Optical Spectroscopic Study of the ADP‐Myosin Interaction
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Abstract:
The binding of ADP to heavy meromyosin, and the separated subfragment 1 components S-1(A1) and S-1 (A2), has been observed by ultraviolet spectrophotometry. The results are compatible with the presence of spectroscopically equivalent and independent sites, one per head, at both 10 degrees C and 25 degrees C. We do not observe the heterogeneity of binding and of the spectroscopic response that has been reported. The binding has also been followed by other methods sensitive to the effect of ligand on the aromatic residues of the protein, viz. intrinsic fluorescence of heavy meromyosin and changes in the near-ultraviolet Cotton effects of myosin, and its active fragments. Within the limits of our experimental precision, the binding profiles, based on concentration of myosin heads, are the same for myosin as for subfragment 1. A perturbation in the circular dichroism is also generated by pyrophosphate, which competes with ADP. The spectra suggest that subsites for the purine ring and the diphosphate can be recognized. The sensitivity of binding profiles obtained by methods of the kind used here to cooperative or antagonistic interactions between the binding sites has been analysed. It is clear that sizeable effects of this nature could be concealed by the binding curves, even for high experimental precision.Keywords:
Heavy meromyosin
Adenosine diphosphate
Journal Article Presence of a Unit for Actin-Myosin Interaction during the Superprecipitation of Actomyosin Get access Tetsu HOZUMI, Tetsu HOZUMI Department of Physiology, Nagoya City University Medical SchoolMizuho-ku, Nagoya, Aichi 467 Search for other works by this author on: Oxford Academic PubMed Google Scholar Ken HOTTA Ken HOTTA Department of Physiology, Nagoya City University Medical SchoolMizuho-ku, Nagoya, Aichi 467 Search for other works by this author on: Oxford Academic PubMed Google Scholar The Journal of Biochemistry, Volume 81, Issue 4, April 1977, Pages 1141–1146, https://doi.org/10.1093/oxfordjournals.jbchem.a131539 Published: 01 April 1977 Article history Received: 04 October 1976 Published: 01 April 1977
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Myosin ATPase
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Background & Aims : Myosin II is a typical motor protein and is classified either as non-regulated myosin, for example, skeletal muscle myosin, or as myosin regulated by phosphorylation or Ca-binding. Myosin II from Physarum polycephalum is a Ca-binding myosin. Although expression of myosin II as a recombinant protein is essential for the analysis of its structure, function and regulation, it is not easy. Successful examples are smooth muscle myosin II and Dictyostelium myosin II, both of which are regulated by phosphorylation. Myosin II regulated by Ca-binding has not yet been obtained. Here, I report the expression of heavy meromyosin (HMM), i.e., the motor domain of Physarum myosin II.Method : I used baculovirus expression system. Sf9 cells were infected with the virus constructs.Results and Conclusions : The recombinant HMM showed basal Mg2+-ATPase activity, which was activated by actin to a similar extent to native myosin purified from Physarum. Ca-binding activity of HMM was also comparable to that of the native myosin. In vitro motility assay showed that motor activity of HMM was sensitive to Ca2+. The present study is the first report of expression of the motor domain of a myosin II that is regulated by Ca-binding.
Heavy meromyosin
Meromyosin
Physarum
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The relationship between the myosin ability to bind pyrophosphate and the protective effect of pyrophosphate on the activity of Ca 2+ ATPase during inactivation by 2′‐hydroxyethyl 2,4‐dinitrophenyl disulphide was studied. The diminution of myosin ability to bind pyrophosphate when myosin is treated with 2′‐hydroxyethyl 2,4‐dinitrophenyl disulphide is correlated with the decrease of Ca 2+ ‐ATPase activity and is more rapid if myosin is modified by this disulphide without pyrophosphate. The presence of ADP diminishes the protective effect of pyrophosphate on myosin Ca 2+ ‐ATPase when myosin is treated with 2′‐hydroxyethyl 2,4‐dinitrophenyl disulphide. A correlation has been observed between the influence of ADP on the ability of myosin to bind pyrophosphate as well as on the protective effect of myosin Ca 2+ ‐ATPase when myosin is treated with 2′‐hydroxyethyl 2,4‐dinitrophenyl disulphide. The protective effect of pyrophosphate was observed only when the pyrophosphate binding sites of myosin seemed to be saturated.
Dinitrophenyl
Myosin ATPase
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Purified skeletal muscle myosin was labeled with iodoacetamidofluorescein and microinjected into cultured chick myotubes. The fluorescent myosin analogue became incorporated within 10-15 min after injection, into either periodic (mean periodicity = 2.23 +/- 0.02 micron) bands or apparently continuous fibrillar structures. Comparison of rhodamine-labeled alpha-actinin with coinjected fluorescein-labeled myosin suggested that myosin fluorescence was localized at the A-bands of myofibrils. In addition, close examination of the fluorescent myosin bands indicated that they were composed of two fluorescent bars separated by a nonfluorescent line that corresponded to the H-zone. Once incorporated, the myosin underwent a relatively slow exchange along myofibrils as indicated by fluorescence recovery after photobleaching. Glycerinated myofibrils were able to bind fluorescent myosin in a similar pattern in the presence or absence of MgATP, indicating that actin-myosin interactions had little effect on this process. Fluorescent heavy meromyosin did not incorporate into myofibrillar structures after injection. Light meromyosin, however, associated with A-bands as did whole myosin. These results suggest that microinjected myosin, even with its relatively low solubility under the cytoplasmic ionic condition, is capable of association with physiological structures in living muscle cells. Additionally, the light meromyosin portion of the molecule appears to be mainly responsible for the incorporation.
Myofibril
Heavy meromyosin
Meromyosin
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Heavy meromyosin
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Heavy meromyosin
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Heavy meromyosin
Meromyosin
Myofibril
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Ca2+ -activated neutral protease (CAF) was capable of degrading myosin over a 200-fold range of protease concentrations. CAF selected the heavy chain of myosin, although either prolonged exposure to or high concentrations of the protease degraded the L1, but not the L2 or L3, light chains of myosin. The following results indicated that during the first hour of digestion, under conditions where native myosin was the substrate, CAF selected for the "head" region of the myosin heavy chain: (a) large heavy chain fragments of identical molecular weight were produced from filamentous and from soluble myosin; (b) light meromyosin was not a substrate; (c) agents known to bind to the head of myosin (actin, MgATP, and L2) had both a qualitative and quantitative effect on degradation; and (d) similar cleavage sites could be demonstrated for myosin and for heavy meromyosin (HMM) despite the fact that HMM was a much poorer substrate than myosin. This observation is interpreted as an indication that the conformation of myosin heavy chain is altered in the preparation of HMM. The principal cleavage sites on the heavy chain of myosin were 20,000, 35,000 and 50,000 D from the N-terminus, producing large fragments with molecular weights of 180,000, 165,000, and 150,000 which comprised a "nicked" species of myosin. This nicked species retained both normal solubility properties and normal hydrolytic activities. For this reason, it is concluded that "nicked myosin" is an important pathophysiological species.
Heavy meromyosin
Meromyosin
Cleavage (geology)
Myofibril
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Heavy meromyosin
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