Uncoupling of allosteric and oligomeric regulation in a functional hybrid enzyme constructed from Escherichia coli and human ribonucleotide reductase.

An N-terminal-domain (NTD) and adjacent catalytic body (CB) make up subunit-α of ribonucleotide reductase (RNR), the rate-limiting enzyme for de novo dNTP biosynthesis. A strong linkage exists between ligand binding at the NTD and oligomerization-coupled RNR inhibition, inducible by both dATP and nucleotide chemotherapeutics. These observations have distinguished the NTD as an oligomeric regulation domain dictating the assembly of inactive RNR oligomers. Inactive states of RNR differ between eukaryotes and prokaryotes (α6 in human versus α4ß4 in Escherichia coli , wherein ß is RNR's other subunit); however, the NTD structurally interconnects individual α2 or α2 and ß2 dimeric motifs within the respective α6 or α4ß4 complexes. To elucidate the influence of NTD ligand binding on RNR allosteric and oligomeric regulation, we engineered a human- E. coli hybrid enzyme (HE) where human-NTD is fused to E. coli -CB. Both the NTD and the CB of the HE bind dATP. The HE specifically partners with E. coli -ß to form an active holocomplex. However, although the NTD is the sole physical tether to support α2 and/or ß2 associations in the dATP-bound α6 or α4ß4 fully inhibited RNR complexes, the binding of dATP to the HE NTD only partially suppresses HE activity and fully precludes formation of higher-order HE oligomers. We postulate that oligomeric regulation is the ultimate mechanism for potent RNR inhibition, requiring species-specific NTD-CB interactions. Such interdomain cooperativity in RNR oligomerization is unexpected from structural studies alone or biochemical studies of point mutants.

Enhanced subunit interactions with gemcitabine-5'-diphosphate inhibit ribonucleotide reductases.

Ribonucleotide reductases (RNRs) catalyze the conversion of nucleotides to deoxynucleotides in all organisms. The class I RNRs are composed of two subunits, alpha and beta, with proposed quaternary structures of alpha2beta2, alpha6beta2, or alpha6beta6, depending on the organism. The alpha subunits bind the nucleoside diphosphate substrates and the dNTP/ATP allosteric effectors that govern specificity and turnover. The beta2 subunit houses the diferric Y* (1 radical per beta2) cofactor that is required to initiate nucleotide reduction. 2',2'-difluoro-2'-deoxycytidine (F2C) is presently used clinically in a variety of cancer treatments and the 5'-diphosphorylated F2C (F2CDP) is a potent inhibitor of RNRs. The studies with [1'-(3)H]-F2CDP and [5-(3)H]-F2CDP have established that F2CDP is a substoichiometric mechanism based inhibitor (0.5 eq F2CDP/alpha) of both the Escherichia coli and the human RNRs in the presence of reductant. Inactivation is caused by covalent labeling of RNR by the sugar of F2CDP (0.5 eq/alpha) and is accompanied by release of 0.5 eq cytosine/alpha. Inactivation also results in loss of 40% of beta2 activity. Studies using size exclusion chromatography reveal that in the E. coli RNR, an alpha2beta2 tight complex is generated subsequent to enzyme inactivation by F2CDP, whereas in the human RNR, an alpha6beta6 tight complex is generated. Isolation of these complexes establishes that the weak interactions of the subunits in the absence of nucleotides are substantially increased in the presence of F2CDP and ATP. This information and the proposed asymmetry between the interactions of alphanbetan provide an explanation for complete inactivation of RNR with substoichiometric amounts of F2CDP.

Detection of a new substrate-derived radical during inactivation of ribonucleotide reductase from Escherichia coli by gemcitabine 5'-diphosphate.

