Human platelet thromboxane A2/prostaglandin H2 (TXA2/ PGH2) receptors are linked to phosphoinositide-specific phospholipase C (PI-PLC) via a G protein tentatively identified as a member of the Gq class. In contrast, platelet thrombin receptors appear to activate PI-PLC via other unidentified G pro-, teins. Platelets from most dogs are TXA2 insensitive (TXA2-); i.e., they do not aggregate irreversibly or secrete although they bind TXA2, but they respond normally to thrombin. In contrast, a minority of dogs have TXA2-sensitive (TXA2+) platelets that are responsive to TXA2. To determine the mechanism responsible for TXA2-platelets, we evaluated receptor activation of PI-PLC. Equilibrium binding of TXA2/PGH2 receptor agonists, 1251IBOP and 13H1U46619, and antagonist, 13H1SQ29,548, revealed comparable high-affinity binding to TXA2-, TXA2+, and human platelets. U46619-induced PI-PLC activation was impaired in TXA2-platelets as evidenced by reduced (a) phosphorylation of the 47-kD substrate of protein kinase C, (b) phosphatidic acid (PA) formation, (c) rise in cytosolic calcium concentration, and (d) inositol 1,4,5 trisphosphate (IP3) formation, while thrombin-induced PI-PLC activation was not impaired. GTPase activity stimulated by U46619, but not by thrombin, was markedly reduced in TXA2-platelets. Antisera to Gq class a subunits abolished U46619-induced GTPase activity in TXA2-, TXA2+, and human platelets. Direct G protein stimulation by GTPyS yielded significantly less PA and IP3 in TXA2-platelets. Immunotransfer blotting revealed comparable quantities of Gq class a-subunits in all three platelet types. Thus, TXA2-dog platelets have impaired PI-PLC activation in response to TXA2/PGH2 receptor agonists secondary to G protein dysfunction, presumably involving a

Methylamine (methylammonium ion) entered Saccharomyces cerevisiae X2180-A by means of a specific active transport system. Methylamine uptake was pH dependent (maximum rate between pH 6.0 and 6.5) and temperature dependent (increasing up to 35 C) and required the presence of a fermentable or oxidizable energy source in the growth medium. At 23 C the Vmax for methylamine transport was-17 nmol/min per mg of cells (dry weight) and the apparent Km was 220 MM. The transport system exhibited maximal activity in ammonia-grown cells and was repressed 60 to 70% when glutamine or asparagine was added to the growth medium. There was no significant derepression of the transport system during nitrogen starvation. Ammonia (ammonium ion) was a strong competitive inhibitor of methylamine uptake, whereas other amines inhibited to a much lesser extent. Mutants selected on the basis of their reduced ability to transport methylamine (MeaR) simultaneously exhibited a decreased ability to transport ammonia.

During recent studies conducted with suspensions of three strains of Saccharomyces cerevisiae, it was observed that ammonia was rapidly liberated when L-asparagine was added to the medium. Subsequent investigation has revealed that these strains of S. cerevisiae have an extemally active asparaginase as well as an internally active one. The appearance of the external asparaginase is stimulated by nitrogen starvation, requires an available energy source, and is prevented by cycloheximide. The internal enzyme appears to be constitutive. The external activity is relatively insensitive to para-hydroxymercuribenzoate inhibtion, whereas the internal activity is highly inhibited by this compound.