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FTIR spectroscopy has been applied to study the coordination structures of Mg2+ and Ca2+ ions bound in Akazara scallop troponin C (TnC), which contains only a single Ca2+ binding site. The region of the COO– antisymmetric stretch provides information about the coordination modes of COO– groups to the metal ions: bidentate, unidentate, or pseudo-bridging. Two bands were observed at 1584 and 1567 cm-1 in the apo state, whereas additional bands were observed at 1543 and 1601 cm-1 in the Ca2+-bound and Mg2+-bound states, respectively. The intensity of the band at 1567 cm-1 in the Mg2+-bound state was identical to that in the apo state. Therefore, the side-chain COO– group of Glu142 at the 12th position in the Ca2+-binding site coordinates to Ca2+ in the bidentate mode but does not interact with Mg2+ directly. A slight upshift of COO– antisymmetric stretch due to Asp side-chains was also observed upon Mg2+ and Ca2+ binding. This indicates that the COO– groups of Asp131 and Asp133 interact with both Ca2+ and Mg2+ in the pseudo-bridging mode. Therefore, the present study directly demonstrated that the coordination structure of Mg2+ was different from that of Ca2+ in the Ca2+-binding site. In contrast to vertebrate TnC, most of the secondary structures remained unchanged among apo, Mg2+-bound and Ca2+-bound states of Akazara scallop TnC, as spectral changes upon either Ca2+ or Mg2+ binding were very small in the infrared amide-I′ region as well as in the CD spectra. Fluorescence spectroscopy indicated that the spectral changes upon Ca2+ binding were larger than that upon Mg2+ binding. Moreover, gel-filtration experiments indicated that the molecular sizes of TnC had the order apo TnC > Mg2+-bound TnC > Ca2+-bound TnC. These results suggest that the tertiary structures are different in the Ca2+- and Mg2+-bound states. The present study may provide direct evidence that the side-chain COO– groups in the Ca2+-binding site are directly involved in the functional on/off mechanism of the activation of Akazara scallop TnC. [ABSTRACT FROM AUTHOR]

SUMMARY:The complete amino acid sequence of phospholipase A2 (PLA2) from the pyloric ceca of the starfish Asterina pectinifera was determined by automated Edman degradation. The A. pectinifera PLA2 (APLA2) consists of 137 amino acids with an unblocked N-terminus and its molecular weight is calculated to be 15 300.1. The enzyme contains 14 cysteine (Cys) residues at the corresponding positions of the same residues which have been shown to be involved in intramolecular disulfide bonds in mammalian pancreatic PLA2. The region involving an active site and a Ca2+-binding loop shows fairly high sequence homology (75%) between the APLA2 and porcine pancreatic PLA2. The APLA2 conserved the amino acid sequence of the loop portion of the porcine pancreatic PLA2 except for the deletion of two amino acids. These features indicate that the APLA2 can be classified into the group 1 type PLA2. In contrast, the homology between the APLA2 and porcine pancreatic PLA2 was calculated to be 47% in the whole region. Further, the insertion of sixteen residues and the deletion of three residues were required in the sequence of the APLA2 to align the corresponding region to the β-wing of porcine pancreatic PLA2. These differences in amino acid sequence of the APLA2 may account for its specific properties such as the higher activity and the characteristic substrate specificity. [ABSTRACT FROM AUTHOR]

Scallop troponin C (TnC) binds only one Ca2+/mol and the single Ca2+-binding site has been suggested to be site IV on the basis of the primary structure [K. Nishita, H. Tanaka, and T. Ojima (1994) J. Biol. Chem. 269, 3464-3468; T. Ojima, H. Tanaka, and K. Nishita (1994) Arch. Biochem. Biophys. 311, 272-276]. In the present study, the functional role of Ca2+-binding site IV of akazara scallop (Chlamys nipponensis akazara) TnC in Ca2+-regu-lation was investigated using a site-directed mutant with an inactivated site IV (TnC-ZEQ), N- and C-terminal half molecule mutants (TnCN and TnCc), and wild-type TnC (TnCW). Equilibrium dialysis using 45Ca2+demonstrated that TnCw and TnCc bind 0.6-0.8 mol of Ca2+/mol, but that TnC-ZEQ and TnCN bind virtually no Ca2+. The UV difference spectra of TnCw and TnCc showed bands at around 280-290 nm due to the perturbation of Tyr and Trp upon Ca2+-binding, while TnC-ZEQ and TnCN did not show these bands. In addition, TnCw and TnCc showed retardation of elution from Sephacryl S-200 upon the addition of 1 mM CaCl2, unlike TnC-ZEQ and TnCN. These results indicate that Ca2+ binds only to site IV and that Ca2+-binding causes structural changes in both the whole TnC molecule and the C-terminal half molecule. In addition, TnCw, TnC-ZEQ, and TnCc, but not TnCN, were shown to form soluble complexes with scallop TnI at physiological ionic strength. On the other hand, the Mg-ATPase activity of reconstituted rabbit actomyosin in the presence of scallop tropomyosin was inhibited by scallop TnI and recovered by the addition of an equimolar amount of TnCw, TnC-ZEQ, or TnCc, but not TnCN. These results imply that the site responsible for the association with TnI is located in the C-terminal half domain of TnC. Ternary complex constructed from scallop TnT, TnI, and TnCw conferred Ca2+-sensitivity to the Mg-ATPase of rabbit actomyosin to the same extent as native troponin, but the TnCN-TnT-TnI and TnC-ZEQ-TnT-TnI complexes conferred no Ca2+-sensitivity, while the TnCc-TnT-TnI complex conferred weak Ca2+-sensi-tivity. Thus, the major functions of scallop TnC, such as Ca2+-binding and interaction with TnI, are located in the C-terminal domain, however, the full Ca2+-regulatory function requires the presence of the N-terminal domain [ABSTRACT FROM AUTHOR]

In order to examine the involvement of troponin-linked Ca2+-regulation, in addition to well-known myosin-linked Ca2+-regulation, in the contraction of molluscan striated muscle, myofibrils from Ezo-giant scallop striated muscle were desensitized to Ca2+ by removing both myosin regulatory light chain and troponin C by treatment with a strong divalent cation chelator, CDTA. The ATPase level in the desensitized myofibrils was about half the maximum level in intact myofibrils regardless of the Ca2+-concentration at 25 and 15*C. In the absence of Ca2+, the ATPase of the desensitized myofibrils was suppressed by myosin regulatory light chain but not affected by troponin C at either temperature. The ATPase was activated at higher Ca2+-concentrations by both myosin regulatory light chain and troponin C, but the activating effects of these two proteins were affected differently by temperature. The activation of ATPase by myosin regulatory light chain was much greater than that by troponin C at 25°C, whereas the activation by troponin C was much greater than that by myosin regulatory light chain at 15*C. The maximum activation was only obtained in the presence of both myosin regulatory light chain and troponin C at these temperatures. These findings strongly suggest that the contraction of scallop striated muscle is regulated through both myosin-linked and troponin-linked Ca2+ -regulation, and that the troponin-linked Ca2+-regulation is more significant at lower temperature. [ABSTRACT FROM AUTHOR]