Ribonucleotide reductases (RNRs) play a central role in replication and repair by catalyzing the conversion of nucleotides to deoxynucleotides. Gemcitabine 5'-diphosphate (F2CDP), the nucleoside of which was recently approved by the FDA for treatment of pancreatic cancer, is a potent mechanism-based inhibitor of class I and II RNRs. Inactivation of the Eschericia coli class I RNR is accompanied by loss of two fluorides and one cytosine. This RNR is composed of two homodimeric subunits: R1 and R2. R1 is the site of nucleotide reduction, and R2 contains the essential diferric-tyrosyl radical cofactor. The mechanism of inactivation depends on the availability of reductant. In the presence of reductant [thioredoxin (TR)/thioredoxin reductase (TRR)/NADPH or dithiothreitol], inhibition results from R1 inactivation. In the absence of reductant with prereduced R1 and R2, inhibition results from loss of the essential tyrosyl radical in R2. The same result is obtained with C754S/C759S-R1 in the presence of TR/TRR/NADPH. In both cases, tyrosyl radical loss is accompanied by formation of a new stable radical (0.15-0.25 equiv/RNR). EPR studies in 2H2O, with [U-2H]R1, and examination of the microwave power saturation of the observed signal, indicate by process of elimination that this new radical is nucleotide-based. In contrast to all previously investigated 2'-substituted nucleotide inhibitors of RNR, inactivation is not accompanied by formation of a new protein-associated chromophore under any conditions. The requirement for reductant in the R1 inactivation pathway, the lack of chromophore on the protein, the loss of two fluoride ions, and the stoichiometry of the inactivation all suggest a unique mechanism of RNR inactivation not previously observed with other 2'-substituted nucleotide inhibitors of RNR. This unique mode of inactivation is proposed to be responsible for its observed clinical efficacy.

Cellular delivery of nucleoside diphosphates: a prodrug approach.

PURPOSE: This study is concerned with cellular delivery/generation of 2'-azido-2'-deoxyuridine and -deoxycytidine diphosphate (N3UDP or N3CDP), potent inhibitors of ribonucleotide reductase. It characterizes the phosphorylation steps involved in the conversion of 2'-azido-2'-deoxyuridine (N3Urd) and 2'-azido-2'-deoxycytidine (N3Cyd) to the corresponding diphosphates and explores a prodrug approach in cellular delivery of the inhibitor which circumvents the requirement of deoxynucleoside kinases. METHODS: Cell growth of CHO and 3T6 cells of known deoxycytidine kinase level was determined in the presence of N3Urd and N3Cyd. Activity of ribonucleotide reductase was determined in the presence of the azidonucleosides as well as their mono- or di-phosphates in a Tween 80-containing permeabilizing buffer. A prodrug of 5'-monophosphate of N3Urd was prepared and its biological activity was evaluated with CHO cells as well as with cells transfected with deoxycytidine kinase. RESULTS: N3Urd failed to inhibit the growth of both cell lines, while N3Cyd was active against 3T6 cells and moderately active against CHO cells. These results correlate with the deoxycytidine kinase levels found in the cells. Importance of the kinase was further established with the finding that the nucleoside analogs were inactive as reductase inhibitors in a permeabilized cell assay system while their mono- and di-phosphates were equally active. The prodrug was active in cell growth inhibition regardless of the deoxycytidine kinase level. CONCLUSIONS: The azidonucleosides become potent inhibitors of the reductase by two sequential phosphorylation steps. The present study indicates that the first step to monophosphate is rate-limiting, justifying a prodrug approach with the monophosphate.

Synthesis and antiproliferative activity of cytidine-5'-alkylphosphonophosphates and structurally related compounds.

The chemical synthesis of cytidine-5'-alkyl- and cytidine-5'-alkyl (acyl)deoxyglycerophosphonophosphates is reported. The compounds obtained represent a novel class of cytostatically active agents based on phospholipids, which inhibit the growth of various tumor cell lines in vitro. They are phosphono analogs of the cytidine-5'-diphosphate-diacylglycerol (CDP-DAG) possessing a structurally modified lipid moiety and a phospholipase C-resistant P-C bond. The antiproliferative efficacy of the cytidine-5'-alkylphosphonophosphates strongly depends on the alkyl chain length. The cytidine-5'-hexadecylphosphonophosphate was found to be the most effective compound tested in this study. Its cytostatic effect was distinctly higher than that of the alkyl(acyl) deoxyglycero derivatives and of the corresponding diphosphates. The structure of the new compounds were confirmed by fast atom bombardment mass spectrometry (FAB). The FAB fragmentation pattern is discussed.

