99 Correlation of rat hepatic phenylalanine hydroxylase with concentrations of tetrahydrobiopterin and of GTP

Abstract

To date, studies on the regulation of biopterin metabolism have concentrated on correlations with the metabolism of catecholamines (1-2) and, with the exception of one study correlating biopterin concentrations and phenylalanine hydroxylase activities in rat hepatomacells (3) there have not been any reports on the regulation of this cofactor in liver, in viva In this study we have also monitored the changes in the concentration of biopterin that ensue in rat liver upon feeding either glycerol or fructose and find that the ratio of cofactor concentration to phenylalanine hydroxylase activity is preserved. In addition, we have observed that the concentrations of GTP in freeze-clamped livers closely correlate with phenylalanine hydroxylase activities and with tetrahydrobiopterin concentrations. The possible mechanisms by which GTP might influence the expression of phenylalanine hydroxylase are considered.Male Sprague-Dawley rats (in groups of at least four) weighing 150-170 g were used throughout and, except where indicated, were allowed ad libitum access to standard laboratory chow. The 40% (wlw) glycerol diet was prepared by mixing in 400 ml of glycerol with 750 g of the standard chow. The fructose diet was prepared as described previously (4) with glucose (in the control diet) being replaced by fructose. As a further control for the glycerol diet, a group of rats were fed a glucose diet containing 10% (wlw) casein. The various diets were consumed at similar rates.The frozen livers were carefully thawed, cut into small pieces and homogenised in 3 volumes of cold 0.15 M KCI and this homogenate was centrifuged at 35,000 g for 45 min at 4OC; the supernatants were collected and unused amounts were stored at -2BC. Protein was determined by the biuret method using bovine serum albumin as standard. Phenylalanine hydroxylase activity was measured by colorimetric determination (5) of tyrosine formed in the presence of 5 mM dithiothreitol (6), 1 mM phenylalanine and 20 pM tetrahydrobiopterin (BI-L+) or 5 mM phenylalanine and 0.5 mM dimethyltctrahydropterin (DMPI-L+) . SDS polyacrylamide gel electrophoresis and western blotting experiments were carried out according to accepted procedures (7). Polyclonal antisera raised against purified rat hepatic phenylalanine hydroxylase were used as source of primary antibody.For biopterin analyses, the tissues were homogenised, at 4OC, in 5 vol. of a solution containing 0.2 M phosphoric acid, 0.2 M perchloric acid and 5 mM ascorbic acid; centrifuged at 20,000 x g for 30 min at 40C and aliquots of those supernatants were subjected to oxidation by MnO.2 for one hour at ambient temperature. Biopterin concentrations were determined by HPLC (8) using fluorescence detection. Statistical evaluations used were one-way ANOVA and Scheffe's test together with correlation and regression analyses.For determination of nucleotide concentrations, liver samples were collected by freeze-clamping. All samples were stored at -7BC. The tissues were powdered using a pre-cooled mortar and pestle; homogenised ...