Biomedical subjects
Publications and source records attributed to F Morita.
The alkali light chain, A2, in subfragment-1 (S-1) was exchanged with A1 added externally in NH4 + -NH3 buffer (pH 9.9). The exchange yield was higher than 80% using only 2-fold molar excess of A1 over S-1 containing A2. The ATPase activities of the exchanged S-1 (A1) were the same as those of untreated S-1 (A1). The method was also applicable to exchanging the alkali light chains in myosin.
F-Actin bindings to subfragment-1 (S-1) and S-1 after limited proteolysis by trypsin (S-1t) were studied in the absence and presence of ATP by means of ultracentrifugation. No significant difference in the affinities for F-actin was observed between S-1 and S-1t in the absence of ATP. In contrast, the affinity for F-actin in the presence of ATP was decreased about 50 times by the limited proteolysis of the S-1 heavy chain. The S-1 whose SH1 and SH2 groups were cross-linked by N,N'-p-phenylenedimaleimide bound F-actin weakly. The affinity for F-actin was similar to that of unmodified S-1 in the presence of ATP and was also decreased markedly by limited proteolysis of the cross-linked S-1. Reciprocals of the dissociation constant of acto-S-1 complex decreased markedly with increase of ionic strength in the presence of ATP, but decreased only slightly at the rigor state. All these results are consistent with our proposal that S-1 has two different actin binding sites, as reported previously (Katoh, T., Imae, S., & Morita, F. (1984) J. Biochem. 95, 447-454). The mechanism of activation of S-1 ATPase by F-actin is discussed.
Tyr 180 of chicken breast muscle alkali light chain A1 was nitrated with tetranitromethane. The nitroA1 was incorporated into chicken breast muscle subfragment-1 (S-1) by exchange with the intrinsic alkali light chain. In the presence of adenylylimidodiphosphate (AMPPNP) or ADP, the S-1 containing nitroA1 showed a difference visible absorption spectrum by Mg2+ or Ca2+. The difference spectrum has a trough around 435 nm, indicating a blue shift of the absorption spectrum due to the nitrophenol chromophore of the modified A1. The plot of delta A at 435 nm versus concentration of free Mg2+ fitted a single binding curve, independent of the total concentration of AMPPNP. These results reveal that free Mg2+ binds to the active site of S-1 ATPase, but not as Mg-AMPPNP complex. The dissociation constants of magnesium from S-1 complex were different with the two nucleotides and were 1.25 X 10(-8) M and 1.24 X 10(-7) with AMPPNP and ADP, respectively. The difference spectrum was also obtained in the presence of ATP. The delta epsilon value after adding ATP changed with the ATPase reaction. The steady state rate of S-1 ATPase was measured at various concentrations of free Mg2+. The dissociation constant of magnesium from the steady state complex, EPADP(a), was estimated as 6 X 10(-8) M. These results suggest that the affinity of magnesium at the active site of ATPase changes with the intermediate states of ATPase reaction. The affinity of calcium was lower than that of magnesium.
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Myosin purified from the smooth muscle of scallop adductor contains two kinds of regulatory light chain, regulatory light chain a (RLC-a) and regulatory light chain b (RLC-b) (Kondo, S. & Morita, F. (1981) J. Biochem. 90, 673). The myosin was fractionated by salting out with ammonium sulfate, and samples containing the two regulatory light chains with different molar ratios were obtained. ATPase activities of the myosin fractions were determined. From the analysis of the dependence of ATPase activity on molar ratio of the two regulatory light chains, we concluded that myosin purified from the smooth muscle of scallop contains three species of myosin having different combinations of regulatory light chains: one has two RLC-a (aa), another has two RLC-b (bb), and the third one each of RLC-a and RLC-b (ab). The order of ATPase activities of these three myosin species was estimated as (aa) less than (bb) less than (ab). Distribution of the two regulatory light chains in the smooth muscle from the inside, translucent portion to the outside, opaque portion was examined by means of one- and two-dimensional gel electrophoreses. The content of RLC-b was about 1 mol per mol of SH-light chain independent of the portion of muscle. The content of RLC-a was markedly dependent on the portion of muscle--about 0.2 mol per mol of SH-light chain in the innermost portion and 0.7 mol per mol of SH-light chain in the outside, opaque portion. The sum of both regulatory light chain contents was about 1.5 mol per mol of SH-light chain in the opaque portion where the catch contraction is notable. Myosin species in the catch muscle are discussed.
