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Mitochondrial enzymes contribute to several discrete areas of metabolism in the cell. In particular, a member of the electron transport chain, dihydroorotate dehydrogenase (DHODH), in the inner mitochondrial membrane, contributes to compartmentation and metabolic channelling in the cytosolic enzymes, clustered around the mitochondria, which catalyse de novo synthesis of UMP. It is clear that the cytosolic pyrimidine pools must respond to demand for membrane synthesis, RNA synthesis and DNA replication as a prerequisite for the proliferation of normal and malignant cells. Mitochondria are also important in the salvage of nucleosides and deoxynucleosides, whose interconversion and catabolism are crucial to maintenance of mitochondrial DNA. A new theme emerges from recent research on the deoxynucleotide pools in mitochondria. In turn, the integrity of mtDNA is essential for that of the genome.

Nicotinic acid–adenine dinucleotide phosphate (NAADP) is fast emerging as a new intracellular Ca2+-mobilizing messenger. In sea urchin egg homogenates, binding of NAADP to its receptor is not readily reversible; hence, prior incubation with low concentrations of NAADP is more effective in inhibiting subsequent binding of radiolabelled NAADP than incubating the preparation with the two ligands simultaneously [Patel, Churchill and Galione (2000) Biochem. J. 352, 725–729]. We extend this finding to show that NAADP is more effective still in inhibiting the subsequent radioligand binding at lower homogenate concentrations, an effect again quite probably due to the non-reversible nature of the receptor–ligand interaction. Enhanced sensitivity of the preparation to NAADP afforded by simple manipulation of the experimental conditions has been applied to determine low levels of NAADP in acid extracts from human red blood cells, rat hepatocytes and Escherichia coli without interference from NADP breakdown. Our improved method for the quantification of NAADP should prove useful in the further assessment of its signalling role within cells.

Our aim was to identify an unusual nucleotide accumulating, with precursors, in erythrocytes in uraemia and to establish its relationship to the candidate uraemic toxin, N1-methyl-2-pyridone-5-carboxamide (M2Py).This nucleotide, plasma creatinine and M2Py were measured in four categories of patients: mild chronic renal failure (CRF), end-stage renal failure (ESRF), haemodialysis (HD) and continuous ambulatory peritoneal dialysis (CAPD); also in healthy controls and patients after successful kidney transplantation (Post-Tx).The nucleotide was identified using liquid chromatography and mass spectrometry as 2-pyridone-5-carboxamide ribonucleoside triphosphate (2PyTP). In the patient groups, mean 2PyTP concentrations are lowest in CRF (21.8 micromol/l), increasing with degree of renal failure through ESRF (55.1 micromol/l) and HD patients (70.9 micromol/l) and being highest in CAPD patients (216.7 micromol/l). Following successful transplantation, 2PyTP concentrations fall to the control range. Plasma concentrations of M2Py correlate with 2PyTP in the erythrocytes; both are undetectable in patients lacking molybdenum cofactor, needed to oxidise N-methylnicotinamide.Our evidence indicates that M2Py accumulates in parallel with the nucleotide 2PyTP in renal failure: either may be a uraemic toxin, since both increase with the degree of renal failure. Elevated concentrations of 2PyTP in CAPD patients reflect a longer half-life for erythrocytes than in HD patients.

It is timely to consider the many facets of the small molecule orotic acid (OA), which is well-known as an essential intermediate of pyrimidine de novo synthesis. In addition, it can be taken up by erythrocytes and hepatocytes for conversion into uridine and for use in the pyrimidine recycling pathway. We discuss the link between dietary orotate and fatty liver in rats, and the potential for the alleviation of neonatal hyperbilirubinaemia. We address the development of orotate derivatives for application as anti-pyrimidine drugs, and of complexes with metal ions and organic cations to assist therapies of metabolic syndromes. Recent genetic data link human Miller syndrome to defects in the dihydroorotate dehydrogenase (DHODH) gene, hence to depleted orotate production. Another defect in pyrimidine biosynthesis, the orotic aciduria arising in humans and cattle with a deficiency of UMP synthase (UMPS), has different symptoms. More recent work leads us to conclude that OA may have a role in regulating gene transcription.