Characterization and biological significance of sialyl alpha 2-3galactosyl beta 1-4xylosyl beta 1-(4-methylumbelliferone) synthesized in cultured human skin fibroblasts.

Human skin fibroblasts were incubated in the presence of a fluorogenic xyloside, 4-methyl-umbelliferyl-beta-D-xyloside (Xyl-MU), then the cultured medium was recovered, concentrated with a lyophilizer, and dialyzed against distilled water. The structures of the Xyl-MU derivatives purified from the dialyzable fraction were investigated. In addition to established glycosaminoglycans-MU (GAGs-MU), Gal-Gal-Xyl-MU, Gal-Xyl-MU, sulphate-GlcA-Xyl-MU, GlcA-Xyl-MU, and Xyl-Xyl-MU, which were induced by Xyl-MU, an oligosaccharide having fluorescence was purified using a combination of gel filtration, ion-exchange chromatography and high-performance liquid chromatography, then subjected to carbohydrate composition analysis, enzyme digestion, Smith degradation, 1H-NMR, and ion-spray mass spectrometric analysis. From the data obtained, the oligosaccharide was considered to have the structure SA alpha 2-3Gal beta 1-4Xyl beta 1-MU. The amount of MU-oligosaccharide in the cell culture increased with time and was dependent on the amount of Xyl-MU added. Its production was also different from that of Gal-Gal-Xyl-MU and Gal-Xyl-MU, which are biosynthetic intermediates of GAG-MU. Addition of CDP, an inhibitor of sialytransferase, to the cell culture medium increased the secretion of GAG-MU. These results suggest that SA-Gal-Xyl-MU production may be related to the regulation of GAG-MU biosynthesis.

Differential scanning calorimetric study of the complexes of myosin subfragment 1 with nucleoside diphosphates and vanadate or beryllium fluoride.

It has been recently shown by differential scanning calorimetry (DSC) that the formation of stable complexes of myosin subfragment 1 (S1) with Mg-ADP and orthovanadate (Vi) or beryllium fluoride (BeFx) causes a global conformational change in the S1 molecule which is reflected in a pronounced increase of S1 thermal stability and in a significant change of S1 domain structure [Shriver, J. W., & Kamath U. (1990) Biochemistry 29, 2556-2564; Levitsky, D. I., Shnyrov, V. L., Khvorov, N. V., Bukatina, A. E., Vedenkina, N. S., Permyakov, E. A., Nikolaeva, O. P., & Poglazov, B. F. (1992) Eur. J. Biochem. 209, 829-835; Bobkov, A. A., Khvorov, N. V., Golitsina, N. L., & Levitsky, D. I. (1993) FEBS Lett. 332, 64-66]. In this work, which continues the previous investigations, we report on a DSC study of the complexes of S1 with various nucleoside diphosphates (NDP). In the absence of Vi or BeFx the various Mg(2+)-NDP and Mg(2+)-PPi had a similar effect on the S1 conformation. All of them had practically no influence on the temperature of the thermal transition but increased its sharpness. However, in the presence of Vi or BeFx the effects of Mg(2+)-NDP complexes were quite different from each other and strongly depended on the base structure of NDP; their effectiveness in inducing conformational changes in S1 and the stability of these complexes decreased in the following order: ADP > CDP >> UDP >> IDP > GDP.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of glycogenin-catalyzed glucosyl and xylosyl transfer by cytidine 5'-diphosphate and related compounds.

