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J Olate

Publications and source records attributed to J Olate.

29 records · Page 2Linked to original sources
PubMed1988

Newly identified brain potassium channels gated by the guanine nucleotide binding protein Go.

Potassium channels in neurons are linked by guanine nucleotide binding (G) proteins to numerous neurotransmitter receptors. The ability of Go, the predominant G protein in the brain, to stimulate potassium channels was tested in cell-free membrane patches of hippocampal pyramidal neurons. Four distinct types of potassium channels, which were otherwise quiescent, were activated by both isolated brain G0 and recombinant Go alpha. Hence brain Go can couple diverse brain potassium channels to neurotransmitter receptors.

Adenylyl Imidodiphosphate↗
PubMed1988

Reticulocyte lysates synthesize an active alpha subunit of the stimulatory G protein Gs.

We placed the cDNAs encoding one of the short types of alpha s (alpha s-1) with Asp-Ser in positions 70 and 71 and one of the long types of alpha s (alpha s-2) in which Asp-Ser are substituted with a string of 16 amino acids, into the pGEM-3 transcription vector downstream from its T7 RNA polymerase promoter, obtained transcripts and translated the mRNAs using a rabbit reticulocyte lysate system, to determine if the molecules would be synthesized and, if so, whether they would be active as assessed in cyc- reconstitution assays. The translation products obtained from both alpha s RNAs were a mixture of primarily three polypeptides of which one (approximately 40-50% of total) represented the complete translation product and the other two appeared to be due to internal translation starts at Met60, before the splice difference between the RNAs, and the other at the first Met after the splice difference. Lysates incubated with short or long alpha s RNA when added to cyc- membranes reconstituted fluoride and GTP[gamma S]-stimulated activities. Thus, in vitro synthesized alpha s subunits are active in interacting both with guanine nucleotides and the adenylyl cyclase enzyme. On incubation without and with the receptor agonist isoproterenol, using GTP as sole added guanine nucleotide, both types of alpha s subunits reconstituted the isoproterenol-stimulated adenylyl cyclase activity. Thus, the synthetic alpha s also interact with receptors, and by inference with beta-gamma dimers, shown previously to be needed for activation by receptor. Quantitative assays in which the activity of the synthetic alpha s-1 was compared to that of native purified human erythrocyte type-1 Gs, indicated that the two products are equipotent within a 2-fold margin of error. Thus, the lysate made fully active alpha s subunits, and alpha s subunits require no post-translational modifications dependent on microsomal processes. This approach may be useful in studying biological functions of other cloned alpha subunits of G proteins.

Animals↗
PubMed1988

Alpha i-3 cDNA encodes the alpha subunit of Gk, the stimulatory G protein of receptor-regulated K+ channels.

cDNA cloning has identified the presence in the human genome of three genes encoding alpha subunits of pertussis toxin substrates, generically called "Gi." They are named alpha i-1, alpha i-2 and alpha i-3. However, none of these genes has been functionally identified with any of the alpha subunits of several possible G proteins, including pertussis toxin-sensitive Gp's, stimulatory to phospholipase C or A2, Gi, inhibitory to adenylyl cyclase, or Gk, stimulatory to a type of K+ channels. We now report the nucleotide sequence and the complete predicted amino acid sequence of human liver alpha i-3 and the partial amino acid sequence of proteolytic fragments of the alpha subunit of human erythrocyte Gk. The amino acid sequence of the proteolytic fragment is uniquely encoded by the cDNA of alpha i-3, thus identifying it as alpha k. The probable identity of alpha i-1 with alpha p and possible roles for alpha i-2, as well as additional roles for alpha i-1 and alpha i-3 (alpha k) are discussed.

Amino Acid Sequence↗
PubMed1988

Screening of cDNA libraries with oligonucleotides as applied to signal transducing G proteins, receptors and effectors.

Screening of cDNA libraries constructed in phage or plasmids with oligonucleotide probes has become one of the preferred cloning techniques with the least number of false positive failures. In this article we present our current protocols for designing the procedure to detect cDNA inserts and isolate them. We illustrate with primary screens for G protein subunits and membrane receptors.

Bacteriophage lambda↗
PubMed1985

Treatment of oocyte membranes with the 2',3'-dialdehyde of guanosine triphosphate reduces progesterone inhibition of adenylyl cyclase.

Treatment of Xenopus laevis membranes with the 2',3'-dialdehyde of GTP (dial GTP) drastically inhibits their adenylyl cyclase activity. Optimal inhibition is obtained by treatment with 1 mM dial GTP for 1h at 32 degrees C. Using guanyl-5'-yl imidodiphosphate, F-, forskolin and Mn2+ as activators of the enzyme it can be concluded that dial GTP preferentially reacts with the stimulatory subunit (Ns) and slightly with the catalytic subunit. Dial GTP treatment greatly reduces the inhibition of adenylyl cyclase by progesterone. Pure exogenous Ns stimulates the enzyme but does not restore progesterone inhibition. Treatment with dial [alpha-32P]GTP labels several membrane proteins some of which have similar Mr to Ns and Ni.

Adenylyl Cyclase Inhibitors↗
PubMed1984

Oocyte adenylyl cyclase contains Ni, yet the guanine nucleotide-dependent inhibition by progesterone is not sensitive to pertussis toxin.

