Thymidylate synthase and fluorouracil.
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Deoxyuracil Nucleotides↗
Biomedical subjects
K M Ivanetich
Publications and source records attributed to K M Ivanetich.
Kinetics and thermodynamics of the interaction of 5-fluoro-2'-deoxyuridylate with thymidylate synthase.
Thymidylate synthase (TS), 5-fluorodeoxyuridylate (FdUMP), and 5,10-methylenetetrahydrofolate (CH2-H4folate) form a covalent complex in which a Cys thiol of TS is attached to the 6-position of FdUMP and the one-carbon unit of the cofactor is attached to the 5-position. The kinetics of formation of this covalent complex have been determined at several temperatures by semirapid quench methods. Together with previously reported data the results permit calculation of every rate and equilibrium constant in the interaction. Conversion of the noncovalent ternary complex to the corresponding covalent complex proceeds at a rate of 0.6 s-1 at 25 degrees C, and the dissociation constant for loss of CH2-H4folate from the noncovalent ternary complex is approximately 1 microM. Activation parameters for the formation of the covalent complex were shown to be Ea = 20 kcal/mol, delta G+ = 17.9 kcal/mol, delta H+ = 19.3 kcal/mol, and delta S+ = 0.005 kcal/(mol.deg). The equilibrium constant between the noncovalent and covalent ternary complexes is approximately 2 X 10(4), and the overall dissociation constant of CH2-H4folate from the covalent complex is approximately 10(-11) M. The conversion of the noncovalent ternary complex to the covalent adduct is about 12-fold slower than kcat in the normal enzymic reaction. However, because the dissociation constant for CH2-H4folate from the noncovalent ternary complex is about 10-fold lower than that from the TS-dUMP-CH2-H4folate Michaelis complex, the terms corresponding to kcat/Km are nearly equal. We propose that some of the intrinsic binding energy of CH2-H4folate may be used to facilitate formation of a 5-iminium ion intermediate.(ABSTRACT TRUNCATED AT 250 WORDS)
Buffers↗
Determination of erythrocyte protoporphyrin by magnetic circular dichroism.
A method for the determination of erythrocyte protoporphyrin by magnetic circular dichroism is reported. The method was sufficiently sensitive to distinguish normal from elevated levels of erythrocyte protoporphyrin and may be useful for diagnostic screening purposes. Erythrocyte protoporphyrin levels determined by magnetic circular dichroism correlated well with levels determined by the fluorimetric method of Piomelli, viz. a plot of MCD versus fluorimetry yielded a straight line relationship with a slope of 0.98 +/- 0.01, y-intercept of 0.05 +/- 0.35 and correlation coefficient of 0.99.
Circular Dichroism↗
Prizidilol. Metabolism by cytochrome P-450 and acetyltransferase.
The hepatic microsomal cytochrome P-450 enzyme system bound and metabolized the experimental drug prizidilol. Prizidilol bound to two distinct sites on cytochrome P-450. At low concentrations (less than ca 20 microM), prizidilol bound to the substrate binding site of the enzyme and produced a Type I difference spectrum. At higher concentrations (25-190 microM), prizidilol bound to the oxygen binding site of the enzyme and produced a type II difference spectrum. Prizidilol stimulated hepatic microsomal CO-inhibitable NADPH oxidation. Prizidilol metabolism by hepatic microsomes assessed by prizidilol disappearance was inhibited by CO:O2 (80:20; v/v), SKF 525-A and metyrapone. Prizidilol disappearance was monitored using a newly developed TLC assay for prizidilol following derivatization with quinolin-3-al. The apparent binding constants (Ks), maximum extents of binding (delta Amax), Michaelis constants (Km) and maximum velocities (Vmax) for the interaction of prizidilol with hepatic microsomal cytochrome P-450 were assessed in rats pretreated or not with the inducing agents phenobarbital, beta-naphthoflavone and pregnenolone-16 alpha-carbonitrile. For the differently pretreated rats the apparent Ks values for the type I site and the type II site and the apparent Km were ca 3 microM, 150 microM and 2 microM, respectively. Apparent Vmax values varied from 20 to 70 pmol per min per mg microsomal protein. The observed effects of induction on the apparent equilibrium constants and maximum extents of binding and metabolism of prizidilol indicate that the forms of cytochrome P-450 induced by phenobarbital, pregnenolone-16 alpha-carbonitrile or beta-naphthoflavone do not play a major role in the metabolism of prizidilol. Prizidilol was also metabolized by hepatic cytosolic N-acetyltransferase. The apparent Km values for prizidilol and acetyl CoA were 0.8 and 22 microM. Apparent Vmax values were 50 and ca 2 pmol per min per mg protein for partially purified transferase and cytosol, respectively. It is concluded that the rates of oxidation and acetylation of this drug would be expected to be relatively low, being limited by low apparent Vmax values for both oxidation and acetylation.
