Kinetic, thermodynamic and statistical studies on the inhibition of adenosine deaminase by aspirin and diclofenac

The kinetic and thermodynamic effects of aspirin and diclofenac on the activity of adenosine deaminase (ADA) were studied in 50 mM phosphate buffer pH = 7.5 at 27 and 37 degrees C, using UV-Vis spectrophotometry and isothermal titration calorimetry (ITC). Aspirin exhibits competitive inhibition at 27 and 37 degrees C and the inhibition constants are 42.8 and 96.8 microM respectively, using spectrophotometry. Diclofenac shows competitive behavior at 27 degrees C and uncompetitive at 37 degrees C with inhibition constants of 56.4 and 30.0 microM, at respectively. The binding constant and enthalpy of binding, at 27 degrees C are 45 microM, - 64.5 kJ/mol and 61 microM, - 34.5 kJ/mol for aspirin and diclofenac. Thermodynamic data revealed that the binding process for these ADA inhibitors is enthalpy driven. QSAR studies by principal component analysis implemented in SPSS show that the large, polar, planar, and aromatic nucleoside and small, aromatic and polar non-nucleoside molecules have lower inhibition constants.     

The measurement of membrane potential during photosynthesis and during respiration in intact cells of Rhodopseudomonas capsulata by both electrochromism and by permeant ion redistribution.

Adenosine deaminase was purified 3038-fold to apparent homogeneity from human leukaemic granulocytes by adenosine affinity chromatography. The purified enzyme has a specific activity of 486 mumol/min per mg of protein at 35 degrees C. It exhibits a single band when subjected to sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, non-denaturing polyacrylamide-gel electrophoresis and isoelectric focusing. The pI is 4.4. The enzyme is a monomeric protein of molecular weight 44000. Both electrophoretic behaviour and molecular weight differ from those of the low-molecular-weight adenosine deaminase purified from human erythrocytes. Its amino acid composition is reported. Tests with periodic acid-Schiff reagent for associated carbohydrate are negative. Of the large group of physiological compounds tested as potential effectors, none has a significant effect. The enzyme is specific for adenosine and deoxyadenosine, with Km values of 48 microM and 34 microM respectively. There are no significant differences in enzyme function on the two substrates. erythro-9-(2-Hydroxy non-3-yl) adenine is a competitive inhibitor, with Ki 15 nM. Deoxycoformycin inhibits deamination of both adenosine and deoxyadenosine, with an apparent Ki of 60-90 pM. A specific antibody was developed against the purified enzyme, and a sensitive radioimmunoassay for adenosine deaminase protein is described.     

Adenosine deaminase activity of chicken erythrocyte and heart cytosols

1. The adenosine deaminase (ADA) activities of chicken erythrocyte and heart cytosols had pH optima of 6.5. The temperature optima for erythrocyte and heart ADA were 30 and 35 degrees C, respectively. 2. The deoxyadenosine/adenosine deamination ratios ranged from 0.75 to 0.84 for both ADA activities. 3. For erythrocyte ADA, Km values were 8.9-12.9 microM adenosine (range) and 8.3 microM 2'-deoxyadenosine. For heart ADA, Km values were 6.7-12.0 microM adenosine (range) and 5.3 microM 2'-deoxyadenosine. 4. Inosine was a competitive inhibitor of both erythrocyte (Ki = 73 microM) and heart (Ki = 109 microM) ADA.     

A product inhibition study on adenosine deaminase by spectroscopy and calorimetry

Kinetic and thermodynamic studies have been made on the effect of the inosine product on the activity of adenosine deaminase in a 50 mM sodium phosphate buffer, pH 7.5, at 27 degrees C using UV spectrophotometry and isothermal titration calorimetry (ITC). A competitive inhibition was observed for inosine as a product of the enzymatic reaction. A graphical-fitting method was used for determination of the binding constant and enthalpy of inhibitor binding by using isothermal titration microcalorimetry data. The dissociation-binding constant is equal to 140 microM by the microcalorimetry method, which agrees well with the value of 143 microM for the inhibition constant that was obtained from the spectroscopy method     

Conformational and Structural Analysis of Bovine β Lactoglobulin-A Upon Interaction with Cr+3

