Assay of ribonucleotide reduction in nucleotide-permeable hamster cells

Ribonucleotide reduction was measured in Chinese hamster ovary cells made permeable to nucleotides by treatment with the detergent Tween-80. When compared to the respective ribonucleotide reductase activity in partially purified cell extracts, CDP and GDP reductase activities in permeabilized cells responded in a similar fashion to dithiothreitol, pH, MgCl2, FeCl3, substrate concentration and the presence of positive or negative allosteric effectors. At low protein concentrations both CDP and GDP reduction with whole cells increased linearly with cell number and was greater than the activity in corresponding cell extracts. Permeabilized cells were used to measure the level of CDP and GDP reductase in a hamster cell line resistant to the cytotoxic effects of hydroxyurea. The hydroxyurea-resistant cell line contained four to ten times more CDP and GDP reductase activity compared to parental or revertant cell lines. The permeabilized cell assay was also used to measure CDP and GDP reductase activities in Chinese hamster ovary cells synchronized by isoleucine starvation. CDP reductase activity was low in G1 arrested cells but increased 10-fold by 16 hours after the readdition of isoleucine to the growth medium. GDP reductase, which is present at much higher levels, is similarly induced after isoleucine addition, but only by 2-fold. The maximum activity of both CDP and GDP reductase occurred from 14 to 16 hours after isoleucine addition, which corresponded to the period of maximum DNA synthesis.     

Studied on ribonucleoside-diphosphate reductase in permeable animal cells. I. Reversible permeabilization of mouse L cells with dextran sulfate

The treatment of mouse L cells with sodium dextran sulfate 500 for 20 min at 4 °C rendered them selectively permeable to small molecules. The degree of permeabilization was reproducible and depended on dextran sulfate concentration: as the dextran sulfate concentration increased, the cells became more permeable to the dye erythrosin B, lost the ability to incorporate [³H]thymidine into DNA, and acquired the capacity for nucleotide-dependent incorporation of [³H]dTTP into DNA and for K⁺- and ATP-dependent incorporation of [³H]lysine into protein. The ability to engage in macromolecular synthesis indicated that the permeabilized cells had retained a considerable amount of integrity, and this was confirmed by the observation that the cells could regain viability after an appropriate repair treatment. Permeabilization was rapidly reversed by incubating the cells in suspension culture medium plus 10% calf serum or in a defined medium composed of NaCl, NaH₂PO₄, glucose, glutamine, and CaCl₂, calcium ion being the most critical component. Using this minimally disruptive permeabilization procedure, it was possible to assay the enzyme ribonucleoside-diphosphate reductase in situ. The enzymatic conversion of CDP and GDP to the corresponding deoxyribonucleotides was studied; enzyme activity was almost entirely intracellular and responded to the allosteric activators and inhibitors that are known to modulate the activity of ribonucleotide reductase in vitro.     

Inhibition of mammalian ribonucleotide reductase by a dinucleotide produced in eucaryotic cells.

HS3, a highly phosphorylated dinucleoside originally purified from the fungus Achlya, has been isolated from Chinese hamster ovary cells undergoing glutamine starvation. The HS3 compounds obtained from the fungal and mammalian sources exhibited similar physical and chemical properties. This unusual dinucleotide may be an important regulator of eucaryotic ribonucleoside diphosphate reductase activity; for 50 micrometer HS3, isolated from either mammalian or fungal cells, significantly inhibited CDP reduction in Achlya or hamster cell preparations, but only marginally affected the activity of the enzyme from E. coli. Studies with HS3 isolated from Achlya and partially purified mammalian ribonucleotide reductase indicated that the compound noncompetitively inhibited the reduction of varying concentrations of the substrates CDP, ADP and GDP with Ki values of 23 micrometer, 14 micron and 16 micron respectively. These inhibitor concentrations are well below the estimated intracellular levels of HS3 in glutamine starved cells and suggest that HS3 inhibition of ribonucleotide reduction may be responsible for the rapid inhibition of DNA synthesis seen under these culture conditions.     

