Binding of myotoxin a to sarcoplasmic reticulum Ca(2+)-ATPase: a structural study.
The interaction of myotoxin alpha with intact sarcoplasmic reticulum (SR) components was investigated, and two SR proteins were identified that associated with myotoxin a. One of the proteins has an apparent molecular weight similar to the Ca(2+)-ATPase, the major SR protein responsible for calcium loading. Ca(2+)-ATPase was purified, and its interaction with myotoxin a was studied. Evidence for specific binding of myotoxin a to Ca(2+)-ATPase was established by isolating chemically cross-linked myotoxin a-Ca(2+)-ATPase complexes and further proving their association with anti-myotoxin a antibodies. The binding region of myotoxin a was further delineated by cleaving the protein with cyanogen bromide (CNBr) into two fragments, a larger N-terminal fragment of 28 residues and a smaller C-terminal fragment of 14 residues. Competition experiments with 125I-myotoxin a showed that the C-terminal fragment competed better against 125I-myotoxin a than the N-terminal fragment for SR protein binding. Two overlapping peptides covering the sequence of the N-terminal fragment were synthesized to clarify the interaction of the N-terminal fragment of myotoxin a with SR proteins. A 16-residue peptide corresponding to residues 1-16 competed strongly with 125I-myotoxin a, while a second peptide (residues 13-28) did not.
Utaisincharoen
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Department of Biochemistry, Colorado State University, Fort Collins 80523.
Baker
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D000595:Amino Acid Sequence; D000818:Animals; D001667:Binding, Competitive; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D003435:Crotalid Venoms / Q000737:chemistry / Q000378:metabolism*; D003488:Cyanogen Bromide; D004797:Enzyme-Linked Immunosorbent Assay; D006868:Hydrolysis; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism; D011485:Protein Binding; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology / Q000378:metabolism*; D013329:Structure-Activity Relationship
D003435:Crotalid Venoms; D010446:Peptide Fragments; C018932:myotoxin A; D000252:Calcium-Transporting ATPases; D003488:Cyanogen Bromide