Citrulline synthesis

The underlying biochemical defect is a failure of mitochondrial uptake of ornithine. This results in a failure of citrulline synthesis and a consequent hyperammonemia. Urinary orotic acid is high, presumably because of underutilization of carbamyl phosphate. In contrast, excretion of creatine is low, reflecting the inhibition of glycine trans-amidinase by excessive levels of ornithine. 

Biotin is a growth factor for many bacteria, protozoa, plants, and probably all higher animals. In the absence of biotin, oxalacetate decarboxylation, oxalosuccinate carboxylation, a-ketoglutarate decarboxylation, malate decarboxylation, acetoacetate synthesis, citrulline synthesis, and purine and pyrimidine syntheses, are greatly depressed or absent in cells (Mil, Tl). All of these reactions require either the removal or fixation of carbon dioxide. Together with coenzyme A, biotin participates in carboxylations such as those in fatty acid and sterol syntheses. Active C02 is thought to be a carbonic acid derivative of biotin involved in these carboxylations (L10, W10). Biotin has also been involved in... 

The large concentrations of acyl phosphatase present in most tissues led to the belief that it prevented the demonstration of acetyl-P biosynthesis and accumulation in animal tissues. We purified extensively, some years ago, what- we first thought to be a specific carba-myl-P phosphatase. This enzyme interested us as a tool for better understanding the mechanism of citrulline synthesis however, on testing for specificity, we found that it attacked acetyl-P just as readily as carbamyl-P. 

This relatively rapid type of activation explains the autocatalytic curves obtained when initial rates of citrulline synthesis are compared with those obtained after several minutes incubation. Table IV demonstrates that by increasing the acetyl glutamate concentration and the length of incubation for the over-all synthesis, the activation was obscured. This is why the activation effect remained undetected until extremely short incubation times and low temperatures were used. Bringing a complicated reaction mixture to incubation temperature, waiting for equilibration, and completing with a last component, as is often done, may obscure effects such as the ones described here. 

Coenzyme A does not appear to be involved, since the passage of the enzyme preparation through a Dowex-2 column, or the addition of coenzyme A to the incubation mixtures, does not change the velocity of the reaction. Coenzyme A is not involved in citrulline synthesis (13). 

Recent investigations have shed light on peculiarities of the NOS action mechanism the role of the H4B cofactor and CaM, and cooperativity in kinetic and thermodynamic properties of different components of the nitric oxide synthesis system. Stop flow experiments with eNOS (Abu-Soud et al., 2000) showed that calmodulin binding caused an increase in NADH-dependent flavin reduction from 0.13 to 86 s 1 at 10 °C. Under such conditions, in the presence of Arg, heme is reduced very slowly (0.005 s 1). Heme complex formation requires a relatively high concentration ofNO (>50 nM) and inhibits the entire process NADH oxidation and citrulline synthesis decreases 3-fold and Km increases 3-fold. NOS reactions were monitored at subzero temperatures in the presence of 50% ethylene glycol as an anti-freeze solvent (Bee et al., 1998). 

This rate is probably underestimated,. since citrulline synthesis in liver mitochondria is faster. 

According to Halestrap [98] activation of mitochondrial electron transport not only increases the proton-motive force but also the intramitochondrial ATP concentration which is important for intramitochondrial ATP utilising reactions like pyruvate carboxylation and citrulline synthesis, processes known to be activated by glucagon. Siess et al. [35], however, showed that in hepatocytes glucagon not only increased mitochondrial ATP, but also the sum of ATP, ADP and AMP at the expense of the cytosolic pool of adenine nucleotides, a phenomenon to which no attention has been paid in the literature. Exchange between ADP and ATP cannot increase the mitochondrial adenine nucleotide pool. Possibly net influx of adenine nucleotides can occur via exchange between ADP and mitochondrial phosphoenol-pyruvate (see [4] for literature). 

After it became apparent that carbamyl aspartate was an intermediate in orotate synthesis, Reichard 20) investigated the synthesis of carbamyl aspartate in rat liver preparations and demonstrated its formation from aspartate, carbon dioxide, and ammonia, in the presence of ATP and iV-acetylglutamate. Previously, Grisolia and Ck)hen 21) had proposed that an active carbamyl was involved in citrulline synthesis in mammalian liver preparations ... 

Citrulline Synthesis. The first syntheses of citrulline and arginine by the addition of NHa and CO2 to ornithine were achieved in the laboratory of Cohen, where soluble enzymes of liver supplemented with cell particles were found to carry out these syntheses. The soluble extract was found to contain the several activities involved in the reactions of the urea cycle, while the particles were found to function solely as a source of ATP. The synthesis of citrulline was found to require ornithine, CO2, NH3, and... 

For descriptive information on the principles of the phosphor imaging process, see Klein and Clark (1993) and Clark and Klein (1996) and literature available from the companies named above. An example of a commercial instrument is shown in Figure 13.5. The method was applied to studies of drug metabolism (Nagatsuka et al., 1993 Ueda et al., 1993), iodothyronine deiodinase activity (Koopdonk-Kool et al., 1993), and citrulline synthesis by nitric oxide synthase activity (Vyas et al., 1996). The synthesis of cholesterol in rat liver was studied using a combination of bioimaging and liquid scintillation counting (Okuyama et al., 1995). 

Vyas, P., Attur, M., Ou, G.-M., Haines, K. A., Abramson, S. B., and Amin, A. R. (1996). Thin layer chromatography an effective method to monitor citrulline synthesis by nitric oxide synthase activity. Portland Press Proc. JO (Biology of Nitric Oxide, Part 5) 44. 

Calculated from the values of citrulline synthesis after 5 hours of incubation. 

Under many conditions the rate-limiting step in urea synthesis is the carbamoyl phosphate synthase reaction. Isolated liver mitochondria will synthesize citrulline from added ammonia under appropriate conditions. The rate of citrulline synthesis can be shown to depend on the intramitochondrial content of acetylglutamate, and both the mitochondrial acetylglutamate content and the rate of citrulline synthesis increase on adding glutamate to the mitochondria. 

Grisolia, S. and Cohen, P. P. (1952) The catalytic role of carbamyl glutamate in citrulline synthesis. /. Biol. Chem. 198, 561-571. 

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