Partial characterization of a novel oestrogen-induced protein in the rat adenohypophysis X Casabiell, J L Zugaza, C M Pombo, L Bokser, N Mulet and FF Casanueva ABSTRACT In order to detect putative markers of prolactin\x=req-\ secreting pituitary tumours, adult rats were sub- jected to long-term oestrogenization with oestradiol benzoate (OE2) on a monthly basis. At 6 months, anterior pituitaries were dissected and incubated either as tissue fragments or as dispersed cells with a S]methionine mix for labelling. Proteins released into the incubation medium and from tissue extracts were further analysed by sodium dodecyl sulphate\x=req-\ polyacrylamide gel electrophoresis and fluoro- graphy. Oestrogen induced the appearance in the incuba- tion medium of a protein (OE2 band) with an Mr of 38 000 under reducing conditions, and high specific activity. Surprisingly, such a protein was not detected in tissue extracts. The OE2 band was detectable by 7 days after the first dose of oestrogen, and remained throughout 1 year of treatment. The tumour cell line GH3 showed a similar OE2 band which was further enhanced by oestrogens. The pro- tein was observed similarly in both female and male pituitary donors, either intact or gonadectomized, and also in rats of different strains, suggesting that its appearance was independent of the strain of rat and gonadal status. Furthermore, the OE2 band was specific for pituitary cells and not produced by other oestrogenized tissues. No alteration in the rate of generation or the elec- trophoretic pattern of the OE2 band was observed when pituitary cells from oestrogenized rats were metabolically labelled while being incubated with tunicamycin. Furthermore, a system for glycan detection, adsorption to Concanavalin A or incuba- tion with endoglycosidase F also failed to show a clear amount of glycosylation of the oestrogen\x=req-\ induced protein. Both immunoprecipitation experi- ments and time-limited proteolysis with V8 protease ruled out the possibility that the OE2 band could be structurally related to either GH or prolactin. In conclusion, oestrogens induce the generation of a new monocatenary protein with an apparent Mr of 38 000, which has at least one intramolecular disul- phide loop and is not glycosylated. The OE2 band was detected only in incubation medium and never in tissue extracts. Journal of Molecular Endocrinology (1993) 10, 345-357 INTRODUCTION Prolactin-secreting pituitary adenomas are the most frequent pituitary tumours in man (von Werder, 1984). Although our understanding of this patholog¬ ical entity has progressed significantly from the time of the discovery of prolactin as a human hormone (Hwang et al. 1971), basic pathophysiological facts and biochemical markers still remain elusive (Kovacs & Stefaneanu, 1991). Several groups have taken advantage of the observation that oestrogens in pharmacological doses are able to induce prolactin- secreting pituitary adenomas in rats (McEuen et al. 1936; Lloyd et al. 1973; Neill, 1980). They have used this to gain an insight into their pathophysiology and even to develop methods allowing early tumour detection (Nakagawa et al. 1980; Casanueva et al. 1982). Molecular and Cellular Endocrinology Laboratory, Faculty of Medicine, Santiago de Compostela University, Santiago de Compostela 15780, Spain *Department of Medicine and Endocrine Section, General Hospital of Galicia, Santiago de Compostela, Spain (X Casabiell is now at Cellular Biochemistry and Biophysics Program, Sloan-Kettering Institute, New York, New York, USA) (Requests for offprints should be addressed to FF Casanueva at PO Box 563, Santiago de Compostela 15780, Spain)