Ornithine transcarbamylase in liver mitochondria

Summary Ornithine transcarbamylase (ornithine carbamoyltransferase, EC 2.1.3.3), the second enzyme of urea synthesis, is localized in the matrix of liver mitochondria of ureotelic animals. The enzyme is encoded by a nuclear gene, synthesized outside the mitochondria, and must then be transported into the organelle. The rat liver enzyme is initially synthesized on membrane-free polysomes in the form of a larger precursor with an amino-terminal extension of 3 400–4 000 daltons. In rat liver slices and isolated rat hepatocytes, the pulse-labeled precursor is first released into the cytosol and is then transported with a half life of 1 2 min into the mitochondria where it is proteolytically processed to the mature form of the enzyme. The precursor synthesized in vitro exists in a highly aggregated form and has a conformation different from that of the mature enzyme. The precursor has an isoelectric point (pI = 7.9) higher than that of the mature enzyme (pI = 7.2).The precursor synthesized in vitro can be taken up and processed to the mature enzyme by isolated rat liver mitochondria. The mitochondrial transport and processing system requires membrane potential and a high integrity of the mitochondria. The transport and processing activities are conserved between mammals and birds or amphibians and is presumably common to more than one precursor. Potassium ion, magnesium ion, and probably a cytosolic protein(s), in addition to the transcarbamylase precursor and the mitochondria, are required for the maximal transport and processing of the precursor.A mitochondrial matrix protease which converts the precursor to a product intermediate in size between the precursor and the mature subunit has been highly purified. The protease has an estimated molecular weight of 108 000 and an optimal pH of 7.5–8.0, and appears to be a metal protease. The protease does not cleave several of the protein and peptide substrates tested. The role of this protease in the precursor processing remains to be elucidated.Rats subjected to different levels of protein intake and to fasting show significant changes in the level of enzyme protein and activity of ornithine transcarbamylase. The dietary-dependent changes in the enzyme level are due mainly to an altered level of functional mRNA for the enzyme. In contrast, during fasting, the increase in the enzyme level is associated with a decreased level of translatable mRNA forthe enzyme.Pathological aspects of ornithine transcarbamylase including the enzyme deficiency and reduced activities of the enzyme in Reye's syndrome are also described. A possibility that impaired transport of the enzyme precursor into the mitochondria leads to a reduced enzyme activity, is proposed.Abbreviation pOTC precursor of ornithine transcarbamylase     

Chicken ornithine transcarbamylase: purification and some properties

Ornithine transcarbamylase [EC 2.1.3.3] has been purified from chick kidney to homogeneity. The molecular weight is 110,000 as determined by gel filtration. Sodium dodecylsulfate polyacrylamide gel electrophoresis of the enzyme showed that the enzyme exists as a trimer of identical subunits of 36,000 daltons like other mammalian species ornithine transcarbamylases. In 0.1 M triethanolamine/HCl, the apparent optimum pH of the purified enzyme was 7.5 in the presence of 5 mM ornithine. The curve shifted toward a more alkaline region with a decrease in ornithine concentration. The specific activity of the purified enzyme as 77 units at pH 7.5. The Km for carbamyl phosphate was 0.11 mM and the Km for ornithine was 1.21 mM. With an increase in pH, a decrease in Km values for ornithine and an increase in the extent of inhibition by ornithine were observed. On using antibody against bovine liver ornithine transcarbamylase, the precipitin lines for the chick and bovine enzymes showed a spur pattern. Even when excess amounts of the antibody were added, the chick enzyme did not lose the activity while the bovine enzyme activity was inhibited completely.     

