Actin-binding site of pig cardiac myosin.

An actin-binding site is also present in the tryptic 20 kDa peptide fragment of the subfragment-1 heavy chain of pig cardiac myosin. As previously reported for skeletal myosin (Katoh, T., Katoh, H., and Morita, F. (1985) J. Biol. Chem. 260, 6723-6727), the site was further narrowed down to the 10 kDa peptide containing the reactive SH1 and SH2 groups. Thus it appears that the actin-binding site around the two thiols found in skeletal myosin is common to different types of myosin.

Different modes of interaction of two peptide fragments from subdomain 4 of rabbit skeletal muscle actin with actin promoters.

Previously, we reported that the 2.6 kDa peptide fragment extending from Arg-177 to Tyr-198 in rabbit skeletal muscle actin bound to actin itself and inhibited its polymerization, while the 9.1 kDa peptide extending from Ser-199 to Tyr-279 in actin did not. The 2.6 kDa segment of actin was reported to contain one of the important actin-actin contacts (Hori, K. and Morita, F. (1992) J. Biochem. 112, 401-408). In this paper, we show additional evidence that the rate of salt-induced increase in the fluorescence of pyrene-labeled actin was decreased in the presence of the 2.6 kDa peptide. Conventional actin filaments were only scarcely observed in the presence of the 2.6 kDa peptide under an electron microscope with a steady state of fluorescence increase. Furthermore, the 2.6 kDa peptide was found to sever F-actin into short filament fragments. The 9.1 kDa peptide, on the other hand, neither inhibited the fluorescence increment of pyrene-actin nor severed actin filaments. However, the 9.1 kDa peptide was found to increase the viscosity and fluorescence intensity of pyrene-G-actin and to form short actin filaments in the absence of salts. Contact sites in the 9.1 kDa segment in actin may have a different mode of interaction with adjacent actin promoters in actin filaments from that of the 2.6 kDa segment.

Protective effect of a prosaposin-derived, 18-mer peptide on slowly progressive neuronal degeneration after brief ischemia.

SUMMARY: Slowly progressive degeneration of the hippocampal CA1 neurons was induced by 3-minute transient global ischemia in gerbils. Sustained degeneration of hippocampal CA1 neurons was evident 1 month after ischemia. To investigate the effects of an 18-mer peptide comprising the hydrophilic sequence of the rat saposin C domain (18MP) on this sustained neuronal degeneration, an intracerebroventricular 18MP infusion was initiated 3 days after ischemia. Histopathologic and behavior evaluations were conducted 1 week and 1 month after induction of ischemia. When compared with the vehicle infusion, 18MP treatment significantly increased the response latency time in a passive avoidance task. Increased neuronal density was also evident, as was the number of intact synapses in the hippocampal CA1 region at 1 week and 1 month after ischemia. 18MP treatment also significantly decreased the number of TUNEL-positive CA1 neurons 1 week after ischemia. Subsequent in vitro experiments using cultured neurons demonstrated that the 18MP at optimal extracellular concentrations of 1 to 100 fg/mL prevented nitric oxide-induced neuronal damage as expected and significantly up-regulated the expressions of bcl-x(L) mRNA and its translated protein. These results suggest that the gerbil model of 3-minute ischemia is useful in studying the pathogenesis of slowly progressive neuronal degeneration after stroke and in evaluating effects of novel therapeutic agents. It is likely that the 18MP at low extracellular concentrations prevents neuronal apoptosis possibly through up-regulation of the mitochondrial antiapoptotic factor Bcl-x(L).

Molecular cloning of a novel phosphorylation-dependent inhibitory protein of protein phosphatase-1 (CPI17) in smooth muscle: its specific localization in smooth muscle.

The cDNA encoding a phosphorylation-dependent inhibitory protein of protein phosphatase-1 (PP1) was isolated from a porcine aorta library. The coding region represented the complete amino acid sequence of this protein comprised of a novel 147-residue polypeptide, which we termed CPI17, a 17-kDa PKC-potentiated inhibitory protein of PP1. As well as the native CPI17 from porcine aorta, the recombinant protein completely suppressed the PP1 activity (IC50 = 0.18 nM) by the stoichiometric thiophosphorylation. The CPI17 mRNA is expressed in smooth muscle tissues such as aorta and bladder, whereas little expression was observed in heart, skeletal muscle, and non-muscle tissues. These results suggest a specific regulatory mechanism of the PP1 activity through CPI17 in smooth muscle.

