Half-of-sites binding of orotidine 5'-phosphate and alpha-D-5-phosphorylribose 1-diphosphate to orotate phosphoribosyltransferase from Saccharomyces cerevisiae supports a novel variant of the Theorell-Chance mechanism with alternating site catalysis

A ping-pong bi-bi kinetic mechanism ascribed to yeast orotate phosphoribosyltransferase (OPRTase) [Victor, J., Greenberg, L. B., and Sloan, D. L. (1979) J. Biol. Chem. 254, 2647-2655] has been shown to be inoperative [Witte, J. F., Tsou, R., and McClard, R. W. (1999) Arch. Biochem. Biophys. 361, 106-112]. Radiolabeled orotidine 5'-phosphate (OMP), generated in situ from [7-(14)C]-orotate and alpha-d-5-phoshorylribose 1-diphosphate (PRPP), binds tightly enough to OPRTase (a dimer composed of identical subunits) that the complex survives gel-filtration chromatography. When a sample of OMP.OPRTase is extensively dialyzed, a 1:1 (per OPRTase dimer) complex is detected by (31)P NMR. Titration of the apoenzyme with OMP yields a (31)P NMR spectrum with peaks for both free and enzyme-bound OMP when OMP is in excess; the complex maintains an OMP/enzyme ratio of 1:1 even when OMP is in substantial excess. A red shift in the UV spectrum of the OMP.OPRTase complex was exploited to measure K(d(OMP)) = 0.84 muM and to verify the 1:1 binding stoichiometry. PRPP forms a Mg(2+)-dependent 1:1 complex with the enzyme as observed by (31)P NMR. Isothermal titration calorimetry (ITC) experiments revealed 1:1 stoichiometries for both OMP and Mg(2+)-PRPP with OPRTase yielding K(d) values of 0.68 and 10 microM, respectively. The binding of either 1 equiv of OMP or PRPP is mutually exclusive. ITC experiments demonstrate that the binding of OMP is largely driven by increased entropy, suggesting substantial distal disordering of the protein. Analytical gel-filtration chromatography confirms that the OMP.OPRTase complex involves the dimeric form of enzyme. The off rate for release of OMP, determined by magnetization inversion transfer, was determined to be 27 s(-)(1). This off rate is somewhat less than the k(cat) in the biosynthetic direction (about 39 s(-)(1)); thus, the release of OMP from OMP.OPRTase may not be kinetically relevant to the steady-state reaction cycle. The body of available data can be explained in terms of alternating site catalysis with either a classical Theorell-Chance mechanism or, far more likely, a novel "double Theorell-Chance" mechanism unique to alternating site catalysis, leading us to propose co-temporal binding of orotate and the release of diphosphate as well as the binding of PRPP and the release of OMP that occur via ternary complexes in alternating site fashion across the two highly cooperative subunits of the enzyme. This novel "double Theorell-Chance" mechanism yields a steady-state rate equation indistinguishable in form from the observed classical ping-pong bi-bi kinetics.     

Genetic complementation and enzyme correlates at the locus encoding the last two steps of de novo pyrimidine biosynthesis in Drosophila melanogaster

Summary Eight mutations of the rudimentary-like (r-l) locus were isolated following mutagenesis with ethylmethanesulfonate and inter se crosses revealed three basic complementation groups, using the wing phenotype as an index of complementation. One group consists of three entirely noncomplementing mutants that each specify severe reductions in levels of both r-l-encoded enzymes, orotate phosphoribosyltransferase (OPR-Tase) and orotidylate decarboxylase (ODCase). The other two groups consist of complementing mutants, such that any member of one group fully complements all members of the other group. One of these groups consists of two mutants that each specify severly reduced OPRTase, but normal ODCase. The other group consists of three mutants that specify severe OPRTase and OD-Case reductions in homoallelic flies, but that appear to contribute OPRTase in certain heteroallelic genotypes. It is concluded that the reciprocal and complementing enzymatic phenotypes of mutants in these two groups account for most instances of genetic trans complementation among r-l mutants. These findings are discussed relative to extant information on OPRTase and OD-Case in animals and an hypothesis is developed that the r-l locus encodes a single polypeptide product that contains both enzyme activities.     

