Purification, characterization, and partial sequence analysis of a newly identified EF-hand type 13-kDa Ca(2+)-binding protein from smooth muscle and non-muscle tissues.
A novel Ca(2+)-binding protein, tentatively designated calgizzarin, has been purified to apparent homogeneity from chicken gizzard smooth muscle by W-7 (N-(6-aminohexyl-5-chloro-1-naphthalenesulfonamide))-Sepharose affinity chromatography and ion-exchange chromatography. Application of W-7-Sepharose affinity chromatography to various tissues revealed that calgizzarin-like proteins were abundant in bovine aorta and rabbit lung. Using the same procedure, we could purify a calgizzarin-like protein from rabbit lung. Calgizzarin has a Mr of 13,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and approximately 30,000 as determined by gel filtration on a TSK G 3000SW high performance liquid chromatography column, suggesting that calgizzarin seems to be a rodlike protein. The isoelectric point of calgizzarin was found to be pH 5.8. Calgizzarin can exist as a dimer by forming a disulfide bridge. The 45Ca autoradiographic technique showed that the protein binds to Ca2+. On an alkaline/urea gel, calgizzarin migrated faster in the presence of EGTA than in the presence of CaCl2, thereby indicating a Ca(2+)-dependent conformational change in this protein. The partial amino acid sequence (65 amino acid residues) of calgizzarin was seen to be SLLAVFQRYAGREGDNLKLSKKEFRTFMNTELASFTKNQKDPAVVDRMMKRLDINSDGQLDFQEF, and two putative Ca(2+)-binding sites (GREGDNLKLSKKE and D INSDGQLDFQE) were detected. So far as the obtained 65-amino acid sequence is concerned, calgizzarin has approximately a 50% sequence homology with S-100 alpha, 47% with S-100 beta, and 39% with pEL-98 protein.
Department of Pharmacology, Nagoya University School of Medicine, Japan.
H
H
Todoroki
R
R
Kobayashi
M
M
Watanabe
H
H
Minami
H
H
Hidaka
D000595:Amino Acid Sequence; D000818:Animals; D002645:Chickens; D002846:Chromatography, Affinity; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D008168:Lung / Q000737:chemistry*; D008969:Molecular Sequence Data; D009130:Muscle, Smooth / Q000737:chemistry*; D011817:Rabbits; D009418:S100 Proteins / Q000737:chemistry / Q000235:genetics*; D016415:Sequence Alignment
D009418:S100 Proteins; C072337:S100A11 protein, human