Human hypoxanthine-guanine phosphoribosyltransferase. IMP-GMP exchange stoichiometry and steady state kinetics of the reaction.

Steady state kinetics of hypoxanthine guanine phosphoribosyltransferase-catalyzed reactions are studied. The results obtained suggest that IMP, GMP, P-Rib-PP, and pyrophosphate bind to the same enzyme form, while hypoxanthine and guanine bind to a different form. Guanine activates IMP-pyrophosphorolysis. During the reaction guanine and IMP are consumed with formation of GM     

Steady-state kinetics of the schistosomal hypoxanthine-guanine phosphoribosyltransferase.

Schistosomiasis is a trematode infection of some 200 million people. The hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) of the major etiologic agent, Schistosoma mansoni, has been proposed as a potential target for antischistosomal chemotherapy [Dovey, H. F., McKerrow, J. H., & Wang, C. C. (1984) Mol. Biochem. Parasitol, 11, 157-167]. The steady-state kinetic mechanism for the schistosomal HGPRTase has been determined by including both hypoxanthine and guanine in the forward and reverse reactions under identical conditions. Double-reciprocal plots of initial velocity versus the concentration of one substrate, at a series of fixed concentrations of the other, give groups of intersecting straight lines indicating a sequential mechanism for the schistosomal HGPRTase-catalyzed reactions. In product inhibition studies, the results show that magnesium pyrophosphate (MgPPi) is a noncompetitive inhibitor with respect to dimagnesium phosphoribose pyrophosphate (Mg2PRPP), hypoxanthine, and guanine. Also, magnesium inosine monophosphate (MgIMP) and magnesium guanosine monophosphate (MgGMP) are noncompetitive inhibitors with respect to hypoxanthine or guanine, respectively, but are competitive inhibitors to Mg2PRPP. Furthermore, Mg2PRPP is a competitive inhibitor with respect to MgIMP and MgGMP but is a non-competitive inhibitor to MgPPi. The minimum kinetic model which fits the experimental data is an ordered bi-bi mechanism, where the substrates bind to the enzyme in a defined order (first Mg2PRPP followed by the purine bases), while products are released in sequence (first MgPPi followed by MgIMP or MgGMP).(ABSTRACT TRUNCATED AT 250 WORDS)     

31P nuclear magnetic resonance study of phosphoribosyldiphosphate and its interaction with magnesium ions

31P NMR has been used to study phosphoribosyldiphosphate (P-Rib-PP) over a wide range of pH values, both in the absence and presence of MgCl2. In the absence of MgCl2, the chemical shift variations of the three 31P nuclei in the molecule, over the pH range 4 to 9, were found to be largest for the terminal 1-diphosphate (1P beta) oxyanion and the 5-phosphate (5P) moiety. Apparent pK alpha values of approximately 6.1 and 6.3 were estimated for protonation of the 1P beta and 5P groups, respectively. Variations in the apparent pK alpha values associated with 1P beta and 5P oxyanions in the presence of various concentrations of MgCl2 were consistent with P-Rib-PP having two independent metal ion binding sites with different affinities for Mg2+ ions. The binding of Mg2+ reduced the apparent pK alpha of the 1P beta moiety by approximately 1.6 units and the apparent pK alpha of the 5P group by approximately 0.7 unit. This behavior is analogous to the situation reported for the terminal phosphooxyanion of ADP and observed for the phosphate group of ribose 5-phosphate, respectively. In the presence of an equimolar concentration of added MgCl2, the 1P alpha and 1P beta resonances of P-Rib-PP were shifted downfield and the 31P-31P coupling constant was decreased. Changes in both these parameters were very similar to those reported for the MgADP- complex. The observed chemical shifts and spin-spin coupling constants suggest that the diphosphate and monophosphate moieties of P-Rib-PP act as independent binding sites for Mg2+ in a manner similar to the phosphooxyanion groups of ADP and ribose 5-phosphate, respectively.     

Salt inactivation as a mechanistic probe of membrane-bound chloroplast coupling factor 1.

