Shotaro Kitagawa

The initial phase of actomyosin-adenosinetriphosphitase

Abstract 1. 1. By means of an apparatus constructed for the measurement of rapid reactions, ATPase activity in the first few seconds (the initial phase) of the reaction has been quantitatively investigated. 2. 2. In the initial stage the rate of P-liberation was about 5 times as high as the rate in the stationary state. 3. 3. Under our experimental conditions, the maximum amount observed in the initial, additional burst of P-liberation was even equivalent to about 1 3 of the terminal P of added ATP. Therefore, this phenomenon cannot be attributed to an impurity of the ATP. 4. 4. From an experiment using 32P-labelled ATP as substrate, it has been confirmed that the initial burst of P does not originate in P bound previously to the protein. 5. 5. The Michaelis constant of the initial ATPase is 1.3·10−4M. The activation energy is 11.0 kcal, which is smaller than that of the stationary ATPase, 13.7 kcal. No difference has been observed between the pH-dependency of the initial and the stationary states. 6. 6. EDTA prevents the transition of ATPase activity. DNP and PCMB enhance both the initial and the stationary ATPase.

Binding of p-nitrothiophenol to myosin a

Abstract The p -nitrothiophenol-myosin A compound was produced by reaction of p -nitrothiophenol with myosin A in the presence of Mg 2+ and ATP, and was isolated by gel filtration through a Sephadex column. The change in absorption spectrum by the binding of p -nitrothiophenol to myosin A was similar to that of the formation of a thiolester bond between p -nitrothiophenol and succinic anhydride. The p -nitrothiophenol-myosin A compound was stable at acidic and neutral pH, but was unstable at alkaline pH. The stability was not affected by replacement of air with N 2 gas. The p -nitrothiophenol bound to myosin A was easily displaced by cysteine and H 2 S. p -Nitrothiophenyl-peptides were isolated with 24% recovery after proteolysis and chromatography on talc, since the proteolysis did not accelerate the liberation of p -nitrothiophenol. The hydromate was formed quantitatively by the addition of NH 2 OH to p -nitrothiophenyl-peptides. The amino acid composition of the p -nitrothiophenyl-peptides was determined, and Arg, Asp, Ser, Glu, Ileu and Leu were found as amino acid residues whose mole ratios to p -nitrothiophenol were nearly equal to or higher than 1. These results establish the covalent binding of p -nitrothiophenol to myosin A in the presence of Mg 2+ and ATP, and strongly suggest the binding of p -nitrothiophenol on a carboxyl side-chain of Glu or Asp in myosin A as an acylthiol bond. Effects of divalent cations, pH and modifiers of the ATPase activity of the p -nitrothiophenyl-myosin A were also investigated. The pH-activity curve of the p -nitrothiophenyl-myosin A was similar to those of the p -chloromercuribenzoate-myosin A compound and the trinitrophenyl-myosin A, and showed no depression at neutral pH. The ATPase (ATP phosphohydrolase, EC 3.6.1.3) of the p -nitrothiophenyl-myosin A was activated by p -chloromercuribenzoate or EDTA, whereas the ATPase of the trinitrophenyl-myosin A was not activated. Actomyosin reconstituted from the p -nitrothiophenyl-myosin A showed ATPase activity of the typical actomyosin type.

The initial phase of actomyosin-adenosinetriphosphatase: II. Factors influencing activity

Abstract 1. 1. The same initial rapid splitting of ATP by myosin B is repeatedly obtained upon adding fresh ATP, if the ATP added previously has been completely hydrolyzed. 2. 2. The quantity of the initial rapid splitting of ITP is very small, as compared with that of ATP. 3. 3. The initial ATP hydrolysis by non-dialysed myosin B preparations is only slightly influenced by the addition of Ca ++ or Mg ++ . 4. 4. The initial burst of P-liberation is annihilated by dialysis of the myosin. It is restored by adding Mg ++ or Mn ++ , but not by adding Ca ++ . 5. 5. It is also abolished by adding small amounts of EDTA, capable of chelating only a negligible part of the Mg ++ present. It is also decreased by the addition of p -chloromercuribenzoate. 6. 6. When sufficient Mg ++ is added to a reaction mixture containing ATP and EDTA, a burst of P-liberation is observed.

The effect of EDTA on the interaction between actomyosin and adenosine triphosphate

Abstract 1. 1. In the presence of 0.6M of KCl, EDTA increases concomitantly the Vmax and Km of myosin B ATPase. Especially when concentration of EDTA is higher than 5mM, the line 1/v versus 1/[S] passes through the origin. 2. 2. EDTA decreases both grade and rate of the optical change of myosin B caused by ATP addition. 3. 3. The recovery step from the physically changed myosin B to the original myosin B follows strictly monomolecular kinetics. 4. 4. On the basis of these results the reaction mechanism of EDTA with the myosin B-ATP system is discussed.

Interaction between synthetic ATP analogues and actomyosin systems. II

Abstract The following compounds were synthesized chemically as analogues of ATP; 9-(2′,3′- di -O- acetyl )- D - erythrityladenine 4′-triphosphate (erytrityladenine-TP), 9-β- D -glucopyranosyladenine 6′-triphosphate (glucosyladenine-TP), I -β- D -ribofuranosyl 4-amino-benzimidazole 5′-triphosphate (4-aminobenzimidazoleriboside-TP) and N6-monomethylcytidine 5′-triphosphate (methyl CTP). The reactions of these analogues with myosin B or myofibrils were investigated. The strength of binding to myosin B, as judged by the change in light scattering of myosin B solution, was in the decreasing order of ATP >/ methyl CTP > glucosyladenine-TP > 4-aminobenzimidazole-riboside-TP > erythrityladenine-TP. The velocity at the steady state of Pi-liberation by myosin B was in the order of methyl CTP > ATP > erthrityladenine-TP > 4-aminobenzimidazole-riboside-TP > glucosyladenine-TP in 0.6 M KCl-7 mM Ca2+, and in the order of ATP > methyl CTP > glucosyladenine-TP > erythrityladenine-TP > >4-aminobenziimidazole-riboside-TP in 0.075 M KCl-2 mM Mg2+, respectively. The contracting ability of the analogues for myofibrils was in the order of ATP > 4-aminobenzimidazole-riboside-TP ≈ methyl CTP > erthyrityladenine-TP. Glucosyl-adenine-TP did not cause myofibrils to contract.