Reciprocal periodicity in cyclic AMP binding and phosphorylation of differentiating Dictyostelium discoideum cells.

The binding of [³H]cyclic AMP to cell surface receptors of differentiated D. discoideum cells at 25° is an oscillatory process with a periodicity of 2 min. This alternating change in the cells' binding capacity for cyclic AMP may be the basis for the refractory period to cyclic AMP stimulation, an essential feature of the chemotactic system. The incorporation of [³²P] by whole cells from [γ³²P]ATP is also oscillatory with a periodicity identical to that of [³H]cyclic AMP binding. However, the two processes are inversely related in time such that periods of maximal cyclic AMP binding correspond to periods of minimal cellular phosphorylation. These results suggest a receptor kinase/phosphatase mediated desensitization of the cyclic AMP receptor.     

Uptake and metabolism of 3′,5′-cyclic adenosine monophosphate and N,O′-dibutyryl 3′,5′-cyclic adenosine monophosphate in isolated bovine thyroid cells

1.

1. Accumulation of intracellular radioactivity was measured during incubation of isolated bovine thyroid cells with cyclic [³²P]AMP, cyclic [8-³H]AMP and dibutyryl cyclic [8-³H]AMP. With cyclic [³²P]AMP, ³²P cell/medium ratios ranged from 0 to to 0.04 compared to a maximum ³H cell/medium ratio of 0.29 with cyclic [³H]AMP and 0.16 with dibutyryl cyclic [³H]AMP. The excess of intracellular cyclic [³H] over cyclic [³²P]AMP radioactivity was due to extracellular formation of more penetrable dephosphorylated cyclic AMP metabolites which probably served as precursor of intra-cellular cyclic AMP.     

Identification and characterization of the adenosine 3′,5′-cyclic monophosphate binding proteins appearing during the development of Dictyostelium discoideum

A photosensitive, radioactive analogue of cyclic adenosine monophosphate, 8-azido-adenosine 3′,5′-[³²P]monophosphate (8-N₃-cyclic AMP), was used to label the cyclic AMP binding proteins of Dictyostelium discoideum. During development cytosolic proteins appear which are specifically labeled by the photoaffinity agent. The proteins are developmentally regulated since they are only found in starved, developing cells. Unlabeled cyclic AMP competes specifically with the labeled analogue for protein binding sites in contrast to unlabeled 5′-AMP which does not compete. A mutant which develops spores but is deficient in stalk cell production produces a different set of cyclic AMP binding proteins from the parent strain.     

Cyclic adenosine 3',5'-monophosphate phosphodiesterase from Mucor rouxii: regulation of enzyme activity by phosphorylation and dephosphorylation.

Crude preparations of cyclic adenosine 3′, 5′-monophosphate phosphodiesterase were activated 1.5 to 2 fold by incubation with ATP, Mg²⁺ and cyclic AMP in a reaction which was both, time and temperature dependent. Cyclic AMP phosphodiesterase remained in an activated state upon filtration of the enzymatic preparation through Sephadex G-25 and ion-exchange chromatography. Activation of the enzyme in the presence of [γ ³²P]ATP resulted in a significant amount of [³²P] protein-bound radioactivity. Reversible deactivation of cyclic AMP phosphodiesterase was enhanced by Mg²⁺ and was accompanied by the release of [³²P] protein bound radioactivity. The evidence is consistent with a mechanism for controlling cyclic AMP phosphodiesterase through phosphorylation-dephosphorylation sequence.     

Adenylate cyclase assay with [alpha-32P]ATP as substrate. A simple modification for lowering blanks

In the assay of adenylate cyclase using [α-³²P]ATP as the substrate and alumina chromatography as the separating procedure for labeled nucleotides, blank levels are dependent on the quality of the labeled ATP and also on that of the alumina. In order to lower the blanks by eliminating the radioactive material contaminating the commercial [α-³²P]ATP preparations, the following treatment is proposed: The reaction mixture resulting from the incubation is heated for 4 min at 95°C in 0.165 n HCl, then it is chromatographed on a selected alumina (Woelm) column. In the conditions used, cyclic AMP was unaffected, while blank values were low. The detection limit of [³²P]cyclic AMP was thus higher and the precision of enzyme activity determination was improved, while the advantages of one-step chromatography were retained.     