Ribonucleotide reductase from wild type and hydroxyurea-resistant chinese hamster ovary cells

The kinetic properties of partially purified ribonucleotide reductase from Chinese hamster ovary cells have been investigated. Double reciprocal plots of velocity against substrate concentration were found to be linear for three the substrates tested, and yielded apparent Km values of 0.12 mM for CDP, 0.14 mM for ADP and 0.026 mM for GDP. Hydroxyurea, a potent inhibitor of ribonucleotide reduction, was tested against varying concentrations of ribonucleotide substrates and inhibited the enzyme activity in an uncompetitive fashion. Intercept replots were linear and exhibited Ki values for hydroxyurea of 0.08 mM for CDP reduction, 0.13 mM for ADP reduction and 0.07 mM for GDP reduction. Guanazole, another inhibitor of ribonucleotide reductase, interacted with the enzyme in a similar manner to hydroxyurea showing an uncompetitive pattern of inhibition with CDP reduction and yielding a Ki value of 0.57 mM. Partially purified ribonucleotide reductase from hydroxyurea-resistant cells was compared to enzyme activity from wild type cells. Significant differences were observed in the hydroxyurea Ki values with the three ribonucleotide substrates that were tested. Also, CDP reductase activity from the drug-resistant cells yielded a significantly higher Ki value for guanazole inhibition than the wild type activity. The properties of partially purified ribonucleotide reductase from a somatic cell hybrid constructed from wild type and hydroxyurea-resistant cells was also examined. The Ki value for hydroxyurea inhibition of CDP reductase was intermediate between the Ki values of the parental lines and indicated a codominant expression of hydroxyurea-resistance at the enzyme level. The most logical explanation for these results is that the mutant cells contain a structurally altered ribonucleotide reductase whose activity is less sensitive to inhibition by hydroxyurea or guanazole.     

Hydroxyurea-resistant mouse L cells with elevated levels of drug-resistant ribonucleotide reductase activity

We describe the isolation and partial characterization of a mouse L-cell line which is resistant to normally highly cytotoxic concentrations of hydroxyurea. A detailed analysis of the target enzyme ribonucleotide reductase in both wild-type and hydroxyurea-resistant enzyme preparations suggests that the drug-resistant cells form a ribonucleotide reductase enzyme which contains a structural alteration, rendering it less sensitive to inhibition by hydroxyurea. K₁ values for hydroxyurea inhibition of ribonucleotide reduction in enzyme preparations from hydroxyurea-resistant cells were significantly higher than corresponding values from preparations from wild-type cells. The K_m for CDP reduction in enzyme preparations of drug-resistant cells was approximately threefold higher than the corresponding parental wild-type value. In addition, in vivo enzyme assays detected a major difference between the temperature profiles of ribonucleotide reduction in nucleotide-permeable drug-resistant and wild-type cells. When levels of ribonucleotide reductase activity were measured in vivo, it was found that the drug-resistant cells contained approximately 3 times the wild-type level of CDP reductase activity and twice wild-type level of GDP reductase activity. This combination of enhanced enzyme levels plus an altered sensitivity to drug inhibition can easily account for the drug-resistance phenotype. The properties of these hydroxyurea-resistant cells indicate that they will be useful for genetic and biochemical studies.This work was supported by the N.S.E.R.C. of Canada and the Muscular Dystrophy Association of Canada through research funds (J. A. W.) and by the N.R.C. of Canada through a graduate scholarship (B. A. K.).     

Isolation and initial characterization of a series of Chlamydia trachomatis isolates selected for hydroxyurea resistance by a stepwise procedure.

