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Biomedical subjects

R I Christopherson

Publications and source records attributed to R I Christopherson.

At least 73 records · Page 4Linked to original sources
PubMed1989

Antimalarial action of nitrobenzylthioinosine in combination with purine nucleoside antimetabolites.

The infection of human erythrocytes by two strains of the human malarial parasite, Plasmodium falciparum (FCQ-27 or the multi-drug-resistant strain K-1), markedly changed the transport characteristics of the nucleosides, adenosine and tubercidin, compared to uninfected erythrocytes. A component of the transport of these nucleosides was insensitive to the classical mammalian nucleoside transport inhibitor nitrobenzylthioinosine (NBMPR). In vitro studies with tubercidin demonstrated ID50 values of 0.43 and 0.51 microM for FCQ-27 and K-1, respectively. In addition, the nucleoside transport inhibitors NBMPR, nitrobenzylthioguanosine (NBTGR), dilazep and dipyridamole also independently exhibited antimalarial activity in vitro. The combination of tubercidin and NBMPR or NBTGR in vitro demonstrated synergistic activity, whilst tubercidin together with dilazep or dipyridamole showed subadditive activity. Analysis by HPLC indicated that NBMPR could permeate the infected cell membrane and provided evidence for the catabolism of NBMPR in vitro, with subsequent alteration of the purine pool in the infected erythrocyte. These observations further indicated the possibility of the utilization of cytotoxic nucleosides against P. falciparum infection in conjunction with a nucleoside transport inhibitor to protect the host tissue.

Aminoglycosides↗
PubMed1986

Effects of acivicin and dichloroallyl lawsone upon pyrimidine biosynthesis in mouse L1210 leukemia cells.

Acivicin (NSC 163501) and dichloroallyl lawsone (NSC 126771) are potent inhibitors of nucleotide biosynthesis with consequent anti-cancer activity against certain experimental tumors. To determine in detail the metabolic events induced by each inhibitor, we have devised a new two-dimensional chromatographic procedure for measurement of the concentrations of all pyrimidine intermediates and some purine nucleotides from 100 microliter of an extract of cells grown in the presence of [14C]bicarbonate. Addition of acivicin (25 microM) to mouse L1210 leukemia cells causes severe depletion in the cellular levels of CTP and GTP, accumulation of uridine nucleotides, and abrupt but transient increases in the concentrations of the early intermediates of both the pyrimidine and purine pathways. Addition of dichloroallyl lawsone (25 microM) results in a rapid depletion of uridine and cytidine nucleotides; carbamyl aspartate and dihydroorotate accumulate to high levels in an equilibrium ratio of 20.5:1, and orotate, orotidine, and UMP increase transiently before decreasing to levels approaching their original steady states. The predominant inhibitory effects of acivicin are upon the reactions UTP----CTP and XMP----GMP, but there is also an initial transient activation of both the pyrimidine and purine pathways by acivicin. The data obtained with dichloroallyl lawsone are consistent with inhibition of the conversion of UMP----UDP initially followed by potent inhibition of dihydroorotate----orotate.

Animals↗
PubMed1985

Chorismate mutase-prephenate dehydrogenase from Escherichia coli: cooperative effects and inhibition by L-tyrosine.

The effects of a variety of structural analogs of L-tyrosine on the mutase and dehydrogenase activities of hydroxyphenylpyruvate synthase have been investigated. From these studies it is concluded that the alpha-NH3+ alpha-COO-, and the 4-OH groups are essential for binding of L-tyrosine as an inhibitor of the dehydrogenase and that the L configuration is also essential. Dixon plots for inhibition of the dehydrogenase activity by some of these analogs were nonlinear and could be described by a velocity equation that is the ratio of quadratic polynomials (a 2/1 function). Dixon plots for inhibition of the mutase by prephenate at low concentrations of chorismate could also be described by a 2/1 function, but at low concentrations of prephenate chorismate acts as an apparent hyperbolic activator of the dehydrogenase activity. Up to concentrations of 300 microM, L-tyrosine activates the mutase but acts as a potent inhibitor of the dehydrogenase. Such data for the dehydrogenase could not be described by a 2/1 function in 1/[prephenate] but could be fitted to the Hill equation with increasing concentrations of L-tyrosine in the presence of 1.0 mM NAD yielding increasing values for the Hill number (n): in the absence of L-tyrosine, n = 1.6 +/- 0.1; at 150 microM L-tyrosine, n = 2.1 +/- 0.1; at 300 microM L-tyrosine, n = 2.3 +/- 0.4. L-Tyrosine bears a close structural resemblance to both prephenate and hydroxyphenylpyruvate, and evidence is presented which is consistent with L-tyrosine acting as a competitive inhibitor with respect to prephenate of the dehydrogenase.