Carbamoyl phosphate is the product of carbamoyl phosphate synthetase (CPS II) activity and the substrate of the aspartate transcarbamoylase (ATCase) activity, each of which is found in CAD, a large 240-kDa multienzyme polypeptide in mammals that catalyses the first three steps in pyrimidine biosynthesis. In our study of the transfer of the labile intermediate between the two active sites, we have used assays that differentiate the synthesis of carbamoyl phosphate from the overall reaction of CPS II and ATCase that produces carbamoyl aspartate. We provided excess exogenous carbamoyl phosphate and monitored its access to the respective active sites through the production of carbamoyl phosphate and carbamoyl aspartate from radiolabelled bicarbonate. Three features indicate interactions between the folded CPS II and ATCase domains causing reciprocal conformational changes. First, even in the presence of approximately 1 mM unlabelled carbamoyl phosphate, when the aspartate concentration is high ATCase uses endogenous carbamoyl phosphate for the synthesis of radiolabelled carbamoyl aspartate. In contrast, the isolated CPS II forward reaction is inhibited by excess unlabelled carbamoyl phosphate. Secondly, the affinity of the ATCase for carbamoyl phosphate and aspartate is modulated when substrates bind to CPS II. Thirdly, the transition-state analogue phosphonacetyl-L-aspartate is a less efficient inhibitor of the ATCase when the substrates for CPS II are present. All these effects operate when CPS II is in the more active P state, which is induced by high concentrations of ATP and magnesium ions and when 5′-phosphoribosyl diphosphate (the allosteric activator) is present with low concentrations of ATP; these are conditions that would be met during active biosynthesis in the cell. We propose a phenomenon of reciprocal allostery that encourages the efficient transfer of the labile intermediate within the multienzyme polypeptide CAD. In this model, binding of aspartate to the active site of ATCase causes a conformational change at the active site of the liganded form of CPS II, which protects it from inhibition by its product, carbamoyl phosphate; reciprocally, the substrates for CPS II affect the active site of ATCase by increasing the affinity for its substrates, endogenous carbamoyl phosphate and aspartate, and thus impede access of exogenous carbamoyl phosphate or the transition-state analogue. Reciprocal allostery justifies the close association of the enzyme activities within the polypeptide and ensures that carbamoyl phosphate is efficiently synthesised and is dedicated to the second step of pyrimidine biosynthesis. These conditions fulfill those required for metabolic channeling in the cell.

5-azidouridine-5′-triphosphate and UDP-pyridoxal have been synthesized using modified synthetic methods. The two synthetic UTP analogues, with commercially available analogues and chemicals have been tested for their effect on CPSase II in the mammalian multienzyme polypeptide CAD. Structure activity relationships are discussed.

CAD, the key enzyme in pyrimidine biosynthesis The first step dedicated to pyrimidine biosynthesis in mammals is catalysed by carbamoylphosphate synthase I1 (CPS 11; EC6.3.5.5). This activity is regulated through feedback inhibition by UTP, the end-product of the pathway, and activated by phosphoribosylpyrophosphate (PRib-PP), a substrate of a later step in the pathway [ l ] . The CPS I1 activity is part of the 240 kDa multienzyme polypeptide CAD, named after the activities it contains in discretely folded domains: CPS 11, aspartate transcarbamoylase (ATCase; EC 2.1.3.2) and dihydro-orotase (EC 3.5.2.3).

