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Abalone myosin was digested with proteases, (trypsin, chymotrypsin, nagarse, papain) in the presence of CaCl2 or EDTA. The time course of the digestion and the properties of degradation products were investigated by SDS-polyacrylamide gel electrophoresis. In the presence of CaCl2, myosin light chains were not digested, but in the presence of EDTA, regulatory light chains disappeared at first, followed by SH light chains. Heavy meromyosin (HMM) was obtained by tryptic and chymotryptic digestion of abalone myosin. These HMM had multiple cleavages in the heavy chain (HC) region. Subfragment-1 (S1) was also obtained by chymotryptic, nagarse and papain digestion and by tryptic digestion in the presence of EDTA. The molecular weight of S1 HC was 90-100 kilodalton (kD). Papain S1 obtained in the presence of CaCl2 had a single HC (94kD) and two light chains, but other S1 had intra-cleavages in the HC region. These results indicate that abalone myosin has protesae sensitive region whose cleavage leads to HMM or S1, like skeletal muscle myosin, homogeneous S1 preparation is obtained by diges-tion with papain.

Actomyosin was extracted from smooth muscle of molluscan abalone with 0.1 M PPit pH 6.4. Myosin was separated from the actomyosin by centrifugation at 100,000 X g in the presence of 5 mM ATP and 10 mM MgCl2. Myosin in the supernatant was further purified by gel filtration on a Sepharose 4B column. Paramyosin contamination of the actomyosin preparation interfered with the isolation of myosin and complete removal of actin and paramyosin from the myosin has not been accomplished. The myosin appeared to consist of a single f-chain and a single g-chain, as examined by SDS-disc electrophoresis in 8 or 13.7% acrylamide gel. The ATPase [EC 3.6.1.3] activity of this myosin in 0.5 M KCL at neutral pH and at 0 degrees was rather unstable and decreased by 10-20% per day. The effects of rho-chloromercuribenzoate and EDTA on the ATPase activity were similar to those observed with other smooth muscle myosin but the dependence upon pH or KCL concentration was different.

Myosin subfragment-1 (S1), which has one heavy chain (HC) (93 kDa) and two light chains (LC1 and LC2), was prepared by papain digestion of myosin from abalone-smooth muscle in the presence of Ca2+. The Ca-sensitivity of abalone S1 itself was not lost completely (about 30%). The tryptic digestion of S1 showed that in the presence of EDTA, S1 HC was split into 68, 55, and 23 kDa fragments, as in the presence of Ca2+, but 23 kDa was further degraded into 19 kDa. In contrast to the result in the presence of Ca2+, LCs disappeared in the early stage of reaction and Ca-ATPase activity decreased rapidly to about 70% of that of intact S1. This rapid decrease of Ca-ATPase activity seemed to be accompanied with the digestion of LCs. Therefore, LCs contribute to the protection of 23 kDa fragment from further digestion, to the maintenance of Ca-ATPase activity by stabilizing the structure of S1 to some extent in the presence of Ca2+. Since F-actin suppressed the cleavage of S1 HC to 68 and 23 kDa during tryptic digestion, it might be that 23 and 68 kDa corresponded to 20 kDa (C-terminal fragment) and to 50 + 25 kDa (N-terminal fragment) of skeletal myosin S1, respectively.

Abalone myosin was prepared by a more speedy method. The properties of this myosin were studied under various conditions with regard to their Mg-, Ca-, EDTA-ATPase and actin-activated ATPase activities. The properties of abalone myosin ATPase were almost similar to those of scallop myosin, though the following differences were observed. 1. Ca-sensitivity of abalone myosin was higher than that of scallop myosin. 2. At neutral pH and 25°C, enzymatic activities were lower than those of scallop myosin, except in EDTA-ATPase activity. 3. PH activity curve of Mg-ATPase activity at low ionic condition was an U-shaped curve in contrast to the biphasic curve in scallop myosin. PH activity curves of EDTA-ATPase activity were bell-shaped curves with a peak at pH around 8.0, which was higher than that of scallop my-osin. 4. Arrhenius plots of abalone myosin ATPase activities showed no bending between 5-30°C which was different from that of scallop myosin. The spccific cystein residue, so-called SHI was not dotected by the treatment of abalone myosin with NEM or PCMB.