The self-glucosylation of beef kidney glycogenin was inhibited by the following pyrimidine nucleotides and nucleotide sugars, listed in order of decreasing effectiveness: CDP-glucose, CDP, UDP-xylose, UDP-N-acetylglucosamine, UDP-galactose, UDP, CTP, CDP-choline, UDP-glucuronic acid, beta-S-UDP-glucose, and CMP. In contrast, the purine nucleotide sugars, ADP-glucose and GDP-glucose, were essentially ineffective, as was the pyrimidine nucleoside, cytidine. UDP-Xylose may be utilized by glycogenin as an alternative sugar donor instead of UDP-glucose (Rodén, L., Ananth, S., Campbell, P., Manzella, S., and Meezan, E. (1994) J. Biol. Chem. 269, 11509-11513) and therefore presumably inhibited the glucosyl transfer reaction by being a competitive substrate. Like glucosyl transfer, xylosyl incorporation into glycogenin was also inhibited effectively by CDP. On the other hand, UDP-xylose:proteoglycan core protein xylosyltransferase (EC 2.4.2.26) was not affected by CDP, nor was it inhibited by UDP-glucose. Addition of CDP or UDP-glucose to reaction mixtures containing both enzymes therefore made it possible to assay xylosyltransferase EC 2.4.2.26 reliably without the extensive product characterization that is otherwise necessary. The CDP effect on glycogenin further allowed the development of an improved procedure for the purification of this enzyme, in which specific elution of an affinity matrix (UDP-glucuronic acid-agarose) was carried out with CDP as the eluant.

The interaction of serotonin depletion with anxiolytics and antidepressants on reticular-elicited hippocampal RSA.

Hippocampal rhythmical slow activity (RSA) can be elicited by stimulation of the midbrain reticular formation. Buspirone, chlordiazepoxide and imipramine are all anxiolytic and have all been shown to decrease the frequency of RSA. All these compounds have been suggested to affect, directly or indirectly, 5-HT metabolism and function. The present experiments tested the possibility that buspirone, chlordiazepoxide and imipramine reduce RSA frequency via 5-HT1A autoreceptors. Rats received buspirone (10 mg/kg), chlordiazepoxide (5 mg/kg) and imipramine (30 mg/kg) after 5-HT depletion with p-chlorophenylalanine (pCPA, 100 mg/kg/day for 3 days or 350 mg/kg/day for 2 days) or after pretreatment with 5-HTP (40 mg/kg, to replete 5-HT) as well as pCPA. The frequency-reducing effects produced by buspirone and chlordiazepoxide were unchanged by either dose of pCPA, whereas the frequency-reducing effect of imipramine was completely eliminated by the high dose of pCPA. Pindolol, but not beta-blockers (a combination of metoprolol and ICI118,551), was able to block the effect of imipramine on RSA frequency. Pindolol has been reported to block the effects of buspirone but not chlordiazepoxide. These data suggest that: (1) buspirone obtains its frequency-reducing effects via pre- or post-synaptic 5-HT1A receptors rather than 5-HT1A autoreceptors; (2) chlordiazepoxide obtains its frequency-reducing effect via benzodiazepine receptors and GABA with no direct or indirect involvement of 5-HT systems; and (3) imipramine obtains its frequency-reducing effect by increasing the availability of 5-HT at 5-HT1A receptors which are not autoreceptors.

The AAPT1 gene of soybean complements a cholinephosphotransferase-deficient mutant of yeast.

Aminoalcoholphosphotransferases (AAPTases) utilize diacylglycerols and cytidine diphosphate (CDP)-aminoalcohols as substrates in the synthesis of the abundant membrane lipids phosphatidylcholine and phosphatidylethanolamine. A soybean cDNA encoding an AAPTase that demonstrates high levels of CDP-choline:sn-1,2-diacylglycerol cholinephosphotransferase activity was isolated by complementation of a yeast strain deficient in this function and was designated AAPT1. The deduced amino acid sequence of the soybean cDNA showed nearly equal similarity to each of the two characterized AAPTase sequences from yeast, cholinephosphotransferase and ethanolaminephosphotransferase (CDP-ethanolamine:sn-1,2-diacylglycerol ethanolaminephosphotransferase). Moreover, assays of soybean AAPT1-encoded enzyme activity in yeast microsomal membranes revealed that the addition of CDP-ethanolamine to the reaction inhibited incorporation of 14C-CDP-choline into phosphatidylcholine in a manner very similar to that observed using unlabeled CDP-choline. Although DNA gel blot analysis suggested that AAPT1-like sequences are represented in soybean as a small multigene family, the same AAPT1 isoform isolated from a young leaf cDNA library was also recovered from a developing seed cDNA library. Expression assays in yeast using soybean AAPT1 cDNAs that differed only in length suggested that sequences in the 5'leader of the transcript were responsible for the negative regulation of gene activity in this heterologous system. The inhibition of translation mediated by a short open reading frame located 124 bp upstream of the AAPT1 reading frame is one model proposed for the observed down-regulation of gene activity.