Thermodynamic studies have been made on the effect of Cr⁺³ on the conformation and structure of bovine β lactoglobulin-A, (Blg-A) in 50 mM sodium chloride solution at 27°C using isothermal titration calorimetry (ITC), circular dichroism (CD) and fluorescence spectroscopy. There is a set of six identical and independent binding sites for Cr⁺³ by a dissociation binding constant of 124 μM and the molar enthalpy of binding −17.8 kJ/mol. Circular dichroism studies do not show any significant change in the secondary structure of the protein after the binding of chromium ion on the Blg-A. Fluorescence spectroscopy studies do not show any considerable change in the tertiary structure of Blg-A due to the increasing of Cr⁺³ in low concentration. However, occupation of fourth and fifth binding sites for chromium ions induce partially unfolding in the tertiary structure of the protein resulted from solvent – exposed hydrophobic patches on BLG-A.     

Inhibition of 5-aminoimidazole-4-carboxamide ribotide transformylase, adenosine deaminase and 5''-adenylate deaminase by polyglutamates of methotrexate and oxidized folates and by 5-aminoimidazole-4-carboxamide riboside and ribotide.

With the use of a continuous spectrophotometric assay and initial rates determined by the method of Waley [Biochem. J. (1981) 193, 1009-1012] methotrexate was found to be a non-competitive inhibitor, with Ki(intercept) = 72 microM and Ki(slope) = 41 microM, of 5-aminoimidazole-4-carboxamide ribotide transformylase, whereas a polyglutamate of methotrexate containing three gamma-linked glutamate residues was a competitive inhibitor, with Ki = 3.15 microM. Pentaglutamates of folic acid and 10-formylfolic acid were also competitive inhibitors of the transformylase, with Ki values of 0.088 and 1.37 microM respectively. Unexpectedly, the pentaglutamate of 10-formyldihydrofolic acid was a good substrate for the transformylase, with a Km of 0.51 microM and a relative Vmax. of 0.72, which compared favourably with a Km of 0.23 microM and relative Vmax. of 1.0 for the tetrahydro analogue. An analysis of the progress curve of the transformylase-catalysed reaction with the above dihydro coenzyme revealed that the pentaglutamate of dihydrofolic acid was a competitive product inhibitor, with Ki = 0.14 microM. The continuous spectrophotometric assay for adenosine deaminase based on change in the absorbance at 265 nm was shown to be valid with adenosine concentrations above 100 microM, which contradicts a previous report [Murphy, Baker, Behling & Turner (1982) Anal. Biochem. 122, 328-337] that this assay was invalid above this concentration. With the spectrophotometric assay, 5-aminoimidazole-4-carboxamide riboside was found to be a competitive inhibitor of adenosine deaminase, with (Ki = 362 microM), whereas the ribotide was a competitive inhibitor of 5'-adenylate deaminase, with Ki = 1.01 mM. Methotrexate treatment of susceptible cells results in (1) its conversion into polyglutamates, (2) the accumulation of oxidized folate polyglutamates, and (3) the accumulation of 5-aminoimidazole-4-carboxamide riboside and ribotide. The above metabolic events may be integral elements producing the cytotoxic effect of this drug by (1) producing tighter binding of methotrexate to folate-dependent enzymes, (2) producing inhibitors of folate-dependent enzymes from their tetrahydrofolate coenzymes, and (3) trapping toxic amounts of adenine nucleosides and nucleotides as a result of inhibition of adenosine deaminase and 5'-adenylate deaminase respectively.     

Thermal denaturation of Micrococcus lysodeikticus adenosine triphosphatase. Influence of temperature on the circular dichroism, fluroescence and enzymic activity of the protein.