1. Difference UV absorption spectra of chicken breast g1 induced by magnesium and calcium ions were compared with those of rabbit skeletal alkali light chains. The value of delta epsilon due to the burial of Tyr 180 of domain 4 was about one-fourth of that of rabbit alkali light chains. Regions other than domain 4 appear to affect the environment of Tyr 180, since the first-order structure of domain 4 of chicken g1 is present in rabbit g1 without significant alteration (Matsuda, G. et al. (1981) FEBS Lett. 126, 111). Movement of a phenylalanyl residue was suggested to be larger in chicken g1 than rabbit g1. 2. CD spectra of alkali light chains were measured in the wavelength region down from 260 nm. Divalent metal ions increased the helix content of alkali light chains about 2%. CB1 peptide of rabbit skeletal g1 containing domain 1 showed a slight change of CD spectrum in the presence of divalent ions. These results suggest that domain 1 of alkali light chains binds divalent ions. 3. Binding of calcium ions to rabbit skeletal g1 was directly measured by means of a calcium ion selectrode. More than 3 mol of calcium was bound per mol of g1. The concentration of free calcium ions required to give a half-maximal binding was 5 mM assuming that the maximum binding number is 4 mol of calcium per mol of g1. 4. Nitration of tyrosyl residues in rabbit skeletal g1 may slightly affect the affinity for divalent metal ions and the backbone structure of the polypeptide chain of g1.
Smooth muscle myosin purified from the small adductor of scallop, Patinopecten yessoensis, showed four distinct bands in the region of low molecular weight upon urea-gel electrophoresis. The two components showing the lowest mobilities in the electrophoresis were assigned as regulatory light chains and named as RLC-a and RLC-b. The component showing the highest mobility was SH-light chain, and the other component was not identified. Regulatory light chains of myosin prepared from the opaque portion of smooth muscle were estimated to be 40% RLC-a and 60% RLC-b, while those of myosin from the translucent portion were 20% RLC-a and 80% RLC-b. The rates of both myosin and actomyosin ATPases and of superprecipitation of actomyosin appear to be lower with myosin having RLC-a than with myosin RLC-b. Myosin having RLC-a might be involved in catch contraction, which is seen mainly in the opaque portion of smooth muscle. Amino acid analysis revealed a marked difference in Arg contents between the two regulatory light chains. The amino acid composition of SH-light chains of smooth myosin was almost the same as that of striated myosin of scallop except for a slight difference in Asp content.
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Binding of divalent metal ions to the g1 subunit isolated from myosin was shown by the difference UV-absorption spectrum. The difference spectrum indicated movement of a tyrosyl residue of g1. The residue responsible for the difference spectrum was assigned to be 180 Tyr.
A difference UV-absorption spectrum induced by ATP was observed with scallop myosins purified from both striated and smooth adductor muscles. The difference spectra showed a positive peak at 289 nm with a shoulder around 295 nm, and two small negative troughs around 280 nm. Tryptophanyl movement similar to that in rabbit skeletal myosin is indicated. Some tyrosyl movements in scallop myosins, however, may be in the opposite direction.
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The pH-activity curve of heavy meromyosin ATPase [EC 3.6.1.3] was measured at various temperatures. The pH-activity curve at higher temperatures showed a maximum at low pH and a minimum at pH 7 to 8 as has been already reported. At lower temperatures it was sigmoidal in shape, similar to a simple dissociation curve of pKa 6 to 7. The pH-activity curve at intermediate temperatures appeared to be inbetween the two extreme shapes. These changes in pH-activity curve with temperature were found to be common in the presence of divalent cations such as Mg2+, Mn2+, and Ca2+. The ATPase mechanism may be identical in the presence of any divalent cation, and the rate determining step revealing the steady state rate alters by changing the temperature. The transition temperatures estimated at pH 8 were 10 degrees, 8 degrees, and about 5 degrees in the presence of MnCl2, CaCl2, and MgCl2, respectively. The difference in the temperature coefficients above and below the transition temperature was most distinct in the presence of MnCl2, and vague in the presence of CaCl2. A similar change of pH-activity curve with temperature was found with heavy meromyosin ITPase in the presence of MgCl2.
