Pig heart calpastatin: identification of repetitive domain structures and anomalous behavior in polyacrylamide gel electrophoresis.
Isolation and nucleotide sequencing of the complementary DNA for pig heart calpastatin have been completed. The amino acid sequence of 713 residues predicted from the nucleotide sequence contains five domains, each composed of approximately 140 amino acid residues. A unique N-terminal domain is followed by four mutually homologous domains. The best fit alignment of these four domains gives residue identities between any two domains of 22.5-36.0%. The analysis of the sequence similarities by several methods also suggests the existence of additional shorter repeats at intervals of 60-80 residues. The calculated molecular weight of pig calpastatin of 713 amino acid residues (Mr 77,122) is significantly lower than the value of purified pig heart calpastatin (Mr 107,000) estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS-PAGE). The expression of the calpastatin genes in Escherichia coli and the detection of the translation products of 713, 366, and 140 amino acid residues by the specific anti-calpastatin antibody indicate that the products always migrate considerably slow on SDS-PAGE, giving an average of 1.53 for the ratio of the molecular weight estimated by SDS-PAGE to the value calculated from the amino acid sequences. It is most likely that the discrepancy in the molecular weight is caused by an anomalous behavior of calpastatin in SDS-PAGE.
Takano
E
E
Department of Clinical Science and Laboratory Medicine, Faculty of Medicine, Kyoto University, Japan.
Maki
M
M
Mori
H
H
Hatanaka
M
M
Marti
T
T
Titani
K
K
Kannagi
R
R
Ooi
T
T
Murachi
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002135:Calcium-Binding Proteins / Q000235:genetics*; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics; D004262:DNA Restriction Enzymes; D004926:Escherichia coli / Q000235:genetics; D005796:Genes; D008969:Molecular Sequence Data; D009206:Myocardium / Q000378:metabolism*; D010957:Plasmids; D011480:Protease Inhibitors / Q000235:genetics*; D013552:Swine
D002135:Calcium-Binding Proteins; D011480:Protease Inhibitors; C033668:calpastatin; D004247:DNA; D004262:DNA Restriction Enzymes