Purification and properties of orotate phosphoribosyltransferases from Escherichia coli K-12, and its derivative purine-sensitive mutant

Orotate phosphoribosyltransferase (OPT) was purified from both Escherichia coli K-12 strain and its derivative, a purine-sensitive mutant. The wild-type OPT had a molecular weight (M.W.) of 47,000 and was composed of two identical subunits (M.W. 23,500). The wild-type OPT showed maximum activity at pH 9.5, and no activity was seen in the absence of Mg2+ or Mn2+ ion. It also catalyzed a reverse reaction, namely orotidine-5'-monophosphate (OMP) pyrophosphorolysis. In this reverse reaction, tripolyphosphate, tetrapolyphosphate, and trimetaphosphate were also effective as pyrophosphate donors. The apparent Km values of the wild-type OPT were 30 microM for orotate and 40 microM for 5-phosphoribosyl 1-pyrophosphate (PRib-PP), and also 3.6 microM for OMP and 13 microM for PPi. On the other hand, the mutant OPT showed increased apparent Km values for all four substrates, 440 microM for orotate, 360 microM for PRib-PP, 33 microM for OMP, and 250 microM for PPi. The mutant OPT required a higher concentration of Mg2+ ion for maximum activity than the wild-type OPT. The nature of the purine-sensitive phenotype of the mutant is discussed from the standpoint of the reactivity of the mutant OPT, which has an increased Km value for PRib-PP (about 9-fold).     

Membrane binding induces destabilization of cytochrome c structure.

The effect of membranes binding on the structure and stability of ferricytochrome c was studied by Fourier-transform infrared spectroscopy and differential scanning calorimetry. Association of cytochrome c with phospholipid membranes containing phosphatidylglycerol as a model acidic phospholipid results in only slight, if any, perturbation of the protein secondary structure. However, upon membrane binding, there is a considerable increase in the accessibility of protein backbone amide groups to hydrogen-deuterium exchange, which suggests a lipid-mediated loosening and/or destabilization of the protein tertiary structure. A lipid-induced conformational perturbation of ferricytochrome c is also indicated by a marked decrease in the thermodynamic stability of the membrane-bound protein. Upon binding to membranes containing dimyristoylphosphatidylglycerol (DMPG) or dioleoylphosphatidylglycerol (DOPG) as a single lipid component, the denaturation temperature of ferricytochrome c decreases by approximately 30 degrees C. This is accompanied by a decrease in the calorimetric enthalpy of denaturation, particularly for the DMPG-associated protein. With ferricytochrome c bound to membranes containing a mixture of DMPG (or DOPG) and zwitterionic phosphatidylcholine, the extent of structural perturbation depends on the surface density of the negatively charged lipid head groups, becoming smaller with decreasing proportions of acidic phospholipid in the membrane. The observed destabilization of protein structure mediated by acidic phospholipids (and possibly formation of folding intermediates at the membrane surface) may represent a general property of a larger class of water-soluble proteins for which membrane binding is governed by electrostatic forces.     

Effects of multiple ligand binding on kinetic isotope effects in PQQ-dependent methanol dehydrogenase