Brain N-acetylaspartate is elevated in Pelizaeus-Merzbacher disease with PLP1 duplication.

OBJECTIVE: To assess alterations in brain metabolites of patients with Pelizaeus-Merzbacher disease (PMD) with the proteolipid protein gene 1 (PLP1) duplications using quantitative proton MRS. METHODS: Five unrelated male Japanese patients with PMD with PLP1 duplications were analyzed using automated proton brain examination with the point resolved spectroscopy technique (repetition and echo time of 5,000 and 30 msec). Localized spectra in the posterior portion of the centrum semiovale were acquired, and absolute metabolite concentrations were calculated using the LCModel. RESULTS: Absolute concentrations of N-acetylaspartate (NAA), creatine (Cr), and myoinositol (MI) were increased by 16% (p < 0.01), 43% (p < 0.001), and 31% (p < 0.01) in patients with PMD as compared with age-matched controls. There was no statistical difference in choline concentration. CONCLUSION: The increased concentration of NAA, which could not be detected by previous relative quantitation methods, suggests two possibilities: axonal involvement secondary to dysmyelination, or increased cell population of oligodendrocyte progenitors. Elevated Cr and MI concentrations may reflect the reactive astrocytic gliosis. Our study thus emphasizes the importance of absolute quantitation of metabolites to investigate the disease mechanism of the dysmyelinating disorders of the CNS.

Distinctly abnormal brain metabolism in late-onset ornithine transcarbamylase deficiency.

OBJECTIVE: To assess alterations in brain metabolites in patients with late-onset ornithine transcarbamylase deficiency (OTCD). METHODS: Six unrelated, asymptomatic Japanese late-onset OTCD patients were analyzed by proton MRS ((1)HMRS) using a point-resolved spectroscopy technique (repetition and echo times, 5000 and 30 ms). Localized spectra for the centrum semiovale were acquired and absolute metabolite concentrations were calculated using an LCModel. RESULTS: Compared with age-matched controls, N-acetylaspartate and creatine concentrations were normal in all patients. The glutamine (Gln) plus glutamate concentration was increased in four patients, which progressed in proportion to the clinical stage. myo-inositol (mI) could not be detected in five symptomatic patients. A decreased choline (Cho) concentration was detected in two clinically severe patients. (1)HMRS after liver transplantation in one patient revealed the normalization of all metabolites. CONCLUSION: These findings suggest progression of neurochemical events in OTCD, i.e., mI depletion and Gln accumulation followed by Cho depletion, which is reverse of that in hepatic encephalopathy, i.e., Cho depletion followed by mI depletion and Gln accumulation.

Rapid HLA class I DNA typing using microtiter plate-reverse hybridization assay (MRHA) by simple thermoregulation: high-resolution subtyping of the HLA-A2 and -B40 antigen groups.

We have established a precise, rapid, simple and economical subtyping method for alleles encoding the HLA-A2 and -B40 antigens using microtiter plate-reverse hybridization assay (MRHA), which is based on the general principle of HLA oligotyping by reverse dot blot hybridization. Amino-modified sequence-specific oligonucleotide (SSO) probes were immobilized covalently onto a carboxylate-modified microtiter plate. In order to perform high-resolution subtyping of the HLA-A2 and -B40 antigen groups, the alpha1 and alpha2 domain regions were amplified using a pair of group-specific primers composed of an unlabeled sense primer and a biotinylated antisense primer. PCR-amplified products were hybridized with SSO probes in hybridization buffer containing formamide for 1 hour at 37 degrees C. After washing with 2 X SSC at room temperature, the bound PCR products were detected by alkaline phosphatase-conjugated streptavidine followed by color development. All of 8 HLA-B40 suballeles, all of 2 HLA-B47 suballeles (B40 group-specific primers used in this study allowed also B47 amplification) and 17 out of 21 HLA-A2 suballeles were discriminated. The remaining four HLA-A2 suballeles were determined by analysis after exon 4 amplification. HLA-DNA typing by this method was easily and exactly performed regardless of sample number. The greatest advantages of this technique are strong positive signals obtained, reproducibility and the ease of thermoregulation for hybridization and washing as compared to previously reported microtiter plate hybridization methods.

