A critical reexamination of the continous spectrophotometric assay for adenosine deaminase

The continuous spectrophotometric assay for adenosine deaminase has been reinvestigated, using both adenosine and 9-β-d-arabinofuranosyladenine as substrates. This assay is based on the reported decrease in absorbance at or near 265 nm between the adenine nucleoside substrate and the hypoxanthine nucleoside product. In the substrate concentration range 1,5 – 8.0 × 10^?4m, the progress of the reaction is associated with an anomalous sigmoidal dependence of absorbance on time, and the overall change in absorbance decreases with increasing substrate concentration. Near 8 × 10^?4m substrate, the deamination proceeds with no change in absorbance, while at higher concentrations, small increases in absorbance are observed. These effects, if ignored, generate initial “rate” data exhibiting an apparent substrate inhibition whieh, however, is completely an artifact induced by the spectral anomalies. Over the entire concentration range 5 × 10^?6–1 × 10^?3m, alternative assay methods (e.g., discontinuous detection of the product, ammonia) yeld normal Michaelis-Menten kineties. The anomalous behavior manifested in the continuous spectrophotometric assay is due to large negative deviations from Beer's law. These deviations are observed for all four of the nucleosides tested, viz., adenosine, 9-β-d-arabinofuranosyladenine, inosine, and 9-β-d-arabinofuranosylhypoxanthine. The departure from Beer's law is detectable anywhere in the concentration range 5 × 10^?6–1 × 10^?3m, but is most marked at concentrations above 1 × 10^?4m. Thus, the continuous spectrophotometric assay for adenosine deaminase should be utilized withextreme caution, and should not be employed at concentrations exceeding 1 × 10^?4m, irrespective of the K_m value for the substrate. Specific recommendations are given for future assays.     

A coupled optical enzyme assay for phosphopentomutase

Published assays for phosphopentomutase activity are based on acid lability differences between ribose 1-phosphate and ribose 5-phosphate. The present work describes a new method in which the isomerization of ribose 5-phosphate to ribose 1-phosphate is followed spectrophotometrically at 265 nm by coupling it with the following two-stage enzymatic conversion: ribose 1-phosphate + adenine ? phosphate + adenosine (adenosine phosphorylase); adenosine + H₂O → inosine + NH₃ (adenosine deaminase). The method has been used to show some properties of Escherichia coli phosphopentomutase.     

Coupling of glucose oxidase and Fenton's reaction for a simple and inexpensive assay of β-glucosidase

A simple and inexpensive assay for β-glucosidase, based on the coupling of glucose oxidase and Fenton's reagent has been described. Hydrogen peroxide formed as a result of the action of glucose oxidase on glucose (derived from the action of β-glucosidase on cellobiose) oxidizes ferrous sulphate, resulting in an increase in absorbance. The oxidation products produced a peak of maximum absorbance at 340 nm. Using this assay system, a linear relationship between glucose concentration in the range 5.55–27.78 mmol l^?1(100–500 mg dl^?1) and absorbance was obtained, indicating conformity to Beer's law. The preciseness of the glucose oxidase/Fenton's reagent for the assay of glucose was shown to be satisfactory. β-Glucosidase was assayed using the hexokinase assay reagent and the glucose oxidase/ferrous sulphate reagent. The values obtained using both reagents did not differ significantly. Although 2.6 times less sensitive than the hexokinase reagent when absorbance is measured at 340 nm, the glucose oxidase/Fenton's reagent is 10 times cheaper and could be used satisfactorily for routine assays of β-glucosidase and other carbohydrases including cellulase and amylase. In this respect, fructose, mannose, xylose, sucrose and cellobiose did not affect the sensitivity of the reagent. Of several metals tested, only aluminium interfered with the reagent, decreasing its sensitivity.     

