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The identification and subcellular localization of thrombosthenin "M", the myosin-like component of pig platelets
Abstract:
A protein has been studied which spontaneously precipitates from stored fractions of platelet soluble phase prepared by density gradient centrifugation. It is rich in a Ca2+ ATPase activity which displays an activity/pH profile resembling that of skeletal muscle myosin. Adjustment of freshly prepared soluble phase fractions to 0.6 M with respect to KCl and dilution 1 in 3 results in the precipitations of a protein fraction with essentially the same enzymatic properties as the spontaneously precipitable protein. These two similar proteins represent between 9 and 13% of the soluble phase total protein and each account for almost the whole of divalent cation activated ATPase activity of the soluble phases from which they were derived. The Mg2+ ATPase activity is only about twice purified with respect to the soluble phase enzyme activity, but the Ca2+ ATPase shows a 10-13-fold enrichment. Synthetic actomyosins can be prepared from the two proteins by addition of either platelet or skeletal muscle actin. These show significant increases in Mg2+ ATPase at the most favourable combination ratios. The ratio between the yield of soluble phase protein obtained by dilution precipitation and the lactate dehydrogenase activity of the soluble phase remains constant under a wide range of homogenization and sonication conditions applied to the original whole platelet suspensions. This confirms our earlier view that the soluble phase is a valid intracellular compartment for a considerable proportion of the platelet contractile protein and that in the complex the myosin-like component predominates.
Insights
Researchers identified a myosin-like contractile protein in platelet soluble fractions. This protein possesses Ca2+ ATPase activity, similar to skeletal muscle myosin, and plays a significant role in platelet function.
Area of Science:
- Biochemistry
- Cell Biology
- Platelet Physiology
Background:
- Platelets contain contractile proteins, including a myosin-like component.
- The soluble phase of platelets is a compartment containing cellular proteins.
- Understanding platelet contractile proteins is crucial for platelet function research.
Purpose of the Study:
- To characterize a spontaneously precipitating protein from platelet soluble fractions.
- To investigate the enzymatic properties and purification of this platelet protein.
- To confirm the presence and significance of contractile proteins in the platelet soluble phase.
Main Methods:
- Density gradient centrifugation of platelet soluble fractions.
- KCl precipitation and dilution techniques for protein isolation.
- Enzymatic assays for Ca2+ and Mg2+ ATPase activity.
- Preparation of synthetic actomyosins with platelet or skeletal muscle actin.
- Lactate dehydrogenase activity assay for compartment validation.
Main Results:
- A protein rich in Ca2+ ATPase activity was identified, resembling skeletal muscle myosin.
- Two similar proteins, representing 9-13% of soluble phase protein, were isolated.
- Ca2+ ATPase activity showed a 10-13-fold enrichment, while Mg2+ ATPase had a twofold purification.
- Synthetic actomyosins demonstrated increased Mg2+ ATPase activity.
- Lactate dehydrogenase ratio confirmed the platelet soluble phase as a valid compartment for contractile proteins.
Conclusions:
- The identified protein is a significant myosin-like contractile component within the platelet soluble phase.
- This contractile protein possesses distinct Ca2+ ATPase characteristics.
- The platelet soluble phase serves as a key intracellular compartment for these contractile proteins.