Guanine Triphosphate Research Articles

Characterization of a Goalpha mutant that binds xanthine nucleotides.

Several GTP binding proteins, including EF-Tu, Ypt1, rab-5, and FtsY, and adenylosuccinate synthetase have been reported to bind xanthine nucleotides when the conserved aspartate residue in the NKXD motif was changed to asparagine. However, the corresponding single Goalpha mutant protein (D273N) did not bind either xanthine nucleotides or guanine nucleotides. Interestingly, the introduction of a second mutation to generate the Goalpha subunit D273N/Q205L switched nucleotide binding specificity to xanthine nucleotide. The double mutant protein GoalphaD273N/Q205L (GoalphaX) bound xanthine triphosphate, but not guanine triphosphate. Recombinant GoalphaX (GoalphaD273N/Q205L) formed heterotrimers with betagamma complexes only in the presence of xanthine diphosphate (XDP), and the binding to betagamma was inhibited by xanthine triphosphate (XTP). Furthermore, as a result of binding to XTP, the GoalphaX protein underwent a conformational change similar to that of the activated wild-type Goalpha. In transfected COS-7 cells, we demonstrate that the interaction between GoalphaX and betagamma occurred only when cell membranes were permeabilized to allow the uptake of xanthine diphosphate. This is the first example of a switch in nucleotide binding specificity from guanine to xanthine nucleotides in a heterotrimeric G protein alpha subunit.

Resting and stimulated human polymorphonuclear leucocytes from type-2 diabetic patients: change in purine nucleotide pattern.

It is known that polymorphonuclear leucocytes are deeply involved in the inflammatory complications of diabetes mellitus, showing many functional and biochemical abnormalities. Because adenine and guanine metabolites exert an important role in many metabolic aspects of phagocytic cells, we have investigated the pattern of purine metabolites during the respiratory burst of polymorphonuclear leucocytes in order to characterize any difference that may be significantly correlated with the abnormal neutrophil function of diabetic patients. The results obtained show clearly that polymorphonuclear leucocytes from diabetic patients are characterized by an abnormal pattern of purine nucleotides and their metabolites. In particular, the concentration of adenine and guanine triphosphates and the net amount of adenosine triphosphate hydrolysed during neutrophil stimulation by phorbol ester is higher in diabetic than in control cells. Moreover, higher values of adenosine monophosphate, inosine monophosphate and inosine have been found in diabetic cells. The behaviour of guanosine triphosphate is highly interesting. In fact, in addition to the higher concentration found in diabetic polymorphonuclear leucocytes, stimulation by phorbol ester induces a net decrease in guanosine triphosphate whereas control neutrophils show a slight increase. These findings have been associated with the ease with which diabetic neutrophils undergo metabolic activation and sustain an inflammatory response.

G Protein-mediated Activation of a Nonspecific Cation Current in Cultured Rat Retinal Pigment Epithelial Cells

We used whole-cell patch-clamp recording techniques to investigate G protein-activated currents in cultured rat retinal pigment epithelial (RPE) cells. Using 140 mM KCl intracellular and 130 mM NaCl extracellular solutions, rat RPE cells possessed both inward and outward K+ currents. Upon addition of the nonhydrolyzable guanine triphosphate analogue, guanosine-5'-O-(3-thiophosphate) (GTPgammaS, 0.1 mM), to the recording electrode, a nonspecific cation (NSC) current was elicited. The NSC current had a mean reversal potential of +5.7 mV in 130 mm extracellular NaCl with Cs+-aspartate in the pipette, and was not affected by alterations in the extracellular Ca2+ or Cl- concentration. The GTPgammaS-activated current was found to be permeable to several monovalent cations (K+, Na+, choline, TRIS, and NMDG). Addition of fluoroaluminate, an activator of large molecular weight heterotrimeric GTP-binding proteins (G proteins), to the intracellular recording solution activated the NSC current. The G protein involved was pertussis toxin (PTX)-sensitive, since GTPgammaS failed to activate the NSC current in cells pretreated with PTX. Further investigation of second messenger molecules suggested that activation of the NSC current was not affected by alterations in intracellular Ca2+ or ATP. From these results, we conclude that a G protein-regulated NSC current is present in rat RPE cells. Activation of the NSC current may sufficiently depolarize RPE cells to activate outward K+ currents. This would provide a mechanism by which these cells could rid themselves of accumulated K+.