A coupled spectrophotometric enzyme assay for methyltransferases

Adenosine deaminase (EC 3.5.4.4), purified from Aspergillus oryzae, is active in deaminating S-adenosylhomocysteine and its related thioethers, whereas the related sulfonium compound, S-adenosylmethionine, is not deaminated. By taking advantage of the different reactivity of the two compounds, a coupled optical enzyme assay for methyl transfer reactions has been developed. The amount of Ado-Hcy formed is calculated from the decrease in optical density at 265 nm, after addition of an excess of adenosine deaminase. The validity of the method has been tested with three purified enzymes, i.e., homocysteine methyltransferase, histamine methylase, and acetylserotonin methyltransferase. Some kinetic constants of these enzymes have been obtained. The procedure is highly accurate, reproducible, and relatively simple compared to the conventional radio-chemical methods currently in use.     

Colorimetric determination of arginine residues in proteins by p-nitrophenylglyoxal

A new method is presented for the colorimetric determination of arginine residues in proteins. Under mildly alkaline conditions, p-nitrophenylglyoxal reacted with arginine to produce a stable colored solution in the presence of 0.15 m sodium ascorbate. Complete color development was obtained after 30 min at pH 9.0 and 30°C. The color produced at 475 nm obeyed Beer's law in the range 0.03–0.33 mm arginine. This color reaction was used to determine the number of arginine residues in several proteins of known arginine content. Best results were obtained when the protein samples were digested with a mixture of trypsin and subtilisin prior to assaying. The arginine contents obtained by this method agreed well with either the published values or with the results of amino acid analysis.     

Effects of adenosine analogs on rat cerebral cortical neurons

Adenosine and the adenosine 5'-phosphates (5'-AMP, 5'-ADP and 5'-ATP) depress the spontaneous firing of cerebral cortical neurons. In this study adenosine analogs, adenosine transport blockers and adenosine deaminase inhibitors have been used to gain further insight into the nature of the adenosine receptor and the likely routes of metabolism of extracellularly released adenosine. The firing rate of cortical neurons, including identified corticospinal neurons, was depressed by 2-substituted derivatives of adenosine. 2-Halogenated derivatives of adenosine were potent depressors while 2-aminoadenosine and 2-hydroxyadenosine (crotonoside) were slightly less potent than adenosine. The α,β-methylene isosteres of 5'-ADP and 5'-ATP were almost devoid of agonist activity while the β,γ-methylene analog was an active agonist. This suggests that ADP and ATP must be converted to AMP or possibly adenosine before they can activate the adenosine receptor. 2'-, 3'- and 5'- deoxyadenosine depressed spontaneous firing without antagonizing the effect of adenosine. Adenosine deaminase inhibitors, deoxycoformycin and $\text{erythro}$-9-(2-hydroxy-3-nonyl) adenine had potent, long lasting depressant actions on the spontaneous firing of cortical neurons and concurrently potentiated the actions of adenosine or 5'-AMP. Inhibitors of adenosine transport, papaverine and 2-hydroxy-5-nitrobenzylthioguanosine, prolonged the duration of action of adenosine and 5'-AMP. Intracellular recordings show that 5'-AMP hyperpolarizes cerebral cortical neurons and suppresses spontaneous and evoked excitatory postsynaptic potentials, in the absense of any pronounced alterations in membrane resistance.     

Cysteine 265 Is in the Active Site of, But Is Not Essential for Catalysis by tRNA-Guanine Transglycosylase (TGT) from Escherichia coli

Site-directed mutagenesis and X-ray absorption spectroscopy studies have previously shown that the tRNA-guanine transglycosylase (TGT) from Escherichia coli is a zinc metalloprotein and identified the enzymic ligands to the zinc [Chong et al. (1995), Biochemistry 34, 3694–3701; Garcia et al. (1966), Biochemistry 35, 3133–3139]. During these studies one mutant, TGT (C265A), was found to exhibit a significantly lower specific activity, but was not found to be involved in the zinc site. The present report demonstrates that TGT is inactivated by treatment with thiol reagents (e.g., DTNB, MMTS, and N-ethylmaleimide). Further, this inactivation is shown to be due to modification of cysteine 265. The kinetic parameters for the mutants TGT (C265A) and TGT (C265S), however, suggest that this residue is not performing a critical role in the TGT reaction. We conclude that cysteine 265 is in the active site of TGT, but is not performing a critical catalytic function. This conclusion is supported by the recent determination of the X-ray crystal structure of the TGT from Zymomonas mobilis [Romier et al. (1966), EMBO J. 15, 2850–2857], which reveals that the residue corresponding to cysteine 265 is distant from the putative catalytic site, but is in the middle of a region of the enzyme surface proposed to bind tRNA.     

