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Abstract
Abstract Myosin was extracted from guinea pig polymorphonuclear leukocytes (granulocytes) homogenized in either 0.6 m KCl or 0.34 m sucrose solutions. It was purified by precipitation at low ionic strength, ammonium sulfate fractionation, and gel filtration with buffers containing KI and ATP. Myosin isolated from sucrose extracts of granulocytes consisted of essentially one polypeptide that co-migrated with the heavy chain of skeletal muscle myosin on sodium dodecyl sulfate acrylamide gel electrophoresis. Antibody against this protein was prepared in rabbits and reacted with it to yield one precipitin line by immunodiffusion. Myosin isolated from extracts of cells prepared with 0.6 m KCl was heterogenous by electrophoresis and immunodiffusion probably because of partial degradation by lysosomal enzymes solubilized by KCl but not by sucrose. All myosin preparations reacted with antiserum to yield at least two arcs on immunoelectrophoresis. Granulocyte myosin, like other myosins, had EDTA- and calcium- but not magnesium-activated ATPase activity in 0.6 m KCl, and it bound skeletal muscle F-actin in the absence but not the presence of ATP. At low ionic strength the Mg2+ATPase activity of granulocyte myosin was activated by skeletal muscle F-actin. This activation was not influenced by calcium. However, under the influence of skeletal muscle troponin-tropomyosin, activation of granulocyte myosin Mg2+ATPase by skeletal muscle F-actin occurred only after addition of calcium. In 0.12 m KCl, granulocyte myosin formed thin short bipolar filaments in the presence of divalent cations. Actin was identified in granulocyte extracts as a protein which comigrated with skeletal muscle actin on sodium dodecyl sulfate acrylamide gel electrophoresis and which formed filaments that bound granulocyte myosin to form polarized arrowhead structures.
Highlights
Myosin was extracted from guinea pig polymorphonuclear leukocytes homogenized in either 0.6 M KC1 or 0.34 M sucrose solutions
Myosin isolated from extracts of cells prepared with 0.6 M KC1 was heterogenous by electrophoresis and immunodiffusion probably because of partial degradation by lysosomal enzymes solubilized by KC1 but not by sucrose
Actin was identified in granulocyte extracts as a protein which comigrated with skeletal muscle actin on sodium dodecyl sulfate acrylamide gel electrophoresis and which formed filaments that bound granulocyte myosin to form polarized arrowhead structures
Summary
Granulocytes-Acute peritoneal exudates were induced in guinea pigs. Twenty to thirty female Hartley strain guinea pigs weighing 400 to 800 g each received intraperitoneal injections of 15 ml of autoclaved sodium caseinate, 120 mg per ml in 0.15 M NaCl [5]. Sixteen to twenty hours later the animals were killed, and the exudate cells were washed out of the peritoneal cavities with 0.15 M NaCl (25”). The cell suspensions were thereafter kept at ice bath temperature and were washed thrice with 10 to 21 volumes of 0.15 M NaCl by centrifugation at less than 100 x g. After the last wash the cell pellets were suspended in 10 to 15 volumes of distilled water, mixed by inversion for 30 s, and immediately centrifuged at 250 x g. In initial experiments 20 to 30 ml of packed granulocytes were suspended in 2 volumes of a solution containing 0.6 M KCI, 10 mM dithiothreitol, 2.5 mM ATP, 20 mM
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