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Biomedical subjects

P Greengard

Publications and source records attributed to P Greengard.

At least 19 recordsLinked to original sources
PubMed1979

Multiple phosphorylation sites in protein I and their differential regulation by cyclic AMP and calcium.

The phosphorylation of protein I, a specific neuronal protein, has been found to be regulated both by cyclic AMP (cAMP) and by calcium, in intact as well as in lysed synaptosome preparations from rat brain. In order to determine the phosphorylation site(s) of protein I that were regulated by cAMP and calcium, protein I was purified after it was phosphorylated under various conditions. This purified protein I was then subjected either to peptide mapping after limited proteolysis in sodium dodecyl sulfate/polyacrylamide gels or to tryptic fingerprinting. 8-Br-cAMP selectively increased the phosphorylation of the same protein I peptide fragment in both intact and lysed synaptosomes. Depolarization-induced calcium influx into intact synaptosomes, or the addition of calcium to lysed synaptosomes, caused a stimulation of the phosphorylation not only of this peptide but also of other distinct peptides. Differential regulation by cAMP and calcium of the phosphorylation of multiple sites on the same neuronal protein may provide a molecular basis for interactions between these two second-messenger systems in certain nerve terminal functions.

Animals↗
PubMed1979

Widespread distribution of protein I in the central and peripheral nervous systems.

Protein I, a naturally occurring substrate for cyclic AMP-dependent and calcium-dependent protein kinases, previously has been found only in mammalian brain, where it has been demonstrated to be located in neurons. Various tissues and organs outside the brain have now been examined for the possible occurrence of protein I, by using both an immunohistochemical approach and a chemical procedure involving radioimmunolabeling of polyacrylamide gels. Protein I has been found in the inner plexiform layer of the retina, in the posterior pituitary, and in the autonomic nervous system. In tissue composed predominantly of cells other than nerve cells, immunoreactivity was present only where innervation was present. Protein I appeared to be localized in some, but not all, nerve terminals and synapses.

Animals↗
PubMed1979

Immunocytochemical localization, in synapses, of protein I, an endogenous substrate for protein kinases in mammalian brain.

Protein I is a principal endogenous substrate for cyclic AMP- and Ca2+-dependent protein kinases of mammalian brain. Antibodies raised against purified protein I have been used to localize this protein in the rat central nervous system. At the light microscope level, immunoreactivity was detected in punctate deposits in selected zones of synaptic termination. These deposits varied in density among brain regions. At the electron microscope level, immunoreactivity was observed at some but not all synaptic regions and was restricted to the perimeter of synaptic vesicles and to submembranous material in the postsynaptic neuron.

Animals↗
PubMed1979

Ca2+ and cyclic AMP regulate phosphorylation of same two membrane-associated proteins specific to nerve tissue.

It was shown previously that addition of cyclic AMP (cAMP) to a synaptic membrane fraction incubated with [gamma-32P]ATP stimulated the phosphorylation of two proteins, designated proteins Ia and Ib, found only in nerve tissue. Addition of Ca2+ plus veratridine to synaptosomes preincubated with 32Pi stimulated the phosphorylation of two proteins with similar apparent molecular weights. Various techniques have now been used to determine whether the two proteins phosphorylated in synaptosomes in the presence of Ca2+ plus veratridine are the same as proteins Ia and Ib phosphorylated in synaptic membranes in the presence of cAMP. The proteins phosphorylated by the two procedures were extracted under similar conditions, had similar apparent molecular weights and charges, and were digested by collagenase at similar rates and to the same radioactive intermediates and end products. Furthermore, the two sets of proteins were digested by three other proteolytic enzymes to phosphopeptides with similar molecular weights. The results indicate that Ca2+ and cAMP are each capable of regulating the phosphorylation of proteins Ia and Ib.

Adenosine Triphosphate↗
PubMed1979

Presence of free cyclic AMP receptor protein and regulation of its level by cyclic AMP in neuroblastoma-glioma hybrid cells.

Neuroblastoma-glioma hybrid cells of line 108CC-5 were found to contain high levels of soluble adenosine 3',5'-cyclic monophosphate (cAMP)-dependent protein kinase activity and high levels of two specific cAMP receptor proteins, RI and RII. Treatment of the hybrid cells with dibutyryl cAMP increased the level of RI but did not significantly affect the level either of RII or of cAMP-dependent protein kinase activity. The effect of dibutyryl cAMP could be mimicked by prostaglandin E1 and 3-isobutyl-1-methylxanthine, both of which are known to raise cAMP levels in neuroblastoma-glioma hybrid cells. Both in control as well as in dibutyryl cAMP-treated cells, RII but not RI was associated with cAMP-dependent protein kinase. Several lines of evidence suggest that RI represents the free regulatory subunit of type I cAMP-dependent protein kinase. The presence of this regulatory subunit as free cAMP receptor protein in neuroblastoma-glioma hybrid cells may be of significance with respect to the regulation of growth and differentiation in tumor cells.

