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In Vitro Conditions Modify Immunoassayability of Bovine Pituitary Prolactin and Growth Hormone: Insights into Their Secretory Granule Storage Forms^*

Endocrine Metabolism Unit, Department of Medicine, and the Clinical Research Center, University of Rochester School of Medicine and Dentistry Rochester, New York 14642

Address requests for reprints to: Dr. Mary Y. Lorenson, Endocrine Metabolism Unit, Department of Medicine, University of Rochester School of Medicine and Dentistry, 601 Elmwood Avenue, Rochester, New York 14642.

Abstract

The amount of immunoassayable intracellular bovine (b) PRL and GH varies depending on treatment conditions. The present studies were designed to characterize the mechanisms involved and to compare immunoassayability of both hormones under similar conditions. Pituitary homogenate and secretory granule hormones displayed both time- and temperature-dependent increases when incubated at pH 10.5 with reduced glutathione; for example, 180 min values were 156% (GH) and 439% (PRL) at 22 C compared with control homogenate values. Under these conditions, PRL in granule cores (the sedimentable fraction after hypotonic lysis of granules) increased the most, changing 120-fold from 2.9 ± 0.25 µg/ml (pH 8.3 control) to 349 ± 22.8 µg/ml. Isolated oligomeric PRL increased significantly after exposure to mercaptoethanol, indicating that changes in assayability do not require nonhormonal granule components. No change in monomeric PRL, or GH in either form, was observed. Pretreatment with iodoacetamide resulted in a 33% decrease in maximal PRL values, indicating the presence and functional importance of granule thiols. Cysteamine inhibition of bPRL values in cores was modulated by urea, EDTA, and iodoacetamide. Though 5 M urea or 0.5% sodium dodecyl sulfate (SDS) increased granule PRL at pH 8.3, maximal values required thiols at pH 10.5. In contrast, maximal GH values were obtained with SDS at pH 8.3 without thiols.

Changes in immunoassayability seem to reflect conversion from poorly immunoactive tissue hormone oligomers to monomeric hormone. The data indicate that oligomeric bPRL is stabilized primarily by intermolecular disulfide bonds, although it is also susceptible to urea, SDS, and EDTA; granule thiols may also influence the conversion to monomer. The storage form of bGH appears to be stabilized differently. Maneuvers demonstrated in these studies to influence immunoassayability correlate very well with their previously established effects on hormone release and secretion, strengthening the likelihood that a functional link exists between assayability and secretion. (Endocrinology 116: 1399–1407, 1985)

Footnotes

^* This work was supported in part by PHS Grants AM-21783 and AM-31326 from the NIADDK and Grant RR-00044 from the Division of Research Resources, NIH. A preliminary report of this work was presented in part at the IVth International Congress on Prolactin, Charlottesville, VA, June 1984.

Received June 20, 1984.

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