Chlamydiae are obligate intracellular bacteria that are dependent on eukaryotic host cells for ribonucleoside triphosphates but not deoxyribonucleotide triphosphates. Ribonucleotide reductase is the only enzyme known to catalyze the direct conversion of a ribonucleotide to a deoxyribonucleotide. Hydroxyurea inhibits ribonucleotide reductase by inactivating the tyrosine free radical present in the small subunit of the enzyme. In this report, we show that Chlamydia trachomatis growth is inhibited by hydroxyurea in both wild-type mouse L cells and hydroxyurea-resistant mouse L cells. Hydroxyurea was used as a selective agent in culture to isolate, by a stepwise procedure, a series of C. trachomatis isolates with increasing levels of resistance to the cytotoxic effects of the drug. One of the drug-resistant C. trachomatis isolates (L2HR-10.0) was studied in more detail. L2HR-10.0 retained its drug resistance phenotype even after passage in the absence of hydroxyurea for 10 growth cycles. In addition, L2HR-10.0 was cross resistant to guanazole, another inhibitor of ribonucleotide reductase. Results obtained from hydroxyurea inhibition studies using various host cell-parasite combinations indicated that inhibition of host cell and C. trachomatis DNA synthesis by hydroxyurea can occur but need not occur simultaneously. Crude extract prepared from highly purified C. trachomatis reticulate bodies was capable of reducing CDP to dCDP. The CDP reductase activity was not inhibited by monoclonal antibodies to the large and small subunits of mammalian ribonucleotide reductase, suggesting that the activity is chlamydia specific. The CDP reductase activity was inhibited by hydroxyurea. Crude extract prepared from drug-resistant L2HR-10.0 reticulate bodies contained an elevation in ribonucleotide reductase activity. In total, our results indicate that C. trachomatis obtains the precursors for DNA synthesis as ribonucleotides with subsequent conversion to deoxyribonucleotides catalyzed by a chlamydia-specific ribonucleotide reductase.     

Adenylate cyclase activation by GTP analogs

Benznidazole (a nitroimidazole derivative used for the treatment of Chagas' disease) is reduced by rat liver microsomes to the nitro anion radical, as indicated by ESR spectroscopy. Addition of benznidazole to rat liver microsomes produced an increase of electron flow from NADPH to molecular oxygen, and generation of both superoxide anion and hydrogen peroxide. The benznidazole-stimulated O₂ consumption and O₂^? formation was greatly inhibited by NADP⁺ and p-chloromercuribenzoate but not by SKF-525-A and metyrapone. The former inhibitions indicated the involvement of NADPH-cytochrome P-450 (c) reductase, while the lack of inhibition by SKF-525-A and metyrapone ruled out any major role for cytochrome P-450 in benznidazole reduction. In contrast to nifurtimox, a nitrofuran derivative (R. Docampo and A. O. M. Stoppani, 1979, Arch. Biochem. Biophys.197, 317–321), benznidazole was not reduced to the nitro anion radical, nor did it stimulate oxygen consumption, O₂^? production, and H₂O₂ generation by Trypanosoma cruzi cells or microsomal fractions. A different mechanism of benznidazole toxicity in T. cruzi and the mammalian host is postulated.     

Production of 2-Methylisocitric Acid from n-Paraffins by Mutants of Candida lipolytica

Vitamin B₁₂-dependent ribonucleotide reductase purified from Rhizobium meliloti catalyzes the reduction of 5′-diphosphates of guanosine, adenosine, cytidine and uridine (GDP, ADP, CDP and UDP). The enzyme activities were regulated by Mg²⁺ and deoxyribonucleoside triphosphate effectors as follows: in the presence of Mg²⁺, allosteric effector deoxyguanosine triphosphate (dGTP) had the most stimulatory effect on reduction of ADP and UDP; deoxyadenosine triphosphate (dATP) on reduction of CDP; and thymidine triphosphate (dTTP) on reduction of GDP. These stimulatory effectors were active at a low concentration of 10 μm. Other deoxyribonucleotides may be negative or weakly positive effectors. Without effectors, the rate profile of ADP and GDP reduction showed a sigmoidal curve. In the absence of Mg²⁺, the activities of the reductase showed nearly maximal levels, and the addition of effectors rather decreased the activities, except in the case of UDP reduction which was most strongly stimulated by dGTP. The effect of Mg²⁺ can be replaced by Ca²⁺. Monovalent cations such as Na⁺ and K⁺ had a negligible effect on the activities of ribonucleotide reductase.     