Conditions are reported under which ATP protects membrane-bound coupling factor 1 against sodium bromide inactivation. The presence of Mg2+ was found to be obligatory for this protection. ADP and GTP also protected the enzyme against salt inactivation but to a much smaller extent. Other nucleotides tested were ineffective. At low ATP concentrations ADP prevented the effect of ATP and modified the saturation curve for ATP from hyperbolic to sigmoidal. Treatment of chloroplasts with 0.4 M MgCl2 or 2 M LiCl resulted in inactivation of photophosphorylation. In contrast to NaBr-depleted particles the MgCl2 or LiCl-depleted chloroplasts can be reconstituted by purified coupling factor 1. A binding site for Mg2+ and two different sites for ATP upon the coupling factor 1 are suggested to explain the mechanism of their protection against salt inactivation.     

Modulation of the response of bovine adrenocortical adenylate cyclase to corticotropin.

An assessment was made of some of the basic parameters responsible for the modulation of adenylate cyclase activity in a bovine adrenocortical plasma-membrane preparation. When determined at 0.1 mM-ATP, basal adenylate cyclase activity increased with increasing MgCl2 concentrations, whereas in the presence of corticotropin activity was essentially maximal at 10mM-MgCl2; high concentrations (25mM) of MgCl2 inhibited adenylate cyclase activity determined in the presence of both corticotropin and GTP. At all MgCl2 concentrations, corticotropin and GTP activated the enzyme in a synergistic fashion. The magnitude of the stimulation of basal activity produced by corticotropin was a function of Mg2+ concentration, whereas that produced by GTP appeared largely independent of Mg2+ concentration. Adenylate cyclase activity in the bovine adrenal membrane was half-maximally stimulated by corticotropin concentrations in the range 0.3--1.0 nM. The concentration of corticotropin evoking half-maximum response was not significantly affected by raising the free Mg2+ concentration from 0.4 to 4.9 mM, nor by the presence of GTP. In the presence of GTP, high concentrations (over 1 micrometer) of corticotropin inhibited adenylate cyclase activity, although no inhibition was apparent in the absence of guanine nucleotide.     

Regulation of purine de novo synthesis in cultured human fibroblasts: the role of P-ribose-PP.

Procedures for assaying the rate of purine de novo synthesis in cultured fibroblast cells have been compared. These were (i) the incorporation of [(14)C]-glycine or [(14)C]formate in alpha-N-formylglycinamide ribonucleotide (an intermediate in the purine synthetic pathway) and (ii) the incorporation of [(14)C]-formate into newly synthesised cellular purines and purines excreted by the cell into the medium. Fibroblast cells, derived from patients with a deficiency of hypoxanthine phosphoribosyltransferase (HPRT-) (EC 2.4.2.8) and increased rates of purine de novo synthesis, were compared with fibroblasts from healthy subjects (HPRT+). Fetal calf serum, which was used to supplement the assay and cell growth medium, was found to contain sufficient quantities of the purine base hypoxanthine to inhibit purine de novo synthesis in HPRT+ cells. This inhibition was the basis of differentiation between HPRT- and HPRT+ cells. In the absence of added purine base, both cell types had similar capacities for purine de novo synthesis. This result contrasts with the increased rates of purine de novo synthesis reported for a number of human HPRT- cells in culture but conforms recent studies made on human HPRT- lymphoblast cells. The intracellular concentration and utilisation of 5-phosphoribosyl-1-pyrophosphate (P-Rib-PP), a substrate and potential controlling factor for purine de novo synthesis, were determined in HPRT- and HPRT+ cells. The rate of utilisation of P-Rib-PP in the salvage of free purine bases was far greater than that in purine de novo synthesis. Although HPRT- cells had a 3-fold increase in P-Rib-PP content, the rate of P-Rib-PP generation was similar to HPRT+ cells. Thus, in fibroblasts, the concentration of P-Rib-PP appears to be critical in the control of de novo purine synthesis and its preferential utilisation in the HPRT reaction limits its availability for purine de novo synthesis. In vivo, HPRT+ cells, in contrast to HPRT- cells, may be operating purine de novo synthesis at a reduced rate because of their ability to reutilise hypoxanthine.     

