Fragmentation of rabbit skeletal muscle calsequestrin: spectral and ion binding properties of the carboxyl-terminal region.
Rabbit skeletal muscle calsequestrin was fragmented by using trypsin in the presence and absence of calcium. Calcium ion was found to protect calsequestrin from proteolysis, and the peptides produced in the presence of calcium were stable to further digestion. Peptides produced in the presence or absence of calcium had a decreased helical content but maintained their ability to bind calcium. The amino acid sequence of a 59-residue carboxyl-terminal tryptic peptide was determined by automated Edman degradation and carboxypeptidase Y digestion of carboxyl-terminal tryptic, chymotryptic, and cyanogen bromide peptides. This peptide is highly acidic (Asp + Glu = 42%, Lys + Arg = 0), and it bound a total of 15 calcium ions per mole of peptide (Kd = 8.5 mM). The intrinsic tryptophan fluorescence of the peptide was enhanced by 10% upon binding Ca2+ with the dissociation constant of 1 mM. Analyses of the circular dichroism spectra of the peptide showed that it was primarily in a random-coil conformation with little helical (2%) and moderate beta-structure (25%) regardless of the calcium concentration. This peptide also bound 7 mol of terbium per mole of peptide with high affinity (Kd = 7.5 microM).
Department of Biochemistry, University of Alberta, Edmonton, Canada.
M
M
Ohnishi
R A
RA
Reithmeier
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism; D002155:Calsequestrin / Q000302:isolation & purification / Q000378:metabolism*; D002268:Carboxypeptidases; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D007700:Kinetics; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000378:metabolism*; D009132:Muscles / Q000378:metabolism*; D010446:Peptide Fragments / Q000302:isolation & purification; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D013050:Spectrometry, Fluorescence; D014357:Trypsin / Q000378:metabolism
D002155:Calsequestrin; D009124:Muscle Proteins; D010446:Peptide Fragments; D002268:Carboxypeptidases; C022633:serine carboxypeptidase; D014357:Trypsin; D002118:Calcium