Rat liver glycogen metabolism in the perinatal period

The correlation between blood glucose levels, the concentration of glycogen, the activities of glycogen sythase and phosphorylase and their respective kinases and phosphatases was examined in liver of rat fetuses between day 18 of gestation and one day after birth. Between day 18 and 21 there is a rapid increase in the concentration of glycogen and in the activity of synthase a and a much slower increase in the activity of phosphorylase a. The activity of the respective kinases increased rapidly during this period and reached maximun on day 21. The activity of synthase phosphatase and phosphorylase phosphatase increased after day 18, to reach a maximum on day 19 and 20, respectively, but decreased again towards day 21. The possibility that the changes in glycogen concentration and enzyme activities were related to an effect of glucose of AMP on the respective phosphatases was considered. It was found that the Km of phosphatase for glucose in the prenatal period was 5–7 mM, as in the adult. Since the level of blood glucose during this period was constant (2.8 mM), an effect of glucose on phosphatase activity seems unlikely. AMP concentration increased between day 18 and 21 from 6–15 nmol/g. In view of the low level of phosphorylase a activity during this period, the increase in AMP concentration is not considered to be important in the regulation of glycogen breakdown at this time.Immediately after birth blood glucose levels dropped to 5 mg/dl. This was accompanied by a rapid decrease in glycogen concentration and in the activity of glycogen synthase and a rise in phosphorylase activity. Blood glucose levels returned to the initial level within 1 h after birth, whereas the changes in glycogen concentration and enzyme activities continued for at least 3 h after birth. On day 22 all parameters examined had reached the level found in adult rat liver.It is suggested that the rapid changes observed immediately after birth are due to an effect of hypoglycemia mediated by hormones and cannot be ascribed to direct effects of metabolites on the enzyme systems involved.     

Rat liver glycogen metabolism in the perinatal period.

The correlation between blood glucose levels, the concentration of glycogen, the activities of glycogen synthase and phosphorylase and their respective kinases and phosphatases was examined in liver of rat fetuses between day 18 of gestation and one day after birth. Between day 18 and 21 there is a rapid increase in the concentration of glycogen and in the activity of synthase a and a much slower increase in the activity of phosphorylase a. The activity of the respective kinases increased rapidly during this period and reached maximum on day 21. The activity of synthase phosphatase and phosphorylase phosphatase increased after day 18, to reach a maximum on day 19 and 20, respectively, but decreased again towards day 21. The possibility that the changes in glycogen concentration and enzyme activities were related to an effect of glucose or AMP on the respective phosphatases was considered. It was found that the Km of phosphorylase phosphatase for glucose in the prenatal period was 5--7 mM, as in the adult. Since the level of blood glucose during this period was constant (2.8 mM), an effect of glucose on phosphatase activity seems unlikely. AMP concentration increased between day 18 and 21 from 6--15 nmol/g. In view of the low level of phosphorylase a activity during this period, the increase in AMP concentration is not considered to be important in the regulation of glycogen breakdown at this time. Immediately after birth blood glucose levels dropped to 5 mg/dl. This was accompanied by a rapid decrease in glycogen concentration and in the activity of glycogen synthase and a rise in phosphorylase activity. Blood glucose levels returned to the initial level within 1 h after birth, whereas the changes in glycogen concentration and enzyme activities continued for at least 3 h after birth. On day 22 all parameters examined had reached the level found in adult rat liver. It is suggested that the rapid changes observed immediately after birth are due to an effect of gypoglycemia mediated by hormones and cannot be ascribed to direct effects of metabolites on the enzyme systems involved.     

Ornithine transcarbamylase from Neurospora crassa: purification and properties

Ornithine transcarbamylase catalyzes the synthesis of citrulline from carbamyl phosphate and ornithine. This enzyme is involved in the biosynthesis of arginine in many organisms and participates in the urea cycle of mammals. The biosynthetic ornithine transcarbamylase has been purified from the filamentous fungus, Neurospora crassa. It was found to be a homotrimer with an apparent subunit molecular weight of 37,000 and a native molecular weight of about 110,000. Its catalytic activity has a pH optimum of 9.5 and Km's of about 5 and 2.5 mM for the substrates, ornithine and carbamyl phosphate, respectively, at pH 9.5. The Km's and pH optimum are much higher than those of previously characterized enzymes from bacteria, other fungi, and mammals. These unusual kinetic properties may be of significance with regard to the regulation of ornithine transcarbamylase in this organism, especially in the avoidance of a futile ornithine cycle. Polyclonal antibodies were raised against the purified enzyme. These antibodies and antibody raised against purified rat liver ornithine transcarbamylase were used to examine the structural similarities of the enzyme from a number of organisms. Cross-reactivity was observed only for mitochondrial ornithine transcarbamylases of related organisms.     

Arginine catabolism in Neurospora: cycling of ornithine.

We measured the metabolism of ornithine in Neurospora during the transition from minimal medium to arginine-supplemented medium. Within an hour after arginine supplementation, the amount of intracellular ornithine (95% of which had been stored in vesicles) dropped by 65%, even though the catabolism of arginine produces as much ornithine as had been produced on minimal medium. The arginine level in the cell rose 10-fold. Ornithine flux through the catabolic enzyme ornithine aminotransferase increased fivefold, but flux through the mitochondrial enzyme ornithine transcarbamylase (leading to arginine synthesis) was only 20% of the rate seen on minimal medium. During this transition to arginine catabolism, the enzymes of the arginine pathway operate as an ornithine cycle, but at a restricted rate. We suggest the hypothesis that high levels of arginine may inhibit the movement of ornithine into the vesicles and into the mitochondria.     

