Presence Of Enzymes Research Articles

Intestinal phytase I: Detection and preliminary characterization of activity in the intestinal brush border membrane of hybrid striped bass Morone saxatilis × M. chrysops

While numerous studies have evaluated intestinal phytase activity in terrestrial monogastric species, little work has been conducted investigating the enzyme's activity in finfish. This experiment was conducted to determine if hybrid striped bass Morone saxatilis × M. chrysops possess intestinal phytase activity, and to perform a preliminary characterization of this activity in terms of pH, metal ion dependence and substrate kinetics. Hybrid bass do exhibit low levels of intestinal phytase activity. The activity has an acidic pH optimum between 3.5 and 4.5. Low concentrations of Mn2+ slightly enhance this activity. The divalent cations Mg2+ and Zn2+ were shown to either have no effect on the enzyme activity or to be inhibitory, particularly at high concentrations. The present results indicate that intestinal phytase activity in hybrid bass does follow Michaelis-Menten kinetics, with estimated Km and Vmax values of 2.5 mM phytate and 4.8 units mg−1 protein, respectively. The physiologic significance of the enzyme's presence in hybrid striped bass is questionable due to the low activity as compared to terrestrial monogastrics, the acidic pH optimum and the low affinity the enzyme appears to have for phytate. These observations also raise the question of whether the intestinal phytase activity detected in hybrid bass is merely a manifestation of non-specific acid phosphatase activity. Further investigation is needed to determine the practical significance of phytase activity in hybrid bass in terms of phytate phosphorus utilization. Additional experiments should also be conducted which evaluate intestinal phytase activity in other important aquacultured finfish species.

Structural features underlying selective inhibition of protein kinase CK2 by ATP site-directed tetrabromo-2-benzotriazole.

Two novel crystal structures of Zea mays protein kinase CK2alpha catalytic subunit, one in complex with the specific inhibitor 4,5,6,7-tetrabromobenzotriazole (TBB) and another in the apo-form, were solved at 2.2 A resolution. These structures were compared with those of the enzyme in presence of ATP and GTP (the natural cosubstrates) and the inhibitor emodin. Interaction of TBB with the active site of CK2alpha is mainly due to van der Waals contacts, with the ligand fitting almost perfectly the cavity. One nitrogen of the five-membered ring interacts with two charged residues, Glu 81 and Lys 68, in the depth of the cavity, through two water molecules. These are buried in the active site and are also generally found in the structures of CK2alpha enzyme analyzed so far, with the exception of the complex with emodin. In the N-terminal lobe, the position of helix alphaC is particularly well preserved in all the structures examined; the Gly-rich loop is displaced from the intermediate position it has in the apo-form and in the presence of the natural cosubstrates (ATP/GTP) to either an upper (with TBB) or a lower position (with emodin). The selectivity of TBB for CK2 appears to be mainly dictated by the reduced size of the active site which in most other protein kinases is too large for making stable interactions with this inhibitor.

Prévention du carcinome rénal : l’approche nutrigénétique

The development of renal cell carcinoma (RCC) has been associated with both genetic and environmental factors, with somatic and germline mutations in the von Hippel-Lindau (VHL) tumor suppressor gene and with tobacco smoking, obesity, long term exposure to some nutrients, pollutants, and industrial solvents such as trichloroethylene. Intra and interfamilial variability of expression of germline mutations in the VHL gene and variable susceptibility to carcinogens in the sporadic forms strongly suggest the involvement of conditional modifier genes. In order to identify sub groups of individuals at increased risk because of susceptibility genotypes, we have collected a series of 460 patients who developed an RCC and 79 families with the von Hippel Lindau disease. To collect clinical and mutational data for correlation analysis we have developed a unique tool the Universal Mutation Database. Comparison of the spectrum of germline and somatic mutations in the VHL gene showed that: 1) in sporadic RCC mutations lead more often to truncated proteins (83%), while the remaining mutations (17%), include 3/4 of transversions and 1/4 of transitions. This high proportion of transversions supports the involvement of carcinogens the impact of which is conditioned by the genetic variability of xenobiotic metabolizing enzymes; 2) whereas in familial cases missense mutations are more common; this difference allowed us to define a prognostic factor for the occurrence of RCC in a VHL context. In order to look for genotypes conferring a higher risk we genotyped the RCC patients for 8 different genes (50 genotypes). A significant relationship was observed for several combinations of alleles including CYP1A1 ("variant"), NAT2 and NAT1 (slow) and GSTM1 (null allele). Associations between specific mutational profiles and at risk genotypes at different tumoral stages should allow us to: 1) define more precisely the nature of specific patterns of mutations in relation with the deficiency or overexpression of such or such enzymes in presence of particular carcinogens; 2) demonstrate that certain combinations of genotypes confer a particular risk to develop a specific type of tumor in VHL patients. Thus tracking of potentially carcinogenic substances, through their footprints and through identification of conditionally detrimental genotypes of genes participating in their detoxification should permit a better prevention through an appropriate nutrition adapted to each individual.

