In vitro poly(ADP-ribosyl)ation of seminal ribonuclease

The site of in vitro ADP-ribosylation of seminal ribonuclease was determined. Seminal enzyme was found to be a good receptor of [14C]ADP-ribose residues under the reaction conditions used. The recovery of [14C]ADP-ribosylated RNase was about 65% after purification. After tryptic digestion of modified enzyme, a fraction containing [14C]ADP-ribosylated peptides was separated from the others by ion-exchange chromatography on M82 resin. Radioactive peptides were then purified by affinity chromatography on anti-poly(ADP-ribose)IgG-Sepharose. High performance liquid chromatography of a mixture obtained after pronase digestion of purified ADP-ribosylated peptides revealed only one radioactive peptide whose amino acid composition corresponded to a peptide that has equimolar quantities of aspartic acid, serine, and glycine. Carboxypeptidase Y digestion of this peptide showed that its amino acid sequence was Asp-Ser-Gly. Only position 14-16 of seminal RNase corresponded to this sequence. The chemical stability of the ADP-ribose/enzyme linkage indicated that aspartic acid 14 is the modification site in seminal RNase.     

A radiochemical enzymatic endpoint assay for GTP and GDP

We present here a radiochemical enzymatic endpoint assay for the guanine nucleotides GTP and GDP that is suitable for use with cell extracts. The major coupling enzyme used is phosphoenolpyruvate carboxykinase purified from chicken liver. The ancillary coupling enzyme, aspartate aminotransferase, was used to generate a low steady-state concentration of oxalacetate. GTP was determined by the overall conversion of [U-14C]aspartate into [14C]phosphoenolpyruvate. This reaction was also scaled-up as a preparative method for [U-14C]phosphoenolpyruvate. This was used with the same coupling enzymes in reverse to measure GDP by the formation of [14C]aspartate. The assay method was applied to isolated rat hepatocytes. The total GTP and GDP concentrations found were within the range reported by others for rat liver. The advantages of this assay are its sensitivity, specificity, and applicability to large numbers of samples.     

Acid sphingomyelinase of human placenta: purification, properties, and 125iodine labeling

Human placental acid sphingomyelinase was highly purified in the presence of Triton X-100. DEAE-Sephacel chromatography and chromatofocusing were the most effective steps in the purification procedure. Enzyme purification was 380,000 nmol/mg protein/h. Characterization and radioiodination were carried out with the chromatofocusing fraction containing highly purified enzyme. The purified enzyme contained no activity of eleven other lysosomal hydrolases but hydrolyzed bis-p-nitrophenyl phosphate slowly compared with [14C]sphingomyelin and chromogenic substrates. SDS-gel electrophoresis revealed two distinct protein bands with molecular weights of 70,500 and 39,800. This enzyme had a molecular weight of 200,000 as determined by analytical gel filtration. The pH optimum was 5.0 and Km was 52.6 x 10(-5) M for [14C]sphingomyelin. Highly purified sphingomyelinase was labeled with 125iodine by the use of Enzymobeads. Labeled sphingomyelinase preparation was rapidly cleared from blood with t1/2 of 1 min. It was absorbed mostly into the liver and presumably largely excreted from there. This labeled enzyme may be useful in metabolic studies in normal animals and animal models of genetic lysosomal storage disorders.     

Purification and characterization of an alpha-ketoisocaproate oxygenase of rat liver

