Peleg I, Muhlrad A, Eldor A, Groschel-Stewart U, Kahane I
Arch Biochem Biophys. 1984 Nov 1;234(2):442-53. doi: 10.1016/0003-9861(84)90291-1.
Myosin was purified from the membrane fraction and the cytoplasm of human platelets, and the K+(EDTA)- and Ca2+-dependent ATPase activities were studied under various experimental conditions. The ATPase activity of the myosin from the membrane fraction was slightly lower than that of its cytoplasmic counterpart, regardless of the different assay conditions (pH, ionic strength, and temperature). Both myosins showed the same pH optima and a similar ionic strength dependence for the two ATPase activities measured. In addition, they exhibited the same substrate specificity using ATP, CTP, and GTP as substrates. The activation energy of the Ca2+-dependent ATPase activity was essentially the same for the two myosins, while the activation energy of the K+(EDTA)-dependent ATPase activity of the membrane myosin was higher than that of the cytoplasmic myosin. The ATPase activity of the membrane myosin was found to be more sensitive to freezing and thawing than the cytoplasmic myosin. The alkylation of the thiol groups by N-ethylmaleimide or N-iodoacetyl-N-(5-sulfo-1-naphtyl)ethylenediamine, and the trinitrophenylation of the lysyl residues by 2,4,6-trinitrobenzenesulfonate caused a significant decrease in the K+(EDTA)-dependent ATPase activity of the two myosins. However, the membrane myosin was much less affected than the cytoplasmic myosin. Actin induced inhibition of the K+ (EDTA) ATPase of both myosins, and much smaller quantities of actin were needed to inhibit the cytoplasmic myosin ATPase compared to quantities needed to inhibit the myosin ATPase from the membrane fraction. This indicates that the membrane myosin has a lower affinity toward actin. The observed variations in the ATPase activity of the myosins isolated from the membrane and the cytoplasm fractions of human platelets may reflect differences in their respective physiological functions.
从人血小板的膜部分和细胞质中纯化肌球蛋白，并在各种实验条件下研究其K⁺（EDTA）依赖性和Ca²⁺依赖性ATP酶活性。无论测定条件（pH、离子强度和温度）如何，膜部分的肌球蛋白的ATP酶活性略低于其细胞质对应物。两种肌球蛋白对所测的两种ATP酶活性均表现出相同的pH最适值和相似的离子强度依赖性。此外，它们以ATP、CTP和GTP作为底物时表现出相同的底物特异性。两种肌球蛋白的Ca²⁺依赖性ATP酶活性的活化能基本相同，而膜肌球蛋白的K⁺（EDTA）依赖性ATP酶活性的活化能高于细胞质肌球蛋白。发现膜肌球蛋白的ATP酶活性比细胞质肌球蛋白对冻融更敏感。N-乙基马来酰亚胺或N-碘乙酰-N-（5-磺基-1-萘基）乙二胺对巯基的烷基化，以及2,4,6-三硝基苯磺酸对赖氨酰残基的三硝基苯化导致两种肌球蛋白的K⁺（EDTA）依赖性ATP酶活性显著降低。然而，膜肌球蛋白受影响的程度远小于细胞质肌球蛋白。肌动蛋白诱导两种肌球蛋白的K⁺（EDTA）ATP酶活性受到抑制，与抑制膜部分的肌球蛋白ATP酶活性所需的量相比，抑制细胞质肌球蛋白ATP酶活性所需的肌动蛋白量要少得多。这表明膜肌球蛋白对肌动蛋白的亲和力较低。从人血小板的膜部分和细胞质部分分离得到的肌球蛋白的ATP酶活性的观察到的变化可能反映了它们各自生理功能的差异。
