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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.

The oxidation of proline to glutamate in mitochondria requires two enzymes, proline oxidase and pyrroline 5-carboxylate (P5C) dehydrogenase. In this paper we report an 800-fold purification P5C dehydrogenase from rat liver mitochondria to yield an essentially homogenous protein. The protein, whose Mr is 59,000, is an alpha 2 dimer (Mr = 115,000) in solution with an isoionic point at pH 5.7. The substrates P5C and NAD+ have apparent dissociation constants of 0.16 and 1.0 mM, respectively. Studies have been conducted to see if the conversion of glutamate and NADH to P5C and NAD+ is catalyzed by this enzyme. These studies have established that if the reverse reaction occurs the rate is 1/15,000th of the rate at which P5C is oxidized to glutamate. The concentration of the substrates needed in the assay results in a high background that interferes with accurate spectrophotometric analysis of the rate of NADH production; therefore a radiochemical (2) or a new colorimetric (3) assay was used here. A number of aldehydes were tested as substrates. It was found that the rat and human enzymes (4) have similar requirements for an aldehyde to be a substrate. Both of these proteins interacted with a polyclonal rabbit anti-rat P5C dehydrogenase serum.

Orotic aciduria (type 1) results from a mutation in the gene for UMP synthase, a bifunctional protein containing the two enzyme activities which convert orotic acid and 5-phosphoribosyl-1-pyrophosphate to UMP and CO2. In fibroblasts from individuals with orotic aciduria, these two enzymatic activities are about 1% of normal but increase dramatically when deficient cells are grown in the presence of 6-azauridine. Using a polyclonal antiserum to sodium dodecyl sulfate-denatured, pure human UMP synthase, we show that fibroblasts from a patient with orotic aciduria have a low level of immunoreactive UMP synthase protein. Pulse-chase analysis reveals that the UMP synthase is degraded rapidly in the deficient cells. Growth of deficient cells in 6-azauridine leads to an increase in UMP synthase protein and its two enzymatic activities via a decreased rate of proteolytic degradation of UMP synthase. UMP synthase in extracts from deficient cells is more readily denatured by heat and is stabilized after growth of cells in 6-azauridine. These data suggest that the detrimental deficiency of this one patient results from a structurally altered UMP synthase that is probably present in low steady-state amounts due to proteolysis and that this labile protein can be stabilized against heat denaturation and proteolytic degradation by 6-aza-UMP.

Orotidine-5'-monophosphate decarboxylase (OD-Case) catalyzes the conversion of orotidine 5'-monophosphate to UMP. In mammals, ODCase is present as part of a bifunctional protein which also contains orotate phosphoribosyltransferase; the preceding enzyme in the de novo UMP biosynthetic pathway. We have isolated a plasmid (pMEJ) which contains a cDNA for the ODCase domain of UMP synthase. Insertion of this sequence into an Escherichia coli expression vector (pUC12) has allowed for the expression of ODCase and not orotate phosphoribosyltransferase in E. coli. The molecular weight of the expressed protein is 26,000-27,300 from immunoblot analysis which corresponds closely to the molecular weight of the ODCase domain (28,500) isolated by tryptic digestion of UMP synthase. We have sequenced the cDNA insert of pMEJ and deduced the amino acid sequence. The molecular weight of the ODCase domain calculated from the amino acid sequence in 28,654. Comparison of the deduced amino acid sequence from pMEJ with that for yeast ODCase (a monofunctional protein) demonstrated that 52% of the amino acids were identical when the two sequences are compared. Furthermore, several stretches of the amino acid sequence have 80% or greater absolute homology.

Effective repair of a vascular injury depends on establishment of a stable fibrin patch at the injury site. Data presented in this study demonstrate that structural modification of fibrin occurs as a result of fibrin interaction with naturally occurring components of the vascular basement membrane and subendothelial structures. Of the basement membrane components, type IV collagen produces the greatest structural modification, generating thick fibrin fibers; a 3-fold increase in the fiber mass/length ratio occurs when type IV collagen is increased from 0 to 100 ng/ml. Laminin and dermatan sulfate decrease the fibrin fiber mass/length ratio resulting in thinner fibers. However, the overall effect of the basement membrane on fibrin is to increase the fibrin fiber diameter. Electrophoretic light scattering and the binding of type IV collagen by fibrinogen-Sepharose further establish the interaction between type IV collagen and fibrinogen. Incorporation of laminin with type IV collagen onto coated surfaces decreases the ability of type IV collagen to bind fibrinogen. These studies emphasize that the final fibrin structure is influenced by the milieu in which the clot is assembled.