Effects of hypoxia and acute osmotic stress on intermediary metabolism in Leishmania promastigotes.

This study further explores the effects of hypoxia and acute osmotic stress on intermediary metabolism of Leishmania major and Leishmania donovani. Late log phase promastigotes were washed and incubated with glucose as the sole exogenous carbon source, and rates of glucose consumption and product formation were measured as a function of osmotic strength (610, 305, and 167 mOsm kg-1) and pO2 (95, 10, and 0% O2) in the presence of 5% CO2. Very mild hypoxia dramatically altered flux through the pathways of intermediary metabolism and increased the rates of production of the major metabolites, thus confirming the presence of a low-affinity O2 sensor which was active under all osmolalities tested. The data also require that as pO2 is lowered towards anoxia an endogenous carbohydrate source is mobilized. Under aerobic conditions, acute hypo-osmotic stress had little effect on product formation, whereas acute hyperosmotic stress altered metabolism in a manner similar to mild hypoxia, with the exception of decreasing the rates of acetate and succinate production. It was also shown in L. donovani promastigotes that the effects of anoxia and hyperosmolality were not additive. Thus, separate sensors with partially overlapping actions are involved in the metabolic responses to hypoxia and hyperosmolality. There was no apparent species-specificity for the responses to pO2 and osmotic stress. Uncoupling with carbonyl cyanide p-trifluoromethoxyphenylhydrazone caused changes in metabolite flux patterns which differed from the changes caused by either hypoxia or acute osmotic stress, while rotenone and calcium ionophore A23187 had no significant effects. The identity of the sensors responsive to pO2 and osmolality, and the mechanisms by which they regulate flux through the pathways of intermediary metabolism, require further study.

5'-CMP stimulates phospholipase A-mediated hydrolysis of phosphatidylinositol in permeabilized pituitary GH3 cells.

We showed previously that 5'-CMP activates PtdIns-Ins base exchange and reversal PtdIns synthase in permeabilized rat pituitary GH3 cells. Here we report another effect of 5'-CMP on PtdIns metabolism in these cells. In permeabilized GH3 cells prelabelled with [3H]Ins and incubated in buffer with LiCl and a free Ca2+ concentration of 0.1 microM but without added Ins, 5'-CMP stimulated formation of glycerophospho[3H]inositol (GroP[3H]Ins) after a lag period of at least 5 min. This effect was concentration-dependent; the apparent Km was 0.30 +/- 0.02 mM. CDP and CTP stimulated GroPIns formation less effectively than did 5'-CMP, but cytidine, 2'-CMP, 3'-CMP, 5'-AMP and 5'-GMP had no effect. 5'-CMP stimulated formation of lysoPtdIns also. In permeabilized GH3 cells prelabelled with [3H]arachidonic acid, 5'-CMP stimulated release of [3H]arachidonic acid without a measurable lag period. These data show that 5'-CMP stimulates a phospholipase A activity in permeabilized GH3 cells that hydrolyses PtdIns.

Effects of deoxyadenosine on ribonucleotide reductase in adenosine deaminase-deficient lymphocytes.