In situ staining procedure for the detection of ornithine transcarbamylase activity in polyacrylamide gels

Ornithine transcarbamylase deficiency is a human genetic disease potentially susceptible to gene therapy. A murine model system exists for the disease in the sparse-fur (spf) mouse. Before gene therapy studies can be performed it is necessary to have practical methods which could detect successful gene transfer. Therefore we have developed an in situ staining procedure for the detection of ornithine transcarbamylase activity in polyacrylamide gels. Following electrophoretic separation under nondenaturing conditions inorganic phosphate cleaved from carbamyl phosphate in gels as a result of enzymatic activity was precipitated as phosphomolybdic acid and visualized by reduction with ascorbic acid. Results from the procedure correlated with ornithine transcarbamylase activity as measured by solution assay for citrulline, the other product of the reaction. This procedure readily distinguished mutant forms of ornithine transcarbamylase as exemplified by the murine spf mutation and resolved ornithine transcarbamylases of all animals tested into multiple forms. The procedure further distinguished ornithine transcarbamylases of animals of several different genera while yielding virtually identical patterns of the enzyme from species within the same genus. This procedure also suggested that the human enzyme was more labile than murine ornithine transcarbamylase; direct thermolability studies confirmed this finding.     

Two variants of ornithine decarboxylase activity in the mouse liver. The nature of enzyme induction

Ornithine decarboxylase activity in mouse liver is predominantly located in the cell nuclei. After injection of some inducing agents (thioacetamide, diethylnitrosamine, hydrocortisone) the enzyme leaves the nucleus for cytosol. A circadian rhythm of ornithine decarboxylase activity has been observed in nucleus and cytosol, the decrease of enzyme activity in the nucleus being accompanied by its increase in cytosol. The enzyme obtained from intact mice with a minimal level of ornithine decarboxylase activity in the cytosol differs in ion-exchange properties, pH-optimum and Km for ornithine from the thioacetamide stimulated (nucleus enzyme).     

Roles of arginine and canavanine in the synthesis and repression of ornithine transcarbamylase by Escherichia coli

Conditions were found under which the processes of repression and derepression of ornithine transcarbamylase were separated from the process of enzyme synthesis. After 10 min of arginine deprivation followed by the addition of 2 to 200 mug of l-arginine per ml, a number of strains of Escherichia coli exhibited a significant burst of ornithine transcarbamylase synthesis which lasted 3 to 4 min before the onset of repression. The rapid increase of enzyme activity was shown to require protein synthesis, and was not due to a slow uptake of arginine or induction of an arginine-inducible ornithine transcarbamylase. The capacity of E. coli to synthesize the burst of ornithine transcarbamylase reached a maximum after 10 min of arginine deprivation and then remained constant. The observed increase in enzyme synthesis may reflect the level of unstable messenger ribonucleic acid (RNA) for ornithine transcarbamylase present in the cell at the time protein synthesis was reinitiated. After the addition of arginine in the absence of protein synthesis, the burst of ornithine transcarbamylase decayed with a half-life of about 3 min. The data implied that arginine prevents synthesis of new messenger RNA that can translate this enzyme. Repression of ornithine transcarbamylase by l-canavanine (100 to 200 mug/ml) was observed, and no active enzyme was formed in the presence of this analogue. The action of canavanine as a repressor was distinguished from the inhibitory effect of this compound on protein synthesis.     

Ornithine transcarbamylase of rat liver. Kinetic, physical, and chemical properties.

Ornithine transcarbamylase of rat liver has been purified to homogeneity. The purified enzyme of specific activity 870 to 920 focuses as a single protein at pH 7.2. At pH 7.7, the Km for carbamyl phosphate is 0.026 mM, and the Km for ornithine is 0.04 mM. The inhibition constants of a number of amino acids that act as competitive inhibitors of the enzyme are reported. The native enzyme of Mr = 112,000 is composed of three subunits of Mr = 39,600 +/- 1,000. Chemical evidence indicates that the subunits are identical in amino acid composition and amino acid sequence. The amino acid sequence of the NH2-terminal region of ornithine transcarbamylase is Ser-Gln-Val-Gln-Leu-Lys-Gly-Ser-Asp-Leu-Leu-Thr-Leu-Lys-Asn-(Phe)-X-Thr-X-Glu-Ile-Gln-Tyr-Met-.     