Adamantane↗
PubMed1985

Regulation of hamster carbamoyl-phosphate synthase II by 5-phospho-alpha-D-ribosyl 1-diphosphate and uridine 5'-triphosphate.

In mammals, carbamoyl phosphate for utilization in pyrimidine biosynthesis is synthesized by a glutamine-dependent carbamoyl-phosphate synthase II which is subject to regulation by 5-phospho-alpha-D-ribosyl 1-diphosphate (PRib-PP), a positive effector, and MgUTP, a negative effector [Mori, M., Ishida, H. and Tatibana, M. (1975) Biochemistry 14, 2622-2630]. We have found that Lineweaver-Burk plots of carbamoyl phosphate synthase activity versus 1/[MgATP] are described by a velocity equation which is a ratio of quadratic polynomials, consistent with a positive homotropic interaction between two catalytic sites for the binding of MgATP (Ks = 16.6 +/- 3.1 mM, interaction factor a = 0.00538 +/- 0.00245). The activating effect of PRib-PP upon carbamoyl-phosphate synthase is consistent with PRib-PP binding at an allosteric site (Ka = 31.4 +/- 6.4 microM) and promoting the binding of a first molecule of MgATP as substrate (interaction factor l = 0.0437 +/- 0.0063). Thus MgATP and PRib-PP bind to the E X MgATP complex with respective dissociation constants of a X Ks = 0.089 mM and l X Ka = 1.4 microM while MgATP binds to the E X PRib-PP complex with a dissociation constant of l X Ks = 0.73 mM. Data for the inhibitory effect of MgUTP upon carbamoyl-phosphate synthase indicate that MgUTP competes with MgATP for binding at the catalytic site (Ki = 0.203 +/- 0.016 mM). A computer model has recently been developed which enables quantitative stimulation of the time-dependent effects of blockade of the pyrimidine pathway by a tight-binding enzyme inhibitor [Duggleby, R.G. and Christopherson, R.I. (1984) Eur. J. Biochem. 143, 221-226]. The velocity equation derived in the present paper provides a quantitative basis for predicting changes in the flux through the de novo pyrimidine pathway in growing cells.

Adenosine Triphosphate↗
PubMed1985

Chorismate mutase-prephenate dehydrogenase from Escherichia coli: positive cooperativity with substrates and inhibitors.

Investigations have been made at pH 6.0 of the effect of chorismate and adamantane derivatives on the mutase and dehydrogenase activities of hydroxyphenylpyruvate synthase from Escherichia coli. When used over a wide range of concentrations, chorismate 5,6-epoxide, chorismate 5,6-diol, adamantane-1,3-diacetate, adamantane-1-acetate, adamantane-1-carboxylate, and adamantane-1-phosphonate give rise to nonlinear plots of the reciprocal of the initial velocity of each reaction as a function of the inhibitor concentration. The inhibitors do not induce the enzyme to undergo polymerization and have only a small effect on the S20,w value of the enzyme as determined by using sucrose density gradient centrifugation. At low substrate concentration, low concentrations of adamantane-1-acetate cause activation of both the mutase and dehydrogenase activities while at higher concentrations this compound functions as an inhibitor. When chorismate and prephenate are varied over a wide range of concentrations, double-reciprocal plots of the data indicate that the reactions exhibit positive cooperativity. The addition of albumin eliminates the cooperative interactions associated with substrates but has little effect on those associated with inhibitors.

Adamantane↗
PubMed1984

Metabolic resistance to tight-binding inhibitors of enzymes involved in the de novo pyrimidine pathway. Simulation of time-dependent effects.

