic nucleotides by treatment of intact arterial smooth muscle with 50% ethanol. In (32)P-labeled smooth muscle the actin-bound ADP and phosphate readily exchanged with the cytoplasmic [gamma,beta-(32)P]ATP; the specific radioactivity of actin-bound ADP was equal to that of the beta-phosphate of cytoplasmic ATP and the specific radioactivity of actin-bound phosphate was equal to that of the gamma-phosphate of cytoplasmic ATP. In contrast, the exchange of the actin-bound ADP in skeletal muscle was very slow. The presence of cytoplasmic ATP was required for the exchange of the actin-bound ADP and phosphate; if ATP synthesis was inhibited the exchange was also inhibited. The extent of exchange was reduced in muscles contracted by histamine or K(+), as compared with resting muscles. The exchange was also shown in other mammalian smooth muscles, uterus, urinary bladder, and stomach. The data indicate a dynamic state of actin in smooth muscle. The data also suggest that polymerization-depolymerization of actin is part of the contraction-relaxation cycle of smooth muscle.     

Elevation of cAMP in cultured mesangial cells diminishes vasopressin-stimulated increases of phosphate uptake and 32P-specific activity in ATP but has no effect on phosphoinositide metabolism

Agents known to elevate intracellular cyclic AMP (cAMP) in cultured mesangial cells (e.g., isoproterenol with and without isobutylmethylxanthine (MIX] inhibit vasopressin-induced contraction. Since contraction of these cells in response to vasopressin is accompanied by release of inositol trisphosphate and increased intracellular ionized calcium, we wanted to determine whether cAMP is exerting its relaxing effect by altering phosphoinositide metabolism. Isoproterenol and MIX did not diminish the release of inositol trisphosphate in response to vasopressin. However, the stimulated 32P incorporation into phospholipids seen with vasopressin treatment was diminished by prior treatment with isoproterenol-MIX. Since incorporation of 32P into phospholipids is not only dependent on phospholipid synthesis but also on the amount of label in the gamma-phosphate of ATP, we determined the specific activity of 32P in ATP. We found that suppression of 32P incorporation into phospholipids in cells treated with isoproterenol-MIX was paralleled by a decline of specific activity of 32P in ATP. Furthermore, the changes in ATP specific activity were paralleled by similar changes in phosphate uptake into the cells. Thus, diminished phosphate uptake (transport) could account for the decline of 32P content in phospholipids and ATP following treatment of mesangial cells with isoproterenol-MIX.     

Alterations in nicotinamide and adenine nucleotide systems during mixed-function oxidation of p-nitroanisole in perfused livers from normal and phenobarbital-treated rats

When rat liver nuclei were incubated with [adenine-3H]NAD, besides histone 1, histone 2A and especially histone 2B accepted 3H radioactivity. 3H radioactivity was also found on the non-histone proteins and on the small amounts of histones 1 and 3 released into the supernatant during incubation. [14C]Adenine uptake in vivo by liver and thymus nuclei showed radioactivity in histones 1 and 3. After digestion with Pronase and leucine aminopeptidase 14C- or 32P-labelled histone 3 released a serine phosphate-containing nucleotide, which on acid hydrolysis yielded ADP-ribose and serine phosphate. Serine phosphate was also found in the material from the nucleotide peaks from histones 2A and 2B. ADP-ribosylated histones 1 and 3 were more easily released from nuclei than their unmodified forms and showed higher [32P]Pi and [3H]lysine uptakes in vivo [Ord & Stocken (1975) FEBS Meet. Proc. 34, 113-125].     

