The effect of folate and folate analogs upon dihydrofolate reductase and DNA synthesis in kidneys of normal mice

A single subcutaneous injection of folate, homofolate or MTX resulted in the inhibition of the activity of dihydrofolate reductase in homogenates prepared from the kidneys of normal mice. Stimulation of ³H-thymidine uptake occurred in the kidneys of treated animals approximately 30 hr after administration of either folate or homofolate, and reached a peak 72 hr after administration. The effects of folate and MTX on dihydrofolate reductase activity $\text{in}$$\text{vivo}$ were also determined. One hr after administration of 15 mg/kg methotrexate (MTX) or 300 mg/kg folate, enzyme activity $\text{in}$$\text{vivo}$ was inhibited by 90%.³H-deoxyuridine uptake was neither stimulated nor depressed after treatment with MTX. After administration of folate, uptake of ³H-deoxyuridine was stimulated at approximately 30 hr after drug-treatment and reached a peak at 72 hr after folate administration. Treatment with xanthopterin had no effect on the activity of dihydrofolate reductase $\text{in}$$\text{vitro}$. Xanthopterin stimulated uptake of both deoxyuridine and thymidine in an identical manner.The increased DNA synthesis that occurs in animals after treatment with agents that cause renal damage is distinct from the effect these agents have upon dihydrofolate reductase. Nucleoside incorporation after treatment with folate, homofolate, MTX or xanthopterin cannot be predicted on the basis of enzyme inhibition. Treatment with MTX, folate or homofolate results in enzyme inhibition which is not correlated with the uptake of deoxyuridine into DNA.     

Thyroxine stimulation of rate liver microsomal NADH-cytochrome c reductase in vitro

Rat liver microsomal NADH-cytochrome $\text{c}$ reductase activity is stimulated by 20 μM thyroxine $\text{in}$$\text{vitro}$. Thyroxine does not influence microsomal NADH-dichlorophenolindophenol reductase, NADPH-cytochrome $\text{c}$ reductase, or NADPH-dichlorophenolindophenol reductase activity. Stimulation of NADH-cytochrome $\text{c}$ reductase activity is not mediated by super-oxide and is likely due to enhanced reduction or oxidation of cytochrome $\text{b}$5.     

Toluene dioxygenase: purification of an iron-sulfur protein by affinity chromatography

Toluene dioxygenase, from $\text{Pseudomonas}\text{putida}$, oxidizes toluene to (+)-$\text{cis}$-1(S),2(R)-dihydroxy-3-methylcyclohexa-3,5-diene. The oxygenase-component of this multienzyme system was purified to homogeneity by a two-step procedure that utilized affinity and ion exchange chromatography. The purified enzyme would oxidize toluene only in the presence of NADH, ferrous iron and partially purified preparations of NADH cytochrome c reductase and an iron-sulfur protein (ferredoxin_TOL). Spinach NADPH cytochrome c reductase and NADPH could substitute for the $\text{Pseudomonas}$ reductase and NADH. The molecular weight of the oxygenase-component was determined to be 151,000 and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicated that the enzyme is composed of two subunits with molecular weights of 52,500 and 20,800. The absorption spectrum showed maxima at 550 (Shoulder), 450, 326 and 278 nm and preliminary experiments have indicated the presence of 2 gram atoms of iron and 2 gram atoms of acid-labile sulfur per mole of protein. The results indicate that the oxygenase-component of the toluene dioxygenase enzyme system is an iron-sulfur protein that has been designated ISP_TOL.     

Characterization of a membrane-bound nitrate reductase from Azotobacter chroococcum.

Nitrate reductase from the aerobic bacterium $\text{Azotobacter chroococcum}$ is a soluble enzyme with the characteristic features of Pichinoty's type B nitrate reductase. When cell suspensions of $\text{A. chroococcum}$ are repeatedly subcultured in liquid medium with nitrate as the nitrogen source, most of the nitrate-reducing activity is incorporated into the cytoplasmic membrane. The properties of the particulate nitrate reductase closely resemble Pichinoty's type A enzyme.     

Aldehyde content and cross-linking of type III collagen.

Three phosphorylated dinucleosides designated HS1, HS2, and HS3, isolated from the water-mould $\text{Achlya}$, were shown to significantly inhibit ribonucleotide reductase activity from $\text{Achlya}$. All three compounds decreased CDP reduction in fungal extracts by 50% at concentrations of 0.1mM. At the same concentration HS3 also inhibited partially purified CDP reductase from Chinese hamster ovary cells by at least 80% but showed only 10% inhibition with enzyme from $\text{E.}\text{coli}$. ADP reductase activity from $\text{Achlya}$ was inhibited 50% by both HS1 and HS3 at 0.1mM. HS2 however, showed no inhibitory effect on purine reduction. The levels of ribonucleotide reductase during the asexual growth cycle of $\text{Achlya}$ correlated with thymidine uptake into DNA and with the synthesis of HS compounds.     

Solubilization and partial purification of 4,16-androstadien-3-one synthesizing enzyme from boar testis microsomes and requirements for the enzymatic activity

The enzyme related to the synthesis of 4,16- androstadien-3-one from progesterone was investigated using boar testis. To elucidate the activity, in the soluble form, the lyophilized powder of 12,000 g supernatant from homogenate was first treated with n-butanol after which the enzyme could be extracted with 1 mM ethylenediaminete-traacetic acid(EDTA), 1 mM dithiothreitol(DTT) and 20 % glycerol. The enzyme was stable in this medium.The enzyme filtered through a column of Sephacryl S-200 super fine gel exhibited requirements of NADPH-cytochrome C reductase and phosphatidylcholine for maximum enzymatic activity. The requirements of reductase and phosphatidylcholine were not observed in the crude extract fraction. The enzyme separated by column chromatography with DEAE-cellulose required phosphatidylcholine for the synthesis but the reductase had no effect. These lines of evidence suggest that the activity of the enzyme, as related to synthesis of 4,16-androstadien-3-one from progesterone might be regulated by phosphatidylcholine and reductase, $\text{in situ}$.     

The obligatory requirement of cytochrome b5 in the p-nitroanisole O-demethylation reaction catalyzed by cytochrome P-450 with a high affinity for cytochrome b5

The role of cytochrome $\text{b}$₅ in the $\text{p}$-nitroanisole O-demethylation was studied with a reconstituted system containing a unique cytochrome P-450, isolated from rabbit liver microsomes as a species with a high affinity for cytochrome $\text{b}$₅. The maximal activity was obtained in the complete system consisting of cytochrome P-450, NADPH-cytochrome P-450 reductase, NADH-cytochrome $\text{b}$₅ reductase, and Triton X-100 in addition to cytochrome $\text{b}$₅. The omission of cytochrome $\text{b}$₅ from the complete system entirely abolished the activity. These results clearly show that cytochrome $\text{b}$₅ is obligatory in the reconstitute p-nitroanisole O-demethylation system, and this cytochrome P-450 probably interacts with cytochrome $\text{b}$₅ in such a way that the second electron is transferred from cytochrome $\text{b}$₅ and thus exhibits the demethylase activity.     

Liver microsomal electron transport systems. II. The involvement of cytochrome b5 in the NADH-dependent hydroxylation of 3,4-benzpyrene by a reconstituted cytochrome P-448-containing system

An enzyme system in $\text{rat liver microsomes}$ which catalyzes the NADH-dependent $\text{hydroxylation}$ of 3,4-benzpyrene has been reconstituted. The essential components of this NADH-mediated $\text{electron transport chain}$ are cytochrome b₅, NADH-cytochrome b₅ reductase, lipid, and cytochrome P-448.     

Interaction between the nitrate respiratory system of Escherichia coli K12 and the nitrogen fixation genes of Klebsiella pneumoniae.

Hybrids were constructed between $\text{E. coli}$ K12 $\text{chl}$^? mutants defective in nitrate respiration and an F′ plasmid carrying nitrogen fixation genes from $\text{K. pneumoniae}$. Examination of these hybrids showed that expression of $\text{nif}$_Kp⁺ genes does not require a functional nitrate respiratory system, but that nitrate reductase and nitrogenase do share some Mo-processing functions. For nitrate repression of nitrogenase activity, reduction of nitrate to nitrite is not necessary, but the Mo-X cofactor encoded by $\text{chl}$ genes is essential. Nitrate probably inhibits nitrogen fixation by affecting the membrane relationship of the nitrate and fumarate reduction systems such that the membrane cannot be energized for nitrogenase activity.     

Electron transport-linked nitrous oxide synthesis and reduction by Paracoccusdenitrificans monitored with an electrode

The nitrous oxide reductase activity of $\text{Paracoccus}\text{denitrificans}$ can be conveniently measured using an electrochemical method for determining N₂O. Introduction of this procedure has shown that (i) N₂O reductase activity is reversibly inhibited by oxygen; (ii) antimycin strongly inhibits electron flow to N₂O and that the inhibition is bypassed by tetramethyl-p-phenylenediamine; (iii) ascorbate plus tetramethyl-p-phenylenediamine, presumably by donating electrons to cytochrome c, is an effective reductant for nitrous oxide reductase; (iv) in the presence of the nitrous oxide reductase inhibitor, acetylene, N₂O is promptly produced from nitrite, consistent with the product of nitrite reductase being N₂O.     

Functional arginine residues involved in coenzyme binding by dihydrofolate reductase

Reaction of dihydrofolate reductase from amethopterin-resistant $\text{Lactobacillus}\text{casei}$ with phenylglyoxal results in a complete loss of enzyme activity. This inactivation is concomitant with the modification of five of a total of eight arginine residues per mole of enzyme. In the presence of the reduced coenzyme, NADPH, two of the five reactive arginines are protected from chemical modification with complete retention of enzyme activity. The results suggest the involvement of essential arginine residues at or near the coenzyme binding site and thus at or near the active center of the enzyme.     

NADPH-dependent reductase solubilized from microsomes by peroxidation and its activity

Isolated rat liver microsomes were subjected to enzymatic or non-enzymatic lipid peroxidation $\text{in vitro}$. NADPH-dependent cytochrome $\text{c}$ reductase activity was released from the microsomes into the media during peroxidation. This activity could be recovered from the media by DEAE-cellulose chromatography. The recovered enzyme retained high activity for the reduction of cytochrome $\text{c}$ and a lower level of activity for the reduction of cytochrome P-450. The active fractions were capable of enzymatically supporting the peroxidation of isolated mitochondria in the presence of organically complexed Fe⁺³ and NADPH, and in this respect the specific activity was found to be about ten times higher than in microsomes.     

The interaction of vinyl chloride with rat hepatic microsomal cytochrome P-450 in vitro.

In the presence of hepatic microsomes, vinyl chloride produces a ‘type I’ difference spectrum and stimulates carbon monoxide inhibitable NADPH consumption. A comparison of the binding and Michaelis parameters for the interaction of vinyl chloride with uninduced, phenobarbital and 3-methylcholanthrene induced microsomes indicates that the binding and metabolism of vinyl chloride is catalyzed by more than one type P-450 cytochrome, but predominantly by cytochrome P-450. Metabolites of vinyl chloride from this enzyme system decrease the levels of cytochrome P-450 and microsomal heme, but not cytochrome $\text{b}$₅ or NADPH-cytochrome $\text{c}$ reductase $\text{in vitro}$.     

Presence of peptide hormones that control gonadotropin secretion in extrahypothalamic areas of the brain.

The hypocholesterolemic drug clofibrate (ethyl-α-$\text{p}$-chlorophenoxyisobutyrate) was found to strongly suppress 3-hydroxy-3-methylglutaryl coenzyme A reductase (EC 1.1.1.34) activity in cultured mouse L cells at concentrations of 20 – 50 μg/ml. The half-life ($\text{t1}\text{2}$) of the reductase (approximately 120 min) was strongly reduced when L cells were incubated with cycloheximide plus a maximal inhibitory concentration of clofibrate (50 μg/ml), resulting in a $\text{t1}\text{2}$ value of 10 min. Preliminary kinetic analysis of the inhibition suggested that clofibrate increased the rate of inactivation (or degradation) of the reductase without affecting the rate of enzyme synthesis.     

EPR studies on a Hipip type iron-sulfur center in the succinate dehydrogenase segment of the respiratory chain

In addition to the two species of ferredoxin-type iron-sulfur centers (Centers S-1 and S-2), a Hipip-type iron-sulfur center (Center S-3) has been detected in the reconstitutively active soluble succinate dehydrogenases. E_m7,4 determined in a particulate, antimycin A sensitive succinate-cytochrome $\text{c}$ reductase is +60 ± 15 mV. This center is extremely labile towards oxygen in a manner similar to the reconstitutive activity of the dehydrogenase. Even freshly prepared reconstitutively active enzyme shows a considerably diminished content of Center S-3 relative to flavin and displays a partly modified spectra. All reconstitutively inactive dehydrogenases give rise to a highly modified or no Center S-3 spectra at all. These observations indicate that Center S-3 is a constituent of succinate dehydrogenase and plays a role in the physiological function of the enzyme, $\text{i.e.}$ transferring electrons most probably to ubiquinone.     

Regulation of 24,25-dihydroxyvitamin D-3 production by 1,25-dihydroxyvitamin D-3 and synthetic human parathyroid hormone fragment 1–34 in a cloned monkey kidney cell line (JTC-12)

Regulation of 25-hydroxyvitamin D-3 24-hydroxylase by 1,25-dihydroxyvitamin D-3 and synthetic human parathyroid hormone fragment 1–34 (PTH_1–34) was investigated using a cloned monkey kidney cell line, JTC-12. Treatment of the cells with 1,25-dihydroxyvitamin D-3 markedly enhanced the conversion of [³H]-25-hydroxyvitamin D-3 into a more polar metabolite. The metabolite was identified as 24,25-dihydroxyvitamin D-3 by normal phase and reverse phase high-performance liquid chromatography and periodate oxidation. The 24-hydroxylae activity appeared to follow Michaelis-Menten kintics, and 1,25-dihydroxyvitamin D-3 treatment increased the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ of 24-hydroxylase from 33 to 95 pmol/h per 10⁶ cells without affecting the apparent $\text{K}{}_{\mathrm{<mtext>m</mtext>}}$ value of the enzyme (220 nM in control vs. 205 nM in 1,25-dihydroxyvitamin D-3 treated cells). The enzyme activity reached a maximum between 4 and 8 h of treatment with 1,25-dihydroxyvitamin D-3. The dose of 1,25-dihydroxyvitamin D-3 required to cause a half-maximal stimulation was about 3 · 10^?10 M. The 1,25-dihydroxyvitamin D-3-induced increase in 24-hydroxylase was almost completely inhibited by the presence of 1 μM cycloheximide. Treatment of the cells with PTH_1–34 caused a dose-dependent increase in cyclic AMP production. Half-maximal stimulation of cyclic AMP production was obtained at about 5 · 10^?9 M PTH_1–34. When 2.4 · 10^?9 M PTH_1–34 was added after 1,25-dihydroxyvitamin D-3 treatment, the 1,25-dihydroxyvitamin D-3-stimulated 24-hydroxylase was inhibited to $\text{70.7 ± 2.9\%}$ of control. Higher concentrations of PTH_1–34 caused less inhibition of the enzyme activity. When cyclic AMP was added instead of PTH_1–34, the enzyme activity was also suppressed significantly. These results indicate that, in JTC-12 cells, 1,25-dihydroxyvitamin D-3 stimulates 24-hydroxylase in a dose- and time-dependent manner by increasing the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$ of the enzyme through a mechanism dependent upon new protein synthesis, and suggest that PTH_1–34 inhibits the 1,25-dihydroxyvitamin D-3-induced stimulation of 24-hydroxylase through its effect on cyclic AMP production.     