To explore the relationship between ribonucleotide reductase and immunodysfunction in adenosine deaminase deficiency, the effects of deoxyadenosine on ribonucleotide reductase in ADA-deficient lymphocytes was investigated. An assay system for ribonucleotide reductase in intact permeabilized lymphocytes was developed to approximate physiological conditions. The activity of cytidine diphosphate (CDP) reductase in resting but not in proliferating lymphocytes in culture was inhibited by 1 to 10 mumol/L deoxyadenosine. The resting cells were protected from the toxicity of 1 mumol/L deoxyadenosine by 5 mmol/L nicotinamide or 30 mumol/L deoxycytidine and from that of 10 mumol/L deoxyadenosine by 30 mumol/L deoxycytidine. These findings suggest that depletion of nicotinamide adenine dinucleotide might be the principal cause of death in resting lymphocytes with ADA deficiency. It is concluded that the mechanism of deoxyadenosine toxicity on non-replicating lymphocytes, which may not be mediated by ribonucleotide reductase inhibition, is closely related to the mechanism of immunodysfunction in patients with ADA deficiency.

CMP activates reversal of phosphatidylinositol synthase and base exchange by distinct mechanisms in rat pituitary GH3 cells.

CMP is known to activate phosphatidylinositol (PtdIns)/inositol (Ins) base exchange and has been reported to activate reversal of PtdIns synthase also. Because it is possible that PtdIns synthase acting in the reverse direction, followed by re-incorporation of ambient Ins, could be responsible for base-exchange activity, we characterized these processes in rat pituitary GH3 cells. In permeabilized GH3 cells prelabelled with [3H]Ins and incubated in buffer with LiCl but without added Ins, CMP stimulated rapid accumulation of [3H]Ins and decreases in [3H]PtdIns; the Km for CMP was 1.7 mM. CDP and CTP were less effective, whereas 2'-CMP, 3'-CMP, other nucleoside monophosphates and cytidine did not influence this process. In permeabilized cells prelabelled to isotopic equilibrium with [3H]Ins and [32P]Pi, CMP stimulated decreases in both the 32P and 3H labelling of PtdIns, but did not increase that of [32P]phosphatidic acid. These findings demonstrate that in the absence of added Ins the effect of CMP is not via activation of base exchange nor via a phospholipase D, but by reversal of PtdIns synthase. In permeabilized cells prelabelled with [3H]Ins and [32P]Pi, unlabelled Ins inhibited loss of 32P labelling of PtdIns caused by CMP while markedly stimulating loss of 3H labelling of PtdIns and release of [3H]Ins. These data demonstrate that Ins inhibits reversal of PtdIns synthase, but stimulates base exchange. We conclude that in GH3 cells reversal of PtdIns synthase and PtdIns/Ins base exchange are both stimulated by CMP, but are distinct processes.

Fosfadecin and fosfocytocin, new nucleotide antibiotics produced by bacteria.

Two new nucleotide antibiotics, fosfadecin and fosfocytocin, have been isolated from the culture filtrates of Pseudomonas viridiflava PK-5 and Pseudomonas fluorescens PK-52, respectively. These antibiotics were purified by column chromatographies using adsorption, gel filtration and ion exchange resins. On the basis of the spectroscopic and degradation studies, the chemical structures of fosfadecin and fosfocytocin were determined. These antibiotics were either enzymatically or chemically hydrolyzed to generate fosfomycin and a new antibiotic, fosfoxacin, which are also produced in the culture filtrates. They showed antibacterial activity against Gram-positive and Gram-negative bacteria. The antibacterial activity of these nucleotide antibiotics was weaker than that of fosfomycin and fosfoxacin.

Herpes simplex virus type 1 ribonucleotide reductase: selective and synergistic inactivation by A1110U and its iron complex.

