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P J Bechtel

Publications and source records attributed to P J Bechtel.

60 records · Page 4Linked to original sources
PubMed1975

Reversibility of adenosine 3':5'-monophosphate-dependent protein kinase reactions.

Using a homogeneous enzyme from rabbit skeletal muscle, it has been demonstrated that the cyclic adenosine 3':5'-monophosphate (cyclic AMP)-dependent protein kinase reaction is reversible. In addition to the phosphorylated protein substrate, the reverse reaction requires Mg2+, ADP, and cyclic AMP when the holoenzyme is used as the source of enzyme. It is independent of cyclic AMP when the catalytic subunit of the protein kinase is used. The optimum pH for the reverse reaction with 32P-labeled casein as the substrate is 5.7, essentially the same as that for the forward reaction. Among the nucleotide subtrates tested, ADP serves as the best phosphoryl group acceptor. The Km of the enzyme for ADP is 3.3 mM and that for 32P-casein is 1.7 mg/ml. The equilibrium constant at 30 degrees is approximately 0.042 at a magnesium concentration of 10 mM and a pH of 6.9. This result indicates that the free energy of hydrolysis (deltaG0obs) of the phosphorylated protein substrate is relatively high, i.e. approximately -6.5 kcal/mol under these conditions.

Adenosine Diphosphate↗
PubMed1975

Mechanisms of control for cAMP-dependent protein kinase from skeletal muscle.

A number of properties of homogeneous cyclic 3',5'-AMP (cAMP)-dependent protein kinase from rabbit skeletal muscle were determined. The enzyme is shown to be a tetramer consisting of one regulatory subunit dimer and two catalytic subunit monomers. Skeletal muscle protein kinase interacts with cAMP and MgATP as illustrated in the following equilibrium expression: R2C2 - (MgATP)2 + 2 cAMP in equilibrium R2 - (cAMP)2 + 2C + 2 MgATP. MgATP is shown to decrease the affinity of the enzyme for cAMP and to be necessary for recombination of the subunits. The concentration of the enzyme in tissue relative to that of cAMP is high enough to influence kinetic parameters of the activation process by cAMP. The cumulative effects of MgATP and high enzyme concentration are to increase the apparent activation constant for cAMP so that in vivo the enzyme would not be highly activated under basal conditions but would be greatly stimulated by increases in cAMP concentration. As a result, it is not necessary to invoke the concept of compartmentalization of cAMP to explain how it could regulate protein kinase activity in vivo. Finally, data are presented which indicate that a possible function of the heat-stable protein inhibitor of cAMP-dependent protein kinases may be to suppress the activity of protein kinase due to basal concentrations of cAMP. As such, the inhibitor could indirectly change the amount of cAMP needed to allow expression of protein kinase activity.

Adenosine Triphosphate↗
PubMed1974

Activation of protein kinase by physiological concentrations of cyclic AMP.

When determined under the usual conditions of an excess of ligand over protein, the concentration of cyclic AMP necessary to activate pure preparations of cyclic AMP-dependent protein kinase (EC 2.7.1.37; ATP:-protein protein phosphotransferase) half-maximally is in the range of 0.2-0.3 muM when casein or glycogen synthetase is used as the substrate, i.e., essentially the same as the concentration of the nucleotide that is found in resting skeletal muscle. The apparent dissociation constant for cyclic AMP bound to the protein kinase is also about 0.2-0.3 muM when measured under similar conditions. The concentration of the protein kinase in muscle is relatively high (0.23 muM), however, and under these conditions the apparent activation constant of the enzyme for cyclic AMP is raised so that an increase in cyclic AMP levels in the tissue would cause a concomitant increase in protein kinase activity over a wide range of nucleotide concentration. As a result, it is unnecessary to invoke compartmentalization of cyclic AMP to explain how it can control protein kinase activity in vivo. Another factor that may increase the effectiveness of changes in cyclic AMP concentration is the heat-stable protein inhibitor of protein kinase that may function to inhibit the activity of nearly all the protein kinase catalytic subunit dissociated by basal concentrations of cyclic AMP. Finally, the near equity between the concentration of cyclic AMP binding sites and the ligand itself provides a potential mechanism whereby agents can affect the total cyclic AMP content without directly affecting adenylate cyclase, cyclic AMP phosphodiesterase, or cyclic AMP transport.

Animals↗