Inhibition and activation of mannan synthesis in Saccharomyces cerevisiae spheroplast lysates

Abstract

Mannan synthetase activity in spheroplast lysates prepared from Saccharomyces cerevisiae was measured by following the incorporation of [14C]mannose from guanosine 5'-diphosphate-[14C]mannose into material precipitable with cold 0.3 M perchloric acid. When enzyme activity was assayed at high concentrations of spheroplast lysate protein (10 mg/ml) in the presence of 7.5 mM MnCl2, a severe inhibition was observed. This inhibition could be relieved by preincubation of the spheroplast lysate at 4 degrees C for 16 to 32 h before assay, by repeated freezing and thawing of the spheroplast lysate, or by the omission of MnCl2 from assay mixtures. The addition of ethylenediaminetetraacetic acid or monovalent cations removed inhibition in the presence of Mn2+. No similar inhibition was observed when a washed membrane fraction was substituted for spheroplast lysate as the source of mannan synthetase. The supernatant fluid obtained by centrifuging spheroplast lysate at 100,000 x g, when added to assay mixtures containing either spheroplast lysate preincubated at 4 degrees C or washed membrane fraction, also caused inhibition of enzyme activity. This inhibition required 7.5 mM MnCl2 and was destroyed by heating the supernatant fluid at 60 degrees C for 10 min, or by trypsin treatment at 30 degrees C. These results indicate the existence of a protein inhibitor of mannan synthesis whose inhibitory activity in spheroplast lysates may be modulated by preincubation at low temperature or by varying the available Mn2+ concentration.