Temperature induced analog reaction of adenylyl imidodiphosphate to an intermediate step of heavy meromyosin adenosine triphosphatase.

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.

A study of the binding of adenosine diphosphate to myosin subfragment-1.

The binding of ADP to subfragment-1 was investigated by the gel filtration method. The amount of bound ADP was determined as a function of free ADP concentration. Linear Scatchard plots were obtained. The maximum binding number, 0.55 mole of ADP per 10(5) g of protein, and the dissociation constant, 1.6 x 10(-6) M, were obtained, using subfragment-1 prepared by tryptic digestion, in the presence of 0.083 M KCl-10 mM MgCl2-0.02 M Tris-HCl (pH 8), at 25 degrees. Similar maximum numbers, about 0.5 mole per 10(5) g of protein, were obtained with subfragment-1 prepared by chymotryptic digestion of myosin or papain digestion of myofibrils. The maximum number did not depend on the KCl concentration or the temperature, while the dissociation constant decreased on decreasing either the KCl concentration or the temperature. Adenylyl imidodiphosphate binding to subfragment-1 prepared by chymotryptic digestion was also measured by the gel filtration method. The maximum binding number, 0.41 mole per 10(5) g of subfragment-1, and the dissociation constant, less than 10(-7) M, were obtained in the presence of 0.7 M KCl-10 mM MgCl2-0.02 M Tris-HCl (pH 8), at 8 degrees. The difference absorbance at 288 nm of the difference absorption spectrum induced by ADP of subfragment-1 prepared by tryptic digestion was proportional to the amount of bound ADP. The steady-state ATPase rate of subfragment-1 prepared by tryptic digestion was inhibited competitively by ADP in the presence of MgCl2. The extent of the initial burst of ATPase [EC 3.6.1.3] decreased from 0.46 +/- 0.06 to 0.30 +/- 0.09 mole of Pi per 10(5) g of subfragment-1 on adding ADP to a level of 0.6 mM. Subfragment-1 prepared by tryptic digestion bound F-actin with a mole ratio of 1/0.96 of actin monomer. The binding was depressed by the addition of ADP. On the basis of these results, subfragment-1 preparations were assumed to be a half-and-half mixture of two kinds of protein, and properties of each protein are discussed.

Divalent metal ion binding to g1 subunit of myosin and movement of 180 tyrosyl residue.

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.

Smooth muscle of scallop adductor contains at least two kinds of myosin.

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.

Regulatory light chain contents and molecular species of myosin in catch muscle of scallop.

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.

Interaction between alkali light chains of myosin and divalent metal ions.

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.

An activating factor (tropomyosin) for the superprecipitation of actomyosin prepared from scallop adductor muscles.

An activating factor for the superprecipitation of actomyosin reconstructed from scallop smooth muscle myosin and rabbit skeletal muscle F-actin was purified from thin filaments of scallop smooth and striated muscles. Two components were obtained from the smooth muscle and one from the striated muscle. All three components similarly affected the actomyosin ATPase activity. According to the results of analysis involving double reciprocal plotting of the ATPase activity versus F-actin concentration, the activating factor for superprecipitation decreased the apparent dissociation constants of actomyosin about 30 to 110 times. The activation of the superprecipitation by the factor, therefore, may be due to the enhancement of the affinity between F-actin and myosin in the presence of ATP. The activating factor was identified as tropomyosin based on it mobility on polyacrylamide gel electrophoresis and on the recovery of the Ca2+-sensitivity of purified rabbit skeletal actomyosin in the presence of troponin.

Purification of a protein kinase phosphorylating myosin regulatory light chain-a (RLC-a) from smooth muscle of scallop, Patinopecten yessoensis.