Kinetic characteristics of the enzymatic conversion in presence of cyclodextrins: study of the oxidation of polyunsaturated fatty acids by lipoxygenase

The capability of cyclodextrins (CDs) to greatly enhance the solubility in water of poorly water-soluble substances makes them an ideal alternative system for studying the expression of enzyme activity with such substrates in aqueous solution. In order to evaluate the behaviour of the enzymes in presence of CDs a study of the lipoxygenase (LOX)-catalyzed oxidation of polyunsaturated fatty acids (PUFA) as model reaction has been carried out. This was done by using LOX from two different sources (soybean and potato tuber), at two pH values (6.3 and 9.0), with two substrates (linoleic acid and arachidonic acid) and three types of CD ( β-CD, methyl- β-CD and monoglucosyl- β-CD). PUFA have been shown to form inclusion complexes of 1:2 stoichiometry which are in equilibrium with free PUFA and free CD, thus complexation is governed by two equilibrium constants, K 1 and K 2 (J.M. López-Nicolás et al., Biochem. J. 308 (1995) 151–154; R. Bru et al., Colloids Surf. 97 (1995) 263–269). For the oxidation of PUFA by LOX in the presence of β-CD we propose a model in which free PUFA is the only effective substrate, thus the oxidation of the complexed substrate requires the previous dissociation of the complex. The equilibrium constants of complex formation are determined by both a physico-chemical and an enzymatic method. In spite of giving quite similar results, the second was proven to be more accurate so it was employed in further studies. CD was shown to slow down the reaction rate of LOX, specifically due to the increase of K m, V max remaining unchanged. That apparent inhibition is due to removal of effective (free) substrate in the form of inclusion complexes. This `sequestered' substrate can, however, be converted since it is in equilibrium with the free. The feasibility of realizing a CD-mediated accurate control over the conversion rate is demonstrated in the experiment called `cyclodextrin assay' in which the concentration of the free substrate is calculated by using the equilibrium constants of complex formation and setting the initial concentrations of total substrate and total CD. From the observation of the reaction progress curves in the conditions of the CD assay, we have studied some characteristic parameters of the oxidation of PUFA by LOX in this new medium, such as enzymatic activity, duration of linear product accumulation and the lag phase.

Enzymatic Racemic Resolution of a Fluorinated Substrate and Syntheses of E and Z Alkenes as Precursors for Some Biologically Active Fluorohexoses

Abstract A fluorinated substrate 6 was prepared and then the enantiomers (6a and 7a) were separated by an enzyme in presence of an acetylating agent. The optical purity of 6a and 7a were determined by derivatising them into their MTPA -esters and then by taking their 19F NMR spectra. It was observed that, PL 266 (enzyme), benzene (solvent), 24 h (time), 40 °C (temp.) and vinyl acetate (acetylating agent) were the ideal conditions for racemic resolution. The optical purity was further improved by changing the solvent (hexane), amount of enzyme (PL 266; 3000 units), reaction time (12 h) and amount of acetylating agent (vinyl acetate; two molar). The optically pure species was used for the preparation of E and Z alkenes.

Effect of Denaturant and Cosolvents on the Stability of Wheat Germ Lipase

Purified wheat germ lipase has a specific activity of 12 ± 1 units, and the addition of urea decreases the activity of the enzyme. Partial specific volume of the enzyme in presence of 8 M urea is 0.723 and 0.717 mL/g for isomolal and isopotential measurements, respectively, and the preferential interaction parameter (ξ3) value is 0.05 g/g. Ultraviolet and fluorescence emission measurements indicated the exposure of aromatic amino acid residues from the interior of the protein, and thermal denaturation temperature (Tm) measurements indicated a decreased stability of the enzyme by 12 °C from a control value of 56 °C. The addition of specific cosolvents was found to increase the thermal stability of the enzyme to different extents (patent pending). The concentration of the cosolvent (10−40%) used has a direct effect on the retention of activity of the enzyme after being exposed to elevated temperatures in the presence of these cosolvents. Keywords: Wheat germ lipase; thermal stabilization; hydration; denatur...

