Kardana A, Cole L A
Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, Connecticut 06520.
J Clin Endocrinol Metab. 1994 Sep;79(3):761-7. doi: 10.1210/jcem.79.3.7521352.
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)
人绒毛膜促性腺激素（hCG）是一种由α亚基和β亚基非共价结合而成的二聚体。除了hCG二聚体，循环中还能检测到未结合的α亚基和β亚基（游离β亚基）以及切口hCG和游离β分子（在44 - 45或47 - 48位裂解）。在这些循环分子中，只有完整的hCG二聚体充分表达生物学活性。孕期中使hCG和β亚基分子解离、产生切口并降解的途径尚不清楚，可能在调节激素水平方面起主要作用。之前已描述了针对完整（无切口）hCG和完整β亚基（切口分子检测率<1%）的免疫测定法以及用于测量切口hCG水平的消减免疫测定系统。本文描述了一种用于测量切口β亚基水平（完整β亚基检测率<6%）的多抗体清除测定法。在本报告中，我们使用这四种测定法来评估对照（健康未孕）、孕妇和癌症患者血清样本中完整hCG或β亚基随时间转化为切口形式（切口酶活性）的情况。将孕妇和对照血清池补充完整hCG及其解离的β亚基，并在37℃孵育。在0 - 48小时之间进行完整和切口分子的测量。在两个不同的对照血清池中，48小时内未检测到完整hCG或完整β亚基的损失，也未检测到切口hCG或切口β亚基的显著增加。这表明对照血清中缺乏切口酶活性。在两个不同的孕早期孕妇血清池中，我们发现48小时内完整hCG没有明显损失，切口hCG水平也没有增加。然而，在同一时间段内，我们发现完整β亚基分别损失了70%和62%（池1和池2），切口β亚基分别增加了51%和39%。我们推断孕妇血清中存在未结合或游离β亚基修饰活性。我们用六种不同浓度的β亚基（0.62 - 29 mg/L）重复了孕妇血清实验。在37℃下，六种浓度在长达6小时内切口百分比与时间呈线性关系（r = 0.97）；之后，切口速率下降。速率与浓度的关系图显示出典型的米氏酶关系（对数回归，r = 0.96）。通过凝胶过滤对孕妇血清β亚基切口活性进行了部分纯化。出现了一个单一的活性峰，在分子量150,000 - 443,000的标准品之间洗脱。（摘要截断于400字）
