The use of monoclonal antibodies to study the structure and function of cytochrome f

Abstract

Monoclonal antibodies (MAbs) were prepared against native cytochrome f (cyt f) isolated from turnip leaves. The two MAbs obtained, designated MAb-JB2 and MAb-ED4, were Western blot positive to purified turnip cytochrome f and also reacted with inside-out (ISO) but not right-side-out (RSO) spinach thylakoid membranes. MAb-ED4 reacted with a covalent adduct formed by crosslinking cyt f and plastocyanin (PC), whereas MAb-JB2 did not. In contrast, MAb-JB2 reacted with the isolated cyt b6/f complex but MAb-ED4 did not. These results indicate that MAb-JB2 binds to cyt f at or near the PC binding site on f, whereas MAb-ED4 binds to a portion of cyt f which is not exposed in the cyt b6/f complex. The location of the epitopes in the primary sequence of cyt f was determined by trypsin hydrolysis, HPLC separation of tryptic peptides, and ELISA identification of the purified peptides. The molecular weights of the purified peptides, determined by gel exclusion chromatography, were found to be 5040 and 3130 Da for MAb-JB2 and MAb-ED4, respectively. Amino acid sequencing showed that the first eight amino acids of the MAb-ED4 positive peptide were L-D-Q-P-L-T-S-N. These results suggest that the 3130-Da peptide has 28 amino acids extending from Leu 223 to Arg 250. This peptide is located on the N-terminal (lumen) side of the postulated membrane-spanning sequence. The first eight amino acids of the MAb-JB2-positive peptide were N-I-L-V-I-G-P-V. This sequence and the peptide molecular weight indicate that the epitope for MAb-JB2 is located within a 44-amino acid peptide extending from Asn 111 to Arg 154.