Isolation and characterization of a calcium-binding protein derived from mRNA termed p9Ka, pEL-98, 18A2, or 42A by the newly synthesized vasorelaxant W-66 affinity chromatography.
W-66 (N-(2-aminoethyl)-N-[2-(4-chlorocinnamylamino) ethyl]-5-isoquinolinesulfonamide), a newly synthesized isoquinolinesulfonamide, was shown to have a potent vasodilatory action and calmodulin (CaM)-antagonizing action. Using the W-66 affinity chromatographic technique, we purified two Ca(2+)-binding proteins from the EGTA-soluble fraction of bovine aorta. One was CaM and the other was an acidic protein with a molecular mass of 11 kDa. It was tentatively named "calvasculin." Calvasculin was a dimeric protein. Equilibrium dialysis showed that 1 mol of calvasculin (dimer) bound to 1.98 +/- 0.30 mol of Ca2+ in the presence of 10(-3) M Ca2+. Calvasculin failed to activate Ca2+/CaM-dependent enzymes such as myosin light chain kinase, Ca2+/CaM-dependent phosphodiesterase, or Ca2+/CaM-dependent protein kinase II and to inhibit the CaM stimulation of these enzymes. The partial amino acid sequence of calvasculin revealed a high homology to the predicted protein derived from mRNA, named pEL-98, 18A2, 42A, or p9Ka. We also examined the physicochemical and biochemical properties of calvasculin. Using the antibody specific for calvasculin, we obtained evidence that calvasculin is present in abundance in bovine aorta but not in brain, lung, heart, or testis.
Watanabe
Y
Y
Department of Pharmacology, Nagoya University School of Medicine, Japan.
Kobayashi
R
R
Ishikawa
T
T
Hidaka
H
H
D000595:Amino Acid Sequence; D000818:Animals; D001011:Aorta / Q000378:metabolism; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000302:isolation & purification*; D002147:Calmodulin / Q000037:antagonists & inhibitors; D002417:Cattle; D002460:Cell Line; D002846:Chromatography, Affinity / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel; D007546:Isoquinolines / Q000494:pharmacology; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009131:Muscle, Smooth, Vascular / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D000071999:S100 Calcium-Binding Protein A4; D009418:S100 Proteins; D012689:Sequence Homology, Nucleic Acid; D013449:Sulfonamides / Q000494:pharmacology; D014664:Vasodilation / Q000187:drug effects; D014665:Vasodilator Agents
D002135:Calcium-Binding Proteins; D002147:Calmodulin; D007546:Isoquinolines; D046911:Macromolecular Substances; D012333:RNA, Messenger; D000071999:S100 Calcium-Binding Protein A4; D009418:S100 Proteins; C499413:S100A4 protein, Bos taurus; D013449:Sulfonamides; D014665:Vasodilator Agents; C072267:W 66; C017967:W 7