Membranes were obtained from Xenopus laevis oocytes after removal of follicular cells by collagenase treatment. [32P]ADP-ribosylation with pertussis toxin showed them to contain a single Mr = 40000 substrate for this toxin that co-migrates on sodium dodecylsufate-polyacrylamide gel electrophoresis with pure human erythrocyte Ni, the inhibitory regulatory component of adenylyl cyclase. [32P]ADP-ribosylation of oocyte membranes with cholera toxin also showed presence of a single substrate but of Mr = 42000. These results indicate, that the adenylyl cyclase system of oocytes, like that of somatic cells and unlike that of spermatozoids, contains the catalytic unit C and both of the known regulatory N components. The possible susceptibility to pertussis toxin of the guanine nucleotide-dependent inhibition of oocyte adenylyl cyclase by progesterone was investigated. This action of progesterone is mediated by a membrane bound receptor as opposed to a receptor of cytosolic or nuclear localization. However, the inhibitory effect of progesterone was unaffected by pertussis toxin, even though the oocyte membrane Ni was fully ADP-ribosylated with pertussis toxin, as revealed by lack of further [32P]ADP-ribosylation on subsequent re-incubation with pertussis toxin. These results indicate that the action of progesterone, in spite of being nucleotide-dependent, is either not mediated by Ni, suggesting the existence of an additional nucleotide regulatory component, or if mediated by Ni, involves a mode of regulation of this coupling protein that is different from that by which all other inhibitory hormones act on adenylyl cyclase.

Adenosine Diphosphate Ribose↗
PubMed1984

Studies on the mechanism of inhibition of amphibian oocyte adenylate cyclase by progesterone.

Progesterone treatment induces the meiotic maturation of Xenopus laevis oocytes. Previous evidence indicates that this hormonal effect may be due to inhibition of oocyte adenylate cyclase. The present work studies several aspects of the mechanism of adenylate cyclase inhibition by this hormone. Forskolin greatly stimulates oocyte adenylate cyclase in the absence of guanine nucleotides and this activity is not sensitive to progesterone inhibition. In addition the forskolin-activated enzyme is not inhibited by a wide range of guanine nucleotide, in the presence or absence of hormone. The time course of cAMP synthesis catalyzed by oocyte adenylate cyclase in the presence of guanyl-5'l-imidodiphosphate (Gpp(NH)p) shows an initial lag period that does not depend on the concentration of Gpp(NH)p. Progesterone causes a very significant increase in the hysteresis of the reaction, at least doubling the half-time of enzyme activation. The hormonal effect on the lag cannot be reversed by saturating concentrations of Gpp(NH)p. Progesterone also decreases the steady-state rates of the reaction. This effect, however, depends on the concentration of Gpp(NH)p. High concentrations of Gpp(NH)p almost completely reverse the inhibition of the steady-state rates. Progesterone does not inhibit if it is added to the reaction after the initial lag period. Guanosine-5'-O-(2-thiodiphosphate) (GDP-beta-S) is an efficient competitive inhibitor of Gpp(NH)p activation of adenylate cyclase. Progesterone inhibition is observed at all concentrations of GDP-beta-S and is potentiated at high ratios of GDP-beta-S to Gpp(NH)p. These data indicate that progesterone inhibits by interfering with the activation of the Ns subunit of the enzyme by guanine nucleotides, rather than through a mechanism involving a separate Ni subunit.

Adenylyl Cyclase Inhibitors↗
PubMed1983

Inhibition by phenothiazine derivatives of the adenylate cyclase of amphibian oocytes.

The adenylate cyclase activity of membranes of Xenopus laevis oocytes and follicle cells was affected by the presence of 2-chloro-10-(3-aminopropyl)phenothiazine (CAPP) and two other antipsychotic drugs, fluphenazine and penfluridol. CAPP, at concentrations of 10 and 100 microM, had opposite effects on the activation of the oocyte adenylate cyclase by effectors that act through the G/F regulatory subunit. Under these conditions, the drug stimulated the activation by fluoride and drastically inhibited the activation by guanyl-5'-yl-imidodiphosphate [Gpp(NH)p] and by cholera toxin and GTP. The activity of the catalytic subunit measured in the presence of either Mn2+ or forskolin was not affected by 100 microM CAPP. however, concentrations of this drug above 100 microM inhibited the adenylate cyclase activated by fluoride or by forskolin and also inhibited the activity of a calmodulin-independent cyclic nucleotide phosphodiesterase present in the same oocyte membrane preparation. Oocyte adenylate cyclase has been shown previously to be inhibited by the hormone progesterone. The inhibitory effect of CAPP is additive to that measured with the hormone, indicating that these compounds act through different mechanisms. CAPP did not modify the concentration of Gpp(NH)p required to yield half-maximal activation and, although the drug inhibited more strongly at lower concentrations of Gpp(NH)p, saturating amounts of the guanine nucleotide did not reverse completely the inhibition caused by CAPP. The effects of these antipsychotic drugs on oocyte adenylate cyclase did not require the presence of free Ca2+ and were not altered by the addition of exogenous calmodulin and calcium.

3',5'-Cyclic-AMP Phosphodiesterases↗