1. The activity of pig heart pyruvate dehydrogenase kinase was assayed by the incorporation of [32P]phosphate from [γ-32P]ATP into the dehydrogenase complex. There was a very close correlation between this incorporation and the loss of pyruvate dehydrogenase activity with all preparations studied. 2. Nucleoside triphosphates other than ATP (at 100μm) and cyclic 3′:5′-nucleotides (at 10μm) had no significant effect on kinase activity. 3. The Km for thiamin pyrophosphate in the pyruvate dehydrogenase reaction was 0.76μm. Sodium pyrophosphate, adenylyl imidodiphosphate, ADP and GTP were competitive inhibitors against thiamin pyrophosphate in the dehydrogenase reaction. 4. The Km for ATP of the intrinsic kinase assayed in three preparations of pig heart pyruvate dehydrogenase was in the range 13.9–25.4μm. Inhibition by ADP and adenylyl imidodiphosphate was predominantly competitive, but there was nevertheless a definite non-competitive element. Thiamin pyrophosphate and sodium pyrophosphate were uncompetitive inhibitors against ATP. It is suggested that ADP and adenylyl imidodiphosphate inhibit the kinase mainly by binding to the ATP site and that the adenosine moiety may be involved in this binding. It is suggested that thiamin pyrophosphate, sodium pyrophosphate, adenylyl imidodiphosphate and ADP may inhibit the kinase by binding through pyrophosphate or imidodiphosphate moieties at some site other than the ATP site. It is not known whether this is the coenzyme-binding site in the pyruvate dehydrogenase reaction. 5. The Km for pyruvate in the pyruvate dehydrogenase reaction was 35.5μm. 2-Oxobutyrate and 3-hydroxypyruvate but not glyoxylate were also substrates; all three compounds inhibited pyruvate oxidation. 6. In preparations of pig heart pyruvate dehydrogenase free of thiamin pyrophosphate, pyruvate inhibited the kinase reaction at all concentrations in the range 25–500μm. The inhibition was uncompetitive. In the presence of thiamin pyrophosphate (endogenous or added at 2 or 10μm) the kinase activity was enhanced by low concentrations of pyruvate (25–100μm) and inhibited by a high concentration (500μm). Activation of the kinase reaction was not seen when sodium pyrophosphate was substituted for thiamin pyrophosphate. 7. Under the conditions of the kinase assay, pig heart pyruvate dehydrogenase forms 14CO2 from [1-14C]pyruvate in the presence of thiamin pyrophosphate. Previous work suggests that the products may include acetoin. Acetoin activated the kinase reaction in the presence of thiamin pyrophosphate but not with sodium pyrophosphate. It is suggested that acetoin formation may contribute to activation of the kinase reaction by low pyruvate concentrations in the presence of thiamin pyrophosphate. 8. Pyruvate effected the conversion of pyruvate dehydrogenase phosphate into pyruvate dehydrogenase in rat heart mitochondria incubated with 5mm-2-oxoglutarate and 0.5mm-l-malate as respiratory substrates. It is suggested that this effect of pyruvate is due to inhibition of the pyruvate dehydrogenase kinase reaction in the mitochondrion. 9. Pyruvate dehydrogenase kinase activity was inhibited by high concentrations of Mg2+ (15mm) and by Ca2+ (10nm–10μm) at low Mg2+ (0.15mm) but not at high Mg2+ (15mm).
Skip Nav Destination
Article navigation
December 1974
-
Cover Image
Cover Image
- PDF Icon PDF LinkFront Matter
- PDF Icon PDF LinkTable of Contents
- PDF Icon PDF LinkAdvertising
Research Article|
December 01 1974
Regulation of heart muscle pyruvate dehydrogenase kinase
Ronald H. Cooper;
Ronald H. Cooper
1Department of Biochemistry, University of Bristol, Medical School, University Walk, Bristol BS8 1TD, U.K.
Search for other works by this author on:
Philip J. Randle;
Philip J. Randle
1Department of Biochemistry, University of Bristol, Medical School, University Walk, Bristol BS8 1TD, U.K.
Search for other works by this author on:
Richard M. Denton
Richard M. Denton
1Department of Biochemistry, University of Bristol, Medical School, University Walk, Bristol BS8 1TD, U.K.
Search for other works by this author on:
Publisher: Portland Press Ltd
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© 1974 London: The Biochemical Society
1974
Biochem J (1974) 143 (3): 625–641.
Citation
Ronald H. Cooper, Philip J. Randle, Richard M. Denton; Regulation of heart muscle pyruvate dehydrogenase kinase. Biochem J 1 December 1974; 143 (3): 625–641. doi: https://doi.org/10.1042/bj1430625
Download citation file:
Sign in
Don't already have an account? Register
Sign in to your personal account
You could not be signed in. Please check your email address / username and password and try again.
Captcha Validation Error. Please try again.