Nucleotides regulate the binding affinity of the recombinant type A cholecystokinin receptor in CHO K1 cells

Cholecystokinin (CCK) receptors on rat pancreatic acinar cells display two binding affinity states in the presence of adeninine and guanine triphosphates with the effect of ATP mediated by the enzyme nucleoside diphosphate kinase. To determine whether this behavior was intrinsic to a single receptor protein we studied the binding affinity of CHO cells stably transfected with a cloned rat CCK A receptor. 125I-CCK binding to intact cells at 37°C revealed two affinity states for CCK of K d values 20 pM and 2.4 nM. Membranes prepared from these cells displayed a single affinity state for CCK but two affinity states could be restored in the presence of GTP[γS], ATP and ATP[γS] but not AMP-PCP. ATP and ATP[γS] but not AMP-PCP were substrates for nucleoside diphosphate kinase present in CHO cell membranes and transferred their terminal phosphate to GDP. These findings indicate that the interconvertible affinity states of the CCK receptor are inherent in a single receptor protein and that nucleoside diphosphate kinase mediates the effect of ATP to regulate these two affinity states

Guanine nucleoside diphosphate and triphosphate modulate [3H]CGS 21680 binding to A2 adenosine receptor in rat striatal membranes

AbstractIn striatum and several other tissue, a guanine nucleotide binding protein (Gs) couples A2 adenosine receptor to activation of adenylyl cyclase. We have examined the effect of guanine nucleoside diphosphate and triphosphate on [3H]CGS 21680 binding to A2A adenosine receptors of rat striatum. Both GDP and GTP inhibited specific [3H]CGS 21680 binding to rat striatal membranes by 50% at about 0.1 mM. GMP was inhibitory only at higher concentrations, and the estimated IC50 value was greater than 1mM. The nonhydrolyzable analog of GTP, GPP (NH)p, was as potent as GTP with an IC50 value of approximately 86 μM. These results suggest that the regulation of A2a adenosine receptor binding properties by guanine nucleotides is independent of Gs activation, since inhibition of agonist binding is achieved by addition of agonist binding is achieved by addition of both guanine nucleoside diphosphate and triphosphate © 1993 Wiley‐Liss, Inc.

Modulation of Guanine Triphosphate Nucleotide Binding to p21ras Immunoprecipitates of Rat Liver Plasma Membranes by Agents Affecting Redox State

GTP-γ-[35S] and GTP-γ-[32P] or GTP-α-[32P] bound to plasma membranes of rat liver was immunoprecipitated using anti p21V-H-ras. Binding was enhanced approximately 2-fold by incubation with an exogenous electron acceptor, potassium ferricyanide (but not with potassium ferrocyanide), or oxidized ubiquinone10 and was inhibited or unaffected by incubation with reduced pyridine nucleotides (NADH or NADPH) or reduced ubiquinone10. The results suggest a mechanism of guanine nucleotide exchange that is responsive to oxidation-reduction.

Single K+ channels in membrane patches of arterial chemoreceptor cells are modulated by O2 tension.

Type I cells of the carotid body are known to participate in the detection of O2 tension in arterial blood but the primary chemotransduction mechanisms are not well understood. Here we report the existence in excised membrane patches of type I cells of a single K+ channel type modulated by changes in PO2. Open probability of the O2-sensitive K+ channel reversibly decreased by at least 50% on exposure to hypoxia but single-channel conductance (approximately 20 pS) was unaltered. In the range between 70 and 150 mmHg (1 mmHg = 133 Pa) the decrease of single-channel open probability was proportional to the PO2 measured in the vicinity of the membrane patch. The inhibition of K+ channel activity by low PO2 was independent of the presence of non-hydrolyzable guanine triphosphate analogues at the internal face of the membrane. The results indicate that the O2 sensor of type I cells is in the plasma membrane and suggest that environmental O2 interacts directly with the K+ channels.

Adenosine and the Control of Adrenergic Regulation of Adipose Tissue Lipolysis During Lactation

Adenosine is a locally active factor that is produced intracellularly and extracellularly in adipose tissue. Adenosine binds to receptors in the plasma membrane of adipocytes; this activates a guanine triphosphate binding protein that inhibits adenylate cyclase activity and, hence, lipolysis. Lactation results in an enhanced responsiveness of adipocytes to β-agonists, which stimulate lipolysis, and, paradoxically, to adenosine, which inhibits lipolysis. These adaptations are partly due to increases in ligand binding and to changes in postreceptor components of the signal transduction systems. Somatotropin is implicated in the chronic adaptations of the β-adrenergic system, whereas insulin, somatotropin, glucocorticoids, and at least one unidentified factor have a role in the chronic control of the adenosine system of adipocytes.