2-Acetylpyridine-5-[(dimethylamino)thiocarbonyl]thiocarbonohydr azone (A1110U) inactivated herpes simplex virus Type 1 ribonucleotide reductase (EC 1.17.4.1) by a first-order process (kinact) which had a maximum value (Mkinact) of 8 hr-1 and a Kd that was less than 1 microM. The stable complex between iron and A1110U, (A1110U)2Fe+i, inactivated this enzyme with a Mkinact of 7 hr-1 and a Kd of 7 microM. Free A1110U and its iron-complex synergized as inactivators of the enzyme. For example, the kinact for the combination of 2 microM A1110U and 1 microM (A1110U)2Fe+i as independent inactivators was calculated to be about 9 hr-1, while the observed value was 32 hr-1. The bimolecular rate constant for inactivation of the viral enzyme by (A1110U)2Fe+i in the presence of a saturating concentration of A1110U was 2.5 10(7) M-1 hr-1 at 30 degrees. Human ribonucleotide reductase was less sensitive to the inhibitory effects of A1110U and its iron-complex. This enzyme was neither inhibited nor inactivated by A1110U and was weakly inhibited by (A1110U)2Fe+i. Furthermore, inactivation required the combination of A1110U and (A1110U)2Fe+i. The bimolecular rate constant for inactivation of human ribonucleotide reductase by (A1110U)2Fe+i in the presence of a saturating concentration of A1110U was considerably smaller (3.8 10(6) M-1 hr-1 at 37 degrees) than the analogous constant for the viral enzyme. Several iron-chelating reagents with unrelated structures substituted for free A1110U in its various roles with both enzymes. However, the iron complexes of these alternative chelators did not substitute for (A1110U)2Fe+i. The rates of inactivation of both enzymes were independent of the oxidation state of iron in (A1110U)2Fe+i and of CDP concentration. The inactivated enzymes were reactivated rapidly by FeSO4, but were not reactivated by CoCl2, CuSO4, or NiCl2. MnCl2 inhibited reactivation of the viral enzyme by FeSO4.

ADP and glucose as possible synergistic partners in the stimulation of liver glycogen synthase activation.

Glucose administered either intravenously or orally causes liver glycogen synthase activation independent of a rise in circulating insulin. In vitro, physiological concentrations of glucose stimulate synthase phosphatase activity but only in the presence of a second effector which reduced the A0.5 for glucose. Caffeine and certain methylxanthines have been in vitro models for a putative natural effector. The present study demonstrates that, in vitro, ADP also reduced the A0.5 for glucose comparable to the effect of caffeine. The maximum stimulation by glucose in the presence of caffeine or ADP was comparable. The effect of ADP was specific among the major nucleoside diphosphates. However, the A0.5 for ADP was greater than the normal liver concentration which does not change in response to either glucose or insulin administration. The effect of ADP appeared distinct from that of the methylxanthines since it was observed that at near saturating concentrations of ADP and of glucose, stimulation was increased by addition of theophylline. Similarly, addition of adenosine, a natural cell constituent, caused increased stimulation. Subsequently, it was shown that adenosine reduced the A0.5 for ADP to a nearly physiological concentration. Thus, while ADP is not the inducible putative effector which has been predicted it may be part of an intracellular amplification system for glycogen synthase activation which increases the sensitivity to an induced effector. The present work suggests that the effective concentration of the natural ligand may be less than originally anticipated. This work also suggests that the putative effector could be structurally related to adenosine. Phosphorylase phosphatase activity known to be stimulated by ADP and glucose is further stimulated by the combination which may be acting in synergy.

Activation of rat liver cytosolic phosphatidic acid phosphatase by nucleoside diphosphates.

