Escherichia coli AspP activity is enhanced by macromolecular crowding and by both glucose-1,6-bisphosphate and nucleotide-sugars.

Escherichia coli ADP-sugar pyrophosphatase (AspP) is a "Nudix" hydrolase that catalyzes the hydrolytic breakdown of ADP-glucose linked to glycogen biosynthesis. Moderate increases of AspP activity in the cell are accompanied by significant reductions of the glycogen content. In vitro analyses showed that AspP activity is strongly enhanced by macromolecular crowding and by both glucose-1,6-bisphosphate and nucleotide-sugars, providing a first set of indicative evidences that AspP is a highly regulated enzyme. To our knowledge, AspP is the sole bacterial enzyme described to date which is activated by both G1,6P(2) and nucleotide-sugars.

The structural basis of the catalytic mechanism and regulation of glucose-1-phosphate thymidylyltransferase (RmlA).

The synthesis of deoxy-thymidine di-phosphate (dTDP)-L-rhamnose, an important component of the cell wall of many microorganisms, is a target for therapeutic intervention. The first enzyme in the dTDP-L-rhamnose biosynthetic pathway is glucose-1-phosphate thymidylyltransferase (RmlA). RmlA is inhibited by dTDP-L-rhamnose thereby regulating L-rhamnose production in bacteria. The structure of Pseudomonas aeruginosa RmlA has been solved to 1.66 A resolution. RmlA is a homotetramer, with the monomer consisting of three functional subdomains. The sugar binding and dimerization subdomains are unique to RmlA-like enzymes. The sequence of the core subdomain is found not only in sugar nucleotidyltransferases but also in other nucleotidyltransferases. The structures of five distinct enzyme substrate- product complexes reveal the enzyme mechanism that involves precise positioning of the nucleophile and activation of the electrophile. All the key residues are within the core subdomain, suggesting that the basic mechanism is found in many nucleotidyltransferases. The dTDP-L-rhamnose complex identifies how the protein is controlled by its natural inhibitor. This work provides a platform for the design of novel drugs against pathogenic bacteria.

ATP-sensitive and inwardly rectifying potassium channels in smooth muscle.

The properties and roles of ATP-sensitive (KATP) and inwardly rectifying (KIR) potassium channels are reviewed. Potassium channels regulate the membrane potential of smooth muscle, which controls calcium entry through voltage-dependent calcium channels, and thereby contractility through changes in intracellular calcium. The KATP channel is likely to be composed of members of the inward rectifier channel gene family (Kir6) and sulfonylurea receptor proteins. The KIR channels do not appear to be as widely distributed as KATP channels in smooth muscle and may provide a mechanism by which changes in extracellular K+ can alter smooth muscle membrane potential, and thereby arterial diameter. The KATP channels contribute to the resting membrane conductance of some types of smooth muscle and can open under situations of metabolic compromise. The KATP channels are targets of a wide variety of vasodilators and constrictors, which act, respectively, through adenosine 3',5'-cyclic monophosphate/protein kinase A and protein kinase C. The KATP channels are also activated by a number of synthetic vasodilators (e.g., diazoxide and pinacidil) and are inhibited by the oral hypoglycemic sulfonylurea drugs (e.g., glibenclamide). Together, KATP and KIR channels are important regulators of smooth muscle function and represent important therapeutic targets.

Stimulation of Ca(2+)-dependent membrane currents in Xenopus oocytes by microinjection of pyrimidine nucleotide-glucose conjugates.

Microinjection, but not extracellular application, of cytidine-5'-diphosphate-D-glucose (CDPG) has been shown to elicit Ca(2+)-dependent currents in Xenopus laevis oocytes. These responses were comparable to those of inositol-1,4,5-trisphosphate (InsP3) in being both rapid and dose dependent. For example, maximal amplitudes of CDPG-induced current were similar (approximately 365 +/- 75 nA at 1 microM CDPG) to those of InsP3. The CDPG currents were insensitive to removal of extracellular Ca2+, indicating the dependence on Ca2+ release from intracellular Ca2+ stores but not on Ca2+ entry through plasma membrane. CDPG-induced currents were reduced or abolished by pretreatment with thapsigargin, by injection of the Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, or by extracellular perfusion of the Cl- channel blocker niflumic acid but were insensitive to injection of the InsP3 antagonist heparin. These results suggest that CDPG induces Ca2+ discharge from intracellular Ca2+ stores via a mechanism distinct from that of InsP3 in Xenopus oocytes. Another pyrimidine nucleotide-glucose derivative, uridine-5'-diphosphate-alpha-D-glucose, also induced Ca(2+)-dependent currents, but the activity was lower than that of CDPG (maximal amplitude, 272 +/- 62 nA). Other nucleotide-glucose compounds (adenosine-5'-diphosphate-D-glucose, guanosine-5'-diphosphate-D-glucose, and thymidine-5'-diphosphate-D-glucose) had no current responses when injected into oocytes. After injection of CDPG, CDPG-induced Ca2+ release appeared to couple to a Ca2+ entry pathway similar to that coupled to InsP3. These results indicate that pyrimidine nucleotide-glucose conjugates may provide novel pharmacological tools for the study of Ca2+ signaling in oocytes.