A nonradioactive high-performance liquid chromatographic microassay for uridine 5'-monophosphate synthase, orotate phosphoribosyltransferase, and orotidine 5'-monophosphate decarboxylase.

A novel nonradioactive, microassay method has been developed to determine simultaneously the two enzymatic activities of orotate phosphoribosyltransferase (OPRTase) and orotidine 5'-monophosphate decarboxylase (ODCase), either as a bifunctional protein (uridine 5'-monophosphate synthase, UMPS) or as separate enzymes. Substrates (orotate for OPRTase or orotidine 5'-monophosphate for ODCase) and a product (UMP) of the enzymatic assay were separated by high-performance liquid chromatography (HPLC) using a reversed-phase column and an ion-pairing system; the amount of UMP was quantified by dual-wavelength uv detection at 260 and 278 nm. This HPLC assay can easily detect picomole levels of UMP in enzymatic reactions using low specific activity UMPS of mammalian cell extracts, which is difficult to do with the other nonradioactive assays that have been described. The HPLC assay is suitable for use in protein purification and for kinetic study of these enzymes.     

Characterization of an Autosomal Rudimentary-Shaped Wing Mutation in DROSOPHILA MELANOGASTER That Affects Pyrimidine Synthesis

A new autosomal mutation, rudimental (ral), which causes rudimentary-shaped wings in Drosophila melanogaster, has been isolated following ethyl methanesulfonate (EMS) mutagenesis. The wing phenotype of rudimental is identical to that of the X-linked rudimentary (r) mutation, which affects the first three enzymes in the pyrimidine biosynthetic pathway. The autosomal mutant maps very close to ebony (3–70.7) at 70.42 on the right arm of chromosome 3. Analysis of the enzyme activities of orotate phosphoribosyltransferase (OPRTase) and orotidylate decarboxylase (ODCase) indicates that the ral^a26a allele has less than wild-type activity for both enzymes. This result is discussed in light of the fact that the OPRTase and ODCase activities are part of an enzyme complex, as are the carbamyl phosphate synthetase (CPSase), aspartate transcarbamylase (ATCase) and dihydroorotase (DHOase) activities, which are encoded by the complex rudimentary locus. We suggest that rudimental is also a complex locus.     

Enzymes of de novo pyrimidine biosynthesis in Babesia rodhaini

The pathway of de novo pyrimidine biosynthesis in the rodent parasitic protozoa Babesia rodhaini has been investigated. Specific activities of five of the six enzymes of the pathway were determined: aspartate transcarbamylase (ATCase: E.C. 2.1.3.2); dihydroorotase (DHOase: E.C. 3.5.2.3); dihydroorotate dehydrogenase (DHO-DHase: E.C. 1.3.3.1); orotate phosphoribosyltransferase (OPRTase: E.C. 2.4.2.10); and orotidine-5'-phosphate decarboxylase (ODCase: E.C. 4.1.1.23). Michaelis constants for ATCase, DHO-DHase, OPRTase, and ODCase were determined in whole homogenates. Several substrate analogs were also investigated as inhibitors and inhibitor constants determined. N-(phosphonacetyl)-L-aspartate was shown to be an inhibitor of the ATCase with an apparent Ki of 7 microM. Dihydro-5-azaorotate inhibited the DHO-DHase (Ki, 16 microM) and 5-azaorotate (Ki, 21 microM) was an inhibitor of the OPRTase. The UMP analog, 6-aza-UMP (Ki, 0.3 microM) was a potent inhibitor of ODCase, while lower levels of inhibition were found with the product, UMP (Ki, 120 microM) and the purine nucleotide, XMP (Ki, 95 microM). Additionally, menoctone, a ubiquinone analog, was shown to inhibit DHO-DHase.     

A method for assaying orotate phosphoribosyltransferase and measuring phosphoribosylpyrophosphate

An orotate phosphoribosyltransferase, OPRTase, assay method which relies upon binding reactant [3H]orotic acid and product [3H]orotidine-5'-monophosphate to polyethyleneimine-impregnated-cellulose resin and collecting on a GFC glass fiber filter is presented. Elution with 2 X 5 ml of 0.1 M sodium chloride in 5 mM ammonium acetate removes all of the orotate and leaves all of the product orotidine monophosphate (OMP) bound so that it may be measured in a scintillation counter. It was found that the addition of 10 microM barbituric acid riboside monophosphate to the reaction mixture prevented the conversion of OMP to UMP and products of UMP. The assay is suitable for measurement of OPRTase activity with purified enzyme or in crude homogenates. A modification of this scheme using commercially available yeast OPRTase and 10 microM of unlabeled OMP provides an assay for phosphoribosylpyrophosphate with a sensitivity such that 10 pmol of PRPP may be measured.     