When a tight-binding inhibitor interacts with a target enzyme of a metabolic pathway, the end products of the pathway are depleted and the substrate(s) for the inhibited reaction accumulate. The accumulating substrate(s) can reach a concentration which is sufficiently high that the inhibition is effectively reversed, with restoration of the original flux through the inhibited reaction. We have recently developed a theoretical model for this phenomenon which we have called 'metabolic resistance' [R. I. Christopherson and R. G. Duggleby (1983) Eur. J. Biochem. 134, 331-335]. In the present communication, we have successfully used the technique of numerical integration to simulate the effects of inhibition of several enzymes in the pathway of pyrimidine biosynthesis. Utilizing appropriate dissociation constants and enzyme concentrations for the inhibited systems, these simulations are consistent with published experimental data for the interactions of N-phosphonacetyl-L-aspartate (P AcAsp) with mammalian aspartate transcarbamoylase in vitro [R. I. Christopherson and M. E. Jones (1980) J. Biol. Chem. 255, 11 381 -11 395] and of 5-fluorodeoxyuridine 5'-phosphate (FdUMP) with thymidylate synthetase in vivo [R. G. Moran, C. P. Spears, and C. Heidelberger (1979) Proc. Natl Acad. Sci. USA 76, 1456- 1460]. In addition we present a simulation of the expected effect in vivo of phosphoribofuranosyl barbituric acid (BMP), a tight-binding inhibitor of OMP decarboxylase [H. L. Levine, R. S. Brody, and F. H. Westheimer (1980) Biochemistry 19, 4993- 4999]. This simulation shows that on addition of BMP, there is a sequential depletion of all pyrimidine intermediates between UMP and dCDP and a concomitant accumulation of OMP. Eventually, OMP reaches a new steady-state concentration and the concentrations of the depleted intermediates rise to their original levels. We have simulated the depletion of dCDP at various concentrations of BMP; since dCDP is comitted to DNA synthesis we can integrate the dCDP concentrations over time to calculate the amount of DNA synthesis and thereby predict the delay in cell division which would be elicited by BMP. This form of analysis may help to explain in quantitative terms why inhibitors of nucleic acid biosynthesis have a selective toxicity for rapidly growing tumour cells.

Carboxy-Lyases↗
PubMed1983

Metabolic resistance: the protection of enzymes against drugs which are tight-binding inhibitors by the accumulation of substrate.

Blockade of a metabolic pathway by interaction of a drug with a particular 'target enzyme' results in depletion of essential end-products of the pathway and accumulation of intermediates prior to the blockade. Metabolic resistance to a particular drug can arise if the substrate of the inhibited enzyme accumulates to levels sufficiently high to compete effectively with the inhibitor, leading to restoration of full activity of the metabolic pathway after a transitory delay. Such resistance has recently been demonstrated in vitro for the interaction of the tight-binding inhibitor N-phosphonacetyl-L-aspartate (PAcAsp) with the aspartate transcarbamoylase activity of the trifunctional protein which initiates pyrimidine biosynthesis in mammals [Christopherson, R. I. and Jones, M. E. (1980) J. Biol. Chem. 255, 11381-11395]. Carbamoyl phosphate, the product of the carbamoyl phosphate synthetase activity of this trifunctional protein, accumulates to a sufficiently high concentration that the inhibitory effect of PAcAsp is effectively abolished. We have developed a theoretical model for metabolic resistance which quantitatively accounts for these experimental data. This model has been used to simulate the interaction between the following potential or proven anti-cancer drugs and their target enzyme, under conditions similar to those which would occur in vivo: PAcAsp with aspartate transcarbamoylase; various OMP analogues [the 5'-monophosphates of 6-azauridine, pyrazofurin and 1-(beta-D-ribofuranosyl)-barbituric acid] with OMP decarboxylase; 5-fluorodeoxyUMP with thymidylate synthase; methotrexate with dihydrofolate reductase; and deoxycoformycin with adenosine deaminase.

Aspartate Carbamoyltransferase↗
PubMed1983

Chorismate mutase-prephenate dehydrogenase from Escherichia coli: spatial relationship of the mutase and dehydrogenase sites.

The inhibition of the bifunctional enzyme chorismate mutase-prephenate dehydrogenase (4-hydroxyphenylpyruvate synthase) by substrate analogues has been investigated at pH 6.0 with the aim of elucidating the spatial relationship that exists between the sites at which each reaction occurs. Several chorismate and adamantane derivatives, as well as 2-hydroxyphenyl acetate and diethyl malonate, act as linear competitive inhibitors with respect to chorismate in the mutase reaction and with respect to chorismate in the mutase reaction and with respect to prephenate in the dehydrogenase reaction. The similarity of the dissociation constants for the interaction of these compounds with the free enzyme, as determined from the mutase and dehydrogenase reactions, indicates that the reaction of these inhibitors at a single site prevents the binding of both chorismate and prephenate. However, not all the groups on the enzyme, which are responsible for the binding of these two substrates, can be identical. At lower concentrations, citrate or malonate prevents reaction of the enzyme with prephenate, but not with chorismate. Nevertheless, the combining sites for chorismate and prephenate are in such close proximity that the diethyl derivative of malonate prevents the binding of both substrates. The results lead to the proposal that the sites at which chorismate and prephenate react on hydroxyphenylpyruvate synthase share common features and can be considered to overlap.

