Mary Ellen Jones

Modified methods for the determination of carbamyl aspartate

Summary Two simple and reproducible modifications of Ceriottis colorimetric method are presented. These procedures may be used to measure carbamyl aspartate and other ureido compounds, and yield somewhat greater activity for carbamyl aspartate than has been possible with other techniques.

Citrulline synthesis in rat tissues.

Abstract The distribution of carbamyl phosphate synthetase (CPS) and/or ornithine transcarbamylase (OTC) was determined in 17 different rat tissues. Both enzymes, but particularly OTC, were found to be located mainly in the liver so that assay for OTC activity can provide a highly specific test for liver parenchymal cells. Optimal conditions for treatment of small samples of liver to maintain enzyme activity on storage are described. The significance of the absence of detectable CPS activity in most tissues for the presumed role of carbamyl phosphate in pyrimidine biosynthesis is discussed. In addition, it was found that (a) both CPS and OTC first appeared late in fetal life in the rat and did not reach adult levels until after birth, (b) OTC activity in rat and mouse hepatomas varied from undetectable to the normal level of adult liver, and (c) cells derived from liver and grown in culture were devoid of OTC activity.

The cellular location of dihydroorotate dehydrogenase: Relation to de novo biosynthesis of pyrimidines

Abstract Dihydroorotate dehydrogenase from rat liver is found to be located on the outer surface of the inner membrane of mitochondria. Dihydroorotate can diffuse freely from the cytosol into the mitochondria. Orotate can also diffuse freely from the mitochondria into the cytosol for futher conversion to UMP. Therefore, no active transport of either dihydroorotate or orotate is required in pyrimidine biosynthesis. The K m for l -dihydroorotate is 5.2 ± 0.6 μ m . pd -Dihydroorotate is not a substrate for the enzyme but is a competitive inhibitor with a K i of 1.4 m m . Of the compounds tested as analogs for dihydroorotate or metabolites related to pyrimidine biosynthesis, orotate is the strongest inhibitor, with a K i of 8.4 μ m . The K i values for 2,4-dinitrophenol and barbiturate are 180 and 56 μ m , respectively.

END-PRODUCT INHIBITION OF ASPARTATE TRANSCARBAMYLASE IN VARIOUS SPECIES.

Abstract The activity of aspartate transcarbamylase (ATCase) from Escherichia coli is known to be regulated by feed-back inhibition. The catalytic and end-product inhibition characteristics of this enzyme from various species were compared. The enzyme from lettuce seedlings is best inhibited by uridylic acid. The inhibition is noncompetitive with regard to both aspartate and carbamyl phosphate, the substrates for the enzyme; it can be relieved specifically by ribose-5-phosphate without enzyme activity being impaired. The ATCase from Pseudomonas fluorescens is inhibited by various pyrimidine and purine derivatives, both in crude and 50-fold purified extracts. The most potent inhibitor is uridine-5′-triphosphate (UTP) but adenosine-5′-triphosphate also inhibits strongly. The degree of inhibition by UTP increases with decreasing carbamyl phosphate concentration and is independent of aspartate concentration. The enzyme could not be “desensitized” toward the inhibition by UTP by a variety of treatments without a parallel decrease in enzyme activity. ATCase from Bacillus subtilis is not inhibited by a large number of pyrimidine derivatives. The enzyme is inhibited by phosphate (product inhibition) competitively with regard to CAP. In this bacterium the synthesis of the ATCase is regulated by feed-back repression. The possible meaning of the differences in the characteristics of ATCase from the various organisms was discussed.

Purification, composition, and some properties of rat liver carbamyl phosphate synthetase (ammonia)

Abstract A procedure for the purification of rat liver carbamyl phosphate synthetase (ammonia) to homogeneity is described. The molecular weight of isolated active enzyme is 222,000 ± 10,000; that of the subunits obtained by denaturation with sodium dodecyl sulfate in the presence of dithiothreitol is 120,000 ± 5,000. The enzyme appears to be composed of two subunits of identical size. The amino acid composition of the rat liver enzyme is reported and shown to be very similar to that of the bovine enzyme. The concentration of carbamyl phosphate synthetase I in the matrix of rat liver mitochondria is about 5 × 10 −4 m , very similar to that of acetylglutamate; on this basis, it is suggested that one-third to one-half of the total enzyme may be in the active form in the matrix. Calculations also indicate that normally, ATP is unlikely to limit the rate of carbamyl phosphate synthesis in vivo . Carbamyl phosphate synthetase I appears to constitute 22 to 26% of the total matrix protein of liver mitochondria; the significance of this fact is briefly examined.