The amphiphilic drug-induced tryptophan fluorescence change of ion-transporting ATPases

The tryptophan (Trp) fluorescence of Na +,K +-ATPase, Ca 2+,Mg 2+-ATPase, Ca 2+-ATPase enriched microsomal membranes have been found to be changed by two amphiphilic drugs, i.e., choroquine (anti-malarial) and chlorpromazine (anti-psychotic) alone or in combination with ligands and/or substrate. The findings suggest that some conformational change possibly in E 1 and E 2 state of the enzymes in presence of these drugs and/or ions and substrate have taken place. The emission maxima of Trp residues were found to be at 335 nm irrespective of experimental conditions. A different level of fluorescence quenching was observed in presence of drugs and in combination with ions and/or substrate. By the use of modified Stern Volmer equation, f a, the effective fraction of tryptophan most exposed to the drug and effective quenching constant, K a, have been calculated. The non-linearity of the Stern-Volmer plots indicate that a fraction of Trop residues remain accessible to the quencher, which may correspond to highly hydrophobic regions that are normally buried in the membranes. The differences in f a and K a values calculated from the modified Stern-Volmer plots under various conditions indicate the different extent of exposure of Trp residues to the quencher.

The characteristics and effect on catalysis of nucleotide binding to noncatalytic sites of chloroplast F1-ATPase

The recent finding that the presence of ATP at non-catalytic sites of chloroplast F1-ATPase (CF1) is necessary for ATPase activity (Milgrom, Y. M., Ehler, L. L., and Boyer, P. D. (1990) J. Biol. Chem. 265,18725-18728) prompted more detailed studies of the effect of noncatalytic site nucleotides on catalysis. CF1 containing at noncatalytic sites less than one ADP or about two ATP was prepared by heat activation in the absence of Mg2+ and in the presence of ADP or ATP, respectively. After removal of medium nucleotides, the CF1 preparations were used for measurement of the time course of nucleotide binding from 10 to 100 microM concentrations of 3H-labeled ADP, ATP, or GTP. The presence of Mg2+ strongly promotes the tight binding of ADP and ATP at noncatalytic sites. For example, the ADP-heat-activated enzyme in presence of 1 mM Mg2+ binds ADP with a rate constant of 0.5 x 10(6) M-1 min-1 to give an enzyme with two ADP at noncatalytic sites with a Kd of about 0.1 microM. Upon exposure to Mg2+ and ATP the vacant noncatalytic site binds an ATP rapidly and, as an ADP slowly dissociates, a second ATP binds. The binding correlates with an increase in the ATPase activity. In contrast the tight binding of [3H]GTP to noncatalytic sites gives an enzyme with no ATPase activity. The three noncatalytic sites differ in their binding properties. The noncatalytic site that remains vacant after the ADP-heat-activated CF1 is exposed to Mg2+ and ADP and that can bind ATP rapidly is designated as site A; the site that fills with ATP as ADP dissociates when this enzyme is exposed to Mg2+ and ATP is called site B, and the site to which ADP remains bound is called site C. Procedures are given for attaining CF1 with ADP at sites B and C, with GTP at sites A and/or B, and with ATP at sites A, B, and/or C, and catalytic activities of such preparations are measured. For example, little or no ATPase activity is found unless ATP is at site A, but ADP can remain at site C with no effect on ATPase. Maximal GTPase activity requires ATP at site A but about one-fifth of maximal GTPase is attained when GTP is at sites A and B and ATP at site C. Noncatalytic site occupancy can thus have profound effects on the ATPase and GTPase activities of CF1.

