Regulation of the pyruvate dehydrogenase complex by Ca2+ within toluene-permeabilized heart mitochondria.
(1) Rat heart mitochondria, permeabilized to all low Mr solutes by toluene treatment, have been used to study the regulation in situ of the phosphatase and kinase components of the pyruvate dehydrogenase complex (PDH) by Ca2+. (2) Inactivation of the complex, resulting from phosphorylation by the kinase, and reactivation induced by the phosphatase, were both apparent first-order processes. This behaviour of the phosphatase differs from that observed with toluene-permeabilized adipose tissue mitochondria (Midgley, P.J.W., Rutter, G.A. and Denton, R.M. (1987) Biochem. J. 241, 271-377) where a 'lag phase' preceded reactivation of inactive complex. Further, reactivation due to phosphatase activity was stimulated by Ca2+ only at subsaturating Mg2+ concentrations, in contrast with the extracted enzyme which is stimulated by Ca2+ at all Mg2+ concentrations. (3) Maximum values of half-times observed for inactivation and reactivation were about 10 and 15 s, respectively, at 30 degrees C. (4) At Mg2+ concentrations where effects of Ca2+ on the activity of the phosphatase were apparent, no effect of Ca2+ on the activity of the kinase could be detected. (5) The sensitivity of the phosphatase to [Ca2+] was essentially unchanged in the presence of either ADP or ATP, with half-maximal effects at 0.7 microM in each case.
Rutter
G A
GA
Department of Biochemistry, School of Medical Sciences, University of Bristol, U.K.
Midgley
P J
PJ
Denton
R M
RM
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D002118:Calcium / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008929:Mitochondria, Heart / Q000187:drug effects / Q000201:enzymology*; D009243:NAD / Q000032:analysis; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases; D011769:Pyruvate Dehydrogenase (Lipoamide)-Phosphatase / Q000378:metabolism; D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D011768:Pyruvate Dehydrogenase Complex / Q000037:antagonists & inhibitors / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D014050:Toluene / Q000494:pharmacology
D000081382:Pyruvate Dehydrogenase Acetyl-Transferring Kinase; D011768:Pyruvate Dehydrogenase Complex; D009243:NAD; D014050:Toluene; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D011769:Pyruvate Dehydrogenase (Lipoamide)-Phosphatase; D008274:Magnesium; D002118:Calcium