Human chorionic gonadotropin beta-subunit nicking enzymes in pregnancy and cancer patient serum.

Abstract

hCG is a dimer composed of an alpha- and a beta-subunit, joined noncovalently. In addition to the hCG dimer, uncombined alpha- and beta-subunits (free beta) and nicked hCG and free beta molecules (cleaved at 44-45 or 47-48) can be detected in the circulation. Of these circulating molecules, only the intact hCG dimer fully expresses biological activity. The pathways that dissociate, nick, and degrade hCG and beta-subunit molecules in pregnancy are unknown and could have a major role in regulating hormone levels. Immunoassays for intact (nonnicked) hCG and intact (beta-subunit (with < 1% detection of nicked molecules) and a subtractive immunoassay system for measuring nicked hCG levels have been described previously. A multiantibody scavenger assay is described here for measuring nicked beta-subunit levels (< 6% detection of intact beta-subunit). In this report we use these four assays to assess conversion of intact hCG or beta-subunit to nicked forms over time (nicking enzyme activities) in control (healthy nonpregnant), pregnant, and cancer patient serum samples. Pools of pregnancy and control sera were supplemented with intact hCG and its dissociated beta-subunit and incubated at 37 C. Intact and nicked molecule measurements were made between 0-48 h. In two different pools of control sera, no loss of intact hCG or intact beta-subunit and no significant gain in nicked hCG or nicked beta-subunit were detected over 48 h. This indicated a lack of nicking enzyme activity in control serum. In two different pools of first trimester pregnancy sera, we found no obvious loss of intact hCG or gain of nicked hCG levels over 48 h. However, we found 70% and 62% losses (pools 1 and 2) of intact beta-subunit and 51% and 39% gains of nicked beta-subunit over the same time period. We inferred that an uncombined or free beta-subunit-modifying activity was present in pregnancy serum. We repeated the pregnancy serum experiment with six different concentrations of beta-subunit (0.62-29 mg/L). A linear relationship, percent nicking against time, existed for the six concentrations for up to 6 h at 37 C (r = 0.97); after that, the rate of nicking declined. A plot of rate against concentration against revealed a classical Michaelis-Menten enzyme relationship (logarithmic regression, r = 0.96). The pregnancy serum beta-subunit nicking activity was partially purified by gel filtration. A single peak of activity emerged, eluting between the 150,000-443,000 mol wt standards.(ABSTRACT TRUNCATED AT 400 WORDS)