Nucleoside 5'-triphosphate analogs as positive and negative effectors of mammalian ribonucleotide reductase

Ribonucleotide reductase activity is strongly regulated by nucleoside 5'-triphosphates acting as positive and negative effectors. With the use of dGTP analogs, araGTP and dITP, it was found that the structural requirements of dGTP to serve as a positive effector of ADP reductase were not the same as the requirements for dGTP to serve as a negative effector of CDP and ADP reductase activities. The dTTP analogs methylenedTTP and dideoxyTTP also gave different responses in terms of activating GDP reductase activity and inhibiting CDP and ADP reductase activities. Etheno-ATP and etheno-dATP were inactive as positive and negative effectors, respectively, of CDP reductase activity. DideoxyATP was less active than dATP as a negative effector. Formycin ATP was a very poor substitute for ATP as a positive effector of CDP reductase. These studies indicate that the effector sites are very specific in terms of binding nucleoside triphosphates as positive or negative modulators of ribonucleotide reductase activity.     

Irreversible inhibition of ribonucleotide reductase from Ehrlich tumor cells by a modulator analog

Periodate-oxidized ATP (ATP-PI) was prepared and studied as a modulator analog of ATP in the ribonucleotide reductase system from Ehrlich tumor cells. ATP-PI could not replace ATP as an activator of CDP reduction, but was found to be an effective inhibitor of both CDP and ADP reduction. The inhibition was time dependent with 1 μM ATP-PI causing 100% inhibition after 20 hrs. The inhibition was shown to be irreversible by the Ackermann-Potter plot and enzyme activity was not restored by passage of the ATP-PI-treated enzyme over a Sephadex G-25 column. ¹⁴C-ATP-PI eluted with the protein peak on Sephadex G-25 chromatography.     

Ferredoxin:NADP+ oxidoreductase as a target of Cd2+ inhibitory action--biochemical studies

The ferredoxin:NADP+ oxidoreductase (FNR) catalyses the ferredoxin-dependent reduction of NADP+ to NADPH in linear photosynthetic electron transport. The enzyme also transfers electrons from reduced ferredoxin (Fd) or NADPH to the cytochrome b₆f complex in cyclic electron transport. In vitro, the enzyme catalyses the NADPH-dependent reduction of various substrates, including ferredoxin, the analogue of its redox centre - ferricyanide, and the analogue of quinones, which is dibromothymoquinone. This paper presents results on the cadmium-induced inhibition of FNR. The K_i value calculated for research condition was 1.72 mM.FNR molecule can bind a large number of cadmium ions, as shown by the application of cadmium-selective electrode, but just one ion remains bound after dialysis. The effect of cadmium binding is significant disturbance in the electron transfer process from flavin adenine dinucleotide (FAD) to dibromothymoqinone, but less interference with the reduction of ferricyanide. However, it caused a strong inhibition of Fd reduction, indicating that Cd-induced changes in the FNR structure disrupt Fd binding. Additionally, the protonation of the thiol groups is shown to be of great importance in the inhibition process. A mechanism for cadmium-caused inhibition is proposed and discussed with respect to the in vitro and in vivo situation.     

Dihydrofolate reductase from amethopterin-resistant Lactobacillus casei. Effects of pH, salts, temperature, and source of NADPH on enzyme activity and substrate specificity studies.

