Kiyoyoshi Nishita

Bacterial expression and characterization of starfish phospholipase A2

Phospholipase A(2) (PLA(2)) from the pyloric ceca of the starfish Asterina pectinifera showed high specific activity and characteristic substrate specificity, compared with commercially available PLA(2) from porcine pancreas. To investigate enzymatic properties of the starfish PLA(2) in further detail, we constructed a bacterial expression system for the enzyme. The starfish PLA(2) cDNA isolated previously (Kishimura et al., 2000b. cDNA cloning and sequencing of phospholipase A(2) from the pyloric ceca of the starfish Asterina pectinifera. Comp. Biochem. Physiol. 126B, 579-586) was inserted into the expression plasmid pET-16b and the PLA(2) protein was expressed in Escherichia coli BL21 (DE3) by induction with isopropyl-beta-D(-)-thiogalactopyranoside. The recombinant PLA(2) produced as inclusion bodies was dissociated with 8 M urea and 10 mM 2-mercaptoethanol and renatured by dialyzing against 10 mM Tris--HCl buffer (pH 8.0). Renatured PLA(2) was purified by subsequent column chromatographies on DEAE--cellulose (DE-52) and Sephadex G-50. Although an N-terminal Ser in the native starfish PLA(2) was replaced by an Ala in the recombinant PLA(2), the recombinant enzyme showed essentially the same properties as did the native PLA(2) with respect to specific activity, substrate specificity, optimum pH and temperature, and Ca(2+) requirement.

cDNA cloning and sequencing of phospholipase A2 from the pyloric ceca of the starfish Asterina pectinifera

Three cDNA from the pyloric ceca of the starfish Asterina pectinifera, (namely, cDNA 1, 2, and 3), encoding phospholipase A2 (PLA2), were isolated and sequenced. These cDNAs were composed of 415 bp with an open reading frame of 414 bp at nucleotide positions 1-414, which encodes 138 amino acids including N-terminal Met derived from the PCR primer. The amino acid sequence deduced from the cDNA 1 was completely consistent with the sequence determined with the starfish PLA2 protein, while those deduced from cDNA 2 and cDNA 3 differed at one and twelve amino acid residual positions, respectively, from the sequence of the PLA2 protein, suggesting the presence of multiple forms in the starfish PLA2. All of the sequences deduced from cDNA 1, 2, and 3 required two amino acid deletions in pancreatic loop region, and sixteen insertions and three deletions in beta-wing region when aligned with the sequence of mammalian pancreatic PLA2. In phylogenetic tree, the starfish PLA2 should be classified into an independent group, but hardly to the established groups IA and IB. The characteristic structure in the pancreatic loop and beta-wing regions may account for the specific properties of the starfish PLA2, e.g. the higher activity and characteristic substrate specificity compared with commercially available PLA2 from porcine pancreas.

Primary structure of myosin heavy chain from fast skeletal muscle of Chum salmon Oncorhynchus keta

The nucleotide sequence of the cDNA encoding myosin heavy chain of chum salmon Oncorhynchus keta fast skeletal muscle was determined. The sequence consists of 5,994 bp, including 5,814 bp of translated region deducing an amino acid sequence of 1,937 residues. The deduced sequence showed 79% homology to that of rabbit fast skeletal myosin and 84-87% homology to those of fast skeletal myosins from walleye pollack, white croaker and carp. The putative binding-sites for ATP, actin and regulatory light-chains in the subfragment-1 region of the salmon myosin showed high homology with the fish myosins (78-100% homology). However, the Loop-1 and Loop-2 showed considerably low homology (31-60%). On the other hand, the deduced sequences of subfragment-2 (533 residues) and light meromyosin (564 residues) showed 88-93% homology to the corresponding regions of the fish myosins. It becomes obvious that several specific residues of the rabbit LMM are substituted to Gly in the salmon LMM as well as the other fish LMMs. This may be involved in the structural instability of the fish myosin tail region.

