Francis T. Kenney

Turnover of Rat Liver Tyrosine Transaminase: Stabilization after Inhibition of Protein Synthesis

Turnover of the rat liver tyrosine transaminase in vivo was measured by a label and chase procedure under conditions where the amount of enzyme undergoes no change. Half-life of the 14C-labeled enzyme in this basal condition was found to be 1.5 � 0.3 hours. Inhibitors of protein synthesis (cycloheximide or puromycin) do not appreciably influence the basal enzyme level over a 5-hour period, although these drugs will block hormonal induction of this enzyme. In pulse-labeling experiments, cycloheximide blocked transaminase synthesis almost completely. The conclusion that enzyme degradation, as well as synthesis, must be blocked when protein synthesis is stopped was confirmed in experiments showing that labeled enzyme is stable in the liver of rats treated with cycloheximide The participation of a continuously synthesized polypeptide in the degradative phase of transaminase turnover is suggested.

Hormonal stimulation of hepatic ornithine decarboxylase

Abstract Hepatic ornithine decarboxylase activity in adrenalectomized rats was increased by a variety of hormones, including hydrocortisone, growth hormone, insulin, glucagon, and thyroxin. Repeated treatment with either hydrocortisone or growth hormone, the most effective of the hormones tested, failed to maintain the elevated enzyme levels attained within a few hours after the initial injection.

RNA Synthesis in Rat Seminal Vesicles: Stimulation by Testosterone

Within 70 minutes after the administration of testosterone to rats castrated 12 to 15 hours previously, the rate of synthesis of RNA in the seminal vesicle is increased by 50 percent and continues to rise until approximately 50 minutes after injection when a two- to threefold increase was attained. No further increase was detected for as long as 240 minutes after hormone administration. The base composition of the pulse-labeled RNA was intermediate between that of the total seminal vesicle RNA and DNA-like RNA. No change in this composition was detected at any interval after injection.

Regulation of yolk protein synthesis in amphibian liver. I. Induction of lipovitellin synthesis by estrogen.

Abstract Synthesis of the yolk protein, lipovitellin, in the livers of estrogen-treated Xenopus laevis males was followed by isotopic-immunochemical techniques. Synthesis of this protein begins between 9 and 12 h after estrogen administration, following a general stimulation of liver protein synthesis that begins between 3 and 6 h after hormone treatment.Both the general and specific effects of estradiol were sensitive to actinomycin D.

Effects of alterations in hormonal status on ribosomes of rat uterus

Abstract A ribosome-supernatant system capable of active amino acid incorporation into protein in vitro was prepared from rat uterus. Requirements and properties of this system do not differ markedly from those of similar preparations from other rat tissues. Manipulation of estrogen levels in vivo primarily affected the ribosome component of the system. Following ovariectomy, the incorporating activity of the ribosomes at 1 day was transiently lowered but was restored to normal in the second and subsequent days. The lowered activity of ribosomes was associated with an increased response to addition of polyuridylic acid, whereas a short (4 hours before killing) treatment with estrogen increased ribosomal activity while decreasing the response to the synthetic messenger. The yield of ribosomes obtained in a standard fractionation dropped sharply after ovariectomy, and was restored by a short-term treatment with estrogen. The results are interpreted as indicating that both the number and the capacity of the ribosomes are estrogen-dependent.

Insulin enhances transcription of the tyrosine aminotransferase gene in rat liver.

The mechanism of insulin-mediated induction of tyrosine aminotransferase in rat liver was investigated using a cloned cDNA probe. The level of aminotransferase mRNA increases about fourfold following administration of the hormone. This induced mRNA accumulation does not require de novo protein synthesis. Nuclear runoff transcription assays in isolated liver nuclei demonstrate that insulin has a rapid and time-dependent stimulatory effect on aminotransferase gene transcription. The magnitude of enhanced transcription can fully account for the increase in the mRNA. We conclude that the induction of tyrosine aminotransferase in rat liver by insulin is primarily a consequence of a selective increase in the rate of transcription of the aminotransferase gene.