Activity analyses of pure dihydrofolate reductase from amethopterin-resistant Lactobacillus casei conducted with commercial sources of NADPH yielded a progression of nonlinear assay tracings whose shapes were both pH dependent and reminiscent of classical product inhibition. The extent of curving of the assay tracings was dependent on the source and age of the commercial NADPH and was enhanced as the pH was decreased from 7.5 to 5.0. Under these conditions a “pseudo”-pH-activity profile, exhibiting a maximal specific activity of 9 units/mg of protein between pH 7.0 and 7.5, was found. In contrast, freshly prepared NADPH provided strictly linear assay tracings over the pH range of 8.5 to 5.0, yielding uniformly higher specific activities than those observed with commercial NADPH. The new pH-activity profile was characterized by a broad optimum between pH 5.0 and 6.0, with a maximal specificity activity of 24.9 units/ mg in 0.1m potassium phosphate in the absence of added salt. The curving phenomenon and pseudo-pH optimum observed with commercial NADPH is attributed to the presence of minor but potent inhibitory impurities in these coenzyme preparations. Optimal concentrations of monovalent (~0.1 m) and divalent (~0.05 m) salts activated the enzyme between 1.5- and 1.7-fold, resulting in maximal specific activities in the range of 34 to 39 units/mg. A similar extent of activation was observed in 0.8 m Tris-acetate buffer, pH 5.5. At concentrations of monovalent salts above 0.5 m and of divalent salts above 0.2 m a reduction in salt-dependent activation and, in some cases, inhibition of activity were obtained. Substrate specificity studies indicated that the V for folate at saturating levels is 1% of that for dihydrofolate. Deamino-NADPH yielded V values 1.4-fold higher than that for NADPH, while acetylpyridine-NADPH and thio-NADPH provided values 6.5- and 235-fold lower, respectively, than the value with the natural coenzyme. Gel electrophoresis studies reflected a similar trend of selectivity in the interaction of NADPH and its analogs to form stable binary complexes. Stable ternary complexes of enzyme and amethopterin were formed with NADPH, deamino-NADPH, thio-NADPH, and acetylpyridine-NADPH. Although neither dihydrofolate nor NADP⁺ and its analog form stable complexes with L. casei dihydrofolate reductase, both NADP⁺ and deamino-NADP⁺ interact with enzyme and dihydrofolate to generate stable ternary complexes.     

Induction of hemoglobin-hydrolase activity by the thiol-protease inhibitors leupeptin and antipain in adult rat liver cells in primary culture

Addition of the inhibitors of thiol-protease leupeptin and antipain to adult rat hepatocytes in primary culture caused rapid inhibition of α-N-benzoylarginine-β-naphthylamide (BANA)²-hydrolase and induction of the acid-protease, hemoglobin (Hb)-hydrolase. This induction was inhibited completely by cycloheximide and puromycin, and partially by actinomycin D. Addition of leupeptin did not affect the activity of acid phosphatase, cytochrome c oxidase, or NADPH cytochrome c reductase or protein synthesis. Pepstatin, an inhibitor of acid-protease, did not induce BANA-hydrolase activity. These findings suggest that inhibitors of thiol-protease specifically induced acid-protease in lysosomes by some unknown mechanism.     

Redox interconversion of Escherichia coli glutathione reductase. A study with permeabilized and intact cells

Summary The redox interconversion of Escherichia coli glutathione reductase has been studied both in situ, with permeabilized cells treated with different reductants, and in vivo, with intact cells incubated with compounds known to alter their intracellular redox state.The enzyme from toulene-permeabilized cells was inactivated in situ by NADPH, NADH, dithionite, dithiothreitol, or GSH. The enzyme remained, however, fully active upon incubation with the oxidized forms of such compounds. The inactivation was time-, temperature-, and concentration-dependent; a 50% inactivation was promoted by just 2 M NADPH, while 700 M NADH was required for a similar effect. The enzyme from permeabilized cells was completely protected against redox inactivation by GSSG, and to a lesser extent by dithiothreitol, GSH, and NAD(P)⁺. The inactive enzyme was efficiently reactivated in situ by physiological GSSG concentrations. A significant reactivation was promoted also by GSH, although at concentrations two orders of magnitude below its physiological concentrations. The glutathione reductase from intact E. coli cells was inactivated in vivo by incubation with DL-malate, DL-isocitrate, or higher L-lactate concentrations. The enzyme was protected against redox inactivation and fully reactivated by diamide in a concentration-dependent fashion. Diamide reactivation was not dependent on the synthesis of new protein, thus suggesting that the effect was really a true reactivation and not due to de novo synthesis of active enzyme. The glutathione reductase activity increased significantly after incubation of intact cells with tert-butyl or cumene hydroperoxides, suggesting that the enzyme was partially inactive within such cells. In conclusion, the above results show that both in situ and in vivo the glutathione reductase of Escherichia coli is subjected to a redox interconversion mechanism probably controlled by the intracellular NADPH and GSSG concentrations.     

