State and reactivity of tryptophyl residues in two bacterial proteases from Sorangium sp.

The state and reactivity of tryptophyl residues in two proteolytic enzymes from Sorangium sp. were investigated by means of the following methods: spectrophotometric oxidation of tryptophans with N-bromosuccinimide, 2-hydroxy-5-nitrobenzyl bromide, and H2O2 in dioxane, optical rotatory dispersion, ultraviolet difference spectrophotometry, solvent perturbation and viscosity measurements. Out of two tryptophyl residues/molecule of alpha-lytic protease, one appears to be completely buried, while the other seems to be exposed. None of these two residues seem to be responsible for the activity of the enzyme. The beta-lytic protease undergoes an irreversible conformational transition between pH 5.0 and 3.5. Out of total four tryptophyl residues/molecule, only one is fully exposed at neutral pH. The other three are gradually exposed in the pH transition region. The degree of exposure and the dimensions of "cavities" shielding tryptophyl residues were estimated. The tryptophyl residues of of beta-lytic protease do not seem to participate in substrate binding or the active site; they are rather one of the determinants of the conformational state of the enzyme.

External yeast beta-fructosidase. The role of tryptophyl residues in catalysis.

1. In native invertase at pH 4.6, 23 out of a total of 34 tryptophyl residues are "exposed" to oxidation with N-bromosuccinimide, the other residues being apparently shielded from the oxidant within the molecule. 2. Oxidation of 5-6 tryptophyl residues/molecule with N-bromosuccinimide is proportional to the complete inactivation of the enzyme, and appears to be specific for indole chromophore only. The ligand binding and fluorescence measurements indicate that the oxidation of native enzyme, up to 50% inhibition, apparently does not change the conformation and topography of enzymes surface. 3. Invertase is inhibited by diazonium-1-H-tetrazole. Since tyrosine residues can be excluded by nitration studies as catalytically unimportant, it appears that a mocification of a single histidyl residue/molecule with diazonium-1-H-tetrazole is sufficient to abolish the enzymic activity. However, the absence of inhibition with diethyl pyrocarbonate indicates that the inhibition with diazonium-1-H-tetrazole may be mediated through steric hindrance or other indirect effects. 4. The absence of inhibition with 2,4-dinitrophenylhydrazine, trinitro benzenesulfonic acid and 5,5'-dithiobis-(2-nitrobenzoate) indicates that the carbonyl groups of the carbohydrate moiety, free amino and -SH groups are not essential for activity.

Glucose oxidase from Aspergillus niger: the mechanism of action with molecular oxygen, quinones, and one-electron acceptors.

Glucose oxidase from the mold Aspergillus niger (EC 1.1.3.4) oxidizes beta-D-glucose with a wide variety of oxidizing substrates. The substrates were divided into three main groups: molecular oxygen, quinones, and one-electron acceptors. The kinetic and chemical mechanism of action for each group of substrates was examined in turn with a wide variety of kinetic methods and by means of molecular modeling of enzyme-substrate complexes. There are two proposed mechanisms for the reductive half-reaction: hydride abstraction and nucleophilic attack followed by deprotonation. The former mechanism appears plausible; here, beta-D-glucose is oxidized to glucono-delta-lactone by a concerted transfer of a proton from its C1-hydroxyl to a basic group on the enzyme (His516) and a direct hydride transfer from its C1 position to the N5 position in FAD. The oxidative half-reaction proceeds via one- or two-electron transfer mechanisms, depending on the type of the oxidizing substrate. The active site of the enzyme contains, in addition to FAD, three amino acid side chains that are intimately involved in catalysis: His516 with a pK(a)=6.9, and Glu412 with pK(a)=3.4 which is hydrogen bonded to His559, with pK(a)>8. The protonation of each of these residues has a strong influence on all rate constants in the catalytic mechanism.

Simple and rapid chromatographic method for the separation of major classes of ribosomal ribomononucleotides.