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 biogenesis of muscle glycogen: regulation of the activity of the autocatalytic primer protein.

Glycogen synthesis in rabbit muscle is initiated by an autocatalytic, self-glucosylating protein (SGP). This creates a maltosaccharide primer on itself that in turn primes glycogen synthesis. Here we describe the powerful allosteric inhibition of autocatalysis by ATP and ADP, sufficient, at the physiological concentration of ATP in muscle, to inhibit autocatalysis. We also examined inhibition of self-glucosylation by analogues of the substrate UDPglucose. One of them, UDPxylose, acts as an alternative substrate and serves to block glucosylation. An improved purification procedure for the SGP is also described.

Inhibition of prolyl hydroxylase by poly(ADP-ribose) and phosphoribosyl-AMP. Possible role of ADP-ribosylation in intracellular prolyl hydroxylase regulation.

Poly(ADP-ribose) prepared by incubating NAD+ with rat liver nuclei inhibited the hydroxylation reaction catalyzed by purified prolyl hydroxylase (proline,2-oxoglutarate dioxygenase, EC 1.14.11.2) in vitro. Near complete inhibition of the enzyme was seen in the presence of 6 nM (ADP-Rib)18 with a Ki(app) of 1.5 nM. The monomer unit of poly(ADP-ribose), adenosine diphosphoribose (ADP-Rib), was found to be a weak inhibitor. On the other hand, poly(ADP-ribose)-derived phosphoribosyl-AMP (PRib-AMP) and its dephosphorylated product, ribosyl-ribosyl-adenine (Rib-RibA), inhibited the enzyme in nanomolar concentrations (Ki(app) 16.25 nM). The order of inhibition was (ADP-Rib)18 greater than PRib-AMP, Rib-RibA much greater than ADP-Rib. These results suggested that the 1"----2' ribosyl-ribosyl moiety in these compounds was involved in the inhibition of the enzyme. The possibility that intracellular prolyl hydroxylase is regulated by the involvement of ADP-ribosylation reactions was examined in confluent cultures of skin fibroblast treated with 20 mM lactate. The activity of prolyl hydroxylase was stimulated by 145% over that of untreated cultures. In the lactate-treated cells, the level of NAD+ was lowered and the total ADP-ribosylation of cellular proteins reduced by 40%. These observations imply that the lactate-induced activation of cellular prolyl hydroxylase is mediated by a reduction in ADP-ribosylation and that the synthesis and degradation of ADP-ribose moiety(ies) may possibly regulate prolyl hydroxylase activity in vivo.

The effect of poly(ADP-ribose) on interactions of DNA with histones H1, H3 and H4.

The competition between poly(ADP-ribose) and DNA for binding of the histones H1, H3 and H4 was studied, using a membrane filter-binding test. Poly(ADP-ribose) differently affected the interaction between DNA and the individual histones. While poly(ADP-ribose) effectively competed with DNA for binding of histone H4, it equally competed with DNA for binding of histone H3 and only inefficiently competed with DNA for binding of histone H1. Moreover, preformed complexes were correspondingly affected by the addition of competing polynucleotides, thereby also indicating the reversibility of complex formation. The competition capacity of DNA for histone H4 binding did not depend on DNA size. Competition experiments with poly(A) also indicated that poly(ADP-ribose) preferentially affected DNA-histone H4 interaction. The significance of the differing binding properties is discussed with regard to the possible molecular function of poly(ADP-ribose), especially with regard to its potential effect on nucleosome structure.