A protein kinase activity phosphorylating regulatory light chain-a (RLC-a) of scallop smooth muscle myosin was found to be present in scallop smooth muscle homogenate. The kinase was purified to homogeneity and named RLC-a myosin kinase (aMK). aMK was extracted from the muscle homogenate with a low salt solution and was purified by successive DE-32 ion exchange chromatography, gel filtration on Ultrogel AcA 44, and affinity chromatography on Sepharose 4B-6-aminohexyl-1-pyrophosphate. The molecular weight of aMK was estimated to be 40-kDa from the mobility on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and 35-kDa from the elution volume on Sephadex G-150 gel filtration. The phosphorylation site of RLC-a by aMK was determined to be Ser residue(s). Only RLC-a was phosphorylated; the other regulatory light chain, RLC-b, was not. The phosphorylatable Ser of RLC-a is, therefore, considered to be Ser-11, which is located in the N-terminal region having a different amino acid sequence from that of RLC-b. RLC-a was phosphorylated by aMK 3 times faster in the free state than in the bound state to myosin. aMK does not require calmodulin and is rather inhibited by CaCl2.

Two phosphorylations specific to the tail region of the 204-kDa heavy chain isoform of porcine aorta smooth muscle myosin.

In a porcine aorta extract, we observed two protein kinase activities which specifically phosphorylate the 204-kDa heavy chain isoform of aorta myosin in the absence of conventional kinase activators. We referred to these two protein kinases, eluted at 0.15 and 0.2 M KCl from a DEAE-column, as myosin kinases I (MKI) and II (MKII), respectively. The phosphorylation site for MKI was determined using a purified phosphopeptide derived from porcine aorta myosin phosphorylated with MKI. By comparison with the deduced amino acid sequence for smooth muscle myosins, the site corresponded to a Ser located at 3 amino acids upstream from a Pro, the putative end of the alpha-helical segment of the 204-kDa heavy chain tail. A homologous Ser is only present in smooth muscle myosins, i.e. not in nonmuscle myosins. MKI was purified 130-fold, but not separated from a kinase activity phosphorylating Ser1 or Ser2 in the 20-kDa regulatory light chain of aorta myosin. In contrast, MKII was purified to near homogeneity. MKII phosphorylated the porcine aorta myosin heavy chain at a Ser 19 amino acids downstream from the MKI site. The amino acid sequence around the Ser shared a consensus sequence of the phosphorylation site. The amino acid sequence around the Ser shared a consensus sequence of the phosphorylation site for casein kinase II and was homologous to that reported for bovine aorta myosin [Kelley, C.A. and Adelstein, R.S. (1990) J Biol. Chem. 265, 17876-17882]. MKII was identified as a multifunctional protein kinase, casein kinase II.

Binding of myosin subfragment 1 to actin.

The dissociation constant for the binding of myosin subfragment 1 (S1) and chromatographed actin in the presence and absence of nucleotide was measured at various ionic strengths and various temperatures. The dissociation constant was of nM order in the absence of nucleotide and increased by approximately 100- and approximately 100,000-fold in the presence of ADP and ATP, respectively. The dissociation constant also increased with increasing ionic strength, irrespective of the presence of nucleotide, and its dependence on the ionic strength was increased by the presence of ATP but decreased by the presence of ADP. The standard enthalpy change and entropy change for the binding of S1 to actin were both positive, irrespective of the presence of nucleotide, indicating that the binding was entropy-driven. The standard entropy change was essentially unaffected by the presence of ADP but was greatly decreased by ATP, suggesting that the large increase in the dissociation constant in the presence of ATP was due to the decrease of hydrophobic interactions. On the other hand, the increase in the dissociation constant for acto-S1 in the presence of ADP might be induced by the decrease of electrostatic interactions.

Mapping myosin-binding sites on actin probed by peptides that inhibit actomyosin interaction.