Purification and active site modification studies on glyoxalase I from monkey intestinal mucosa

Glyoxalase I (( R)- S-lactoylglutathione methylglyoxal-lyase (isomerizing), EC 4.4.1.5) from monkey intestinal mucosa was purified to homogeneity. The purified enzyme had a molecular weight of 48 000, composed of two apparently identical subunits. Active-site modification was carried out on the purified enzyme in presence and absence of S-hexylglutathione, a reversible competitive inhibitor of glyoxalase I. Modification by tetranitromethane and N-acetylimidazole caused inactivation of the enzyme. Inactivation by N-acetylimidazole was reversible with hydroxylamine treatment, suggesting the importance of tyrosine residues for the activity of the enzyme. The enzyme was inactivated by 2-hydroxy-5-nitrobenzyl bromide, N-bromosuccinimide, 2,4,6-trinitrobenzenesulphonic acid, pyridoxal phosphate and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, indicating the importance of tryptophan, lysine and glutamic acid/aspartic acid residues for the activity of the enzyme. The enzyme was inactivated by diethyl pyrocarbonate and the activity was not restored by hydroxylamine treatment, suggesting that histidine residues may not be important for activity. Modification by N-ethylmaleimide and p-hydroxymercuribenzoate did not affect its activity, indicating that sulphydryl groups may not be important for activity. These studies indicated that the amino acids present in the active site of glyoxalase I from intestinal mucosa which may be important for activity are tyrosine, tryptophan, lysine and glutamic acid/aspartic acid residues.

Photoinactivation of ?-aminolevulinic acid dehydratase from maize by flavin mononuclotide

The δ-aminolevulinic acid dehydratase activity was irreversibly inactivated by irradiation of the enzyme in presence of flavin mononucleotide. The loss of enzyme activity was dependent on time of irradiation, concentration of FMN and intensity of irradiance. It required oxygen and was markedly enhanced in heavy water. The presence of levulinic acid (a competitive inhibitor of δ-ALAD) during irradiation prevented the inactivation considerably indicating photooxidative damage at or near the active site. Superoxide dismutase, sodium benzoate and sodium formate offered no protection, but singlet oxygen quenchers like azide and tryptophan were effective. NADH, electron donor to excited flavins, also prevented the loss of enzyme activity. These results indicate that singlet oxygen produced by light absorption of FMN was responsible for the photooxidative inhibition of the enzyme.

Rat intestinal maltase--glucoamylase. Purification of the detergent-solubilized enzyme in the presence of protease inhibitors: properties and identification of a protease-sensitive subunit.

Failure to develop clear-cut, distinguishing characteristics for hydrophobic and hydrophilic forms of maltase-glucoamylase led us to attempt the purification of the detergent-extracted enzyme in the continuous presence of protease inhibitors (phenylmethylsulfonyl fluoride and N-ethylmaleimide). The enzyme was purified by molecular exclusion, anion-exchange, and affinity column chromatography to a final specific maltase activity of 80 U/mg protein, comparable to previously solubilized enzymes. Both detergent (d-maltase) and proteolytically (p-maltase) solubilized enzymes had identical Km's for maltose and similar glycogenase activity. d-Maltase was clearly amphipathic. Whereas 95% of p-maltase was eluted with aqueous buffer from an octyl-Sepharose CL-4B column, the elution of d-maltase required solutions containing Triton X-100 and ethylene glycol. On density gradient centrifugation and sodium dodecyl sulfate (SDS)--polyacrylamide gels, p-maltase migrated as one high molecular weight species of 500,000. In contrast d-maltase migrated heterogeneously and the smallest maltase-active forms delineated by these two techniques, as well as by high pressure liquid chromatography, had molecular weights which ranged from 120,000 to 15,0000. Both p- and d-maltase were dissociated by heat in SDS, forming five prominent species as we have previously described. In contrast to p-maltase, in which the smallest species, band 1, equalled 36.7% of the total mass, band 1 of d-maltase accounted for 66.5%. Band 1 was separable when smaller amounts of enzyme were applied to slab gels and stained with silver, into two proteins of 130,000 and 145,000 daltons. The 145,000 dalton protein was absent in p-maltase and was replaced by a faint band of 140,000 daltons.(ABSTRACT TRUNCATED AT 250 WORDS)

Kinetics of succinic dehydrogenase inhibition by methyl parathion and its recovery by oximes and L-cysteine in hepatopancreas of Lamellidens marginalis.