Production of S-adenosyl-l-homocysteine by bacterial cells with a high content of S-adenosylhomocysteine hydrolase: Utilization of a racemic mixture of homocysteine as the substrate

Alcaligenes faecalis cells contain a high level of S-adenosylhomocysteine hydrolase (EC 3.3.1.1) and are the useful catalyst for the preparative production of S-adenosyl-l-homocysteine (Shimizu et al., 1984, Eur. J. Biochem. 141, 385–392; 1986, J. Biotechnol. 4, 81–90). A problem with this production was that the d-isomer of homocysteine was not utilized. To solve this problem, we screened for microbial strains capable of synthesizing S-adenosyl-l-homocysteine from d-homocysteine and adenosine, and found that Pseudomonas putida cells catalyze this conversion. Although P. putida S-adenosylhomocysteine hydrolase catalyzed only the condensation of l-homocysteine with adenosine, the bacterium also produced a racemase which interconverts the d- and l-homocysteine isomers. For practical purposes. A. faecalis was still superior in showing high S-adenosylhomocysteine hydrolase activity and low adenosine deaminase activity. Therefore, P. putida was used just as a source of the racemase. With 200 mM adenosine and 200 mM dl-homocysteine, the molar conversion to S-adenosyl-l-homocysteine was 86%, when a mixture of A. faecalis cells (36 mg ml⁻¹) and P. putida cells (4 mg ml⁻¹) was used as the catalyst.     

Identification of a New Class of Adenosine Deaminase from Helicobacter pylori with Homologs among Diverse Taxa

Early studies of Helicobacter pylori''s nutritional requirements alluded to a complete purine salvage network in this organism. Recently, this hypothesis was confirmed in two strains of H. pylori, whose purine requirements were satisfied by any single purine base or nucleoside. Most of the purine conversion enzymes in H. pylori have been studied using mutant analysis; however, the gene encoding adenosine deaminase (ADD) in H. pylori remained unidentified. Through stepwise protein purification followed by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF), we discovered that H. pylori ADD is encoded by hp0267, an apparently essential gene. Hp0267 shares no sequence homology with previously characterized ADDs, yet both are members of the amidohydrolase superfamily. Hp0267 is grouped within cog0402, while other ADDs studied to date are found in cog1816. The hp0267 locus was previously misannotated as encoding a chlorohydrolase. Using purified recombinant Hp0267, we determined the enzyme''s pH optimum, temperature optimum, substrate specificity, and estimated kinetic constants. In contrast to other known ADDs, Hp0267 contains Fe(II) as the relevant metal ligand. Furthermore, Hp0267 exhibits very low deaminase activity on 2′-deoxyadenosine, a substrate that is readily hydrolyzed by cog1816 ADDs. Our preliminary comparative genomic analysis suggests that Hp0267 represents a second enzyme class of adenosine deaminase whose phyletic distribution among prokaryotes is broad.     