Rat liver contains a cytosolic alpha-ketoisocaproate oxygenase which oxidatively decarboxylates and hydroxylates alpha-ketoisocaproate to form beta-hydroxyisovalerate. This oxygenase was purified to near homogeneity. The oxygenase is unstable during purification, unless 5% monothioglycerol is added. The purified enzyme is stable in the presence of 5% monothioglycerol for 3 weeks at 4 degrees C and at least 10 weeks at -80 degrees C. The molecular weight of the alpha-ketoisocaproate oxygenase as determined to be 46,000 and 51,000 using denaturing and nondenaturing conditions, respectively, indicating a monomer. The alpha-ketoisocaproate oxygenase requires Fe2+; other metal ions did not replace Fe2+. Ascorbate activates the enzyme at subsaturating levels of Fe2+, by regenerating Fe2+. The activity is markedly affected by the type of buffer used. For example, the oxygenase activity increased 2- to 3-fold when 0.1 M maleate was used. Iron chelators, such as ADP and EDTA, are inhibitory. The ratio of decarboxylation of 1 mM alpha-[1-14C] ketoisocaproate (as measured by 14CO2 release) to decarboxylation of 1 mM alpha-[1-14C]keto-gamma-methiolbutyrate is 1.0 for all purification fractions, indicating that a single enzyme catalyzes the decarboxylation of both substrates. The apparent Km and Vmax values of the alpha-ketoisocaproate oxygenase using optimized assay conditions are 0.32 mM and 130 nmol/min/mg of protein for alpha-ketoisocaproate and 1.9 mM and 247 nmol/min/mg of protein for alpha-keto-gamma-methiolbutyrate. The principal product of the purified alpha-ketoisocaproate oxygenase, using alpha-ketoisocaproate as a substrate, is beta-hydroxyisovalerate, although small amounts of a compound, which has the chromatographic properties of isovalerate, are also produced.     

Canine thyroid fucokinase.

A radiometric assay was developed for fucokinase (ATP:6-deoxy-L-galactose 1-phosphotransferase, EC 2.7.1.52) based on the conversion of L-[14C]fucose to L-[14C]fucose 1-phosphate which is trapped and counted on ion exchange paper. This assay was used to detect the presence of a fucokinase in canine thyroid tissue which was subsequently purified 2754-fold over the crude tissue extracts. The product of the fucokinase was identified as the beta-anomer. The pH versus activity curve for the enzyme appears biphasic with optima at pH 6.5 and pH 8.25. The enzyme was shown to be highly specific for L-fucose with a Km of 2.6 - 10(-5) M at pH 8.25. It was shown to be absolutely specific for ATP as a phosphate donor with a Km of 6.3 - 10(-4) M at pH 8.25. The enzyme requires a divalent cation. Mg2+ is slightly more effective than Mn2+ in meeting this need. The molecular weight of the enzyme has been determined to be 494 000 +/- 12 400.     

Stereospecificity of hepatic L-tryptophan 2,3-dioxygenase.

Tryptophan 2,3-dioxygenase [L-tryptophan--oxygen 2,3-oxidoreductase (decyclizing), EC 1.13.11.11] has been reported to act solely on the L-isomer of tryptophan. However, by using a sensitive assay method with D- and L-[ring-2-14C]tryptophan and improved assay conditions, we were able to demonstrate that both the D- and L-stereoisomers of tryptophan were cleaved by the supernatant fraction (30000 g, 30 min) of liver homogenates of several species of mammals, including rat, mouse, rabbit and human. The ratio of activities toward D- and L-tryptophan was species variable, the highest (0.67) in ox liver and the lowest (0.07) in rat liver, the latter being hitherto exclusively used for the study of hepatic tryptophan 2,3-dioxygenase. In the supernatant fraction from mouse liver, the ratio was 0.23 but the specific activity with D-tryptophan was by far the highest of all the species tested. To identify the D-tryptophan cleaving enzyme activity, the enzyme was purified from mouse liver to apparent homogeneity. The specific activities toward D- and L-tryptophan showed a parallel rise with each purification step. The electrophoretically homogeneous protein had specific activities of 0.55 and 2.13 mumol/min per mg of protein at 25 degrees C toward D- and L-tryptophan, respectively. Additional evidence from heat treatment, inhibition and kinetic studies indicated that the same active site of a single enzyme was responsible for both activities. The molecular weight (150000), subunit structure (alpha 2 beta 2) and haem content (1.95 mol/mol) of the purified enzyme from mouse liver were similar to those of rat liver tryptophan 2,3-dioxygenase. The assay conditions employed in the previous studies on the stereospecificity of hepatic tryptophan 2,3-dioxygenase were apparently inadequate for determination of the D-tryptophan cleaving activity. Under the assay conditions in the present study, the purified enzyme from rat liver also acted on D-tryptophan, whereas the pseudomonad enzyme was strictly specific for the L-isomer.     

