Purification and properties of Ca2+-regulated thin filaments and F-actin from sheep aorta smooth muscle

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Abstract
Summary We have investigated the conditions for isolation of Ca2+-regulated thin filaments from sheep aorta. Inhibition of proteolysis by 2 µg ml−1 leupeptin and chymostatin and of oxidation with 5mm dithiothreitol were essential. Washed homogenates were extracted in 10mm ATP of low ionic strength at pH 6.1 to minimize coextraction of myosin with thin filaments. Thin filaments were separated from myosin by high speed sedimentation; 20% glycol was added to prevent loss of regulatory factors and tropomyosin. The resulting thin filaments (yield 2.5 mg protein g−1 artery wet weight) were made up of actin, tropomyosin and a 120 000M r protein (molar ratio 1:1/5:1/29) and were up to 4 µm long. They activated skeletal muscle myosin at least 50 times in presence of Ca2+. Up to 80% inhibition was observed in the absence of Ca2+. We also prepared pure arterial F-actin, which activated skeletal myosin more than the thin filaments, but was similar to skeletal F-actin. We conclude that Ca2+ regulation is negative, involves cooperative interactions between actin, myosin and tropomyosin and suggest that it is mediated by the 120 000M r protein.