Changes in histone phosphorylation and associated early metabolic events in pig lymphocyte cultures transformed by phytohaemagglutinin or 6-N,2′-O-dibutyryladenosine 3′:5′-cyclic monophosphate

1. Pig lymphocytes were transformed by dibutyryl cyclic AMP (6-N,2'-O-dibutyryladenosine 3':5'-cyclic monophosphate) at concentrations of 0.01-0.1mum. The pattern of incorporation of label from [5-(3)H]uridine and [6-(3)H]thymidine into RNA and DNA respectively was identical with that obtained with unpurified phytohaemagglutinin. 2. Chlorpromazine (0.1mum) prevented the stimulation of [5-(3)H]uridine incorporation into RNA by phytohaemagglutinin, but only slightly lowered the lymphocyte response to dibutyryl cyclic AMP. 3. An increase in the size and specific radioactivity of the intracellular P(i) pool was found immediately after stimulation by both phytohaemagglutinin and dibutyryl cyclic AMP. This was followed after some 30min by a rise in the specific radioactivity and concentration of ATP. 4. There was an immediate increase in the specific radioactivity of phosphate groups of histones; by about 45min after stimulation only the histones remaining after extraction of histone fraction F1 continued to incorporate (32)P from [(32)P]P(i). 5. Histone kinase activity increased in the first 30min after stimulation; subsequently histone F1 kinase activity decreased, but activity with the other histones as substrate continued to increase for a further 30min. Kinase activation was effected by cyclic AMP (adenosine 3':5'-cyclic monophosphate). 6. Histone phosphatase activity behaved similarly to that of the kinase.     

A phosphatase resistant substrate for the assay of protein kinase C in crude tissue extracts.

Protein kinase C (PKC) is routinely assayed, after it is partially purified over DEAE-cellulose chromatography to eliminate any interfering protein kinases and phosphatases, by measuring the transfer of gamma-phosphate of [gamma-32P]ATP to H1 histone. Recently, it has been shown that a synthetic peptide, comprising residues 4-14 of myelin basic protein (MBP4-14), is a very selective PKC substrate which is not phosphorylated effectively by cyclic AMP-dependent protein kinase, casein kinase I and II, Ca2+/calmodulin dependent protein kinase II or phosphorylase kinase [Yasuda, I., Kishimoto, A., Tanaka, S-I., Tominaga, M., Sakurai, A. and Nishizuka, Y. (1990) BBRC 166, 1220-1227]. We report here that once MBP4-14 is phosphorylated, it is not dephosphorylated by okadaic acid-sensitive phosphatases (protein phosphatases 1, 2A and 3) or other protein phosphatases such as calcineurin and/or PP 2C present in hippocampal homogenates. Therefore, MBP4-14 can be used for PKC assay in crude extracts of neural tissue.     

The reactive serine residue in phosphoglucomutase of Micrococcus lysodeikticus (Short Communication)

1. Phosphoglucomutase of Micrococcus lysodeikticus was labelled at the active site by exchange with (32)P-labelled substrates of high specific radioactivity. 2. Partial acid hydrolysis gave rise to radioactive peptides; serine phosphate was identified as one of the derivatives. 3. Comparison of the other (32)P-labelled peptides with the peptides obtained from the (32)P-labelled rabbit muscle phosphoglucomutase indicates that the sequence around the reactive serine residue is identical in both enzymes.     

Kinetics of Phosphate Incorporation into Adenosine Triphosphate and Guanosine Triphosphate in Bacteria

Escherichia coli cells were labeled with (33)PO(4) for several generations and then (32)PO(4) was added, samples were taken at various times, and adenosine triphosphate (ATP) and guanosine triphosphate (GTP) were purified from an acid extract. The ratio of (32)P/(33)P in each of the six phosphate species was determined and compared to the ratio expected at isotopic steady state. The rate constants of the simplest network of pools which could explain the data, and were consistent with metabolic pathways, were then calculated. In the range of times studied (30 to 3,000 s at 21.5 C) the following generalizations can be made. (i) The gamma-phosphate at ATP requires 2,200 s to achieve 50% of its final specific activity and behaves as if it were exchanging with another species 4.4 times as large, with this other species turning over in 540 s. (ii) The beta-phosphate of ATP and the gamma-phosphate of GTP are in equilibrium with the gamma-phosphate of ATP. (iii) The specific activity of the gamma-phosphate of GTP lags 66 s behind the gamma-phosphate of ATP. (iv) The alpha-phosphates of ATP and GTP have equal specific activities which lag far behind the other four phosphates. A single precursor pool with a turnover time of 416s fits the data best, although a fair fit can be obtained assuming several sequential precursor pools with much shorter turnover times. These findings are consistent with known biosynthetic pathways and the probable flows through them, including the relationship of nucleotide biosynthesis to ribonucleic acid synthesis and turnover.     