Phosphatidic acid phosphatase (EC 3.1.3.4) was purified 30-fold by ammonium sulfate fractionation and hydroxyapatite chromatography from the soluble fraction of rat liver. ADP was found to stimulate the enzyme activity with half-maximal stimulation at 0.2 mM. Similar effects were seen when ADP was replaced by GDP or CDP. In contrast, ATP inhibited the enzyme; half-maximal inhibition observed at 0.2 mM. Again, the degree of inhibition did not differ when GTP or CTP replaced ATP. Thus, the structure of the base part of the nucleotide was not critical for mediating these effects. The positions of the phosphate groups in the nucleotide structure were however found to be of importance for the enzyme activity. Variations in the structure of the phosphate ester bound at the 5'-position had a pronounced effect on phosphatidic acid phosphatase activity. The effect of nucleotides depended on pH, and the inhibition by ATP was more pronounced at pH levels lower than 7.0, whereas the stimulatory effect of ADP was virtually the same from pH 6.0 to pH 8.0. The enzyme showed substrate saturation kinetics with respect to phosphatidic acid, with an apparent Km of 0.7 mM. Km increased in the presence of ATP, whereas both apparent Vmax and Km increased in the presence of ADP, suggesting different mechanisms for the action of the two types of nucleotides. The results indicated that physiological levels of nucleotides with a diphosphate or a triphosphate ester bound at the 5'-position of the ribose moiety influenced the activity of phosphatidic acid phosphatase. The possibility is discussed that these effects might be of importance for the regulation of triacylglycerol biosynthesis.

Mechanism-based inhibition of a mutant Escherichia coli ribonucleotide reductase (cysteine-225----serine) by its substrate CDP.

The B1 subunit of Escherichia coli ribonucleotide reductase (EC 1.17.4.1) has been overexpressed using the pT7-5/pGP1-2 system developed by Tabor and Richardson [Tabor, S. & Richardson, C. (1985) Proc. Natl. Acad. Sci. USA 82, 1074-1078]. This method has allowed the preparation of two mutant B1 subunits in which two of the four thiols postulated to be within the active site of the enzyme, Cys-225 and Cys-759, have been changed to serines. Incubation of the [Ser225]B1 mutant with the B2 subunit, [U-14C]CDP, and the allosteric effector ATP results in production of cytosine, destruction of the tyrosyl radical in B2, radiolabeling of the protein, and cleavage of the B1 subunit into two pieces of 26 and 61.5 kDa. This process is independent of the identity of reductant. The [Ser759]B1 mutant reduces CDP in the presence of thioredoxin/thioredoxin reductase at 7.7% the rate of wild-type B1. When dithiothreitol is utilized as reductant, however, the rate of CDP reduction with [Ser759]B1 is identical to that observed with wild type.

Pyrimidine nucleotides impair phosphoribosylpyrophosphate (PRPP) synthetase subunit aggregation by sequestering magnesium. A mechanism for the decreased PRPP synthetase activity in hereditary erythrocyte pyrimidine 5'-nucleotidase deficiency.

The activity of phosphoribosylpyrophosphate (PRPP) synthetase (ATP: D-ribose-5-phosphate pyrophosphotransferase, EC 2.7.6.1) is decreased in the erythrocyte in hereditary pyrimidine 5'-nucleotidase (P5N) deficiency. Given the increased pyrimidine nucleotide content of the P5N-deficient erythrocyte, we evaluated the effects of prototypic pyrimidine nucleotides on the activity of PRPP synthetase. In normal hemolysate a 1.0 mM combination of cytidine tri-, di- and monophosphate (CTP/CDP/CMP) inhibited PRPP synthetase activity and changed the ribose 5-phosphate (R5P) saturation curve from a hyperbola to a biphasic shape. Untreated crude hemolysate from P5N-deficient erythrocytes showed a biphasic R5P kinetic curve. Since the activity of PRPP synthetase is dependent on its state of subunit aggregation, we examined PRPP synthetase subunit aggregation using gel permeation chromatography. P5N-deficient erythrocytes had a decreased absolute amount of aggregated PRPP synthetase and almost a total loss of disaggregated PRPP synthetase. Using normal hemolysate, 1 mM CTP/CDP/CMP interfered with the ability of 1.0 mM ATP and 2.0 mM MgCl2 to promote PRPP synthetase subunit aggregation. Increasing the MgCl2 to 6.0 mM overcame the inhibitory effect of CTP/CDP/CMP. Thus, the decreased PRPP synthetase activity of the P5N-deficient erythrocyte is due, at least in part, to the ability of the accumulated pyrimidine nucleotides to sequester magnesium and to interfere with the subunit aggregation of PRPP synthetase.