Cysteine 265 Is in the Active Site of, But Is Not Essential for Catalysis by tRNA-Guanine Transglycosylase (TGT) from Escherichia coli

Site-directed mutagenesis and X-ray absorption spectroscopy studies have previously shown that the tRNA-guanine transglycosylase (TGT) from Escherichia coli is a zinc metalloprotein and identified the enzymic ligands to the zinc [Chong et al. (1995), Biochemistry 34, 3694–3701; Garcia et al. (1966), Biochemistry 35, 3133–3139]. During these studies one mutant, TGT (C265A), was found to exhibit a significantly lower specific activity, but was not found to be involved in the zinc site. The present report demonstrates that TGT is inactivated by treatment with thiol reagents (e.g., DTNB, MMTS, and N-ethylmaleimide). Further, this inactivation is shown to be due to modification of cysteine 265. The kinetic parameters for the mutants TGT (C265A) and TGT (C265S), however, suggest that this residue is not performing a critical role in the TGT reaction. We conclude that cysteine 265 is in the active site of TGT, but is not performing a critical catalytic function. This conclusion is supported by the recent determination of the X-ray crystal structure of the TGT from Zymomonas mobilis [Romier et al. (1966), EMBO J. 15, 2850–2857], which reveals that the residue corresponding to cysteine 265 is distant from the putative catalytic site, but is in the middle of a region of the enzyme surface proposed to bind tRNA.     

Purification and characterization of adenosine deaminase from Klebsiella sp. LF 1202

Adenosine deaminase was induced when the cells of Klebsiella sp. LF 1202 were cultured in the medium containing adenosine as a sole source of carbon and nitrogen. The induction was partially repressed by the addition of ammonium sulfate in the medium. The amount of adenosine deaminase reached approximately 4.6% of the total intracellular soluble proteins. The enzyme was purified approximately 22-fold with a 25% activity yield. The enzyme was a monomer with a molecular weight of 26,000. The optimal activity was obtained at pH 8.0, 37°C, and the K_m value for adenosine was 37 μM. Metal ions such as Zn²⁺, Co²⁺, Fe² and Ni⁺ inhibited the activity of the enzyme. Sulfhydryl blocking agents such as p-chloromercuribenzoate and HgCl₂ were also found to be potent inhibitors for adenosine deaminase.     

Purine catabolism in isolated rat hepatocytes. Influence of coformycin

1. The catabolism of purine nucleotides was investigated by both chemical and radiochemical methods in isolated rat hepatocytes, previously incubated with [¹⁴C]adenine. The production of allantoin reached 32±5nmol/min per g of cells (mean±s.e.m.) and as much as 30% of the radioactivity incorporated in the adenine nucleotides was lost after 1h. This rate of degradation is severalfold in excess over values previously reported to occur in the liver in vivo. An explanation for this enhancement of catabolism may be the decrease in the concentration of GTP. 2. In a high-speed supernatant of rat liver, adenosine deaminase was maximally inhibited by 0.1μm-coformycin. The activity of AMP deaminase, measured in the presence of its stimulator ATP in the same preparation, as well as the activity of the partially purified enzyme, measured after addition of its physiological inhibitors GTP and Pi, required 50μm-coformycin for maximal inhibition. 3. The production of allantoin by isolated hepatocytes was not influenced by the addition of 0.1μm-coformycin, but was decreased by concentrations of coformycin that were inhibitory for AMP deaminase. With 50μm-coformycin the production of allantoin was decreased by 85% and the formation of radioactive allantoin from [¹⁴C]adenine nucleotides was completely suppressed. 4. In the presence of 0.1μm-coformycin or in its absence, the addition of fructose (1mg/ml) to the incubation medium caused a rapid degradation of ATP, without equivalent increase in ADP and AMP, followed by transient increases in IMP and in the rate of production of allantoin; adenosine was not detectable. In the presence of 50μm-coformycin, the fructose-induced breakdown of ATP was not modified, but the depletion of the adenine nucleotide pool proceeded much more slowly and the rate of production of allantoin increased only slightly. No rise in IMP concentration could be detected, but AMP increased manyfold and reached values at which a participation of soluble 5′-nucleotidase in the catabolism of adenine nucleotides is most likely. 5. These results are in agreement with the hypothesis that the formation of allantoin is controlled by AMP deaminase. They constitute further evidence that 5′-nucleotidase is inactive on AMP, unless the concentration of this nucleotide rises to unphysiological values.     

