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Characterization of Luteinizing Hormone Synthesized and Released by Rat Pituitaries in Vitro: Dissociation of Immunological and Biological Activities^*

TSUEI-CHU LIU, ROY L. AX and G. L. JACKSON

Departments of Veterinary Biosciences and Animal Sciences (R.L.A.), University of Illinois at Urbana Champaign, Illinois 61801

Address requests for reprints to: Dr. Tsuei-Chu Liu, Department of Veterinary Biosciences, University of Illinois, 261 Veterinary Medicine Building, Urbana, Illinois 61801.

Abstract

These studies were designed to characterize the LH synthesized de novo by rat anterior pituitary glands in vitro. Pituitaries from ovariectomized rats were incubated with [^H]- glucosamine ([³H]GA), [¹⁴C]alanine, ([¹⁴C]A), and synthetic gonadotropin- releasing hormone for 4 h. At the end of incubation, pituitaries were separated from medium and then homogenized, centrifuged, and dialyzed. Dialyzed medium and supernatant were chromatographed separately on Sephadex G-100. Fractions were monitored for total immunoreactive LH (IR-LH) by RIA. Newly synthesized radiolabeled LH was measured by monitoring the radioactivity of LH immunoprecipitated with anti-LH-β serum. About 4% of the total IR-LH in the pituitary supernatant eluted in the void volume (peak A), whereas 96% eluted at the same position as [¹²⁵I]rat LH (peak B). Seventy-six percent of total [¹⁴C]A-LH and 37% of total [³H]GA-LH eluted in peak A, whereas the remainder eluted in peak B. The ratio of [¹⁴C]A-LH to [³H]GA-LH in peak A varied from 2.5 to 3 5, whereas that in peak B was 0.5. Peak A material was treated with 6 M guanidine HC1 and 0.25% Triton-X, then sonicated and rechromatographed. Two major IR-LH peaks emerged: one in the void volume, the other at the same position as [^l2I]LH subunits. AH [¹⁴C]A-LH and [³H]GA-LH eluted in the void volume. After chromatography of the medium all IR-LH, [¹⁴C]A-LH, and [³H]GA-LH eluted in peak B. The [¹⁴C]-A-LH to [³H]GA-LH ratio was 0.5. Equivalent amounts of immunoreactive material in peaks A and B were tested for their ability to induce testosterone secretion by minced cockerel testes. Peak A material from the pituitary had little activity compared to NIH-LH-S18 standard, whereas peak B material from both pituitary and medium exhibited potencies of 56% and 46%, respectively. Doseresponse curves from peak B materials paralleled the standard curve. In pituitaries pulsed for 20 min with [³H]proline, all [³H]proline-LH was of the high molecular weight species. These results demonstrate synthesis of two kinds of I -LH. One appears to be of high molecular weight, to appear early in the biosynthetic pathway, to contain relatively little carbohydrate, and to possess little biological activity. The other is native LH.

Footnotes

^* This work was supported by USPH Grant HD-09659. It was presented in part at the 60th Annual Meeting of The Endocrine Society, Miami, FL, June 14–16, 1978 (Abstract 59).

Present address: Department of Dairy Science, University of Wisconsin, Madison, Wisconsin 53706.

Received December 28, 1978.