Identification, Development, and Regional Distribution of Ribonucleotide Reductase in Adult Rat Brain

The development and regional distribution of ribonucleotide reductase (EC 1.17.4.1) were determined in rat brain. Ribonucleotide reductase was partially purified by ammonium sulfate fractionation (20-40% saturation). Enzyme activity was measured by a specific radiochemical assay. This method involved the reduction of [¹⁴C]cytidine diphosphate (CDP) to [¹⁴C]deoxy-cytidine diphosphate with subsequent hydrolysis and separation of the product ([¹⁴C]deoxycytidine) from substrate ([¹⁴C]cytidine) by Dowex-1-borate ion-exchange chro-matography. The specific activity of ribonucleotide reductase in whole brain of newborn rats was 3.78 ± 0.55 units (pmol/h)/mg protein (SEM; n = 6) and declined to 0.17 ± 0.01 units/mg protein (n = 7) at 10-12 weeks of age, with a further decline to 0.11 ± 0.01 units/mg protein (n = 3) at 1 year. Ribonucleotide reductase activity in rat liver decreased from 4.58 ± 0.62 units/mg protein (n = 3) in newborn animals to 0.06 ± 0.01 units/mg protein (n = 7) at 10-12 weeks and was present at trace levels at 6 months of age. The decline in specific activity with age was not due to a change in the K_m for CDP. The K_m for CDP in brain of newborn and adult rats was 80-90 μM. In 10- to 12-week-old rats, the specific activity of ribonucleotide reductase was similar in the various regions of the brain tested except for the brainstem, which had 50% lower specific activity than the whole brain. These results indicate that ribonucleotide reductase activity is present and widely distributed in adult rat brain.     

Isolation of dihydroxyacetone phosphate reductase from dunaliella chloroplasts and comparison with isozymes from spinach leaves

A dihydroxyacetone phosphate (DHAP) reductase has been isolated in 50% yield from Dunaliella tertiolecta by rapid chromatography on diethylaminoethyl cellulose. The activity was located in the chloroplasts. The enzyme was cold labile, but if stored with 2 molar glycerol, most of the activity was restored at 30°C after 20 minutes. The spinach (Spinacia oleracea L.) reductase isoforms were not activated by heat treatment. Whereas the spinach chloroplast DHAP reductase isoform was stimulated by leaf thioredoxin, the enzyme from Dunaliella was stimulated by reduced Escherichia coli thioredoxin. The reductase from Dunaliella was insensitive to surfactants, whereas the higher plant reductases were completely inhibited by traces of detergents. The partially purified, cold-inactivated reductase from Dunaliella was reactivated and stimulated by 25 millimolar Mg²⁺ or by 250 millimolar salts, such as NaCl or KCl, which inhibited the spinach chloroplast enzyme. Phosphate at 3 to 10 millimolar severely inhibited the algal enzyme, whereas phosphate stimulated the isoform in spinach chloroplasts. Phosphate inhibition of the algal reductase was partially reversed by the addition of NaCl or MgCl₂ and totally by both. In the presence of 10 millimolar phosphate, 25 millimolar MgCl₂, and 100 millimolar NaCl, reduced thioredoxin causes a further twofold stimulation of the algal enzyme. The Dunaliella reductase utilized either NADH or NADPH with the same pH maximum at about 7.0. The apparent K_m (NADH) was 74 micromolar and K_m (NADPH) was 81 micromolar. Apparent V_max was 1100 μmoles DHAP reduced per hour per milligram chlorophyll for NADH, but due to NADH inhibition highest measured values were 350 to 400. The DHAP reductase from spinach chloroplasts exhibited little activity with NADPH above pH 7.0. Thus, the spinach chloroplast enzyme appears to use NADH in vivo, whereas the chloroplast enzyme from Dunaliella or the cytosolic isozyme from spinach may utilize either nucleotide.     

Characterization of ribonucleotide reductase activity from mouse L cells

We describe some fundamental properties of the cytidine 5'-diphosphate (CDP) and guanosine 5'-diphosphate (GDP) reductase activity from mouse L cells. Both activities increased in a nonlinear fashion at low protein concentrations; this may be due to dissociation of two protein subunits of the enzyme at very low concentrations. CDP reductase activity was greatly stimulated in the presence of ATP and required magnesium and iron for maximum activity. GDP reductase required 2'-deoxythymidine 5'-triphosphate for maximum activity. Also apparent Km values of 0.14 mmol/l for CDP and 0.05 mmol/l for GDP were determined from double reciprocal plots of velocity against substrate concentrations. Activity in extracts of logarithmically growing mouse L cells was very high indicating that attempts to purify the enzyme from this source should be rewarding.     

Partial Purification and Characterization of d-Ribose-5-phosphate Reductase from Adonis vernalis L. Leaves

This study presents evidence for a new enzyme, d-ribose-5-P reductase, which catalyzes the reaction: d-ribose-5-P + NADPH + H⁺ → d-ribitol-5-P + NADP⁺. The enzyme was isolated from Adonis vernalis L. leaves in 38% yield and was purified 71-fold. The reductase was NADPH specific and had a pH optimum in the range of 5.5 to 6.0. The Michaelis constant value for d-ribose-5-P reduction was 1.35 millimolar. The enzyme also reduced d-erythrose-4-P, d-erythrose, dl-glyceraldehyde, and the aromatic aldehyde 3-pyridinecarboxaldehyde. Hexoses, hexose phosphates, pentoses, and dihydroxyacetone did not serve as substrates. d-Ribose-5-P reductase is distinct from the other known ribitol synthesizing enzymes detected in bacteria and yeast, and may be responsible for ribitol synthesis in Adonis vernalis.     

A fluorescent derivative of methotrexate as an intracellular marker for dihydrofolate reductase in L1210 cells

Fluorescein isothiocyanate coupled via a diaminopentyl-linking group to methotrexate (G.R. Gapski, J. M. Whiteley, J. I. Rader, P. L. Cramer, G. B. Henderson, V. Neef, and F. M. Huennekens, 1975, J. Med. Chem.18, 526–528) produces a fluorescent compound which is a strong inhibitor of dihydrofolate reductase (K_i = 60 nM) purified from L1210 murine leukemia cells. The fluorescent methotrexate derivative is preferentially taken up by methotrexate-resistant rather than wild-type L1210 cells grown in culture and acts as a visual marker for dihydrofolate reductase (K_D = 50 nM) during both purification and polyacrylamide electrophoresis. Uptake, which is proportional to the level of dihydrofolate reductase (often an indicator of the degree of acquired cellular methotrexate resistance), occurs slowly and via a route that is distinct from the carrier-mediated system utilized by these cells to transport methotrexate.     

Effects of purine nucleotides on photosynthetic electron transport in isolated chloroplasts

The effects of guanylates and inosinates (and adenylates) on phosphorylation, ferricyanide reduction, and light-induced H⁺ uptake in spinach chloroplasts were studied. GDP, GTP, IDP, and ITP (but not GMP and IMP) stimulated the light-induced H⁺ uptake and partially inhibited ferricyanide reduction. Phosphate, arsenate, and phlorizin increased the extent of inhibition by these nucleotides and decreased the values of their apparent dissociation constants for the inhibition process. In the presence of phosphate (or arsenate), restoration of ferricyanide reduction from the level inhibited by guanylates and inosinates was observed as phosphorylation (or arsenylation) proceeded. These results suggest that phosphorylation of GDP and IDP as well as ADP takes place after two steps of nucleotide binding to the chloroplast coupling factor 1. The apparent dissociation constants of GDP and IDP for these two binding steps were estimated to be about 34 and 38 µM for the first and 110 and 160 µM for the second step, respectively (at pH 8.3, 15°C). Above pH 9, the ratio (P/e) of the extent of phosphorylation to the increment of electron transport from the basal level measured in the presence of [ATP + Pi] or [ADP + Pi + phlorizin], became increasingly large. When the electron transport level inhibited by dicyclohexylcarbodiimide was taken to be the basal activity, the P/e ratio remained almost constant ( 1) from pH 7.0 up to 10.