Biology of Reproduction, Vol 24, 380-392, Copyright © 1981 by Society for the Study of Reproduction

Big Luteinizing Hormone (BLH): Possible Precursor of Native LH (NLH) in Anterior Pituitary Glands of Rats

¹ Department of Veterinary Biosciences, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801

In this study we attempted to dissociate big LH (BLH) into low molecular weight forms by treatment with either ribonuclease or mercaptoethanol. We compared the molecular nature of BLH and native LH (NLH) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Also, we explored the relationship between BLH and NLH by both continuous and pulse-chase labeling techniques on the premise that BLH represents a precursor to NLH. During the pulse periods, [¹⁴C]-alanine-labeled BLH was detected in the tissue before [¹⁴C]-NLH, and then the amounts of both forms increased linearly with time. At all times, the percentage of total [¹⁴C]-LH as BLH remained very high. [³H]-Glucosamine-labeled BLH and NLH were detected coincidentally in the tissue, and then [³H]-BLH increased at a constant rate, whereas [³H]-NLH increased at an increasing rate with time. The percentage of total [³H]-LH as BLH declined with time. The magnitude of change of radiolabeled LH varied with conditions during the chase periods, but similar trends were observed in all experiments. During the chase, [¹⁴C]-BLH decreased slightly (6-30%), whereas [¹⁴C]-NLH increased (50-160%). In contrast, [³H]-BLH increased 18% to 200%, whereas [³H]-NLH increased 112% to 500%. For dissociation studies, pooled aliquots of BLH fractions obtained from pulse-labeled and gel-filtered anterior pituitary extracts were treated with either ribonuclease or mercaptoethanol and rechromatographed and monitored. Such treatments did not release smaller forms of radiolabeled LH. However, both treatments released considerable amounts of total immunoreactive LH which eluted from Sephadex G-100 columns at positions near NLH and LH subunits. Coincubation with unlabeled ovine LH specifically displaced the binding of both BLH and NLH to the anti-LH- serum. Sodium dodecyl sulfate-gel electrophoresis of the antibody-precipitated NLH revealed two glycosylated forms of and subunits with apparent molecular weights of 21,000 and 17,000, respectively. In contrast, the antibody-precipitated BLH revealed a major glycosylated subunit and three minor unglycosylated forms with apparent molecular weights of 52,000, 22,000, and 13,000. These results suggest that BLH is the earliest detectable biosynthetic form of LH. BLH may represent a pool of immature LH or subunit which is attached to other molecules prior to conversion into NLH.

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ACKNOWLEDGMENTS We are grateful to Dr. D. N. Ward for providing ovine-LH- and ovine-LH-; Dr. G. D. Niswender for anti-serum GDN-15; Dr. L. E. Reichert, Jr. for the purified ovine LH used for radioiodination; NIAMDD Rat Pituitary Distribution Program for supply of rat LH-I-4, rat LH subunits, and synthetic GnRH; and Mr. David Kuehl and Miss Sara White for technical assistance.