We have described a simple and rapid chromatographic method for the analytical and preparative separation of major types of ribosomal ribomononucleotides with Dowex 1-X10 (HCOO-, 37-74 microns) and Dowex 2-X10 (HCOO-, 37-74 microns) columns, by desorption with formiate solutions in 1-2 h. The separation has been achieved for Cp, Ap, Up and Gp, while a mixture of 2'-, and 3'-nucleoside phosphates desorbs as a single peak; with both resins, a successful separation was achieved with a load from 25 micrograms to 1 mg of ribomononucleotide mixture per ml of packed resin. A complete separation was achieved with Dowex 1, while the separation with Dowex 2 resin was even better. The resins cannot separate unusual nucleosides; therefore, our method is suitable for studies of ribonucleic acids with a low content of unusual nucleosides. Our method has been applied for the quantitative determination of the ribomononucleotide composition of 18S and 28S rRNAs, isolated from mammalian tissues: rat liver, mouse kidney and Ehrlich ascites cells. Dowex 1 and Dowex 2 resins afforded similar or identical ribomononucleotide compositions in all cases; analytical data were in agreement with the literature data. Our method is competitive, in several respects, with modern HPLC techniques for the separation of ribomononucleotides.

Primary toxic effects of anthraquinone-2-sulfonic acid in rat liver microsomes.

(1) The endogenous, NADPH-supported production of H2O2 and of O2-.-radicals in rat liver microsomes was very strongly enhanced in the presence of anthraquinone-2-sulfonic acid (AQSA). (2) This induction of H2O2 and of O2-.-radicals was catalyzed by the microsomal NADPH:cytochrome P450 oxidoreductase (EC 1.6.2.4). (3) AQSA was reduced to AQSA radicals by reductase; AQSA radicals reduce molecular oxygen to O2-.-radicals, which are readily dismutated to H2O2 by the microsomal superoxide dismutase. (4) O2-.-radicals are the sole precursors of all AQSA-induced production of H2O2 in liver microsomes.

Inhibition of Na,K-dependent adenosine triphosphatase by adenosine in intact pigeon erythrocytes.

In intact pigeon erythrocytes, adenosine is a potent inhibitor of Na,K-dependent adenosine triphosphatase. In purified cell-membrane preparations, adenosine is only a weak competitive inhibitor of Na,K-ATPase, with respect to ATP. This indicates that adenosine must not be a direct inhibitor of the sodium pump in intact red cells per se; instead, adenosine exerts its inhibitory effect via endogenous cell factors.

Yeast alcohol dehydrogenase catalyzed reduction of p-nitroso-N, N-dimethylaniline by NADH.

The steady-state kinetics, product identification, stoichiometries, and solvent isotope effects of yeast alcohol dehydrogenase catalyzed reduction of p-nitroso-N,N-dimethylaniline (NDMA) by NADH, are reported. NDMA is enzymatically reduced to p-hydroxylamine-N,N-dimethylaniline, which is further enzymatically dehydrated to corresponding quinonediimine cation (QDI+). QDI+ undergoes nonenzymatic transformations. QDI+ is rapidly reduced by NADH to p-amino-N,N-dimethylaniline (ADMA). Also, QDI+ is readily dismutated with ADMA to form N,N-dimethyl-p-phenylenediamine radicals; radicals are stable under steady-state conditions, below pH 7.5. A complete kinetic mechanism for above reactions has been proposed.

Kinetic mechanism of yeast alcohol dehydrogenase activity with secondary alcohols and ketones.

The kinetic mechanism of yeast alcohol dehydrogenase (EC 1.1.1.1) activity with the redox pair 2-propanol/acetone has been probed in detail by the application of initial rate studies in the absence and in the presence of products, and a dead-end inhibitor pyrazole. An overall steady-state random Bi Bi mechanism in both directions, with the formation of both abortive ternary complexes, enzyme.NADH.2-propanol and enzyme.NAD+.acetone has been observed. A complete list of steady-state kinetic constants are also reported for the redox pair (S)-(+)-2-butanol/2-butanone.

Use of pH studies to determine the kinetic and chemical mechanism of yeast alcohol dehydrogenase with primary aliphatic alcohols and aldehydes.

A complete list of all steady-state kinetic constants for the yeast alcohol dehydrogenase (EC 1.1.1.1) catalyzed oxidation of ethanol, propan-1-ol and butan-1-ol, and for the reduction of acetaldehyde and propionaldehyde was collected in the pH range 6-10, and an appropriate pH profile for each constant was constructed. A common minimal mechanism with all these substrates has been postulated and pKa values and the pH independent limiting values have been assigned for the rate constants.

Redox-elimination reaction catalyzed by yeast alcohol dehydrogenase.

Yeast alcohol dehydrogenase (EC 1.1.1.1) catalyzed reduction of N,N-dimethyl-4-nitrosoaniline by NADH. The stoichiometry of reaction, steady-state kinetic parameters, and the pH-profile for this reaction were estimated. On that basis, the minimal mechanism of the above reaction was postulated.