Enzymes of de novo Pyrimidine Biosynthesis in Babesia rodhaini1

ABSTRACT. The pathway of de novo pyrimidine biosynthesis in the rodent parasitic protozoa Babesia rodhaini has been investigated. Specific activities of five of the six enzymes of the pathway were determined: aspartate transcarbamylase (ATCase: E.C. 2.1.3.2): dihydroorotase (DHOase: E.C. 3.5.2.3): dihydroorotate dehydrogenase (DHO-DHase: E.C. 1.3.3.1); orotate phosphoribosyltransferase (OPRTase: E.C. 2.4.2.10); and orotidine-5′-phosphate decarboxylase (ODCase: E.C. 4.1.1.23). Michaelis constants for ATCase, DHO-DHasc. OPRTase, and ODCase were determined in whole homogenates. Several substrate analogs were also investigated as inhibitors and inhibitor constants determined. N-(phosphonacetyl)-L-aspartate was shown to be an inhibitor of the ATCase with an apparent K, of 7μM. Dihydro-5-azaorotate inhibited the DHO-DHase (K, 16 μM) and 5-azaorotate (K_i, 21 μM) was an inhibitor of the OPRTase. The UMP analog, 6-aza-UMP (K_i, 0.3 μM) was a potent inhibitor of ODCase, while lower levels of inhibition were found with the product. UMP (K_i, 120 μM) and the purine nucleotide, XMP (K₁, 95 μM). Additionally, menoctone, a ubiquinone analog, was shown to inhibit DHO-DHase.     

Hydrogen isotope tracing in the reaction of orotidine-5'-monophosphate decarboxylase

The mechanism of the enzyme orotidine-5(')-monophosphate decarboxylase (OMP decarboxylase, ODCase) is not fully characterized; some of the proposed mechanisms suggest the possibility of hydrogen rearrangement (shift from C5 to C6 or loss of H5 to solvent) during catalysis. In this study, we sought mechanistic information for the ODCase reaction by examining the extent of hydrogen exchange in the product uridine-5(')-monophosphate, in combination with ODCase, at the H5 and H6 positions. In a subsequent experiment, partially deuterated OMP was prepared, and the extent of 2H5 rearrangement or loss to solvent was examined by integration of 1H nuclear magnetic resonance signals in the substrate and the resulting enzymatically decarboxylated product. The absence of detectable hydrogen exchange in these experiments limits somewhat the possible mechanisms for ODCase catalysis.     

Orotidine Monophosphate Decarboxylase——A Fascinating Workhorse Enzyme with Therapeutic Potential

Orotidine 50-monophosphate decarboxylase(ODCase) is known as one of the most proficient enzymes. The enzyme catalyzes the last reaction step of the de novo pyrimidine biosynthesis, the conversion from orotidine 50-monophosphate(OMP) to uridine 50-monophosphate. The enzyme is found in all three domains of life, Bacteria, Eukarya and Archaea. Multiple sequence alignment of 750 putative ODCase sequences resulted in five distinct groups. While the universally conserved Dx Kxx Dx motif is present in all the groups,depending on the groups, several characteristic motifs and residues can be identified. Over 200 crystal structures of ODCases have been determined so far. The structures, together with biochemical assays and computational studies, elucidated that ODCase utilized both transition state stabilization and substrate distortion to accelerate the decarboxylation of its natural substrate. Stabilization of the vinyl anion intermediate by a conserved lysine residue at the catalytic site is considered the largest contributing factor to catalysis, while bending of the carboxyl group from the plane of the aromatic pyrimidine ring of OMP accounts for substrate distortion. A number of crystal structures of ODCases complexed with potential drug candidate molecules have also been determined, including with 6-iodouridine, a potential antimalarial agent.     