Animals↗
PubMed1979

Regulation of the state of phosphorylation of specific neuronal proteins in mouse brain by in vivo administration of anesthetic and convulsant agents.

The effect of drug treatment in vivo on the state of phosphorylation of two specific neuronal proteins, proteins Ia and Ib, has been studied in mouse brain. For this purpose, animals were killed by immersion into liquid nitrogen, and proteins Ia and Ib were extracted by a procedure designed to prevent alterations in their state of phosphorylation. Several anesthetic agents (pentobarbital, chloral hydrate, and urethane) each caused a decrease in the state of phosphorylation of these proteins. Conversely, the convulsant agents pentylenetetrazol and picrotoxin each caused an increase in the state of phosphorylation of these proteins. Neither the anesthetic nor the convulsant agents affected the total amount of these proteins. The results are compatible with a role for proteins Ia and Ib in neuronal function.

Anesthetics↗
PubMed1979

Hormonally regulated phosphoprotein of turkey erythrocytes: localization to plasma membrane.

The catecholamine-stimulated cotransport of sodium and potassium ions across the plasma membrane of the turkey erythrocyte was previously found to be associated with increased 32P incorporation into a high molecular weight protein. To determine the subcellular localization of this phosphorylated protein, which we have termed goblin, a new method has been developed for isolation of pure plasma membranes from turkey erythrocytes. With this method, it has been demonstrated that goblin is located in the plasma membrane. Goblin is not extracted by solutions of low or high ionic strength but is partially extracted by nonionic detergents, indicating that it is not a component of turkey erythrocyte spectrin and suggesting that it may be an intrinsic protein of the plasma membrane. The data are compatible with a possible role for goblin in the hormonal control of ion movements across the plasma membrane.

Animals↗
PubMed1979

Subcellular distribution in cerebral cortex of two proteins phosphorylated by a cAMP-dependent protein kinase.

The subcellular distribution of Proteins Ia and Ib, two proteins which serve as specific substrates for protein kinases present in mammalian brain, was studied in the dog cerebral cortex. Proteins Ia and Ib were found to be most highly enriched in synaptic vesicle fractions; they were also present in postsynaptic density and synaptic membrane fractions in significant amounts. Proteins Ia and Ib present in the synaptic vesicle fraction appear to be similar, if not identical, to those present in the postsynaptic density fraction as judged by several criteria: (a) the ability to serve as substrate for cAMP-dependent protein kinase, (b) electrophoretic mobility in the presence of sodium dodecyl sulfate, (c) extractability with NH4Cl or EGTA, and (d) fragmentation to electrophoretically similar peptides by a purified Staphylococcus aureus protease. In addition, the postsynaptic density fraction has been found to contain cAMP-dependent Protein Ia and Protein Ib kinase activity. The subcellular localization of Proteins Ia and Ib suggests a role for these proteins in the physiology of the synapse.

Animals↗
PubMed1979

Cyclic nucleotides, phosphorylated proteins, and the nervous system.

Some postsynaptic effects of several classes of neurotransmitters appear to be mediated or modulated through the cyclic nucleotides, cyclic AMP and cyclic GMP. Available evidence suggests that the molecular mechanism by which the cyclic nucleotides carry out this second messenger role in nerve cells involves regulation of the state of phosphorylation of specific neuronal proteins. Phosphorylated proteins also appear to be involved in mediating certain of the actions of several other classes of regulatory agents, including calcium and the steroid hormones.

Animals↗
PubMed1978

Regulation of synthesis of guanosine 3':5'-cyclic monophosphate in neuroblastoma cells.

The increase in intracellular cyclic GMP concentrations in response to muscarinic-receptor activation in N1E-115 neuroblastoma cells is dependent on extracellular Ca2+ ion. The calcium ionophore A23187 can also evoke an increase in cyclic GMP in the presence of Ca2+ ion. Most (about 85%) of the guanylate cyclase activity of broken-cell preparations is found in the soluble fraction. The soluble enzyme can utilize MnGTP (Km = 55 micrometer), MgGTP (Km = 310 micrometer) and CaGTP (Km greater than 500 micrometer) as substrates. Free GTP is a strong competitive inhibitor (Ki approximately 20 micrometer). The enzyme possesses an allosteric binding site for free metal ions (Ca2+, Mg2+ and Mn2+). The membrane-bound guanylate cyclase is qualitatively similar to the soluble form, but has lower affinity for the metal-GTP substrates. Entry of Ca2+ into cells may increase cyclic GMP concentration by activating guanylate cyclase through an indirect mechanism.

Calcimycin↗