The role of C-4-substituted mannose analogues in protein glycosylation. Effect of the guanosine diphosphate esters of 4-deoxy-4-fluoro-D-mannose and 4-deoxy-D-mannose on lipid-linked oligosaccharide assembly.

The effects of the guanosine diphosphate esters of 4-deoxy-4-fluoro-D-mannose (GDP-4FMan) and 4-deoxy-D-mannose (GDP-4dMan) on reactions of the dolichol pathway in chick-embryo cell microsomal membranes were investigated by studies with chick-embryo cell microsomal membranes in vitro and in baby-hamster kidney (BHK) cells in vivo. Each nucleotide sugar analogue inhibited lipid-linked oligosaccharide biosynthesis in a concentration-dependent manner. GDP-4FMan blocked in vitro the addition of mannose to Dol-PP-(GlcNAc)2Man from GDP-Man (where Dol represents dolichol), but did not interfere with the formation of Dol-P-Man, Dol-P-Glc and Dol-PP-(GlcNAc)2. Although GDP-4FMan and Dol-P-4FMan were identified as metabolites of 4FMan in BHK cells labelled with [1-14C]4FMan, GDP-4FMan was a very poor substrate for GDP-Man:Dol-P mannosyltransferase and Dol-P-4FMan could only be synthesized in vitro if the chick-embryo cell membranes were primed with Dol-P. It therefore appears that the inhibition of lipid-linked oligosaccharide formation in BHK cells treated with 4FMan [Grier & Rasmussen (1984) J. Biol. Chem. 259, 1027-1030] is due primarily to a blockage in the formation of Dol-PP-(GlcNAc)2Man2 by GDP-4FMan. In contrast, GDP-4dMan was a substrate for those mannosyltransferases that catalyse the transfer of the first five mannose residues to Dol-PP-(GlcNAc)2. In addition, GDP-4dMan was a substrate for GDP-Man:Dol-P mannosyltransferase, which catalysed the formation of Dol-P-4dMan. As a consequence of this, the formation of Dol-P-Man, Dol-P-Glc and Dol-PP-(GlcNAc)2 may be inhibited through competition for Dol-P. In BHK cells treated with 10 mM-4dMan, Dol-PP-(GlcNAc)2Man9 was the major lipid-linked oligosaccharide detected. Nearly normal extents of protein glycosylation were observed, but very little processing to complex oligosaccharides occurred, and the high-mannose structures were smaller than in untreated cells.

Inhibition of simian virus 40 DNA replication in vitro by poly(ADP-ribosyl)ated diadenosine tetraphosphate.

Poly(ADP-ribosyl)ated diadenosine tetraphosphate was found to inhibit the in vitro replication of SV40 DNA. This inhibition was sensitive to preincubation of the polymer with either poly(ADP-ribose) glycohydrolase, diadenosine tetraphosphate (Ap4A):ADP phosphohydrolase, or an excess of free Ap4A. In contrast, the general catalytic activity of DNA polymerase was not inhibited by the poly(ADP-ribosyl)ated Ap4A when activated salmon sperm DNA was used as a template. These data suggest that inhibition of SV40 DNA replication by poly(ADP-ribosyl)ated Ap4A requires both the intact polymer and intact Ap4A moiety and is specific to events occurring during the initiation or elongation of a double-stranded template. Since both poly(ADP-ribose) and Ap4A accumulate in cultured mammalian cells following stresses which are accompanied by DNA strand breaks, these data are consistent with a model in which poly(ADP-ribosyl)ated Ap4A inhibits DNA replication following DNA damage.

Covalent modification of Escherichia coli ADPglucose synthetase with 8-azido substrate analogs.

Two photoaffinity labeling agents, 8-azido-ATP and 8-azido-ADPglucose, are substrate site specific probes of the Escherichia coli ADPglucose synthetase. In the presence of light (254 nm), the analogs specifically and covalently modify the enzyme with concomitant loss of catalytic activity. The substrate ADPglucose completely protects the enzyme from covalent modification by these 8-azido analogs. ATP, another substrate, also provides nearly 100% protection from 8-azido-ATP inactivation but is less efficient in protection of inactivation by 8-azido-ADPglucose. In the absence of light, however, ADPglucose synthetase can utilize either 8-azido-ATP or 8-azido-ADPglucose as substrates.

ADP-ribosylation induced changes in the conformation of the chromatin of the brain of developing rats.