Acetylation↗
Reversible inhibition and activation of hepatic GSH S-transferases by ethylene dibromide.
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Animals↗
Chlorinated ethylenes: their metabolism and effect on DNA repair in rat hepatocytes.
The major initial metabolites of the chlorinated ethylenes in hepatocyte suspensions isolated from phenobarbital treated rats were as follows (rates of metabolite production in nmol/10(6) cells/min are given in brackets): vinylidene chloride, dichloroacetic acid (0.015); cis-1,2-dichloroethylene, 2,2-dichloroethanol (0.24); trans-1,2-dichloroethylene, dichloroacetic acid (0.005); trichloroethylene, chloral hydrate (2.7); tetrachloroethylene, trichloroacetic acid (0.08). Comparison of the metabolism of the chlorinated ethylenes by isolated hepatocyte suspensions and hepatic microsomes indicates that the initial products of the three dichlorinated ethylenes from cytochrome P-450 in hepatic microsomes are rapidly and extensively metabolized in the hepatocyte, where the Phase II enzymes are present. In contrast, the initial metabolites of trichloroethylene and of tetrachloroethylene in the two systems are identical. The abilities of the chlorinated ethylenes to induce unscheduled DNA synthesis was assessed in isolated hepatocytes using a method which does not require the blocking of semi-conservative DNA synthesis. Vinylidene chloride, cis-1,2-dichloroethylene and trichloroethylene induced unscheduled DNA synthesis, while trans-1,2-dichloroethylene and tetrachloroethylene did not.
Acetaldehyde↗
Metabolism of chloroethanes by rat liver nuclear cytochrome P-450.
1,2-Dichloroethane, 1,1,1-trichloroethane and 1,1,2,2-tetrachloroethane appear to be metabolized by hepatic nuclear cytochrome P-450. All of these compounds are converted to chlorinated metabolites after incubation with hepatic nuclei and an NADPH-generating system plus EDTA, with the omission of any component eliminating metabolite production. In addition, CO, an inhibitor of cytochrome P-450, diminished the production of the chlorinated metabolites by hepatic nuclear preparations. The major metabolites of the chlorinated ethanes from hepatic microsomal cytochrome P-450, viz. chloroacetaldehyde from 1,2-dichloroethane, 2,2,2-trichloroethanol from 1,1,1-trichloroethane, and dichloroacetic acid from 1,1,2,2-tetrachloroethane, were also produced from the three chloroalkanes by hepatic nuclear cytochrome P-450. The levels of the metabolites produced were 65, 0.09 and 4.4 nmol/nmol cytochrome P-450/60 min. It is proposed that the pathways for the formation of these metabolites by hepatic nuclear cytochrome P-450 are as for their production by hepatic microsomal cytochrome P-450. Chloral hydrate was produced from 1,1,1-trichloroethane by hepatic nuclei plus NADPH, but not by hepatic microsomes. The presence of reactive species or transient enzyme bound intermediates in the pathways for the cytochrome P-450 dependent metabolism of the chloroethanes in hepatic nuclei is suggested by the observation that nuclear cytochrome P-450 is degraded in the presence of the chloroethanes in a NADPH dependent process which is inhibited by CO. It is proposed that, although the cytochrome P-450 dependent metabolism of the chloroethanes in microsomes can greatly exceed that in nuclei, the metabolism of 1,2-dichloroethane and 1,1,2,2-tetrachloroethane by nuclear cytochrome P-450 may in part mediate the mutagenicity and carcinogenicity of parent compounds.