Comparative studies on the functional roles of N- and C-terminal regions of molluskan and vertebrate troponin-I

Vertebrate troponin regulates muscle contraction through alternative binding of the C‐terminal region of the inhibitory subunit, troponin‐I (TnI), to actin or troponin‐C (TnC) in a Ca2+‐dependent manner. To elucidate the molecular mechanisms of this regulation by molluskan troponin, we compared the functional properties of the recombinant fragments of Akazara scallop TnI and rabbit fast skeletal TnI. The C‐terminal fragment of Akazara scallop TnI (ATnI232−292), which contains the inhibitory region (residues 104–115 of rabbit TnI) and the regulatory TnC‐binding site (residues 116–131), bound actin‐tropomyosin and inhibited actomyosin‐tropomyosin Mg‐ATPase. However, it did not interact with TnC, even in the presence of Ca2+. These results indicated that the mechanism involved in the alternative binding of this region was not observed in molluskan troponin. On the other hand, ATnI130−252, which contains the structural TnC‐binding site (residues 1–30 of rabbit TnI) and the inhibitory region, bound strongly to both actin and TnC. Moreover, the ternary complex consisting of this fragment, troponin‐T, and TnC activated the ATPase in a Ca2+‐dependent manner almost as effectively as intact Akazara scallop troponin. Therefore, Akazara scallop troponin regulates the contraction through the activating mechanisms that involve the region spanning from the structural TnC‐binding site to the inhibitory region of TnI. Together with the observation that corresponding rabbit TnI‐fragment (RTnI1−116) shows similar activating effects, these findings suggest the importance of the TnI N‐terminal region not only for maintaining the structural integrity of troponin complex but also for Ca2+‐dependent activation.

Akazara scallop troponin C: Ca2+-induced conformational change and interaction with rabbit troponin subunits

The number of specific Ca2+ bound to Akazara scallop troponin C was estimated to be 0.7 with an apparent binding constant of 5 x 10(5) M-1 (T. Ojima and K. Nishita, 1986, J. Biol. Chem. 261, 16749-16754). In the present paper, we report on the Ca(2+)-induced conformational changes in the troponin C and the interaction of the troponin C with rabbit troponin subunits. The Ca2+ binding to the troponin C caused a marked change in difference uv absorption spectra and a retardation of elution on Sephacryl S-200 gel filtration. However, its circular dichroism spectrum was hardly changed by the Ca2+ binding. These results suggest that the Ca2+ binding to the troponin C induced changes predominantly in tertiary structure rather than in secondary structure. Akazara scallop troponin C was shown to be able to bind to rabbit troponin I-Cellulofine affinity column, but the affinity was not greatly increased by Ca2+ unlike the case of rabbit troponin C. On hybridizing with rabbit troponin T and I, Akazara scallop troponin C was shown to be incapable of substituting rabbit troponin C; i.e., the hybrid troponin strongly inhibited the Mg-ATPase activity of rabbit actomyosin-tropomyosin irrespective of the presence or absence of Ca2+, thus recovering no Ca2+ sensitivity.

Amino acid sequence of squid troponin C

The complete amino acid sequence of squid Todarodes pacificus troponin C (TnC), which was shown to bind only 1 mol Ca(2+)/mol, was determined by both the Edman and cDNA methods. The squid TnC is composed of 147 amino acids including an unblocked Pro at the N-terminus and the calculated molecular weight is 17003.9. Among the four potential Ca(2+)-binding sites, namely sites I-IV from the N-terminus, only site IV completely satisfied the consensus amino acid sequence for the active Ca(2+)-binding loop. This indicates that squid TnC possesses a single Ca(2+)-binding site at the site IV as scallop TnCs [Nishita et al., J. Biol. Chem. 269 (1994) 3464-3468; Ojima et al., Arch. Biochem. Biophys. 311 (1994) 272-276). The sequence homology of squid TnC to TnCs of scallop, arthropods, and rabbit was 61%, 31-38%, and 31%, respectively. In the sequence of the central D/E-helix region of squid and scallop TnCs, a deletion of three amino acids was required to maximize the homology with the other TnCs.

