Level Of 32P Incorporation Research Articles

Phosphorylation of Connexin43 and the Regulation of Neonatal Rat Cardiac Myocyte Gap Junctions

The functional state of gap junctions and the state of phosphorylation of connexin43 (Cx43), the major gap junction protein in rat heart, were evaluated in primary cultures of neonatal rat cardiocytes. Functional coupling was greatly reduced after treatment with staurosporine (ST), a protein kinase inhibitor. The ST-induced reduction in cell coupling was reversed by activation of protein kinase C (PKC) with 12-O-tetradecanoylphorbol 13-acetate (TPA). The cellular distribution of Cx43, as detected by immunofluorescence, was not grossly affected by either ST alone or ST plus TPA. Although immunoblot analysis did not detect significant changes in the relative amounts of the unphosphorylated and individual phosphorylated forms of Cx43 after each treatment, the level of32P-incorporation into Cx43 of radiolabeled cells was significantly affected. Consistent with their known properties, treatment with ST reduced, and combined treatment with TPA and ST increased, the level of32P-incorporation into Cx43. Two-dimensional tryptic phosphopeptide maps of32P-labeled Cx43 indicated that a distinct subset of the phosphopeptides that are present under basal conditions were affected by ST or ST/TPA treatments, with TPA-induced phosphorylation occurring at the ST-sensitive sites. However, the ST/TPA-sensitive tryptic phosphopeptides did not comigrate with others that were derived fromin vitrophosphorylation by PKC of a recombinant C-terminal Cx43 peptide (Cx43[243–382]). Although a PKC-dependent mechanism appears to be involved in the regulation of functional coupling between neonatal rat cardiocytes, PKC itself may not be the final mediator of Cx43 phosphorylation.

Regulatory phosphorylation of banana fruit phosphoenolpyruvate carboxylase by a copurifying phosphoenolpyruvate carboxylase-kinase.

Phosphoenolpyruvate (P-pyruvate) carboxylase from ripened banana fruit was purified to near homogeneity and a final specific activity of 20-23 U/mg protein; P-pyruvate carboxylase-kinase copurified with P-pyruvate carboxylase throughout the purification. Gel filtration FPLC indicated that the two proteins form a tightly associated 425-kDa complex. Both the 103-kDa and 100-kDa subunits of the P-pyruvate carboxylase alpha2beta2 hetetrotetramer were phosphorylated and subsequently dephosphorylated in vitro in a time-dependent manner when the final preparation was incubated with 0.1 mM [gamma-32P]ATP followed by rabbit muscle protein phosphatase type 2A1. Phosphoamino acid and phosphopeptide map analyses indicated that in vitro phosphorylation of both subunits likely occurs at an identical Ser residue. Maximal stoichiometry of 32P incorporation was 0.2 and 0.4 mol/mol 103-kDa and 100-kDa subunit, respectively. The level of 32P incorporation was correlated with the enzyme's activation state when assayed under suboptimal assay conditions (pH 7.3, 75 microM P-pyruvate, 0.2 mM L-malate). The main kinetic effect of phosphorylation was to decrease the enzyme's Km(P-pyruvate), as well as its sensitivity to inhibition by L-malate and L-glutamate. Banana P-pyruvate carboxylase-kinase: (a) also phosphorylated maize leaf P-pyruvate carboxylase, histone III-S, and dephosphorylated casein; (b) demonstrated Mg2+ dependence and Ca2+ independence, (c) exhibited a broad pH activity optimum of pH 8.0-8.5, and (d) was inhibited by L-malate and activated by Ba2+ and Co2+. Time-course kinetic studies suggested that P-pyruvate carboxylase exists mainly in the dephosphorylated form in preclimacteric, climacteric and postclimacteric fruit, but that its kinase is expressed throughout ripening. In situ 32P-labeling indicated that, while both subunits of ripe banana P-pyruvate carboxylase are phosphorylated in vivo, it is primarily the 100-kDa subunit that is radiolabeled. The results suggest that similar to the enzyme from leaves, root nodules and seeds, a fruit P-pyruvate carboxylase may be subject to regulatory seryl phosphorylation by an endogenous P-pyruvate carboxylase-kinase.

Starch Phosphorylation in Potato Tubers Proceeds Concurrently with de Novo Biosynthesis of Starch.