Diminished inhibition of succinate-cytochrome c reductase activity of resolved reductase complex by thenoyltrifluoroacetone in the presence of antimycin.

Antimycin, when added to resolved succinate-cytochrome $\text{c}$ reductase complex in amounts sufficient to partially inhibit succinate-cytochrome $\text{c}$ reductase activity, causes a decrease in inhibition of the residual succinate-cytochrome $\text{c}$ reductase activity by 2-thenoyltrifluoroacetone. Antimycin has no effect on the inhibition of succinate-ubiquinone reductase activity by 2-thenoyltrifluoroacetone. We propose that antimycin increases the steady state concentration of ubisemiquinone in the reductase complex, and that 2-thenoyltrifluoracetone is competitive with ubisemiquinone.     

A Novel function of cytochrome C (555, Chlorobium thiosulfatophilum) in oxidation of thiosulfate

Thiosulfate-cytochrome c-551 reductase derived from $\text{Chlorobium}\text{thiosulfatophilum}$ has been highly purified. The enzyme reduces cytochrome $\text{c}\text{-551 of}\text{C}\text{.}\text{thiosulfatophilum}$ in the presence of thiosulfate while cytochrome $\text{c}$-555 of the organism is not reduced by the enzyme. Cytochrome $\text{c}$-555 reacts with the enzyme at an appreciable rate only in the presence of cytochrome $\text{c}$-551. However, the reduction rate of cytochrome $\text{c}$-551 by the enzyme is greatly enhanced on addition of a catalytic amount of cytochrome $\text{c}$-555. Therefore, cytochrome $\text{c}$-555 seems to function as an effector on thiosulfate-cytochrome $\text{c}$-551 reductase as well as it acts as the electron donor to the light-excited chlorobium chlorophylls.     

Reconstituted hamster liver microsomal enzyme system for N-hydroxylation of the carcinogen 2-acetylaminofluorene

A procedure is described for the isolation of cytochrome P-450 fraction from hamster liver microsomes. It involves removal of NADPH-cytochrome c reductase activity by treatment with bacterial protease before solubilization with Triton X-100 and precipitation with ammonium sulfate. Reconstitution studies indicate that 2-acetylaminofluorene $\text{N}$-and ring-hydroxylation require both cytochrome P-450 fraction and the reductase fraction. $\text{N}$-hydroxylation activity of cytochrome P-450 fraction from 3-methylcholanthrene pretreated hamsters is different and severalfold greater than that of cytochrome P-450 fraction from controls. These results demonstrate for the first time an activation of a chemical carcinogen by a reconstituted cytochrome P-450 enzyme system.     

Spectral intermediates in the reaction of oxygen with purified liver microsomal cytochrome P-450

Stopped flow spectrophotometry has shown the occurrence of two distinct spectral intermediates in the reaction of oxygen with the reduced form of highly purified cytochrome P-450 from liver microsomes. As indicated by difference spectra, Complex I (with maxima at 430 and 450 nm) is rapidly formed and then decays to form Complex II (with a broad maximum at 440 nm), which resembles the intermediate seen in steady state experiments. In the reaction sequence, P-450_LM^red$\text{→}\text{O}{}_2$Complex I→Complex II→P-450_LM^ox the last step is rate-limiting. The rate of that step is inadequate to account for the known turnover number of the enzyme in benzphetamine hydroxylation unless NADPH-cytochrome P-450 reductase or cytochrome $\text{b}{}_5$ is added. The latter protein does not appear to function as an electron carrier in this process.