The yeast Saccharomyces cerevisiae is shown to be equipped with cyclic GMP, the level of which ranges from 6 pmol/10(9) cells with pressed baker's yeast to 21 pmol/10(9) cells with exponentially growing cells. In extracts from synchronized growing yeast, cyclic GMP increases stepwise, being doubled at the time of each mitosis. Theophylline and 3-isobutyl-1-methylxanthine induce a rapid increase of cyclic GMP, followed by a premature formation of the septal cell wall between mother cell and bud. The effects of 3-isobutyl-1-methylxanthine are reversible. Dibutyryl-cyclic GMP, and, after a pronounced lag, also dibutyryl-cyclic AMP, induce a premature cell division, too. Cholera toxin induces premature cell divisions without a preceding increase in cyclic GMP. Neither theophylline nor 3-isobutyl-1-methylxanthine, cholera toxin or one of the dibutyryl-cyclic nucleotides modify the growth rate of the culture. None of the agents has significant effects on the level of cyclic AMP. The results suggest that cyclic GMP possibly controls an early step of mitosis, whereas ADP-ribosylation might govern a subsequent event.

3',5'-Cyclic-GMP Phosphodiesterases↗

PubMedDate not supplied

A particulate guanylate cyclase (EC 4.6.1.2) from growing yeast cells (Saccharomyces cerevisiae).

The detection of cGMP in yeast (Eckstein 1988), but lacking hints at guanylate cyclase from sequencing of the yeast genome, raised questions about existence, isoform, and regulation of guanylate cyclase from this organism. We found a particulate guanylate cyclase activity in yeast extracts, exhibiting properties of an integral membrane protein. Characteristics are: pH-optimum at pH 6.8, temperature-optimum around 60 degrees C, only slight stimulation by Mn2+. Sigmoidal enzyme kinetics indicate allosteric regulation, ATP and Ca2+ act as negative allosteric effectors. The enzyme activity is increased by yeast alpha-1 mating factor, and by sodium nitrite, thus showing properties of particulate as well as of soluble isoforms from other eukaryotes. The activation by alpha-1 mating factor suggests receptor functions, and a role in ascospore conjugation.

Cyclic GMP↗

PubMedDate not supplied

Guanosine 3':5'-cyclic monophosphate-dependent particulate protein kinase activity from yeast (Saccharomyces cerevisiae).

Continuing our studies on cGMP in growing yeast we detected a particulate cGMP dependent protein kinase (Pk-G), which was solubilized by detergents and NaCl. It achieves maximum activity at 25 degrees C and pH = 6.8, high concentrations of substrate proteins or cGMP produce saturation. Casein and histones are appropriate substrates, phosphatase-pretreated histone H-2a provokes outstandingly high activity. Pk-G differs from cAMP-dependent protein kinase (Pk-A) with respect to pH optimum, temperature tolerance above 50 degrees C, and stability. Partial purification is achieved by chromatography with DEAE-cellulose, Sepharose, and cGMP-substituted Sepharose. The latter step also markedly removes Pk-A. At least three proteins with Pk-G-activity and high cGMP-affinity are separated by polyacrylamide-gel-electrophoresis. Their apparent molecular masses, as deduced from comigrating marker proteins, differ considerably from those of other Pk-G's, but also of Pk-A's.

Chromatography, DEAE-Cellulose↗

PubMedDate not supplied

Inhibition cyclic guanosine 3':5'-monophosphate of the soluble DNA polymerase activity, and of partially purified DNA polymerase A (DNA polymerase I) from the yeast Saccharomyces cerevisiae.

DNA polymerase activity from extracts of growing yeast cells is inhibited by cGMP. Experiments with partially purified yeast DNA polymerases show, that cGMP inhibits DNA polymerase A (DNA polymerase I from Chang), which is the main component of the soluble DNA polymerase activity in yeast extracts, by competing for the enzyme with the primer-template DNA. Since the enzyme is not only inhibited by 3', 5'-cGMP, but also by 3',5'-cAMP, the 3'--:5'-phosphodiester seems to be crucial for the competition between cGMP and primer. This would be inconsistent with the concept of a 3'-OH primer binding site in the enzyme. The existence of such a site in the yeast DNA polymerase A is indicated from studies with various purine nucleoside monophosphates. When various DNA polymerases are compared, inhibition by cGMP seems to be restricted to those enzymes, which are involved in DNA replication, DNA polymerases with an associated nuclease activity are not inhibited, DNA polymerase B from yeast is even activated by cGMP. Though some relations between the cGMP effect and the presumed function of the enzymes in the living cell are apparent, the biological meaning of the observations in general remains open.

Animals↗