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Characterization of soluble and microsomal adenosine 3',5'-monophosphate-dependent protein kinases from rabbit heart
Abstract:
Cardiac microsomes contained an intrinsic adenosine 3',5'-monophosphate (cyclic AMP)-dependent protein kinase which stimulated phosphorylation of serine residue(s) of microsomal protein. The phosphorylated residues were associated with a microsomal protein component of 20,000 molecular weight as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Intrinsic phosphoprotein phosphatase activity of the microsomal membrane resulted in rapid dephosphorylation of these residues. Microsomes phosphorylated in the presence of cyclic AMP (10(-6) M) exhibited enhanced calcium uptake. We conclude that: 1) cardiac microsomes contain intrinsic cyclic AMP-dependent protein kinase(s) which phosphorylate a specific microsomal protein and phosphoprotein phosphatase(s) capable of dephosphorylating this protein, 2) phosphorylation of this protein enhances calcium uptake, 3) reversible phosphorylation of microsomal membrane may be an important mechanism for the regulation of calcium uptake of cardiac microsomes by cyclic AMP.
Insights
Cardiac microsomes possess a cyclic AMP-dependent protein kinase that phosphorylates a specific protein, enhancing calcium uptake. This reversible phosphorylation regulates cardiac microsome calcium uptake via cyclic AMP.
Area of Science:
- Biochemistry
- Cell Biology
- Cardiovascular Physiology
Background:
- Cardiac microsomes are critical for calcium storage and release in heart muscle.
- Cyclic AMP (cAMP) is a key second messenger involved in cardiac function regulation.
- Protein phosphorylation plays a vital role in cellular signaling pathways.
Purpose of the Study:
- To investigate the presence and function of cyclic AMP-dependent protein kinase in cardiac microsomes.
- To determine the effect of protein phosphorylation on calcium uptake by cardiac microsomes.
- To elucidate the role of reversible phosphorylation in regulating cardiac microsome calcium handling.
Main Methods:
- Isolation of cardiac microsomes.
- In vitro phosphorylation assays using cyclic AMP (cAMP).
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to identify phosphorylated proteins.
- Measurement of calcium uptake in phosphorylated and non-phosphorylated microsomes.
Main Results:
- Cardiac microsomes contain intrinsic cyclic AMP-dependent protein kinase activity.
- Phosphorylation targets a specific microsomal protein component of 20,000 molecular weight.
- Phosphorylation of this protein significantly enhances calcium uptake by cardiac microsomes.
- Microsomal membranes exhibit phosphoprotein phosphatase activity, leading to dephosphorylation.
Conclusions:
- Cardiac microsomes possess both cyclic AMP-dependent protein kinase and phosphoprotein phosphatase activities.
- Phosphorylation of a specific 20 kDa microsomal protein by cAMP-dependent kinase enhances calcium uptake.
- Reversible phosphorylation of the cardiac microsomal membrane is a key mechanism for cAMP-mediated regulation of calcium uptake.