The in vivo phosphorylation of starch was studied in Solanum tuberosum cv Dianella and Posmo. Small starch granules contain 25% more ester-bound phosphate per glucose residue than large starch granules. The degree of phosphorylation was found to be almost constant during tuber development. Isolated tuber discs synthesize starch from externally supplied glucose at a significant rate. Tuber discs supplied with glucose and [32P]orthophosphate incorporate radiolabeled phosphorus into the starch. The level of 32P incorporation is proportional to the amount of starch synthesized. The incorporation of 32P from orthophosphate is correlated to de novo synthesis of starch, since the incorporation of 32P is diminished upon inhibition of starch synthesis by fluoride. Based on the amount of [14C]glucose phosphate isolated after hydrolysis of purified starch from tuber discs incubated in the presence of [U-14C]glucose, approximately 0.5% of the glucose residues of the de novo-synthesized starch are phosphorylated. This value is in general agreement with the observed levels of phosphorus in starch accumulated during tuber development. Thus, the enzyme system responsible for starch phosphorylation is fully active in the isolated tuber discs, and the starch phosphorylation proceeds as an integrated part of de novo starch synthesis.

In vivo phosphorylation of isocitrate lyase inEscherichia coli andAcinetobacter calcoaceticus

Isocitrate lyase inEscherichia coli and inAcinetobacter calcoaceticus is phosphorylated when the cells are grown with acetate as the sole carbon source in low-phosphate mineral salts medium containing32P inorganic phosphate. The level of32P incorporation into the enzyme in both microorganisms appears to be constant throughout the entire growth cycle. Further, theresults of immunoblots and rocket immunoelectrophoresis suggest that the amount of isocitrate lyase protein, although at different levels in each microorganism, also remains constant throughout the growth cycle.

In vivo phosphorylation of NADP+ glutamate dehydrogenase inEscherichia coli

The NADP+-specific glutamate dehydrogenase inEscherichia coli K12 has been shown to be phosphorylated in vivo when the cells are grown in a low-phosphate minimal salts medium containing32P inorganic phosphate. The amount of radioactivity incorporated into the enzyme is different depending on the growth phase of the culture, with the highest level of32P incorporation occurring during the mid-exponential growth phase. Previously reported studies have demonstrated that the enzyme is also phosphorylated in vitro in an ATP-dependent reaction.

The Effect of Neurocatin on Protein Phosphorylation in Striatal Synaptosomes from Rat Brain

Neurocatin, a neuroregulatory factor isolated from mammalian brain, is a powerful affector of protein phosphorylation in rat striatal synaptosomes. Two major synaptosomal phosphoproteins of approximately 80 and approximately 60 kDa, possibly synapsin I and tyrosine hydroxylase, were especially sensitive to neurocatin. Immunoprecipitation experiments confirmed that the 60-kDa protein is the enzyme tyrosine hydroxylase. At low concentrations of neurocatin (to approximately 7.5 ng/100 microliters of suspension), incorporation of 32P orthophosphate into these proteins increased with increasing neurocatin concentration. At 7.5 ng of neurocatin, incorporation of the label into the two proteins increased by 22 and 26%, respectively. Concentrations of neurocatin > 7.5 ng/100 microliters caused progressive decrease in incorporation of 32P into many synaptosomal proteins; by a concentration of neurocatin of approximately 45 ng/100 microliters, the level of 32P incorporation into many proteins was < or = 70% of control. The effects of neurocatin on synaptosomal protein phosphorylation were also dependent on the time of incubation. At a constant concentration of approximately 7.5 ng/100 microliters of neurocatin, increased incorporation of 32P into many proteins was measurable within 0.5 min and was maximal by 1 min. Incubation times > 2.0 min, showed progressive decrease in 32P incorporation. Removing extrasynaptosomal Ca2+ with EGTA attenuated the increased 32P incorporation induced by low neurocatin concentrations, suggesting that calcium plays a role in neurocatin-induced phosphorylation of rat striatal synaptosomal proteins. The reduced incorporation of label induced by high neurocatin concentrations, however, was not calcium dependent. The effects of neurocatin on the level of 32P incorporation into proteins were observed only in intact synaptosomes, consistent with this compound acting through receptors on the plasma membrane.