Purification of a reconstitutively active K+/H+ antiporter from rat liver mitochondria

We describe purification of three different states of the 82-kDa K+/H+ antiporter from rat liver mitochondria. The denatured 82-kDa protein, identified by its selective labeling with [14C]dicyclohexylcarbodiimide (DCCD), was purified by preparative two-dimensional gel electrophoresis. This purified product was used to raise and immunopurify monospecific polyclonal antibodies. Western blot analysis showed that the [14C] DCCD-labeled 82-kDa protein is not a DCCD-crosslinked product. The native, [14C]DCCD-labeled, 82-kDa protein was purified by (NH4)2SO4 fractionation and column chromatography, using 14C labeling and gel electrophoresis to track the protein. The native, non-DCCD-labeled 82-kDa protein was purified by similar procedures, using immunopurified antibodies to track the protein. DCCD binding had no effect on chromatographic behavior of the antiporter protein. This protocol resulted in purification of the 82-kDa protein to apparent homogeneity. The purified, native 82-kDa protein was reconstituted into proteoliposomes and assayed for K+ transport with the new fluorescent probe, PBFI. K+ transport was electroneutral and was inhibited by DCCD, Mg2+, and timolol. The turnover number for K+ transport was about 1000 s-1, very similar to the value previously estimated in intact mitochondria.     

A radioisotope assay for 1-aminocyclopropane-1-carboxylic acid synthase: S-adenosylhomocysteine analogs as inhibitors of the enzyme involved in plant senescence

A simple and rapid radioisotopic assay for 1-aminocyclopropane-1-carboxylic acid (ACC) synthase was developed, an enzyme involved in the biosynthesis of the plant hormone ethylene. The assay utilizes an AG50W-X4(NH+4) column which separates S-adenosyl-L-[carboxyl-14C]methionine (AdoMet) from the product [14C]ACC, since the latter is not bound to the resin while [14C]AdoMet is. As opposed to other assays, this procedure measures ACC directly and does not require further conversion to ethylene. When an enzyme preparation from ripe tomato fruits (Lycopersicon esculentum Mill). was assayed, an I50 of 2.5 +/- 0.8 microM for sinefungin and a Km of 27 +/- 2 microM for AdoMet were obtained; these values were in good agreement with previous determinations made with a gas chromatographic assay. When other nucleosides were tested as inhibitors, the following order of decreasing activity was found: sinefungin greater than S-adenosylhomocysteine (AdoHcy) greater than AdoHcy sulfoxide greater than S-n-butyladenosine greater than 3-deaza-adenosylhomocysteine greater than S-isobutyladenosine greater than S-isobutyl-1-deazaadenosine. In contrast, S-isobutyl-3-deazaadenosine, S-isobutyl-7-deazaadenosine, 3-deazaadenosine, and adenosine were not inhibitory.     

Purification and properties of the methanol dehydrogenase from Methylophilus methylotrophus.

