Administration Of Puromycin Research Articles

Induction of collagenolytic and proteolytic activities by anti-inflammatory drugs in the skin and fibroblast

The administration of cortisol, oxyphenylbutazone and indomethacin to rats results in a marked and abrupt loss of cutaneous collagen from these animals. This collagen loss was associated with the appearance of both collagenolytic and proteolytic activities in the extracellular, extrafibrillar compartment of the skin. Cycloheximide and 5,5-diphenylhydantoin pretreatment inhibited both the cutaneous collagen losses and the appearance of these enzyme activities in the skin. Kinetic studies have shown that within 4 hr after anti-inflammatory drug administration, peak concentrations of both collagenolytic and proteolytic activities were reached in the skin. These enzymatic activities were profoundly depressed by simultaneous administration of puromycin, cycloheximide and actinomycin-D. Monolayers of cultured strain-L fibroblasts neither contained nor released either proteolytic or collagenolytic activities. Within 4 hr after administration of cortisol, idomethacin or oxyphenylbutazone, both types of enzymatic activities appeared within these cell cultures.

Studies on the development of ornithine-keto acid aminotransferase activity in rat liver.

1. During the normal development of the rat, the specific activity of liver ornithine-keto acid aminotransferase exhibits a transient elevation around term, and subsequently increases to adult activity levels during the third postnatal week. 2. The synthetic glucocorticoid triamcinolone, administered as a single injection, produces a marked elevation of the ornithine-keto acid aminotransferase activity within 24hr. if given postnatally before the natural increase in ornithine-keto acid aminotransferase activity has occurred. In foetal and adult animals, triamcinolone does not induce any increase in this enzyme activity. 3. The repeated administration of puromycin completely prevents the rise in ornithine-keto acid aminotransferase activity that follows triamcinolone administration. 4. If adult rats are fed with a protein-free carbohydrate diet, or one free of arginine, the ornithine-keto acid aminotransferase activity diminishes to a fraction of the normal. When such diets are given, a single injection of triamcinolone does not increase the enzyme activity within 24hr. 5. Partial hepatectomy, and repeated injections of growth hormone, depress the ornithine-keto acid aminotransferase activity in adult rats. 6. The findings are discussed in relation to the mechanisms concerned with developmental and adaptive changes in enzyme activities in the liver.

Factors influencing the development of urea-synthesizing enzymes in rat liver

1. The activities of enzymes of the urea cycle, carbamoyl phosphate synthetase, ornithine transcarbamoylase, argininosuccinate synthetase, argininosuccinase (the last two comprising the arginine synthetase system) and arginase, were measured in the liver during development of the rat. All five enzymes exhibited relatively low activities in foetal liver and a rapid postnatal increase was found. The rate-limiting enzyme of urea synthesis in the rat, the condensing enzyme of the arginine synthetase system, showed the lowest activity at birth and the most rapid postnatal increase, a fivefold increase within 24hr. after birth. A second increase of activity was noted after the tenth day. These results suggest that the postnatal increase of arginine synthetase activity initiates the ability for urea synthesis in the rat. 2. Some factors influencing the development of the rate-limiting arginine synthetase system were studied in more detail. (a) Intraperitoneal administration of puromycin inhibited the postnatal increaseof the enzyme activity. (b) Starvation of newborn animals for 24hr. after birth had no effect on the postnatal development of the enzyme. (c) Bilateral adrenalectomy at birth caused a marked diminution in the postnatal increase of the enzyme activity and injections of triamcinolone were effective in preventing the effect of adrenalectomy. (d) Administration of triamcinolone alone had a marked stimulatory effect on the postnatal development of this enzyme. (e) Premature and postmature birth had virtually no effect on the developmental pattern of the arginine synthetase activity, suggesting that the increase of this enzyme activity after birth is not initiated by the birth process.

Noradrenalin-induced increase in hepatic cholesterol synthesis and its blockade by puromycin

Noradrenalin administration is shown to result in an increased cholesterol synthesis by rat liver slices. The increase does not occur until 12 h after the injection of noradrenalin, however. Noted at earlier time intervals are increased conversion of acetate to CO 2 and ketone bodies and decreased conversion to fatty acid. The delayed increase in cholesterol synthesis suggests that enzyme induction may be a major mechanism underlying this control. Concomitant administration of puromycin with the noradrenalin abolished the increased cholesterol formation, thereby supporting the hypothesis of enzyme induction.

Inhibition of the Lymphopenic Effect of Cortisol by Puromycin Injection in Mice.

