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Biomedical subjects

J B Gibbs

Publications and source records attributed to J B Gibbs.

96 records · Page 6Linked to original sources
PubMed1984

Intrinsic GTPase activity distinguishes normal and oncogenic ras p21 molecules.

The 21-kilodalton protein (p21) encoded by normal cellular Harvey-ras has been expressed in Escherichia coli as a fusion protein by using the pUC8 vector and has been purified to greater than 95% homogeneity by ion-exchange chromatography and gel filtration. The purified protein molecules possess intrinsic GTPase activity on the basis of the following criteria: (i) elution of the GTPase activity with p21 GDP-binding activity in two different chromatography systems, (ii) parallel thermal inactivation of GTPase activity and p21 GTP-binding activity, and (iii) immunoprecipitation of the GTPase activity with monoclonal antibodies to p21. At 37 degrees C, the rate of GTP hydrolysis by the purified normal p21 assayed in solution was 5.3-6.6 mmol/min per mol of p21. The rate of GTP hydrolysis by a form of p21 [Val12] encoded by a human oncogene was significantly lower (1.4-1.9 mmol/min per mol of p21). The presence of a threonine phosphate acceptor site at residue 59 also decreased p21 GTPase activity. For regulatory proteins that use GTP as part of their biochemical mechanism, the hydrolysis of GTP to GDP reverses the biological activity of the respective proteins. The observation that oncogenic forms of p21 lose GTPase activity suggests that GTP hydrolysis may be a biochemical event that inactivates the growth-promoting effects of a p21 X GTP complex.

Animals↗
PubMed1984

Autophosphorylation of v-Ha-ras p21 is modulated by amino acid residue 12.

The 21,000-dalton protein (p21) encoded by the ras oncogene of Harvey murine sarcoma virus (v-Ha-ras) becomes phosphorylated (pp21) in vivo and in vitro on threonine residue 59. p21 molecules encoded by cellular ras genes (c-Ha-ras-1) contain an alanine at position 59, and thus these p21 molecules are not phosphorylated. In this investigation, recombinant ras genes have been constructed between the 5' p21 coding region of normal (EC) or oncogenically activated (EJ) human c-Ha-ras-1 and the 3' p21 coding region of v-Ha-ras to generate p21 molecules containing a threonine phosphoacceptor site at position 59 and a glycine (EC/v-Ha) or valine (EJ/v-Ha) at residue 12. In transformed NIH 3T3 mouse fibroblast cells labeled with [35S]methionine, the ratio of pp21 to p21 was strikingly modulated by the amino acid at residue 12. v-Ha-ras p21 has an arginine at position 12, and 24% of total p21 was in the phosphorylated form. A glycine at residue 12 decreased the amount of pp21 to 14% of total p21, and a valine at residue 12 dramatically increased this value to 50%. In vitro, the valine form of p21 had 2.4- and 2.7-fold greater autophosphorylating activity than the glycine and arginine forms of p21, respectively, using [gamma-32P]GTP as phosphate donor, but the three p21 species had similar Km values for GTP (0.20-0.27 microM). These results indicate that a biochemical activity of p21 distinguishes between previously observed biological differences of normal and activated human ras genes.

Amino Acid Sequence↗
PubMed1980

Calcium and microtubule dependence for increased ornithine decarboxylase activity stimulated by beta-adrenergic agonists, dibutyryl cyclic AMP, or serum in a rat astrocytoma cell line.

Ornithine decarboxylase (ODCase; L-ornithine carboxy-lyase; EC 4.1.1.17) activity was increased 5-20 fold above basal activity by N(6),O(2')-dibutyryl cyclic AMP, isoproterenol, epinephrine, or fetal calf serum in confluent C6-2B rat astrocytoma cells. Serum increased ODCase activity by a mechanism apparently independent of cyclic AMP because it decreased intracellular cyclic AMP. Calcium ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) buffers (pCa = 6.4) reduced basal ODCase activity and blocked the increase in activity by beta-adrenergic agonists, dibutyryl cyclic AMP, or serum. Readdition of calcium to pCa = 4.0 restored the ability of the cells to respond to these agents. The rise in ODCase activity stimulated by isoproterenol, dibutyryl cyclic AMP, or serum was also blocked by the microtubule disrupting agents, vinblastine and colchicine, and by the microfilament disrupting agent, cytochalasin B. Lumicolchicine, an analog of colchicine that does not disrupt microtubules, was ineffective. The microtubule and microfilament disrupting agents and EGTA had no effect on the intracellular accumulation of cyclic AMP. Cycloheximide inhibited protein synthesis >95% within 30 min, and caused an immediate decline of stimulated ODCase activity with a half-time of 20-30 min. The inhibition of ODCase activity by colchicine or EGTA was distinct from that seen with cycloheximide and could not be correlated to their effects on general protein synthesis. Colchicine or EGTA each caused identical rates of decline in ODCase activity with a half-time of 20-30 min after an initial lag period of about 60 min.

Adrenergic beta-Agonists↗
PubMedDate not supplied

Yeast and mammalian ras proteins have conserved biochemical properties.

Mammalian ras oncogenes encode polypeptides of relative molecular mass (Mr) 21,000 (p21) which bind GTP and GDP. Oncogenic ras-encoded proteins differ from their normal homologues by an amino acid substitution for Gly 12, Ala 59 or Gln 61. Recently, we and others have observed that normal p21, encoded by the Ha-ras gene, has a GTP hydrolytic activity that is reduced by the oncogenic substitutions Val 12 or Thr 59. The yeast Saccharomyces cerevisiae contains two ras-related genes, RASsc1 and RASsc2, the expression of either of which is sufficient for viability. RASsc1 and RASsc2 encode proteins of 309 (SC1) and 322 (SC2) residues which are 62% homologous to mammalian p21 in their 172-amino acid N-terminal sequences. We report here that the N-terminal domain of SC1 binds GTP and GDP and has a GTP hydrolytic activity that is reduced in the variants SC1[Thr 66] and SC1[Leu 68] which are analogous to oncogenic Ha[Thr 59] and Ha[Leu 61], respectively. These results suggest that yeast and mammalian ras proteins have similar biochemical and possibly biological functions.

Fungal Proteins↗