REGULATION OF ACID TREHALASE ACTIVITY BY ASSOCIATION-DISSOCIATION IN SACCHAROMYCES-CEREVISIAE

Acid trehalase (AT) has always been reported to be copurified with inv ertase (I) and a 40 kDa additional protein. Glucose grown stationary p hase cells of Saccharomyces cerevisiae contained least I activity. So, it was attempted to purify AT from these cells (I:AT = 10.83). Studie s on specific activity, percent recovery and I:AT ratio of different p ools, collected during purification of AT, indicated that samples cont aining ratio I:AT < 2.2 were unstable. Purification methodology favour ing association (DEAE-Sephadex chromatography) resulted in gaining tot al activity while methodology favouring dissociation (HPGPLC) resulted in tremendous loss in recovery. Active pool (Pool 1X) appeared to be electrophoretically homogeneous but dissociated into 175, 90, 68, 61, 57 (minor bands) and 37-41 (major band) molar mass (kDa) bands on SDS- PAGE. Inactive pools (Pools 1Y, 3X, 3Y) did not contain the 37-41 kDa major band. So, association of both I and a 37-41 kDa protein with AT appeared to be essential. Two bands of isoelectric pH (pi) 4.6 and 4.7 were present in pool 1X enzyme preparation. All SDS-PAGE-resolved ban ds of pool 1X, in an average, contained high aspartate/asparagine and low cysteine residues. AT activity appeared to be highly sensitive to the change in pH and also to agents affecting ionisation of protein, e g., betaine, NaCl, acetate, etc. Association of AT components in prese nce of NaCl was demonstrated spectrophotometrically. Specific activity of AT decreased with dilution. Substrate mediated allosterism for thi s enzyme preparation suggested that AT existed as an equilibrium mixtu re of protomer-oligomer. It was suggested that reversible association- dissociation was a mechanism for the regulation of AT activity. (C) 19 98 Elsevier Science B.V.