PUTATIVE RAT SPERM LIPID-BINDING PROTEIN - ISOLATION AND PARTIAL CHARACTERIZATION

Previous work has identified a prominent 22-24-kD protein that is pres ent in rat male reproductive tissues, including epididymis and testis (Brooks, 1985; Jones and Brown, 1987; Moore et al., 1987). Using a mon oclonal antibody (designated mAb-B109) against this 24-kD antigen (ref erred to as B109), we have isolated the protein using a combination of chromatofocusing and electroelution from SDS-PAGE gels, and reverse p hase HPLC. B109 (pl = 4.8) is amino-terminal blocked. To obtain intern al amino acid sequences, the isolated protein was cleaved either with cyanogen bromide in 70% formic acid or with TLCK-treated chymotrypsin. With cyanogen bromide treatment, two peptides, 17.8 kD and 11.9 kD, w ere isolated and partial amino acid sequences obtained. Chymotryptic p eptides were isolated by reverse-phase HPLC and two were chosen for se quence analysis. A computer search for sequence homology through the p rotein identification resource (PIR) matched B109 to a basic 21-kD cyt osolic protein (pl = 7.4) found in bovine brain (>80% homology). When peptide sequence differences obtained in the present study were substi tuted into the 21-kD cytosolic protein sequence obtained from the PIR using Intelligenetics(TM) software, the calculated pi dropped from 7.4 to 5.8, suggesting that pl differences between the bovine and rat mol ecules are the result of amino acid substitutions in the testis protei n and not tissue-specific posttranslational processing. It has been po stulated that the 21-kD bovine brain protein is associated with phosph olipid transport, although the function of B109 is unknown. (C) 1994 W iley-Liss, Inc.