SummaryIn acute experiments, administration of puromycin to adrenalectomized or intact mice inhibited the lymphopenic action of cortisol, whereas injection of 6-dimethylaminopurine, a puromycin analogue, did not prevent this response to cortisol. It is suggested that protein synthesis is an essential preliminary or accompanying process for the manifestation of the lymphopenic activity of glucocorticoids.

δ-Aminolevulinic Acid Synthetase: II. INDUCTION IN RAT LIVER

Abstract Administration of allylisopropylacetamide to rats causes a marked increase in the hepatic ribonucleic acid-dependent synthesis of δ-aminolevulinic acid synthetase as evidenced by the fact that this increase is markedly inhibited by administration of actinomycin D or 5-fluorouracil. The half-life of hepatic δ-aminolevulinic acid synthetase following puromycin administration is 67 to 72 min and is independent of the level of the enzyme. The administration of actinomycin D or 5-fluorouracil to rats previously treated with allylisopropylacetamide results in a rapid decline of the hepatic levels of induced δ-aminolevulinic acid synthetase. This suggests that the turnover of the messenger RNA for hepatic δ-aminolevulinic acid synthetase is considerably more rapid than the bulk of rat liver messenger RNA. Glucose markedly inhibits induction of hepatic δ-aminolevulinic acid synthetase and also the increase of δ-aminolevulinic acid dehydratase produced by allylisopropylacetamide administration.

Proof of the hepatic synthesis of a sex-dependent protein in the rat

An α 2-globulin ( α 2u), previously purified from the urine of male rats, has been identified immunologically in the liver, kidneys and serum. This protein was associated with the microsomal fraction of the liver but not that of the kidneys. Total nephrectomy yielded in 6 h a 30-fold accumulation of α 2u above the normal plasma concentration of 3 mg per 100 ml. Simultaneous partial hepatectomy or administration of puromycin inhibited its accumulation. The perfusion of rat liver in vitro with blood containing [ 14C]glycine led to the production of radioactively labeled α 2u. These studies demonstrate the hepatic synthesis of the sex-related α 2-globulin originally isolated from male rat urine.

Insulin-stimulated ribonucleic acid synthesis and RNA polymerase activity in alloxan-diabetic rat liver

Administration of insulin to alloxan-diabetic rats stimulated equally the incorporation of [ 14C]orotic acid or 32P 1 into liver RNA within 30–60 min after injection. RNA polymerase (nucleosidetriphosphate: RNA nucleotidyltransferase, EC 2.7.7.6) activity of isolated liver nuclei was also stimulated by insulin in vivo. This activity rose measurably within 2 h after insulin injection and reached peak levels (2.3-fold increase) after 36 h of insulin treatment. Addition of insulin to nuclei in vitro did not affect RNA polymerase activity. The rise in RNA polymerase activity after insulin was almost completely blocked by the simultaneous administration of puromycin, suggesting a requirement for protein synthesis in this response.

Control of Enzymatic Synthesis of Adrenaline in the Adrenal Medulla by Adrenal Cortical Steroids

The activity of phenylethanolamine N-methyltransferase, the enzyme that catalyzes the N-methylation of noradrenaline to form adrenaline, falls following hypophysectomy. Enzyme activity can be restored by injections of adrenocorticotropic hormone (ACTH) or of dexamethasone, a potent synthetic glucocorticoid. The effect of ACTH on the adrenaline-forming enzyme is not direct, but involves stimulation of the release of endogenous glucocorticoids from the adrenal cortex. These steroids then act to elevate the activity of the transferase, which is principally localized in the adrenal medulla. The stimulation by glucocorticoids of phenylethanolamine N-methyltransferase activity can be blocked by the concurrent administration of puromycin or actinomycin D. Glucocorticoids do not stimulate the activity of other adrenal enzymes involved in catecholamine biosynthesis or metabolism, such as tyrosine hydroxylase, catechol O-methyltransferase, or monoamine oxidase.

Effect of puromycin on hepatic glycogen phosphorylase activity.

SummaryLiver glycogen phosphorylase activity was studied in mice at early times subsequent to in vivo administration of puromycin, and following puromycin addition to incubations of rat liver slices. No effect of puromycin on phosphorylase could be detected following injection of unanesthetized mice. Puromycin treatment of pentobarbital-anesthetized mice, however, resulted in an enhancement of the phosphorylase activity as compared to control values. Phosphorylase activity in rat liver slices incubated with puromycin was greater than those incubated without puromycin. These results indicate that puromycin can elevate liver phosphorylase activity, a finding consistent with the hepatic glycogenolytic action of the antibiotic in vivo.

