Studies on the metabolism of ATP by isolated bacterial membranes: solubilization and phosphorylation of a protein component of the diglyceride kinase system

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.     

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.     

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.     

Determination of phosphorylase kinase activity in crude homogenates by affinity chromatography on 5′-AMP Sepharose

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.     

Regulation of hepatic acetyl coenzyme A carboxylase by phosphorylation and dephosphorylation

Acetyl CoA carboxylase, in a partially purified preparation, was inactivated by ATP in a time- and temperature-dependent reaction. Adenosine 3′,5′-monophosphate did not affect the inactivation. Further purification separated the carboxylase from a protein fraction which could greatly enhance the inactivation of the enzyme.Inactivation of the enzyme with [γ-³²P]ATP resulted in the incorporation of ³²P which copurified with the enzyme. No label was incorporated when [U-¹⁴C]ATP was used. When carboxylase inactivated by exposure to [γ-³²P]ATP was precipitated with antibody, isotope incorporation into the precipitate paralleled enzyme inactivation. The phosphate was bound to serine and threonine residues by an ester linkage.Sodium fluoride completely inhibited the activation of partially purified enzyme by magnesium ions. Activation by magnesium, accompanied by the release of protein-bound ³²P, was antagonistic to inactivation of the enzyme by ATP.The data presented in this communication are consistent with a mechanism for controlling acetyl CoA carboxylase activity by interconversion between phosphorylated and dephosphorylated forms. Phosphorylation of the enzyme by a portein kinase decreases enzyme activity, whereas dephosphorylation by a protein phosphatase reactivates the enzyme.     

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.     

A rapid assay for ribokinase.

A rapid method capable of detecting low levels of ribokinase is given. [γ-³²P]ATP is converted to ribose 5-[³²P]phosphate which is not absorbable onto charcoal. The assay is linear in enzyme concentration to a lower limit of at least 4 × 10^?2 mg of enzyme/ml.     

A rapid method for the measurement of [γ-32P]ATP specific radioactivity in tissue extracts and its application to the study of 32Pi uptake in perfused rat heart

(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) ³²P_i uptake in perfused rat heart was found to be a relatively slow process, with a K_m of 0.084 mm, whereas equilibration between intracellular ³²P_i and [γ-³²P]ATP occurred rapidly.     

Formation of palmityl-[13'-32P]coenzyme A from [gamma-32P]ATP in mitochondrial extracts of guinea pig liver.

Investigations of the incorporation of ³²P into acyl-coenzyme A (CoA) in incubation mixtures containing a soluble protein preparation derived from mitochondria, [γ-³²P]ATP, and palmityl-CoA have led to the discovery of an enzymatic activity which catalyzes the exchange of palmityl groups between molecules of CoA: CoA¹ + palmityl-CoA ? palmityl-CoA¹ + CoA. The preparation also contains dephospho-CoA kinase and palmityl-CoA thiolester hydrolase activities. The initial detection of the exchange reaction resulted from the formation of [3′-³²P]CoA via the dephospho-CoA kinase reaction with exogenous [γ-³²P]ATP. The described preparation of palmityl-[3′-³²P]CoA and palmityl-[³⁵S]CoA facilitated demonstration of the reversibility of the reaction and ruled out the possibility that the exchange of fragments of the CoA molecule mediated the observed incorporation. The reversible palmityl group exchange does not appear to be catalyzed by a previously described enzyme. None of the possible acyl group acceptors considered in these studies participated in the reaction as efficiently as CoA itself. The possibility is discussed that the exchange reaction may explain reports of an unknown lipid formed by an oligomycin-sensitive mitochondrial ATPase preparation.     

An assay for pyrimidine deoxyribonucleoside kinase using gamma-32P-labeled ATP.

A rapid, simple and sensitive assay for pyrimidine deoxyribonucleoside kinase is described. After phosphorylation of unlabeled nucleoside substrate through the transfer of the γ-phosphate of [γ-³²P]ATP, the reaction mixture is subjected to 1 n HCl at 100°C. The β- and γ-phosphates of unreacted ATP are hydrolyzed to inorganic phosphate while the 5′-phosphate of the pyrimidine deoxyribonucleotide product is not hydrolyzed. Radioactive phosphate remaining in the supernatant fluid after precipitation of inorganic phosphate corresponds to product and is measured by liquid scintillation spectrometry. Evidence is presented suggesting that pyrimidine and purine ribonucleoside kinase activity can also be determined by this assay.     