Pathways of adenine nucleotide catabolism in primary rat muscle cultures

The pathways of AMP degradation and the metabolic fate of adenosine were studied in cultured myotubes under physiological conditions and during artificially induced enhanced degradation of ATP. The metabolic pathways were gauged by tracing the flow of radioactivity from ATP, prelabelled by incubation of the cultures with [¹⁴C]adenine, into the various purine derivatives. The fractional flow from AMP to inosine through adenosine was estimated by the use of the adenosine deaminase (EC 3.5.4.4) inhibitors, coformycin and 2′-deoxycoformycin. The activities of the enzymes involved with AMP and adenosine metabolism were determined flow of label from ATP to diffusible bases and nucleosides, most of which are effluxed to the incubation medium. This catabolic flow is mediated almost exclusively by the activity of AMP deaminase (EC 3.5.4.6), rather than by AMP 5′-nucleotidase (EC 3.1.3.5), reflecting the markedly higher V_max/K_m ratio for the deaminase. Enhancement of ATP degradation by inhibition of glycolysis or by combined inhibition of glycolysis and of electron transport resulted in a markedly greater flux of label from adenine nucleotides to nucleosides and bases, but did not alter significantly the ratio between AMP deamination and AMP dephosphorylation, which remained around 19:1. Combined inhibition of glycolysis and of electron transport resulted, in addition, in accumulation of label in IMP, reaching about 20% of total AMP degraded. In the intact myotubes at low adenosine concentration, the anabolic activity of adenosine kinase was at least 4.9-fold the catabolic activity of adenosine deaminase, in accord with the markedly higher V_max/K_m ratio of the kinase for adenosine. The results indicate the operation in the myotube cultures, under various rates of ATP degradation, of the AMP to IMP limb of the purine nucleotide cycle. On the other hand, the formation of purine bases and nucleosides, representing the majority of degraded ATP, indicates inefficient activity of the IMP to AMP limb of the cycle, as well as inefficient salvage of hypoxanthine under these conditions.     

ECTO-enzyme activity of human erythrocyte adenosine deaminase

Summary Adenosine deaminase is found primarily in the cytoplasm of many cell types. In the human erythrocyte, about 30 per cent of the total adenosine deaminase activity is membrane associated, and about two-thirds of this is inactivated by treatment of intact erythrocytes with the nonpenetrating reagent diazotized sulfanilic acid, without affecting lactate dehydrogenase, a soluble cytoplasmic enzyme. This indicates that within the cell membranes, the catalytic site of about two-thirds of the adenosine deaminase faces the external medium, i.e., ecto adenosine deaminase. Localization of adenosine deaminase activity at the cell membrane is demonstrated directly by electron microscopy by use of the substrate 6-Chloropurine ribonucleoside, which is dechlorinated by adenosine deaminase to produce Cl^–, which is precipitated at its locus of formation by added Ag⁺, and the precipitated AgCl converted into the electron dense Ag⁰ upon exposure to light.From the Hydropathic Profile of the amino acid sequence of adenosine deaminase it is evident that there are two hydrophobic domains of sufficient length to span a biological membrane, and it is proposed that these domains could function to anchor the enzyme to the membrane.The importance of adenosine deaminase is indicated by the fatal immuno-deficiency which results from untreated genetic adenosine deaminase deficiency. It may be important to determine whether the amount of ecto adenosine deaminase activity is better suited to assess the clinical status of adenosine deaminase deficient patients that the currently used total cellular enzyme activity.Abbreviations ADA Adenosine Deaminase - LDH Lactate Dehydrogenase - HEPES N-2-Hydroxyethylpiperazine-N-2-ethanesulfonic acid - CPR 6-Chloropurine Ribonucleoside - SDS Sodium Dodecyl Sulfate - NAD -Nicotinamide Adenine Dinucleotide - HBSS Hank's Balanced Salt Solution - DASA Diazotized Sulfanilic Acid     

A coupled optical assay for adenine phosphoribosyltransferase and its extension for the spectrophotometric and radioenzymatic determination of 5-phosphoribosyl-1-pyrophosphate in mixtures and in tissue extracts