Studies on the energy metabolism of opossum (Didelphis virginiana) erythrocytes: V. Utilization of hypoxanthine for the synthesis of adenine and guanine nucleotides in vitro

High pressure liquid radiochromatography was used to test the ability of opossum erythrocytes to incorporate tracer amounts of [G-3H] hypoxanthine (Hy) into [3H] labelled triphosphates of adenine and guanine. In the presence of supraphysiologic (30 mM) phosphate which is optimal for PRPP synthesis, both ATP and GTP are extensively labelled. When physiologic (1 mM) medium phosphate is used, red cells incubated under an atmosphere of nitrogen accumulate [3H] ATP in a linear fashion suggesting ongoing PRPP synthesis in red cells whose hemoglobin is deoxygenated. In contrast, a lesser increase of labelled ATP is observed in cells incubated under oxygen, suggesting that conditions for purine nucleotide formation from ambient Hy are more favorable in the venous circulation.

Somatostatin-14 and somatostatin-28 inhibit calcium currents in rat neocortical neurons

The prosomatostatin-derived peptides, somatostatin-14 and somatostatin-28, are believed to function as neurotransmitters or neuromodulators in the cerebral cortex. To investigate the molecular mechanisms by which these peptides induce their physiological effects in the cerebral cortex, we have examined the effects of somatostatin-14 and somatostatin-28 on voltage-dependent Ca 2+ currents in rat neocortical neurons in culture. Ca 2+ currents were recorded using whole-cell patch-clamp techniques under conditions in which K + and Na + currents were blocked. Ca 2+ currents were induced by depolarization from the holding potential of -80 mV. Somatostatin-14 (100 nM) and somatostatin-28 (100 nM) did not significantly affect low-voltage activated Ca 2+ currents, but blocked high-voltage activated Ca 2+ currents and slowed the activation of this current. The effects of both peptides were concentration-dependent and reversible. Furthermore, the effects of somatostatin-14 and somatostatin-28 on the high-voltage activated Ca 2+ currents were not additive, suggesting that both peptides regulate this ionic current through similar cellular mechanisms. When patch pipettes used to record the Ca 2+ currents contained 100 μ M cAMP and 0.5 mM isobutylmethylxanthine, a phosphodiesterase inhibitor, somatostatin-14 and somatostatin-28 still inhibited Ca 2+ currents, indicating that the effects of these peptides on the Ca 2+ currents were cAMP-independent. Inclusion of the non-hydrolysable guanine triphosphate analogue, guanine triphos-phate-gamma-thiol (500 μM) in the recording electrode produced effects similar to those induced by somatostatin-14 or somatostatin-28, suggesting the involvement of guanine nucleotide binding proteins in the actions of the peptides on the Ca 2+ currents. This hypothesis is supported by the finding that pretreatment of the neurons with pertussis toxin greatly reduced the abilities of somatostatin-14 and somatostatin-28 to inhibit Ca 2+ currents. Our results suggest that somatostatin-14 and somatostatin-28 inhibit Ca 2+ currents of rat neocortical neurons through a cAMP-independent mechanism involving pertussis toxin-sensitive guanine nucleotide binding proteins.

Characterization of the calcitonin gene-related peptide receptor in mouse T lymphocytes

We have identified and characterized specific binding for calcitonin gene-related peptide (CGRP) in mouse T lymphocytes. The binding of 125I-CGRP to mouse lymphocytes was reversible and the rate of dissociation of 125I-CGRP increased with the addition of the guanine triphosphate nucleotide analog, Gpp(NH)p. Saturation experiments, and Scatchard analysis indicated two classes of binding sites for CGRP; the apparent dissociation constants (K D) were 3.5 × 10 −10 M for high affinity binding sites (Bmax, 265 sites/cell) and 4.8 × 10 −8 M for low affinity binding sites (Bmax, 13000 sites/cell). The K D value for the high affinity binding sites is roughly comparable to the IC 50 and ED 50 values for inhibition of T lymphocyte proliferation and increase in the cyclic AMP concentration in these cells, respectively. 125I-CGRP bound to mouse T lymphocytes was displaced by unlabeled CGRP with an IC 50 value of 3.2 × 10 −10 M; salmon or human calcitonins did not inhibit the specific binding up to 1 × 10 −6 m. Our studies suggest that CGRP may be an important immunoregulatory molecule, and implicate it in the regulation of T cell function.

