1988 Fiscal Year Final Research Report Summary
Study on the structural and functional significance of the double-heads of myosin with the use of selective and reversible affinity labeling.
| Project/Area Number |
60440107
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| Research Category |
Grant-in-Aid for General Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Research Field |
分子遺伝学・分子生理学
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| Research Institution | Kyushu University |
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Principal Investigator |
TAWADA Katsuhisa Kyushu University, Faculty of Science, Department of Biology., 理学部, 助教授 (20029507)
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| Project Period (FY) |
1985 – 1988
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| Keywords | muscle contraction / myosin / double-heads / myosin subfragment-1 / ATPase / active site / heterogeneity / ADP親和性カラム |
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| Research Abstract |
A myosin molecule consists of two globular heads (S1): a conserved structure. Each head has an active site for ATPase activity. The final goal of our research project is to elucidate the significance of this double-headed structure. We invented a kinetic method to test whether the active site of myosin is enzymatically homogeneous or not, by using vanadate plus ADP as a reversible affinity labelling. The method revealed that S1, modified by an SH-reagent, contains at least two (1:1) different species. Later, we succeeded in separating these two species of S1 into two fractions by an ADP-column chromatography. Subsequent studies showed that these two S1s manifest "two states" S1 takes before the SH-modification. Recent electron microscopic observation of myosin heads by other research group has shown that myosin heads take two (1:1 different) distinct shapes, which may correspond to our two fractions of S1. Each myosin head has one highly reactive lysin residue (RL), which can be modified by TNBS. It was shown previously that the number of RL modified by TNBS is reduced to 0.5 (M/M) in the presence of MgPPi, and this observation suggested that two "structurally different" S1s exist. To see the relation of this observation with our above-mentioned finding, we stated to re-investigate the TNBS-modification of S1. To do it, we constructed a system for the precise data-collection and analysis of the TNBS reaction with a combination of a spectrophotometer with a computer. We found that RL of only half of added sl is modified by TNBS in the presence of mgADP as in the presence of MgPPi. This result suggests the existence of two distinct S1s which are different from each other with regards to the ADP-binding effect. We are now attempting to separate these two S1s by column chromatography, to elucidate the relationship of these two different S1s with the double-headed structure of the myosin molecule.
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