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Purification, phosphate content and phosphorylation of myosin from human vascular smooth muscle
S Fazekas1, G L Nádasy, E Monos
12nd Institute of Biochemistry, Semmelweis University Medical School, Budapest, Hungary.
Abstract:
Myosin was prepared from human umbilical artery and some of its properties were studied. The yield of myosin after ultracentrifugation was 4.3-13.6 mg/gr wet weight of tissue (mean: 8.6 mg/gr). A considerable amount of phosphate bound to the myosin was found in all preparations. Phosphate content of umbilical arterial myosin was higher than what had been found in our earlier works in skeletal muscle myosin. Phosphate content of umbilical arterial myosin also could be further increased by incubation in ATP containing media. While the rate of phosphorylation of human umbilical arterial myosin was slower than that of skeletal muscle myosin, the saturated phosphate levels reached with 0.5 mM ATP concentration were higher for vascular myosin. The slower phosphate uptake rate of vascular myosin may be related to the slower contraction rate of this muscle type. After alkaline hydrolysis of lipid free myosin, 7 or 8 peaks of N-phosphoryl amino acids and phosphorylated amino acid derivates could be separated by ion exchange chromatography.
Insights
Human umbilical artery myosin exhibits higher phosphate content than skeletal muscle myosin. This vascular myosin
Area of Science:
- Biochemistry
- Molecular Biology
- Vascular Physiology
Background:
- Myosin, a key contractile protein, plays a crucial role in muscle function.
- Understanding myosin properties in vascular tissues is essential for comprehending vascular tone and function.
- Previous studies have characterized skeletal muscle myosin, but vascular myosin properties remain less explored.
Purpose of the Study:
- To isolate and characterize myosin from human umbilical artery.
- To investigate the phosphorylation properties of vascular myosin.
- To compare the phosphorylation characteristics of umbilical arterial myosin with skeletal muscle myosin.
Main Methods:
- Myosin isolation from human umbilical artery via ultracentrifugation.
- Determination of myosin yield and bound phosphate content.
- Incubation of myosin in ATP-containing media to assess phosphorylation.
- Analysis of phosphorylated amino acids using ion-exchange chromatography after alkaline hydrolysis.
Main Results:
- Myosin yield from human umbilical artery was 4.3-13.6 mg/gr wet weight.
- Umbilical arterial myosin demonstrated higher intrinsic phosphate content compared to skeletal muscle myosin.
- Vascular myosin showed a slower rate of phosphorylation but reached higher saturated phosphate levels with ATP.
- Ion-exchange chromatography revealed 7-8 peaks of N-phosphoryl amino acids and derivatives.
Conclusions:
- Human umbilical artery myosin is a phosphoprotein with distinct phosphorylation characteristics.
- The observed phosphorylation patterns may correlate with the slower contraction rate of vascular smooth muscle.
- These findings contribute to the understanding of vascular myosin's biochemical properties and functional implications.