Conformational changes in the chromatin of the cerebral hemisphere of 3-, 14- and 30-day old developing rats were studied before and after its ADP-ribosylation using DNase I and micrococcal nuclease (MNase). The rate and extent of digestion of chromatin by DNase I are the highest at 3-day and decline progressively thereafter. The rate and extent of digestion by MNase do not change during development. ADP-ribosylation of chromosomal proteins was carried out by incubating nuclei with NAD+ for 30 min and was followed by endonuclease digestion. Both the rate and extent of digestion by DNase I and MNase were enhanced after ADP-ribosylation which was the maximum for 3-day rats.

Poly(ADP-ribose) and the recovery from damage in Chinese hamster cells due to 5-bromodeoxyuridine photolysis.

Exposure of Chinese hamster cells to near-u.v. light, following the uniform incorporation of 5-bromodeoxyuridine (BrdUrd) into their DNA, resulted in cell killing that was close to exponential. An inhibitor of poly(ADP-ribose) synthesis, 3-aminobenzamide (3-ABA), enhanced the cytotoxic effect of this treatment when present for 2 h at 20 mM after light exposure. The dose modifying factor was 1.4. Under conditions that resulted in a sigmoidal survival curve (a 30 min BrdUrd pulse in S phase, followed 90 min later by light exposure) the effect of 3-ABA was to remove the shoulder of the survival curve with very little change in its final slope. Using various inhibitors of ADP-ribosyl transferase (ADPRT) the enhanced cell killing was found to correlate with the inhibitors' relative potency. Cellular NAD+, the substrate for poly(ADP-ribose) synthesis, was rapidly depleted after exposure. This depletion was largely prevented by 3-ABA; the activity of ADPRT increased with the fluence of near-u.v. light; and the concentration of cellular NAD+ decreased with exposure. ADPRT activity was maximal immediately after exposure to near u.v. light and then decayed to pre-exposure levels within 30 min (37 degrees C). The enhanced cytotoxicity of BrdUrd + near-u.v. light, when followed by 3-ABA treatment, disappeared at a rate similar to that of the decay in ADPRT activity. We conclude from these results that poly(ADP-ribose) synthesis is important for the recovery from BrdUrd photolysis damage in DNA. Because this damage and its repair are relatively specific (e.g. compared to ionizing radiation) and relatively easy to manipulate, it could serve as a model system for the study of the role of poly(ADP-ribose) in the repair of DNA damage.

Inhibition of platelet phosphatidylinositol synthetase by an analog of CDP-diacylglycerol.

Unpublished portions of the synthesis of a phosphinate-phosphonate diether analog of CDPdiacylglycerol are reported. The liponucleotide analog was found to be a very powerful inhibitor of platelet PI synthetase; kinetic data suggest a competitive inhibition mechanism. The structural specificity of CDPdiacylglycerol for liponucleotide-mediated biosynthetic reactions is discussed.

Activation of DNA ligase by poly(ADP-ribose) in chromatin.

To elucidate the molecular mechanism by which poly(ADP-ribose) participates in DNA excision repair, we examined the effect of poly(ADP-ribose) on DNA ligase activity in DNA/histone and reconstituted chromatin systems. The ligase activity was markedly inhibited by histones; the inhibition varied depending on histone subfraction and DNA/histone ratio. Poly(ADP-ribose), either exogenous or synthesized in situ by poly(ADP-ribose) synthetase, reversed this inhibition by histone almost completely. This effect was specific for poly(ADP-ribose); polyanions such as mRNA, rRNAs, tRNA, and synthetic poly(A) were less effective or ineffective. The ligase activity with reconstituted chromatin as the substrate was about half of that with free DNA whereas the activities with these two substrates were almost the same in the presence of poly(ADP-ribose) synthesized in situ. The polymers synthesized under these conditions were exclusively bound to the synthetase. Together with our previous finding that the enzyme is the main acceptor of the polymer in DNA-damaged cells, these results suggest that poly(ADP-ribose) in the synthetase-bound form counteracts inhibition by histones and activates DNA ligase to rejoin DNA strands in polynucleosomal structures.

Inhibition of DNA ligase activity by histones and its reversal by poly(ADP-ribose).