Binding Sites↗
PubMed1983

Synthesis and separation of tritium-labeled intermediates of the shikimate pathway.

[5-3H]Shikimate (sp radioact 2000 Ci/mol) has been synthesized by reduction of the methyl ester of 5-dehydroshikimate with NaB3H4 and subsequent hydrolysis of the ester group (M. M. Leduc, P. M. Dansette, and R. G. Azerad (1970) Eur. J. Biochem. 15, 428-435). The [5-3H]shikimate has been converted enzymatically to [5-3H]chorismate and [5-3H]prephenate of similar high specific radioactivity by using a cell-free extract of Aerobacter aerogenes 62-1. In addition, a chromatographic procedure, which utilizes polyethyleneimine-cellulose thin-layer chromatograms, has been developed for the separation of intermediates along the shikimate pathway between shikimate and hydroxyphenylpyruvate or phenylpyruvate. Since the method allows quantitative measurement of tritium-labeled intermediates, it provides the basis for sensitive radioassays of the individual enzymes and allows study of the reaction flux along the overall pathway. The same intermediates can be separated on a large scale by use of a column of DEAE-Sephacel.

Chorismic Acid↗
PubMed1980

The overall synthesis of L-5,6-dihydroorotate by multienzymatic protein pyr1-3 from hamster cells. Kinetic studies, substrate channeling, and the effects of inhibitors.

In mammals, a trifunctional protein (ME pyr1-32) synthesizes L-5,6-dihydroorotate in three sequential reactions catalyzed by carbamyl phosphate synthetase (EC 2.7.2.9), aspartate transcarbamylase (EC 2.1.3.2), and dihydroorotase (EC 3.5.2.3). 14C-labeled HCO3- has been used as a precursor for the synthesis of L-5,6-dihydroorotate by purified ME pyr1-3, and when this product is converted enzymatically to orotidine 5'-monophosphate, the concentrations of the two intermediates of ME pyr1-3, carbamyl phosphate, and N-carbamyl-L-aspartate, reach steady state concentrations of approximately 0.20 microM and 7.1 microM, respectively. At pH 7.4 in the presence of 0.1 mM 5-phosphoribosyl 1-pyrophosphate and 10% (v/v) glycerol, pure ME pyr1-3 has a Michaelis constant for HCO3- of 0.61 mM and a maximal specific activity of 329 pmol of L-5,6-dihydroorotate synthesized/min/microgram, equivalent to a turnover number of 65.8 mol min-1 (mol of subunit)-1. Consideration of the Km and Vmax values of aspartate transcarbamylase and dihydroorotase determined under the same conditions as the overall rate of synthesis of L-5,6-dihydroorotate by ME pyr1-3, indicates that the local concentrations of carbamyl phosphate at the active site of aspartate transcarbamylase and of N-carbamyl-L-aspartate at the dihydroorotase site must be 2.2-fold and 3.1-fold higher, respectively, than their average concentrations in the bulk solvent. Similar concentrations are predicted by calculation of steady state concentrations from ratios of the rate constants for the three activities. A high local concentration of N-carbamyl-L-aspartate at the third site is also indicated by a 3.6-fold reduction in the transient time for dihydroorotase activity from that predicted. Competition experiments performed with exogenous carbamyl phosphate and N-carbamyl-L-aspartate indicate only partial channeling of these intermediates. The inhibitory effect of N-phosphonacetyl-L-aspartate (PALA), at concentrations up to at least 2.4 microM, upon the aspartate transcarbamylase activity of ME pyr1-3 can be overcome by accumulated carbamyl phosphate. This mechanism for resistance to PALA could be manifest in cells which lack an effective phosphatase activity to hydrolyze carbamyl phosphate. L-Cysteine, a slow acting but potent inhibitor of dihydroorotase in the absence of substrates (Christopherson, R. I., and Jones, M. E. (1980) J. Biol. Chem. 255, 3358-3370), also inactivates dihydroorotate when ME pyr1-3 is synthesizing L-5,6-dihydroorotate.

Amidohydrolases↗
PubMed1980

The effects of pH and inhibitors upon the catalytic activity of the dihydroorotase of multienzymatic protein pyr1-3 from mouse Ehrlich ascites carcinoma.