The soluble ATPase (adenosine triphosphatase) from Micrococcus lysodeikticus underwent a major unfolding transition when solutions of the enzyme at pH 7.5 were heated. The midpoint occurred at 46 degrees C when monitored by changes in enzymic activity and intrinsic fluorescence, and at 49 degrees C when monitored by circular dichroism. The products of thermal denaturation retained much secondary structure, and no evidence of subunit dissociation was detected after cooling at 20 degrees C. The thermal transition was irreversible, and thiol groups were not involved in the irreversibility. The presence of ATP, adenylyl imidodiphosphate, CaCl2 or higher concentrations of ATPase conferred stability against thermal denaturation, but did not prevent the irreversibility one denaturation had taken place. In the presence of guanidinium chloride, thermal denaturation occurred at lower temperatures. The midpoints of the transition were 45 degrees C in 0.25 M-, 38 degrees C in 0.5 M-and 30 degrees C in 0.75 M-denaturant. In the highest concentration of guanidinium chloride a similar unfolding transition induced by cooling was observed. Its midpoint was 9 degrees C, and the temperature of maximum stability of the protein was 20 degrees C. The discontinuities occurring the the Arrhenius plots of the activity of this enzyme had no counterpart in variations in the far-u.v. circular dichroism or intrinsic fluorescence of the protein at the same temperature.     

Purification and characterization of adenosine deaminase from a genetically enriched mouse cell line

Mammalian adenosine deaminase has been shown by genetic and biochemical evidence to be essential for the development of the immune system. For the purpose of studying the function and structure of this enzyme, we have isolated by genetic selection a mouse cell line, B-1/50, in which adenosine deaminase levels were increased 4,300-fold over the parent cell line. The enzyme was purified from these cells in large quantity and high yield by a simple two-step purification scheme. The enzyme derived from the B-1/50 cells was indistinguishable from that of the parental cells as judged by several biochemical criteria. The Km (30 microM) and Ki (4 nM) values using adenosine as substrate and 2'-deoxycoformycin as inhibitor, respectively, were identical for the enzyme derived from the parental cells as well as the adenosine deaminase gene amplification mutants. The enzyme from both cell types exhibited multiple isoelectric focusing forms which co-purified using our purification protocol. Electrophoretic analysis using sodium dodecyl sulfate-polyacrylamide gels showed that adenosine deaminase migrated with an apparent molecular weight of 41,000 or 36,000 depending on whether the enzyme was reduced or oxidized, respectively. This shift was reversible, indicating that proteolysis was not responsible for the faster migrating form. Monospecific antibodies raised against purified adenosine deaminase cross-reacted with the enzyme derived from the parental cells and precipitated 37% of the total soluble protein in the B-1/50 cells. Continued genetic selection resulted in the isolation of cells in which adenosine deaminase was overproduced by 11,400-fold and accounted for over 75% of the soluble protein.     

Structure-function studies of Murraya koenigii trypsin inhibitor revealed a stable core beta sheet structure surrounded by alpha-helices with a possible role for alpha-helix in inhibitory function

Structure-function studies of Murraya koenigii trypsin inhibitor revealed a compact structure made of central beta-sheet surrounded by alpha-helices with differences in structure and functional stability. Proteolysis studies, of native and heat-treated protein, demonstrated that inhibitor exhibited strong resistance to proteolysis by many proteases. However, the inhibitory activity gradually decreased with increasing temperature and was completely lost at 90 degrees C. CD studies, under native conditions, showed that inhibitor contains approximately 46% beta-strand, 30.1% alpha-helical, 16.2% turn and 6.9% random coil structure. At increasing temperatures, however, helix to coil transition was observed. The ANS fluorescence study showed linear increase of fluorescence intensity without showing any melting transition. Correlating decrease in inhibitory activity and helical content at increasing temperatures suggest a possible role for alpha-helical structure in inhibitory function of the protein.     

Conformational changes in bovine lactoferrin induced by slow or fast temperature increases

Lactoferrin (LF) is an iron-binding protein present in several secreted substances, such as milk, and has broad antimicrobial and physiological properties. Because high temperatures may affect protein stability and its functional properties, we investigated the effect of heat on bovine LF structure and stability. The effects of temperatures used during the pasteurization process on LF and its relationship to protein functionality were studied. Conformational changes were monitored using spectroscopic techniques, such as circular dichroism (CD) and fluorescence spectroscopy. The CD data at 70 degrees C showed that LF's secondary structure is drastically and irreversibly affected when the temperature is gradually increased. The same effect is observed when the temperature is gradually raised from 25 degrees C to 105 degrees C and changes are monitored by tryptophan fluorescence emission. We also verified the effects of simulating the pasteurization process; LF remained well structured during the entire process and this result was not time-dependent. Owing to preservation of the secondary structure with changes in the tertiary structure, we thus believe that pasteurization might cause LF to change into an intermediate partially folded state. A better understanding of heat stability is important for the use of LF as a bioactive component in food.     

