A parvalbumin, with its characteristic low molecular weight (∼ 12000), acidic isoelectric point (∼ 5.5), ultraviolet spectrum (maxm 259 nm) and Ca2+‐binding capacity (2 mol/mol protein) has been isolated from rabbit (Oryctolagus cuniculus) muscle. Its primary structure has been determined from a study of its tryptic peptides and of overlapping peptides generated by limited tryptic digestion and by chymotryptic and thermolytic digestions of the protein. The amino acid sequence so obtained is considered in comparison with those known for other parvalbumins and for rabbit troponin C.
The primary structure of the most acidic ( P I = 4.50) of the two major parvalbumins from frog (Rana esculenta) has been determined from a study of its trypsic peptides and of overlapping peptides generated by limited trypsic digestion, chymotrypsic digestion and N-bromosuccinimide cleavage of the protein.The amino acid sequence so obtained is considered in comparison with those known for other parvalbumins and for rabbit troponin-C.Previous work on the primary structure of muscular parvalbumins, isolated from the white muscles of lower vertebrates [l], has dealt exclusively with components as obtained from different species of fish, namely hake [2 -41, carp [5] and pike [6]. It therefore appeared of interest to study the amino acid sequence of an amphibian parvalbumin, the higher class of organisms where such proteins are found abundantly. The present report is thus concerned with a study of one of the two major components from frog muscle, which former work [7,8] had shown to be easily obtainable in the pure state. MATERIALS AND METHODS Preparation of the ProteinFrogs (Rana esculenta) were provided by the Elevages Couitard (St-Hilairede Rier, Vendee, France). The two major parvalbumins were prepared as described previously [8] starting with 123 animals which yielded 1354 g of hind-leg muscles from which 514 mg of the most acidic component (PI 4.50) and 1100 mg of the less acidic component (PI 4.88) could be isolated. The two components were pure as judged by disc electrophoresis in 12 % polyacrylamide gel at pH 5.5 [9] indicated that only the most acidic component was homogeneous. This was therefore chosen for the sequence studies. ChemicalsBio-gel P4 and P150 were obtained from Biorad Laboratories. Thermolysin was from Daiwa Kasei, KK (Japan). N-Bromosuccinimide was obtained from Pierce Chemical Co, phthalic anhydride from Prolabo, succinic anhydride and guanidinium chloride from B.D.H.Polyamide sheets for thin-layer chromatography of dansyl-amino acid were from Chen Ching Corp. (Taiwan).Other chemicals were as described previously [3,41. Trypsic and Chymotrypsic DigestionsThe protein was first dissolved (25 mg/ml) in 6 M guanidinium chloride being 0.1 M in sodium phosphate, pH 8, and incubated for 1 h at 50 "C. It was afterwards desalted on a column of Sephadex G-25 (0.005 M ammonium bicarbonate) in the cold, lyophilized, and redissolved in water (10 mgiml). After heating under stirring for 5 min on a boiling water bath, the solution, which had become abundantly turbid, was cooled to room temperature, ammonium bicarbonate was added to a final concentration of 0.2 M, then enzyme solution (5 mg/ml in 0.001 M HC1) so as to reach an E/S = 1 % (w/v) and the mixture was Eur. J. Biochem. 56 (1975)
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