The reaction of PQQ-dependent methanol dehydrogenase (MDH) from Methylophilus methylotrophus has been studied by steady-state and stopped-flow kinetic methods, with particular reference to multiple ligand binding and the kinetic isotope effect (KIE) for PQQ reduction. Phenazine ethosulfate (PES; an artificial electron acceptor) and cyanide (a suppressant of endogenous activity), but not ammonium (an activator of MDH), compete for binding at the catalytic methanol-binding site. Cyanide does not activate turnover in M. methylotrophus MDH, as reported previously for the Paracoccus denitrificans enzyme. Activity is dependent on activation by ammonium but is inhibited at high ammonium concentrations. PES and methanol also influence the stimulatory and inhibitory effects of ammonium through competitive binding. Reaction profiles as a function of ammonium and PES concentration differ between methanol and deuterated methanol, owing to force constant effects on the binding of methanol to the stimulatory and inhibitory ammonium binding sites. Differential binding gives rise to unusual KIEs for PQQ reduction as a function of ammonium and PES concentration. The observed KIEs at different ligand concentrations are independent of temperature, consistent with their origin in differential binding affinities of protiated and deuterated substrate at the ammonium binding sites. Stopped-flow studies indicate that enzyme oxidation is not rate-limiting at low ammonium concentrations (<4 mM) during steady-state turnover. At higher ammonium concentrations (>20 mM), the low effective concentration of PES in the active site owing to the competitive binding of ammonium lowers the second-order rate constant for enzyme oxidation, and the oxidative half-reaction becomes more rate limiting. A sequential stopped-flow method is reported that has enabled, for the first time, a detailed study of the reductive half-reaction of MDH and comparison with steady-state data. The limiting rate of PQQ reduction (0.48 s(-1)) is less than the steady-state turnover number, and the observed KIE in stopped-flow studies is unity. Although catalytically active, we propose reduction of the oxidized enzyme generated in stopped-flow analyses is gated by conformational change or ligand exchange. Slow recovery from this trapped state on mixing with methanol accounts for the slow reduction of PQQ and a KIE of 1. This study emphasizes the need for caution in using inflated KIEs, and the temperature dependence of KIEs, as a probe for hydrogen tunneling.     

Membrane destabilization by ricin

Ricin is a promising candidate for the treatment of cancer because it can be selectively targeted to tumor cells via linkage to monoclonal antibodies. Biochemical evidence suggests that escape of ricin or its ribosome-inactivating subunit from an intracellular compartment is mediated by retrograde transport to the endoplasmic reticulum and subsequent direction into the ER-associated degradation pathway. Alternatively, lipase activity of ricin may facilitate leakage from endocytic vesicles. We have observed ricin-mediated release of macromolecular dyes from lipid vesicles that mimic the composition of endosomal membranes. Release of small molecules occurs to the same extent, suggesting an all-or-none mechanism due to bilayer destabilization. The level of accompanying membrane fusion depends on vesicle composition. Since it takes 24 h of incubation before the first traces of lysolipids are detectable by matrix-assisted laser desorption/ionization mass spectrometry, membrane destabilization is not due to the lipase activity of ricin.Abbreviations CF Carboxyfluorescein - DPhPC Diphytanoyl-phosphatidylcholine - DPA Dipicolinic acid - EDTA Ethylendiamine-tetracetate - ER Endoplasmic reticulum - ERAD ER-associated degradation - FRET Fluorescence-resonance energy transfer - GM₁ Monosialoganglioside - MALDI-MS Matrix-assisted laser desorption/ionization mass spectrometry - MES 2-Morpholino-ethanesulfonic acid - NBD-PE N-(7-nitro-2,1,3-benzoxadiazol-4-yl)-phosphatidylethanolamine) - PC Phosphatidylcholine - PE Phosphatidylethanolamine - PG Phosphatidylglycerol - Rh-PE N-(lissamine rhodamine B sulfonyl)-phosphatidylethanolamine - RIP Ribosome-inactivating protein - RTA A-chain of ricin - RTB B-chain of ricin - TES N-[Tris-(hydroxymethyl)-methyl]-2-aminoethansulfonic acid - TOF Time-of-flight     

Deuterium isotope effects in mutagenesis by nitroso compounds.

Nitrosamines which have deuterium instead of hydrogen in the position alpha to the nitroso group have been reported to have reduced activity in carcinogenicity tests. This result implies that cleavage of a carbon--hydrogen bond is a limiting step in the reaction mechanism leading to tumor formation. Mutagenicity tests were undertaken with nitrosamines, which require metabolic activation, and with nitrosamides, which are directly acting mutagens, to determine the effect of deuterium substitution on the activity of each type of compound. Two nitrosamides (N-methyl-N'-nitro-N-nitrosoguanidine and methylnitrosourea) and three nitrosamines (dimethylnitrosamine, nitrosomorpholine, and dinitrosopiperazine) and their deuterium-containing analogs were tested for reversion of a nonsense mutation in the tyr locus of Escherichia coli WU 3610 (tyr-, leu-). Nitrosamines activated by rat-liver microsomes, but not nitrosamides, were less active as mutagens when the deuterium atom was present. The results suggest that the metabolic activation of nitrosamines to a mutagenic species involves the loss of hydrogen, a reaction which the nitrosamides, in the absence of enzyme, do not undergo.     