Effects in vitro of methyl parathion on some kinetic constants of succinic dehydrogenase (SDH) in hepatopancreas of freshwater mussel, L. marginalis were studied. Altered pH vs. specific activity curves for SDH demonstrated significant inhibition by methyl parathion in buffered acidic, neutral and alkaline ranges. At high pH ranges IC50 (12.5 microM) of methyl parathion did not cause 50% inhibition enzyme as it did at neutral and acidic pHs. Activation energies (delta E) were found to be increased suggesting decreased efficiency of enzyme in presence of methyl parathion. Non-competitive inhibition with respect to activation by succinate was indicated by decreased maximal velocity (V) without change in Michaelis Menten constant (Km). Pyridine-2-aldoxime (25 microM), pyridine-4-aldoxime (15 microM) and L-cysteine (40 microM) neutralized the inhibition of SDH by methyl parathion (12.5 microM). The kinetic data suggests that inhibition of SDH by methyl parathion was pH and temperature independent.

Effect of sodium cholate on the catalytic and structural properties of phosphorylase b.

Sodium cholate at millimolar concentration is able to induce activity in rabbit muscle phosphorylase b in the absence of AMP. The maximum activation of the enzyme in presence of 7 mM sodium cholate was 24% of that achieved by 1 mM AMP. Other bile salts tested showed a negligible activating effect. The Ka for AMP was lowered fivefold by 5 mM of the steroid detergent, while the cooperative binding of the nucleotide was abolished. Phosphorylase b', a modified form of phosphorylase in which the phosphorylation site has been removed by limited tryptic attack, presented an activation profile similar to that of phosphorylase b. In contrast, phosphorylase a was inhibited by the bile salt, while the activity of liver phosphorylase b was not significantly affected. Modification of the AMP site of the enzyme with 2,3-butanedione could not inhibit sodium-cholate-induced activity. tert-Butanol, an organic solvent activator of phosphorylase b, was found to enhance the activity induced by sodium cholate. The interaction of sodium cholate and phosphorylase b was also followed by difference spectroscopy using a fluorescein isothiocyanate--phosphorylase b conjugate. Furthermore, measurements of electron spin resonance demonstrated that the mobility of a spin-label bound at buried--NH2 groups of phosphorylase b decreases cooperatively with increasing bile salt concentration.

Allosteric properties of rat lung phosphofructokinase.

Rat lung phosphofructokinase is purified 250-fold to a specific activity of about 10 by using ATP-sepharose affinity chromatography. The enzyme is activated by cyclic AMP, 5'-AMP, ADP, Pi, NH4+ and K+ ions. Depending upon the concentration of these effectors, the enzyme can exist in several interconvertible forms, differing widely in their affinity for fructose-6-P. These activators also overcome the inhibition of the enzyme by ATP and citrate, thus increasing the glycolytic rate in lung during hypoxia. Unlike the enzyme from other sources, the lung phosphofructokinase is not inhibited by cyclic GMP or phosphoenolpyruvate. The enzyme is very sensitive to inactivation by trypsin and this inactivation is completely reversed by assaying the proteolyzed enzyme in presence of its activators.

Control of the potassium ion-stimulated adenosine triphosphatase of pig gastric microsomes: Effects of lipid environment and the endogenous activator

The K +-stimulated ATPase activity associated with the purified gastric microsomes from the pig gastric mucosa can be completely inactivated by treatment with 15% ethanol for 60 s at 37 °C but not at 25 °C. Sequential exposure of the microsomes to 15% ethanol at 25 and 37 °C caused the release of 2.9 and 4.3% of the total membrane phospholipids, respectively, consisting entirely of phosphatidyl choline and phosphatidyl ethanolamine. The ethanol-treated (37 °C) membrane had high basal (with Mg 2+ as the only cation in the assay mixture) activity, which was further enhanced during reconstitution with phosphatidyl choline or phosphatidyl ethanolamine. The high basal activities could be reduced to the normal control level by assaying the enzyme in presence of the “activator protein,” partially purified from the soluble supernatant of the pig gastric cells. Phosphatidyl choline was somewhat more effective than phosphatidyl ethanolamine in the restoration of the activity of the ethanol-treated enzyme while phosphatidyl serine, phosphatidyl inositol, and sphingomyelin were without any effect. Synthetic phosphatidyl choline with various fatty acid substitutions were tested for their effectiveness in the restoration of the ethanol-inactivated enzyme. The distearoyl (18:0), dioleoyl (18:1), and dilinoleoyl (18:2) derivatives of phosphatidyl choline were almost equally effective while dipalmitoyl (16:0) phosphatidyl choline was somewhat less effective in the reconstitution process. Cholesterol appeared to interfere with phosphatidyl choline in the restoration of the activity of ethanol-treated enzyme. The fatty acid composition of phosphatidyl choline and phosphatidyl ethanolamine extracted by 15% ethanol at 37 °C was clearly different than those of the total microsome. Our data suggest that the phospholipids extracted by 15% ethanol at 37 °C are derived primarily from the immediate lipid environment of the enzyme and ATP together with Mg 2+ and K + help the partially delipidated enzyme to retain the appropriate conformation for the subsequent reconstitution. Furthermore, ethanol appears to either release or inactivate the membrane-associated activator protein, demonstrated to be essential for the K +-stimulated activity of the pig gastric ATPase.