Animals↗
Inhibition of rat GSH S-transferases by ethylene dibromide.
Incubation of ethylene dibromide (EDB) (37 mM) with a mixture of rat hepatic cytosol GSH S-transferases at 25 degrees resulted in diminished activity towards 1-chloro-2,4-dinitrobenzene (CDNB) and 3,4-dichloronitrobenzene (DCNB). The loss of both activities followed pseudo first order kinetics with a rate constant of 0.13 +/- 0.03 min-1. The concentration of EDB required for half maximal loss of enzymic activity towards CDNB was 3.2 mM. Removal of EDB from the enzyme by lyophilization or gel filtration did not result in the return of activity towards CDNB. GSH partially prevented the loss of activity, but could not reverse the loss. EDB decreased the activity of forms A(YbYb) and C(YbYb) of rat liver GSH S-transferases but not of forms AA(YcYc), B(YaYc) or B(YaYa). It is concluded that EDB inhibits forms A and C of the GSH S-transferases via a mechanism not involving suicide inhibition.
Animals↗
Isoflurane: a comparison of its metabolism by human and rat hepatic cytochrome P-450.
Isoflurane is metabolized by both rat and human hepatic microsomal cytochrome P-450 in vitro to fluoride ion and organofluorine metabolites. The forms of rat liver microsomal cytochrome P-450 induced by phenobarbital and pregnenolone-16 alpha-carbonitrile appear to be involved in the metabolism of isoflurane, while the forms induced by beta-naphthoflavone do not. Different pathways are favored for the metabolism of isoflurane by rat and by human liver microsomes: trifluoroacetaldehyde appears to be produced from isoflurane by rat liver microsomal cytochrome P-450, while trifluoroacetate or other nonvolatile fluorinated metabolites were not. The trifluoroacetaldehyde so produced binds tightly to microsomal constituents. Human liver microsomes converted isoflurane extensively to nonvolatile fluorinated products, one of which appears to be trifluoroacetate. The proposed pathways for the metabolism of isoflurane are considered in view of the above results.
Adult↗
Rapid semiquantitative measurement of total porphyrins in urine and feces by magnetic circular dichroism.
Magnetic circular dichroism is a suitable technique for semiquantitative determination of total porphyrins in urine and feces, being rapid, reliable, and involving little sample preparation. The sensitivity of the assay suffices to distinguish normal from above-normal concentrations of total porphyrins in urine and feces. Results for urine compare well with those obtained by thin-layer chromatography of porphyrin esters (n = 42, r = 0.92) or ion-exchange column chromatography of porphyrin acids (n = 19, r = 0.80).
Chromatography, Ion Exchange↗
Hepatic microsomal metabolism of the dichloroethanes.
The binding of 1,1-dichloroethane (1,1-DCE) to the substrate binding site of hepatic microsomal cytochrome P-450, and the stimulation of hepatic microsomal CO-inhibitable NADPH oxidation by 1,1-DCE and 1,2-dichloroethane (1,2-DCE) were enhanced by induction with phenobarbital but not with beta-naphthoflavone. Incubation of the dichloroethanes with hepatic microsomes from phenobarbital-treated rats, NADPH-generating system and EDTA resulted in the conversion of 1,1-DCE to acetic acid and to a lesser extent to 2,2-dichloroethanol and probably also mono- and dichloroacetic acid and the conversion of 1,2-DCE to chloroacetaldehyde and to a lesser extent to chloroacetic acid and probably 2-chloroethanol. In addition, reaction mixtures constituted as described above resulted in slight but significant losses (ca. 13%) of hepatic microsomal cytochrome P-450. The omission of dichloroethane or the NADPH-generating system from incubation mixtures eliminated the above effects, and SKF-525A or CO diminished or eliminated the effects. Pathways for the metabolism of 1,1-DCE and 1,2-DCE are proposed.
Animals↗
Vinylidene chloride: its metabolism by hepatic microsomal cytochrome P-450 in vitro.
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Animals↗
The 1,2-dichloroethylenes: their metabolism by hepatic cytochrome p-450 in vitro.
Cis- and trans-1,1-dichloroethylene bound to the active site of hepatic microsomal cytochrome P-450 with the production of a Type I difference spectrum and stimulated CO-inhibitable hepatic microsomal NADPH oxidation. Incubation of cis- and trans-1,2-dichloroethylene plus hepatic microsomes, NADPH-generating system-EDTA resulted in the production of measurable levels of 2,2-dichloroethanol and dichloroacetaldehyde but not of 2-chloroethanol, chloroacetaldehyde or chloroacetic acid and, also, resulted in decreased levels of hepatic microsomal cytochrome P-450 and heme. In addition, dichloroacetic acid was produced from trans-dichloroethylene under these experimental conditions. The omission of any component of the incubation mixture eliminated the above effects, while the inclusion of SKF-525A, metyrapone or CO:O2 (80, v/v) diminished these effects. The effects of beta-naphthoflavone and phenobarbital pretreatment on the values of Ks, delta Amax, Km and Vmax for the binding and metabolism of the 1,2-dichloroethylenes are reported. The binding and metabolism of the 1,2-dichloroethylenes and the 1,2-dichloroethylene-mediated inactivation of cytochrome P-450 were enhanced per mg of microsomal protein, but generally not per nmole of cytochrome P-450 by prior induction with beta-naphthoflavone or phenobarbital. It is concluded that multiple forms of hepatic microsomal cytochrome P-450 bind and metabolize the 1,2-dichloroethylenes. The role of cytochrome P-450 in the metabolic activation of the dichloroethylenes is considered.
Animals↗
Limitations on the metyrapone assay for the major phenobarbital inducible form of cytochrome P-450.
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Animals↗
1,1,1-Trichloropropene-2,3-oxide: an alternate mechanism for its inhibition of cytochrome P-450.
Incubation of hepatic microsomes from phenobarbital treated rats, 1,1,1-trichloropropene-2,3-oxide (TCPO) (3 mM), EDTA (0.2 mM) and NADPH-gene-rating system in vitro decreased the levels of cytochrome P-450 by 40% and equivalently decreased microsomal heme. Appreciable losses of cytochrome P-450 were not observed [1] if metyrapone (2.3 mM) or CO:O2 (80:20; v/v) were included in incubation mixtures, [2] if the TCPO or NADPH-gene-rating system were omitted from reaction mixtures, or [3] if microsomes from untreated or beta-naphthoflavone treated rats were utilized. The time course for the TCPO mediated loss of hepatic microsomal cytochrome P-450 showed a time lag of 5 min, before the levels of cytochrome P-450 were significantly altered, followed by a 10-15 min period in which the levels of cytochrome P-450 rapidly decreased to a non-zero plateau level. The concentration of TCPO required for half maximal loss of cytochrome P-450 was ca. 0.5 mM. In the absence of cytochrome P-450 degradation, TCPO (2 mM) was an effective inhibitor of aminopyrine demethylase and benzpyrene-3-hydroxylase but not of p-nitroanisole demethylase or ethoxyresorufin deethylase. In contrast, the degradation of cytochrome P-450 by TCPO strikingly decreased ethoxyresorufin deethylase and to a lesser extent p-nitroanisole demethylase. A reaction mechanism is proposed for the TCPO mediated degradation of cytochrome P-450 in vitro.
Animals↗
Limitations of the sodium fusion assay for fluorinated metabolites.
A method involving sodium fusion and analysis by fluoride electrode, which has been used for the determination of total fluorinated metabolites of fluorine-containing drugs in physiologic fluids, is shown to be inapplicable to volatile fluorinated metabolites of volatile fluorinated anesthetic agents. The yields of inorganic fluoride from the volatile metabolites 2,2,2-trifluoroethanol and trifluoroacetaldehyde were less than 2% of expected values, whether these metabolites were present in water, buffer, urine, or hepatic microsomes, although reliable results were obtained for the relatively nonvolatile sodium trifluoroacetate. It is proposed that the assay does not provide a valid method for determining total fluorinated metabolites of volatile or nonvolatile fluorine-containing drugs, but may be of some use in determining a single nonvolatile fluorinated metabolite of a volatile fluorinated drug.
Anesthetics↗
Chloroethanes : their metabolism by hepatic cytochrome P-450 in vitro.
1,1,1- and 1,1,2-trichloroethane and 1,1,2,3-tetrachloroethane stimulate CO- inhibitable NADPH oxidation by hepatic microsomes. Incubation of the chloroethanes, hepatic microsomes, an NADPH-generating system and EDTA results in the production of chlorinated metabolites. Both NADPH oxidation and metabolite production are inhibited by CO, SKF 525A and/or metyrapone The induction of cytochrome P-450 with phenobarbital enhances the binding and metabolism of the chloroalkanes, while the induction of cytochrome P-448 with beta-naphthoflavone does not. 1,1,1-trichloroethane is converted to 2,2,2-trichloroethanol by hepatic microsomal cytochrome P-450, while the major metabolites of 1,1,2-trichloroethane and 1,1,2,2-tetrachloroethane from this enzyme system are mono- and dichloroacetate, respectively. Mechanisms for the production of these metabolites are suggested. The relative mutagenic and carcinogenic potentials of the three chloroethanes are considered with respect to their relative extents of metabolism and their proposed metabolic pathways.
Animals↗
Influence of two haloalkanes on the redox behavior of hepatic microsomal cytochrome b-5 and its possible relationship to stearate desaturase.
The possible interaction of two haloalkanes - bromotrichloromethane and 1,2-dibromo-1,2-dichlorethane - with stearate desaturase was assessed in hepatic microsomes from rats fed a high carbohydrate diet which elevates the levels of stearate desaturase. Both compounds shifted the redox steady state of NADPH reduced hepatic microsomal cytochrome b-5 towards ferricytochrome b-5 and enhanced the re-oxidation of NADH reduced hepatic microsomal cytochrome b-5. The equilibrium constants for the enhancement of microsomal electron transfer by the haloalkanes in these preparations were 2.2 +/- 0.3 mM and 0.46 +/- 0.1 mM for bromotrichloromethane and 1,2-dibromo-1,2-dichlorethane, respectively. The haloalkane mediated enhancement of the oxidation of cytochrome b-5 in hepatic microsomes from rats fed a high carbohydrate diet was diminished by KCN and the inhibitors of cytochrome P-450, CO and/or metyrapone, as well as by fasting of the experimental animals. The I50 values for KCN inhibition of the effects of the haloalkanes on the re-oxidation of cytochrome b-5 (01 mM) were identical to the I50 for KCN inhibition of stearate desaturase (Oshino et al., 1966). The haloalkanes did not affect the activity of hepatic microsomal NADH- or NADPH-cytochrome c reductase, the autoxidation of purified trypsin-cleaved ferrocytochrome b-5 or the conversion of stearoyl CoA to oleate. It is concluded that bromotrichloromethane and 1,2-dibromo-1,2-dichloroethane stimulate hepatic microsomal electron transfer from NADH via cytochrome b-5 by interacting with cytochrome P-450 and with stearate desaturase.
Animals↗