Functional significance of four successive glycine residues in the pyrophosphate binding loop of fungal 6-oxopurine phosphoribosyltransferases

Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is a key enzyme of the purine recycling pathway that catalyzes the conversion of 5-phospho-ribosyl-α-1-pyrophosphate and guanine or hypoxanthine to guanosine monophosphate (GMP) or inosine monophosphate (IMP), respectively, and pyrophosphate (PPi). We report the first crystal structure of a fungal 6-oxopurine phosphoribosyltransferase, the Saccharomyces cerevisiae HGPRT (Sc-HGPRT) in complex with GMP. The crystal structures of full length protein with (WT1) or without (WT2) sulfate that mimics the phosphate group in the PPi binding site were solved by molecular replacement using the structure of a truncated version (Δ7) solved beforehand by multiwavelength anomalous diffusion. Sc-HGPRT is a dimer and adopts the overall structure of class I phosphoribosyltransferases (PRTs) with a smaller hood domain and a short two-stranded parallel β-sheet linking the N- to the C-terminal end. The catalytic loops in WT1 and WT2 are in an open form while in Δ7, due to an inter-subunit disulfide bridge, the catalytic loop is in either an open or closed form. The closure is concomitant with a peptide plane flipping in the PPi binding loop. Moreover, owing the flexibility of a GGGG motif conserved in fungi, all the peptide bonds of the phosphate binding loop are in trans conformation whereas in nonfungal 6-oxopurine PRTs, one cis-peptide bond is required for phosphate binding. Mutations affecting the enzyme activity or the previously characterized feedback inhibition by GMP are located at the nucleotide binding site and the dimer interface.     

Synaptic destabilization by neuronal Nogo-A

Formation and maintenance of a neuronal network is based on a balance between plasticity and stability of synaptic connections. Several molecules have been found to regulate the maintenance of excitatory synapses but nothing is known about the molecular mechanisms involved in synaptic stabilization versus disassembly at inhibitory synapses. Here, we demonstrate that Nogo-A, which is well known to be present in myelin and inhibit growth in the adult CNS, is present in inhibitory presynaptic terminals in cerebellar Purkinje cells at the time of Purkinje cell-Deep Cerebellar Nuclei (DCN) inhibitory synapse formation and is then downregulated during synapse maturation. We addressed the role of neuronal Nogo-A in synapse maturation by generating several mouse lines overexpressing Nogo-A, starting at postnatal ages and throughout adult life, specifically in cerebellar Purkinje cells and their terminals. The overexpression of Nogo-A induced a progressive disassembly, retraction and loss of the inhibitory Purkinje cell terminals. This led to deficits in motor learning and coordination in the transgenic mice. Prior to synapse disassembly, the overexpression of neuronal Nogo-A led to the downregulation of the synaptic scaffold proteins spectrin, spectrin-E and β-catenin in the postsynaptic neurons. Our data suggest that neuronal Nogo-A might play a role in the maintenance of inhibitory synapses by modulating the expression of synaptic anchoring molecules. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Studying the effects of actin cytoskeletal destabilization on cell cycle by cofilin overexpression

