Philip Reyes

Properties and substrate specificity of a purine phosphoribosyltransferase from the human malaria parasite, Plasmodium falciparum.

The properties of a purine phosphoribosyltransferase from late trophozoites of the human malaria parasite, Plasmodium falciparum, are described. Enzyme activity with hypoxanthine, guanine and xanthine as substrates eluted in parallel during hydroxylapatite, size exclusion and DEAE-Sephadex chromatography as well as during chromatofocusing experiments. Furthermore, enzyme activity with all three purine substrates changed in parallel during heat inactivation of enzyme preparations and upon cold storage (4 degrees C) of the enzyme. When considered together, these results support the view that the phosphoribosyltransferase is capable of utilizing all three purine bases as substrates. Additional characterization revealed that the apparent molecular weight and isoelectric point of this enzyme are 55,500 and 6.2, respectively, and that the apparent Km for 5-phosphoribosyl-1-pyrophosphate ranges from 13.3 to 21.4 microM, depending on the purine base serving as substrate. The apparent Km values for hypoxanthine, guanine and xanthine were found to be 0.46, 0.30 and 29 microM, respectively. Other experiments showed that several divalent cations and sulfhydryl reagents produce a marked reduction of enzyme activity whereas dithiothreitol activates the enzyme. It should be noted that the ability to utilize xanthine as a substrate serves to distinguish the P. falciparum enzyme from its counterpart in the parasites host cell, the human erythrocyte. The human enzyme shows only barely detectable activity with xanthine while the parasite enzyme displays similarly high levels of activity with all three purine substrates. Thus, the parasite enzyme might prove to be selectively susceptible to inhibition by xanthine analogs and related compounds.

PEI-cellulose thin-layer chromatography: A highly versatile system for separating purine and pyrimidine nucleotides from nucleosides and free bases☆

Abstract A simple, rapid, and highly versatile thin-layer system is described for separating purine and pyrimidine nucleotides from their respective nucleosides and free bases. This system, which employs anion-exchange chromatography on PEI-cellulose, retains both ribonucleotides and deoxyribonucleotides at the origin and permits ribonucleosides, deoxyribonucleosides, and free bases to migrate away. Distilled water serves as the developing solvent. The ability to retain nucleotides at the origin makes PEI-cellulose ideally suited for use in the assay of nucleotide synthesizing enzymes. These enzymes include purine phosphoribosyl-transferases, purine nucleoside kinases, and pyrimidine nucleoside kinases.

A new and highly sensitive radiotracer assay for orotate phosphoribosyltransferase

Abstract A new radiotracer assay is described for the measurement of orotate phosphoribosyltransferase. The method is based upon the separation of unreacted orotate from the newly synthesized radioactive product (orotidine 5′-phosphate). This is accomplished by thin-layer chromatography of reaction mixtures on polyethyleneimine (PEI)-cellulose using a borate-containing solvent system. Under these conditions, the radioactive product remains at the origin. Several advantages of this assay over previously available methods include its simplicity, high sensitivity, and versatility. In the latter regard, the present PEI-cellulose system is capable of measuring not only orotate but also uracil and 5-fluorouracil phosphoribosyltransferase activity. That is, both UMP and 5-fluoro-UMP are retained at the origin whereas unreacted substrates, uracil and 5-fluorouracil, respectively, are removed upon thin-layer chromatography on the above system. Unlike earlier methods, the addition of orotidine-5′-phosphate decarboxylase is not required, although the presence of endogenous orotidine-5′-phosphate decarboxylase does not interfere with the assay of orotate phosphoribosyltransferase. The high sensitivity of the present assay (0.1 nmole of product is easily detected) permits its use with crude enzyme extracts.

