Renal metabolism of the beta-subunit of human choriogonadotropin in the rat.

We analyzed the immunoreactive renal metabolites of the beta-subunit moieties of unlabeled highly purified hCG, hCG beta, and desialylated hCG (as-hCG) in rats by RIA and Sephadex G-100 chromatography. Infusions of hCG beta, as-hCG, or intact hCG resulted in accumulation in the kidney of a large quantity of small mol wt peptides lacking the immunological determinants of the carboxy-terminal peptide (CTP) of the beta-subunit. In the case of as-hCG, renal accumulation of these beta-core fragments was greatly enhanced when as-hCG binding to hepatic galactose receptors was inhibited by infusion of as-fetuin. The beta-core fragments in kidney had the same immunological and G-100 chromatographic characteristics as beta-core fragments in liver, suggesting similar intracellular catabolic mechanisms in these tissues. The kinetics of beta-core fragment turnover in kidney were studied after injection of hCG beta, which is cleared from the circulation within 1 h. Loss of beta CTP immunoreactivity was the initial event in hCB beta catabolism by the kidney; most of this process occurred between 7 and 30 min after injection. This was followed by a gradual reduction of the size of accumulated hCG beta metabolites over the next 60 min. The beta-core fragments that accumulated had a Kav of approximately 0.57 and a very slow degradation rate over the next 5 h (half-life greater than 6 h). Chromatographic analysis of urine obtained 6 h after beginning a continuous infusion of hCG, hCG beta, or as-hCG displayed in each case a major peak corresponding to the infused molecule, apparently intact, and a minor peak of beta CTP immunoreactivity of small mol wt. Relative to the beta CTP fragments apparent in urine, there were few beta-core fragments. These data indicate that separate fates exist for immunoreactive fragments generated by hCG beta metabolism in the rat kidney. One appears to be intracellular and similar to the liver pathway of as-hCG degradation in that it leads to the formation of long-lived beta-core fragments. The other takes place within ready access to the urinary compartment and leads to the accumulation in urine of beta-CTP fragments.

Evidence that desialylation and uptake by hepatic receptors for galactose-terminated glycoproteins are immaterial to the metabolism of human choriogonadotropin in the rat.

It is widely known that removal of sialic acid from the carbohydrate chains of glycoproteins in vitro drastically reduces their survival time in the circulation; however, it is not known whether desialylation plays a significant role in the metabolism of sialylated serum glycoproteins in vivo. We have studied the metabolism of hCG and desialylated hCG (as-hCG) in rat serum and liver in vivo to assess this putative metabolic pathway for glycoprotein hormones. A single injection of as-hCG into rats was followed by its rapid removal from the circulation, principally by the liver, and its degradation into fragments of the hCG beta-subunit that lacked the carboxy-terminal peptide antigenic determinant. Continuous infusion of desialylated fetuin (as-fetuin) at a high rate along with as-hCG dramatically reduced accumulation of the beta-subunit fragments in liver and resulted in increased serum levels of as-hCG. Consequently, the MCR of as-hCG was reduced from 301 +/- 10.3 ml/h (mean +/- SE) to 13.4 +/- 1.15 ml/h by infusion of as-fetuin, which is known to compete for hepatic receptors for galactose-terminated glycoproteins. In contrast, coinfusion of as-fetuin with hCG did not influence the MCR of hCG. Furthermore, there was no serum accumulation of hCG desialylated in its hCG beta carboxy-terminal portion, with or without as-fetuin, and the quantity and pattern of immunoreactive hCG products in liver homogenate were not affected by coinfusion of as-fetuin with hCG. Thus, blockade of hepatic receptors for galactose-terminated glycoproteins did not impede hCG turnover in the circulation, impair hepatic catabolism of hCG, or lead to the accumulation of desialylated products of hCG in plasma. These data indicate that in the rat, there is negligible catabolism of glycoproteins, such as hCG, by a pathway that involves peripheral desialylation and subsequent hepatic uptake via receptors for galactose-terminated glycoproteins.

Relevance of the low and high affinity thyrotropin-binding sites of human thyroid membranes to the stimulation of adenylate cyclase.

TSH is known to interact on thyroid membranes with two classes of binding sites that differ in affinity and capacity. To assess the relevance of the class of TSH-binding sites characterized by low affinity and high capacity to the stimulation of adenylate cyclase, we studied the interactions of desialylated hCG (as-hCG) and its beta-subunit (as-hCG beta) with human thyroid membranes. In low ionic strength buffer, pH 7.8, where both classes of sites are operant, as-hCG fully inhibited and as-hCG beta partially inhibited [125I] bovine (b) TSH binding. Scatchard analysis of the [125I]bTSH binding inhibition curve in the presence of 1.0 X 10(-5) M as-hCG beta clearly indicated that as-hCG beta interacted only with the low affinity class of binding sites, leaving the high affinity class unaffected. In the presence of 140 mM NaCl, [125I]bTSH interacted predominantly with the high affinity class of binding sites; as-hCG fully inhibited [125I]bTSH binding to this class of sites, whereas as-hCG beta displayed essentially no interaction. Scatchard analysis of [125I]as-hCG beta binding to human thyroid membranes in low ionic strength buffer revealed a single apparent class of sites with low affinity (Kd = approximately 1.0 X 10(-6) M) and high capacity (Q = approximately 300 pmol/mg membrane protein). The bTSH preparation (Thytropar) showed a 10-fold greater binding inhibition potency at these sites than either the as-hCG or the as-hCG beta preparation, in keeping with the inference that as-hCG beta interacts with the low affinity class of TSH-binding sites. At a concentration more than 3 times that necessary to inhibit TSH binding to the low affinity class of sites, the as-hCG beta molecule neither stimulated adenylate cyclase nor inhibited the ability of TSH to do so. In contrast, the as-hCG molecule, which interacts with both classes of TSH-binding sites, fully inhibited TSH stimulation of adenylate cyclase. We conclude that the low affinity class of TSH-binding sites is not the class of sites through which TSH stimulates adenylate cyclase, and that this role is best ascribed to the high affinity class of TSH-binding sites.

Metabolic clearance rate of dopamine beta-hydroxylase in the rat.

The metabolic clearance rate (MCR) of both bovine and rat dopamine beta-hydroxylase (DBH) preparations was measured using two complementary procedures, pulse-dose injection and constant infusion of enzyme into the rat circulation. Rats that received injections of DBH activity had plasma DBH activity levels similar to those of controls by 24 hr after a pulse dose of rat DBH. The DBH MCR computed by stochastic analysis of the disappearance curve of injected DBH activity was about 1.0 ml/hr/100 g body weight; the mean transit time of DBH was about 8 hr. The disappearance curve of heterologous enzyme (bovine DBH) was more rapid than that of the rat, yielding an MCR of about 8 ml/hr/100 g body weight. MCR values obtained with the constant-infusion technique were similar to those obtained with the pulse-dose technique. These kinetic parameters are consistent with the time frame for altered plasma DBH activity observed with pharmacological and endocrinological factors. These data support the conclusion that plasma DBH turnover time is measured in hours, not days.