1. A dye-linked methanol dehydrogenase, resembling many others from a variety of methylotrophic bacteria, was purified to homogeneity from extracts of methanol-grown Methylophilus methylotrophus. 2. The enzyme was very stable in the presence of methanol; in the absence of methanol it had a half-life of 1-2 days at 4 degrees C. 3. The value of A1% 1cm,280 was 17.5. 4. The enzyme retained bound methanol after passage through Sephadex G-25. This tightly-bound methanol slowly exchanged with free [14C]-methanol from a value of 0.27 mol of [14C]methanol/mol of enzyme after 48 h incubation at 4 degrees C to a limiting value of approx. 2.5 mol of [14C]methanol/mol of enzyme after 3 weeks incubation at 4 degrees C. 5. One mol of this enzyme reduced 89.4 mol of 2,6-dichlorophenol-indophenol (via phenazine methosulphate) in the absence of any additional methanol in the assay mixture. The source of the electrons involved in this reduction is not known.     

Serine Transhydroxymethylase: A Simplified Radioactive Assay; Purification and Stabilization of Enzyme Activity Employing Affi-Gel Blue

An improved radioactive assay has been developed for serine transhydroxymethylase. This assay involves the direct measurement of the [¹⁴C]HCH0 which is generated when [3-¹⁴C]-serine is employed as the substrate. The new assay eliminates 14 the need for a solvent extraction of a [C]HCHO-dimedon adduct which is the basis of the assay devised by Taylor and Weissbach.

The enzyme has been purified employing Affi-Gel Blue. The purified enzyme retains full activity when bound to this affinity chromatography matrix and can be stored in this state at 4° indefinitely.     

A sensitive radiometric assay to measure D-xylulose kinase activity

A radiometric test system for D-xylulose kinase (XK) was developed for the measurement of enzyme activity in crude cell extracts and to minimize the volume of reaction mixtures besides increasing the sensitivity. [U-14C]xylulose 5-phosphate was produced from commercially available [U-14C]xylose in a coupled assay system containing D-xylose isomerase, which yields [U-14C]xylulose, the substrate of ATP-dependent D-xylulose kinase. Separation of products and substrates was achieved by thin layer chromatography, identification of radioactive spots by radioscanning followed by quantitative scintillation counting. The protocol was validated through determination of kinetic constants of a purified His-tagged enzyme from Escherichia coli and comparison with the spectrophotometric method. The radiometric assay was applied to determine xylulose kinase activity in crude cell extracts from a variety of eukaryotic and prokaryotic organisms.     

Preparation of bromo[1-14C]acetyl-coenzyme A as an affinity label for acetyl-coenzyme A binding sites

Bromo[1-14C]acetyl-CoA has been prepared from CoASH and the N-hydroxysuccinimide ester of bromo[1-14C]acetic acid, and unlabeled bromoacetyl-CoA by reaction of CoASH with bromoacetyl bromide. The products were purified by high-pressure liquid chromatography. Purified bromoacetyl-CoA was characterized, and found to be a potent alkylating agent with a substantial stability in aqueous solution: it decomposed at 30 degrees C and pH 6.6 and 8.0 with halftimes of 3.3 and 2.5 h, respectively. The major breakdown products were CoASH and CoAS X CO X CH2 X SCoA. Bromo[1-14C]acetyl-CoA has been used to affinity label the acetyl-CoA binding site of 3-hydroxy-3-methylglutaryl-CoA synthase from ox liver. It was found to irreversibly inhibit the enzyme activity and bind covalently with a stoichiometry for complete inhibition of about 0.8 mol/mol enzyme dimer.     

Purification and partial amino-acid sequence of gibberellin 20-oxidase from Cucurbita maxima L. endosperm