No metal cofactor in orotidine 5'-monophosphate decarboxylase.

Orotidine 5'-monophosphate decarboxylase (OMP decarboxylase, ODCase) is an important enzyme that catalyzes the final step of de novo pyrimidine nucleotide biosynthesis. The mechanism of this unique enzyme and whether metal ions play any role in catalysis have been topics of intense research interest. In this report, the role of Zn in ODCase was reexamined. Atomic absorption (AA) and X-ray absorption (XAS) spectroscopic studies did not detect zinc in active enzyme samples at high concentration. The XAS results also indicated the absence of other transition metal ions in ODCase.     

Significance of the enzyme complex that synthesizes UMP in ehrlich ascites cells

The de novo biosynthesis of pyrimidine nucleotides is completed by two sequential enzyme activities that convert orotate plus 5-phosphoribosyl-1-pyrophosphate to orotidine-5′-monophosphate (OMP) and PP_i and then decarboxylate OMP to produce 5′-uridylic acid. In mammalian cells the two enzyme activities, orotate phosphoribosyltransferase and orotidine-5′-phosphate decarboxylase, form a normally inseparable enzyme complex. It was previously reported that this complex is able to channel the intermediate product, OMP (Traut, T. W., and Jones, M. E., 1977, J. Biol. Chem.252, 8374–8381). The studies reported here indicate that one advantage of this channeling of OMP is to spare OMP from being degraded to orotidine by a potentially competitive nucleotidase activity. Yeast cells have two separate enzymes instead of an enzyme complex, and lack the ability to channel OMP. The OMP formed in yeast cells is not degraded because these cells lack significant nucleotidase activity. These results suggest that the capability for channeling OMP may have been important in evolving the enzyme complex found in mammalian cells.     

Ternary complex formation and induced asymmetry in orotate phosphoribosyltransferase

Orotate phosphoribosyltransferase (OPRTase, EC 2.4.2.10) catalyzes the Mg2+-dependent condensation of orotic acid (OA) with PRPP (5-alpha-d-phosphorylribose 1-diphosphate) to yield diphosphate (PPi) and the nucleotide OMP (orotidine 5'-monophosphate). We have determined the structures of three forms of Saccharomyces cerevisiae OPRTase representing different structural and enzymatic intermediates. The structures include the apoenzyme (2.35 A resolution); a ternary complex of enzyme, Mg2+-PRPP, and OA (1.74 A resolution); and the binary product complex of enzyme with OMP (1.89 A resolution). While the overall structure of the S. cerevisiae OPRTase is similar to that of the Salmonella typhimurium enzyme, as judged by comparison of the two apoenzymes, large conformational transitions occur proceeding from the apoenzyme structure to those of the substrate and product complexes. Comparison of these structures reveals a rotation of the upper hood domain onto the bound ligands by an average of 19.5 degrees in the OMP structure and an average of 24.6 degrees in the OA/Mg2+-PRPP ternary complex. As expected, the conserved loop, composed of residues 104-116, moves extensively and adopts a single stable conformation during the catalytic cycle in order to sequester the substrates from bulk solvent in the ternary complex. The OA and Mg2+-PRPP molecules bound in the ternary complex are oriented for proper attack of the N1 atom of OA onto the C1 atom of the ribose ring. This orientation of substrates, combined with the positioning of the flexible loop, provides a clear picture of a catalytically poised reaction complex for type I phosphoribosyltransferases. The structural asymmetry present in these structures, as well as that found in a recent structure of the S. typhimurium enzyme, combined with the closure of the flexible loop from one subunit into the active site of the opposing subunit in the ternary complex is consistent with the kinetic data [McClard, R. W., et al. (2006) Biochemistry 45, 5330-5342] that demonstrate induced nonequivalence and cooperativity of OPRTase.     