Phosphorylation, a factor controlling the synthesis of L-erythrodihydrobiopterin (BH 2)

The enzyme, D-erythrodihydroneopterin triphosphate synthetase from rat brain was observed to have a significantly lower specific activity than that from liver due to their degree of dephosphorylation during preparation. The brain enzyme could be phosphorylated in vitro in presence of [ 32P]-ATP and protein kinase, resulting in an increased specific activity. Isolation of brain enzyme in presence of 0.8 M NaF allowed recovery of the enzyme phosphorylated at residue 67 (serine) as determined by a new assay for phosphate. This enzyme is present in synaptosomes and its state of phosphorylation may regulate the rate at which dihydrobiopterin, the precursor of the hydroxylase cofactor (tetrahydrobiopterin, BH 4), is synthesized by synaptosomes.

On the Modification of Adenosine Kinase by Thiols

The catalytic activity of adenosine kinase (EC 2.7.1.20) from yeast is very labile. Even incubation with thiols provokes a loss of two thirds of its enzymatic activity. Concomitantly, two SH-groups appear on the enzyme in addition to the single SH-group already present in the untreated enzyme, the latter being absolutely essential for activity. Treatment of adenosine kinase with thiols does not substantially affect the binding of the substrates adenosine and ATP-Mg2. The reactivity of the two newly formed SH-groups is diminished in the presence of ATP-Mg2, whereas adenosine has no influence. The opposite holds for the reactivity of the single SH-group essential for enzymatic activity. Complete reactivation of the enzymatic activity after incubation of adenosine kinase with thiols can be achieved by reoxidation of the enzyme in presence of high concentrations of adenosine. These observations suggest the notion that adenosine kinase contains an essential SH-group close to the adenosine-binding site and a disulfide bridge near to the binding site of ATP-Mg2, the latter being easily accesible to the reduction by thiols.

Purification and characterization of ( [formula omitted])-ATPase. VI. Differential tryptic modification of catalytic functions of the purified enzyme in presence of NaCl and KCl

1. 1. Two distinct patterns of tryptic modification of the catalytic functions of purified ( Na + + K + )-ATPase can be related to the two previously described patterns of enzyme inactivation and cleavage of the large chain seen with NaCl and KCl (Jørgensen, P.L. (1975) Biochem. Biophys. Acta 401, 399–415). 2. 2. With NaCl, in phase A, the rapid inactivation of 50–55% of the ( Na + + K + )-ATPase activity is associated with loss of 85% of the K +-phosphatase activity and an increase in Na +-ADP-ATP exchange activity to 150% of control. ATP binding and phosphorylation are unchanged and the inactivation may result from cleavage of bonds within the large chain which are involved in dephosphorylation reactions. In phase B with NaCl, ATP binding and phosphorylation are lost slowly in parallel to inactivation of ( Na + + K + )-ATPase and cleavage of the large chain to a fragment with M r = 78 000 . 3. 3. With KCl, cleavage of the large chain to almost equal fragments abolish ATP binding and phosphorylation in parallel to the inactivation of ( Na + + K + )-ATPase. An additional split seems required for inactivation of the K +-pNPPase activity. 4. 4. After completion of the digestion in phase A with NaCl a stable preparation can be isolated in which the activity of ( Na + + K + )-ATPase is 40%. ATP binding and phosphorylation are 90%, K +-phosphatase is 15%, and Na +-ADP-ATP exchange is 150% of control. We currently examine if these levels are related to changes in phosphorylation kinetics. 5. 5. The ATP binding area is much more stable to trypsin with NaCl than with KCl, but loss of the binding capacity is in both cases correlated to a distinct cleavage of the large chain. The relationship between the fractional loss of ATP binding and cleavage of the large chain suggests that the nucleotide binding area is confined to one of the two large chains in the protein complex with M r = 270 000 which binds one molecule of ATP. 6. 6. The data also suggest that the phosphatase site is remote from the ATP binding area. It is proposed that the protein complex with M r = 270 000 contains two large chains with different catalytic functions and that each chain forms a cation channel.