Dual Mg2+ control of formyl‐peptide‐receptor–G‐protein interaction in HL 60 cells

In neutrophils and several other phagocytic cell types, a pertussis- and cholera-toxin-sensitive form of the guanine-nucleotide-binding protein (G-protein) Gp couples receptors for N-formylmethionine-containing chemotactic peptides to stimulation of phospholipase C. Using membranes of myeloid differentiated HL 60 cells, we have examined the role of Mg2+ and guanine nucleotides in regulating (a) the interaction of the formyl-peptide receptor with the chemotactic agonist N-formylmethionyl-leucyl-phenylalanine (fMet-Leu-Phe) and (b) the receptor-mediated activation of Gp. Mg2+ markedly enhanced the number of receptors with high affinity for the radiolabeled oligopeptide fMet-Leu-[3H]Phe. At the same time, Mg2+ largely increased the potency of guanosine-5'-(3-O-thio)triphosphate, but not of GDP or guanosine-5'-(2-O-thio)diphosphate, to inhibit binding of the peptide. Comparison of the potency of Mg2+ in eliciting these two effects and analysis of the specificities of the relevant divalent cation sites revealed that Mg2+ interacts with at least two independent sites on the receptor-Gp complex. One site is specific for Mg2+ and exhibits affinity in the micromolar range, the other site interacts with millimolar concentrations of several divalent cations in a non-selective fashion. It is suggested that the former site is located on Gp and that interaction of Mg2+ with this site is necessary for the receptor-mediated G-protein activation, whereas interaction of divalent cations with the latter site is necessary for high affinity agonist binding. The regulation of the formyl-peptide receptor binding properties by guanine nucleotides is independent of Gp activation, since inhibition of peptide binding is achieved by addition of both guanine nucleoside diphosphates and triphosphates and is readily seen both in the presence and in the absence of Mg2+. The latter finding, together with the observation that, at micromolar concentrations of Mg2+, high-affinity GTPase activity is stimulated by fMet-Leu-Phe primarily via low affinity receptors, suggests that, contrary to widely held opinions, (a) divalent cations are not required for a functional receptor--G-protein interaction and (b) high-affinity agonist binding is not a prerequisite for the receptor-mediated activation of the G-protein.

Biochemical properties of brain somatostatin receptors

The physical properties of brain and pituitary somatostatin receptors were characterized using photocrosslinking techniques. Somatostatin receptors in rat corpus striatum and anterior pituitary membranes were covalently bound to the non-reducible somatostatin analog, [ 125I]CGP 23996, using the crosslinking agent n-hydroxysuccinimidyl-4-azidobenzoate and ultraviolet light. In striatal membranes, a protein of 60,000 mol. wt was labeled by [ 125I]CGP 23996. The binding was potently inhibited by somatostatin analogs but not by other biologically active peptides. The labeling of the 60,000 mol. wt protein by [ 125I]CGP 23996 was diminished by guanine triphosphate gamma thiol, which is consistent with the labeling of a somatostatin receptor coupled to guanine triphosphate binding proteins. The migration of the [ 125I]CGP 23996 labeled 60,000 mol. wt protein in native sodium dodecyl sulfate-gels was not affected by the reducing agent dithiothreitol, indicating that there is a general lack of disulfide bridges in the striatal somatostatin receptor. The striatal somatostatin receptor was solubilized with the detergent 3-[(3-cholamidopropyl)-dimethylaminoio]-1-propanesulfonate and specifically bound to the lectin wheat germ agglutinin, suggesting that the striatal somatostatin receptor is a glycoprotein. [ 125I]CGP 23996 also labeled a 60,000 mol. wt protein in anterior pituitary membranes. The characteristics of [ 125I]CGP 23996 binding to anterior pituitary membranes were consistent with the labeling of a somatostatin receptor. Interestingly, a comparison of the [ 125I]CGP 23996 labeled material from striatal and anterior pituitary membranes by two-dimensional polyacrylamide gel electrophoresis revealed the presence of several striatal somatostatin receptors of varying charge (pI values between 6 and 6.5) but only a single pituitary receptor. These findings indicate that physical differences may exist between subtypes of somatostatin receptors.

