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Purification of myosin light chain kinase from rabbit polymorphonuclear leukocytes
Abstract:
To investigate the regulation of actin-myosin interaction in rabbit phagocytic cells, purified myosin and a partially purified cofactor protein were obtained from polymorphonuclear leukocytes (PMN) and alveolar macrophages (ALM) by molecular sieve filtration over an agarose column. ALM cofactor enhanced the Mg++-ATPase activity of ALM myosin by actin to 0.15 +/- 0.04 mumoles Pi per mg myosin per min and PMN cofactor enhanced PMN myosin to 0.43 +/- 0.03 mumoles Pi per mg myosin per min. The crude cofactor preparations isolated from the two types of leukocyte extracts were interchangeable with the leukocyte myosins. When ALM cofactor was added to a PMN actomyosin complex, the Mg++-ATPase activity of the PMN myosin was 3-fold higher than with ALM cofactor and its own actomyosin complex. In contrast, PMN cofactor did not enhance ALM actomyosin Mg++-ATPase activity beyond that observed with ALM cofactor and ALM actomyosin. Cofactor protein from the PMN was further purified on a DEAE-Sephagel column. After electrophoresis on sodium dodecyl sulfate-polyacrylamide gel, the isolated fraction weighed 70,000 daltons. This fraction stimulated the actinmediated myosin Mg++-ATPase. In the presence of Mg++ and [gamma 32P]ATP, the 70,000 dalton protein phosphorylated the 20,000 dalton light chain of PMN myosin, leading to the incorporation of 0.62 +/- 0.09 moles of Pi per mole myosin. On the basis of these results, we propose that phagocytic cofactor is a kinase which regulates the enzymatic activity of phagocytic cell myosin.
Insights
Phagocytic cofactor protein acts as a kinase, regulating the enzymatic activity of myosin in immune cells like polymorphonuclear leukocytes and alveolar macrophages by phosphorylating myosin light chains.
Area of Science:
- Cell Biology
- Biochemistry
- Immunology
Background:
- Actin-myosin interaction is crucial for cellular functions, including phagocytosis.
- Regulation of myosin ATPase activity by cofactor proteins in phagocytic cells is not fully understood.
Purpose of the Study:
- To investigate the role of cofactor proteins in regulating actin-myosin interaction in rabbit phagocytic cells.
- To identify and characterize the cofactor protein involved in myosin activity regulation.
Main Methods:
- Purification of myosin and cofactor proteins from polymorphonuclear leukocytes (PMN) and alveolar macrophages (ALM) using molecular sieve filtration and ion-exchange chromatography.
- Assay of Mg++-ATPase activity of leukocyte myosins in the presence of actin and cofactor proteins.
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) to determine the molecular weight of the purified cofactor protein.
- In vitro phosphorylation assays using [gamma-32P]ATP to identify the target of the cofactor protein.
Main Results:
- Cofactor proteins from ALM and PMN enhanced the Mg++-ATPase activity of their respective myosins.
- Crude cofactor preparations were interchangeable between leukocyte myosins.
- Purified PMN cofactor (70,000 daltons) stimulated actin-mediated myosin Mg++-ATPase activity.
- The 70,000 dalton cofactor protein phosphorylated the 20,000 dalton light chain of PMN myosin.
Conclusions:
- Phagocytic cofactor protein functions as a kinase.
- This kinase activity regulates the enzymatic activity of phagocytic cell myosin through phosphorylation of its light chain.