부산대학교 도서관

To link to full-text access for this article, visit this link: http://dx.doi.org/10.1016/j.bbrc.2005.09.137 Byline: Liron Boyman, Reuben Hiller, Beni Shpak, Erika Yomtov, Chagit Shpak, Daniel Khananshvili Keywords: Calcium; Sodium-calcium exchange; Endogenous inhibitor; NCX1; Cardiac muscle Abstract: A low molecular weight inhibitor (NCX.sub.IF) of the cardiac Na/Ca exchanger, isolated from the calf ventricle tissue, is capable of regulating the muscle strip's contractility and relaxation without involving the [beta]-activation pathway. The structural analysis of NCX.sub.IF requires highly purified preparations that fulfill the demanding requirements for mass spectra and NMR analyses. No such preparation is yet available. To this end, new HPLC procedures were developed by a combination of the reverse phase, normal phase, and HILIC (hydrophilic liquid chromatography) techniques. The specific activity of NCX.sub.IF is 10.sup.5 times higher in the purified preparations (as compared to the crude extract) showing a 2-5% yield of total inhibitory activity and 20-100[mu]g content of final material. The purification yield reveals that 1kg ventricle muscle contains 0.1-0.2mg NCX.sub.IF, meaning that the tissue concentrations of NCX.sub.IF may reach 10.sup.-7-10.sup.-6 M. The diode-array scanning of purified preparations of NCX.sub.IF shows a homogeneous 3D peak with a maximal absorption at 202nm. These spectral properties may represent a five-membered ring (e.g., proline, histidine) and/or simple chemical groups (like amine, carbonyl, ester, etc.), but not an aromatic ring or complex conjugates (alkyne, alkene, aldehyde, etc.). NCX.sub.IF does not respond to phenol/sulfur reagent, suggesting that it lacks reducing (aldo) sugar. NCX.sub.IF shows a faint response to fluorescamine, meaning that it may contain an amino group (or its derivative). It is believed that a combination of presently developed procedures with LC/MS and LC/MS/MS may provide a useful tool for structural analysis of NCX.sub.IF. Author Affiliation: Department of Physiology and Pharmacology, Sackler School of Medicine, Tel-Aviv University, Ramat-Aviv 69978, Israel Article History: Received 20 September 2005 Article Note: (footnote) [star] This work was supported in part from the Israeli Academy of Sciences (Grant No. 16.0-424) and the USA Israeli Binational Science Foundation, BSF (Grant No. 2003-372)., [star][star] Abbreviations: Mops, 3-(N-morpholino)propanesulfonic acid; Tris, Tris(hydroxymethyl)aminomethane; EGTA, ethylene glycol bis([beta]-aminoethyl ether)-N,N,N',N'-tetraacetic acid; PMSF, phenylmethanesulfonyl fluoride; FRCRCFa, cyclic hexapeptide Phe-Arg-Cys-Arg-Cys-Phe-NH2 with intramolecular SS bond; TFA, trifluoroacetic acid; HFBA, heptafluorobutyric acid; LC/MS, liquid chromatography coupled with MS; LC/MS/MS, liquid chromatography coupled with MS/MS; GC/MS/MS, gas chromatography coupled with mass spectroscopy.