Effects of metals on acetyl-coenzyme A carboxylase activity of Saccharomyces cerevisiae.

Previous reports from this laboratory (R. K. Rasmussen and H. P. Klein, Biochem. Biophys. Res. Commun. 28:415, 1967) indicated that many organic compounds can stimulate the acetylcoenzymeA (CoA) carboxylase of Saccharomyces cerevisiae strain LK2G12. The enzyme from this organism appears to differ from that reported by Matsuhashi et al. (Biochem. Z. 340:243, 1964; Biochem. Z. 340:263, 1964), who found no such effects on acetyl-CoA carboxylase prepared from brewers' yeast (Lowenbrau strain). The enzyme used in our studies appears to be similar to those enzymes prepared from animal sources in its susceptibility to activation by citrate, aglycerophosphate, and related compounds, although it did not appear to undergo profound molecular weight changes during activation by these compounds, as has been reported for the enzyme from animal sources (D. B. Martin and P. R. Vagelos, J. Biol. Chem. 237:1787, 1962). Matsuhashi et al. showed that the enzyme prepared from brewers' yeast required magnesium, but they gave no data on other metals. In view of recent reports of unusual stimulations by magnesium on carboxylases from different animal sources (M. Greenspan and J. M. Lowenstein, Arch. Biochem. Biophys. 118:260,1967; B. Keech and G. J. Barritt, J. Biol. Chem. 242:1983, 1967), and because our enzyme seems more like the enzymes prepared from animal tissues, we wish to report our findings on the effects of metals, particularly magnesium, on this acetyl-CoA carboxylase. Information on the cultivation, harvesting, disruption, and subsequent centrifugal fractionation of the organism used in this study was summarized in an earlier publication (D. White and H. P. Klein, J. Bacteriol. 91:1218, 1966). We obtained acetyl-CoA carboxylase from a 0 to 35% saturated ammonium sulfate fraction of a dialyzed high-speed supernatant fluid. This fraction was stable for several months when stored at -70 C. Acetyl-CoA carboxylase activity was usually measured by the incorporation of radioactive bicarbonate according to the method of Martin and Vagelos (J. Biol. Chem. 23 7:1787 1962), with the exception that radioactivity was determined by scintillation counting. In experiments designed to identify the products formed when the radioactive precursor was "4C-bicarbonate or '4C-acetyl-CoA, the CoA compounds were hydrolyzed by the method of Morgan et al. (Federation Proc. 22:414, 1964). The organic acids were then extracted and identified by chromatography on washed cellulose thin-layer chromatographic plates or washed Whatman 3 MM chromatographic paper, using ethyl alcohol (95 %)-ammonia-water (20:1:4) as the solvent system. Radioactive products were located by exposing the chromatograms to Kodak X-ray film. Then the area of cellulose or paper containing the radioactivity was removed to a scintillation vial for counting. A number of different cations, as the chlo-