We have studied factors affecting the catalytic activity of dihydroorotase (EC 3.5.2.3), purified as part of a multienzymatic protein which contains carbamyl phosphate synthetase, aspartate transcarbamylase, and dihydroorotase (ME pyr1-3) and which initiates de novo pyrimidine biosynthesis in mouse Ehrlich ascites carcinoma. The apparent Km value for N-carbamyl-L-aspartate increases by 2 orders of magnitude as the pH increases from 7.0 to 8.3, consistent with equilibration of dihydroorotase (E) between four states of protonation (E in equilibrium EH in equilibrium EH2 equilibrium EH3), where EH3 is the only catalytically active form of dihydroorotase for the biosynthetic reaction, having a Km for N-carbamyl-L-aspartate of 30 micro M. The apparent Km for L-dihydroorotate shows a converse dependence upon pH, remaining relatively constant at alkaline pH and increasing progressively as the pH is decreased below 7.0. These data are consistent with the above model if E and EH are catalytically active for the degradative reaction, both having Km values of 4.4 micro M for L-5,6-dihydroorotate. The D isomers of carbamylaspartate and dihydroorotate are also substrates for dihydroorotase. At pH 7.33, the apparent Km values for N-carbamyl-L-aspartate and N-carbamyl-D-aspartate are 247 and 204 micro M, respectively, but the Vmax for N-carbamyl-D-aspartate is only 1.7% of that obtained with N-carbamyl-L-aspartate. Orotate and a series of 5-substituted derivatives are competitive inhibitors of dihydroorotase. At pH 7.27, the apparent Ki for orotate using N-carbamyl-L-aspartate as substrate is 170 micro M and with L-5,6-dihydroorotate as substrate, the apparent Ki value is 9.6 micro M, suggesting that the enzyme exists in different forms in the presence of each substrate. Dihydroorotase is inhibited in a time-dependent manner by 50 mM L-cysteine and the presence of N-carbamyl-L-aspartate or L-5,6-dihydroorotate protects against this ultimately complete inactivation. 2-Mercaptoacetate, 2-mercaptoethylamine, 3-mercaptopropionate, and L-2,3-diaminopropionate have a similar although less potent inhibitory effect. To account for the data obtained, we propose a model for the equilibria existing between various protonated forms of dihydroorotase which is consistent with the pH dependencies of the apparent Km values observed and the Vmax values observed previously (Christopherson, R.I., and Jones, M.E. (1979) J. Biol. Chem. 254, 12506-12512). In addition, a catalytic mechanism is presented for the interconversion of N-carbamyl-L-aspartate and L-5,6-dihydroorotate.

Amidohydrolases↗
PubMed1978

Response of the pyrimidine pathway of Escherichia coli K 12 to exogenous adenine and uracil.

The effect of exogenous adenine or uracil upon the de novo pathway for synthesis of pyrimidine nucleotides in Escherichia coli K12 was investigated. Parameters studied were levels of the enzymes carbamoyl phosphate synthase (EC 2.7.2.9), aspartate carbamoyltransferase (EC 2.1.3.2) and orotate phosphoribosyltransferase (EC 2.4.2.10) and the intermediates carbamoyl phosphate, aspartate and orotate, together with the contributions of exogenous uracil and aspartate to intracellular pyrimidine nucleotide. Taken with earlier data [Bagnara, A.S. & Finch, L. R. (1974) Eur. J. Biochem- 41, 421--430] on contents of UTP, CTP and 5-phosphoribosyl 1-diphosphate in cultures of this strain after the addition of adenine or uracil, the results obtained provide new insights into the regulatory mechanisms operating on the pathway in vivo. These insights enable evaluation of the contributions of such factors as limitation for a substrate, feed-back allosteric control by end products and enzyme repression/depression mechanisms. The evidence presented indicates that depressed levels of orotate phosphoribosyltransferase in E. coli K12 result in the wasteful ultilization of asparatate for excess synthesis of pyrimidine nucleotide precursors during balanced growth of the strain in minimal medium. Exogenous adenine increases the excessive accumulation of these precursors by lowering the intracellular content of 5-phosphoribosyl 1-diphosphate (Bagnara and Finch, 1974). This causes a decrease in the conversion of orotate to orotidine 5'-monophosphate, thus lowering the utilization or orotate and its precursors for synthesis of pyrimidine nucleotides. Further, since the contents of these nucleotide end products are thereby decreased (Bagnara nad Finch, 1974), theri feed-back on the early steps in the pathway is diminished and the production of the precursors is increased. It is postulated that growth of E. coli K12 under these conditions is limited by a compound that is metabolically related to precursors to aspartate.

Adenine↗