Studies on the mechanism of carbohydrate repression in rat liver

In earlier published studies we have shown that the amino acid catabolizing enzymes, threonine dehydrase and ornithine transaminase, can be rapidly induced in the livers of protein depleted rats force-fed casein hydrolysate or injected with glucagon. This induction, which is inhibited by the administration of gamma irradiation, actinomycin, puromycin, or fluoroorotic acid, is also inhibited by the forced feeding of glucose or fructose. The kinetic picture of the inhibition by carbohydrate feeding appears to resemble that produced by puromycin administration rather than by actinomycin or fluoroorotic acid administration, suggesting that the repressive effect of carbohydrate is exerted at the stage of protein synthesis involving peptide formation. The feeding of carbohydrates is accompanied by the rapid accumulation of large quantities of glycogen in the liver, and when glucagon is injected into rats receiving dietary glucose the resultant enzyme induction is accompanied by a decrease in liver glycogen concentration. During the course of these studies we have evolved the working hypothesis that the metabolic control phenomena which we have observed (induction and repression) arise through the interaction of enzyme forming systems with intracellular lipoprotein membranes such as the endoplasmic reticulum. Agents which alter the configuration of these membranes and/or their association with the enzyme forming systems (polysomes) might be expected correspondingly to affect induction and repression. For example, when glycogen is deposited in the liver cell after feeding carbohydrate, it appears to become associated with the endoplasmic reticulum. Numerous workers have shown that this endoplasmic reticulum is morphologically quite different (tubular network, no ribosomes attached to membranes) from that not associated with glycogen (lamellar network, ribosomes attached to membranes) and it is possible that such morphological differences may be accompanied by differences in metabolic properties. In the further exploration of this hypothesis we have tested the effects of a variety of hormones on the induction and repression of certain amino acid catabolizing enzymes (serine dehydrase, ornithine transaminase, and tyrosine transaminase), the rationale being that hormones may exert their metabolic action through an effect on membrane structure. We observed that in protein depleted rats which have been pretreated with cortisone (5 mg/day for 3 days) glucose repression of enzyme induction is significantly less than that in rats not given cortisone. We also found, however, that induction by casein hydrolysate without glucose is substantially less in rats treated with cortisone as compared to those not receiving cortisone. The administration of cortisone alone does not produce significant enzyme induction under the conditions of these experiments. These results suggest that cortisone exerts a “protective” effect on the induction process, permitting induction to occur in the face of carbohydrate, but not itself causing induction. Similar effects are produced by triamcinolone, but deoxycorticosterone has no effect on the induction or repression phenomena. Experiments were also conducted to study the possibility that carbohydrate repression may in fact be a consequence of increased insulin production stimulated by the carbohydrate feeding. No suppression of induction occurs when rats receiving casein hydrolysate are injected with insulin at dosages just below lethality.

INDUCTION OF DDT-METABOLIZING ENZYMES IN MICROSOMES OF RAT LIVER AFTER ADMINISTRATION OF DDT

DDT is metabolized by rat liver microsomes to a phenolic compound and to a reduced derivative similar to DDD. When rats were injected intraperitoneally with DDT, the microsomal DDT-metabolizing activity was greatly increased. This effect was blocked by the administration of puromycin. DDT administration also increases the content of liver microsomal protein. The results show that the insecticide probably increases the DDT-metabolizing activity of mammalian liver by inducing enzyme synthesis.

STUDIES ON FACTORS AFFECTING THE INDUCTION OF ATP D-HEXOSE 6-PHOSPHOTRANSFERASE IN RAT LIVER.

The administration of carbohydrate elicits a rapid increase of rat liver ATP d-hexose 6-phosphotransferase when this enzyme activity has been decreased by feeding a high-fat, carbohydrate-free diet for several days. Several monosaccharides were tested for their ability to induce the increase of phosphotransferase. The results indicate a preferential response of this enzyme to glucose as compared to other very closely related sugars. The administration of puromycin and actinomycin prevents the response, and suggests a de novo synthesis of protein to explain the increase in enzyme activity caused by carbohydrate. The administration of high doses of commercial insulin to intact animals increases slightly the activity of phosphotransferase already decreased by a carbohydrate-free diet. It appears that a minimum amount of circulating insulin as well as a certain level of glucose in the liver are necessary to maintain adequate enzyme activities. The adrenal glands do not seem to be essential for the induction of phosphotransferase; nevertheless, hydrocortisone seemed to facilitate the inductive effect of exogenous glucose.

REGULATION OF XANTHINE DEHYDROGENASE IN CHICK LIVER. EFFECT OF STARVATION AND OF ADMINISTRATION OF PURINES AND PURINE NUCLEOSIDES.