Stabilization of ornithine decarboxylase and N1-spermidine acetyltransferase in rat liver by methylglyoxal bis(guanylhydrazone)

The activities of ornithine decarboxylase and spermidine N1-acetyltransferase started to rise in normal rat liver 4 h after the intraperitoneal injection of methylglyoxal bis(guanylhydrazone) (MGBG; 80 mg/kg). Ornithine decarboxylase had its greatest activity 24 h after a single injection of MGBG and the acetyltransferase peaked 8 h after the injection. Measurement of the apparent half-life of ornithine decarboxylase after MGBG treatment revealed a clear decrease in the decay rate of the enzyme in both normal and regenerating rat liver. MGBG slowed the decay of the transferase also in normal rat liver, as well as inhibiting its activity in vitro. The stabilization by MGBG of these two short-lived proteins involved in metabolism of polyamines should lead to their accumulation in liver, thus explaining their increased activities. In the case of ornithine decarboxylase, studies with a specific antibody against mouse kidney ornithine decarboxylase showed that the rise in ornithine decarboxylase activity after MGBG application was not due to the appearance of an immunologically different isozyme.     

Modulation of ornithine decarboxylase activity and ornithine decarboxylase-antizyme complex in rat heart by hormone and putrescine treatment

Ornithine decarboxylase was present in a cryptic, complexed form in an amount approximately equivalent to that of free ornithine decarboxylase activity in adult rat heart. Addition of isoproterenol (10 mg/kg) caused a notable rise in ornithine decarboxylase activity and a simultaneous decrease in the amount of the complexed enzyme. During the period of ornithine decarboxylase decay, when cardiac putrescine content had reached high values, the level of the complex increased above that of the control. Administration of putrescine (1.5 mmol/kg, twice) or dexamethasone (4 mg/kg) produced a decrease of heart ornithine decarboxylase activity, while it did not remarkably affect the level of complexed ornithine decarboxylase, therefore raising significantly the ratio of bound to total ornithine decarboxylase. Putrescine also elicited the appearance of free antizyme, concomitantly with the disappearance of free ornithine decarboxylase activity after 3-4 h of treatment. These results indicate that a significant amount of ornithine decarboxylase occurs in an inactive form in the heart under physiological conditions and that its absolute and relative levels may vary following stimuli which affect heart ornithine decarboxylase activity.     

Mirex induces ornithine decarboxylase in female rat liver

Ornithine decarboxylase, the rate-limiting enzyme in polyamine synthesis, was significantly induced in female rat liver following oral administration of the pesticide mirex. After dual oral exposure (120 mg/kg of mirex; 21 and 4 hr prior to sacrifice), ornithine decarboxylase activity in rat liver cytosol was 70-fold higher than control values. A single oral dose of mirex (180 mg/kg) induced hepatic ornithine decarboxylase activity 55-fold over controls. After a single oral dose of mirex the maximal induction of ODC activity occurred at 36 hr. Mirex is an unusually potent and long-lasting inducer of rat hepatic ornithine decarboxylase activity.     

Growth hormone and rat liver mitochondria effects on urea cycle enzymes

Effects of hypophysectomy and subsequent growth hormone administration on mitochondrial enzymes of the urea cycle were investigated in rat liver. Hypophysectomy increased the activities of the two mitochondrial enzymes, carbamyl phosphate synthetase and ornithine transcarbamylase but not of the cytosolic enzyme, argininosuccinate synthetase. The activity of mitochondrial phosphate dependent glutaminase was not affected. Administration of bovine growth hormone (100 μg/100 g body weight) for two weeks decreased the activities of carbamyl phosphate synthetase and ornithine transcarbamylase almost to the normal level. These results suggest a specific effect of growth hormone on mitochondrial enzymes of the urea cycle and serve to explain the increased urea formation in hypopituitarism.     