A highly sensitive adenylate cyclase assay

A highly sensitive adenylate cyclase assay method has been developed which employs sequential chromatography on columns of Dowex cation exchange resin and aluminum oxide. With the use of [α-³²P]ATP as substrate, this method permits the nearly complete separation of cyclic [³²P]AMP formed from the substrate and other ³²P-containing compounds, i.e., ³²P in the assay blanks was barely detectable. In comparative studies, this method was found to be considerably more sensitive than previously reported methods. The high sensitivity of this method permits detection of the small amounts of cyclic AMP formed at low enzyme concentrations or at early time points in kinetic studies.     

Abnormally high rate of cyclic AMP excretion from an Escherichia coli mutant deficient in cyclic AMP receptor protein

By labeling adenosine 3′, 5′-cyclic monophosphate (cyclic AMP) with [³²P] phosphate and chromatographing it on a thin-layer alumina plate, we have determined the extra- and intracellular amounts of cyclic AMP in an $\text{Escherichia coli}$ CRP^? mutant (deficient in a cyclic AMP receptor protein) and its isogenic CRP⁺ cell. The CRP^? cell was found to excrete cyclic AMP at an abnormally high rate as compared to the CRP⁺ cell when growing on glucose or glycerol, which can be correlated with the abnormally high intracellular levels of cyclic AMP in the CRP^? cell.     

Improved synthesis of 32P-labelled 3′,5′-cyclic AMP, 3′,5′-cyclic GMP and other 3′,5′cyclic ribo- and deoxyribonucletodies of high specific activity

A general procedure is described for the two-step chemical synthesis from [³²P]orthophosphoric acid of the eight common ribo- and deoxyribonucleoside 3′,5′-cyclic monophosphates. The method is simple and reliable and both steps are carried out in the same reaction flask without an intermediate purification step. ³²P-labelled cyclic nucleotides are obtained after paper chromatography in yields of 20–60% relative to starting [³²P]orthophosphoric acid and with a specific activity of greater than 1 mCi/μmole. Alternative methods for the purification of reaction mixtures and for the preparation of ³²P-labelled 3′,5′-cyclic AMP and 3,′,5′-cyclic GMP are described.     

Cyclic AMP synthesis in Xenopus laevis oocytes inhibition by progesterone

[α-³²P] ATP was microinjected into Xenopus oocyte and neosynthesized cyclic AMP was isolated. Cholera toxin inhibited progesterone-induced maturation and stimulated after 3 h of preincubation the amount of neosynthesized cyclic AMP. Progesterone decreased the neosynthesis of cyclic AMP during the first hour following addition of the hormone.     

Cyclic AMP,cyclic GMP,and bean rust uredospore germlings

Ten millimolar cyclic AMP (cAMP) or cyclic GMP (cGMP) induced bean rust uredospore germlings to undergo one round of mitosis and to form septa, processes normally associated with appressorium formation. To assess the possibility of cyclic nucleotide regulation of bean rust development, we used an 8-azido-[³²P]cAMP photoaffinity probe to identify three cyclic nucleotide binding peptides. The peptides bound either cAMP or cGMP. The phosphorylation of one peptide in uredospore germling extracts by [γ-³²P]ATP was stimulated by either 1 μM cAMP or cGMP, but only in the presence of 10 mM Na₂MoO₄, a phosphatase inhibitor. Uredospores contain about 1500 and 23 pmol cAMP and cGMP/g dry wt, respectively, as determined by radiobinding assays.     

Etude du mechanisme de la regulation hormonale dans le placenta humain: Mise en evidence d'une activite adenyl cyclasique dans les homogenats de placenta a terme

Human full-term placentas possess adenyl cyclase activity which was assayed by the transformation of [α-³²P]ATP to cyclic 3′,5′-[³²P]AMP according to the method of Krishnaet al. (G. Krishna, B. Weiss and B. B. Brodie), J. Pharmacol. Exptl. Therap., 163 (1968) as modified by Rodbell (M. Rodbell, J. Biol. Chem., 242 (1967) 5744).     

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.     