In Vitro Phosphorylation of Bovine Adrenal Chromaffin Cell Tyrosine Hydroxylase by Endogenous Protein Kinases

Under phosphorylating conditions, addition of Ca2+ or cyclic AMP to the 100,000 g supernatant of purified bovine adrenal chromaffin cells increases both the incorporation of 32P into tyrosine hydroxylase and the activity of the enzyme. Combining maximally effective concentrations of each of these stimulating agents produces an additive increase in both the level of 32P incorporation into tyrosine hydroxylase and the degree of activation of the enzyme. The increased phosphorylation by Ca2+ is due to stimulation of endogenous Ca2+-dependent protein kinase activity and not inhibition of phosphoprotein phosphatases. When the chromaffin cell supernatant is subjected to diethylaminoethyl (DEAE) chromatography to remove calmodulin and phospholipids, tyrosine hydroxylase is no longer phosphorylated or activated by Ca2+; on the other hand, phosphorylation and activation of tyrosine hydroxylase by cyclic AMP are not affected. Subsequent replacement of either Ca2+ plus calmodulin or Ca2+ plus phosphatidylserine to the DEAE-fractionated cell supernatant restores the phosphorylation, but not activation of the enzyme. Reverse-phase HPLC peptide mapping of tryptic digests of tyrosine hydroxylase from the 100,000 g supernatant shows that the Ca2+-dependent phosphorylation occurs on three phosphopeptides, whereas the cyclic AMP-dependent phosphorylation occurs on one of these peptides. In the DEAE preparation, either cyclic AMP alone or Ca2+ in the presence of phosphatidylserine stimulates the phosphorylation of only a single phosphopeptide peak, the same peptide phosphorylated by cyclic AMP in the crude supernatant. In contrast, Ca2+ in the presence of calmodulin stimulates the phosphorylation of three peptides having reverse-phase HPLC retention times that are identical to peptides phosphorylated by Ca2+ addition to the crude unfractionated 100,000 g supernatant. Rechromatography of the peaks from each of the in vitro phosphorylations, either in combination with each other or in combination with each of the seven peaks generated from phosphorylation of tyrosine hydroxylase in situ, established that cyclic AMP, Ca2+/phosphatidylserine, and Ca2+/calmodulin all stimulate the phosphorylation of the same reverse-phase HPLC peptide: in situ peptide 6. Ca2+/calmodulin stimulates the phosphorylation of in situ peptides 3 and 5 as well. Thus, tyrosine hydroxylase can be phosphorylated in vitro by protein kinases endogenous to the chromaffin cell. Phosphorylation occurs on a maximum of three of the seven in situ phosphorylated sites, and all three of these sites can be phosphorylated by a Ca2+/calmodulin-dependent protein kinase.

Progesterone and cAMP-dependent protein kinase regulate in vivo the level of phosphorylation of two proteins ( Mr 20,000 and Mr 32,000) in Xenopus oocytes

The [ 32P]phosphoproteins and [ 35S]thiophosphoproteins were analyzed by electrophoresis and autoradiography after microinjection of [γ- 32P]ATP or of [ 35S]ATP-γ-S into living Xenopus oocytes. The level of 32P incorporation into a 20-kDa protein was decreased following progesterone treatment (between 1 and 2 hr). This 20-kDa protein was partially thiophosphorylated in vivo by [ 35S]ATP-γ-S. Furthermore it was found that this phosphoprotein was partially purified by TCA (1%) extraction and heat treatment. Microinjection of the C-subunit of cAMP-dependent protein kinase (0.6 to 1.2 pmole) inhibited maturation and provoked an increase in the level of phosphorylation of the 20-kDa protein and of a 32-kDa protein, indicating that both proteins were in vivo substrates (directly or indirectly) for cAMP-dependent protein kinase. When inhibitor-1 of protein phosphatase-1 was microinjected (5 to 10 pmole per oocyte) meiotic maturation was inhibited and the level of phosphorylation of the 32-kDa protein was increased; the same result was obtained following ATP-γ-S (1 m M) microinjection. Altogether these results suggest that a 20-kDa phosphoprotein, whose level of phosphorylation is decreased by progesterone, could be involved in the regulation of maturation by lowering the level of phosphorylation of a 32-kDa phosphoprotein. An attractive hypothesis would be that the 20-kDa phosphoprotein is an inhibitor of protein phosphatase-1.