Effects of NaCl concentration on adenylate cyclase regulation by prostaglandins and guanine nucleotides

Na+ has been implicated as a requirement for the inhibition of adenylate cyclase by hormones and neurotransmitters. This study examines effects of salt concentration on neuroblastoma plasma membranes that occur in the absence of an inhibitory hormone. The adenylate cyclase response to stimulatory agonists (GTP plus PGE1 (3), PGI2 or PGE2) was influenced by NaCl. As the [NaCl] increased to 150 mM, an increase in maximal activity and a decrease in apparent affinity was observed. At concentrations above 150 mM, NaCl decreased prostaglandin affinity and progressively decreased maximal activation. The GTP requirement was not altered by 30 or 150 mM NaCl in the presence of PGE1 or PGI2. The rate of Gpp(NH)p stimulated activity increased as the [NaCl] was increased in the assay. This increased rate was conserved when membranes activated in the presence of Gpp(NH)p and NaCl were reassayed in the absence of guanine nucleotide or salt. The salt evoked rate increase was proportionally greater at submaximal MgCl2 concentrations. The concentration requirement for Mg2+ was reduced by salt for adenylate cyclase in the presence of GTP or Gpp(NH)p. However, the enzyme stimulated by hormone exhibited a Mg2+ requirement that was low in the absence of salt and could not be further reduced by increased [NaCl]. Alternative monovalent cations (150 mM Li+, K+, Cs+, but not choline or tetramethylammonium) and anions (SO4=) substituted for NaCl. The observed effects were reversible upon washing the membranes and neither ouabain nor tetrodotoxin altered the response. These effects may result from a conformational alteration of a protein particularly sensitive to neutral salts in the assay.     

Phosphorothioate analogues of 5-phosphoribosyl 1-diphosphate: synthesis, purification, and partial characterization

The 1-phosphorothioate analogues of 5-phosphoribosyl 1-diphosphate (P-Rib-PP) have been prepared enzymatically, in reactions catalyzed by P-Rib-PP synthetase from Salmonella typhimurium. 5-Phosphoribosyl 1-O-(2-thiodiphosphate) (P-Rib-PP beta S) was synthesized from ribose 5-phosphate (Rib-5-P) and the Mg2+ complex of adenosine 5'-O-(3-thiotriphosphate). The SP and RP diastereomers of 5-phosphoribosyl 1-O-(1-thiodiphosphate) (P-Rib-PP alpha S) were synthesized from Rib-5-P and the Mg2+ complex of adenosine 5'-O-(2-thiotriphosphate) (ATP beta S) (SP diastereomer, delta-configuration) and the Cd2+ complex of ATP beta S (RP diastereomer, delta-configuration), respectively. The strategy for the synthesis and stereochemical assignment of the P-Rib-PP alpha S diastereomers was based on the specificity of P-Rib-PP synthetase for the (delta)-beta, gamma-bidentate metal-nucleotide substrate and the stereochemical course of the synthetase reaction, leading to inversion of configuration at the P beta atom of the nucleotide [Li, T. M., Mildvan, A. S., & Switzer, R. L. (1978) J. Biol. Chem. 253, 3918-3923], and the known configurations of the Mg2+ and Cd2+ beta, gamma-bidentate complexes of the ATP beta S diastereomers [Jaffe, E. K., & Cohn, M. (1979) J. Biol. Chem. 254, 10839-10845]. The P-Rib-PP analogues were purified by gradient elution from DEAE-Sephadex and characterized by chemical analysis and 31P nuclear magnetic resonance [Smithers, G. W., & O'Sullivan, W. J. (1984) Biochemistry (following paper in this issue)]. A preliminary account of their interaction with human brain hypoxanthine phosphoribosyltransferase and yeast orotate phosphoribosyltransferase (OPRTase) is described.(ABSTRACT TRUNCATED AT 250 WORDS)     

Guanine salvage by organ cultures of mouse tooth germs

The effect of mycophenolic acid (MPA) which inhibits the biosynthesis of guanosine monophosphate (GMP) in organ cultures of mouse tooth germs can be partially counteracted by adding guanine to the MPA cultures. This may be due to salvaging guanine by the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT), or to competition for a common membrane carrier involved in mediated transport of both guanine and hypoxanthine in normal biosynthesis and also of MPA. Experiments were carried out to compare the effect of either hypoxanthine or guanine on the MPA-caused inhibition. While addition of guanine to the MPA cultures (MPAG) supports growth equal to controls and development of dental-enamel junction (DEJ) to a level intermediate between control and MPA the addition of hypoxanthine (MPAHX) supports growth and DEJ development not better than MPA. This indicates that guanine is salvaged by HGPRT to GMP while hypoxanthine, salvaged to inosinic acid (inosinic monophosphate, IMP) is ineffective because the MPA inhibition is on the pathway from IMP to GMP.     

