Purification of myosin light chain kinase from rabbit polymorphonuclear leukocytes.
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.
D000205:Actomyosin / Q000378:metabolism; D000818:Animals; D008264:Macrophages / Q000032:analysis / Q000201:enzymology; D009218:Myosins / Q000378:metabolism*; D009504:Neutrophils / Q000032:analysis / Q000201:enzymology*; D010770:Phosphotransferases / Q000302:isolation & purification*; D011494:Protein Kinases / Q000378:metabolism; D011650:Pulmonary Alveoli / Q000032:analysis / Q000166:cytology; D011817:Rabbits
D000205:Actomyosin; D010770:Phosphotransferases; D011494:Protein Kinases; D009218:Myosins