Rapid determination of parvalbumin amino acid sequence from Rana catesbeiana (pI 4.78) by combination of ESI mass spectrometry, protein sequencing, and amino acid analysis.
The complete amino acid sequence of beta-type parvalbumin (PA) from bullfrog Rana catesbeiana (pI 4.78) was determined by tandem mass spectrometry in combination with amino acid analysis and peptide sequencing following Arg-C and V(8) protease digestion. The primary structure of the protein was compared with that of beta-type PA from R. esculenta (pI 4.50), with which it is highly homologous. Compared with R. esculenta beta-type PA4.50, R. catesbeiana beta-type parvalbumin (PA 4.78) differed in 15 out of 108 amino acid residues (14% displacement), PA4.78 had Cys at residue 64 and was acetylated at the amino terminus, but 25 residues of the carboxyl terminus were completely conserved. Several amino acid displacements were found between residues 51 and 80 (30% displacement), although the functionally important sequence of PA was completely conserved. The amino acids residues of putative calcium-binding sites were Asp-51, Asp-53, Ser-55, Phe-57, Glu-59, Glu-62, Asp-90, Asp-92, Asp-94, Lys-96, and Glu-101, which were conserved in all a and b-types of R. catesbeiana as well as other parvalbumins. In addition, Arg-75 and Glu-81, which are thought to form a salt bridge located in the interior of the molecule [Coffee, C.J. et al. (1976) Biochim. Biophys. Acta 453, 67-80], were also conserved in PA4.78.
Taka
H
H
Division of Biochemical Analysis, Central Laboratory of Medical Sciences, Juntendo University School of Medicine, Hongo, Bunkyo-ku, Tokyo 113-8421, Japan.
Kaga
N
N
Fujimura
T
T
Mineki
R
R
Imaizumi
M
M
Suzuki
Y
Y
Suzuki
R
R
Tanokura
M
M
Shindo
N
N
Murayama
K
K
D000595:Amino Acid Sequence; D000818:Animals; D002851:Chromatography, High Pressure Liquid; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010320:Parvalbumins / Q000737:chemistry* / Q000145:classification; D010446:Peptide Fragments / Q000737:chemistry; D010802:Phylogeny; D011892:Rana catesbeiana; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D012697:Serine Endopeptidases / Q000378:metabolism
D010320:Parvalbumins; D010446:Peptide Fragments; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase