Turnover of membrane proteins: kinetics of induction and degradation of seven forms of rat liver microsomal cytochrome P-450, NADPH-cytochrome P-450 reductase, and epoxide hydrolase

The in vivo turnover of several rat liver microsomal proteins was studied using techniques designed to maximize antibody recognition specificity and minimize reutilization of radioactive labels. The kinetics of degradation of seven cytochrome P-450 isozymes, NADPH-cytochrome P-450 reductase, and epoxide hydrolase were determined in untreated rats and rats treated with phenobarbital or beta-naphthoflavone. In the cases where induction of these enzymes occurred with the above chemicals, rates of synthesis of the proteins were also estimated. In general, the degradation rates of the different proteins were rather similar to each other, and the effects of phenobarbital and beta-naphthoflavone on these rates were not very great. However, in the case of cytochromes P-450, a general trend was observed in which the heme moiety was degraded more rapidly than the apoprotein. Changes in the rates of synthesis of the individual proteins appear to contribute more to the altered steady-state levels which are expressed than do the rates of degradation, and profiles of steady-state enzyme concentrations predicted by the kinetic constants approximate those observed in vivo.     

Interaction between NADPH-cytochrome P-450 reductase and hepatic microsomes

Solubilized NADPH-cytochrome P-450 reductase has been purified from liver microsomes of phenobarbital-treated rats. When added to microsomes, the reductase enhances the monoxygenase, such as aryl hydrocarbon hydroxylase, ethoxycoumarin O-dealkylase, and benzphetamine N-demethylase, activities. The enhancement can be observed with microsomes prepared from phenobarbital- or 3-methylcholanthrene-treated, or non-treated rats. The added reductase is believed to be incorporated into the microsomal membrane, and the rate of the incorporation can be assayed by measuring the enhancement in ethoxycoumarin dealkylase activity. It requires a 30 min incubation at 37°C for maximal incorporation and the process is much slower at lower temperatures. The temperature affects the rate but not the extent of the incorporation. After the incorporation, the enriched microsomes can be separated from the unbound reductase by gel filtration with a Sepharose 4B column. The relationship among the reductase added, reductase bound and the enhancement in hydroxylase activity has been examined. The relationship between the reductase level and the aryl hydrocarbon hydroxylase activity has also been studied with trypsin-treated microsomes. The trypsin treatment removes the reductase from the microsomes, and the decrease in reductase activity is accompanied by a parallel decrease in aryl hydrocarbon hydroxylase activity. When purified reductase is added, the treated microsomes are able to gain aryl hydrocarbon hydroxylase activity to a level comparable to that which can be obtained with normal microsomes. The present study demonstrates that purified NADPH-cytochrome P-450 reductase can be incorporated into the microsomal membrane and the incorporated reductase can interact with the cytochrome P-450 molecules in the membrane, possibly in the same mode as the endogenous reductase molecules. The result is consistent with a non-rigid model for the organization of cytochrome P-450 and NADPH-cytochrome P-450 reductase in the microsomal membrane.     

NADPH-cytochrome P-450 reductase of yeast microsomes.

NADPH-cytochrome c reductase of yeast microsomes was purified to apparent homogeneity by solubilization with sodium cholate, ammonium sulfate fractionation, and chromatography with hydroxylapatite and diethylaminoethyl cellulose. The purified preparation exhibited an apparent molecular weight of 83,000 on polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The reductase contained one molecule each of flavin-adenine dinucleotide and riboflavin 5′-phosphate, though these were dissociative from the apoenzyme. The purified reductase showed a specific activity of 120 to 140 μmol/min/mg of protein for cytochrome c as the electron acceptor. The reductase could reduce yeast cytochrome P-450, though with a relatively slow rate. The reductase also reacted with rabbit liver cytochrome P-450 and supported the cytochrome P-450-dependent benzphetamine N-demethylation. It can, therefore, be concluded that the NADPH-cytochrome c reductase is assigned for the cytochrome P-450 reductase of yeast. The enzyme could also reduce the detergent-solubilized cytochrome b₅ of yeast. So, this reductase must contribute to the electron transfer from NADPH to cytochrome b₅ that observed in the yeast microsomes.     

Selective inactivation of NADPH-cytochrome P-450 (c) reductase by diazotized 3-aminopyridine adenine dinucleotide phosphate

Cyanogen-bromide cleaved glucagon has been extensively purified in yields of 80–85% by the use of gel filtration and by cation-exchange chromatography at pH 4.5–5.2. This pH range maintains a charge difference between the holohormone and its cleavage product, the truncated homoserine lactone derivative, yet maintains the integrity of the lactone ring. Purity is determined by the lack of methionine and the presence of homoserine following peptide hydrolysis. The homoserine lactone is opened by treatment with 0.2 n triethylamine at pH 9.5. The lactone can be reformed by treatment with trifluoroacetic acid for 1 h at room temperature although protection against photooxidation of tryptophan-25 must be provided. The homoserine lactone form binds less well to glucagon receptors than does the homoserine form. Adenylate cyclase is activated by the lactone to an extent comparable to that obtained by native hormone but at elevated concentrations. The procedures described may be useful for purification of other cyanogen bromide cleavage products and is useful for semisynthetic methods based upon cyanogen bromide-cleaved derivatives of glucagon.     

Hydroxylation of para-chlorotoluene by model complexes of cytochrome P-450

Hydroxylation of p-chlorotoluene with heminthiol complexes, Fenton's system and Udenfriend's system was studied and the complexes assessed as models of cytochrome P-450 monooxygenases. Five species of possible hydroxylation products of p-chlorotoluene, namely, p-chlorobenzyl alcohol, 2-chloro-5-methylphenol, p-chlorobenzaldehyde, 4-chloro-2-methylphenol and 5-chloro-2-methylphenol, were studied using high performance liquid chromatography. The oxidation reactions were characterized by the yields of hydroxylation products and the product ratio. The system consisting of hemin and cysteine ethyl ester as well as Udenfriend's system gave relatively high hydroxylation yields and the former only induced a methyl migration during hydroxylation (methyl NIH shift). However, neither Fenton's nor Udenfriend's systems induced a methyl NIH shift. The hemin-thiol complex is thus concluded to be a good chemical model of cytochrome P-450 monooxygenases.     

Spectroscopic evidence for anthracenedione antineoplastic agent self-association and complex formation with flavin nucleotides

Dihydroxyanthraquinone (DHAQ) and ametantrone (anthraquinone) are two new anthracenedione antineoplastic agents which were found by proton NMR spectroscopy to self-associate in aqueous media. Self-association was consistent with a bimolecular model, with average association constant values of 3400 and 2900 m^?1 determined for DHAQ and ametantrone, respectively. Both anthracenediones interacted with the flavin nucleotides FMN and FAD to produce concentration-dependent upfield shifts of the flavin isoalloxazine ring proton signals, as observed by proton NMR spectroscopy. Average association constant values obtained for FMN-DHAQ, FAD-DHAQ, FMN-metantrone, and FAD-ametantrone complexation were 5100, 2600, 4300, and 1600 m^?1, respectively. Optical difference spectroscopy confirmed FMN-DHAQ complexation, which resulted in a hyperchromic, bathochromic shift of the DHAQ spectrum following addition of FMN. These results were consistent with the formation of a ππ bimolecular ring-stacking complex. Information obtained on anthracenedione self-association and complexation with flavins may be of consequence in the interpretation of anthracenedione-DNA binding data and flavoprotein-mediated anthracenedione metabolic activation.     

Bis(bipyridine)ruthenium(II) cyanobridge polymeric cations

Polymeric cations made up of Ru(bpy)₂ units linked by cyanide bridges can be prepared by reacting Ru(bpy)₂(CN)₂ with Ru(bpy)₂C₂O₄. The reaction yields a mixture of polymeric cations of various chain lengths. Polymeric cations of this type have emitting excited states in solution, with lifetimes in the 50–100 ns range.     

The enhancement of cytochrome P-450 catalyzed methoxyflurane metabolism by a heat-stable microsomal protein

We report the existence of a microsomal, heat-stable, trypsin-sensitive factor that stimulates the O-demethylation of methoxyflurane (CHCl₂CF₂OCH₃) by partially purified preparations of rabbit hepatic cytochrome P-450. The factor is able to stimulate by five to twelve-fold the methoxyflurane metabolizing activity of cytochrome P-450. In contrast, the metabolism of benzphetamine is not affected by the presence of the factor. The factor is inactivated by extraction with methanol, chloroform, butanol and ethanol. It remains intact after treatment with 6M guanidine hydrochloride and is soluble in trifluoroethanol. Thus, the weight of evidence indicates that this factor is a rather hydrophobic protein.     

Superoxide generation by NADPH-cytochrome P-450 reductase: the effect of iron chelators and the role of superoxide in microsomal lipid peroxidation

Superoxide generation, assessed as the rate of acetylated cytochrome c reduction inhibited by superoxide dismutase, by purified NADPH cytochrome P-450 reductase or intact rat liver microsomes was found to account for only a small fraction of their respective NADPH oxidase activities. DTPA-Fe3+ and EDTA-FE3+ greatly stimulated NADPH oxidation, acetylated cytochrome c reduction, and O(2) production by the reductase and intact microsomes. In contrast, all ferric chelates tested caused modest inhibition of acetylated cytochrome c reduction and O(2) generation by xanthine oxidase. Although both EDTA-Fe3+ and DTPA-Fe3+ were directly reduced by the reductase under anaerobic conditions, ADP-Fe3+ was not reduced by the reductase under aerobic or anaerobic conditions. Desferrioxamine-Fe3+ was unique among the chelates tested in that it was a relatively inert iron chelate in these assays, having only minor effects on NADPH oxidation and/or O(2) generation by the purified reductase, intact microsomes, or xanthine oxidase. Desferrioxamine inhibited microsomal lipid peroxidation promoted by ADP-Fe3+ in a concentration-dependent fashion, with complete inhibition occurring at a concentration equal to that of exogenously added ferric iron. The participation of O(2) generated by the reductase in NADPH-dependent lipid peroxidation was also investigated and compared with results obtained with a xanthine oxidase-dependent lipid peroxidation system. NADPH-dependent peroxidation of either phospholipid liposomes or rat liver microsomes in the presence of ADP-Fe3+ was demonstrated to be independent of O(2) generation by the reductase.     

The role of iron chelates in hydroxyl radical production by rat liver microsomes,NADPH-cytochrome P-450 reductase and xanthine oxidase

Uninduced rat liver microsomes and NADPH-Cytochrome P-450 reductase, purified from phenobarbital-treated rats, catalyzed an NADPH-dependent oxidation of hydroxyl radical scavenging agents. This oxidation was not stimulated by the addition of ferric ammonium sulfate, ferric citrate, or ferric-adenine nucleotide (AMP, ADP, ATP) chelates. Striking stimulation was observed when ferric-EDTA or ferric-diethylenetriamine pentaacetic acid (DTPA) was added. The iron-EDTA and iron-DTPA chelates, but not unchelated iron, iron-citrate or iron-nucleotide chelates, stimulated the oxidation of NADPH by the reductase in the absence as well as in the presence of phenobarbital-inducible cytochrome P-450. Thus, the iron chelates which promoted NADPH oxidation by the reductase were the only chelates which stimulated oxidation of hydroxyl radical scavengers by reductase and microsomes. The oxidation of aminopyrine, a typical drug substrate, was slightly stimulated by the addition of iron-EDTA or iron-DTPA to the microsomes. Catalase inhibited potently the oxidation of scavengers under all conditions, suggesting that H₂O₂ was the precursor of the hydroxyl radical in these systems. Very high amounts of superoxide dismutase had little effect on the iron-EDTA-stimulated rate of scavenger oxidation, whereas the iron-DTPA-stimulated rate was inhibited by 30 or 50% in microsomes or reductase, respectively. This suggests that the iron-EDTA and iron-DTPA chelates can be reduced directly by the reductase to the ferrous chelates, which subsequently interact with H₂O₂ in a Fenton-type reaction. Results with the reductase and microsomal systems should be contrasted with results found when the oxidation of hypoxanthine by xanthine oxidase was utilized to catalyze the production of hydroxyl radicals. In the xanthine oxidase system, ferric-ATP and -DTPA stimulated oxidation of scavengers by six- to eightfold, while ferric-EDTA stimulated 25-fold. Ferric-desferrioxamine consistently was inhibitory. Superoxide dismutase produced 79 to 86% inhibition in the absence or presence of iron, indicating an iron-catalyzed Haber-Weiss-type of reaction was responsible for oxidation of scavengers by the xanthine oxidase system. These results indicate that the ability of iron to promote hydroxyl radical production and the role that superoxide plays as a reductant of iron depends on the nature of the system as well as the chelating agent employed.     

NADH: (acceptor) oxidoreductase from bovine adrenal medulla chromaffin granules

The NADH:(acceptor) oxidoreductase from membranes of bovine adrenal medulla chromaffin granules has been purified by column chromatography. After solubilization of the membranes with emulphogen, a nonionic detergent, the enzyme was purified by dye-ligand chromatography and gel filtration. The oxidoreductase appeared essentially homogeneous on two gel electrophoretic systems. On polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the enzyme revealed a dimeric structure with a combined molecular weight of about 55,000. The enzyme eluted as a detergent-lipid-protein aggregate with a Stoke's radius of 43 Å on gel filtration columns in the presence of emulphogen. The amino acid composition of the oxidoreductase was found to be distinct from that of similar enzymes from other organelles. Topographical experiments indicated that the enzyme is a transmembrane protein.     

Multifunctionality of lipoamide dehydrogenase: Role of histidine residues in reductase reaction

Rose bengal sensitizes photoinactivation of lipoamide dehydrogenase from pig heart to a constant residual reductase activity resulting from specific destruction of histidine residues. The rate of sensitized photoinactivation is pH dependent and is associated with an ionizable group with pK 6.6 ± 0.2. All steady-state kinetic parameters are markedly reduced by photooxidation. Spectroscopic studies indicate the contribution of oxidized flavin/dithiol to the half-reduced form of the photooxidized enzyme. The proton magnetic resonance spectrum of lipoamide dehydrogenase shows resolved histidine C₂ proton peak at δ9.18 ppm and a shoulder at δ9.23 ppm. The shoulder protons are eliminated by the sensitized photooxidation and shifted upfield on deprotonation. At high pH, the characteristic Faraday A term also disappears. These observations suggest that the essential histidine stabilizes the nascent thiolate via the ion pair formation to facilitate the reductase reaction catalyzed by lipoamide dehydrogenase.     

Cytochrome P-450 lowering effect of alkyl halides,correlation with decrease in arachidonic acid

Treatment of male rats with carbon tetrachloride, bromotrichloromethane, chloroform, 1,2-dibromoethane, 1-bromo-2-chloroethane, and 1,2-dibromo-3-chloropropane results in a decrease in cytochrome P-450 content and alterations in the relative content of fatty acids in hepatic microsomes. A high correlation was found between the loss of cytochrome P-450, the decrease in arachidonic acid (r=0.93), and the increases in linoleic (r=?0.91) and oleic acids (r=?0.89).     

Purification and comparative properties of NADPH-cytochrome P-450 reductase from rat and rainbow trout: Differences in temperature optima between reconstituted and microsomal trout enzymes

NADPH-cytochrome P-450 reductase has been purified to apparent homogeneity from liver microsomes of β-naphthoflavone-treated rats and rainbow trout. The apparent monomeric molecular weights were 75,000 and 77,000 for the rat and trout, respectively. Differences in amino acid composition were observed, particularly for lysine, glycine, threonine, and tyrosine. Analysis of the flavin composition showed that there were 0.97 mol of FAD and 0.92 mol of FMN per mol of rat reductase, whereas the values for the trout enzyme were 1.06 and 0.76 for FAD and FMN, respectively. Trout NADPH-cytochrome c reductase was inhibited by anti-rat antibody, but not to the same extent as was the rat enzyme. No precipitin lines between the trout reductase and rat antibody were observed on Ouchterlony plates. Peptide patterns, on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, following limited proteolysis were also markedly different. The trout enzyme was as effective, catalytically, as the rat enzyme in a reconstituted system that contained purified rat cytochrome P-448 and lipid. Comparison of ethoxyresorufin-O-deethylase temperature profiles with various combinations of purified trout and rat P-448, reductase, and lipid, in membranous and nonmembranous reconstitution systems, demonstrated that the lower temperature optimum in trout microsomes could only be reproduced when all three trout components were incorporated into liposomes. These results suggest that it is the structural organization of the mixed-function oxidase enzymes and lipid within trout microsomes which were responsible for the lower temperature optimum compared to rat.     

Destruction of cytochrome P-450 by 2-isopropyl-4-pentenamide and methyl 2-isopropyl-4-pentenoate: mass spectrometric characterization of prosthetic heme adducts and nonparticipation of epoxide metabolites.

Incubation of hepatic microsomes from phenobarbital-treated rats with methyl 2-isopropyl-4-pentenoate results in rapid destruction of the microsomal cytochrome P-450. The destruction does not occur in the absence of NADPH or with methyl 2-isopropylpentanoate. Administration of methyl 2-isopropyl-4-pentenoate to phenobarbital-pretreated rats leads to hepatic accumulation of a “green” pigment which, after methylation and purification, yields an abnormal porphyrin chromatographically and spectroscopically indistinguishable from that similarly obtained with 2-isopropyl-4-pentenamide (allylisopropylacetamide). Field desorption mass spectrometry showed that both abnormal porphyrins exhibited molecular ions at $\text{m}\text{e}$ 730. The mass spectrum of the zinc and copper complexes confirmed this value. Esterification in deuterated methanol of the amide-derived porphyrin showed that only two methyl esters were formed. Finally, methyl 4,5-epoxy-2-isopropylpentanoate and the known metabolites of 2-isopropyl-4-pentenamide were shown not to destroy cytochrome P-450. These results clearly establish that the carbonyl groups of the two destructive substrates are intimately involved in formation of the isolated porphyrin adducts, and exclude participation of the corresponding epoxide metabolites in the destruction of cytochrome P-450.     

Denitrosation of carcinostatic nitrosoureas by purified NADPH cytochrome P-450 reductase and rat liver microsomes to yield nitric oxide under anaerobic conditions

The nitrosoureas, CCNU (1-(2-chloroethyl)-3-(cyclohexyl)-1-nitrosourea) and BCNU (1,3-bis(2-chloroethyl)-1-nitrosourea) are representatives of a class of N-nitroso compounds which undergo denitrosation in the presence of NAD(P)H and deoxygenated hepatic microsomes from rats to yield nitric oxide (NO) and the denitrosated parent compound. Formation of NO during microsomal denitrosation of CCNU and BCNU was determined by three methods. With one procedure, NO was measured and concentration shown to increase over time in the head gas above microsomal incubations with BCNU. Two additional methods utilized NO binding to either ferrous cytochrome P-450 or hemoglobin to form distinct Soret maxima at 444 and 415 nm, respectively. Incubation of either BCNU or CCNU in the presence of NAD(P)H and deoxygenated microsomes resulted in the formation of identical cytochrome P-450 ferrous · NO optical difference spectra. Determination of the P-450 ferrous · NO extinction coefficient by the change in absorbance at 444 minus 500 nm allowed measurement of rates of denitrosation by monitoring the increase in absorbance at 444 nm. The rates of BCNU and CCNU denitrosation were determined to be 4.8 and 2.0 nmol NO/min/mg protein, respectively, for phenobarbital (PB) induced microsomes. For the purpose of comparison, the rate of [¹⁴C]CCNU (1-(2-[¹⁴C]chloroethyl)-3-(cyclohexyl)-1-nitrosourea turnover was examined by the isolation of [¹⁴C]CCU (1-(2-[¹⁴C] chloroethyl)-3-(cyclohexyl)-1-urea) from incubations that contained NADPH and deoxygenated PB-induced microsomes. These analyses showed stoichiometric amounts of NO and [¹⁴C]CCU being formed at a rate of 2.0 nmol/min/mg protein. Denitrosation catalysis by microsomes was enhanced by phenobarbital pretreatment and partially decreased by cytochrome P-450 inhibitors, SKF-525A, α-naphthoflavone (ANF), metyrapone, and CO, suggesting a cytochrome P-450-dependent denitrosation. However, in the presence of NADPH and purified NADPH cytochrome P-450 reductase reconstituted in dilauroylphosphatidylcholine, [¹⁴C]CCNU was shown to undergo denitrosation to [¹⁴C]CCU. Thus, NADPH cytochrome P-450 reductase could support denitrosation in the absence of cytochrome P-450.     

Purification of characterization of a minor form of hepatic microsomal cytochrome P-450 from rats treated with polychlorinated biphenyls

A minor form of hepatic microsomal cytochrome P-450 has been purified to apparent homogeneity from rats treated with the polychlorinated biphenyl mixture, Aroclor 1254. This newly isolated hemoprotein, cytochrome P-450_e, is inducible in rat liver by Aroclor 1254 and phenobarbital, but not by 3-methylcholanthrene. Two other hemoproteins, cytochromes P-450_b and P-450_c, have also been highly purified during the isolation of cytochrome P-450_e based on chromatographic differences among these proteins. By Ouchterlony double-diffusion analysis with antibody to cytochrome P-450_b, highly purified cytochrome P-450_e is immunochemically identical to cytochrome P-450_b but does not cross-react with antibodies prepared against other rat liver cytochromes P-450 (P-450_a, P-450_c, P-450_d) or epoxide hydrolase. Purified cytochrome P-450_e is a single protein-staining band in sodium dodecyl sulfate-polyacrylamide gels with a minimum molecular weight (52,500) slightly greater than cytochromes P-450_b or P-450_d (52,000) but clearly distinct from cytochromes P-450_a (48,000) and P-450_c (56,000). The carbon monoxide-reduced difference spectral peak of cytochrome P-450_e is at 450.6 nm, whereas the peak of cytochrome P-450_b is at 450 nm. Ethyl isocyanide binds to ferrous cytochromes P-450_e and P-450_b to yield two spectral maxima at 455 and 430 nm. At pH 7.4, the 455:430 ratio is 0.7 and 1.4 for cytochromes P-450_b and P-450_e, respectively. Metyrapone binds to reduced cytochromes P-450_e and P-450_b (absorption maximum at 445–446 nm) but not cytochromes P-450_a, P-450_c, or P-450_d. Metabolism of several substrates catalyzed by cytochrome P-450_e or P-450_b reconstituted with NADPH-cytochrome c reductase and dilauroylphosphatidylcholine was compared. The substrate specificity of cytochrome P-450_e usually paralleled that of cytochrome P-450_b except that the rate of metabolism of benzphetamine, benzo[a]pyrene, 7-ethoxycoumarin, hexobarbital, and testosterone at the 16α-position catalyzed by cytochrome P-450_e was only 15–25% that of cytochrome P-450_b. In contrast, cytochrome P-450_e catalyzed the 2-hydroxylation of estradiol-17β more efficiently (threefold) than cytochrome P-450_b. Cytochrome P-450_d, however, catalyzed the metabolism of estradiol-17β at the greatest rate compared to cytochromes P-450_a, P-450_b, P-450_c, or P-450_e. The peptide fragments of cytochromes P-450_e and P-450_b, generated by either proteolytic or chemical digestion of the hemoproteins, were very similar but not identical, indicating that these two proteins show minor structural differences.     

Assay of ribonucleotide reductase activity in intact permeabilized hamster cells: an evaluation

An intact cell assay system, based on Tween-80 permeabilization can be used to investigate ribonucleotide reductase activity in a variety of mammalian cell lines. An important consideration in the use of intact cells is the presence of other nucleotide metabolizing activities. The influence of these activities on estimates of pyrimidine (CDP) and purine (ADP) reductase in permeabilized hamster cells has been examined. Studies on the incorporation of label from CDP and ADP into RNA indicated that a very small proportion of the reductase substrates was eventually incorporated into RNA during routine enzyme assays, and would have no detectable effect on activity estimates. The possibility that the products of the reaction (dCDP and dADP) were eventually phosphorylated and incorporated into DNA was also examined, and it was found that proper permeabilization of the cells eliminated or greatly reduced loss of deoxyribonucleotides to DNA. An analysis by HPLC of nucleotides present during CDP and ADP reductase reactions showed that various kinases and phosphatases were active in permeabilized cells, as all levels of phosphorylation of nucleotide substrates and allosteric effectors were detected. The base composition of the nucleotides added to the assay systems were not altered. Although movement of phosphates occurred during the assay, the concentrations of substrates quickly reached equilibrium (within 1 min) with their respective nucleosides and nucleotides, resulting in a relatively constant although reduced concentration of CDP or ADP substrates during the 20-min assay. Similarly the levels of allosteric effectors, ATP for pyrimidine and dGTP for purine reductase activities, declined within the first minute of the assays and quickly reached an equilibrium with their respective adenine or guanine containing nucleotides during most of the reaction time. Although useful approximations of intracellular reductase activity can be obtained without correcting for modified nucleotide concentrations, precise determinations can be calculated when these alterations are taken into consideration. For example, estimates of intracellular K_m values for CDP closely resembled those reported with highly purified mammalian enzyme preparations in other studies. Clearly, the intact cell assay system provides worthwhile information about mammalian ribonucleotide reductase in its physiologically relevant environment.     

Metal(II) complexes of 1-formylisoquinoline thiosemicarbazone: their preparation,characterization and antitumour activity

Complexes of Co(II), Ni(lI), Cu(II), Zn(II) and Pt(II) with 1-formylisoquinoline thiosemicarbazone (1-iqtsc-H) were prepared and characterized by elemental analyses, conductance measurement and spectral studies. On the basis of these studies a distorted octahedral structure for [Co(1-iqtsc)₂]·2H₂O, a distorted trigonal-bipyramidal structure for [Ni- (1-iqtsc-H)Cl₂], [Cu(1-iqtsc-H)Cl₂] and [Zn(1-iqtsc- H)(OAc)₂]·H₂O and a square-planar structure for [Pt(1-iqtsc)Cl] are suggested. All these metal(II) complexes were screened for their antitumour activity in the P388 lymphocytic leukaemia test system in mice. Except for Pt(Il), the complexes were found to possess significant activity; the Ni(II) complex showed a T/C value of 161 at the optimum dosage.