Kinetic characteristics of the ATP-pyrophosphate isotope exchange catalyzed by RNA ligase from T4 bacteriophage

The dependence of initial rate v0 of ATP--PPi exchange reaction catalyzed by RNA-ligase of bacteriophage T4 on the concentration of ATP(s), pyrophosphate (z) and Mgcl2 has been determined. The dependence of v0 on s and z described by the equation v0 = k-1k2E0/(k-1 + K2) (1 + K1/s + k2/z) has been obtained for the reaction of E + S in equilibrium ES in equilibrium E1 + Z, where E--enzyme, E1--adenylylenzyme, S--ATP, Z--pyrophosphate, K1 and K2--constants of equilibrium, k-1, k2--velocity constants of transition of ES to E + S and E1 + Z, E0--complete concentration of enzyme. The low inhibition of the ATP--PPi exchange by the acceptor A(pA)2 and donors pAp, p(Ap)3, pCp has been shown. The dependence of v0 on the concentration of MgCl2 is consent with the incorporation of only dimagnesium salts of substrates in the isotope-exchange reaction.     

Hypoxanthine phosphoribosyltransferase and hypoxanthine uptake in human erythrocytes.

A system of hypoxanthine uptake and IMP retention was studied and characterized in human erythrocytes. It follows closely the system already described for rabbit erythrocytes[7]. IMP formation and retention are dependent on the activity of hypoxanthine phosphoribosyl-transferase and on intracellular availability of phosphoribosyl pyrophosphate (P-Rib-PP), which is one of the substrates. In the extrecellular medium, neither P-Rib-PP nor GMP -- a potent inhibitor of the enzyme in vitro -- has any influence on IMP retention. The amount of residual hypoxanthine phosphoribosyltransferase in erythrocyte ghost preparations is directly related to the residual hemoglobin content. Thus the enzyme is characterized as typically soluble and "loosely bound" to membranes. There is a slight difference in the kinetic properties of the ghost-bound and the free soluble enzyme. The possible importance of these results for purine uptake and utilization in human red cells is discussed.     

Purification and properties of the deoxyuridine triphosphate nucleotidohydrolase enzyme derived from HeLa S3 cells. Comparison to a distinct dUTP nucleotidohydrolase induced in herpes simplex virus-infected HeLa S3 cells

Deoxyuridine triphosphate nucleotidohydrolase (dUTPase) (EC 3.6.1.23) derived from HeLa S3 cells has been purified to near homogeneity as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified enzyme has a specific activity of about 16,000 nmol of dUMP hydrolyzed per min/mg of protein. The dUTPase enzyme derived from HeLa S3 cells appears to be composed to two equal molecular mass subunits, each being about 22,500 daltons. Association of these subunits to produce a 45,000-dalton protein is promoted by MgCl2. In the presence of EDTA enzyme activity is abolished and the enzyme dissociates into its monomeric form. MgCl2 will completely reverse the inhibition caused by EDTA and promote subunit association. MnCl2 will also promote association of the dUTPase subunits. However, MnCl2 will not completely reverse inhibition by EDTA. In addition, purified dUTPase, extensively dialyzed to remove contaminating ions, is activated almost 2-fold by the addition of 5 mM MgCl2. In contrast, addition of 5 mM MnCl2 to the dialyzed enzyme preparation will cause more than a 50% decrease in enzyme activity. This data indicates that Mg2+ is the natural prosthetic group for this enzyme. The Km value of dUTP for the purified enzyme is 3 X 10(-6) M in the presence of MgCl2. The turnover number for this enzyme has been calculated to be 550 molecules of dUTP hydrolyzed per min under standard assay conditions. Infection of HeLa S3 cells with herpes simplex type 1 virus induces a distinct species of dUTPase. This new species of dUTPase has an isoelectric point of 8.0, compared to an isoelectric point in the range of 5.7 to 6.5 for the HeLa S3 dUTPase. Molecular weight determinations of this new species of dUTPase indicate that the native enzyme is monomeric with a molecular weight of about 35,000. The virally induced dUTPase is inhibited by EDTA and this inhibition is reversed by MgCl2. Unlike the HeLa S3 dUTPase, the virally induced enzyme does not appear to be composed of subunits.     