Characterization of adenine phosphoribosyltransferase from the human malaria parasite, Plasmodium falciparum

Because of their inability to synthesize purines de novo, malaria parasites rely on purine phosphoribosyltransferases (PRTases) to convert purine bases salvaged from the host cell (the erythrocyte) into the corresponding purine nucleoside monophosphates. Our studies with late trophozoites of the human malaria parasite, Plasmodium falciparum, showed that virtually all of the purine PRTase activity is accounted for by two distinct enzymes. One enzyme utilizes hypoxanthine, guanine and xanthine (Queen, S.A., Vander Jagt, D. and Reyes, P. (1988) Mol. Biochem. Parasitol. 30, 123-134). The second enzyme utilizes only adenine and is the subject of this paper. This latter enzyme exhibits a biphasic pH-activity profile and is moderately to weakly inhibited by several divalent metal ions. Several of the properties of the P. falciparum enzyme were found to differ significantly from those of human erythrocyte adenine PRTase. (1) The molecular weight (18,000) of the parasite enzyme is smaller than that of the host cell enzyme. (2) The parasite enzyme, unlike the erythrocyte enzyme, is not significantly inhibited by sulfhydryl reagents. (3) 6-Mercaptopurine and 2,6-diaminopurine proved to be competitive inhibitors of the parasite enzyme (Ki 0.70 and 1.0 mM, respectively); on the other hand, the human enzyme is not inhibited by these agents. (4) The Km for adenine (0.80 microM) and 5-phosphoribosyl-1-pyrophosphate (0.70 microM) displayed by the parasite enzyme are significantly smaller than the corresponding Km values shown by the erythrocyte enzyme. These distinctions between the parasite and host enzymes point to the possibility that adenine PRTase of P. falciparum may represent a potential target for chemotherapeutic attack.

Coordinate behavior of orotate phosphoribosyltransferase and orotidylate decarboxylase in developing mouse liver and brain.

Abstract A large body of data has accumulated in recent years supporting the view that orotate phosphoribosyltransferase and orotidylate decarboxylase exist as a bifunctional enzyme complex in adult mammalian tissues. This paper presents evidence that such a complex also occurs in mouse liver and brain, regardless of the developmental stage of the animal. Orotate phosphoribosyltransferase and orotidylate decarboxylase activities remained coordinate in fetal, neonatal, immature and adult liver and brain. In addition, these two enzymes routinely cosedimented during centrifugation of cell-free extracts in sucrose gradients. The sedimentation coefficient of the enzyme complex did not change significantly during mouse development. However, the liver complex exhibited a sedimentation coefficient (5.0 ± 0.2) that differed from that of the brain complex (4.3 ± 0.1)

The rapid separation of orotate, orotidylate, and uridylate by thin-layer chromatography: Utility in the assay of orotate phosphoribosyltransferase and orotidylate decarboxylase☆

Abstract A thin-layer chromatographic method is described that achieves the complete separation of orotidine 5′-monophosphate (OMP), uridine 5′-monophosphate (UMP), and orotate. This is accomplished by developing polyethyleneimine (PEI)-cellulose with 0.2 m LiCl. OMP is retained at or near the origin whereas UMP and orotate migrate with R f values of 0.33 and 0.59, respectively. This method is well suited for studying the conversion of orotate into OMP by orotate phosphoribosyltransferase (PRTase) and the subsequent conversion of OMP into UMP by OMP decarboxylase. The synthesis of OMP and UMP by enzyme preparations from murine leukemia P1534J, mouse liver, and bakers yeast has been quantitated with this method. The results of this study showed that OMP does not normally accumulate in reaction mixtures containing either the mouse liver or P1534J enzyme preparations. This finding is compatible with the presence of a bifunctional enzyme complex comprised of orotate PRTase and OMP decarboxylase. Such a complex is known to exist in P1534J cells and is currently thought to exist in most if not all mammalian tissues. The stoichiometric conversion of orotate into UMP by this complex could easily account for the observed lack of OMP.

Pyrimidine nucleoside monophosphate kinase from rat bone marrow cells: A kinetic analysis of the reaction mechanism

A kinetic analysis of the reaction mechanism of pyrimidine nucleoside monophosphate kinase was carried out with a highly purified enzyme preparation from rat bone marrow cells. The results of initial rate and product inhibition studies provided insight into the mode of action of the enzyme. The data support the views that the reaction mechanism is sequential and nonequilibrium in nature. Substrates bind to the enzyme in a random order. Substrate binding is cooperative. That is, the binding of the first substrate facilitates the binding of the second substrate. UMP can bind to the purine site on the enzyme, resulting in substrate inhibition. Product inhibition can result from the binding of UDP to either the pyrimidine or purine site, or from the binding of ADP to the purine site.

Pyrimidine nucleoside monophosphate kinase from rat bone marrow cells: purification to high specific activity by a two-step affinity chromatography procedure.

