Abstract
In Saccharomyces cerevisiae, trehalase activity in crude extracts obtained from wild type cells was activated about 3-fold by preincubation with cAMP and ATP. The inactive trehalase fractionated by DEAE-Sephacel chromatography was activated by the addition of the cAMP-dependent protein kinase fraction from wild type cells in the presence of cAMP and ATP. Using the crude extract obtained from bcy1 mutant cells which were deficient in the regulatory subunit of cAMP-dependent protein kinase, the stimulation of trehalase activity was observed in the absence of cAMP. The cAMP-dependent protein kinase of CYR3 mutant cells which had a high Ka value for cAMP in the phosphorylation reaction required a high cAMP concentration for activation of trehalase. Increased activation of partially purified inactive trehalase (Mr = 320,000) was observed to correlate with increased phosphorylation of a protein (Mr = 80,000) identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The assay results using various mutants altered in cAMP metabolism indicated that the activation and phosphorylation of inactive trehalase fractions depended on the cAMP concentration accumulated in mutant cells. Inactivation and dephosphorylation of active trehalase fractions were observed by treatment with alkaline phosphatase or crude cell extracts. The results indicated that the conversion of inactive form of trehalase to the active form is regulated by cAMP through cAMP-dependent protein kinase.
Highlights
From the $Instituteof Applied Microbiology, University of Tokyo, Bunkyo-ku, Tokyo 113, Japan and the SDepartmenotf Industrial Chemistry, Tottori University, Tottori-shi, Tottori 680,Japan
Usingthe crude extract obtained from bcyl mutant cells which were deficientin the regulatory subunit of CAMP-dependent protein kinase,the stimulation of the CYR3 mutants produced altered CAMP-dependent protein kinase which had significantly high K, values for CAMP [2, 3]
Activation of Trehalase-Trehalaseactivityincrude extracts preparedfromwild typeand CYR3-I cells was not affected by the presence of CAMP,but activated about3-fold by preincubation with10 PM cAMP and0.1 mM ATP at 30 "C for 5 min (Table I)
Summary
Activation of Trehalase-Trehalaseactivityincrude extracts preparedfromwild typeand CYR3-I cells was not affected by the presence of CAMP,but activated about3-fold by preincubation with PM cAMP and0.1 mM ATP at 30 "C for 5 min (Table I). The bcyl mutant cells had extremely low levels of CAMPbindingprotein,butproduced a highlevel of CAMP-independent protein kinase (Table I) It has been shown in our previous study that the bcyl mutation resulted in simultaneous deficiency of CAMP-binding and CAMP-dependent protein kinaseand an increase of CAMP-independentprotein kinase [2]. These resultsindicate that CAMP-independent protein kinase found in the bcyl cells may be responsible for the high trehalase activity and the insensitivity of trehalase in this mutant to the addition of cAMP andATP. Crude extracts from the wild type and mutant strainswere preincubated in the absence or presence of 0.1 mM ATP, 10p~ CAMP,and 0.1 mM AMP-PNP a t 30 "C for 5 min and assayed for trehalase activity. The photoaffinity labeling of crude extracts was done under the standard conditions except that 8-N3-[3H]cAMPwas added at 10 p ~Pr.otein kinase activity was measured with and without 10 p~ CAMP
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