Inward fluxes of adenosine in erythrocytes and cultured cells measured by a quenched-flow method.

Dilazep, a vasodilator previously recognized as an inhibitor of adenosine permeation, very rapidly blocked the uptake of adenosine by cultured L5178Y cells, and accordingly was used as a quencher in a simple quenched-flow system for measuring cellular uptake of nucleosides during very short intervals. Time courses of cellular uptake of adenosine, assayed during intervals between 0.05 and 0.5s with the quenched-flow system, were linear and defined initial rates of adenosine uptake. The latter are rates of inward transport of adenosine. Kinetic constants for that process in cultured S49 cells determined with the quenched-flow procedure were similar to those determined with an assay dependent on manual timing. In studies of adenosine uptake kinetics in human erythrocytes at 22 degrees C and 37 degrees C in which the quenched-flow procedure was used, time courses of adenosine uptake were linear at both temperatures and defined initial uptake rates; kinetic constants (means +/- S.E.M.) at 22 degrees C (n = 8) were Km 25 +/- 14 microM and Vmax. 15 +/- 5 pmol/s per microliter of cell water and at 37 degrees C (n = 3) were Km 98 +/- 17 microM and Vmax. 80 +/- 9 pmol/s per microliter of cell water.     

Effect of probucol on the physical properties of low-density lipoproteins oxidized by copper

Human plasma low-density lipoproteins (LDL) were incubated with 10 microM probucol for 1 h at 37 degrees C. Probucol incorporation into the LDL was complete as judged by filtration through a 0.2-micron filter, ultracentrifugation, and gel filtration. LDL with and without probucol were incubated for up to 24 h with 5 microM Cu2+ at 37 degrees C. Copper oxidation increased the content of random structure in the LDL protein from 30% to 36% at the expense of beta-structure (which decreased from 22% to 16%) without a change in alpha-helical content as measured by circular dichroism spectroscopy. This loss of beta-structure was prevented by the presence of probucol in the LDL during the copper incubation. Probucol reduced the rate of increase of fluorescence during copper oxidation at 37 degrees C. After 6 h, the fluorescence intensity at 360-nm excitation and 430-nm emission was 30% less in probucol-containing samples. Probucol had no effect on the circular dichroic spectrum of LDL and only minimal effects (less than 5%) on the fluorescence emission spectrum at wavelengths below 500 nm. Two fluorescence peaks, with emission at 420 nm and excitation at 340 and 360 nm, are resolved in three-dimensional fluorescence spectra of oxidized LDL. Probucol reduces the intensity of both peaks equally. The binding of a highly reactive heparin (HRH) fraction to LDL was measured by titration of LDL with HRH in the presence of fluoresceinamine-labeled HRH. The decrease in fluorescence anisotropy of the labeled HRH is proportional to the concentration of bound HRH.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformational studies of a hyperthermostable enzyme

The structural features of the hyperthermophilic endo-beta-1,3-glucanase from Pyrococcus furiosus were studied using circular dichroism, steady-state and time-resolved fluorescence spectroscopy and anisotropy. Upon heat and chemical treatment the folded and denatured states of the protein were characterized by distinguishable spectral profiles that identified a number of conformational states. The fluorescence methods showed that the spectral differences arose from changes in the local environment around specific tryptophan residues in the native, partially folded, partially unfolded and completely unfolded state. A structural resemblance was observed between the native protein and the structurally perturbed state which resulted after heat treatment at 110 degrees C. The enzyme underwent disruption of the native secondary and tertiary structure only after incubation at biologically extremely high temperatures (i.e. 150 degrees C), whilst in the presence of 8 m of guanidine hydrochloride the protein was partially unfolded.     