Comparative studies on biochemical characteristics of troponins from ezo-giant scallop (patinopecten yessoensis) and akazara scallop (chlamys nipponesis akazara)

Abstract 1. 1. Tropinins were isolated from the striated adductor muscle of ezo-giant scallop and akazara scallop, and biochemical properties were comparatively studied. 2. 2. The ezo-giant scallop and akazara scallop troponins conferred the same extent of Ca 2+ -sensitivity to rabbit reconstituted actomyosin Mg-ATPase activity, and the Ca 2+ concentration required for a half-maximal activation, with either troponin, was estimated as 1 μM. 3. 3. The ezo-giant scallop troponin consisted of three subunits of M r , 52,000, 40,000 and 20,000, and they were regarded as troponin-I, -T and -C, respectively, from the hybridization test with the akazara scallop troponin subunits which we previously identified [Ojima and Nashita (1988) J. Biochem. 104 , 207–210].

NMR Structural Study of Troponin C C-Terminal Domain Complexed with Troponin I Fragment from Akazara Scallop

Scallop muscle has been demonstrated to possess both myosin-linked and actin-linked systems1-3 (Fig. 1), even though molluscan muscles were known to be regulated only by the myosin-linked regulatory system mediated through Ca2+-binding to myosin light chains4-6. Recently, the physiological significance of the coexistence of the two systems in scallop adductor muscle was investigated using CDTA-treated scallop myofibrils1. Actin-linked (Troponin-linked) system has been well known as the regulatory system in the muscle contraction of vertebrate striated muscles7. It is regulated by troponin in a Ca2+ dependent manner. Troponin contains three distinct components, i.e., a Ca2+ binding component (TnC), an inhibitory component troponin I (Tnl), and a tropomyosin-binding component troponin T (TnT). TnC contains two independent Ca2+ binding domains, each of which consists of two EF-hand motifs8. Vertebrate striated muscle TnCs bind three or four Ca2+ ions in a molecule and act as the Ca2+ sensor of muscle contraction associated with the binding and release of one or two Ca2+ ion(s) in the N-terminal domain9, 10, 11. The N-terminal domain has, thus, been called the regulatory domain and contains one or two low affinity Ca2+-binding sites (Sites I and II)12. On the other hand, the C-terminal domain has been called the structural domain and contains two high-affinity sites (Sites III and IV). They also bind Mg2+ and are called as Ca2+/Mg2+ sites.

Comparative studies on heat stability and autolysis of scallop (Patinopecten yessoensis) calpain II-like proteinase and rabbit calpain II.

1. The scallop calpain-like proteinase is about five times more labile than the rabbit calpain II upon heat treatment at 35 degrees C. 2. By autolysis of the scallop proteinase of two 100 kDa subunits, 90, 45 and 30 kDa fragments were formed. Thereby the activity decreased monophasically in the presence of millimolar order of Ca2+, but did not increase in the presence of micromolar order of Ca2+ unlike the rabbit calpain II.

Calpain II-like proteinase of scallop (Patinopecten yessoensis) striated adductor muscle.

1. A Ca(2+)-dependent cysteine proteinase was purified from scallop striated adductor muscle by ammonium sulfate fractionation and column chromatography on DEAE-cellulose and Sephacryl S-300. 2. The enzyme is of Mr approximately 200,000, composed of two Mr 100,000 subunits. 3. The enzyme is a cysteine proteinase with optimum activity at pH 6.8 and about 18 degrees C. In addition, it requires 1.7 mM Ca2+ for half-maximal activity and more than 10 mM Ca2+ for maximal activity. Thus the enzyme can be classified as calpain II.