Gibberellin (GA) 20-oxidase was purified to apparent homogeneity from Cucurbita maxima endosperm by fractionated ammonium-sulphate precipitation, gel-filtration chromatography and anion-exchange and hydrophobic-interaction high-performance liquid chromatography (HPLC). Average purification after the last step was 55-fold with 3.9% of the activity recovered. The purest single fraction was enriched 101-fold with 0.2% overall recovery. Apparent relative molecular mass of the enzyme was 45 kDa, as determined by gel-filtration HPLC and sodium dodecyl sulphate-polyacrylamide gel electrophoresis, indicating that GA 20-oxidase is probably a monomeric enzyme. The purified enzyme degraded on two-dimensional gel electrophoresis, giving two protein spots: a major one corresponding to a molecular mass of 30 kDa and a minor one at 45 kDa. The isoelectric point for both was 5.4. The amino-acid sequences of the amino-terminus of the purified enzyme and of two peptides from a tryptic digest were determined. The purified enzyme catalysed the sequential conversion of [¹⁴C]GA₁₂ to [¹⁴C]GA₁₅, [¹⁴C]GA₂₄ and [¹⁴C]GA₂₅, showing that carbon atom 20 was oxidised to the corresponding alcohol, aldehyde and carboxylic acid in three consecutive reactions. [¹⁴C]Gibberellin A₅₃ was similarly converted to [¹⁴C]GA₄₄, [¹⁴C]GA₁₉, [¹⁴C]GA₁₇ and small amounts of a fourth product, which was preliminarily identified as [¹⁴C]GA₂₀, a C₁₉-gibberellin. All GAs except [¹⁴C]GA₂₀ were identified by combined gas chromatography-mass spectrometry. The cofactor requirements in the absence of dithiothreitol were essentially as in its presence (Lange et. al, Planta 195, 98–107, 1994), except that ascorbate was essential for enzyme activity and the optimal concentration of catalase was lower.     

N,N'-dicyclohexylcarbodiimide-binding proteolipid of the vacuolar H+-ATPase from oat roots

The inhibitor N,N'-dicyclohexylcarbodiimide (DCCD) was used to probe the structure and function of the vacuolar H+-translocating ATPase from oat roots (Avena sativa var. Lang). The second-order rate constant for DCCD inhibition was inversely related to the concentration of membrane, indicating that DCCD reached the inhibitory site by concentrating in the hydrophobic environment. [14C]DCCD preferentially labeled a 16-kDa polypeptide of tonoplast vesicles, and the amount of [14C]DCCD bound to the 16-kDa peptide was directly proportional to inhibition of ATPase activity. A 16-kDa polypeptide had previously been shown to be part of the purified tonoplast ATPase. As predicted from the observed noncooperative inhibition, binding studies showed that 1 mol of DCCD was bound per mol of ATPase when the enzyme was completely inactivated. The DCCD-binding 16-kDa polypeptide was purified 12-fold by chloroform/methanol extraction. This protein was thus classified as a proteolipid, and its identity as part of the ATPase was confirmed by positive reaction with the antibody to the purified ATPase on immunoblots. From the purification studies, we estimated that the 16-kDa subunit was present in multiple (4-8) copies/holoenzyme. The purification of the proteolipid is a first step towards testing its proposed role in H+ translocation.     

Evaluations of tyrosine apodecarboxylase assays for pyridoxal phosphate

The alternate procedures used in the tyrosine apodecarboxylase assays for pyridoxal 5'-phosphate were evaluated to determine optimal conditions. Two preparations of tyrosine apodecarboxylase from Streptococcus faecalis were used: a cell suspension and a partially purified cell-free form. The activity of the decarboxylase was measured in two different assays using [14C]tyrosine or [3H]tyrosine as substrate. The presence of serum proteins caused greater inhibition of the assay for serum pyridoxal phosphate using [14C]tyrosine as substrate than the assay with [3H]tyrosine. In contrast, addition of deproteinized serum extract did not appear to inhibit either assay. The rate of reconstitution of the apodecarboxylase in the cell suspension was at least four times slower than that of the cell-free enzyme. The rate of reconstitution of the cell-free enzyme was faster in acetate than in citrate buffer. Inorganic sulfate or phosphate, at normal plasma concentrations, did not alter either the reconstitution rate of tyrosine decarboxylase or the final activity obtained in the assays using either substrate. The tyrosine apodecarboxylase assay for pyridoxal phosphate can be optimized by using deproteinized sera or plasma and incubating the cell-free apoenzyme with the coenzyme in acetate buffer for a time sufficient to obtain maximum reconstitution.     