A 2-kDa peptide (2K peptide) which was derived from the neck region of porcine aorta smooth muscle myosin heavy chain binds to actin competitively with skeletal myosin subfragment 1 (S1) in the absence of ATP and inhibits acto-S1 ATPase activity [Katoh, T. and Morita, F. (1993) J. Biol. Chem. 268, 2380-2388]. Using this and other peptides, myosin-binding sites on actin were mapped and their functions were studied. The 2K peptide inhibited the acto-S1 ATPase activity without inhibiting the binding of S1 to actin in the presence of ATP. On the other hand, the dansylated 2K peptide (DNS-2K peptide) inhibited not only the acto-S1 ATPase activity but also the binding of S1 to actin in the presence of ATP. Then, DNS-2K peptide was crosslinked to actin with 1-ethyl-3[3-(dimethylamino)propyl] carbodiimide. Amino acid composition and sequencing analyses of the fluorescent lysylendopeptidase-peptides of the crosslinked product indicated that DNS-2K peptide was crosslinked to acidic residues within residues 1-18 (Asp1, Glu2, Asp3, Glu4, and/or Asp11), 19-50 (Asp25), and 85-113 (Glu99 or Glu100) of actin. A competition experiment for the crosslinking with unlabeled 2K peptide showed that the crosslinking to residues 85-113 of actin was specific for DNS-2K peptide. In addition, isolated actin peptide 85-113 was found to show the competitive inhibition of actin-activated ATPase activity of S1 with respect to actin. These results suggest that the site within residues 1-28 of actin participates in the actin-activation of myosin ATPase activity, and the site within residues 85-113 of actin participates in the weak binding of myosin to actin in the presence of ATP.

Actin-actin contact: inhibition of actin-polymerization by subdomain 4 peptide fragments.

F-Actin was digested with alpha-chymotrypsin in 6 M urea, and two peptide fragments from subdomain 4 of actin molecule [Kabsch, W., Mannherz, H.G., Suck, D., Pai, E.F., & Holmes K.C. (1990) Nature 347, 37-44] were purified by reverse-phase HPLC and Sephadex G-50 gel filtration. The peptide fragments were identified as segments from Arg-177 to Tyr-198 (2.6-kDa peptide) and from Ser-199 to Tyr-279 (9.1-kDa peptide). Their effects on actin polymerization induced by 50 or 100 mM KCl were studied by measuring the increase in viscosity by the falling ball method. The 2.6-kDa peptide decreased the rate of actin polymerization and increased the critical concentration for the polymerization. Based on the atomic model of the actin filament [Holmes, K.C., Popp, D., Gebhard, W., & Kabsch, W. (1990) Nature 347, 44-49], the peptide is presumed to bind to the barbed end of the actin filament and inhibit the polymerization. By assuming that the peptide affected the rate of association of the actin monomer to the end of the actin filament, well-fitting curves for the polymerization kinetics were calculated. Computer-assisted results indicated that the dissociation constant of the 2.6-kDa peptide for F-actin is 200 to 260 microM. In contrast, the 9.1-kDa peptide only slightly inhibited actin polymerization. These results suggest that the actin-actin interface in the region between Arg-177 and Tyr-198 has a stronger interaction than those between Ser-199 and Tyr-279. The amino acid sequence L-T-D-Y-L present in the 2.6-kDa segment is homologous to a common sequence in the F-actin capping domain of various actin-binding proteins.

Conformation and activity of smooth muscle myosin probed by various essential light chains.

Porcine aorta smooth muscle myosin contains two essential light chain (LC17) isoforms and the light chain was replaced with one of the LC17 isoforms, rabbit skeletal muscle myosin alkali light chain 2 (A2), or scallop striated muscle myosin essential light chain (SHLC). The myosin containing either an LC17 isoform or A2 showed phosphorylation-dependent properties in the monomer conformation, filament formation, ATPase activities, and superprecipitation, behaving in essentially the same way as native myosin. On the other hand, the replacement of LC17 with SHLC destabilized the 10S conformation and the myosin was predominantly filamentous under physiological conditions, irrespective of the phosphorylation state. This myosin containing dephosphorylated regulatory light chain showed higher actin-activated ATPase activity than native dephosphorylated myosin and was further activated by Ca2+, resulting in a decrease of phosphorylation-dependent regulation. Superprecipitation for the myosin was observed only when the regulatory light chain was phosphorylated. Superprecipitation for myosin containing SHLC was significantly slow in the absence of Ca2+ in comparison with that for myosin containing LC17, and was further activated by the presence of Ca2+. On the basis of the differences in amino acid sequences of these essential light chains, it appears that the N-terminal domain of LC17 may be implicated in these phosphorylation-dependent properties of smooth muscle myosin.