Purification and catalytic properties of glutathione transferase from the hepatopancreas of crayfish macrobrachium vollenhovenii (herklots)

Glutathione transferase from the hepatopancreas of fresh water crayfish Macrobrachium vollenhovenii was purified to apparent homogeneity by ion‐exchange chromatography on DEAE‐cellulose and by gel filtration on Sephadex G‐100. The enzyme appeared to be a homodimer with molecular weight (Mr) of 46.0 ± 1.4 kDa and a subunit Mr of 24.1 ± 0.35 kDa. Chromatofocusing of the apparently pure enzyme revealed microheterogeneity and resolved it into two isozymic peaks, which were eluted at pH 8.36 and 8.22 respectively. Inhibition studies showed that the I₅₀ value for cibacron blue, S‐hexylglutathione, hematin, and N‐ethylmaleimide (NEM) were 0.01 μM, 340μM, 5 μM and 33 mM respectively. Out of the several substrates tested, only 1‐chloro‐2,4‐dinitrobenzene (CDNB) and 7‐chloro‐4‐nitrobenzo‐2‐oxa‐1,3‐diazole could be conjugated with glutathione. Chemical modification studies with DTNB revealed that two sulphydryl groups per dimer were essential to the activity of the enzymes. On the basis of structural and catalytic characteristics, M. vollenhovenii GST seems close, tentatively, to the omega and zeta classes of GST. Initial‐velocity studies of the enzyme are consistent with a steady‐state random kinetic mechanism. Denaturation and renaturation studies with guanidine HCl (Gdn‐HCl) revealed that though low Gdn‐HCl concentrations (less than 0.5 M) denatured the enzyme, the enzyme was able to renature completely (100%). At higher concentration of the denaturant (0.5–4 M), refolding studies indicated that complete renaturation was not achieved. The extent of renaturation was however a function of protein concentration. Our results are consistent with a three‐state unfolding process. © 2005 Wiley Periodicals, Inc. J Biochem Mol Toxicol 18:332–344, 2004; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/jbt.20044     

Purification and characterization of a novel glutathione S-transferase from Atactodea striata

A novel GST isoenzyme was purified from hepatopancreas cytosol of Atactodea striata with a combination of affinity chromatography and reverse-phase HPLC. The molecular weight of the enzyme was determined to be 24 kDa by SDS-PAGE electrophoresis and 48 kDa by gel chromatography, in combination with GST information from literature revealed that the native enzyme was homodimeric with a subunit of M(r) 24 kDa. The purified enzyme, exhibited high activity towards 1-chloro-2,4-dinitrobenzene (CDNB) and 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl). Kinetic analysis with respect to CDNB as substrate revealed a K(m) of 0.43 mM and V(max) of 0.24 micromol/min/mg and a specific activity of 108.9 micromol/min/mg. The isoelectric point of the enzyme was 5.5 by isoelectric focusing and its optimum temperature was 38 degrees C and the enzyme had a maximum activity at approximately pH 8.0. The amino acid composition was also determined for the purified enzyme.     

Glutathione S-transferase of the malarial parasite Plasmodium falciparum: characterization of a potential drug target

Glutathione S-transferases (GSTs), which occur abundantly in most organisms, are essentially involved in the intracellular detoxification of numerous substances including chemotherapeutic agents, and thus play a major role in the development of drug resistance. A gene encoding a protein with sequence identity of up to 37% with known GSTs was identified on chromosome 14 of the malarial parasite Plasmodium falciparum. It was amplified using gametocyte cDNA and expressed in Escherichia coli as a hexahistidyl-tagged protein of 26 kDa subunit size. The homodimeric enzyme (PfGST) was found to catalyse the glutathione (GSH)-dependent modification of 1-chloro-2,4-dinitrobenzene and other typical GST substrates such as o-nitrophenyl acetate, ethacrynic acid, and cumene hydroperoxide. The Km value for GSH was 164+/-20 microM. PfGST was inhibited by cibacron blue (Ki=0.5 microM), S-hexylglutathione (Ki=35 microM), and protoporphyrin IX (Ki=10 microM). Hemin, a most toxic compound for parasitised erythrocytes, was found to be an uncompetitive ligand of PfGST with a Ki of 6.5 microM. Based on the activity of PfGST in extracts of P. falciparum, the enzyme represents 1 to 10% of cellular protein and might therefore serve as an efficient in vivo buffer for parasitotoxic hemin. Destabilising ligands of GST are thus expected to be synergistic with the antimalarial drug chloroquine, which itself was found to be a very weak inhibitor of PfGST (IC50>200 microM). X-ray quality crystals of PfGST (250x200x50 microm) will serve as starting point for structure-based drug design.     

