Biosynthesis, glycosylation, and secretion of rat luteinizing hormone alpha- and beta-subunits: differential effects of orchiectomy and gonadotropin-releasing hormone

We have studied the de novo biosynthesis and secretion of LH subunits in pituitary quarters from orchiectomized and intact control adult male rats and their regulation by GnRH. After labeling with [35S]cystine ([35S]Cys), [35S]methionine, or [3H]glucosamine ([3H]GlcN) in the presence or absence of 10(-8) M GnRH, tissue lysates and media were immunoprecipitated with antisera to LH beta, then LH alpha (after removal of TSH by immunoprecipitation with anti-TSH beta), and the products were analyzed by sodium dodecyl sulfate gradient gel electrophoresis. During a 12-min pulse labeling with [35S]methionine, three forms of immunoreactive alpha were labeled at 21,000, 18,000, and 12,000 mol wt. After a 30-min chase with excess unlabeled methionine, the 12,000 form decreased from 10% to 3% of total radioactivity, while the 21,000 form increased from 57% to 69%, implying a precursor-product relationship. Neither orchiectomy nor GnRH had any effect on [35S]Cys or [3H]GlcN incorporation into intracellular or secreted total proteins. After a 6-h continuous labeling, incorporation of [35S]Cys into intracellular combined LH alpha in castrates was 158% of the control value, combined LH beta was 304%, and free alpha was 466%. The [3H]GlcN to [35S]Cys ratio, reflecting relative glycosylation, was unchanged in castrates for total proteins or LH alpha and somewhat decreased for LH beta and free alpha. Orchiectomy increased [35S]Cys-labeled secreted LH beta and free alpha to 183% and 231% of control values, respectively. Relative glycosylation of secreted LH alpha, LH beta, and free alpha was unchanged in castrates. Incorporation of [35S]Cys into intracellular combined LH alpha, LH beta and free alpha-subunit was unaffected by GnRH in pituitaries from intact rats. In castrates, LH alpha was unchanged, but LH beta and free alpha were slightly increased. Incorporation of [3H]GlcN into intracellular combined LH alpha, LH beta, and free alpha was increased with GnRH in both intacts and castrates, such that the 3H to 35S ratio, reflecting relative glycosylation, was also increased with GnRH. In castrates, the ratios, as a percentage of the control, were, respectively 250%, 250%, and 223% for LH alpha, LH beta, and free alpha. In intact animals, the ratios were 221%, 281%, and 143%, respectively. Incorporation of both [35S]Cys and [3H]GlcN into secreted subunits was increased in most instances, such that the 3H to 35S ratio was increased only for LH beta.(ABSTRACT TRUNCATED AT 400 WORDS)