Inhibition of thymidine kinase by P1-(adenosine-5')-P5-(thymidine-5')-pentaphosphate

Potential bisubstrate analogs, with adenosine and thymidine joined at their 5' positions by polyphosphoryl linkages of varying lengths (ApndT, where n = the number of phosphoryl groups), were examined as inhibitors of cytosolic thymidine kinase from blast cells of patients with acute myelocytic leukemia. Ki values were 1.2 microM for Ap3dT, 0.31 microM for Ap4dT, 0.12 microM for Ap5dT, and 0.19 microM for Ap6dT. The best inhibitor of the cytosolic enzyme, Ap5dT, was somewhat less effective as an inhibitor of the mitochondrial enzyme (Ki = 0.50 microM). In addition to their inhibitory modes of binding by the cytosolic enzyme, these compounds were bound at considerably lower concentrations (Kd = 0.029 microM for Ap4dT, 0.0025 microM for Ap5dT, and 0.0027 microM for Ap4dT), in such a way as to protect the cytosolic enzyme from thermal inactivation at 37 degrees C in the absence of substrates.     

Adenosine release from isolated rat adipocytes: influence of fat cell concentration and cell size

The release of adenosine by isolated rat adipocytes into the incubation medium was studied in relation to fat cell size and concentration. Incubations were carried out for 60 min at 37 degrees C in Krebs-Ringer bicarbonate-albumin medium containing 6 mM glucose. 2'-Deoxycoformycin was added to inhibit endogenous adenosine deaminase activity (maximal suppression was achieved at 0.8 microM concentration of the inhibitor). The data show that (a) the amount of adenosine released into the medium was similar for the first and second 30-min incubation periods; (b) increasing adipocyte concentration markedly inhibited adenosine release; and (c) large fat cells (volume greater than 300 pl) released significantly more adenosine (per fat cell) into the medium than smaller fat cells (volume less than 180 pl) when incubated at concentrations of less than or equal to 350,000 cells/ml. Above this cell concentration, differences between adenosine release and cell size were not noted. Adenosine release by isolated rat adipocytes appears to be a precisely regulated process which is exquisitly sensitive to the number of fat cells in the incubation medium and, to a certain extent, to the adipocyte size.     

Purification, stability and kinetic properties of highly purified adenosine deaminase from Bacillus cereus NCIB 8122

Adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4) from Bacillus cereus NCIB 8122 has been purified to electrophoretic homogeneity by ammonium sulfate precipitation, gel filtration through Sephadex G-100, DEAE-Sephadex A-50 chromatography and ion-exchange HPLC on DEAE-Polyol. The enzyme activity is stabilized (at temperatures from 0 degrees C to 40 degrees C) by 50 mM NH4+ or K+, while it is irreversibly lost in the absence of these or a few other monovalent cations. Glycerol (24% by volume) helps the cation in stabilizing the enzyme activity above 40 degrees C, but also exerts per se a noticeable protecting effect at room temperature. B. cereus adenosine deaminase displays the following properties: Mr on Sephadex G-200, 68,000; Mr in SDS-polyacrylamide gel electrophoresis, 53,700; optimal pH-stability (in the presence of 50 mM KCl) over the range 8-11 at 4 degrees C, and maximal catalytic activity at 30 degrees C between pH 7 and 10; Km for adenosine around 50 microM over the same pH range and Km for 2'-deoxyadenosine around 400 microM.     

Characterization of thermostable RecA protein and analysis of its interaction with single-stranded DNA.

Thermostable RecA protein (ttRecA) from Thermus thermophilus HB8 showed strand exchange activity at 65 degrees C but not at 37 degrees C, although nucleoprotein complex was observed at both temperatures. ttRecA showed single-stranded DNA (ssDNA)-dependent ATPase activity, and its activity was maximal at 65 degrees C. The kinetic parameters, K(m) and kcat, for adenosine triphosphate (ATP) hydrolysis with poly(dT) were 1.4 mM and 0.60 s-1 at 65 degrees C, and 0.34 mM and 0.28 s-1 at 37 degrees C, respectively. Substrate cooperativity was observed at both temperatures, and the Hill coefficient was about 2. At 65 degrees C, all tested ssDNAs were able to stimulate the ATPase activity. The order of ATPase stimulation was: poly(dC) > poly(dT) > M13 ssDNA > poly(dA). Double-stranded DNAs (dsDNA), poly(dT).poly(dA) and M13 dsDNA, were unable to activate the enzyme at 65 degrees C. At 37 degrees C, however, not only dsDNAs but also poly(dA) and M13 ssDNA showed poor stimulating ability. At 25 degrees C, poly(dA) and M13 ssDNA gave circular dichroism (CD) peaks at around 192 nm, which reflect a particular structure of DNA. The conformation was changed by an upshift of temperature or binding to Escherichia coli RecA protein (ecRecA), but not to ttRecA. The dissociation constant between ecRecA and poly(dA) was estimated to be 44 microM at 25 degrees C by the change in the CD. These observations suggest that the capability to modify the conformation of ssDNA may be different between ttRecA and ecRecA. The specific structure of ssDNA was altered by heat or binding of ecRecA. After this alteration, ttRecA and ecRecA can express their activities at each physiological temperature.     