A reliable assay was developed to characterize crude cell homogenates with regard to their adenine phosphoribosyltransferase activities. The 5-phosphoribosyl-1-pyrophosphate (PRPP)-dependent formation of AMP from adenine is followed spectrophotometrically at 265 nm by coupling it with the following two-stage enzymatic conversion: AMP + H2O----adenosine + Pi (5'-nucleotidase); adenosine + H2O----inosine + NH3 (adenosine deaminase). The same principle was applied to develop a spectrophotometric and a radioenzymatic assay for PRPP. The basis of the spectrophotometric assay is the absorbance change at 265 nm associated with the enzymatic conversion of PRPP into inosine, catalyzed by the sequential action of partially purified adenine phosphoribosyltransferase, commercial 5'-nucleotidase, and commercial adenosine deaminase, in the presence of excess adenine. In the radiochemical assay PRPP is quantitatively converted into [14C]inosine via the same combined reaction. Tissue extracts are incubated with excess [14C]adenine. The radioactivity of inosine, separated by a thin-layer chromatographic system, is a measure of PRPP present in tissue extracts. The radioenzymatic assay is at least as sensitive as other methods based on the use of adenine phosphoribosyltransferase. However, it overcomes the reversibility of the reaction and the need to use transferase preparations free of any phosphatase and adenosine deaminase activities.     

Identification and Characterization of the Missing Pyrimidine Reductase in the Plant Riboflavin Biosynthesis Pathway