The Michaelis constant ( K m ) and the maximum velocity ( v m ) of myosin A-ATPase (EC 3.6.1.3) and of myosin A-ITPase were measured in 0.6 M KCl, and in the presence of 7 mM CaCl 2 , over a wide range of temperature and pH. The following results were obtained: 1. 1. Both v m and K m of ITPase were increased proportionally to each other with increasing pH, and their half-saturation values were obtained at pH 6.8. The Arrhenius plots of v m and K m were readily represented by two reactions of different activation energies and the extrapolations of the linear portions intersected in each case at around 15°. The value of activation energies were 25.6 and 11.9 kcal/mole for v m and 7.9 and 2.1 kcal/mole for K m below and above 15°, respectively. 2. 2. At 20°, the K m of ATPase changed with changing pH in proportion to v m : both K m and v m showed a depression at neutral pH. The pH dependence of the ratio of v m of ITPase to that of ATPase was given by a bell-shaped curve having its maximum at pH 7.5. At 0°, the pH-activity curve of ATPase lacked the neutral depression. At pH 6, the Arrhenius plots of v m and K m were linear and the activation energies of v m and K m were 16.2 and −2.8 kcal/mole, respectively. At pH7, the activation energies of v m and K m , at temperatures higher than 10°, were noticeably different from those at temperatures lower than 10°; these values were for v m , 9.0 and 3.0 kcal/mole, and for K m , −1.9 and −10.4 kcal/mole, below and above 10°, respectively. 3. 3. These results were analyzed according to the mechanism proposed by one of the authors where by, in ATPase, two forms of the Michaelis complex, viz. , an active one (E 1 S) and an inactive one (E 2 S), can exist. It was concluded that the complex E 2 S is not formed in ITPase but in ATPase it is dominant at around pH 7.5, and at higher temperatures. It was further concluded that the rate-constant of dissociation of the Michaelis complex E 1 S into the enzyme and the substrate is much smaller than that of the breakdown of the complex into the products; the rate-constant of the formation of E 1 S is independent of pH.

Superprecipitation was observed with abalone myosin and purified rabbit actin in the presence of calcium ions, but was not observed in the absence of calcium. The Mg-ATPase [EC 3.6.1.3] activity of abalone myosin and rabbit actin in the absence of calcium ions (EGTA present) showed about 60% inhibition as compared with values in the presence of calcium ions. The calcium sensitivity may be attributable to abalone myosin, as in the case of scallop myosin.

The behaviours of three synthetic ATP analogues, 6-dimethylamino-9-β- d -ribofuranosylpurine 5′-triphosphate (dimethyl-ATP), 9-(4′-hydroxybutyl)-6-aminopurine 4′-triphosphate (BTP) and ribose 5-triphosphate (RTP), on hydrolysis by myosin A were investigated mainly in 0.5 M KCl and 5 m M CaCl 2 (pH7.0, 20°), together with those of ATP, ITP and inorganic tripolyphosphate (TP i ). The following results were obtained. 1. 1. The effect of divalent cations, such as Mg 2+ , Mn 2+ , Ca 2+ , Sr 2+ and Ba 2+ , on the rate of hydrolysis of BTP, TP i subtrates, the relations between the ionic radii ( r ) of various added divalent cations and the rates of hydrolysis were expressed by a bell-shaped curve, which showed a maximum at r = 0.95 A . 2. 2. The Michaelis constants of hydrolysis of ITP, TP i , BTP, RTP, dimethyl-ATP and ATP were 3, 2.5, 2.5, 1.7, 1.4 and 0.2 and m M , respectively, while the ratios of the maximum velocity of hydrolysis of ITP, BTP, dimethyl-ATP, TP i and RTP to that of ATP were, 7, 3, 1.3, 1 13.5 and 1 20 , respectively. 3. 3. The Michaelis constant and the maximum velocity of hydrolysis of TP i showed a remarkable increase with decreasing pH, while the rate of hydrolysis of RTP depended slightly on the pH. The dependence of the rate of hydrolysis of BTP on the pH showed a slight depression at the neutral pH and its Michaelis constant was independent of the pH. 4. 4. p -Chloromercuribenzoate and EDTA inhibited the hydrolysis of TP i , RTP, ITP, dimethyl-ATP and BTP, although they activated ATPase. These results have been interpreted on the basis of the reaction between myosin A and nucleoside triphosphates which has been proposed by one of the present authors 7,24 and is further discussed below.