The molecular mechanism by which poly(ADP-ribose) participates in DNA repair was investigated using purified DNA ligase in DNA-histone systems. The ligase activity was markedly inhibited by histones; the inhibition was greater than 80% with histone H1 at concentrations equal to DNA. This inhibition was reversed efficiently by poly(ADP-ribose), either added exogenously or synthesized in situ with poly(ADP-ribose) synthetase. The reversal effect was specific for poly(ADP-ribose); other polyanions such as mRNA, rRNA's, tRNA, and synthetic poly(A) were less effective or totally ineffective. The poly(ADP-ribose) effect appeared to be caused by binding to histones and decreasing DNA-histone interactions. Poly(ADP-ribose) also had high affinity for DNA ligase. These observations, together with the findings of absolute dependence of poly(ADP-ribose) synthetase activity on DNA strand ends and extensive automodification of the synthetase in DNA-damaged cells, suggested a possible mechanism of poly(ADP-ribose) action in DNA repair, in which auto-modified poly(ADP-ribose) synthetase serves as a link between DNA damage and activation of DNA ligase.

Transfer of glucose in the biosynthesis of thyroid glycoproteins. I. Inhibition of glucose transfer to oligosaccharide lipids by GDP-mannose.

Thyroid rough microsomes catalyzed the synthesis of glucose-containing oligosaccharide lipids which were compared to those extracted from labeled thyroid cells and were found to be largely similar. Glucose transfer to these oligosaccharide lipids in the microsomal system was shown to be markedly depressed by an addition of GDPmannose. This sugar nucleotide, already at 1 microM, blocked dolichol-P-glucose synthesis, thus restraining further glucosylation of oligosaccharide lipids. Using this concentration of radioactive GDPmannose in the incubation medium lead to the detection of three glucose containing mannose-labeled oligosaccharide lipids. Double labeling experiments suggested a precursor-product relationship between them. Previously labeled oligosaccharide lipids, containing glucose or not were compared in their efficiency to act as donors of their oligosaccharide chain to an exogenous synthetic Asn-X-Thr containing peptide. It was found that the presence of glucose did not significantly influence the transfer. Free glucose was released during the reaction when using the glucose-labeled oligosaccharide lipid.

Cytotoxic liponucleotide analogs. II. Antitumor activity of CDP-diacylglycerol analogs containing the cytosine arabinoside moiety.

Among events limiting the effectiveness of cancer chemotherapy are the general lack of preferential uptake of anticancer drugs by tumor cells and the occurrence of drug resistance. An approach has been undertaken to explore whether or not such events can be favorably altered or circumvented therapeutically by development of a new class of anticancer molecules, cytotoxic liponucleotide analogs. The design of cytotoxic liponucleotide analogs encompasses both biochemical and biophysical aspects of liponucleotide and glycerophospholipid structure and metabolism. Several cytotoxic liponucleotide analogs of cytidine diphosphate diacylglycerol (CDPdiacylglycerol/dCDPdiacylglycerol), containing the 1-beta-D-arabinofuranosyl moiety, were tested for antitumor activity. Multispecies ara-CDPdiacylglycerol (1-beta-D-arabinofuranosylcytosine 5'-diphosphate diacylglycerol), which contains egg lecithin-derived mixed fatty acyl chains, was more active than 1-beta-D-arabinofuranosylcytosine (ara-C), a clinically used anticancer drug, against leukemia L5178Y and P388 ascites cells in mice. At identical single doses (50 mg/kg per day times 4) administered intraperitoneally, ara-CDPdiacylglycerol prolonged the life spans of L5178Y tumor-bearing mice 93%, while ara-C prolonged life by 18%. Ara-CDPdiacylglycerol increased life spans of P388 tumor-bearing mice by 357% at doses of 50 mg/kg per day times 4; the maximum increase with ara-C was 159% (85 mg/kg per day times 4). Against a P388 ara-C-resistant cell line (P/Ara-C, kinase deficient) in mice, ara-CDPdiacylglycerol prolonged survival times by 34% at a dose of 50 mg/kg per day times 4 and by 55% at 75 mg/kg per day times 4; the drug was not active against two other ara-C-resistant murine leukemia mutants (CA 55, CA5b). With cell line-derived human colon carcinoma HCT-15 grown in mice immunosuppressed with anti-thymocyte serum, ara-CDPdiacylglycerol at a single daily dose of 50 mg/kg per day times 4 significantly reduced tumor weights to 21% of the controls; the same dose schedule of ara-C caused no observable reduction of tumor weights. Results of these preliminary antitumor evaluations indicate that cytotoxic liponucleotide analogs should be investigated further to determine their potential as antineoplastic molecules.