Paul Greengard

Octopamine-Sensitive Adenylate Cyclase: Evidence for a Biological Role of Octopamine in Nervous Tissue

An adenylate cyclase that is activated specifically by very low concentrations of octopamine has been identified both in homogenates and in intact cells of the thoracic ganglia of an insect nervous system. This enzyme appears to be distinct from two other adenylate cyclases present in the same tissue, which are activated by dopamine and by 5-hydroxytryptamine, respectively. The data raise the possibility of a role of octopamine-sensitive adenylate cyclase in the physiology of synaptic transmission.

CHARACTERIZATION OF A DOPAMINE-SENSITIVE ADENYLATE CYCLASE IN THE RAT CAUDATE NUCLEUS

Abstract— An adenylate cyclase present in the caudate nucleus of rat brain, which is selectively stimulated by low concentrations of dopamine, and which is believed to mediate dopaminergic synaptic transmission, has been characterized with respect to several properties. The parameters studied included temperature, pH, ATP concentration, Mg/ATP ratio, and metal ion specificity. The effects of other compounds, including EGTA, NaF and several guanosine nucleotides, were also tested on the dopa‐mine‐sensitive adenylate cyclase. In addition, the subcellular distribution of the enzyme was studied. The highest specific activity was found in subcellular fractions enriched in nerve endings. A half‐maximal increase in the activity of the enzyme in a subcellular fraction occurred in the presence of 4 × 10−6 M dopamine. Fluphenazine, a dopamine antagonist, competitively inhibited the activity of the enzyme in this fraction, with a calculated inhibition constant (Ki) of 8 × 10−9M.

Hormone-Sensitive Adenyl Cyclase: Cytochemical Localization in Rat Liver

An electron microscopic procedure has been developed, using rat liver, for the localization of hormone-sensitive adenyl cyclase. Isoproterenol-sensitive adenyl cyclase is located almost exclusively in the parenchymal cells. In contrast, glucagon-sensitive adenyl cyclase is located primarily in the reticulo-endothelial cells but is also present in parenchymal cells. Sodium fluoride-sensitive adenyl cyclase is found in both cell types.

Cyclic nucleotide-dependent protein kinases. V. Preparation and properties of adenosine 3′,5′-monophosphate-dependent protein kinase from various bovine tissues

Abstract Adenosine 3′,5′-monophosphate (cyclic AMP)_dependent protein kinases, which catalyze the phosphorylation of proteins by ATP, have been found in each of fifteen bovine tissues examined. The enzymes were partially purified from these tissues, and their properties studied. Cyclic AMP increased by 5–15-fold the ability of each of these enzymes to phosphorylate histones. The concentration of cyclic AMP required to give half-maximal activation ranged from 30 to 160 nM. The 3′,5′-monophosphate derivatives of inosine, guanosine, uridine, and cytidine also activated the enzymes, but only at concentrations considerably higher than those required for cyclic AMP. 2′-Deoxythymidine 3′,5′-monophosphate was incapable of stimulating enzyme activity. Of the proteins tested as substrates, histone was the most effective for all of the enzymes studied. Protamine and casein were also phosphorylated by the enzymes. All of the enzymes had an absolute requirement for a divalent metal. Cyclic AMP stimulated enzyme activity in the presence of Mg2+, Mn2+, or Co2+, whereas it inhibited enzyme activity in the presence of Ca2+. The concentration of ATP required to give half-maximal velocity was determined for several of the enzyme preparations in the absence and presence of cyclic AMP; in each case, the apparent K m for ATP was significantly lower in the presence than in the absence of the activator. It was found that adenine, adenosine, AMP, ADP, GDP, riboflavin, FMN, and FAD inhibited enzyme activity to varying degrees. The fifteen enzyme preparations were found to be generally similar, but some important differences were observed.

HORMONE‐SENSITIVE ION TRANSPORT SYSTEMS IN ERYTHROCYTES AS MODELS FOR EPITHELIAL ION PATHWAYS

216. Sci. USA. 78: 1057-1061. 1980. Nature 284: 281-283. Palfrey & Greengard : Hormone-Sensitive Transport 307

Characterization of the inhibition of protein phosphatase-1 by DARPP-32 and inhibitor-2.