The significance of actin cytoskeleton on cell growth was historically studied using toxic drugs, such as cytochalasin. However, it is possible that unpredictable effects of these agents may have influenced the reported observations. In our study, we have established a drug-free system using cofilin overexpression to investigate the relationship between actin filaments and cell cycle progression. Cofilin is a member of the actin depolymerization factor (ADF)/cofilin family, cofilin cDNA was cloned to a tetracycline-inducible gene expression vector and stably transfected to human lung cancer H1299 epithelial cells. Destabilization of actin filaments and morphological change was detected in cofilin overexpressing cells by actin analysis and microscopy, respectively. Measurements of growth rates showed that cell proliferation was retarded in cells with overexpressed cofilin. Also, cell cycle analysis showed that approx 90% of cofilin overexpressing cells were arrested in G1 phase, which is consistent with previous reports that drug-mediated disruption of actin filaments can cause G1 phase arrest. Taken together, cofilin overexpression cell model provides evidence that the effects of actin cytoskeletal destabilization on cell cycle progression can be studied using molecular approach instead of drug.     

Inhibitory effects of orotate on precursor incorporation into nucleic acids.

The influence of orotic acid on the incorporation of precursors into nucleic acids was studied in mice and rats and in isolated cells. In vivo, orotate levels were modified by two diets which are known to increase the rate of pyrimidine nucleotide synthesis in rat liver. Of these diets, a 1% orotate diet had greater inhibitory effects than an arginine-deficient diet on the incorporation of [3H]orotate into RNA of mouse kidney than mouse liver. This contrasted with the situation in the rat where there was a greater effect in the liver than the kidney. The situation in the rat was more readily interpreted than in the mouse in terms of previously established effects of these diets on ribonucleotide pool sizes. However, studies using [3H]adenosine as a precursor for incorporation into RNA suggested that even in the mouse the effects of orotate were on pool sizes rather than an inhibitory effect on RNA synthesis. The incorporation of [3H]thymidine into DNA was inhibited by orotate to a similar degree in cultured HTC hepatoma cells and a line of rat liver epithelial cells. An effect on DNA synthesis rather than solely on pool sizes was suggested by the observation that the pool size of dTTP was not increased by 5 mM orotate under conditions in which there was a four-fold increase in the level of UTP in HTC cells. An inhibitory effect of orotate on DNA synthesis was further supported by an observation of decreased incorporation of [3H]deoxyadenosine into DNA and a lower rate of cellular proliferation.     

Analysis of the galactosyltransferase reaction by positional isotope exchange and secondary deuterium isotope effects

The mechanism of the galactosyltransferase-catalyzed reaction was probed using positional isotope exchange, alpha-secondary deuterium isotope effects, and inhibition studies with potential transition state analogs. Incubation of [beta-18O2, alpha beta-18O]UDP-galactose and alpha-lactalbumin with galactosyltransferase from bovine milk did not result in any positional isotope exchange. The addition of 4-deoxy-4-fluoroglucose as a dead-end inhibitor did not induce any detectable positional isotope exchange. alpha-Secondary deuterium isotope effects of 1.21 +/- 0.04 on Vmax and 1.05 +/- 0.04 on Vmax/KM were observed for [1-2H]-UDP-galactose. D-Glucono-1,5-lactone, D-galactono-1,4-lactone, D-galactono-1,5-lactone, nojirimycin, and deoxynojirimycin, did not inhibit the galactosyl transfer reaction at concentrations less than 1.0 mM. The magnitude of the secondary deuterium isotope effect supports a mechanism in which the anomeric carbon of the galactosyl moiety has substantial sp2 character in the transition state. Therefore, the cleavage of the bond between the galactose and UDP moieties in the transition state has proceeded to a much greater extent than the formation of the bond between the galactose and the incoming glucose. The lack of a positional isotope exchange reaction indicates that the beta-phosphoryl group of the UDP is not free to rotate in the absence of an acceptor substrate.     