A simple modification to an enzymic method for the preparation of[γ-32P]ATP free of salt and buffer

An existing enzymic method for preparing [γ-³²P]ATP from 32P_i 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 ³²P_i 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.     

Nuclear protein phosphorylation in isolated nuclei from HeLa cells. Evidence that 32P incorporation from [γ-32P]GTP is catalyzed by nuclear kinase II

A nuclear system for studying nuclear protein phosphorylation is characterized, using as phosphate donor either low levels of [γ-³²P]GTP, low levels of [γ-³²P]ATP, or low levels of labeled ATP plus excess unlabeled GTP. Since nuclear casein kinase II is the only described nuclear protein kinase to use GTP with high affinity, low levels of GTP should specifically assay this enzyme. ATP should measure all kinases, and ATP plus unlabeled GTP should measure all kinases except nuclear casein kinase II (ATP-specific kinases). The results are consistent with these predictions. In contrast with the ATP-specific activity, endogenous phosphorylation with GTP was enhanced by 100 mM NaCl, inhibited by heparin and quercetin, stimulated by polyamines, and did not use exogenous histone as substrate. The GTP- and ATP-specific kinases phosphorylated different subsets of about 20 endogenous polypeptides each. Addition of purified casein kinase II enhanced the GTP-supported phosphorylation of the identical proteins that were phosphorylated by endogenous kinase. These results support the hypothesis that activity measured with GTP is catalyzed by nuclear casein kinase II, though other minor kinases which can use GTP are not ruled out. Preliminary observations with this system suggest that the major nuclear kinases exist in an inhibited state in nuclei, and that the effects of polyamines on nuclear casein kinase II activity are substrate specific. This nuclear system is used to determine if the C-proteins of hnRNP particles, previously shown to be substrates for nuclear casein kinase II in isolated particles, is phosphorylated by GTP in intact nuclei. The results demonstrate that the C-proteins are effectively phosphorylated by GTP, but in addition they are phosphorylated by ATP-specific kinase activity.     

Use of photoaffinity nucleotide analogs to determine the mechanism of ATP regulation of a membrane-bound,cAMP-activated protein kinase

Using a radioactively tagged, photoaffinity analog of cAMP, 8-azidoadenosine-3′,5′-cyclic monophosphate (8-N₃ cAMP), and [γ³²P] ATP, the membranebinding properties of both the regulatory and catalytic subunits of the cAMP-activated protein kinase of human erythrocyte membranes were investigated. [³²P] 8-N₃ cAMP was used to locate and quantify regulatory subunits. Increased phosphorylation of specific membrane proteins by [γ³²P] ATP was used to determine the presence of the catalytic subunit. The data support a mechanism which operates through a tight membrane-bound regulatory subunit and a catalytic subunit that is released from the membrane when cAMP is present and the Mg · ATP concentration is below approximately 10 μM. The catalytic subunit is not required for the Mg · ATP inhibition of 8-N₃ cAMP binding. Experiments with a photoaffinity analog of ATP, 8-azidoadenosine triphosphate (8-N₃ATP), support the hypothesis that ATP hydrolysis and phosphorylation are not involved in the regulation. The data indicate that the regulatory subunit contains an ATP regulatory site which inhibits 8-N₃ cAMP binding and the release of the catalytic subunit. These results indicate that the membrane-bound type I enzyme (type IM) differs significantly from the soluble (type IS) enzyme studied in other tissues. These enzymes are compartmentalized by being in different cellular locations and are regulated differently by Mg · ATP.     

