The regulation of carbamoyl phosphate synthase activity in rat liver mitochondria

McGivan, J D; Bradford, Norah M.; Mendes-Mourão, J.

doi:10.1042/bj1540415

Cited by 114 publications

(48 citation statements)

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“…The capacity of rat liver mitochondria to synthesize carbamoyl phosphate is tenfold greater than existing studies [31][32][33]441 suggest. The maximal activity of carbamoylphosphate synthetase was carefully determined in mitochondria to obtain a measure of the total enzyme concentration.…”

Section: Discussionmentioning

confidence: 76%

Carbamoylphosphate Synthetase I of Rat‐Liver Mitochondria

Lusty¹

1978

European Journal of Biochemistry

130

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An improved purification of carbamoylphosphate synthetase I from rat liver mitochondria is described. The enzyme is essentially homogeneous with enhanced specific activity and stability. The enzyme is stable at alkaline pH in concentrated solution of salts. Kinetic studies indicate two types of inactivation, reversible and irreversible, depending on pH. The monomeric unit of carbamoylphosphate synthetase I consists of one polypeptide chain. The protein migrates in dodecylsulfate/polyacrylamide gel as a single component corresponding to a molecular weight of about 155000. The molecular weight of the polypeptide chain determined by sedimentation equilibrium in 6 M guanidine hydrochloride containing dithioerythritol is 158 000 5 000. The weight-average molecular weight of the native enzyme, 188000, suggests that the monomeric form is the predominant form of the enzyme, under the conditions described. Glutamine is inactive as a substrate. With ammonium, carbamoyl phosphate synthesis is optimal in the pH range 6.8-7.6. M$+ and Mn2+ are equally effective metal ions in the direction of carbamoyl phosphate synthesis, but excess Mn2 + is inhibitory. At less than saturating concentrations of substrate, considerable activation of the enzyme is observed when metal ions are in excess of ATP; when MgATP2-concentration is held constant at 1 mM and 10 mM, plots of D versus free Mg2+ concentration are hyperbolic, and extrapolate to zero. These results suggest that free divalent metal ion is required in the forward reaction. The apparent K,,, of the substrates is similar to other preparations of the enzyme. The determination of the maximal activity of carbamoylphosphate synthetase in rat liver mitochondria is reported, providing a measure of the total enzyme concentration that is about 1 -1.5 mM.The purification procedure described also provides an easy method to obtain ornithine transcarbamylase in partially purified form.Carbamoylphosphate synthetase I catalyzes the formation of carbamoyl phosphate, the first intermediate in arginine and urea biosynthesis, from ammonia and bicarbonate in a reaction that requires catalytic amounts of acetylglutamate and two molecules of ATP. The mechanism of the reaction is formulated to occur in at least three steps [l], although more recent data [2,3] indicate that the mechanism is still unclarified. The general properties of carbamoylphosphate synthetase I are based on extensive studies of the frog liver enzyme [4] which is isolated in the most stable form. Only a few studies [5-81 have been reported on the kinetic and physicochemical Eirzymes. Carbamoylphosphate synthetase I (EC 2.7.2.5); ornithine transcarbamylase (EC 2.1.3.3); phosphorylase a (EC 2.4.1 1); 8-galactosidase (EC 3.2.1.23).properties of the purified mammalian enzyme, which was first obtained in stable form from rat liver [6], and more recently, from bovine liver [8]. Molecular weight studies [8] suggest a chemical equilibrium between an 11-S and a 7.5-S form of the enzyme. The 11-S form dissociates at low temperature in the pr...

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Section: Discussionmentioning

confidence: 76%

Carbamoylphosphate Synthetase I of Rat‐Liver Mitochondria

Lusty¹

1978

European Journal of Biochemistry

130

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“…Many authors have inclined to the view that N-AG does regulate urea synthesis (McGivan et al 1974(McGivan et al , 1976Saheki et al 1980). Stewart & Walser (1980) gave amino acids intraperitoneally and showed that a rise in N-AG occurs within minutes.…”

Section: The Biochemical Evidencementioning

confidence: 99%

The mysteries of nitrogen balance

Waterlow¹

1999

Nutr. Res. Rev.

113

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The ®rst part of this review is concerned with the balance between N input and output as urinary urea. I start with some observations on classical biochemical studies of the operation of the urea cycle. According to Krebs, the cycle is instantaneous and automatic, as a result of the irreversibility of the ®rst enzyme, carbamoyl-phosphate synthetase 1 (EC 6.3.5.5; CPS-I), and it should be able to handle many times the normal input to the cycle. It is now generally agreed that acetyl glutamate is a necessary co-factor for CPS-1, but not a regulator. There is abundant evidence that changes in dietary protein supply induce coordinated changes in the amounts of all ®ve urea-cycle enzymes. How this coordination is achieved, and why it should be necessary in view of the properties of the cycle mentioned above, is unknown. At the physiological level it is not clear how a change in protein intake is translated into a change of urea cycle activity. It is very unlikely that the signal is an alteration in the plasma concentration either of total amino-N or of any single amino acid. The immediate substrates of the urea cycle are NH 3 and aspartate, but there have been no measurements of their concentration in the liver in relation to urea production. Measurements of urea kinetics have shown that in many cases urea production exceeds N intake, and it is only through transfer of some of the urea produced to the colon, where it is hydrolysed to NH 3 , that it is possible to achieve N balance. It is beginning to look as if this process is regulated, possibly through the operation of recently discovered urea transporters in the kidney and colon. The second part of the review deals with the synthesis and breakdown of protein. The evidence on whole-body protein turnover under a variety of conditions strongly suggests that the components of turnover, including amino acid oxidation, are in¯uenced and perhaps regulated by amino acid supply or amino acid concentration, with insulin playing an important but secondary role. Molecular biology has provided a great deal of information about the complex processes of protein synthesis and breakdown, but so far has nothing to say about how they are coordinated so that in the steady state they are equal. A simple hypothesis is proposed to ®ll this gap, based on the self-evident fact that for two processes to be coordinated they must have some factor in common. This common factor is the amino acid pool, which provides the substrates for synthesis and represents the products of breakdown. The review concludes that although the achievement and maintenance of N balance is a fact of life that we tend to take for granted, there are many features of it that are not understood, principally the control of urea production and excretion to match the intake, and the coordination of protein synthesis and breakdown to maintain a relatively constant lean body mass.

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“…An impaired energy state and low mitochondrial ATP production could suppress carbamylphosphate synthesis (McGivan et al 1976). N-acetylglutamate synthesis, which activates carbamylphosphate synthetase activity, could be inhibited by elevated acyl-CoA levels (Coude et al 1979).…”

Section: Discussionmentioning

confidence: 99%