We have purified mammalian dihydroorotase as a polypeptide fragment of 46 kDa from an elastase digest of CAD, the 240-kDa multienzyme that catalyses the first three reactions of pyrimidine biosynthesis. The thermal unfolding of the domain was analysed through the change in circular dichroism, indicating a sharp transition at 45 degrees C in which most of the native alpha-helix is lost. Although there is good evidence that the fragments associate as dimers in solution, chemical cross-linking was only possible when the dihydroorotase domain was included in a larger proteolytic fragment of 190-195 kDa. Cross-linking of the isolated domain yielded a species that appeared to result from links between two or more sub-domains, and did not yield the expected 90-kDa dimer of dihydroorotase. We speculate that the presence of other folded regions of CAD stabilises the interactions between dihydroorotase domains.

We have measured the 'core' mammalian carbamoyl-phosphate synthetase II (CPSII) activity, using NH4Cl as the nitrogen-donating substrate and trapping carbamoyl phosphate as urea through its reaction with ammonium ions. When ATP and magnesium ion concentrations are close to those found in the cell, the substrate saturation curves for ammonia and bicarbonate are hyperbolic, giving Km (NH3) values of 166 microM at high ATP concentrations and 26 microM at low ATP concentrations, while the Km (bicarbonate) is 1.4 mM at both ATP concentrations used. These values for the Km (NH3) are lower than previously reported for CPS II, and closer to the values for the mitochondrial counterpart. The Km for ammonia and bicarbonate are not altered by phosphorylation of the multienzyme polypeptide CAD, which contains the first three enzyme activities of pyrimidine biosynthesis. The CPS II activity is lower with an excess of either ATP or magnesium ions, causing the apparently sigmoid dependence of activity upon ATP concentration to be enhanced at low concentrations of free magnesium ions. The feedback inhibitor, UTP, acts by stabilising a state with a low affinity for magnesium ions and for ATP. In the presence of the activator, 5-phosphoribosyl diphosphate (PRibPP), the enzyme has a higher affinity for magnesium ions and thus the ATP dependence of the activity is hyperbolic. Phosphorylation of CAD similarly activates the CPS II enzyme by increasing the affinity for magnesium ions and by pushing the equilibrium away from the low-affinity UTP-stabilised state. Using our improved assay procedure, we observe a very large activation by PRibPP of carbamoylphosphate synthesis at low concentrations of magnesium ions, and we find that unlike UTP, the activator PRibPP is able to act on the phosphorylated enzyme.

We have demonstrated biochemically that the conformation of the proteolytic fragment (mammalian aspartate transcarbamoylase) from the C-terminus of the 240-kDa multienzyme polypeptide carrying the activities carbamoyl phosphate synthetase II, aspartate transcarbamoylase and dihydroorotase (CAD) is similar to that of the catalytic subunits from Escherichia coli aspartate transcarbamoylase. We have measured the extent of unfolding of the mammalian aspartate transcarbamoylase in guanidinium chloride solutions, and have also demonstrated that the protein cross-reacts with antibodies raised against the E. coli enzyme. CAD is digested by low concentrations of trypsin in the presence of 0.2 mM UTP to release an active aspartate transcarbamoylase domain and a 195-kDa ‘nicked CAD’ molecule containing active carbamoyl phosphate synthetase. These two products are easily separated by ion-exchange chromatography. Similar proteolytic cleavage and trimming by elastase releases a family of aspartate transcarbamoylase fragments. Direct N-terminal sequencing of the aspartate transcarbamoylase fragments confirms predictions of the most accessible residues in the region linking the aspartate transcarbamoylase and dihydroorotase domains. Only the largest of the four fragments generated by elastase retains phosphorylation site 2. When this largest fragment is phosphorylated, the family of aspartate transcarbamoylase fragments is eluted together from ion-exchange columns in a different fraction from the completely unphosphorylated preparation, demonstrating the affinity of the domains for each other.