Membrane bound and soluble adenosine triphosphatase of Escherichia coli K 12. Kinetic properties of the basal and trypsin-stimulated activities.

Basal and trypsin-stimulated adenosine triphosphatase activities of Escherichia coli K 12 have been characterized at pH 7.5 in the membrane-bound state and in a soluble form of the enzyme. The saturation curve for Mg2+/ATP = 1/2 was hyperbolic with the membrane-bound enzyme and sigmoidal with the soluble enzyme. Trypsin did not modify the shape of the curves. The kinetic parameters were for the membrane-bound ATPase: apparent Km = 2.5 mM, Vmax (minus trypsin) = 1.6 mumol-min-1-mg protein-1, Vmax (plus trypsin) = 2.44 mumol-min-1-mg protein-1; for the soluble ATPase: [S0.5] = 1.2 mM, Vmax (-trypsin) = 4 mumol-min-1-mg protein-1; Vmax (+ trypsin) = 6.6 mumol-min-1-mg protein-1. Hill plot analysis showed a single slope for the membrane-bound ATPase (n = 0.92) but two slopes were obtained for the soluble enzyme (n = 0.98 and 1.87). It may suggest the existence of an initial positive cooperativity at low substrate concentrations followed by a lack of cooperativity at high ATP concentrations. Excess of free ATP and Mg2+ inhibited the ATPase but excess of Mg/ATP (1/2) did not. Saturation for ATP at constant Mg2+ concentration (4 mM) showed two sites (groups) with different Kms: at low ATP the values were 0.38 and 1.4 mM for the membrane-bound and soluble enzyme; at high ATP concentrations they were 17 and 20 mM, respectively. Mg2+ saturation at constant ATP (8 mM) revealed michealian kinetics for the membrane-bound ATPase and sigmoid one for the protein in soluble state. When the ATPase was assayed in presence of trypsin we obtained higher Km values for Mg2+. These results might suggest that trypsin stimulates E. coli ATPase by acting on some site(s) involved in Mg2+ binding. Adenosine diphosphate and inorganic phosphate (Pi) act as competitive inhibitors of Escherichia coli ATPase. The Ki values for Pi were 1.6 +/- 0.1 mM for the membrane-bound ATPase and 1.3 +/- 0.1 mM for the enzyme in soluble form, the Ki values for ADP being 1.7 mM and 0.75 mM for the membrane-bound and soluble ATPase, respectively. Hill plots of the activity of the soluble enzyme in presence of ADP showed that ADP decreased the interaction coefficient at ATP concentrations below its Km value. Trypsin did not modify the mechanism of inhibition or the inhibition constants. Dicyclohexylcarbodiimide (0.4 mM) inhibited the membrane-bound enzyme by 60-70% but concentrations 100 times higher did not affect the residual activity nor the soluble ATPase. This inhibition was independent of trypsin. Sodium azide (20 muM) inhibited both states of E. coli ATPase by 50%. Concentrations 25-fold higher were required for complete inhibition. Ouabain, atebrin and oligomycin did not affect the bacterial ATPase.

Evidence for zinc function in the Mg ++/Ca ++ stimulated ATPase of escherichiacoli membranes

Summary At low Mg ++ concentration the soluble Mg ++ /Ca ++ activated ATPase extracted from E. coli membranes is inhibited by several chelators including bathophenanthroline sulfonate, diphenylthiocarbazone and zincon. Incubation of the enzyme with zincon shows a gradual extraction of zinc. Activity is lost in proportion to zinc removal and is specifically restored by readdition of zinc to the extracted enzyme. The soluble enzyme in presence of high concentration of Mg ++ is not inhibited by chelators. The presence of high Mg ++ concentration also prevents reconstitution of the zinc depleted enzyme activity with zinc. In the membrane bound form the activity is not inhibited by zincon or other water soluble chelators regradless of Mg ++ concentration whereas the activity is inhibited by various lipophilic chelators such as thenoyltrifluoroacetone and tert-octyl catechol. These latter chelators do not inhibit the soluble ATPase. We conclude that the ATPase contains zinc which is protected from chelators by high Mg ++ concentration or by binding to the membrane and that there is another lipophilic chelator site in the membrane which also controls activity of the membrane-bound ATPase.