Thermococcus profundus 2-ketoisovalerate ferredoxin oxidoreductase, a key enzyme in the archaeal energy-producing amino acid metabolic pathway

2-Ketoisovalerate ferredoxin oxidoreductase (VOR) is a key enzyme in hyperthermophiles catalyzing the coenzyme A-dependent oxidative decarboxylation of mainly aliphatic amino acid-derived 2-keto acids. The very oxygen-labile enzyme purified under anaerobic conditions from a hyperthermophilic archaeon, Thermococcus profundus, is a hetero-octamer (alphabetagammadelta)(2) consisting of four different subunits, alpha = 45,000, beta = 31,000, gamma = 22,000 and delta = 13,000, respectively. Electron paramagnetic resonance and resonance Raman spectra of the purified enzyme indicate the presence of approximately three [4Fe-4S] clusters per alphabetagammadelta-protomer, although one of the clusters has a tendency to be converted to a [3Fe-4S] form during purification. The optimal temperature for the enzyme activity is 93 +/- 2 degrees C and the cognate [4Fe-4S] ferredoxin serves as an electron acceptor of the enzyme. The purified enzyme is highly oxygen-labile (t(1/2), approximately 5 min at 25 degrees C), and is partly protected in the presence of magnesium ions, thiamine pyrophosphate and sodium chloride (t(1/2), approximately 25 min at 25 degrees C).     

Purification of human procollagen type III N-proteinase from placenta and preparation of antiserum.

Procollagen type III N-proteinase, of Mr about 70,000, was detected in human placental tissue and purified from this source more than 5800-fold. It was found to be a glycoprotein, which was bound to both concanavalin A-Ultrogel and heparin-Sepharose affinity columns. Binding to a type III pN-collagen-Sepharose affinity column was used as the final step in purification. The purified enzyme accepted only native type III procollagen or [14C]carboxymethylated type III pN-collagen as its substrate; type I, type II and type IV procollagen and heat-denatured type III pN-collagen were not cleaved by the enzyme. Antibodies against this purified enzyme protein raised in rabbits demonstrated a high inhibitory effect on the enzyme activity. Immunoblotting of the denatured protein and immunoelectrophoresis of the native enzyme showed only one major antigenic component, again with an Mr of about 70,000. The antibodies cross-reacted with the enzyme preparation from foetal-calf aorta smooth-muscle cells.     

The gamma-aminobutyrate/benzodiazepine receptor from pig brain. Purification and characterization of the receptor complex from cerebral cortex and cerebellum.

The gamma-aminobutyrate/benzodiazepine-receptor complex has been purified from a Triton X-100 extract of crude synaptic membranes from pig cerebral cortex and cerebellum by a combination of affinity and ion-exchange chromatography. [3H]Flunitrazepam binding activity was purified 2200-fold from cortex with an overall yield of 2%. The dissociation constants for the binding of [3H]muscimol and [3H]flunitrazepam to the receptor complex were 14 +/- 3 nM and 14 +/- 2 nM respectively. The ratio of [3H]muscimol to [3H]flunitrazepam binding sites was in the range 2.2-2.8. There appeared to be no selective inactivation of either binding site during the purification procedure. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis revealed two major polypeptides of Mr 49 000 and 55 000 from both cortex and cerebellum. When the receptor from cortex was photoaffinity labelled with [3H]flunitrazepam, radioactivity was incorporated predominantly into the Mr-49 000 polypeptide, although some radioactivity was detectable in the Mr-55 000 band. The cerebellar receptor was photoaffinity labelled on the 49 000-Mr polypeptide but not on the polypeptide of Mr 55 000. In addition, some radioactivity was detected in a minor polypeptide of Mr 43 000. When purified in the presence of 3-[(3-cholamidopropyl)dimethylammonio]propanesulphonate the same major polypeptide components (Mr 49 000 and 55 000) were isolated, but the receptor now retained its ability to be modulated by secobarbital and by the anaesthetic propanidid.     

