ABSTRACT. To assess the role of genetic regulation as a state levels of GALT sp act (7). In this report, we have assessed modulating factor in the variability of rat tissue galactose-GALT sp act and steady state mRNA during postnatal rat liver I-phosphate uridyltransferase (GALT) sp act, we have development and in various tissues of the adult rat. Our findings determined steady state GALT mRNA and sp act in rat that there is a correlation between the two parameters in these liver during postnatal development. Steady state GALT investigations indicates that genetic regulation plays an impormRNA levels increase from birth to d 5 and subsequently tant role in modulating GALT activity. Our findings form the decrease toward adult levels. GALT sp act mirrors the basis of this report. mRNA pattern. A survey of steady state mRNA and GALT sp act of several adult rat tissues revealed marked tissue differences with a good correlation of the two parameters. MATERIALS A N D M E T H O D SLiver had the highest GALT mRNA and sp act; kidney, ..lnimul.s. Timed pregnant Sprague-Dawley rats from Charles ovary, a_nd heart had similar but lower mRNA and sp act; River Breeding Farms (Wilmington. MA) were housed three per skeletal muscle and testes had the least GALT mRNA and cage with free access to food and water in a room with a 10: 14 enzyme sp act. These findings suggest that genetic regu-h 1ight:dark cycle. All experiments were performed using the lirtion is important in the variable expression of GALT Children' Chomczynski and Sacchi (8). One hundred mg of tissue were homogenized using a Dounce homogenizer in 1 The metabolism of galactose, a major carbohydrate constituent mL of 4 M guanidinium thiocyanate. 25 mM sodium citrate. of almost all animal milk, is controlled by the three enzymes of pH 7, 0.5% SDS, and O.' 2-mercapt0ethan01. The tissue was the Leloir pathway, galactokinase, GALT, and UDP galactose-transferred to a ~ol~pr0pYlene tube and sequentially extracted 4-epimerase ( 1). ~h~~ GALT is a key enzyme in the pathway is with acidified phenol and chloroform-isoamyl alcohol (49: 1). highlighted by human galactosemia due to deficient GALT ac-The RNA was precipitated with isopropanol and resuspended in tivity. Interest in this recessively inherited disorder has fostered 1 mL of dieth~l~~rocarbonate-treated water. The RNA concenefforts to delineate the mechanism responsible for regulation of tration was determined by at 260 nm UV.GALT activity. We have previously described increases in liver Separate Northern blots of RNA obtained from newborn rat GALT sp act in rats fed a highegalactose diet (2). in pregnant liver and adult rat tissues were generated by electrophoresis of female rat liver, possibly related to progesterone (3. 4), and in l o pg RNA a I% agarOse-fomaldehYde gel the postnatal development of rat liver (5). We have also described by a Zetabind membrane (CUNO. variation of GALT sp act in different rat tissues (5) and modu-Meriden, CT). The RNA was fixed to the membrane using a lation by uridine nucleotides (6). ...

ABSTRACT. The sp act of hepatic galactose-metabolizing enzymes, galactokinase, galactose-1-phosphate uridyltransferase, and uridine diphosphate-4-epimerase were measured in female rats during pregnancy and lactation as well as in fetuses and pups after parturition. S p act for transferase and epimerase in pregnant rat liver are about 50% higher than that of virgin females, and with the increase in organ size during pregnancy the total hepatic activity is double that of nonpregnant animals. Galactokinase activity decreases somewhat during pregnancy, but total activity is 25% higher than in virgin liver. A Michaelis-Menten kinetic analysis of liver transferase indicates an increase in the maximum velocity of the reaction without a change in Km. Isoelectricfocusing on a high-resolution IEF gel demonstrated similar isozyme patterns. The sp act of the fetal liver enzymes increase to about twice that of the maternal tissue, but total activities are low due to the very small fetal liver size. Sp act of these enzymes in maternal liver fall after delivery, but sp act of galactokinase and transferase are programmed to increase in liver of the growing neonatal animals, reaching levels almost 5-fold higher than found in nonpregnant adult liver. An understanding of factors contributing to the enhanced transferase activity of the liver of pregnant and neonatal rats may contribute to possible ways of augmenting the residual transferase activitv of ~atients with transferase-deficient galactosemia as therapeutic strategy. (Pediatr Res 25:161-166, 1989) Abbreviations Galactokinase, (EC 2.7.16) Transferase, galactose-1-phosphate uridyltransferase (EC 2.7.7.12) Epimerase, uridine diphosphate galactose-4-epimerase (EC 5.1.32) UDPglucose, uridine 5'-diphosphoglucose Galactose-1-P, a-D-galactose-1-phosphate IEF, isoelectric focusingThe feeding of a high galactose-containing diet to pregnant rats has been used as a model to study in utero galactose toxicity in the fetus and newborn (1, 2). The focus of such research has centered mainly on neonatal toxicity with little regard for galactose effect on the pregnant mothers. Several pertinent findings, however, have been made indicating galactose metabolism may be different in pregnant animals. We have observed that cataracts do not develop in pregnant rats fed a high galactose diet for the 21 days of gestation, whereas they are found in young male rats fed the same diet for the same number of days (2, 3). Others have reported that pregnant rat lenses are clear until 48 h after parturition when cataractous changes precipitously appear (4). These data suggest that pregnancy affords protection of the female from galactose toxicity. Although extensive investigations of glucose metabolism have been made in the pregnant rat (5-7), little is known about galactose metabolism in the gestational state. Therefore, we have examined the sp act of the galactosemetabolizing enzymes galactokinase, transferase, and epimerase in the liver of dams throughout pregnancy and lactation and in the fetus for c...