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Macromolecular characterization of muscle membranes. Endogenous membrane kinase and phosphorylated protein substrate
Abstract:
Light density membranes derived from the "microsomal" fraction of rat skeletal muscle contained an endogenous protein kinase which catalyzed the phosphorylation of an endogenous membrane substrate. No other membrane fraction contained any significant protein kinase activity. The optimal specific activity of the enzyme in these membranes was 350 pmol/mg/min. The endogenous muscle membrane protein kinase required magnesium, was stimulated by micromolar concentrations of calcium, had a pH optimum between 7.0 and 7.5, and demonstrated a K-m for ATP of 2.6 times 10 minus 5 M. The enzyme was markedly heat labile and demonstrated a linear Arrhenius plot with an apparent energy of activation of 12,100 cal/mol. There was no stimulation by cyclic nucleotides; and neither monovalent cations nor various neurotransmitters exerted any effect. It is presently unclear where the membranes exhibiting protein phosphorylation are localized within the muscle fiber. Enzyme markers suggest that these membranes are not derived from sarcolemma or sarcoplasmic reticulum but may originate in transverse tubules. The membrane phosphorylation was largely confined to a polypeptide with an apparent molecular weight of 28,000. Phosphorylation could also be detected in a lower molecular weight substrate as well as two polypeptides with apparent molecular weights of 95,000 and 56,000. The M-r-28,000 endogenous protein kinase substrate was isolated by preparative gel electrophoresis in sodium dodecyl sulfate. High voltage electrophoresis of a partial acid hydrolysate of the phosphorylated M-r-28,000 substrate identified the phosphate bond to be that of phosphoserine. The amino acid composition of the substrate was neither strongly acidic nor basic. It had a high content of glycine, glutamic acid, serine, and lysine. Hydrophobic residues constituted only 45% of the total composition. Following muscle denervation for 10 days, there was a significant decrease in the amount of the M-r-28,000 polypeptide as well as the extent of phosphorylation.
Insights
Rat skeletal muscle microsomes contain a unique protein kinase that phosphorylates a 28,000 molecular weight substrate. Muscle denervation significantly reduces this protein and its phosphorylation, suggesting a role in muscle fiber integrity.
Area of Science:
- Biochemistry
- Cell Biology
- Muscle Physiology
Background:
- Skeletal muscle microsomes possess endogenous protein kinase activity.
- This activity is primarily localized to light density membranes.
- The specific membrane origin within the muscle fiber remains to be fully elucidated.
Purpose of the Study:
- To characterize the endogenous protein kinase and its substrate in rat skeletal muscle light density membranes.
- To investigate the properties and localization of this protein phosphorylation system.
- To assess the impact of muscle denervation on this system.
Main Methods:
- Isolation of light density membranes from rat skeletal muscle.
- Assay of protein kinase activity and characterization of enzyme kinetics (pH optimum, K-m for ATP, Mg2+ and Ca2+ dependence).
- Analysis of phosphorylated substrates using SDS-PAGE, isolation of M-r-28,000 polypeptide, and phosphoserine identification.
- Investigation of denervation effects on protein and phosphorylation levels.
Main Results:
- A specific protein kinase activity was identified in light density microsomal membranes, with optimal activity at pH 7.0-7.5 and a K-m for ATP of 2.6 x 10^-5 M.
- The enzyme required magnesium, was stimulated by calcium, and was heat labile.
- Phosphorylation primarily occurred on a M-r-28,000 polypeptide, identified as phosphoserine, with a distinct amino acid composition.
- Muscle denervation led to a significant decrease in both the M-r-28,000 polypeptide and its phosphorylation.
Conclusions:
- Rat skeletal muscle light density membranes possess a unique endogenous protein kinase system.
- The M-r-28,000 substrate phosphorylated by this kinase is sensitive to muscle denervation.
- These findings suggest a potential role for this protein phosphorylation in maintaining muscle fiber structure or function.