1. The xanthine-dehydrogenase activity of chick liver, expressed per mg. of nitrogen, is increased during starvation. 2. Administration of inosine and possibly of adenine has a comparable effect on the xanthine dehydrogenase, and also induces an elevation of the total quantity of enzyme. Hypoxanthine, xanthine, guanine, xanthosine, guanosine and adenosine are ineffective. Cortisone is equally ineffective. 3. The administration of puromycin abolishes the effect of inosine and reduces that of starvation. It is concluded that inosine induces an increased synthesis of xanthine dehydrogenase, whereas during starvation the enzyme is spared with respect to other liver proteins. 4. The hypothesis is formulated that chick-liver xanthine dehydrogenase is an adaptive enzyme, its activity being regulated by inosine or by one of its metabolites.

Acceleration of the normal and corticoid-induced increase of alkaline phosphatase in the duodenum of the nursling mouse by actinomycin D, puromycin, colchicine and ethionine

1. 1. The alkaline phosphatase activity of the duodenum of the mouse undergoes a sharp rise, with concomitant changes in its properties, at the end of the second week of postnatal life. This rise is accelerated by the administration of puromycin or actinomycin D. 2. 2. Both antibiotics also induce a precocious increase of phosphatse activity within 48 hr, when injected at 9 days. If hydrocortisone, which also promotes a precocious upsurge of duodenal phosphatase activity, is administered at the same time, the effects of hormone and antibiotic are additive. 3. 3. At 4 days actinomycin D induces a small but only transient increase of activity. It does not facilitate the effect of hydrocortisone at this stage. 4. 4. Colchicine is as effective as actinomycin at 13 days, and at 9 days is also as efficacious as the antibiotic in the presence or absence of hydrocortisone. When administered at 4 days, colchicine induced a larger increase than did actinomycin, and also enhanced the effect of hydrocortisone. 5. 5. Colcemid elevates phosphatase activity only slightly when administered at 9 days, and has no effect at 5 days. Both colchicine and colcemid were equally effective in arresting mitoses during a 6-hr period. 6. 6. Ethionine also induces an increase in phosphatase activity at 9 days, but a very large amount is required to produce a relatively small increase. 7. 7. Jejunal phosphatase is not affected by agents that exert strong effects on duodenal phosphatase. Kidney phosphatase is decreased to about half the control values within 48 hr by actinomycin D at all stages tested. Ethionine also markedly depresses kidney phosphatase activity. Puromycin, however, produced only small and irregular effects.

Reversal of thyroxine-induced hypermetabolism by puromycin.

Previous studies have demonstrated that in addition to its effects on metabolic rate, thyroxine stimulates protein biosynthesis. The administration of puromycin, a drug which blocks protein synthesis and, therefore, the thyroxine effect on protein synthesis, acutely reverses the hypermetabolism induced in rats by prior administration of thyroxine and restores the oxygen consumption of the thyrotoxic rats to the euthyroid level. The results suggest that a larger fraction of the total body basal oxygen consumption in hyperthyroidism is related to the process of protein synthesis than in the euthyroid state and that the calorigenic effect of thyroxine is secondary to its effect on protein synthesis.

THE ROLE OF COENZYMES, CORTISONE AND RNA IN THE CONTROL OF LIVER ENZYME LEVELS.

The tryptophan-induced elevation of rat liver tryptophan pyrrolase activity in vivo is associated with two consecutive processes: (a) saturation of the endogenous apotryptophan pyrrolase with its heme cofactor and (b) an increase in the amount of the protein moiety of the enzyme system which can be determined by immunological titration. Process (b) is inhibited by the administration of puromycin (an inhibitor of protein synthesis) but not by that of actinomycin (an inhibitor of RNA synthesis). The induction of tryptophan pyrrolase by cortisone involves process (b alone and is inhibited by puromycin as well as by actinomycin. These agents also inhibit the cortisone-induced elevation of the level of liver tyrosine-α-ketoglutarate transaminase. The results suggest a basic difference between the mechanism of substrate- and hormone-induced stimulation of enzyme synthesis. The level of activity of rat liver threonine dehydrase is decreased by a vitamin B6 deficient diet. The degree of saturation of this enzyme with pyridoxal phosphate and the activity measured in the presence of excess cofactor is not altered by the administration of threonine in vivo. The early postnatal accumulation of tryptophan pyrrolase, unlike that of tyrosine-α-ketoglutarate transaminase, is not inhibited by adrenalectomy or actinomycin. Observations on the ratio of holo- to apotryptophan pyrrolase during development also suggests a common mechanism underlying the developmental and substrate-induced elevation of this enzyme. The present findings exemplify two ways in which factors regulating enzyme synthesis in adult or developing tissues may operate. They may influence the number of potential synthetic sites (RNA template units) or the speed of functioning of a constant number of these sites.