Function of arginine-166 in the active site of Escherichia coli alkaline phosphatase

The function of arginine residue 166 in the active site of Escherichia coli alkaline phosphatase was investigated by site-directed mutagenesis. Two mutant versions of alkaline phosphatase, with either serine or alanine in the place of arginine at position 166, were generated by using a specially constructed M13 phage carrying the wild-type phoA gene. The mutant enzymes with serine and alanine at position 166 have very similar kinetic properties. Under conditions of no external phosphate acceptor, the kcat for the mutant enzymes decreases by approximately 30-fold while the Km increases by less than 2-fold. When kinetic measurements are carried out in the presence of a phosphate acceptor, 1.0 M Tris, the kcat for the mutant enzymes is reduced by less than 3-fold, while the Km increases by more than 50-fold. For both mutant enzymes, in either the absence or the presence of a phosphate acceptor, the catalytic efficiency as measured by the kcat/Km ratio decreases by approximately 50-fold as compared to the wild type. Measurements of the Ki for inorganic phosphate show an increase of approximately 50-fold for both mutants. Phenylglyoxal, which inactivates the wild-type enzyme, does not inactivate the Arg-166----Ala enzyme. This result indicates that Arg-166 is the same arginine residue that when chemically modified causes loss of activity [Daemen, F.J.M., & Riordan, J.F. (1974) Biochemistry 13, 2865-2871]. The data reported here suggest that although Arg-166 is important for activity is not essential. The analysis of the kinetic data also suggests that the loss of arginine-166 at the active site of alkaline phosphatase has two different effects on the enzyme. First, the binding of the substrate, and phosphate as a competitive inhibitor, is reduced; second, the rate of hydrolysis of the covalent phosphoenzyme may be diminished.     

The effective molarity of the substrate phosphoryl group in the transition state for yeast OMP decarboxylase

The second order rate constant (k(cat)/K(m)) for decarboxylation of orotidine by yeast OMP decarboxylase (ODCase), measured by trapping (14)CO(2) released during the reaction, is 2 x 10(-4)M(-1)s(-1). This very low activity may be compared with a value of 3 x 10(7)M(-1)s(-1) for the action of yeast OMP decarboxylase on the normal substrate OMP. Both activities are strongly inhibited by 6-hydroxy UMP (BMP), and abrogated by mutation of Asp-96 to alanine. These results, in conjunction with the binding affinity of inorganic phosphate as a competitive inhibitor (K(i)=7 x 10(-4)M), imply an effective concentration of 1.1 x 10(9)M for the substrate phosphoryl group in stabilizing the transition state for enzymatic decarboxylation of OMP. The observed difference in rate (1.5 x 10(11)-fold) is the largest effect of a simple substituent that appears to have been reported for an enzyme reaction.     

Purification of the putative hormone-sensitive cyclic AMP phosphodiesterase from rat adipose tissue using a derivative of cilostamide as a novel affinity ligand

A "low Km" cAMP phosphodiesterase with properties of a peripheral membrane protein accounts for approximately 90% of total cAMP phosphodiesterase activity in particulate (100,000 X g) fractions from rat fat cells. Incubation of fat cells with insulin for 10 min increased particulate (but not soluble) cAMP phosphodiesterase activity, with a maximum increase (approximately 100%) at 1 nM insulin. Most of the increase in activity was retained after solubilization (with non-ionic detergent and NaBr) and partial purification (approximately 20-fold) on DEAE-Sephacel. The solubilized enzyme from adipose tissue was purified approximately 65,000-fold to apparent homogeneity (yield approximately 20%) by chromatography on DEAE-Sephacel and Sephadex G-200 and affinity chromatography on aminoethyl agarose conjugated with the N-(2-isothiocyanato)ethyl derivative of the phosphodiesterase inhibitor cilostamide (OPC 3689). A 63,800 +/- 200-Da polypeptide (accounting for greater than 90% of the protein eluted from the affinity column) was identified by polyacrylamide gel electrophoresis in sodium dodecyl sulfate (with or without reduction). Enzyme activity was associated with the single protein band after electrophoresis under nondenaturing conditions. On gel permeation, Mr(app) was 100,000-110,000, suggesting that the holoenzyme is a dimer. A pI of 4.9-5.0 was estimated by isoelectric focusing. At 30 degrees C, the purified enzyme hydrolyzed both cAMP and cGMP with normal Michaelis-Menten kinetics; the pH optimum was 7.5. The Km(app) for cAMP was 0.38 microM and Vmax, 8.5 mumol/min/mg; for cGMP, Km(app) was 0.28 microM and Vmax, 2.0 mumol/min/mg. cGMP competitively inhibited cAMP hydrolysis with a Ki of approximately 0.15 microM. The enzyme was also inhibited by several OPC derivatives and "cardiotonic" drugs, but not by RO 20-1724. It was very sensitive to inhibition by agents which covalently modify protein sulfhydryls, but not by diisopropyl fluorophosphate. The activation by insulin and other findings indicate that the purified enzyme, which seems to belong to a subtype of low Km cAMP phosphodiesterases that is specifically and potently inhibited by cGMP, cilostamide, other OPC derivatives, and certain cardiotonic drugs, is likely to account for the hormone-sensitive particulate low Km cAMP phosphodiesterase activity of rat adipocytes.     