Compensation of DNA stabilization and destabilization effects caused by cisplatin is partially disturbed in alkaline medium

Binding of the antitumor compound cisplatin to DNA locally distorts the double helix. These distortions correlate with a decrease in DNA melting temperature (Tm). However, the influence of cisplatin on DNA stability is more complex because it decreases the DNA charge density. In this way, cisplatin increases the melting temperature and partially compensates for the destabilizing influence of structural distortions. The stabilization is stronger at low Na+ ion concentration. Due to this compensation, the total decrease in the DNA melting temperature after cisplatin binding is much lower than the decrease caused by the distortions themselves, especially at low [Na+]. It is shown in this study that, besides Na+ concentration, pH also strongly influences the value of a change in the melting temperature caused by cisplatin. In alkaline medium (pH=10.5-10.8), a fall in the melting temperature caused by platination is enhanced several times with respect to neutral medium. Such a stronger drop in Tm is explained by a decrease in pK values of base pairs caused by lowering the charge density under platination that facilitates proton release. At neutral pH, the proton release is low for both control and platinated DNA and does not influence the melting behavior. Therefore, lowering in the charge density under platination, besides stabilization, gives additional destabilization just in alkaline medium. Destabilization caused by structural distortions due to this pH induced compensation of stabilizing effect is more pronounced. In the presence of carbonate ion, destabilization caused by high pH value is strengthened. As a decrease in DNA charge density, interstrand crosslinking caused by cisplatin also increases the DNA stability due to loss in the entropy of the melted state. However, computer modeling of DNA stability demonstrates that interstrand crosslinks formed by cisplatin do not stabilize long DNA. It is shown that the increase in Tm caused by interstrand crosslinking itself is compensated for by a local destabilization of the double helix at the sites of location of interstrand crosslinks formed by cisplatin.     

Bilayer membrane destabilization induced by dolichylphosphate

Small vesicles containing the fluorescent probe calcein were used to investigate the effect of dolichyl phosphate (Dol-P) on phospholipid bilayer stability. In the absence of Dol-P, phospholipid vesicles retained the fluorescent probe upon the addition of divalent cations. Small vesicles containing Dol-P, however, exhibited calcein leakage when incubated in the presence of divalent cations. This effect was observed in liposomes composed of a mixture of phosphatidylethanolamine (PE), phosphatidylcholine (PC) and Dol-P, but not in PC/Dol-P liposomes. The rate of calcein leakage was proportional to divalent cation concentration and to temperature, but was independent of vesicle concentration. These results demonstrate that Dol-P has significant effects on the stability of PE containing phospholipid bilayers. Vesicle leakage was also promoted by the addition of rat liver Dol-P-mannose synthase (EC 2.4.1.83) to intact PE/PC/Dol-P vesicles. Enzyme induced leakage from phospholipid vesicles required the presence of both unsaturated PE and Dol-P. The phospholipid composition of leaky vesicles could be correlated with the lipid matrix required for maximal transferase activity of the rat liver synthase. The destabilizing effects of Dol-P on phospholipid bilayers may therefore be involved in the translocation of activated sugars across biological membranes.     

Inhibition of purine phosphoribosyltransferases from Ehrlich ascites-tumour cells by purine nucleotides

1. The purine bases adenine, hypoxanthine and guanine were rapidly incorporated into the nucleotide fraction of Ehrlich ascites-tumour cells in vivo. 2. The reaction of 5'-phosphoribosyl pyrophosphate with adenine phosphoribosyltransferase from ascites-tumour cells (K(m) 6.5-11.9mum) was competitively inhibited by AMP, ADP, ATP and GMP (K(i) 7.5, 21.9, 395 and 118mum respectively). Similarly the reactions of 5'-phosphoribosyl pyrophosphate with both hypoxanthine phosphoribosyltransferase and guanine phosphoribosyltransferase (K(m) 18.4-31 and 37.6-44.2mum respectively) were competitively inhibited by IMP (K(i) 52 and 63.5mum) and by GMP (K(i) 36.5 and 5.9mum). 3. The nucleotides tested as inhibitors did not appreciably compete with the purine bases in the phosphoribosyltransferase reactions. 4. It was postulated that the purine phosphoribosyltransferases of Ehrlich ascites-tumour cells may be effectively separated from the adenine nucleotide pool of these cells.     

Regulation of Escherichia coli ornithine transcarbamylase by orotate.

