Citrulline synthesis and

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... 

Figure 24-10 Biosynthesis of citrulline, arginine, and urea. The green arrows indicate reactions directly involved in deamination of amino acids and the synthesis of urea. N from amino acids and C from C02 are traced in green.

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. 

The final step of the urea cycle is the cleavage of arginine to release urea and regenerate ornithine. Ornithine then reenters the mitochondria via the ORNT-1 ornithine-citrulline antiporter. ARG-1 is a cytosolic homotrimeric enzyme of 35-kd monomers that is expressed in fiver and red blood cells. A second mitochondrial arginase (ARG-2) most likely plays a role in nitric oxide synthesis and is most abundant in brain, kidney, and prostate. ARG-1 deficiency is unique among the urea cycle deficiencies as patients do not present with hyperammonemia and encephalopathy but rather develop progressive spasticity of the lower limbs. Biochem-... 

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). 

Covalent immobilization of enzymes increases their stability while lowering their activity. Also, their storage stability is notably higher. The synthesis of arginine from citrulline, ATP and argenino-succinate synthetase may involve a carbodiimide intermediate. ... 

R4. Ratner, S., Urea synthesis and metabolism of arginine and citrulline. Advan. Enzymol. Relat. Subj. Biochem. 16, 319-387 (1954). 

NO has a short half-life (2-30 s) and therefore the direct measurement of NO production is impractical. In aqueous solutions, however, it rapidly reacts with oxygen to form the stable water-soluble metabolites nitrite and nitrate (Figure 1). The concentration of these ions, which can be measured via a variety of methods, is used as a measure of the tissue content of NO and/or the synthesis of NO by cultured cells (Hevel and Marietta, 1994 Archer, 1993). An alternative method used to quantify NOS activity is to directly measure the catalytic activity of a cell or tissue extract (i.e., the conversion of radioactive arginine to citrulline Hevel and Marietta, 1994 Archer, 1993). While both methods are sensitive measurements of the overall capacity of a cell or tissue extract to synthesize NO, neither one provides a true kinetic... 

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). 

Fig. 3. Proline and arginine synthesis and degradation to show interrelationships between the pathways. The structures are glutamic acid (GLU), ornithine (ORN), citrulline (CIT), arginine (ARG), urea. 2-oxo-5-amino valeric acid (OAV), A -pyrroline-2-carboxylic acid (P2C), proline (PRO), A -pyrroline-5-carboxylic acid (P5C), glutamic semialdehyde (GSA).

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