Crystal structures of free,IMP-, and GMP-bound Escherichia coli hypoxanthine phosphoribosyltransferase

Crystal structures have been determined for free Escherichia coli hypoxanthine phosphoribosyltransferase (HPRT) (2.9 A resolution) and for the enzyme in complex with the reaction products, inosine 5'-monophosphate (IMP) and guanosine 5'-monophosphate (GMP) (2.8 A resolution). Of the known 6-oxopurine phosphoribosyltransferase (PRTase) structures, E. coli HPRT is most similar in structure to that of Tritrichomonas foetus HGXPRT, with a rmsd for 150 Calpha atoms of 1.0 A. Comparison of the free and product bound structures shows that the side chain of Phe156 and the polypeptide backbone in this vicinity move to bind IMP or GMP. A nonproline cis peptide bond, also found in some other 6-oxopurine PRTases, is observed between Leu46 and Arg47 in both the free and complexed structures. For catalysis to occur, the 6-oxopurine PRTases have a requirement for divalent metal ion, usually Mg(2+) in vivo. In the free structure, a Mg(2+) is coordinated to the side chains of Glu103 and Asp104. This interaction may be important for stabilization of the enzyme before catalysis. E. coli HPRT is unique among the known 6-oxopurine PRTases in that it exhibits a marked preference for hypoxanthine as substrate over both xanthine and guanine. The structures suggest that its substrate specificity is due to the modes of binding of the bases. In E. coli HPRT, the carbonyl oxygen of Asp163 would likely form a hydrogen bond with the 2-exocyclic nitrogen of guanine (in the HPRT-guanine-PRib-PP-Mg(2+) complex). However, hypoxanthine does not have a 2-exocyclic atom and the HPRT-IMP structure suggests that hypoxanthine is likely to occupy a different position in the purine-binding pocket.     

Aggregation equilibria of Escherichia coli RNA polymerase: evidence for anion-linked conformational transitions in the protomers of core and holoenzyme

The aggregation equilibria of Escherichia coli RNA polymerase core and holoenzyme have been studied by velocity sedimentation as a function of [NaCl] both in the presence and in the absence of MgCl2. Effects of other anions (F- and I-), pH, and temperature have also been examined. Diffusion coefficients obtained by quasi-elastic light scattering (QLS) at high and low salt concentrations were used in conjunction with sedimentation coefficients under these conditions to obtain molecular weights of the protomer and aggregates of the core enzyme. At low salt concentration, core aggregates to a tetramer in the absence of MgCl2 and to an octamer in the presence of MgCl2. Some ambiguity exists in the interpretation of the sedimentation and QLS data for holoenzyme. The sedimentation results are consistent with the formation of dimers at low salt, both in the presence and in the absence of MgCl2. In all cases, equilibrium constants were calculated assuming a simple monomer--j-mer stoichiometry. These equilibrium constants are extremely sensitive functions of the concentration and type of monovalent anion. In Cl-, aggregation of both core and holoenzyme begins abruptly when the salt concentration is reduced below approximately 0.2 M (at a protein concentration of approximately 0.30 mg/mL); for core, substitution of I- for Cl- suppresses aggregation while F- enhances aggregation at a fixed anion concentration. No specific effect of monovalent cations (Na+, NH4+) is observed; Mg2+ has no effect on holoenzyme dimerization and has little effect on the salt range of core aggregation, though the stoichiometries of the core aggregates in the presence and absence of Mg2+ differ. Anion effects on these equilibria were modeled by assuming that a class of anion-binding sites on the protomer is not present in the aggregate, so that anion release accompanies aggregation. Analytical expressions for several models of the effect of anions on the aggregation equilibria were derived by using the method of binding polynomials. The salt dependence of the aggregation equilibria in the absence of Mg2+ appears inconsistent with a model in which the anion-binding sites on the protomer are independent (noncooperative), but it is well described by a model in which anion binding to the protomers occurs in a completely cooperative manner. The molecular basis of this apparent cooperative effect of anions on the aggregation equilibria is proposed to be an allosteric effect of anions on conformational equilibria of the protomers of core polymerase and the holoenzyme. Implications of such a salt-dependent conformational transition for the DNA-binding interactions of the enzyme are considered.     

