The effect of oestradiol-17β on the ribonucleases and ribonuclease inhibitor of immature rat uterus

Brockdorff, Neil Alexander Steven (1985) The effect of oestradiol-17β on the ribonucleases and ribonuclease inhibitor of immature rat uterus. PhD thesis, University of Glasgow.

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The cytoplasmic ribonuclease inhibitor/ribonuclease system has been detected in a wide variety of tissues and species. In most of the tissues studied, cytoplasmic ribonuclease is undetectable due to the presence of excess inhibitor. The observation that tissues with high levels of RNA/protein synthesis have high levels of free inhibitor has been taken to imply some essential role for the inhibitor in the control of protein synthesis. The precise nature of this regulatory function is however unknown. Studies on the enzyme associated with the inhibitor in vivo indicated that it was similar, but not identical to secretory pancreatic RNase A. Previous studies on the inhibitor/ribonuclease system of rat uterus revealed that free inhibitor was detectable in the uteri of immature rats, but disappeared after administration of oestrogen or during normal sexual maturation of the rat. The following studies were undertaken in order to determine the mechanism whereby oestrogen decreased inhibitor activity, and to gain a better understanding of the precise function of the inhibitor/ribonuclease system. The main points to emerge from these studies were as follows (i) Antiserum raised to purified rat liver inhibitor was used to quantitate total inhibitor levels in control, oestrogen-treated, and mature rat uteri. These studies revealed that inhibitor levels increased by up to 50% after hormone-treatment and normal development. It was therefore apparent that the loss of inhibitor activity was not due to reduced synthesis of the protein. (ii) Total and free ribonuclease activity was assayed, and revealed that the total activity increased by up to eight fold after oestrogen-treatment and during normal development. Free enzyme activity became detectable in uterine cytoplasm. Taken together, these results indicated that the loss of inhibitor activity arose through saturation by endogenous ribonuclease. (iii) Activity stain analysis of the uterine cytoplasmic ribonucleases revealed the presence of at least two distinct species with approximate molecular weights of 14,000 and 18,000. The activity of both of these species appeared to increase after oestrogen-treatment and during normal development. An attempt to detect and quantitate these enzymes using antiserum raised to bovine RNase A was not successful. (iv) A cDNA clone encoding rat pancreatic RNase A mRNA was obtained, and used in an attempt to detect the mRNA encoding the endogenous uterine enzymes. It was not possible to detect a clear cut example of an RNase A like mRNA in control or oestrogen-treated uterine RNA. These results did not however rule out the possibility that the uterine enzymes were induced at the transcriptional level by oestrogen. (v) A technique was developed whereby free inhibitor and inhibitor/ribonuclease complexes could be resolved on non- denaturing polyacrylamide gels, and subsequently detected by immunoblotting. These studies confirmed that the loss of inhibitor activity was due to saturation by endogenous enzyme. Furthermore, it was found that 5-6 distinct enzyme species or enzyme variants were present in the cytoplasm of oestrogen-treated uteri. The relative levels of these enzymes were found to differ in a variety of tissues analysed. Ferguson plot analysis revealed that they differed on the basis of charge, and to a lesser extent, molecular weight. (vi) The cytoplasmic ribonucleases of rat liver and mature rat uteri were purified using a relatively rapid procedure giving high yields. The different species were resolved to some extent using chromatography on heparin-sepharose. Thus, in rat liver one major and one minor species were resolved, (RLC I and RLC II), whilst in mature rat uteri three species were resolved, (RUC I, RUC II and RUC III). RLC I was shown to be homogeneous as determined by SDS-PAGE. Insufficient quantities of the other enzymes were available for similar analyses. The relative molecular weight of each activity was determined by activity staining. These studies revealed that each activity consisted of more than one distinct species or variant. It was assumed that heparin-sepharose chromatography did not completely resolve all of the species present. Tentative analysis indicated that the species RUC II and RUC III corresponded to the oestrogen-induced species. (vii) Some of the properties of the purified enzymes were compared. The enzymes were shown to differ on the basis of pH optima, kinetic parameters, and their relative activity towards the homopolymers Poly (u) and Poly (c) relative to yeast RNA. The aforementioned results are discussed in relation to the possible function of the inhibitor/ribonuclease system.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Biochemistry, Endocrinology
Date of Award: 1985
Depositing User: Enlighten Team
Unique ID: glathesis:1985-76560
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 19 Nov 2019 14:09
Last Modified: 19 Nov 2019 14:09

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