The UV absorption difference spectrum of heavy meromyosin induced by ATP was measured at various temperatures. At higher temperatures, the difference spectrum formed rapidly after adding ATP and continued steadily during the steady state which we have called the ATP-form of difference spectrum. At lower temperatures, the ATP-form of difference spectrum decayed into the other form before the steady state was attained. This was identical to the difference spectrum obtained by adding ADP and has been called the ADP-form of difference spectrum. At intermediate temperatures, biphasic decay was observed. The results indicate that the dominant intermediate at the steady state is altered from the one showing the ATP-form of difference spectrum at higher temperatures to that showing the ADP-form at lower temperatures. The population of the two intermediates depends on the temperature between the two extremes. This temperature-induced transition was observed in the presence of any divalent cation such as Mg2+, Mn2+, or Ca2+. A similar transition was observed with the difference spectrum induced by ITP in the presence of MgCl2. The pH dependence of the single early decay of the ATP-induced difference spectrum was measured in the presence of MnCl2 at 1 degree. The apparent rate constant of the decay showed a biphasic pH dependence, having the same shape as the pH activity curve of ATPase [EC 3.6.1.3] observed at higher temperatures. The rate determining step for the steady state ATPase at higher temperatures is thought to be the step of changing from the intermediate complex showing the ATP-form of difference spectrum to that showing the ADP-form. This is inconsistent with our previous mechanism (Yazawa, M. et al. (1973) J. Biochem. 74, 1107-1117). The rate determining step at lower temperatures was assigned as a step of ADP dissociation.
The UV absorption difference spectrum of heavy meromyosin induced by adenylyl imidodiphosphate (AMP-PNP) was found to be changed by temperature. At higher temperatures, the shape of the difference spectrum resembled the ATP-form of difference spectrum induced by ATP. At lower temperatures, a different shape was observed, resembling that induced by ADP. This temperature transition was found in the presence of both MgCl2 and MnCl2. The transition temperatures, were 21 degrees and 9 degrees in the presence of MnCl2 and MgCl2, respectively. A similar temperature dependence was observed with the difference spectrum induced by ATP at the steady state. The transition temperatures in this case were 11 degrees and 4.5 degrees in the presence of MnCl2 and MgCl2, respectively. The similarity of the effects of the two kinds of divalent cation on both transitions indicates that the temperature induced transition between two species of heavy meromyosin-AMP-PNP complex mimics the step in APTase [EC 3.6.1.3] reaction in which the intermediate complex showing the ATP-form of difference spectrum changes to that showing the ADP-form. The equilibrium constant of the decay step of the ATP-form of difference spectrum to the ADP-form in ATPase is, therefore, thought to be highly temperature dependent. Thermodynamic parameters were calculated for the transition between the two species of heavy meromyosin AMP-PNP complex. Large decreases in enthalpy and entropy were observed, while the standard free energy change was small. The results suggest that the intermediate showing the ATP-form of difference spectrum hardly changes to the forward direction in the ATPase reaction at higher temperature. The complex appears to be so stable in the steady state that almost all the myosin is present as this complex. The decay step in ATPase of the difference spectrum from the ATP-form to to the ADP-form may be coupled to muscular contraction. The temperature induced transition of heavy meromyosin AMP-PNP complex may, therefore, provide information concerning the state of myosin in active muscles.
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Subfragment-1 prepared by chymotryptic digestion of myosin was applied to a column of Sepharose-adipic acid hydrazide-ATP in 1 mM EDTA, 10 mM Tris-HCL (PH 7.6), and 40 mM KCL. Ninety-nine per cent of subfragment-1 was adsorbed on the column in this medium. Fourty-three per cent of the applied protein was eluted with 6 mM ADP in the above buffer and then 52% was eluted with 1 mM EDTA, 10 mM Tris-HCL (pH 7.6), AND 0.7 M KCL. The former fraction contained g3 chain and the latter g1 chain. These fractions were apparently the same as the components, p2 and p1, respectively, isolated by ion-exchange chromatography using DEAE-cellulose (Yagi & Otani (1974) J. Biochem. 76, 365-373). No significant difference of ADP binding was found between the two fractions, both could bind about 0.5 mole per 10(5) g of protein. The preparation of the two subfragment-1 fractions is described.