A thermodynamic study on the interaction between magnesium ion and human growth hormone

A thermodynamic study on the interaction between magnesium ion and human growth hormone (hGH) was studied at 27 degrees C in NaCl solution (50 mM) using different techniques. Two techniques of ionmetry using a Mg2+selective membrane electrode and isothermal titration calorimetry were applied to obtain the binding isotherm for hGHMg2+; results obtained by both techniques were found to be in good agreement. There is a set of three identical and noninteracting binding sites for magnesium ions. The intrinsic dissociation equilibrium constant and the molar enthalpy of binding are 46 microM and -17.7 kJ/mol, respectively. Temperature scanning UV-visible spectroscopy was applied to elucidate the effect of Mg2+ binding on the protein stability, and circular dichroism (CD) spectroscopy was used to show the structural change of hGH due to the metal ion interaction. Magnesium ion binding increased the protein thermal stability by increasing the alpha-helix content as well as decreasing both beta and random coil structures. However, the secondary structural change of the protein returns to its native form, including a small change in the tertiary structure, in high concentrations of magnesium ion.     

A left-handed 3(1) helical conformation in the Alzheimer Abeta(12-28) peptide

We show for the first time that the secondary structure of the Alzheimer beta-peptide is in a temperature-dependent equilibrium between an extended left-handed 3(1) helix and a flexible random coil conformation. Circular dichroism spectra, recorded at 0.03 mM peptide concentration, show that the equilibrium is shifted towards increasing left-handed 3(1) helix structure towards lower temperatures. High resolution nuclear magnetic resonance (NMR) spectroscopy has been used to study the Alzheimer peptide fragment Abeta(12-28) in aqueous solution at 0 degrees C and higher temperatures. NMR translation diffusion measurements show that the observed peptide is in monomeric form. The chemical shift dispersion of the amide protons increases towards lower temperatures, in agreement with the increased population of a well-ordered secondary structure. The solvent exchange rates of the amide protons at 0 degrees C and pH 4.5 vary within at least two orders of magnitude. The lowest exchange rates (0.03-0.04 min(-1)) imply that the corresponding amide protons may be involved in hydrogen bonding with neighboring side chains.     

Purification of a 6.5 kDa protease inhibitor from Amazon Inga umbratica seeds effective against serine proteases of the boll weevil Anthonomus grandis

A 6.5 kDa serine protease inhibitor was purified by anion-exchange chromatography from the crude extract of the Inga umbratica seeds, containing inhibitor isoforms ranging from 6.3 to 6.7 kDa and protease inhibitors of approximately 19 kDa. The purified protein was characterized as a potent inhibitor against trypsin and chymotrypsin and it was named I. umbratica trypsin and chymotrypsin inhibitor (IUTCI). MALDI-TOF spectra of the IUTCI, in the presence of DTT, showed six disulfide bonds content, suggesting that this inhibitor belongs to Bowman-Birk family. The circular dichroism spectroscopy indicates that IUTCI is predominantly formed by unordered and beta-sheet secondary structure. It was also characterized, by fluorescence spectroscopy, as a stable protein at range of pH from 5.0 to 7.0. Moreover, this inhibitor at concentration of 75 microM presented a remarkable inhibitory activity (60%) against digestive serine proteases from boll weevil Anthonomus grandis, an important economical cotton pest.