The NAD-dependent glutamate dehydrogenase from Dictyostelium discoideum: purification and properties

The NAD-dependent glutamate dehydrogenase (GDH) from Dictyostelium discoideum was purified 1101-fold with a yield of 23.4%. The enzyme has an apparent Mr of 356 kDa, determined using Sephacryl S400, and a subunit molecular weight of 54 kDa on SDS-polyacrylamide gel electrophoresis. The Kms for alpha-ketoglutarate, NADH, and NH4+ are 0.36 +/- 0.03 mM, 16.0 +/- 0.1 microM, and 34.5 +/- 2.7 mM, respectively. The purified enzyme has a pH optimum of pH 7.25-7.5. At 0.1 mM, ADP and AMP stimulate GDH activity 25 and 102%, respectively. Half-maximal activity in the presence of 0.1 mM AMP for alpha-ketoglutarate, NADH, and NH4+ is reached at 2.3 +/- 0.1 mM, 71.4 +/- 5.5 microM, and 27.9 +/- 3.6 mM, respectively.     

Inhibition of human glutathione S-transferase P1-1 by tocopherols and alpha-tocopherol derivatives.

alpha-Tocopherol inhibits glutathione S-transferase P1-1 (GST P1-1) (R.I.M. van Haaften, C.T.A. Evelo, G.R.M.M. Haenen, A. Bast, Biochem. Biophys. Res. Commun. 280 (2001)). In various cosmetic and dietary products alpha-tocopherol is added as a tocopherol ester. Therefore we have studied the effect of various tocopherol derivatives on GST P1-1 activity. It was found that GST P1-1 is inhibited, in a concentration dependent manner, by these compounds. Of the compounds tested, the tocopherols were the most potent inhibitors of GST P1-1; the concentration giving 50% inhibition (IC(50)) is <1 microM. The esterified tocopherols and alpha-tocopherol quinone also inhibit the GST P1-1 activity at a very low concentration: for most compounds the IC(50) was below 10 microM. RRR-alpha-Tocopherol acetate lowered the V(max) values, but did not affect the K(m) for either 1-chloro-2,4-dinitrobenzene or GSH. This indicates that the GST P1-1 enzyme is non-competitively inhibited by RRR-alpha-tocopherol acetate. The potential implications of GST P1-1 inhibition by tocopherol and alpha-tocopherol derivatives are discussed.     

Partial purification of a farnesyl diphosphate synthase from whole-body Manduca sexta

Farnesyl diphosphate synthase (FPP synthase) is a ubiquitous enzyme that is required for the biosynthesis of sesquiterpenes, dolichols ubiquinones, and prenylated proteins in insects. We report on the partial purification and characterization of an FPP synthase, obtained from whole-body preparations of the lepidopteran insect, Manduca sexta. The larval enzyme was separated from isopentenyl diphosphate (IPP) isomerase, phosphatase, and GGPP synthase by preparative isoelectric focusing, and was further purified by DEAE Sepharose, hydroxyapatite, and size exclusion chromatography. Whole-body M. sexta FPP synthase has a native molecular weight of 60.5+/-3.5 kDa and consists of two subunits of 28.5+/-0.5 kDa. As seen with other prenyltransferases, the enzyme has an absolute requirement for divalent cation and both Mn(2+) and Mg(2+) stimulated activity, although the former was inhibitory at higher concentrations. Insect FPP synthase catalyzes the condensation of IPP (K(m)=2.9+/-1.2 microM) with both dimethylallyl diphosphate and geranyl diphosphate (K(m)=0.8+/-0.4 microM). The enzyme requires the presence of detergent, glycerol, and non-specific protein-protein interactions for stability and maximum catalytic activity.     

MALDI mass sequencing and characterization of filarialglutathione-S-transferase

Glutathione-S-transferase has been detected in the somatic extract and excretory-secretory products of different life stages of Setaria cervi, a bovine filarial parasite. The enzyme was subjected to MALDI-TOF followed by mass spectrometry and the nearest match found was Pleuronectes platessa GST. Molecular mass of the purified enzyme was approximately 26 kDa as determined by SDS-PAGE and MALDI-TOF. Setaria cervi GST exhibited high activity towards 1-chloro-2,4-dinitrobenzene and ethacrynic acid. Kinetic analysis with respect to 1-chloro-2,4-dinitrobenzene and glutathione as substrate revealed a K(m) of 2.22 mM and 0.61 mM, respectively. The activity was inhibited significantly by Cibacron blue and alpha-tocopherol.     