Activation and phosphorylation of the ''dense-vesicle'' high-affinity cyclic AMP phosphodiesterase by cyclic AMP-dependent protein kinase.

Incubation of a hepatocyte particulate fraction with ATP and the isolated catalytic unit of cyclic AMP-dependent protein kinase (A-kinase) selectively activated the high-affinity 'dense-vesicle' cycle AMP phosphodiesterase. Such activation only occurred if the membranes had been pre-treated with Mg2+. Mg2+ pre-treatment appeared to function by stimulating endogenous phosphatases and did not affect phosphodiesterase activity. Using the antiserum DV4, which specifically immunoprecipitated the 51 and 57 kDa components of the 'dense-vesicle' phosphodiesterase from a detergent-solubilized membrane extract, we isolated a 32P-labelled phosphoprotein from 32P-labelled hepatocytes. MgCl2 treatment of such labelled membranes removed 32P from the immunoprecipitated protein. Incubation of the Mg2+-pre-treated membranes with [32P]ATP and A-kinase led to the time-dependent incorporation of label into the 'dense-vesicle' phosphodiesterase, as detected by specific immunoprecipitation with the antiserum DV4. The time-dependences of phosphodiesterase activation and incorporation of label were similar. It is suggested (i) that phosphorylation of the 'dense-vesicle' phosphodiesterase by A-kinase leads to its activation, and that such a process accounts for the ability of glucagon and other hormones, which increase intracellular cyclic AMP concentrations, to activate this enzyme, and (ii) that an as yet unidentified kinase can phosphorylate this enzyme without causing any significant change in enzyme activity but which prevents activation and phosphorylation of the phosphodiesterase by A-kinase.     

Rapid protein kinase assay using phosphocellulose-paper absorption.

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.     

Convenient methods to determine the specific radioactivity of (gamma-32P)ATP of high specific activity.

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.     

A novel method for measurement of the specific radioactivity of gamma-32P ATP.

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 ${}^{32}\text{P}{}^3\text{H}$ 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.     

Assay for adenylate cyclase and cyclic nucleotide phosphodiesterases and the preparation of high specific activity 32-P-labeled substrates.

Simple one step assay methods for adenylate cyclase (ATP pyrophosphate-lyase (cyclizing) EC 4.6.1.1) and cyclic nucleotide phosphodiesterases (3',5'-cyclic nucleotide 5'-nucleotidohydrolase EC 3.1.4.17) have been developed. [alpha-32-P] ATP is used as the substrate for adenylate cyclase. Acid-heat destruction of [32-P] ATP remaining after the cyclase reaction followed by Zn-Ba treatment quantitatively leaves cyclic [32-P] AMP in the supernatant essentially free from other 32-P-containing compounds. This assay method requires no corrections for recovery and routinely yields blank values less than 0.03 per cent. If higher sensitivity is desired, a simple 5 min alumina column step can be introduced into the procedure which quantitatively elutes cyclic [32-P] AMP directly into a liquid scintillation vial and lowers the blank values to less than 0.002 per cent. This method is rapid and easily performed, without sacrificing high reliability, specificity, or sensitivity. One step phosphodiesterase assays are easily accomplished using 32-P-labeled cyclic nucleotides as substrates. Descending paper chromatography of the reaction mixture on individual 2 cm wide paper strips gives a complete and quantitative separation of all possible products including [5'-32-P] AMP and [5'-32-P] GMP from their respective 32-P-labeled 3',5'-cyclic nucleotides in 1-2 h. The paper strips are cut, inserted in scintillation vials without scintillant and the 32-P-products determined by Cerenkov counting. Low blank values of less than 0.5 per cent and the use of high specific activity 32-P-labeled cyclic nucleotide substrates make this method the most reliable and most sensitive phosphodiesterase assay described to date. Because of the simplicity, specificity, and high sensitivity obtainable with these assay methods using 32-P-labeled substrates, we have also devised simple conditions for the preparation and purification of [alpha-32-P] ATP, cyclic [32-P] AMP and cyclic [32-P] GMP with specific activities in excess of 100 Ci/mmol. These high specific activity 32-Plabeled cyclic nucleotides are important for these new assay methods and are also useful to follow purification recovery of endogenous cyclic AMP and cyclic GMP from biological materials before protein binding or radioimmunological isotope displacement assays when performed in the femtomole range.     