The demonstration of the ornithine biosynthesis from glutamate in cell-free homogenates of rat intestinal mucosa by Ross, G., Dunn, D., and Jones, M.E. (1978) Biochem. Biophys. Res. Commun. 85, 140-147 suggested that this tissue might have the capacity to convert glutamate to pyrroline-5-carboxylate (P5C). We have shown in the preceding paper (Wakabayashi, Y., and Jones, M.E. (1983) J. Biol. Chem. 258, 3865-3872) that this is the case. The intracellular distribution of the P5C-synthesizing activity was investigated utilizing a newly developed procedure for subcellular fractionation of the rat intestinal mucosa. We found that the activity resided in the mitochondrial fraction as characterized by marker enzymes and an electron micrograph. The mitochondrial membrane fraction, freed of the soluble matrix and intermembrane space enzymes, retained all of the P5C-synthesizing activity. Addition of the soluble fraction to the membrane fraction did not affect the activity. P5C synthase, the name we have chosen for the protein(s) that catalyzes P5C synthesis from glutamate when ATP and NADPH are present, is susceptible to thermal inactivation in the presence of detergent. By lowering the incubation temperature to or below 20 degrees C, one can obtain a linear production of P5C with respect to time and protein concentration. Lower incubation temperatures are recommended for routine assay of this enzyme(s). Addition of 30% glycerol to the incubation mixture resulted in a linear formation of P5C with time at 30 degrees C; this and other data suggest that polyhydroxylic compounds may protect this protein against denaturation. Preliminary experiments suggest that P5C synthase can be extracted from a mitochondrial membrane in the presence of detergent, a high salt concentration, and glycerol. The possibility that the enzyme(s) is located in the inner mitochondrial membrane is discussed.

In mammals, the bifunctional protein UMP synthase contains the final two enzymatic activities, orotate phosphoribosyltransferase and orotidine-5'-monophosphate decarboxylase (ODCase), for de novo biosynthesis of UMP. The plasmid pMEJ contains a cDNA for the ODCase domain of mouse Ehrlich ascites UMP synthase. The cDNA from pMEJ was joined to the Saccharomyces cerevisiae iso-1-cytochrome c (CYC1) promoter and the first four CYC1 coding nucleotides in the plasmid pODCcyc. ODCase-deficient yeast cells (HF200x1) transformed with pODCcyc expressed an active ODCase domain with a specific activity of 20 nmol/min/mg in cell extracts. The expressed ODCase domain has a lower affinity for the substrate orotidine 5'-monophosphate and the inhibitor 6-azauridine 5'-monophosphate than intact UMP synthase or an ODCase domain isolated after proteolysis of homogenous UMP synthase. Sucrose density gradient sedimentation experiments showed that the expressed ODCase domain forms a dimer in the presence of ligands which bind at the catalytic site. These studies support the existence of an ODCase structural domain which contains the ODCase catalytic site and a dimerization surface of UMP synthase, but the domain may not have the regulatory site required to form the altered dimer form.

Orotate phosphoribosyltransferase (EC 2.4.2.10) and orotidine 5'-monophosphate decarboxylase (EC 4.1.1.23) are the final two of six enzymatic steps required in the de novo biosynthesis of uridine 5'-monophosphate (UMP). Earlier work of this laboratory showed that, in the mouse Ehrlich ascites carcinoma, both of these enzymatic activities were contained on the single multifunctional polypeptide chain, UMP synthase. We report here that the placenta provided an available human source for UMP synthase with 40-fold higher orotate phosphoribosyltransferase and orotidine 5'-monophosphate decarboxylase specific activities than erythrocytes, a human source previously used by others. By using the placenta as a source of UMP synthase and by developing a novel purification procedure different from that used in the purification of UMP synthase from the Ehrlich ascites carcinoma (the only other homogeneous preparation of a mammalian UMP synthase), we achieved the purification of human UMP synthase to apparent homogeneity. This represents the first publication to homogeneity of UMP synthase from a human source as well as from a source other than malignant cell lines. Contrary to earlier reports human placental UMP synthase was found to be a multifunctional protein containing both enzymatic activities on a single polypeptide of 51,000 molecular weight. Preliminary characterization of the human placental UMP synthase showed it to be similar to UMP synthase from the Ehrlich ascites carcinoma in subunit molecular weight, native molecular weight, isozyme pattern (although not absolute pI values), pH optima of enzymatic activities, and kinetic constants for orotidine 5'-monophosphate (Km) and 6-azauridine 5'-monophosphate (Ki) at the decarboxylase site.