The first two steps of de novo pyrimidine synthesis in Saccharomyces cerevisiae are catalyzed by a multifunctional protein, coded by the URA2 gene and which has the carbamoyl-phosphate (CPSase) synthetase and aspartate transcarbamylase (ATCase) activities. The native enzyme purified from protease-B-deficient URA2-transformed cells, was phosphorylated in vitro using catalytic subunits of pure cAMP-dependent protein kinase. After electrophoresis under denaturing conditions, a single 240-kDa species was found to be phosphorylated. Trypsin digestion of this species gave a single, very acidic phosphopeptide upon isoelectric focussing. Purification by HPLC followed by amino acid sequencing of this peptide, showed a phosphoserine at the expected consensus sequence Arg-Arg-Phe-Ser. Knowledge of the URA2 gene sequence allowed the site to be located in the peptide link between dihydroorotase-like and ATCase domains. Such a location may explain why phosphorylation of the URA2 protein changed neither CPSase and ATCase activities nor their sensitivity to UTP, their common specific inhibitor.

We have measured levels and synthesis of proteolipid protein (PLP) and its transport into myelin in female mice heterozygous for the jimpy gene and in their normal female littermates. In both cord and cerebrum, jimpy carriers show deficits in PLP during development followed by compensation in adulthood. Recovery of PLP occurs earlier in cord than in brain. At 13 days levels of PLP in carriers compared to controls are reduced to 0.60 and 0.44, respectively, in cord and cerebrum. By 100 days, normal levels of PLP are attained in cord (1.13) whereas levels of PLP in cerebrum are only 0.78 of control. By 200 days full recovery occurs in cerebrum, with a ratio of 1.21, suggesting a possible over-compensation. The yield of myelin from cerebrum was reduced to 0.78 in carriers compared to controls at 17 days. In brain slices, incorporation of [3H]leucine into homogenate PLP from carriers is the same as in controls, whereas [3H]leucine incorporation into myelin PLP is reduced to 0.68 of control. These results indicate that synthesis of PLP in the carriers is normal at 17 days, but transport of PLP into myelin is reduced. Similarly, acylation of homogenate PLP is normal, whereas acylation of myelin PLP is reduced, as measured by incorporation of [3H]palmitic acid. Transport of PLP into myelin was compared to transport of MBP; transport of both proteins was equally decreased as indicated by the similar ratio of labeled PLP to MBP in myelin from carriers compared to noncarriers.(ABSTRACT TRUNCATED AT 250 WORDS)

Reduced and ‘scrambled’derivatives of NO2-ribnuclease appear to be less stable than the corresponding derivatives of unmodified ribonuclease. In our hands, reduced ribonuclease is stable in solutions of pH4 for 4-5 days at 4”C, whereas reduced NO,-ribonuclease is reoxidized with a half-time of several hours. Likewise, the background enzymic activity of ‘scrambled‘ NO,-ribonuclease increases during storage in solution at pH8.0, 4“C, whereas scrambled ribonuclease is comparatively stable. Although this instability may be a limitation, the nitrotyrosine residue in these derivatives is a useful probe of the conformational changes accompanying disulphide-formation and interchange. The conversion from ‘scrambled‘ to ‘native’ NO,-ribonuclease corresponds to a vertical transition between the titration curves of Fig. 1. As a result of the shift in PIC., if this conversion is carried out between pH6.0 and 7.4, it should be accompanied by a significant change in A428. We find that this conversion is catalysed by protein disulphide isomerase (EC 5.3.4.1), an enzyme catalysing thiol-disulphide interchange, and hence disulphide-bond isomerization in proteins (Freedman & Hawkins, 1977). Incubation of ‘scrambled‘ NO,-ribonuclease with partially purified protein disulphide isomerase (Hawkins & Freedman, 1976) in the standard conditions for assay of this enzyme (Ibbetson & Freedman, 1976) leads to the appearance of increased ribonuclease activity and an increase in A42s. The rate of increase in ribonuclease activity with ‘scrambled’ NO,-ribonuclease is 2-10 % that found with scrambled unmodified r i b nuclease. With ‘scrambled’ NO,-ribonuclease in the range O.l-O.Smg/ml, rates of increase in absorbance were 0.0035-0.0125 AA428 units/min per mg of enzyme. So nitrated ribonculease derivatives may provide useful probes for the kinetic characterization of disulphide-bond formation and isomerization processes, including those catalysed by protein disulphide isomerase.