Purification and characterization of a pyridine nucleotide glycohydrolase from rabbit spleen

A particulate NMN glycohydrolase of rabbit spleen was solubilized with Triton X100 and purified approximately 100-fold. The enzyme was shown to have a pH maximum of 6.5, a Km of 0.25 mM, a Vmax of 5.3 mumol/min/mg protein, an activation energy of 7.9 kcal/mol, and a molecular weight of approximately 400,000. Both of the purified and the particulate enzymes exhibited identical catalytic properties with respect to substrate specificity, activation energy, pH profile and exchange reaction with nicotinic acid, except that the purified enzyme was highly activated with Triton X100 as compared with the particulate enzyme; it appears that the purified enzyme possesses the same catalytic properties as the enzyme present in the tissue and that solubilization does not significantly alter the native protein. In addition to catalytic activity with NMN, the rabbit spleen enzyme catalyzed an irreversible hydrolysis with NAD and NADP, exhibiting catalyzing activity ratios of NMN:NAD:NADP = 1.00:1.45:0.44 and Vmax/Km ratios of 1.00:1.7:2.3, respectively. These ratios of activity remained constant throughout purification of the enzyme and no separation of these activities was detected. Mutually competitive inhibition of the enzyme with Ki values similar to Km, and identical rates of thermal denaturation of the enzyme and activity-pH profiles with NMN or NAD indicated the hydrolysis of the C-N glycosidic linkage of the pyridine nucleotides to be catalyzed by the same enzyme. The enzyme was less specific for the purine structure of the substrate dinucleotides but was stereospecific for the glycosidic linkage cleaved. Nicotinamide riboside, the nicotinic acid analogs and the reduced forms were not hydrolyzed. A linear noncompetitive inhibition of NMN hydrolysis with nicotinamide indicated an ordered Uni-Bi mechanism in which nicotinamide was the first product released from the enzyme. A property that the rabbit spleen enzyme appears to share with other NAD glycohydrolases is the transglycosidation reaction. The ratio of transglycosidation reaction vs. hydrolysis catalyzed by the enzyme in the presence of NMN and nicotinic acid indicated that the enzyme could function as a primary transglycosidase rather than a hydrolytic enzyme in vivo.     

Pyrimidine nucleotide synthesis in the rat kidney in early diabetes.

Early renal hypertrophy of diabetes is associated with increases in the tissue content of RNA, DNA, and sugar nucleotides involved in the formation of carbohydrate-containing macromolecules. We have previously reported an increase in the activity of enzymes of the de novo and salvage pathways of purine synthesis in early diabetes; the present communication explores the changes in the pathways of pyrimidine synthesis. Measurements have been made of key enzymes of the de novo and salvage pathways at 3, 5, and 14 days after induction of diabetes with streptozotocin (STZ), phosphoribosyl pyrophosphate (PPRibP), and some purine and pyrimidine bases. Carbamoyl-phosphate synthetase II, the rate-limiting enzyme of the de novo route, did not increase in the first 5 days after STZ treatment, the period of most rapid renal growth; a significant rise was seen at 14 days (+38%). Dihydroorotate dehydrogenase, a mitochondrial enzyme, showed the most marked rise (+147%) at 14 days. The conversion of orotate to UMP, catalyzed by the enzymes of complex II, was increased at 3 days (+42%), a rise sustained to 14 days. The salvage route enzyme, uracil phosphoribosyltransferase (UPRTase), showed a pattern of change similar to complex II. The effect of the decreased concentration of PPRibP on the activities of CPSII, for which it is an allosteric activator, and on activities of OPRTase and UPRTase, for which it is an essential substrate, is discussed with respect to the relative Ka and Km values for PPRibP and the possibility of metabolite channeling.     