Ornithine transcarbamylase from Escherichia coli, strain W, exhibits negative cooperativity with respect to ornithine, and the enzymatic activity is further regulated by orotate. The effect of orotate on ornithine transcarbamylase is dependent not only upon the carbamylphosphate concentration, but also upon the concentration of ornithine. At high concentrations of carbamylphosphate (10 mM), a conversion from negative cooperativity to positive cooperativity is observed with 10 mM orotate. At 1 mM carbamylphosphate, however, 10 mM orotate activates the enzyme at low ornithine concentrations, but as the ornithine concentration is increased above 5 mM, inhibition is observed. Thus, a regulatory link has been established between the pathways of arginine biosynthesis and pyrimidine biosynthesis, each of which utilizes carbamylphosphate.     

Inhibition of purine phosphoribosyltransferases of Ehrlich ascites-tumour cells by 6-mercaptopurine

1. The formation of adenosine 5′-phosphate, guanosine 5′-phosphate and inosine 5′-phosphate from [8-¹⁴C]adenine, [8-¹⁴C]guanine and [8-¹⁴C]hypoxanthine respectively in the presence of 5-phosphoribosyl pyrophosphate and an extract from Ehrlich ascites-tumour cells was assayed by a method involving liquid-scintillation counting of the radioactive nucleotides on diethylaminoethylcellulose paper. The results obtained with guanine were confirmed by a spectrophotometric assay which was also used to assay the conversion of 6-mercaptopurine and 5-phosphoribosyl pyrophosphate into 6-thioinosine 5′-phosphate in the presence of 6-mercaptopurine phosphoribosyltransferase from these cells. 2. At pH 7·8 and 25° the Michaelis constants for adenine, guanine and hypoxanthine were 0·9 μm, 2·9 μm and 11·0 μm in the assay with radioactive purines; the Michaelis constant for guanine in the spectrophotometric assay was 2·6 μm. At pH 7·9 the Michaelis constant for 6-mercaptopurine was 10·9 μm. 3. 25 μm-6-Mercaptopurine did not inhibit adenine phosphoribosyltransferase. 6-Mercaptopurine is a competitive inhibitor of guanine phosphoribosyltransferase (K_i 4·7 μm) and hypoxanthine phosphoribosyltransferase (K_i 8·3 μm). Hypoxanthine is a competitive inhibitor of guanine phosphoribosyltransferase (K_i 3·4 μm). 4. Differences in kinetic parameters and in the distribution of phosphoribosyltransferase activities after electrophoresis in starch gel indicate that different enzymes are involved in the conversion of adenine, guanine and hypoxanthine into their nucleotides. 5. From the low values of K_i for 6-mercaptopurine, and from published evidence that ascites-tumour cells require supplies of purines from the host tissues, it is likely that inhibition of hypoxanthine and guanine phosphoribosyltransferases by free 6-mercaptopurine is involved in the biological activity of this drug.     

The substrate specificity of purine phosphoribosyltransferases in Schizosaccharomyces pombe

1. The activities of the purine phosphoribosyltransferases (EC 2.4.2.7 and 2.4.2.8) in purine-analogue-resistant mutants of Schizosaccharomyces pombe were checked. An 8-azathioxanthine-resistant mutant lacked hypoxanthine phosphoribosyltransferase, xanthine phosphoribosyltransferase and guanine phosphoribosyltransferase activities (EC 2.4.2.8) and appeared to carry a single mutation. Two 2,6-diaminopurine-resistant mutants retained these activities but lacked adenine phosphoribosyltransferase activity (EC 2.4.2.7). This evidence, together with data on purification and heat-inactivation patterns of phosphoribosyltransferase activities towards the various purines, strongly suggests that there are two phosphoribosyltransferase enzymes for purine bases in Schiz. pombe, one active with adenine, the other with hypoxanthine, xanthine and guanine. 2. Neither growth-medium supplements of purines nor mutations on genes involved in the pathway for new biosynthesis of purine have any influence on the amount of hypoxanthine-xanthine-guanine phosphoribosyltransferase produced by this organism.