The partly unfolded intermediate (state H) of penicillinase from Staphylococcus aureus PC1 is stable in 0.8 M guanidinium chloride at pH 7.0. It has been characterised by measurements of intrinsic viscosity, sedimentation and diffusion coefficients, leading to an equivalent hydrodynamic volume of five times that of the native penicillinase molecule. Values of α-helix content calculated from circular dichroism were 27% for penicillinase and 16% for state H. A multi-domain model is proposed for penicillinase in which the domains can separate without appreciable change in secondary structure. This model is important in understanding the means by which the enzyme activity can be controlled, and corresponds to a kinetic pathway of folding.

The 29 amino acid polypeptide hormone glucagon was cleaved into two large fragments by the enzyme clostripain. The conformational properties of these two fragments were monitored by circular dichroism at pH 2 and 12 in both the presence and absence of sodium dodecyl sulfate. Both glucagon (1-17) and glucagon (19-29) have reduced abilities to fold in aqueous solution. However, both fragments can take on structure of higher apparent helical content in acidic solution in the presence of sodium dodecyl sulfate but only the glucagon (19-29) retains this conformation at high pH. Neither of the two fragments react with dimyristoylphosphatidylcholine as the intact peptide does. Only the carboxyl terminal fragment was capable of reacting with an antibody specific for glucagon. The glucagon (1-17) has markedly reduced affinity for binding to the glucagon receptor as well as markedly reduced ability to stimulate adenylate cyclase activity which is not affected by the presence of glucagon (19-29). It is proposed that the intact sequence provides specific groups required for activity as well as the potential for forming a stable amphipathic helix, both of which are necessary for full biological activity at low peptide concentrations.

We have examined the domain organization, and the locations of the sites phosphorylated by the cyclic-AMP-dependent protein kinase, in the multifunctional polypeptide of the pyrimidine-biosynthetic protein, CAD. Fragments produced after limited proteolysis by elastase or trypsin were separated by SDS/polyacrylamide gel electrophoresis and transferred onto nitrocellulose. The blots were probed with antibodies raised against the core aspartate carbamoyltransferase (ACTase) and dihydroorotase (DHOase) fragments to locate fragments containing these domains, and we also examined the locations of the phosphorylation sites by complete tryptic digestion of blotted, 32P-labelled fragments, followed by analytical isoelectric focussing. Our results are consistent with the domain order glutaminase(GLNase)-carbamoyl-phosphate synthetase-(CPSase)-DHOase-ACTase, as suggested by recently reported homologies between the predicted amino acid sequence for the Drosophila rudimentary gene product, and monofunctional CPSases/ACTases/DHOases. In particular, the finding of a 95-kDa elastase fragment which cross-reacted with both anti-DHOase and anti-ACTase antibodies rules out the previously suggested domain order: DHOase-GLNase-CPSase-ACTase. Phosphorylation by cyclic-AMP-dependent protein kinase accelerates cleavage of native CAD by both elastase and trypsin, and abolishes the protective effect of UTP. Site 1 is located close to the C-terminal end of the 160-kDa GLNase/CPSase region. Comparison with the predicted amino acid sequence of the Drosophila rudimentary gene revealed a strong homology between the tryptic peptide containing site 1 from hamster CAD, and a region at the extreme C-terminal end of the CPSase II domain of the Drosophila enzyme. Alignment of the Drosophila sequence and that of rat liver CPSase I, which is not phosphorylated by cyclic-AMP-dependent protein kinase, revealed that this putative site 1 region is missing in CPSase I. Site 2 could not be located with certainty, either from the limited proteolysis data, or from comparison of the sequence around this site and the sequence of the rudimentary gene. There were also one or more previously undetected minor phosphorylation site(s) located in the protease-sensitive hinge region between the DHOase and ACTase domains.