Protein phosphorylation in a dopamine-sensitive homogenate of rat caudate: Effect of adenosine 3′,5′-cyclic monophosphate and putative neurotransmitters

Adenosine 3′,5′-cyclic monophosphate (cyclic AMP) and its 8-methylthio derivative stimulate the incorporation of ³²P into proteins endogenous to a homogenate of rat caudate nucleus when 4 μM [γ?³²P] ATP is usedas substrate. Higher concentrations of ATP reduced the effect of the cyclic nucleotide until at 400 μM no significant increase in protein phosphorylation was seen.Incubation of the homogenate with 400 μM ATP and 100 μM dopamine resulted in an approx. 2-fold increase in cyclic AMP but did not alter caudate protein phosphorylation suggesting that the catecholamine could not stimulate protein phosphorylation under the experimental conditions used in the present study.     

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.     

Studies of kidney plasma membrane adenosine-3′,5′-monophosphate-dependent protein kinase

Porcine kidney cortex was utilized for the preparation of plasma-membrane-enriched and soluble cytoplasmic (cytosol) fractions for the purpose of examining the relative properties of cyclic [³H]AMP receptor and cyclic AMP-dependent protein kinase activities of these preparations. The affinity, specificity and reversibility of cyclic [³H]AMP interaction with renal membrane and cytosol binding sites were indicative of physiological receptors.Binding sites of cytosol and deoxycholate-solubilized membranes were half-saturated at approx. 50nM and 100 nM cyclic [³H]AMP. Native plasma membranes exhibited multiple binding sites which were not saturated up to 1 mM cyclic [³H]AMP. Modification of the cyclic phosphate configuration or 2′-hydroxyl of the ribose moiety of cyclic AMP produced a marked reduction in the effectiveness of the cyclic AMP analogue as a competitor with cyclic [³H]AMP for renal receptors. The cyclic [³H]AMP interaction with membrane and cytosol fractions was reversible and the rate and extent of dissociation of bound cyclic [³H]AMP was temperature dependent. With the plasma-membrane preparation, dissociation of cyclic [³H]AMP was enhanced by ATP or AMP.Assay of both kidney subcellular fractions for protein kinase activity revealed that cyclic AMP enhanced the phosphorylation of protamine, lysine-rich and arginine-rich histones but not casein. The potency and efficacy of activation of renal membrane and cytosol protein kinase by cyclic AMP analogues such as N⁶-butyryl-adenosine cyclic 3′,5′-monophosphate or N⁶,O²-dibutyryl-adenosine cyclic 3′,5′-monophosphate supported the observations on the effectiveness of cyclic AMP analogues as competitors with cyclic [³H]AMP in competitive binding assays.This study suggested that the membrane cyclic [³H]AMP receptors may be closely associated with the membrane-bound catalytic moiety of the cyclic AMP-dependent protein kinase system of porcine kidney.     

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.     

Partial characterization of protein kinase-catalyzed phosphorylation of low molecular weight proteins in purified preparations of pigeon heart sarcolemma and sarcoplasmic reticulum

Pigeon heart microsomes contain three minor size protein kinase substrates of minimal molecular weights of 22 000, 15 000, and 11 500, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When the microsomes were partially loaded with calcium oxalate and subjected to rate zonal and isopynic centrifugations in sucrose density gradient columns, the 22 000 and the 15 000 dalton proteins settled in the heaviest fraction, which was composed mainly of vesicles of sarcoplasmic reticular membranes; the 11 500 dalton protein was concentrated in the lightest fractions, which consisted chiefly of vesicles of sarcolemmal origin. During incubation of the membrane fractions with Mg[γ-³²P]ATP significant amounts of ³²P were incorporated into all these proteins. Incorporation of ³²P into the 15 000 dalton protein was moderately and ³²P incorporation into the 22 000 dalton protein was markedly enhanced in the presence of exogenous soluble cyclic AMP-dependent protein kinase and cyclic AMP. The phosphorylation of the three proteins was virtually unaffected by CA²⁺ concentrations up to 0.1 mM and by ethyleneglycol-bis(β-aminoethylether)-N,N′-tetraacetic acid in the absence of added Ca²⁺.Phosphorylation of the 22 000 and the 11 500 dalton proteins occurred mainly at serine residues. In the 15 000 dalton protein threonine residues were the main site of endogenous phosphorylation. Nearly equal amounts of [³²P]-phosphate were incorporated into threonine and serine residues of this protein when phosphorylation was supported by exogenous cyclic AMP-dependent protein kinase and cyclic AMP.The 15 000 dalton protein could be removed from its membrane attachment by extraction with an acidic chloroform/methanol mixture. This step opens the way for the purification of this membrane-bound protein kinase substrate.     

