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Combination of the chorionic gonadotropin free beta-subunit with alpha.

The rate-limiting event for combination of hCG alpha- and beta-subunits in JAR choriocarcinoma cells is the rate of disulfide bond formation in the beta-subunit. This is accompanied by a conformational change that produces a combination-competent form of the beta-subunit. The combination reaction, however, is incomplete, and 50% of the synthesized beta molecules remain uncombined (free). In addition, 70% of biosynthetically labeled free beta is degraded in the cell. Possible explanations for incomplete dimer formation include 1) biochemical differences between the free and combined beta-subunits that limit combination of free beta, and 2) an inefficient combination reaction due to low intersubunit affinities or limiting concentrations of combination-competent subunits within the cell. To examine whether the biochemical differences between free and combined beta-subunits that we have previously observed affect the combined beta-subunits that we have previously observed affect the combination competence of free beta, free and dimer beta-subunits were purified from the culture medium and lysates of JAR cells and examined for their ability to combine with alpha purified from pregnancy urine in an in vitro combination assay. Secreted free and dimer beta obtained from culture medium combined to the same extent with urinary alpha. Although the combination efficiencies were lower for the intracellular forms, the free and dimer beta-subunits purified from cell lysates also combined to the same extent with urinary alpha. Thus, biochemical differences that exist between the beta forms do not prevent combination of free beta with alpha in an in vitro combination assay. To examine the second possibility, we speculated that if high concentrations of hCG subunits remained in the rough endoplasmic reticulum (ER) for extended periods of time, the extent of dimer formation would increase in the cell. To increase the residence time of hCG subunits in the ER, JAR cells were treated with carbonyl cyanide trifluoromethoxyphenylhydrazone, an agent that inhibits the translocation of hCG subunits from the ER to the Golgi. Treatment of cells with trifluoromethoxyphenylhydrazone in long and short term pulse-chase labeling studies did not result in an increase in the extent of dimer formation. Thus, the subunit combination reaction in JAR cells may be incomplete due to subtle conformational differences in the free beta-subunit; however, these differences do not inhibit the combination of the free beta-subunit in vitro.