Synthesis and Release of Cyclic Adenosine 3':5'-Monophosphate by Ochromonas malhamensis

The chrysophycean alga, Ochromonas malhamensis Pringsheim, was shown to synthesize cyclic adenosine 3′:5′-monophosphate (cAMP) and to release it into the culture medium. Cells contained 3 to 3,000 picomoles per gram fresh weight; medium contained up to 20 times the amount in the cells. Putative [³²P]cAMP was purified from cultures supplied [³²P]phosphate. The compound was identified as [³²P]cAMP by co-chromatography with authentic cAMP through 10 serial steps; by chemical deamination at the same rate as authentic cAMP, to a ³²P compound with the chromatographic behavior of cIMP; and by its conversion through the action of cyclic nucleotide phosphodiesterase to a ³²P compound with the chromatographic behavior of 5′-AMP. A two-step procedure involving chromatography on alumina and on Dowex 50 purified the unlabeled compound from cells or medium sufficiently for it to be assayable by competitive inhibition of binding of [³H]cAMP to cAMP-binding protein (Gilman assay) or by stimulation of cAMP-dependent protein kinase. The activity was destroyed by cyclic nucleotide phosphodiesterase with the same kinetics as authentic cAMP, provided that an endogenous inhibitor of the phosphodiesterase was first removed by an additional purification step.     

Erratum

RNA polynucleotide kinase has been shown to transfer [γ³²P] from ATP to 5-OH termini of endogenous nuclear RNA. The products of this reaction have been isolated in RNA larger than 125 after in vitro incubation of mouse L cell nuclei. About 20%–30% of these 5′-OH kinase products are polyadenylated. A sizeable fraction of the [γ³²P] label from ATP is also found in internal phosphodiester bonds after 30-minute nuclear incubation in vitro. The possibility of substantial [³²P] recycling via the α position of nucleoside triphosphate was ruled out because: (1) 2mM nucleoside triphosphates in the incubation medium, (2) limited nearestneighbor distribution 3′ and 5′ to the phosphodiester bond compared with that from [α³²P] UTP, (3) different nearest-neighbor distribution for RNA molecules > 12S and 12-3S, (4) relative insensitivity of the [γ³²P] incorporation to α-amanitin as compared with total RNA synthesis, (5) internal [³²P] appearance in RNA > 12S in less than five minutes of incubation, and (6) < 0.03% to 0.6% of the total [³²P] in the α position of nucleoside triphosphates after 30 minutes of incubation. The [γ³²P] incorporation was dependent on high ATP concentration and was insensitive to competition by inorganic phosphate. These results are consistent with the levels of 5′ RNA polynucleotide kinase activity in L cell nuclei and suggest the presence of an RNA ligase that can utilize the termini generated by the 5′-OH RNA kinase in a ligation reaction.     

Calcium-calmodulin dependent phosphorylation of erythrocyte pyruvate kinase

In the red cell incubated with ortho-[32P] phosphate, CaCl₂ and calcium ionophore A 23187, phosphorylation of erythrocyte pyruvate kinase was demonstrated using the double antibody technique and autoradiography. Phosphorylation was inhibited by calmodulin inhibitors, trifluoperazine or ZnCl₂. In the presence of purified erythrocyte calmodulin, CaCl₂ and [γ-³²P] ATP, the partially purified erythrocyte pyruvate kinase containing cytozol protein kinases was phosphorylated. This was also inhibited by trifluoperazine or ZnCl₂. From these results, it was concluded that erythrocyte pyruvate kinase is phosphorylated by a calcium-calmodulin dependent process.     

Use of hydrophilic interaction chromatography for the study of tyrosine protein kinase specificity

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.     

Possible involvement of adenylylation in the modification of a 26 kDa protein in rat parotid acinar cells

• 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.

     

A simple procedure for the synthesis of [32P]phosphoenolpyruvate via the pyruvate kinase exchange reaction at equilibrium

A one step procedure is presented for the preparation of [³²P]phosphoenolpyruvate from [γ-³²P]ATP using pyruvate kinase. The reaction is carried out at chemical equilibrium and involves only an exchange of isotope between ATP and phosphoenolpyruvate. The initial phosphoenolpyruvate/ATP ratio in the reaction mixture determines the degree of ³²P incorporation into phosphoenolpyruvate when isotopic equilibrium is achieved.     

Transient phosphorylation by ATP of a 160 000 dalton protein in rod outer segments of Bufo marinus

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).