The influence of nicergoline on reaction kinetics of synaptosomal adenosine triphosphatases from porcine brain.

10-Methoxy-1,6-dimethylergoline-8beta-methanol-5-bromonicotinate (nicergoline) has in vitro a modulatory effect upon reaction kinetics of two synaptosomal adenosine triphosphatases from porcine brain: Na,K-dependent ATPase and Ca,Mg-dependent ATPase. Nicergoline is a non-competitive inhibitor of synaptosomal Ca,Mg-dependent ATPase in vitro; thus, if its in vivo concentration reaches a sufficiently high level, the rate of exchange of Ca and Mg ions between the intra- and the extracellular spaces of neurons slows down. In vivo effect of nicergoline upon reaction kinetics of synaptosomal Na,K-dependent ATPase is complex; below 80 micromol/l ATP it is an activator, and above 80 micromol/l ATP it is an inhibitor or enzyme. It decreases Km for ATP from 310 micromol/l in the absence, to 70 micromol/l in the presence of 0.3 mmol/l drug. It is believed that, in vivo, nicergoline helps to level or normalize the rate of physiological processes energetically coupled to synaptosomal Na,K-dependent ATPase, by moderately accelerating the slow and strongly inhibiting the fast reaction rates by ATP hydrolysis.

The influence of nicergoline on reaction kinetics of various forms of cyclic AMP phosphodiesterase.

10-Methoxy-1,6-dimethylergoline-8 beta-methanol-5-bromonicotinate (nicergoline) has in vitro a modulatory effect upon reaction kinetics of various forms of cAMP phosphodiesterase (PDE) from beef heart and mice brain. Nicergoline inhibits low Km form of cAMP PDE from hear and brain; concentration of inhibitor required for 50% inhibition is 3-12 mumol/l. It is believe that the observed in vitro effects of nicergoline on reaction kinetics of various forms of cAMP PDE may in vivo help to normalize the level of cAMP, keep its concentration within certain limits, both by opposing large increase and large decrease in its cellular concentration.

Kinetic mechanism of yeast alcohol dehydrogenase with primary aliphatic alcohols and aldehydes.

Yeast alcohol dehydrogenase (EC 1.1.1.1) catalyzes the interconversion of three redox pairs, ethanol/acetaldehyde, propanol/propionaldehyde and butanol/butyraldehyde by the same common mechanism, only with different magnitudes of rate constants. This general mechanism is Ordered Bi Bi in both directions, with the formation of abortive ternary complexes enzyme.NAD+.aldehyde and binary complexes enzyme.aldehyde.

Influence of temperature on hemolysis of erythrocytes by digitonin.

The kinetics of hemolysis of pig erythrocytes by digitonin was continuously monitored by a potassium selective electrode. The following minimal mechanism of hemolysis was postulated, based upon kinetic measurements: 2 D + E (1)in equilibrium E x D2 (2)leads to (E x D2) (3)leads to (E x D2) where D denotes a digitonin molecule and E a specific digitonin binding-site on the membrane. The first step (1) represents a rapid reversible combining of digitonin with specific binding-sites on the membrane. The second step (2) is prelytic, related to the time required for bound digitonin molecules to alter the membrane structure so much that hemolysis may take place; this step has a transition temperature at 26 degrees C, probably related to the "melting" of specific membrane structures at that temperature. The third step (3) is hemolytic, and comprises the changes within the digitonin-altered membrane during hemolysis; it is stongly influenced by temperature. Lowering of temperature slows down the rate of hemolysis and increases the quantity of digitonin required to obtain a fixed extent of hemolysis. It appears that two molecules of digitonin combine with a single binding-site on the outer face of the membrane in a digitonin-membrane complex.

Novel substrates of yeast alcohol dehydrogenase--4. Allyl alcohol and ethylene glycol.

In the present work, we have determined the steady-state kinetic constants for yeast alcohol dehydrogenase-catalyzed oxidation of allyl alcohol (H2C = CH.CH2OH) and ethylene glycol (HOCH2.CH2OH) with NAD+, at pH 8.0; also, a kinetic mechanism for the former reaction was determined at the same pH. In addition, it was found that acrolein is a potent inhibitor of yeast alcohol dehydrogenase.

A simple fluorimetric method for the estimation of ligand binding parameters in ligand-enzyme complexes.