This report describes a two-column scheme for purifying a pyrimidine nucleoside monophosphate kinase from rat bone marrow cells. Purification was achieved by affinity chromatography on Blue Sepharose and cellulose phosphate, with selective elution of the enzyme by substrates (UMP, ATP). The enzyme preparation appeared to be about 90% pure upon polyacrylamide gel electrophoresis, exhibited an exceptionally high specific activity (greater than 600 mumol/min/mg protein), and was obtained with 30-36% recovery of enzyme activity. It was concluded that UMP, dUMP, and CMP serve as phosphate acceptors for the enzyme, based on the parallel behavior displayed by enzyme activity with these substrates both during the purification process and during other procedures. The purified enzyme preparation did not display dTMP kinase activity. This report also describes a simplified radiotracer assay for pyrimidine nucleoside monophosphate kinases. Thin-layer chromatography on polyethyleneimine-cellulose is used to resolve residual substrates and products. Because both nucleoside di- and triphosphates remain at the origin, the assay is insensitive to the action of nucleoside diphosphate kinases and does not require the use of marker compounds. A variety of radiolabeled substrates can be used with this assay, including UMP, dUMP, CMP, and dTMP.

Pyrimidine nucleoside monophosphate kinase from rat bone marrow cells: Chromatographic, electrophoretic, and sedimentation behavior of active and inactive enzyme forms☆

This paper describes the study of a highly purified pyrimidine nucleoside monophosphate kinase from rat bone marrow cells. Short-term storage (24 h at 4 degrees C) of the purified enzyme in the absence of dithiothreitol, a sulfhydryl reducing agent, led to considerable losses of enzyme activity. Most of the lost activity could be regained, however, by incubating the enzyme with 50 mM dithiothreitol. Enzyme stabilization by dithiothreitol and reactivation by dithiothreitol were enhanced in the presence of phosphate buffer. Severe enzyme inhibition was produced by micromolar concentrations of sulfhydryl group reagents. Chromatographic, electrofocusing, and sucrose gradient centrifugation experiments revealed that the enzyme has a molecular weight of about 26,000, an isoelectric point of 4.7, and a sedimentation coefficient of 2.5. These experiments were also carried out with enzyme preparations which had been almost completely inactivated by means of dialysis to remove dithiothreitol. Enzyme preparations of this type displayed at least one additional enzyme form. This form(s) was inactive but capable of being partially reactivated by dithiothreitol. The inactive form(s) exhibited the same apparent molecular weight as the native enzyme but possessed a higher isoelectric point (5.7). A working hypothesis was presented which states (1) that inactive enzyme forms arise because of disulfide bond formation, (2) that enzyme sulfhydryl groups are less susceptible to oxidation in the presence of phosphate buffer, and (3) that enzyme reactivation by dithiothreitol results from the regeneration of critical enzyme sulfhydryls.

[56] Dihydrofolate reductase: A coupled radiometric assay

Publisher Summary This chapter describes a coupled radiometric assay for dihydrofolate reductase based on the oxidative decarboxylation of D-6-phospho[l- 14 C]gluconate. It also illustrates a simple procedure for the synthesis of D-6-phospho[l- 14 C]gluconate from the readily available D-[ 1- 14 C]glucose 6-phosphate. Glucose 6-phosphate labeled with carbon-14 in carbon atom 1 is readily available from commercial sources. Selective oxidation of the aldehyde function of glucose 6-phosphate to a carboxylate group by bromine yields 6-phosphogluconate. It is important to determine the exact specific activity of 6 phosphogluconate, as it has to be used as a substrate in the quantitation of dihydrofolate reductase activity. This is done by using 6-phosphogluconate dehydrogenase to catalyze the conversion of D-6-phospho[l- 14 C]gluconate into ribulose 5-phosphate and 14 CO 2 . Decarboxylation occurs with a simultaneous reduction of nicotinamide adenine dinucleotide phosphate + (NADP + ) to NADPH. The molar stoichiometry between CO 2 release and NADP + reduction is 1:1. The specific activity of the phosphogluconate can be ascertained by determining the microcuries of released CO 2 for each mole of NADPH formed. A molar extinction coefficient of 6200 M -1 cm -1 for NADPH is employed to quantitate absorbance changes at 340 nm.