Riboflavin (vitamin B₂) is the precursor of the flavin coenzymes flavin mononucleotide and flavin adenine dinucleotide. In Escherichia coli and other bacteria, sequential deamination and reduction steps in riboflavin biosynthesis are catalyzed by RibD, a bifunctional protein with distinct pyrimidine deaminase and reductase domains. Plants have two diverged RibD homologs, PyrD and PyrR; PyrR proteins have an extra carboxyl-terminal domain (COG3236) of unknown function. Arabidopsis (Arabidopsis thaliana) PyrD (encoded by At4g20960) is known to be a monofunctional pyrimidine deaminase, but no pyrimidine reductase has been identified. Bioinformatic analyses indicated that plant PyrR proteins have a catalytically competent reductase domain but lack essential zinc-binding residues in the deaminase domain, and that the Arabidopsis PyrR gene (At3g47390) is coexpressed with riboflavin synthesis genes. These observations imply that PyrR is a pyrimidine reductase without deaminase activity. Consistent with this inference, Arabidopsis or maize (Zea mays) PyrR (At3g47390 or GRMZM2G090068) restored riboflavin prototrophy to an E. coli ribD deletant strain when coexpressed with the corresponding PyrD protein (At4g20960 or GRMZM2G320099) but not when expressed alone; the COG3236 domain was unnecessary for complementing activity. Furthermore, recombinant maize PyrR mediated NAD(P)H-dependent pyrimidine reduction in vitro. Import assays with pea (Pisum sativum) chloroplasts showed that PyrR and PyrD are taken up and proteolytically processed. Ablation of the maize PyrR gene caused early seed lethality. These data argue that PyrR is the missing plant pyrimidine reductase, that it is plastid localized, and that it is essential. The role of the COG3236 domain remains mysterious; no evidence was obtained for the possibility that it catalyzes the dephosphorylation that follows pyrimidine reduction.Riboflavin is the substrate for biosynthesis of the essential flavocoenzymes FMN and FAD, which occur in all kingdoms of life and have roles in diverse redox reactions as well as in other processes such as DNA repair, light sensing, and bioluminescence (Fischer and Bacher, 2005). Plants and many microorganisms can synthesize riboflavin, but humans and other animals cannot, so they must obtain it from the diet (Powers, 2003). Plant foods are important sources of riboflavin for humans, and the riboflavin pathway is a target for engineering biofortified crops (Fitzpatrick et al., 2012).Riboflavin biosynthesis proceeds via the same pathway in bacteria and plants (Fischer and Bacher, 2005; Roje, 2007). This pathway starts from GTP, which is converted by GTP cyclohydrolase II (named RibA in Escherichia coli) to the pyrimidine derivative 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5′-P. Deamination of the pyrimidine ring then yields 5-amino-6-ribosylamino-2,4(1H,3H)-pyrimidinedione 5′-P, and subsequent reduction of the ribosyl moiety gives 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione 5′-P. After dephosphorylation, this product is condensed with 3,4-dihydroxy-2-butanone 4-P to give 6,7-dimethyl-8-ribityllumazine, whose dismutation yields riboflavin. Figure 1 shows the first four steps of this pathway.Open in a separate windowFigure 1.The first four steps of the riboflavin biosynthesis pathway in bacteria and plants. The enzymes involved are GTP cyclohydrolase II (RibA), pyrimidine deaminase (Deam), pyrimidine reductase (Red), and a specific phosphatase (Pase). Enzymes for which the plant genes are not known are colored red. Intermediates are as follows: 1, 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5′-P; 2, 5-amino-6-ribosylamino-2,4(1H,3H)-pyrimidinedione 5′-P; 3, 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione 5′-P; 4, 5-amino-6-ribitylamino-2,4(1H,3H)-pyrimidinedione.In E. coli, the deamination and reduction steps are catalyzed by a single bifunctional enzyme, RibD, which has N-terminal deaminase and C-terminal reductase domains that retain their respective activities when expressed separately (Richter et al., 1997; Magalhães et al., 2008). The situation in plants seems superficially similar but is in fact more complex (Gerdes et al., 2012). The bidomain bacterial RibD protein has two types of homologs in plants (Fischer et al., 2004; Chatwell et al., 2006; Chen et al., 2006), here called PyrD and PyrR, both with apparent deaminase and reductase domains (Fig. 2A). Only PyrD, represented by At4g20960, has been studied biochemically; it was found to have pyrimidine deaminase but not reductase activity (Fischer et al., 2004). The function of PyrR, represented by At3g47390, is unknown, although it has been inferred to have reductase activity (Chatwell et al., 2006; Chen et al., 2006; Ouyang et al., 2010) and perhaps to lack deaminase activity (Ouyang et al., 2010). Another mystery surrounding PyrR proteins is the presence of an extra C-terminal domain of unknown function (COG3236 in the Clusters of Orthologous Groups database; Fig. 2A); this domain occurs as a stand-alone protein in many bacteria. One possibility is that it catalyzes the dephosphorylation that follows the pyrimidine reduction step in the pathway (Fig. 1). The phosphatase involved is most likely substrate specific, but it has not been identified in plants or any other organism (Roje, 2007; Gerdes et al., 2012), and genes for enzymes in the same pathway, especially for successive steps, are quite commonly fused (Suhre, 2007). A mutation (phs1) that deleted the COG3236 domain from Arabidopsis (Arabidopsis thaliana) PyrR resulted in a photosensitive phenotype that could be rescued by supplied FAD (Ouyang et al., 2010).Open in a separate windowFigure 2.Structure and phylogeny of plant PyrD and PyrR proteins. A, Domain architectures. The examples shown are Arabidopsis At4g20960 and At3g47390; the predicted plastid targeting peptide (TP) varies in length between species. B, Phylogenetic tree of PyrD and PyrR proteins. Sequences were aligned with ClustalW; the tree was built by the neighbor-joining method with MEGA5. Bootstrap values (%) for 1,000 replicates are next to the nodes. Only the tree topology is shown. Note that the PyrD proteins of green algae (underlined) lack a reductase domain. C, Alignments showing the conservation of the zinc-binding residues (arrowheads) in the deaminase domain of PyrD but not PyrR proteins and the conservation of the predicted substrate-binding residues (asterisks) in the reductase domain of PyrR but not PyrD proteins. The deaminase sequences correspond to residues 45 to 85 of B. subtilis RibD (synonym RibG); the reductase sequences correspond to residues 150 to 210 and (separated by dots) 288 to 292 of B. subtilis RibD. Identical zinc- or substrate-binding residues are black, and conservative replacements are gray. Dashes indicate gaps that maximize the alignment.The plant riboflavin synthesis pathway is considered to be plastidial (Roje, 2007), but this location is based almost solely on bioinformatics and high-throughput proteome analyses (Gerdes et al., 2012). In only one case is there more definitive experimental support: in vitro chloroplast import data for the pathway’s penultimate enzyme, 6,7-dimethyl-8-ribityllumazine synthase (Jordan et al., 1999). Similarly, clear genetic support for the function of most plant riboflavin synthesis enzymes is lacking (Gerdes et al., 2012), the exception being an Arabidopsis RibA homolog (Hedtke and Grimm, 2009).The work reported here established, using maize (Zea mays) and Arabidopsis, that PyrR is indeed the missing pyrimidine reductase, that it lacks deaminase activity, and that its COG3236 domain is not essential for pyrimidine reductase activity and most likely lacks phosphatase activity. We also demonstrated the import of PyrR and PyrD into chloroplasts in vitro and confirmed that the gene for PyrR is essential.     