Phospho-DARPP-32 (where DARPP-32 is dopamine- and cAMP-regulated phosphoprotein, M r 32,000), its homolog, phospho-inhibitor-1, and inhibitor-2 are potent inhibitors (IC50 ∼1 nm) of the catalytic subunit of protein phosphatase-1 (PP1). Our previous studies have indicated that a region encompassing residues 6–11 (RKKIQF) and phospho-Thr-34, of phospho-DARPP-32, interacts with PP1. However, little is known about specific regions of inhibitor-2 that interact with PP1. We have now characterized in detail the interaction of phospho-DARPP-32 and inhibitor-2 with PP1. Mutagenesis studies indicate that within DARPP-32 Phe-11 and Ile-9 play critical roles, with Lys-7 playing a lesser role in inhibition of PP1. Pro-33 and Pro-35 are also important, as is the number of amino acids between residues 7 and 11 and phospho-Thr-34. For inhibitor-2, deletion of amino acids 1–8 (I2-(9–204)) or 100–204 (I2-(1–99)) had little effect on the ability of the mutant proteins to inhibit PP1. Further deletion of residues 9–13 (I2-(14–204)) resulted in a large decrease in inhibitory potency (IC50 ∼800 nm), whereas further COOH-terminal deletion (I2-(1–84)) caused a moderate decrease in inhibitory potency (IC50∼10 nm). Within residues 9–13 (PIKGI), mutagenesis indicated that Ile-10, Lys-11, and Ile-13 play critical roles. The peptide I2-(6–20) antagonized the inhibition of PP-1 by inhibitor-2 but had no effect on inhibition by phospho-DARPP-32. In contrast, the peptide D32-(6–38) antagonized the inhibition of PP1 by phospho-DARPP-32, inhibitor-2, and I2-(1–120) but not I2-(85–204). These results indicate that distinct amino acid motifs contained within the NH2 termini of phospho-DARPP-32 (KKIQF, where italics indicate important residues) and inhibitor-2 (IKGI) are critical for inhibition of PP1. Moreover, residues 14–84 of inhibitor-2 and residues 6–38 of phospho-DARPP-32 share elements that are important for interaction with PP1.

Dissociation and concomitant activation of adenosine 3′,5′-monophosphate-dependent protein kinase by histone☆

Abstract Adenosine 3′,5′-monophosphate-dependent (cAMP-dependent) protein kinase from bovine brain has been examined after sedimentation in a sucrose density gradient. The molecular weight of the catalytic and of the cAMP binding protein decreased in the presence of low concentrations of either histone or cAMP, indicating that the enzyme had dissociated into subunits. The dissociation in the presence of histone was accompanied by conversion of the enzyme activity from a cAMP-dependent to a cAMP-independent form.

Widespread occurrence of a specific protein in vertebrate tissues and regulation by cyclic AMP of its endogenous phosphorylation and dephosphorylation

A protein whose endogenous phosphorylation and dephosphorylation are affected by cAMP has been found in the soluble and particulate fractions of all vertebrate tissues studied. This phosphoprotein, which contained a substantial proportion of the radioactive phosphate observed on SDS-polyacrylamide gels, was estimated to have an apparent molecular weight of 49,000. In the presence of Zn++, cAMP inhibited the endogenous phosphorylation of this protein (protein 49) in the cytosol and microsomal fractions. In the presence of Mg++, cAMP stimulated the phosphorylation of protein 49 in the cytosol fractions, but had only slight effects in the microsomal fractions. The dephosphorylation of protein 49 by an endogenous protein phosphatase was markedly stimulated by cAMP in the cytosol and microsomal fractions of all tissues studied. The binding of 8-azido-cAMP (a photoaffinity analog of cAMP, which reacts specifically with cAMP-binding sites) to subcellular fractions was also studied. This binding was principally to a protein of molecular weight 49,000. These and other data suggest that a cAMP-binding protein with a molecular weight of 49,000 capable of undergoing cAMP-dependent phosphorylation and dephosphorylation, occurs in a variety of tissues.

Autophosphorylation of adenosine 3',5'-monophosphate-dependent protein kinase from bovine brain.

A highly purified adenosine 3′,5′-monophosphate-dependent protein kinase from bovine brain has been found to catalyze its own phosphorylation. The incorporated phosphate was shown to be associated with the cyclic AMP-binding subunit (R-protein) of the protein kinase. The catalytic subunit exhibited no detectable incorporation of phosphate into itself, but was required for the phosphorylation of R-protein. The molecular weight of R-protein was determined by polyacrylamide gel electrophoresis to be about 48,000 in the presence of sodium dodecyl sulfate. Cyclic AMP strikingly inhibited the rate of autophosphorylation observed in the presence of ZnCl2, CaCl2, NiCl2, or FeCl2, but had no significant effect in the presence of MgCl2 or CoCl2. The concentration of cyclic AMP required to give half-maximal inhibition of phosphorylation was 3 × 10−7m in the presence of either CaCl2 or ZnCl2. Guanosine 3′,5′-monophosphate was far less effective under the same experimental conditions than cyclic AMP. R-protein appears to be similar to a phosphoprotein recently discovered in synaptic membrane fractions from rat and bovine cerebral cortex.

Cyclic Nucleotide-Dependent Protein Kinases and Some Major Substrates in the Rat Cerebellum After Neonatal X-Irradiation

Abstract: The levels of cAMP‐dependent protein kinase (type I), of cGMP‐dependent protein kinase, of protein I and of a 23,000 MW substrate for the cGMP‐dependent protein kinase were measured in cerebella from normal rats and in the cerebella from rats in which a selective loss of interneurons in the cerebellar cortex had been produced by X‐irradiation. A decrease was observed in the concentrations of cAMP‐dependent protein kinase and of protein I, whereas an increase was observed in the concentrations of cGMP‐dependent protein kinase and of the 23,000 MW substrate. The data, taken together with the results of other studies, support the interpretation that cAMP‐dependent protein kinase and protein I are distributed throughout the cerebellum, but that cGMP‐dependent protein kinase and the 23,000 MW substrate are highly concentrated in Purkinje cells.