Purification of ornithine transcarbamylase from rat liver by affinity chromatography with immobilized transition-state analog

Ornithine transcarbamylase (EC 2.1.3.3) was purified to homogeneity from rat liver. The basis of the method is the chromatography of a high-speed supernatant fraction of a homogenized rat liver on an affinity column consisting of the transition-state analog of ornithine transcarbamylase, δ-N-(phosphonacetyl)-l-ornithine, immobilized on epoxy-activated Sepharose 6B through the α-amino group. The enzyme was eluted from the column using a gradient of the substrate, carbamyl phosphate, and further purified by gel filtration. The enzyme elutes with a constant specific activity of 250 to 260 μmol min^?1 mg^?1 at pH 8.5, 37°C, and is free of contaminating proteins on sodium dodecyl sulfate gel electrophoresis. Determination of the molecular weight of the purified enzyme by centrifugation (98,000) and by gel electrophoresis in the presence of sodium dodecyl sulfate (35,300) indicates that the enzyme from rat liver is a trimer. The enzyme exhibits conventional Michaelis-Menten kinetics at pH 7.4 and in this respect differs from the enzyme prepared by other methods.     

Regulation of Escherichia coli ornithine transcarbamylase by orotate.

Ornithine transcarbamylase from Escherichia coli, strain W, exhibits negative cooperativity with respect to ornithine, and the enzymatic activity is further regulated by orotate. The effect of orotate on ornithine transcarbamylase is dependent not only upon the carbamylphosphate concentration, but also upon the concentration of ornithine. At high concentrations of carbamylphosphate (10 mM), a conversion from negative cooperativity to positive cooperativity is observed with 10 mM orotate. At 1 mM carbamylphosphate, however, 10 mM orotate activates the enzyme at low ornithine concentrations, but as the ornithine concentration is increased above 5 mM, inhibition is observed. Thus, a regulatory link has been established between the pathways of arginine biosynthesis and pyrimidine biosynthesis, each of which utilizes carbamylphosphate.     

Ornithine decarboxylase activity in insulin-deficient states

The activity of ornithine decarboxylase, the rate-controlling enzyme in polyamine biosynthesis, was determined in tissues of normal control rats and rats made diabetic with streptozotocin. In untreated diabetic rats fed ad libitum, ornithine decarboxylase activity was markedly diminished in liver, skeletal muscle, heart and thymus. Ornithine decarboxylase was not diminished in a comparable group of diabetic rats maintained on insulin. Starvation for 48h decreased ornithine decarboxylase activity to very low values in tissues of both normal and diabetic rats. In the normal group, refeeding caused a biphasic increase in liver ornithine decarboxylase; there was a 20-fold increase in activity at 3h followed by a decrease in activity, and a second peak between 9 and 24h. Increases in ornithine decarboxylase in skeletal muscle, heart and thymus were not evident until after 24–48h of refeeding, and only a single increase occurred. The increase in liver ornithine decarboxylase in diabetic rats was greater than in normal rats after 3h of refeeding, but there was no second peak. In peripheral tissues, the increase in ornithine decarboxylase with refeeding was diminished. Skeletal-muscle ornithine decarboxylase is induced more rapidly when meal-fed rats are refed after a period without food. Refeeding these rats after a 48h period without food caused a 5-fold increase in ornithine decarboxylase in skeletal muscle at 3h in control rats but failed to increase activity in diabetic rats. When insulin was administered alone or together with food to the diabetic rats, muscle ornithine decarboxylase increased to activities even higher than in the refed controls. In conclusion, these findings indicate that the regulation of ornithine decarboxylase in many tissues is grossly impaired in diabetes and starvation. They also suggest that polyamine formation in vivo is an integral component of the growth-promoting effect of insulin or some factor dependent on insulin.     

Stimulation of ornithine decarboxylase activity in rat tissues by growth hormone and by serum growth factor from rats infested with spargana of Spirometra mansonoides

Ornithine decarboxylase activity was studied in heart, kidney, liver, thymus, lung, spleen, skeletal muscle and fat of hypophysectomized rats after growth hormone treatment. A marked increase in enzyme activity was observed in kidney and liver, and a significant increase in heart and thymus at 4 h after injection of growth hormone. The kidney was the most responsive organ with an increase in activity of about 100 fold. The enzyme activity in kidney responded to a dose of 10 μg of growth hormone. Daily injection for 12 days raised activity only in the heart. Infestation for 6–13 days with spargana of Spirometra mansonoides, which also causes growth of hypophysectomized rats, increased enzyme activity in the heart and thymus. Intravenous injection of serum of hypophysectomized rats infested with spargana of Spirometra mansonoides caused a significant increase in the enzyme activity in liver and kidney after 4 h. Growth hormone and the serum growth factor of sparganosis seem to share the characteristic of causing an early increase in ornithine decarboxylase activity in rat tissues. The marked response in kidney and liver raises the possibility that these organs are the primary targets of both substances.