Structural changes in the C-terminal region of human brain creatine kinase studied with monoclonal antibodies.
Epitopes on human brain creatine kinase (B-CK) recognized by three monoclonal antibodies have been located by chemical cleavage methods, followed by peptide synthesis or analysis of specificity for natural variants (isoforms). One antibody, CK-HTB, recognizes a conformational, or assembled, surface epitope on native CK which is also present on partially unfolded forms. It requires an Asn residue at position 300 in the amino acid sequence and will not recognize variants with Lys or His in this position. This results in a striking specificity of the antibody, which binds to B-CK only in chicken and man, but to muscle-form (M-CK) only in the rat. The results suggest that Asn-300 is exposed on the enzyme surface as part of a relatively denaturation-resistant region. Two monoclonal antibodies, CK-END1 and CK-END2, recognise epitopes within 53 amino acids of the C-terminus and bind to a synthetic hexapeptide representing the last six amino acids of human B-CK (Leu-375-Lys-380). The two antibodies show overlapping, but distinct, specificities in their binding to CK variants. CK-END1 requires Met-376 and will not tolerate Ile in this position, whereas CK-END2 requires Leu-375 and will not tolerate Met. Neither antibody binds to native CK, though both will bind to a folding intermediate and to partially unfolded states. This shows that the C-terminus of CK becomes inaccessible to the antibodies during those later stages of protein folding associated with recovery of enzyme activity and suggests that the protein may 'tuck in its tail' during one of the final steps.
Nguyen thi Man
Research Division, N.E. Wales Institute, Deeside, Clwyd, U.K.
Cartwright
A J
AJ
Osborne
M
M
Morris
G E
GE
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal; D001921:Brain / Q000201:enzymology*; D003402:Creatine Kinase / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism; D000939:Epitopes / Q000032:analysis*; D006801:Humans; D007527:Isoenzymes; D007700:Kinetics; D008969:Molecular Sequence Data; D011485:Protein Binding; D012689:Sequence Homology, Nucleic Acid
D000911:Antibodies, Monoclonal; D000939:Epitopes; D007527:Isoenzymes; D003402:Creatine Kinase