Increase in the molecular weight and radius of gyration of apocalmodulin induced by binding of target peptide: evidence for complex formation.

Small-angle X-ray scattering was used to investigate a complex state of apocalmodulin induced by the binding of a Ca(2+)/calmodulin-dependent protein kinase IV calmodulin target site. Upon binding of the peptide, the molecular weight for apocalmodulin increased by 8.4%, which provides direct evidence for the formation of a calmodulin/target peptide complex. Comparison of the radius of gyration and Kratky plots of the apocalmodulin/peptide complex with those of apocalmodulin indicates that the overall conformation remains unchanged but the flexibility of the central linker decreases. An analysis of residue pairs between calmodulin and the target peptides suggests that the complex formation is induced by electrostatic interactions and subsequent van der Waals interactions.

Evidence for calmodulin inter-domain compaction in solution induced by W-7 binding.

Small-angle X-ray scattering and nuclear magnetic resonance were used to investigate the structural change of calcium-bound calmodulin (Ca2+/CaM) in solution upon binding to its antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7). The radius of gyration was 17.4+/-0.3 A for Ca2+/CaM-W-7 with a molar ratio of 1:5 and 20.3+/-0.7 A for Ca2+/CaM. Comparison of the radius of gyration and the pair distance distribution function of the Ca2+/CaM-W-7 complex with those of other complexes indicates that binding of two W-7 molecules induces a globular shape for Ca2+/CaM, probably caused by an inter-domain compaction. The results suggest a tendency for Ca2+/CaM to form a globular structure in solution, which is inducible by a small compound like W-7.

Cloning and expression of the gene for a vanadium-dependent bromoperoxidase from a marine macro-alga, Corallina pilulifera.

The cDNAs for a vanadium-dependent bromoperoxidase were cloned from a marine macro-alga, Corallina pilulifera. The open reading frame of one clone (bpo1) encoded a protein of 598 amino acids with a calculated molecular mass of 65312 Da in good agreement with that of 64 kDa determined for the native enzyme. The deduced amino acid sequence coincided well with partial sequences of peptide fragments of the enzyme. From the same cDNA library we also isolated another cDNA clone (bpo2) encoding a protein of 597 amino acids with an identity of about 90% to BPO1, suggesting a genetic diversity of the bromoperoxidase gene of C. pilulifera growing in a relatively narrow area. The carboxy-terminal 123 residues of the enzyme (BPO1) showed an identity of 45% to that of the marine macro-alga Ascophillum nodosum. The homology search of the sequences of bromoperoxidases from C. pilulifera (this study) and A. nodostum, and chloroperoxidase from the fungus Curvularia inaequalis indicated highly conserved sequences PxYxSGHA and LxxxxAxxRxxxGxHxxxD. Furthermore, it was found that the histidine residue directly bound to vanadium, other residues building up the metal center and catalytic histidine residue forming the active site of the chloroperoxidase from C. inaequalis are conserved in the primary structure of the bromoperoxidase from C. pilulifera. The cloned hpol was introduced into Escherichia coli, and the expressed PO1 was purified from the recombinant strain. The N-terminal amino acid sequence of the purified BPO1 was identical to the deduced sequence from the cDNA except the N-terminal methionine.

Inverse gene expression of prostacyclin and thromboxane synthases in resident and activated peritoneal macrophages.

Prostacyclin and thromboxane A2 produced from prostaglandin H2 are known to be important modulators with opposite biological activities. To examine possible roles of these prostanoids in immune responses, we have studied the gene expression of prostacyclin synthase (PGIS) and thromboxane synthase (TXS) in murine resident macrophages or in macrophages elicited with casein or bacillus Calmette-Guérin (BCG). Northern blot analyses showed that the PGIS mRNA was expressed in a decreasing order in the resident, and casein- and BCG-elicited macrophages. In contrast, the TXS mRNA was expressed in an increasing order in the resident, and casein- and BCG-elicited macrophages. On the other hand, the mRNA for cyclooxygenase-2, which produces PGH2 and participates in the production of prostanoids in inflammation, was expressed in both the resident and BCG-elicited macrophages but barely in the casein-elicited cells. In situ hybridization analysis showed that the expression of mRNAs for PGIS and TXS was ascribable not only to the alteration of the expression levels of both mRNAs in the each macrophage but also to the changes in subpopulations of the cells expressing these mRNAs. These observations suggested that the inverse gene expression of PGIS and TXS in macrophages contributes to immune responses by modulating the relative levels of prostacyclin and thromboxane A2.