gamma-Glutamyltranspeptidase in dimethylbenz[a]anthracene-induced mammary adenocarcinomas and in livers of tumor bearing rats

A single dose of dimethylbenz[a]anthracene (DMBA) at 20 mg/kg resulted in 100% incidence of intraductal mammary adenocarcinomas in Wistar rats, the large tumors averaging 1.87 +/- 0.45 g. gamma-Glutamyltranspeptidase activities were elevated in DMBA-induced mammary adenocarcinomas relative to lactating mammary tissue in all fractions examined: 18.8-fold in homogenates; 22.1-fold in particulate fractions; and 5.7-fold in supernatant fractions. In DMBA-induced mammary adenocarcinomas, gamma-glutamyltranspeptidase was 95% particulate, 5% supernatant, whereas in lactating mammary tissue, gamma-glutamyltranspeptidase was equally distributed between particulate and supernatant fractions. Particulate gamma-glutamyltranspeptidase from DMBA-induced mammary adenocarcinomas as well as lactating mammary tissue displayed classical Michaelis-Menten characteristics: for the adenocarcinoma enzyme Km was 2.5 nM and Vmax 200 nmol mg-1 min-1; for mammary tissue enzyme Km was 2.5 nM and Vmax 11.1 nmol X mg-1 X min-1. Both particulate enzymes were activated at 50 degrees C relative to 37 degrees C to the same extent: 1.37-fold. The activities of gamma-glutamyltranspeptidase were increased 1.8-fold in the livers of rats bearing DMBA-induced mammary adenocarcinomas relative to age-matched controls. Plasma levels of gamma-glutamyltranspeptidase were also increased 1.6-fold in tumor bearing rats. There was no observable sign of liver damage in tumor bearing rats; plasma glutamic pyruvic transaminase levels were normal in these animals. Blood glucose levels were elevated 17% in rats bearing DMBA-induced mammary adenocarcinomas compared to age-matched controls, although plasma insulin levels were the same in both groups: 35.4 +/- 3.5 microIU/ml for the former; 31.9 +/- 3.1 microIU/ml for the latter.     

Purification and characterization of yeast orotidine 5'-monophosphate decarboxylase overexpressed from plasmid PGU2

Orotidine 5'-monophosphate decarboxylase (ODCase) has been overexpressed in yeast 15C cells transformed with a plasmid carrying the URA3 gene that encodes ODCase. Twenty g of cells having ODCase activity equal to 30 mg of pure enzyme per liter of cell culture were obtained after 9 h of galactose induction. To remove yeast proteases, a 60-90% ammonium sulfate fractionation step plus the addition of EDTA as an inhibitor of metallopeptidases was necessary. The purification protocol yielded ODCase that was protease-free and stable to storage at 4 degrees C for 16 months. The pure enzyme had a specific activity of 40 units/mg in 50 mM phosphate buffer, pH 6, and could be stored at -20 degrees C in 20% glycerol with retention of full activity for more than 2 years. The enzyme had a Km for orotidine 5'-monophosphate of 0.7 microM at pH 6 and 25 degrees C. The molecular weight of the plasmid-derived ODCase monomer determined by electrophoresis on denaturing polyacrylamide gels was 29,500. ODCase sedimented through sucrose density gradients as a monomer of about 30 kDa at low protein concentration and in the absence of ligands that bind at the catalytic site. An increase in the sedimentation rate could be induced by increasing the ODCase concentration or by adding ligands that are competitive inhibitors. ODCase sedimented in a single band typical of a protein of 46 kDa at the highest protein concentration studied or in the presence of 50 mM phosphate or 933 microM substrate (orotidine 5'-monophosphate) or product (UMP). A dimer sedimenting as a protein of about 64 kDa occurred in the presence of 50 microM 6-azauridine 5'-monophosphate or 2 microM 1-(5'-phospho-beta-D-ribofuranosyl) barbituric acid, competitive inhibitors of ODCase. These results resemble the ligand-induced subunit association of the ODCase domain of bifunctional UMP synthase and support the use of yeast ODCase as a model for ODCases from other species.