An insulin-sensitive cytosolic protein kinase accounts for the regulation of ATP citrate-lyase phosphorylation.

Purified rat liver ATP citrate-lyase is phosphorylated on serine residues by an insulin-stimulated cytosolic kinase activity partially purified from rat adipocytes [Yu, Khalaf & Czech (1987) J. Biol. Chem. 262, 16677-16685]. The Km for lyase phosphorylation by this hormone-sensitive kinase activity is approx. 3 microM. Two-dimensional tryptic-peptide mapping of the 32P-labelled lyase reveals that the kinase-catalysed phosphorylation occurs primarily on a specific peptide. In intact 32P-labelled adipocytes, insulin enhances the serine phosphorylation of ATP citrate-lyase by 2-3-fold. Tryptic digestion of the 32P-labelled lyase immunopurified from insulin-treated adipocytes also yields one major phosphopeptide. 32P-labelled lyase tryptic peptides derived from labelling experiments in vitro and in vivo exhibit identical electrophoretic and chromatographic migration profiles. Furthermore, radio-sequencing of the phosphopeptide from lyase 32P-labelled in vitro indicates that serine-3 from the N-terminus is phosphorylated by the insulin-stimulated cytosolic kinase, in agreement with previous studies on the position of the phosphoserine residue in ATP citrate-lyase isolated from insulin-treated cells. Taken together, the similarity in site-specific phosphorylation of ATP citrate-lyase from insulin-treated adipocytes to that catalysed by the hormone-activated cytosolic kinase in vitro strongly suggests that this kinase mediates insulin action on lyase phosphorylation in intact cells.     

Endogenous phosphorylation of sarcoplasmic reticulum fragments of rabbit fast skeletal muscles

The purified membrane fragments of sarcoplasmic reticulum (SR) of rabbit fast skeletal muscles were found to incorporate 32P from[gamma-32P]ATP in endogenous membrane substrates and in histone H1. The existence of membrane-bound protein kinase of SR was demonstrated by steady state binding of [3H]-cAMP to the SR membranes. The constant of [3H]cAMP binding to the membranes is 2.5 +/- 0.003 x 10(6) M-1, the number of binding sites is 6.1 +/- 0.8 pmol per 1 mg of protein. The endogenous phosphorylation of SR components was inhibited by cAMP and cGMP at concentrations of 10(-7)-10(-6) and depended on Mg2+ and Ca2+. The thermostable protein inhibitor of cAMP-dependent protein kinase inhibited the endogenous phosphorylation of SR membranes by 30-40%. The protein phosphoproduct of SR membranes revealed the properties of a phosphoester. The membrane-bound protein kinase was active towards the exogenous substrate--histone H1. Phosphorylation in the presence of histones was independent of cyclic nucleotides, Mg2+ and Ca2+. Fractionation of 32P-labelled solubilized membranes in polyacrylamide gel in the presence of Na-SDS showed that the radioactivity is bound to protein zones with molecular weights of 95 000 and 6000.