Characterization of pure human renal renin. Evidence for a subunit structure

Renin was completely purified from human kidney cortex employing a rapid three-step procedure which included homogenization and ammonium sulfate precipitation, aminohexyl-pepstatin affinity chromatography, and affinity chromatography using a synthetic octapeptide renin inhibitor (H-77) with a reduced peptide bond (-CH2-NH- instead of -CO-NH-) between Leu5-Leu6, Three kg of cortex dissected from 10 kg of human cadaver kidney yielded 1.7 +/- 0.5 mg of protein (mean +/- S.E. for five procedures) with a specific activity of 1094 +/- 166 Goldblatt units/mg of protein and an overall recovery of 52 +/- 2%. Both gel filtration high performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a molecular weight of 44,000, although Mr = 22,000 and 18,000 bands were also identified by SDS-PAGE. The pH optima with sheep angiotensinogen were 5.5 and 7.8 and the Km was 0.31 microM. With pure human substrate the pH optimum was 6.0 and the Km was 1.15 microM. Enzyme activity was inhibited by two different anti-human renal renin antibodies. Amino-terminal sequencing demonstrated a leucine residue at the 1-position. Sequencing of 15 additional amino acids agreed with that predicted from the gene sequence and indicated that prorenin is converted to renin following cleavage at the carboxyl end of two basic residues, Lys-2 Arg-1. As with SDS-PAGE analysis, high performance liquid chromatography in the presence of 6 M urea demonstrated Mr = 44,000, 22,000, and 18,000 bands. Immunoblot studies revealed that all of these bands cross-reacted with antihuman renin antibody. Amino-terminal sequencing indicated the Mm = 22,000 band is the amino terminus and the Mr = 18,000 band the carboxyl terminus of Mr = 44,000 renin. In the aqueous phase, these subunits bound to H-77 suggesting that they represent components of the active enzyme complex. Unlike mouse renin, there was no evidence of disulfide bonds. These results raise the question of whether human renin circulates as a subunit aggregation as well as a single chain protein. This may serve as a possible mechanism to regulate renin activity in plasma and tissues.     

Hydrodynamic examination of the dimeric cytoplasmic domain of the human erythrocyte anion transporter, band 3.

Solution studies of the cytoplasmic domain (molecular mass approximately 40kDa) of band 3, the anion exchanger from human erythrocyte membranes, previously suggested a dimeric molecule on the basis of the relative techniques of calibrated gel filtration and calibrated preparative ultracentrifugation. This dimeric behavior is firmly established on an absolute basis by a combination of calibrated gel chromatography and absolute ultracentrifugation techniques. Sedimentation velocity in the analytical ultracentrifuge combined with calibrated gel chromatography give a molecular mass M of (77 +/- 4) kDa, a value confirmed by low-speed sedimentation equilibrium. Velocity sedimentation in the analytical ultracentrifuge gave a single sedimenting species with an s o 20,w of (3.74 +/- 0.07)S. Sedimentation equilibrium analysis was also used to establish the strength of the binding via the dissociation constant Kd, with a value from direct fitting of the concentration distribution curves of (2.8 +/- 0.5) microM, confirmed by a value of approximately 3 microM obtained from fitting a plot of molecular weight Mw,app versus cell loading concentration. Hydrodynamic calculations based on the classical translational frictional ratio showed that the protein was highly asymmetric, with an axial ratio of approximately 10:1, consistent with observations from electron microscopy.     

A gel-electrophoretic analysis of protein ADP-ribosylation in polyoma virus-transformed and non-transformed BHK-21/C13 fibroblasts