Mu-type opioid receptors in rat periaqueductal gray-enriched P 2 membrane are coupled to guanine nucleotide binding proteins

The periaqueductal gray (PAG) region of the midbrain has been implicated in both stimulation-produced and opioid-induced analgesia. High affinity μ-selective opioid binding sites presumably associated with μ-type opioid receptors have been detected in rat PAG-enriched P 2 membrane. In the present study the signal transduction mechanism of μ-type opioid receptors in the PAG was examined utilizing both in vitro radioligand binding and GTPase assays. The non-hydrolyzable guanine triphosphate (GTP) analog g guanyl-5′-ylβ-γ-imidodiphosphate (GppNHp) inhibited specific high affinity [ 3 H][ d-Ala 2,N- MethylPhe 4, Glyol 5] enkephalin (DAGO) binding in rat PAG-enriched P 2 membrane in a dose-dependent manner. DAGO stimulated total GTPase activity in rat PAG-enriched P 2 membrane in a saturable, dose-dependent, ligand-selective, stereoselective, and naloxone-reversible manner. This DAGO stimulation of total GTPase activity was also dependent on Na + and Mg 2+, and was abolished by pertussis toxin pretreatment of the membrane. Overall these data suggest that μ-type opioid receptors in the PAG are coupled to guanine nucleotide binding proteins (G proteins).

Inhibition of varicella-zoster virus-induced DNA polymerase by a new guanosine analog, 9-[4-hydroxy-2-(hydroxymethyl)butyl]guanine triphosphate.

The triphosphates of the antiherpesvirus acyclic guanosine analogs 9-[4-hydroxy-2(hydroxymethyl)butyl] guanine (2HM-HBG), 9-(2-hydroxyethoxymethyl)guanine (acyclovir [ACV]), and 9-(3,4-dihydroxybutyl)guanine (buciclovir) were examined for their effects on partially purified varicella-zoster virus (VZV) DNA polymerase as well as cellular DNA polymerase alpha. The triphosphate of 2HM-HBG competitively inhibited the incorporation of dGMP into DNA catalyzed by the VZV DNA polymerase. 2HM-HBG-triphosphate (2HM-HBG-TP) had a higher affinity for the dGTP-binding site on the VZV DNA polymerase than did dGTP; apparent Km and Ki values of dGTP and 2HM-HBG-TP were 0.64 and 0.034 microM, respectively. ACV-triphosphate (ACV-TP) was found to be the most potent inhibitor of VZV DNA polymerase. ACV-TP had a 14 and 464 times better direct inhibitory effect than 2HM-HBG-TP and buciclovir-triphosphate, respectively. The cellular (human embryonic lung fibroblast) DNA polymerase alpha inhibition was related to viral polymerase inhibition as efficacy ratios: 2HM-HBG-TP had a ratio of more than 1,000, which appeared to be similar to that of ACV-TP.

Insertion and extension of acyclic, dideoxy, and ara nucleotides by herpesviridae, human alpha and human beta polymerases. A unique inhibition mechanism for 9-(1,3-dihydroxy-2-propoxymethyl)guanine triphosphate.

The ability of human alpha and beta DNA polymerases and herpes simplex virus type 2 (HSV-2) and human cytomegalovirus (HCMV) DNA polymerases to insert and extend several nucleotide analogs has been investigated using a variation of Sanger-Coulson DNA sequencing technology. The analogs included the triphosphates of two antiviral nucleosides with incomplete sugar rings: 9-(1,3-dihydroxy-2-propoxymethyl)guanine (dhpG) and 9-(2-hydroxyethoxymethyl)guanine (acyG or acyclovir), as well as dideoxy and arabinosyl nucleoside triphosphates. Three pairs of contrasting behaviors were found, each pair distinguishing the two human polymerases from the two viral ones: first, extension behavior with araNTPs; second, insertion/extension behavior with dhpGTP; and third, the relative preference for insertion of ddGTP versus acyGTP. The relative level of insertion of the nucleotide analogs by HCMV and HSV-2 DNA polymerases was dhpGTP greater than (acyGTP and araNTP) greater than ddGTP, whereas by human alpha polymerase it was araATP greater than ddGTP much greater than (acyGTP and dhpGTP) and by human beta polymerase it was (araATP and ddGTP) much greater than (acyGTP and dhpGTP). Evidence is presented for three mechanisms of inhibition by extendible nucleotides (of dhp and ara types) exhibiting frequent internalization: araATP acted as a simple pseudoterminator of alpha and beta polymerases, but was easily extended past singlet sites by Herpesviridae polymerases and only stalled at sites requiring two or more araATP insertions in a row. Herpesviridae polymerases stalled after adding dhpGMP and one additional nucleotide, suggesting that polymerase translocation problems may be a factor in polymerase inhibition by modified sugar nucleotide analogs. The amino acid sequence of the human alpha DNA polymerase, which is acyGTP resistant, was found to vary by one amino acid from the amino sequences of the Herpesviridae polymerases in a region of significant similarity and probable functional homology. Amino acid differences at that same site differentiate acyclovir-resistant HSV-1 mutants from the acyclovir-sensitive HSV-1 wild type.