Inhibitory effect of Ca2+ on formation of Mg2(+)-mediated two-dimensional hexagonal lattice structure by an R-form lipopolysaccharide from Klebsiella pneumoniae

When the R-form lipopolysaccharide (LPS) from Klebsiella pneumoniae strain LEN-111 (O3-:K1-), from which cationic material had been removed by electrodialysis, was suspended in 50 mM Tris buffer at pH 8.5 containing 0.1 mM or higher concentrations of MgCl2, it formed an ordered two-dimensional hexagonal lattice structure and its center-to-center distance (lattice constant) depended upon the concentration of MgCl2 and reached the shortest value (14 nm) at 10 mM. In contrast, in the presence of 0.1 to 10 mM CaCl2 in place of MgCl2, the electrodialyzed LPS did not form such an ordered hexagonal lattice structure but formed an irregular network structure with a center-to-center distance of 19 to 20 nm. We investigated interaction of Mg2+ and Ca2+ in formation of the hexagonal lattice structure by the electrodialyzed LPS suspended in 50 mM Tris buffer at pH 8.5. When 0.1 mM or higher concentrations of CaCl2 were mixed with 1 mM MgCl2 or when 1 mM or higher concentrations of CaCl2 was mixed with 10 mM MgCl2, the electrodialyzed LPS did not form the hexagonal lattice structure of the magnesium salt type but formed the irregular network structure of the calcium salt type. In the coexistence of equimolar or higher concentrations of CaCl2 together with 1 or 10 mM MgCl2, the binding of Mg to the electrodialyzed LPS was significantly inhibited and, conversely, the binding of Ca was enhanced as compared with when MgCl2 or CaCl2 was present alone. However, the coexistence of 10 times less molar concentrations of CaCl2 did not significantly inhibit the binding of Mg to the electrodialyzed LPS. Therefore, the inhibition of formation of the Mg2(+)-mediated hexagonal lattice structure of the electrodialyzed LPS by equimolar or higher concentrations of CaCl2 accompanied the inhibition of binding of Mg but that by 10 times less molar concentrations of CaCl2 did not accompany it.     

Guanine deaminase in rat liver and mouse liver and brain

1. The guanine deaminase in rat liver supernatant preparations was resolved into two fractions, A and B, on DEAE-cellulose columns. The two differed in electrophoretic mobility and in various properties. The most noteworthy distinction between A and B components was that the enzyme A activity showed a sigmoid dependence on substrate concentration whereas the enzyme B showed classical Michaelis-Menten kinetics. The K(m) value of enzyme A for guanine was 5.3mum and that of enzyme B 20mum. 2. The entire guanine deaminase activity of mouse liver was contained in the 15000g supernatant of iso-osmotic homogenates. 3. A reinvestigation of the behaviour of rat brain 15000g supernatant guanine deaminase isoenzymes revealed that one enzyme had sigmoidal kinetics and the other enzyme showed a hyperbolic response. 4. Of the guanine deaminase in mouse brain iso-osmotic sucrose homogenate 80% was recovered in the 15000g supernatant and the rest from the particles. The supernatant guanine deaminase was resolvable into two fractions on DEAE-cellulose columns. One enzyme showed sigmoidal kinetics whereas the other showed a hyperbolic response to increasing substrate concentration; the K(m) values for the reaction with guanine were respectively 5 and 66mum. 5. The particulate fractions of mouse liver and brain were devoid of any overt inhibitory activity.     

Inhibition of photophosphorylation by ATP and the role of magnesium in photophosphorylation.

ATP and pyrophosphate at high concentration (greater than 1 mM) inhibited photophosphorylation of isolated spinach chloroplasts in the normal salt medium and did not cause stimulation of electron transport. The inhibition of photophosphorylation by ATP or pyrophosphate was shown to be abolished by the addition of excess MgCl2, ADP and phosphate. It has been demonstrated that the rates of photophosphorylation in the absence and presence of ATP or pyrophosphate are determined similarly by the concentrations of magnesium-ADP (Mg - ADP-) and magnesiumphosphate (Mg - Pi) complexes. It is highly probable that Mg - ADP- and Mg - Pi, but not free ADP and free phosphate, are the active form of the substrates of photophosphorylation. This is in support of the view that ATP inhibits photophosphorylation by decreasing the concentration of Mg2+ which is available for the formation of the complex with ADP and phosphate.     

Comparative enzymatic properties of avian liver and skeletal muscle D-fructose-1,6-bisphosphate 1-phosphohydrolase.