We have examined a variety of conditions for solubilizing and electrophoresing cell proteins in order to define optimum conditions for studying proteins modified by ADP-ribosylation. We have identified conditions in which proteins can be quantitatively extracted from cells in an undegraded form with the protein-ADPribose linkages intact. Effective measures include boiling cells briefly (4 min) in the presence of 2% SDS and 2 M urea at pH 6.8. Both SDS and urea were present in the 6-18% gradient polyacrylamide gel matrix used for electrophoresis. Under these conditions good resolution of proteins of a wide molecular-weight range is obtained. This system has been used to compare protein ADP-ribosylation in non-transformed and polyma virus-transformed baby hamster kidney (BHK) fibroblasts, since the latter cells have a greater NAD+ ADP-ribosyltransferase activity (measured in isolated nuclei and permeabilized cells). Addition of DNAase to permeabilized BHK cells over the range 10-150 micrograms led to a progressively greater activation of transferase compared with controls. When PyY cells were used, however, maximum activation was achieved with only 10 micrograms of DNAase, further additions producing a successively smaller activation relative to control cells without added nuclease. There were also differences between these cells in response to salt. Addition of NaCl (to about 0.3 M) to BHK cells resulted in various extents of transferase activation, whereas any addition of NaCl to the incubate of permeabilized PyY cells decreased transferase activity. These different enzyme activities between this transformed and non-transformed cell line are for the most part not reflected in the protein modification profiles seen on autoradiograms of acrylamide gels after electrophoresis 32P-labelled proteins. A variety of proteins are modified and their molecular weights depend on the NA concentration in the permeabilized cell incubation. At 0.5 microM NAD+ there were two major acceptors with Mr values of 14 kDa and 30 kDa, and at 100 microM NAD+, three major acceptors, with Mr values of 19 kDa. 45 kDa and greater than 170 kDa. NAD concentrations of between 1 microM and 100 microM had no further effect on protein ADP-ribosylation profiles, except for the protein(s) of Mr greater than 170 kDa, pointing to a critical difference around 0.5-1.0 microM substrate. In some experiments, however, a difference was observed in the intensity of radioactivity in two bands. This may represent two different proteins, or a single protein modified to different extents.(ABSTRACT TRUNCATED AT 400 WORDS)     

Characterization and molecular cloning of a glutathione S-transferase from the whitefly Bemisia tabaci (Hemiptera: Aleyrodidae)

Glutathione S-transferases (GST) catalyzing the conjugation of reduced glutathione to a vast range of xenobiotics including insecticides were characterized in the whitefly Bemisia tabaci. GST activities were determined in susceptible and resistant strains of B. tabaci towards artificial substrates, i.e. 1-chloro-2,4-dinitrobenzene (CDNB) in a photometric microplate assay and monochlorobimane (MCB) in a fluoroemtric microplate assay and characterized by their Michaelis-Menten kinetics. The inhibitory potential of ethacrynic acid was very effective with IC50-values between 0.9 and 5.8 microM depending on substrate and strain. The inhibitory effect of dicumarol was 10 times lower. Glutathione-affinity chromatography purified GST enzymes of two different B. tabaci strains appeared as a single band on SDS-PAGE and had a molecular mass of 23.5 kDa determined by MALDI mass spectrometry. The N-terminus of the purified enzyme was sequenced by Edman degradation. The nearly full-length cDNA of the enzyme was isolated by RT-PCR using a degenerate primer derived from the N-terminal amino acid sequence and contained an open reading frame encoding a 194-amino-acid protein. Comparison of the deduced amino acid sequence with GSTs from other species revealed that the enzyme is closely related to insect class sigma GSTs.     

Purification, subunit structure and properties of a high-molecular-mass protein phosphatase capable of dephosphorylating mRNP of the brine shrimp Artemia sp

A phosphoprotein phosphatase active towards casein, phosphorylase a and mRNP proteins has been detected in the cytosol of cryptobiotic gastrulae of Artemia sp. This phosphatase has a relative molecular mass (Mr) of 225,000 as measured by gel filtration on Sephadex G-200 and has been purified to near homogeneity by ion-exchange chromatography on different DEAE-substituted matrices, affinity chromatography on polylysine-agarose, histone-Sepharose 4B and protamine-agarose, hydrophobic chromatography on phenyl-Sepharose 4B and gel filtration on Sephadex G-200. Sodium dodecyl sulphate gel electrophoresis of the final purification step revealed that the enzyme contains two types of subunits, alpha and beta, with Mr of 40,000 and 75,000, respectively. These values, in conjunction with the native Mr and the molar ratios of the subunits estimated by densitometric analysis of the gel, suggested that the subunit composition of the enzyme is alpha 2 beta 2. When treated with 1.7% (v/v) 2-mercaptoethanol at -20 degrees C or with ethanol, the enzyme released the catalytic alpha subunit of Mr 40,000. The protein phosphatase was activated by basic proteins e.g. protamine (A 0.5 = 1 microM), histone H1 (A 0.5 = 1.6 microM) and polylysine (A 0.5 = 0.2 microM) and inhibited by ATP (I 0.5 = 12 microM), NaF (I 0.5 = 3.1 mM) and pyrophosphate (I 0.5 = 0.6 mM). The enzyme is a polycation-stimulated protein phosphatase. Purified mRNP proteins, phosphorylated by the mRNP-associated casein kinase type II, are among the substrates used by the enzyme. The function of reversible phosphorylation-dephosphorylation of mRNP as a regulatory mechanism in mRNP metabolism is discussed.     

Non-transferrin-bound iron species in the serum of hypotransferrinaemic mice.

