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Abalone myosin contains two kinds of light chain, regulatory light chain (LC2) and essential light chain (LC1) according to SDS-PAGE. Three distinct light chain bands were observed on polyacrylamide gel electrophoresis of purified abalone myosin in the presence of urea (urea-PAGE). The slower two components showed had mobility on SDS-PAGE and they also showed regulatory activity as the regulatory light chain. They were termed LC2-a and LC2-b in order of increasing mobility on urea-PAGE and isolated by DE-32 ion exchange column chromatography in the presence 8 M urea. The ratio of LC2-a and LC2-b in the central portion of adductor muscle of abalone (LC2-a: LC2-b = 7:3) was different from that (1:1) in the peripheral portion. These results suggest that the two light chains are isoforms of the regulatory light chain. The amino acid compositions of LC2-a and LC2-b were very similar to each other except for the Cys content. The UV absorption spectra were also quite similar, as were the UV difference absorption spectra induced by Ca2+. Phosphorylation was not detectable with the myosin light chain kinase of chicken gizzard. The Ca2+ concentration dependencies of Mg-ATPase activity of LC2-a or LC2-b hybridized abalone myosin (a-myosin, b-myosin) were similar to each other in the absence of rabbit F-actin, but differed in the presence of actin. The b-myosin had a higher maximum value of actomyosin ATPase activity and a lower apparent binding constant of actin and myosin than a-myosin.(ABSTRACT TRUNCATED AT 250 WORDS)

1. Abalone myosin consisted of one kind of heavy chain and two kinds of light chain according to SDS gel electrophoresis. The molecular weights of the light chains were estimated as 16,600 daltons (Light Chain-1: LC-1) and 14,500 daltons (Light Chain-2: LC-2). 2. The amino acid composition of LC-2 was not appreciably different from that of LC-1 except that the valine residue was 1 mol per mol of LC-2. 3. both LC-1 and LC-2 showed a calcium-induced UV absorption difference spectrum through the apparent binding constants for Ca2+ were low (2.5 x 10(-3) M for LC-1 and 3.2 x 10(-4) M for LC-2). 4. The modification of carboxyl groups of LC-2 by 1-ethyl-3(3-dimethylaminopropyl)carbodiimide caused the disappearance of the calcium-induced UV absorption difference spectrum. 5. Manganese ion could also induce a UV absorption difference spectrum with these light chains although the concentration of Mn2+ required to produce the difference spectrum was very high. 6. Abalone LC-2 bound to desensitized scallop myosin and restored the calcium sensitivity of the myosin but LC-1 did not.

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.

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 experimental conditions for release of the regulatory light chain (RLC) of scallop myosin at 30 degrees C were studied. Substantially all RLC was released from myosin by incubation for 5 min in medium containing buffer and KCl. This release of RLC was inhibited strongly by Ca2+, while the effect of Mg2+ was about 10,000 times weaker than that of Ca2+. Even in the absence of Ca2+, MgATP and MgADP inhibited the release of RLC, while the protective effect of AMPPNP was negligible. Other Mg nucleotides also showed some protective effect, though appreciably less than MgATP. The incubation of scallop myosin with abalone regulatory light chain (LC2) at 30 degrees C for 5 min produced a hybrid myosin. In the presence of 5 mM MgCl2, 1 of the 2 mol of RLC per mol of scallop myosin was exchanged with 1 mol of LC2. In the presence of Ca2+ or MgATP, myosin bound 1 extra mole of LC2 besides the 2 mol each of SH-LC and RLC.