The Renal B-Glucuronidase Response to Androgen Stimulation: Hormone Action and Effects on Protein Synthesis

Thesis (Ph.D)--Boston University

The purpose of this study has been to evaluate an enzyme system of the mouse kidney, which exhibits a large increase in activity when the animal is stimulated with testosterone provided exogenously or endogenously (following gonadotropin injection). The primary question is whether or not the increase in enzyme activity represents synthesis of new enzyme protein. The A/Jax male mouse kidney was the organ studied; this was chosen because of the findings of Fishman, that this strain of inbred mice is genetically a "high-responder," exhibiting a 16-fold increase of renal B-glucuronidase above the level of unstimulated mice. Moreover, the enhanced kidney B-glucuronidase activity is accompanied by a parallel urinary excretion of B-glucuronidase. The usefulness of this circumstance is two-fold: the extent of the kidney response may be followed in the urine without sacrificing the animal, and the pooled urine specimens provide a rich source from which enzyme can be isolated more easily and in a purer state than from tissues. Methods of purification of enzyme from liver, kidney and urine have been devised and the pure enzyme preparations have been compared with each other by means of kinetic studies. The liver enzyme has been studied as a reference protein for the isotope incorporation experiments, as its activity does not increase in androgen stimulated mice. The quantities of enzyme protein present in tissue from which it was fractionated are estimated to be about 1-2 milligrams in the richest source, from which 50-100 micrograms are present in the purified enzyme preparation. The procedure employed involves solvent precipitation, acid and alkaline ammonium sulfate fractionation, and incubation of the resulting solution at 38 degrees Celcius, which removes additional inactive protein. Electrophoresis in a polyurethane sponge apparatus has produced some very pure preparations with a specific activity of 100,000 units activity/mgm protein. Other methods investigated have been adsorption and ion exchange chromatography. The major evidence for synthesis of new enzyme protein was provided by the in vivo labelling experiments, in which mice were injected intraperitoneally with glycine-l-C14. The experiments were of three types: (1) steady state experiments, in which a time study was made of the C14 incorporation into enzyme and tissue proteins in maximally stimulated mice (1 mg. testosterone propionate per day for 14 days), (2) incorporation into enzyme and tissue proteins in partially stimulated mice (5 days of testosterone propionate) and (3) reversal of these conditions, in that mice are first labelled with isotope and then stimulated with gonadotropin for 5 days. The results from the steady state experiment provide direct evidence that the kidney enzyme in these stimulated mice is more radioactive than the liver enzyme from the same mice, or the kidney and liver enzyme from control mice. The data from the second and third experiments are best interpreted in the same fashion. Here also, the incorporation rate into stimulated kidney enzyme is greater than that into stimulated liver, or control kidney or liver. The question of hypothetical precursors is discussed. The appearance of new enzyme protein in response to testosterone stimulation is supported directly by the incorporation experiments. These facts now explain the large increase in observed enzyme activity, as well as other findings in the literature. The action of testosterone in this system is concluded to be the one which facilitates the production of more enzyme protein, and not one which increases the observable activity of that already present.