[21] Orotate phosphoribosyltransferase: Orotidylate decarboxylase (Ehrlich Ascites Cell)

Publisher Summary This chapter describes purification procedure related to the orotate phosphoribosyltransferase enzyme. Assay procedures reported include methods measuring the spectrophotometric changes that occur when a pyrimidine base is converted to a nucleotide or when orotidylic acid (OMP) is converted to uridylic acid (UMP), methods that separate the free base from the nucleotides, and methods measuring the release of [14C]O2 from [14C]carboxyl-labeled orotate or orotidylate. This assay is appropriate for both crude or pure enzyme preparations. In this procedure orotate, OMP, and UMP are separated from one another by thin-layer chromatography. It is also useful when one wishes to estimate how effective inhibitors of the decarboxylase are when the OMP level is that maintained at a steady-state level by the complex itself,for example, when orotate and phosphoribosyl pyrophosphate (P-Rib-PP) are the substrates and only the OMP formed by the transferase is present, this assay is ideal

Initial steps in pyrimidine synthesis in ehrlich ascites carcinoma

Abstract The pyrimidine specific carbamyl phosphate synthetase (CPSase) and aspartate transcarbamylase (ATCase) of the Ehrlich ascites carcinoma have been found to exist as a complex with a molecular weight of 750,000–850,000 on sucrose gradients in the presence of 30% dimethyl sulfoxide (DMSO). In the presence of 10% DMSO, the activities separate and the remaining CPSase activity has a molecular weight of 150,000–200,000 while the ATCase now exists as two peaks of activity, with molecular weights of 300,00–400,000 and 525,000–700,000, depending on the exact conditions of the gradient. Dihydroorotase activity was found to co-sediment also with the two ATCase peaks under these conditions. It appears then that ATCase can be associated with CPSase and DHOase and that these first three enzymes of the pyrimidine biosynthetic pathway may exist as a complex.

Regulation of Uridylic Acid Biosynthesis in Eukaryotic Cells

Publisher Summary In fungi and the vertebrates there can be a complete separation of the arginine and pyrimidine biosynthetic pathways because each pathway can derive carbamyl phosphate (CAP) from a specific carbamyl-phosphate synthetase (CAPSase). In some organisms like Neurospora the two pathways normally do not share CAP pools, but in other organisms, such as yeast, CAP can flow one “pool” to the other. The plants differ from other eukaryotes in that a single synthetase has so far been observed, with feedback regulatory characteristics typical of the bacterial synthetase , which can serve both pathways. The critical experiments have not been carried out, however, to test whether a second synthetase exists in plants. The other prominent phenomenon in eukaryotes is the appearance of enzyme complexes. In fungi , one such complex for the pyrimidine pathway exists consisting of the first two enzyme activities, namely, CPSase-ATCase (aspartate transcarbamylase). These two enzymes are coded for by a single polar gene. It is not known with certainty whether this gene codes for one or several polypeptide chains, although the studies suggest that more than one type of polypeptide chain is in the complex.

[124a] Carbamyl phosphate synthesis and utilization

Publisher Summary This chapter focuses on carbamyl phosphate synthesis and utilization. Carbamyl-P is the energy-rich phosphate compound that donates its carbamyl group to ornithine, aspartie acid, and oxamic acid in the enzymatic synthesis of, respectively, citrulline, carbamyl aspartate, and carbamyl oxamic acid. The chemical synthesis of carbamyl-P from acid phosphate and cyanate is detailed in the chapter. The preparations described are the Li 8 , K 8 , NH 4 + , and Na salts. This synthesis is an equilibrium reaction so that the yield with respect to either one of the starting materials, cyanate or acid phosphate, may be increased by the addition of the other reactant several-fold over the limiting reactant, provided that care is taken to maintain the mixture near pH 6. This ability to influence the yield of carbamyl-P synthesized, and particularly to increase the yield with respect to cyanate or phosphate, by varying the amount of the starting materials is valuable for the synthesis of radioactive carbamyl-P. Carbamyl-P incubated in arsenate buffer, pH 6, undergoes a chemical arsenolysis.

Radioassay of orotic acid phosphoribosyltransferase and orotidylate decarboxylase utilizing a high-voltage paper electrophoresis technique or an improved 14CO2-release method

Abstract Orotic acid phosphoribosyltransferase (EC 2.4.2.10) and orotidylate decarboxylase (EC 4.1.1.23) can be assayed independently of one another by the high voltage paper electrophoresis method described here, which separates orotic acid, OMP, and UMP, the substrates and/or products of these enzymes, from each other. The relative migration of other compounds, mainly other nucleotides, their bases, or other intermediates of the UMP biosynthetic pathway, has also been recorded. The method has allowed us to observe that OMP is not released to any significant degree from the enzyme complex of these two enzymes that occurs in Ehrlich ascites cells; rather orotic acid is converted stoichiometrically by the enzyme complex to UMP. For purification of the enzyme complex, we have found the release of 14 CO 2 from [ 14 C]carboxyl-labeled orotic acid (when phosphoribosyl pyrophosphate and Mg 2+ are present) preferable to the HVPE method as a routine assay procedure. The most economical CO 2 -absorbant for the assay of the enzyme complex or for orotidylate decarboxylase (and possibly for other enzymes which release CO 2 ) is an NaOH-soaked paper strip. As detailed here, its use allows one to repeatedly reuse the scintillation vials and fluid.