The theory and practice of a simple fluorimetric method for the estimation of ligand binding parameters in ligand-enzyme complexes is described. In this method, the concentration of a ligand-binding enzyme and the dissociation constant of a ligand-enzyme complex were estimated solely from the total concentration of a ligand and the total fluorescence of the ligand in the absence and in the presence of enzyme.

Hepatic metabolism of artemisinin drugs--I. Drug metabolism in rat liver microsomes.

1. In this communication, metabolism of the semisynthetic antimalarial drugs of the artemisinin class (beta-arteether, beta-artelinic acid and dihydroartemisinin) in rat liver microsomes, is reported. 2. Dihydroartemisinin was the major early metabolite of arteether (57%) and artelinic acid (80%); in addition, arteether was hydroxylated in the positions 9 alpha- and 2 alpha- of the molecule. 3. Dihydroartemisinin was further metabolized by extensive hydroxylation of its molecule; we were able to identify four hydroxylated derivatives of DQHS, but not the exact positions of the hydroxyl groups. 4. The rates of NADPH-supported metabolism of arteether, artelinic acid and dihydroartemisinin in rat liver microsomes were: 4.0, 2.5 and 1.3 nmol/min/mg of microsomal protein, respectively. 5. The apparent affinity constants of arteether and artelinic acid for the microsomal metabolizing system, calculated from the rates of product formation, were 0.54 mM and 0.33 mM (for arteether) and 0.11 mM (for artelinic acid), respectively. The appearance of two affinity constants indicated that arteether was metabolized by two different isoenzymes of cytochrome P-450 in rat liver microsomes.

Hepatic metabolism of artemisinin drugs--II. Metabolism of arteether in rat liver cytosol.

1. In this communication, in vitro metabolism of a semisynthetic antimalarial drug arteether in rat liver cytosol is reported. 2. Whenever 14C-labeled arteether was mixed with rat liver cytosol, a crude postmicrosomal fraction of liver cell homogenates, an appearance of three major 14C-labeled metabolites was always attested: deoxy-dihydroartemisinin, AEM-1 (Baker et al., 1988) and metabolite MW286. 3. Transformation of arteether into deoxyDQHS was catalyzed by an enzyme present in the rat liver cytosol, whose activity depended on the presence of NAD+/NADH and a low molecular, dialyzable factor present in the cytosol. The maximal activity of this enzyme was 0.31 nmol of deoxyDQHS formed/min/mg of cytosolic protein. 4. AEM-1 and metabolite mol. wt 286 have been formed directly from arteether by a chemical interaction of the drug with the cytosolic fraction, probably in a non-enzymatic reaction. 5. Taking together the in vitro data of arteether metabolism in rat liver cytosol, presented in this communication, and in vitro data in rat liver microsomes, presented in the preceding communication (Leskovac and Theoharides, 1991), we were able to postulate an integral pathway of Phase I metabolism of arteether in a whole rat liver cell.

Improved simple chromatographic method for the fractionation of major classes of mammalian nucleic acids.

CL-Sepharose 4B column chromatography has been used for the separation of four major classes of mammalian nucleic acids in a single chromatographic run. Gel filtration at 2.5 M NaCl separated DNAs (containing RNA hybrids) from tRNAs. The 18 S RNA (containing 3-5 wt% of small 5 S RNA and RNA degradative products) was eluted at 0.7 M NaCl, and 28 S RNA (containing hnRNAs) was eluted at 0.1 M NaCl. Poly(A)+ mRNAs were detected in both 18 and 28 S RNA fractions. The present procedure is suitable for both analytical and preparative work and may serve as an initial step for the further isolation of ultrapure nucleic acid preparations.

Hepatic metabolism of artemisinin drugs--III. Induction of hydrogen peroxide production in rat liver microsomes by artemisinin drugs.

1. In this communication, induction of hydrogen peroxide production by the semisynthetic antimalarial drugs of the artemisinin class (beta-arteether, beta-artelinic acid and dihydroartemisinin) in rat liver microsomes, is reported. 2. Endogenous, NADPH-dependent, production of hydrogen peroxide in rat liver microsomes was enhanced in the presence of arteether and artelinic acid, but not in the presence of dihydroartemisinin. 3. NADPH-dependent metabolism of arteether and artelinic acid was closely coupled to the drug-induced production of hydrogen peroxide. 4. The redox cycle of cytochrome P-450 was presented, which describes satisfactorily both the endogenous and the drug-assisted hydrogen peroxide production in rat liver microsomes; also, the rate-limiting step of the cycle was identified.