Reactions of 1-bromo-2-[14C]pinacolone with acetylcholinesterase from Torpedo nobiliana. Effects of 5-trimethylammonio-2-pentanone and diisopropyl fluorophosphate

1-Bromo-2-[14C]pinacolone, (CH3)3C14COCH2Br [( 14C]BrPin), was prepared from [1-14C]acetyl chloride and tert-butylmagnesium chloride with cuprous chloride catalyst, followed by bromination. It was examined as an active-site directed label for acetylcholinesterase (acetylcholine acetylhydrolase, EC 3.1.1.7) (AcChE). AcChE, isolated from Torpedo nobiliana, has k(cat) = (4.00 +/- 0.04).10(3) s-1, Km = 0.055 +/- 0.008 mM in hydrolysis of acetylthiocholine, and k(cat) = (5.6 +/- 0.2).10(3) s-1, Km = 0.051 +/- 0.003 mM in hydrolysis of acetylcholine. BrPin, binding in the trimethyl cavity, acts initially as a reversible competitive inhibitor, Ki = 0.20 +/- 0.09 mM, and, with time, as an irreversible covalently bound inactivator. Introduction of 14C from [14C]BrPin into Torpedo AcChE at pH 7.0 was followed by SDS-PAGE, autoradiography and scintillation counting, in the absence and presence of 5-trimethylammonio-2-pentanone (TAP), a competitive inhibitor (Ki = 0.075 +/- 0.001 mM) isosteric with acetylcholine; 1.8-1.9 14C was incorporated per inactivated enzyme unit at 50% inactivation. TAP retarded inactivation by [14C]BrPin, and prevented introduction of 0.9-1.1 14C per unit of enzyme protected. Prior inactivation of AcChE by BrPin prevents reaction with [3H]diisopropyl fluorophosphate [( 3H]DFP). Prior inactivation by DFP or [3H]DFP does not prevent reaction with [14C]BrPin, and this subsequent reaction with BrPin does not displace the [3H] moiety. [14C]BrPin alkylates a nucleophile in the active site, and this reaction does not alkylate or utilize the serine-hydroxyl.     

Effects of some analgesic anaesthetic drugs on human erythrocyte glutathione reductase: an in vitro study

Inhibitory effects of some analgesic and anaesthetic drugs on human erythrocyte glutathione reductase were investigated. For this purpose, human erythrocyte glutathione reductase was initially purified 2139-fold in a yield of 29% by using 2', 5'-ADP Sepharose 4B affinity gel and Sephadex G-200 gel filtration chromatography. SDS polyacrylamide gel electrophoresis confirmed the purity of the enzyme by sharing a single band. A constant temperature (+4 degrees C) was maintained during the purification process. Diclofenac sodium, ketoprofen, lornoxicam, tenoxicam, etomidate, morphine and propofol exhibited inhibitory effects on the enzyme in vitro using the Beutler assay method. K(i) constants and IC(50) values for drugs were determined from Lineweaver-Burk graphs and plotting activity % versus [I] graphs, respectively. The IC(50) values of diclofenac sodium, ketoprofen, lornoxicam, propofol, tenoxicam, etomidate and morphine were 7.265, 6.278, 0.3, 0.242, 0.082, 0.0523 and 0.0128 mM and the K(i) constants were 23.97 +/- 2.1, 22.14 +/- 7.6, 0.42 +/- 0.18, 0.418 +/- 0.056, 0.13 +/- 0.025, 0.0725 +/- 0.0029 and 0.0165 +/- 0.0013 mM, respectively. While diclofenac sodium, ketoprofen, lornoxicam, tenoxicam etomidate and morphine showed competitive inhibition, propofol displayed noncompetitive inhibition.