Monoclonal antibodies that inhibit the enzyme activity of NAD+-dependent 15-hydroxyprostaglandin dehydrogenase

Abstract

Three hybridoma cell lines secreting antibodies against human placental NAD+-dependent 15-hydroxyprostaglandin dehydrogenase (15-OH-PGDH) were produced. Purified IgG(2b) from these cell lines recognized a distinct band of M(r) 28000 on SDS/PAGE from the purified enzyme as well as a band of M(r) 56000 from the crude enzyme preparation. These three monoclonal antibodies inhibited 15-OH-PGDH activity to different degrees. Inhibition of the enzyme activity could be prevented by prior incubation of the enzyme with NAD+ but not with prostaglandin E2 (PGE2) or NADP+. Inhibition by monoclonal antibodies appears to be non-competitive with respect to NAD+ and PGE2. An increased concentration of antibodies alters the apparent K(m) for NAD+ but not for PGE2, further supporting the notion that the antibodies bind to the coenzyme-binding site. The availability of these monoclonal antibodies should be valuable for probing the structure of the active site.