Phosphorylation and dephosphorylation of the regulatory subunit of cyclic 3′,5′-monophosphate-dependent protein kinase (type II) in vivo and in vitro

A phosphorylated regulatory subunit of cyclic AMP-dependent protein kinase (type II) was purified to homogeneity from inorganic [³²P]phosphate-injected rats.A new method of measuring the phosphorylation reaction was developed. It was found that this regulatory subunit was phosphorylated in cells and comprised 60, 82 and 55% of the total regulatory subunit in brain, heart and liver cytosol fractions from rats, respectively.Dephosphorylation was stimulated by cyclic nucleotides. The K_a values for cyclic AMP and cyclic IMP were 0.30 and 1.0 μM, respectively. Purified phosphoprotein phosphatase could dephosphorylate the regulatory subunit and this reaction was also stimulated by cyclic nucleotides with similar K_a values. The inhibitors of phosphoprotein phosphatase, NaF and ZnCl₂, protected against dephosphorylation unless ADP or cyclic AMP were present.     

Effects of cyclic adenosine 3': 5'-monophosphate on phosphoprotein kinase and phosphatase fractions prepared from rat liver nuclei.

A soluble rat liver nuclear extract containing total RNA polymerase activities also exhibits appreciable amounts of protein kinase activity. This unfractionated protein kinase catalyzes the phosphorylation of both endogenous proteins and exogenous lysine-rich histone in the presence of [γ-³²P]ATP and Mg²⁺. The optimal concentration of Mg²⁺ is 5 mm for histone phosphorylation and 25 mm for the phosphorylation of endogenous proteins. Cyclic AMP has no effect on the phosphorylation of lysine-rich histone by this unfractionated nuclear protein kinase. However, addition of cyclic AMP causes a reduction in the ³²P-labeling of an endogenous protein (CAI) which can be characterized by its mobility during SDS-acrylamide gel electrophoresis and elution in the unbound fraction of a DEAESephadex column. If CAI is first labeled with ³²P and then incubated with 10^?6m cyclic AMP under conditions where protein kinase activity is inhibited, the presence of the cyclic nucleotide causes a loss of the ³²P-labeling of this protein, implying the activation of a substrate-specific protein phosphatase. When rat liver RNA polymerases are purified by DEAE-Sephadex chromatography, protein kinase activity is found in the unbound fraction and in those column fractions containing RNA polymerase I and II. The fractionated protein kinases exhibit different responses to cyclic AMP, the unbound protein kinase being stimulated and the RNA polymerase-associated protein kinases being dramatically inhibited. A second protein (CAII) whose phosphorylated state is modified by cyclic AMP is found within the DEAE-Sephadex column fractions containing RNA polymerase II. The cyclic nucleotide in this case appears to reduce labeling of CAII by inhibition of the protein kinase activity which co-chromatographs with both CAII and RNA polymerase II. Based on molecular weight estimates, neither CAI nor CAII appears to be an RNA polymerase subunit. The identity of CAI as a protein factor whose phosphorylated state influences nuclear RNA synthesis is suggested by the fact that addition of fractions containing CAI to purified RNA polymerase II inhibits the activity of this enzyme, but only if CAI has been previously incubated in the presence of cyclic AMP.     

Thymidine phosphorylation in wheat: analysis of phosphate transfer from ATP to thymidine

Extracts of wheat (Triticum vulgare Vill. [Triticum aestivum L.] var. Lemhi) seedlings contain thymidine-phosphorylating activity with ATP, ADP, or AMP and nucleotide hydrolase activity (ATP → → AMP). The synthesis of [³²P]dTMP exclusively from [α-³²P]ATP with none detectable from [γ-³²P]ATP demonstrates the absence of thymidine kinase and the presence of nucleoside phosphotransferase as the only observable thymidine-phosphorylating enzyme.