SUMMARY During short incubation in vitro of intact Ehrlich ascites cells, 14C-bicarbonate was incorporat,ed into carbon atom 2 of the uracil ring derived from intracellular, acid-soluble uridine nu- cleotides. The incorporation was almost completely inhibited by erotic acid added to t,he medium, with accumulation of radio- activity in carbamylaspartate, thus confirming that bicarbonate incorporation into uridine nucleotides occurs by erotic acid pathway in these cells. The incorporation was increased by glucose and glutamine or ammonia. Glutamine produced maximum incorporation at much lower external concentrations than ammonium chloride. The incorporation was inhibited by the glutamine analogues, 0-carbamyl-n-serine and 6-diazo-5-oxo-r-norleucine. The effectiveness of these analogues and azaserine in inhibit- ing pyrimidine synthesis in Ehrlich ascites cells was compared with their effectiveness in inhibiting the glutamine-dependent carbamyl phosphate synthetase of crude extracts Escherichia coli. The dat,a suggest, and are consistent with, a synthesis of carbamyl phosphate in the Ehrlich ascites cell by an enzyme requiring bicarbonate and glutamine and the subsequent incor- poration of this carbamyl phosphate into uridine nucleotides by the erotic acid pathway. dcknowledgments-The

Aspartate transcarbamylase from a Pseudomonas fluorescens has been purified to homogeneity. The enzyme has a molecular weight of 360,000. It is composed of 2 apparently identical subunits with molecular weights of 180,000. Activity can be recovered from the enzyme denatured with mercaptoethanol and sodium dodecyl sulfate when the dodecyl sulfate is dialyzed away. The renatured enzyme has the molecular weight of the native enzyme. Carbamyl phosphate synthetase activity is lost continuously during purification, so that it is unlikely that this bacterium possesses a carbamyl phosphate synthetase-aspartate transcarbamylase complex such as is found in Neurospora and yeast. The kinetic and regulatory properties of aspartate transcarbamylase from P. fluorescens were also studied. The substrate saturation curves in the absence and presence of all inhibitors tested are hyperbolic. With limiting carbamyl phosphate, all trinucleotides tested, and CDP, pyrophosphate, and orthophosphate inhibit competitively at micromolar concentrations. When carbamyl phosphate is saturating a second binding site for the nucleotides is evident, but pyrophosphate and orthophosphate no longer inhibit. At millimolar concentrations, the nucleotides are noncompetitive inhibitors with aspartate. The function of this regulatory site cannot be separated from the catalytic site by mild heat treatment.

The carbamyl phosphate synthetase of frog liver particles catalyzes the synthesis of 1 mole of carbamyl phosphate, 1 mole of orthophosphate, and 2 moles of adenosine diphosphate from ammonia, bicarbonate, and 2 moles of ATP. It can produce orthophosphate from ATP if ammonia is replaced by either hydroxylamine or hydrazine. Both hydroxylamine and hydrazine are competitive inhibitors of carbamyl phosphate synthesis and, therefore, presumably replace ammonia or decompose the activated carbonate which is a precursor of carbamyl phosphate. Hydrazine replaces ammonia because it is incorporated into a labile phosphate which appears to be N-aminocarbamyl phosphate. It can be shown that the new derivative contains hydrazine, 14C from 14C-bicarbonate, and phosphate, the latter two in equal amounts. N-Aminocarbamyl phosphate migrates more slowly toward the anode at pH 6 in an electrical field than carbamyl phosphate. It is a markedly more acid-labile and base-stable phosphate than carbamyl phosphate. N-Aminocarbamyl phosphate cannot serve as a substrate for ornithine transcarbamylase. Hydroxylamine, however, does not appear to yield a carbamyl phosphate or hydroxylamine phosphate. It can be shown that hydroxylamine can act catalytically, and it is therefore suggested that it decomposes the activated carbonate which is the product of the first mole of ATP utilized by the enzyme. If 1 or 2 of the hydrogen atoms of hydrazine or hydroxylamine are replaced with methyl groups, the derivatives are not substrates. Glutamine will not serve as a substrate.

Experiments with the soluble supernatant fraction of fetal rat liver indicate that it contains a glutamine-dependent carbamyl phosphate synthetase. This enzyme can utilize ammonia or glutamine as substrate, but the Km for glutamine is low (approximately 10-5 m) while the Km for ammonium ion-ammonia is at least 100 times higher. The ATP saturation curve for the enzyme appears to be sigmoidal. The pH optimum is 7.4. Glutamine analogues inhibit the ability of either glutamine or ammonia to serve as substrate. Comparisons of the change in the amounts of the glutamine-dependent carbamyl phosphate synthetase and aspartate transcarbamylase in the soluble fraction of the cell and in the amounts of acetylglutamate-dependent carbamyl phosphate synthetase and ornithine transcarbamylase in the particulate fraction of the cell with development from 4 days prior to gestation to 2 days after birth are reported. They suggest that the new enzyme provides carbamyl phosphate for the pyrimidine pathway. The same enzyme activity has also been found in the soluble fraction of a pigeon liver homogenate.