Ligatin from embryonic chick neural retina inhibits retinal cell adhesion

Ligatin, a filamentous cell-surface protein purified from embryonic chick neural retina, has been found to inhibit the reassociation of dissociated retinal cells. This inhibition was demonstrated using two methods, a single cell disappearance assay and an improved monolayer collection assay utilizing microtiter plates. Monomeric ligatin at approximately 20 μg/ml inhibited rates of adhesion, but polymeric ligatin and tryptic fragments of ligatin were ineffective. Ligatin's inhibitory effect is suggested to be mediated through binding to retinal cell surfaces since preincubation of dissociated retinal cells with monomeric ligatin inhibited the cells' adhesiveness and removed the inhibitory activity from the culture media. Ligatin homologues prepared from mammalian tissues were ineffective in inhibiting retinal cell adhesion, suggesting a tissue and/or species specificity. Similarities in physicochemical and biological properties suggest that ligatin may be the inhibitor of adhesion previously described by Merrell et al.[Merrell, R., Gottlieb, D. I., and Glaser, L. (1975). J. Biol. Chem., 250, 4825].     

On the identity of strychnicine

Evidence is presented for the probable identity of strychnicine, a Strychnos alkaloid isolated by Boorsma in 1902 but no longer available, with vomicine (4-hydroxy-N-methyl-sec.-pseudostrychnine). A product obtained in 1966 by Hifny Saber et al. and identified by them as strychnicine is different from Boorsma's compound and its nature remains uncertain.     

Catabolism of adenine nucleotides in adenosine deaminase deficient erythrocytes

$\text{In vitro}$ incubation studies using fluoride and iodoacetate as glycolytic inhibitors have been carried out on red cells of the two subjects with adenosine deaminase deficiency. For comparison, similar studies have also been carried out on red cells from a normal subject and from a child with severe combined immunodeficiency with normal adenosine deaminase activity. The adenosine formed in the adenosine deaminase deficient red cells is a measure of adenosine 5′-phosphate breakdown initiated by 5′-nucleotidase, whereas inosine 5′-phosphate, inosine and hypoxanthine formation is a measure of adenosine 5′-phosphate breakdown initiated by adenylate deaminase. With fluoride as inhibitor, nearly all of the adenosine 5′-phosphate breakdown proceeded by way of adenylate deaminase, while with iodoacetate as inhibitor, 20–30% of the adenosine 5′-phosphate breakdown was initiated by 5′-nucleotidase acting on adenosine 5′-phosphate. In addition, significant amounts of adenine were produced in adenosine deaminase deficient red cells in the presence of the glycolytic inhibitors. Possible explanations for the findings noted in this study are discussed and related to recent studies on the properties of the pertinent purine nucleotide catabolic enzymes.