PT  - JOURNAL ARTICLE
AU  - G Morris
AU  - J S Mistry
AU  - J P Jani
AU  - J E Mignano
AU  - S M Sebti
AU  - J S Lazo
TI  - Neutralization of bleomycin hydrolase by an epitope-specific antibody.
DP  - 1992 Jul 01
TA  - Molecular Pharmacology
PG  - 57--62
VI  - 42
IP  - 1
4099  - http://molpharm.aspetjournals.org/content/42/1/57.short
4100  - http://molpharm.aspetjournals.org/content/42/1/57.full
SO  - Mol Pharmacol1992 Jul 01; 42
AB  - Bleomycin hydrolase (BH) is a cysteine proteinase that terminates the pharmacological action of bleomycin (BLM). Amino acid sequence data obtained from a tryptic digest fragment of purified rabbit lung BH were used to synthesize a 14-amino acid peptide (LAVLEQEPIVLPAK; BHP14), which was conjugated to horseshoe crab hemocyanin and used to produce rabbit antiserum that was immunoreactive to both BHP14 and rabbit BH. Anti-BHP14 binding to BHP14 could be competitively blocked by the presence of either BHP14 or BH. Anti-BHP14 recognized both purified rabbit liver BH and postmicrosomal fraction from rabbit liver on Western blot, as a single band of M(r) approximately 48,000. Anti-BHP14 inhibited, in a concentration-dependent manner, BH activity in rabbit liver cytosolic fractions, as measured by deamido-BLM A2 formation. Thus, we have generated an epitope-specific neutralizing antibody to rabbit BH, which can block the metabolism of BLM by homogenates from rabbit tissue. These results suggest that the LAVLEQEPIVLPA epitope of rabbit BH is involved in the metabolism of BLM or is topologically near the active site. Furthermore, a BLM-resistant squamous carcinoma (C-10E) exhibited slightly more immunoreactivity, by enzyme-linked immunosorbent assay, to anti-BHP14 than did the parental A-253 cells, and a partially revertant (C-10E ND) cell line had intermediate anti-BHP14 binding. BH activity in these cell lines was in the same rank order as antibody binding, but differences in immunoreactivity were less than differences in enzymatic activity. Our epitope-specific neutralizing antibody should be useful in the further characterization of BH.