title: Regulation of acid trehalase activity by association-dissociation in Saccharomyces cerevisiae.
abstract: Acid trehalase (AT) has always been reported to be copurified with invertase (I) and a 40 kDa additional protein. Glucose grown stationary phase cells of Saccharomyces cerevisiae contained least I activity. So, it was attempted to purify AT from these cells (I:AT = 10.83). Studies on specific activity, percent recovery and I:AT ratio of different pools, collected during purification of AT, indicated that samples containing ratio I:AT < 2.2 were unstable. Purification methodology favouring association (DEAE-Sephadex chromatography) resulted in gaining total 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 bands 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 presence of NaCl was demonstrated spectrophotometrically. Specific activity of AT decreased with dilution. Substrate mediated allosterism for this enzyme preparation suggested that AT existed as an equilibrium mixture of protomer-oligomer. It was suggested that reversible association-dissociation was a mechanism for the regulation of AT activity.
authors.affiliation: Department of Applied Biochemistry, Indian Institute of Chemical Biology, Calcutta, India.
authors.forename: N
authors.initials: N
authors.lastname: Biswas
authors.forename: A K
authors.initials: AK
authors.lastname: Ghosh
mesh_terms: D000596:Amino Acids / Q000032:analysis; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004591:Electrophoresis, Polyacrylamide Gel; D004795:Enzyme Stability; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000302:isolation & purification; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008970:Molecular Weight; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000254:growth & development; D013379:Substrate Specificity; D014198:Trehalase / Q000737:chemistry / Q000302:isolation & purification*; D014199:Trehalose; D043324:beta-Fructofuranosidase
chemical_list: D000596:Amino Acids; D014199:Trehalose; D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase; D014198:Trehalase