Purification of Myosin Light Chain Kinase from Rabbit Polymorphonuclear Leukocytes

Abstract

Summary: 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 μmoles Pi per mg myosin per min and PMN cofactor enhanced PMN myosin to 0.43 × 0.03 μmoles 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 actin-mediated myosin Mg++-ATPase. In the presence of Mg++ and [γ32P]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.

Speculation: The identification of actin, myosin, and related protein in eukaryotic cells raises questions as to the mechanisms involved in the control of these proteins. The results obtained in this study indicate that phosphorylation of the 20,000 daltons light chain may have a crucial role in regulating actin-myosin interaction in phagocytic cells.

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Correspondence to Laurence A Boxer.

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Yang, H., Boxer, L. Purification of Myosin Light Chain Kinase from Rabbit Polymorphonuclear Leukocytes. Pediatr Res 15, 229–234 (1981). https://doi.org/10.1203/00006450-198103000-00006

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Keywords

  • alveolar macrophages
  • kinase
  • myosin
  • PMN myosin light chain
  • polymorphonuclear leukocytes