HEPATIC METABOLISM OF PHENYLALANINE DURING DEVELOPMENT

The phenomenon of substrate induced enzyme formation has been amply documented in studies of microbial systems (1), and similar adaptive changes in enzymic activity in the tissues of vertebrate forms have been described by Knox, Mehler and Lin (2, 3) and by others (4, 5). The suggestion has been made (4, 6) that substrate induction may be a mechanism involved in those changes in enzymatic activities that normally occur during development and differentiation. An extension of this concept might predict that changes in the tissue level of a given substrate during development would be associated with alterations in the activities of enzymes concerned in the metabolism of that substrate. The present studies were initiated to test this hypothesis, using hepatic metabolism of the essential amino acid phenylalanine as a model system. Aside from incorporation into protein, which is common to all amino acids, the known reactions of phenylalanine in mammalian liver include only transamination to yield phenylpyruvate (7-9) and hydroxylation at the para position to form tyrosine (10). A previous report from this laboratory ( 11 ) established that the enzyme system forming tyrosine from phenylalanine is essentially absent during fetal life in the rat, developing to the level of the adult several days after birth. In the present report, this observation has been extended to other mammalian species and the nature of the enzymic deficiency in the liver of fetal animals has been determined. In addition, transamination of phenylalanine in rat liver has been studied, with particular refer-

Regulation of yolk protein synthesis in amphibian liver: II. Elevation of ribonucleic acid synthesis by estrogen

Abstract Changes in RNA synthesis during the course of induction of yolk protein synthesis were determined in the livers of Xenopus laevis males given estrogen. Increased synthesis of rapidly labeled nuclear RNA precedes the changes in protein synthesis induced by the hormone. Base composition analyses of this RNA suggest an initial synthesis of uridylic acid-rich RNA followed by increased synthesis of ribosomal RNA. No significant changes in leucyl-, methionyl-, or seryl-tRNAs were detected, and phosphoserine was not found in seryl-tRNA formed either in vivo or in vitro.

RNA synthesis and enzyme induction by hydrocortisome

Abstract Fractionation and characterization of the pulse-labeled RNA from livers of rats treated with hydrocortisone demonstrates that the hormone increases the synthesis of ribosomal and transfer RNA as well as a DNA-like RNA. The hormone did not affect the rate of labeling of transfer RNA due to turnover of the -pCpCpA terminus of the polynucleotide chain. Labeling of acid-soluble nucleotides (AMP) was similarly unaffected. The general nature of the hormone effect on RNA synthesis suggests that enzyme induction may simply reflect a general increase in hepatic protein synthesis. The rapid turnover enzymes, tyrosine transaminase and tryptophan pyrrolase, can be induced to a limited extent by ethanol in a steroid-independent and actinomycin-sensitive fashion.

Turnover of tyrosine transaminase in cultured hepatoma cells after inhibition of protein synthesis.

Abstract Addition of cycloheximide to hydrocortisone-induced cell cultures of the Reuber (H-35) hepatoma results in an immediate decrease in the levels of tyrosine transaminase (L-tyrosine-2-oxoglutarate aminotransferase, E.C.2.6.1.5). The half-life of the enzyme is about 1.6 hr during the first hour after treatment; however, the rate of inactivation decreases such that the enzyme has a half-life of about 10 hr between 4 and 9 hr after addition of the inhibitor. The cells are functionally viable after 3 hr treatment with cycloheximide and retain their capacity both to inactivate tyrosine transaminase and to have the synthesis of this enzyme reinduced by hydrocortisone after removal of the inhibitor. The loss of 14 C-labeled tyrosine transaminase in H-35 cells is blocked by cycloheximide after a l-hr period of normal turnover. Puromycin also blocks the turnover of the enzyme in the interval between 4 and 6 hr after addition of the inhibitor.