The enzymatic properties of purified preparations of chicken liver and chicken skeletal muscle fructose bisphosphatases (D-fructose-1,6-bisphosphate 1-phosphohydrolase, EC 3.1.3.11) were compared. Both enzymes have an absolute requirement for Mg2+ or Mn2+. The apparent Km for MgCl2 at pH 7.5 was 0.5 mM for the muscle enzyme and 5 mM for the liver enzyme. Fructose bisphosphate inhibited both enzymes. At pH 7.5, the inhibitor constants (Ki) were 0.18 and 1.3 mM for muscle and liver fructose bisphosphatases, respectively. The muscle enzyme was considerably more sensitive to AMP inhibition than the liver enzyme. At pH 7.5 and in the presence of 1 mM MgCl2, 50% inhibition of muscle and liver fructose bisphosphatases occurred at AMP concentrations of 7 X 10(-9) and 1 X 10(-6) M, respectively. EDTA activated both enzymes. The degree of activation was time and concentration dependent. The degree of EDTA activation of both enzymes decreased with increasing MgCl2 concentration. Ca2+ was a potent inhibitor of both liver (Ki, 1 X 10(-4) M) and muscle (Ki, 1 X 10(-5) M) fructose bisphosphatase. This inhibition was reversed by the presence of EDTA. Ca2+ appears to be a competitive inhibitor with regard to Mg2+. There is, however, a positive homeotropic interaction among Mg2+ sites of both enzymes in the presence of Ca2+.     

Lysosomal enzyme leakage during the hypoxanthine/xanthine oxidase reaction

Impairment of lysosomal stability due to reactive oxygen species generated during the oxidation of hypoxanthine by xanthine oxidase was studied in rat liver lysosomes isolated in a discontinuous Nycodenz gradient. Production of O2.- and H2O2 during the hypoxanthine/xanthine oxidase reaction occurred for at least 5 min, while lysosomal damage, indicated by the release of N-acetyl-beta-glucosaminidase, occurred within 30 s, there being no further damage to these organelles thereafter. The extent of lysosomal enzyme release increased with increasing xanthine oxidase concentration. Superoxide dismutase and catalase did not prevent lysosomal damage during the hypoxanthine/xanthine oxidase reaction. Lysosomes reduced xanthine oxidase activity, as assessed in terms of O2 consumption, only slightly but substantially inhibited in a competitive manner the O2.- -mediated reduction of cytochrome c. This inhibition was almost completely reversed by potassium cyanide, thus pointing to the presence of a cyanide-sensitive superoxide dismutase in the lysosomal fraction. However, potassium cyanide did not affect the hypoxanthine/xanthine oxidase-mediated lysosomal damage, thus suggesting an inability of the lysosomal superoxide dismutase to protect the organelles. Negligible malondialdehyde formation was observed in the lysosomes either during the hypoxanthine/xanthine oxidase reaction or with different selective experimental approaches known to produce lipid peroxidation in other organelles such as microsomes and mitochondria.(ABSTRACT TRUNCATED AT 250 WORDS)     

Uracil phosphoribosyltransferase from Acholeplasma laidlawii: partial purification and kinetic properties

Uracil phosphoribosyltransferase was purified 34-fold from sonicated extracts of Acholeplasma laidlawii by ammonium sulfate precipitation, binding to DEAE-Sephadex, Sephadex G-200 chromatography, and hydroxylapatite chromatography. The molecular weight of the enzyme by gel filtration was approximately 80,000. The pH optimum for phosphoribosylation was around 7.5, and the optimum MgCl2 concentration was 5 mM. Initial velocity studies were conducted over a wide range of both uracil and 5-phosphoribosyl-1-pyrophosphate (P-Rib-PP) concentrations, and various equations for biomolecular reaction mechanisms were fitted to the data by nonlinear regression. When the equation for an ordered sequential mechanism was fitted to the data, the Kia thus obtained was not statistically different from zero. This is interpreted as evidence for a nonsequential ("ping-pong") reaction. Graphic analysis of the data by the Hanes-Woolf linear transform supported this conclusion. The enzyme has high affinity for uracil (KmUra = 4.2 microM; KmP-Rib-PP = 66 microM), which provides supporting evidence that this activity is responsible for the incorporation of uracil and uridine into nucleotides.