Jose Sy

Ribosome specificity for the formation of guanosine polyphosphates

Abstract Ribosomes obtained from Bacillus brevis (ATCC 8185) were slightly active in synthesizing guanosine polyphosphates, which activity was greatly stimulated by addition of Escherichia coli stringent factor. Chlamydomonas reinhardtii chloroplast ribosomes did not produce guanosine polyphosphates on incubation but responded with abundant synthesis to addition of the stringent factor from E. coli . In contrast, cytoplasmic ribosomes from the same organism did not respond. Interchange experiments between either subunit from chloroplasts with the E. coli counterparts showed good activity. When the small subunit of cytoplasmic Chlamydomonas ribosomes was combined with the large subunit of E. coli or of chloroplasts, a small but definite response was obtained.

Catabolite inactivation of fructose 1,6-bisphosphatase and cytoplasmic malate dehydrogenase in yeast

Catabolite inactivation of fructose 1,6-bisphosphatase and cytoplasmic malate dehydrogenase was studied using the protease-deficient and vacuole-defective yeast strain pep4-3. The catabolite inactivation of fructose 1,6-bisphosphatase in pep4-3 was found to have a normal first inactivation step but with a defective second proteolytic step. In contrast, catabolite inactivation of cytoplasmic malate dehydrogenase was normal in pep4-3. These results suggest that the proteolytic pathways utilized in the hydrolysis of the two enzymes may be different and that proteolysis of fructose 1,6-bisphosphatase may require functional vacuoles while proteolysis of cytoplasmic malate dehydrogenase may not.

Catabolite inactivation of fructose 1,6-bisphosphatase inKluyveromyces fragilis

Catabolite inactivation of fructose 1,6-bisphosphatase inKluyveromyces fragilis was found to occur as a one-step process with a half-life of approximately 90 min in contrast to the two-step process previously reported forSaccharomyces cerevisiae. No rapid initial 50% loss of activity immediately after a glucose-induced catabolite inactivation was found; nevertheless, fructose 1,6-bisphosphatase was rapidly phosphorylated within 5 min of glucose addition. This result supports the hypothesis that protein phosphorylation serves as a signal for the specific degradation of fructose 1,6-bisphosphatase during catabolite inactivation.

Cyclic AMP-dependent protein kinase of yeast

Cyclic AMP-binding proteins with properties of a protein kinase regulatory subunit have been described in yeast with, however, conflicting reports on Mr-Values. AnM r of 28 000 was reported in [ 1 ] andM r 50 000 in [2]. Using photoaffinity labelling technique, a cAMP binding protein of 54 000 M r was reported [3], while binding proteins o fM r 25 000-58 000 were reported in [4]. We have identified a low M r (25 000) cAMP-binding protein from Kluyveromyces (Saccharomyces) fragilis [5]. The function of this protein was not known. Cyclic AMP inhibits the outgrowth of K. fragilis from lag phase in minimal medium; the inhibition can be prevented by methionine or S-adenosyl methionine [6]. In searching for the underlying biochemical mechanism for this inhibition, we have initiated studies on cAMP-binding proteins of this organism. We found that K. fragilis contained 2 cAMP binding proteins: a 64 000 M r protein which is the regulatory subunit of cAMP-dependent protein kinase and a 37 000M r protein which is derived from the regulatory subunit.

BIOSYNTHESIS OF GUANOSINE TETRAPHOSPHATE IN BACILLUS BREVIS

Two distinct ppGpp synthetic enzymes, a soluble and a ribosome-bound synthetase, have been identified in Bacillus brevis. Both catalyze the transfer of the pyrophosphoryl group from ATP to the 3′OH of GTP or GDP. The ribosome-bound enzyme (mol. wt. 76,000) requires a ribosome-mRNA-uncharged-tRNA complex for activity. It is inhibited by thiostrepton and tetracycline and is therefore similar to the re1A gene product, the stringent factor, of Escherichia coli. The B. brevis stringent factor, in contrast to the E. coli factor, is not stimulated by 20% methanol. The soluble enzyme (mol. wt. 55,000) is neither activated nor inhibited by the ribosomal complex. Both enzymes are present in about equal amounts in log phase cells. However, the ribosome-bound stringent factor activity is drastically reduced in stationary cultures where cells are sporulating.