Insertion and extension of acyclic, dideoxy, and ara nucleotides by herpesviridae, human alpha and human beta polymerases. A unique inhibition mechanism for 9-(1,3-dihydroxy-2-propoxymethyl)guanine triphosphate.

Insertion and extension of acyclic, dideoxy, and ara nucleotides by herpesviridae, human alpha and human beta polymerases. A unique inhibition mechanism for 9-(1,3-dihydroxy-2-propoxymethyl)guanine triphosphate.

Distribution and HPLC study of chromium-51 binding sites in Chinese hamster ovary cells.

Chinese hamster ovary cells were cultured with chromium-51 chromate to study the site of chromium interaction with cell biomolecules. After incubation, cells were homogenized and separated into nuclear, mitochondrial, microsomal, and cytosolic fractions. Greater than 75% of the radioactivity was found in the cytosolic fraction. The supernatant from the centrifuged cell homogenate, which contained greater than 90% of the chromium radioactivity, was subjected to chromatographic investigation. The combination of anion exchange and ion-pair high-performance liquid chromatography (HPLC) showed that at least three different molecular species interact with chromate or its reduced derivative, Cr(III). These species are glutathione, the nucleotides cytosine triphosphate, uridine triphosphate, guanine triphosphate, adenosine triphosphate, and adenosine diphosphate, plus an as yet unknown species of protein or peptide. Preliminary data for the specific activity of nucleoside triphosphates range from 6000 to 18,000 cpm/micrograms ribonucleoside triphosphate. The glutathione accounted for 50% of the observed radioactivity, the nucleotides for 30%, and the metalloprotein accounted for the remainder.

Mutually exclusive inhibition of herpesvirus DNA polymerase by aphidicolin, phosphonoformate, and acyclic nucleoside triphosphates.

Dual inhibitor studies were performed to examine the interaction of aphidicolin, phosphonoformate, 9-(2-hydroxyethoxymethyl)guanine triphosphate, and 9-(1,3-dihydroxy-2-propoxymethyl)guanine triphosphate with herpes simplex virus DNA polymerase. Kinetic data indicated that inhibition by one agent prevents simultaneous inhibition by a second agent, producing a mutually exclusive inhibition pattern. This suggested that binding sites on the DNA polymerase molecule for these compounds are kinetically overlapping. These findings should be taken into consideration for the design of future antiviral compounds and combination chemotherapy protocols.

Isolation and characterization of human liver guanine deaminase

Guanine deaminase (EC 3.5.4.3, guanine aminohydrolate [GAH]) was purified 3248-fold from human liver to homogeneity with a specific activity of 21.5. A combination of ammonium sulfate fractionation, and DEAE-cellulose, hydroxylapatite, and affinity chromatography with guanine triphosphate ligand were used to purify the enzyme. The enzyme was a dimer protein of a molecular weight of 120,000 with each subunit of 59,000 as determined by gel filtration and sodium dodecyl sulfate-gel electrophoresis. Isoelectric focusing gave a p I of 4.76. It was found to be an acidic protein, as evidenced by the amino acid analysis, enriched with glutamate, aspartate, alanine and glycine. It showed a sharp pH optimum of 8.0. The apparent K m for guanine was determined to be 1.53 × 10 −5 m at pH 6.0 and 2 × 10 −4 m for 8-azaguanine as a substrate at pH 6.0. The enzyme was found to be sensitive to p-hydroxymercuribenzoate inhibition with a K i of 1.53 × 10 −5 m and a K i of 5 × 10 −5 m with 5-aminoimidazole-4-carboxamide as an inhibitor. The inhibition with iodoacetic acid showed only a 7% loss in the activity at 1 × 10 −4 m and a 24% loss at 1 × 10 −3 m after 30 min of incubation, whereas p-hydroxymercuribenzoate incubation for 30 min resulted in a 91% loss of activity at a concentration of 1 × 10 −4 m. Guanine was the substrate for all of the inhibition studies. The enzyme was observed to be stable up to 40 °C, with a loss of almost all activity at 65 °C with 30 min incubation. Two p K a values were obtained at 5.85 and 8.0. Analysis of the N-terminal amino acid proved to be valine while the C-terminal residue was identified as alanine.