Gonadotropin Receptors in Rat Testes
INTERACTION OF AN ETHANOL-SOLUBLE TESTICULAR FACTOR WITH HUMAN FOLLICLE-STIMULATING HORMONE AND LUTEINIZING HORMONE
Vinod K. Bhalla 1 and Leo E. Reichert Jr. 1
From the
1 From the Department of Biochemistry, Division of Basic Health Sciences, Emory University, Atlanta, Georgia 30322
Attempts have been made to solubilize gonadotropin receptors from rat testes by extraction with ethanol. This approach is in contrast to earlier studies which have generally employed nonionic detergents such as Triton X-100 or Lubrol PX for receptor solubilization. Extraction of homogenates of mature rat testes with 30% (v/v) ethanol in 0.01 m phosphate pH 7.5 (37°) causes solubilization of factors which appear to interact specifically with iodinated human luteinizing hormone (hLH) or follicle-stimulating hormone (hFSH). Interaction of such ethanol-soluble factor (or factors) with hFSH and hLH was seen with the initial ethanol extract, after heating of the ethanol extract (5 min at 100°) to remove easily denatured contaminants and after fractionation of the heated ethanol extract by gel filtration through Sephadex G-100. Evidence for such interaction was obtained from three different types of experiments. In the first it was shown that the solubility of free, iodinated hFSH or hLH in polyethylene glycol (Carbowax 400, 47% v/v) was enhanced by ethanol-soluble factor (or factors). This phenomenon was interpreted as a reflection of direct interaction of components of the ethanol-soluble testicular extract with hLH or hFSH, and is in contrast with results obtained using detergent extracts of testicular and ovarian tissue wherein polyethylene glycol is used to precipitate the putative hormone-receptor complex. Second, when applied to a column of Sepharose or Sephadex G-25 which have been previously equilibrated with buffer containing radioactive hFSH or hLH until a constant amount of radioactivity emerged from the column, the ethanol-soluble factor concentrated radioactivity upon elution. Passage of ethanol-soluble factor through a similar column equilibrated with radioiodinated human serum albumin produced no such concentration of radioactivity, nor did filtration of extracts of rat testis not containing ethanol. Finally, the interaction of hFSH and hLH with unheated, heated, and Sephadex G-100-fractionated ethanol extracts of testicular tissue were examined by circular dichroism spectroscopy. The circular dichroism spectra of mixtures of hLH or hFSH with each of these three types of ethanol-soluble extracts differed significantly in detail and in sum from the circular dichroism spectra obtained when components of the mixture were analyzed separately. The most pronounced differences in circular dichroism spectra occurred in the near ultraviolet region (250 to 350 nm). In the far ultraviolet region (200 to 250 nm), the sums of the circular dichroism spectra of hLH or hFSH and the various ethanol-soluble extracts tested, were additive. Differences in circular dichroism spectra of mixtures of hormone- and ethanol-soluble factor (or factors), compared to circular dichroism spectra predicted on the basis of analysis of individual components of the mixture, are considered indicative of hormone-factor interaction. Of the seven fractions obtained following gel filtration of heated ethanol-soluble factor through Sephadex G-100, only three gave evidence of interaction with FSH or LH. Of these, the fractions which interacted most strongly with FSH or LH had average distribution coefficients of 0.19 and 0.29, respectively. Based upon the relationship between the average distribution coefficient of standard proteins and the log of their molecular weights, these correspond to approximate molecular weights of 67,000 and 22,500, respectively. The possibility cannot be excluded, however, that the circular dichroism active species is either not protein in nature, or represents an active component formed in a complex with a protein, such as a lipid. Although the ethanol-soluble factor appears necessary for binding of FSH or LH by tissue receptors, the gonadotropin-receptor complex, once formed, can no longer be dissociated with ethanol under conditions found effective for solubilization of the factor (or factors). This may indicate that the ethanol-soluble factor (or factors) is a necessary intermediate for hormone-receptor interaction, but is not required for maintenance of the complex once it is formed.
Submitted on June 12, 1974
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