Serum from homozygous hypotransferrinaemic mice (a mixed group of males and females, aged 6-8 wk) was found to contain low levels of iron (mean 0.9 +/- 0.5 microM (SEM, n = 4), as assayed by conventional serum iron assays. Similarly, low levels of non-transferrin-bound iron were determined with a nitrilotriacetate chelation assay (1.3 +/- 0.4 microM, n = 4) (Singh, S., Hider, R.C. and Porter, J.B. (1990) Analytical Biochemistry 186, 320-323). Mononuclear Fe (citrate) was undectable by electron paramagnetic resonance spectroscopy (EPR). Significantly larger quantities of iron (16 +/- 5 microM, n = 8) were detected by the bleomycin assay (Gutteridge, J.M.C., Rowley, D.A. and Halliwell, B. (1981) Biochemical Journal 199, 263-265), while non-haem iron assay or atomic absorption spectrophotometry revealed up to 96 microM iron. Haemoglobin iron was detectable at approximately 10 microM by spectrophotometry. Ferri-haem was undetectable by EPR spectroscopy. Serum ferritin levels of 641 +/- 128 micrograms/l (n = 14) in hypotransferrinaemic mice (wild-types 44 +/- 6 micrograms/l, n = 14) were observed and these cannot account for the non-transferrin-bound iron. Hypotransferrinaemic mouse serum therefore contains large quantities of non-transferrin-bound iron which is unreactive in some assays used to detect such iron in human iron overload. Fractionation by Sephadex G200 chromatography revealed three distinct species with apparent molecular weights of > or = 150 kDa, 40-80 kDa and 1-5 kDa. The iron may be distinguished from known extracellular iron proteins and haem-proteins by its availability to hot acid extractions.     

Characterization of (Na+, K+)-ATPase in gill microsomes of the freshwater shrimp Macrobrachium olfersii

To better understand the adaptive strategies that led to freshwater invasion by hyper-regulating Crustacea, we prepared a microsomal (Na+, K+)-ATPase by differential centrifugation of a gill homogenate from the freshwater shrimp Macrobrachium olfersii. Sucrose gradient centrifugation revealed a light fraction containing most of the (Na+, K+)-ATPase activity, contaminated with other ATPases, and a heavy fraction containing negligible (Na+, K+)-ATPase activity. Western blotting showed that M. olfersii gill contains a single alpha-subunit isoform of about 110 kDa. The (Na+, K+)-ATPase hydrolyzed ATP with Michaelis Menten kinetics with K5, = 165+/-5 microM and Vmax = 686.1+/-24.7 U mg(-1). Stimulation by potassium (K0.5 = 2.4+/-0.1 mM) and magnesium ions (K0.5 = 0.76+/-0.03 mM) also obeyed Michaelis-Menten kinetics, while that by sodium ions (K0.5 = 6.0+/-0.2 mM) exhibited site site interactions (n = 1.6). Ouabain (K0.5 = 61.6+/-2.8 microM) and vanadate (K0.5 = 3.2+/-0.1 microM) inhibited up to 70% of the total ATPase activity, while thapsigargin and ethacrynic acid did not affect activity. The remaining 30% activity was inhibited by oligomycin, sodium azide and bafilomycin A. These data suggest that the (Na+, K+)-ATPase corresponds to about 70% of the total ATPase activity; the remaining 30%, i.e. the ouabain-insensitive ATPase activity, apparently correspond to F0F1- and V-ATPases, but not Ca-stimulated and Na- or K-stimulated ATPases. The data confirm the recent invasion of the freshwater biotope by M. olfersii and suggest that (Na+, K+)-ATPase activity may be regulated by the Na+ concentration of the external medium.     

A Streptomyces collinus thiolase with novel acetyl-CoA:acyl carrier protein transacylase activity

Acetyl-CoA:acyl carrier protein (ACP) transacylase (ACT) activity has been demonstrated for the 3-ketoacyl-ACP synthase III (KASIII) which initiates fatty acid biosynthesis in the type II dissociable fatty acid synthases of plants and bacteria. Several lines of evidence have indicated the possibility of ACT activity being associated with proteins other than KASIII. Using a crude extract of Streptomyces collinus, we have resolved from KASIII an additional protein with ACT activity and subsequently purified it 85-fold in five chromatographic steps. The 45 kDa protein was shown by gel filtration to have a molecular mass of 185 +/- 35 kDa, consistent with a homotetrameric structure for the native enzyme. The corresponding gene (fadA) was cloned and sequenced and shown to encode a protein with amino acid sequence homology to type II thiolases. The fadA was expressed in Escherichia coli, and the resulting recombinant FadA enzyme purified by metal chelate chromatography was shown to have both ACT and thiolase activities. Kinetic studies revealed that in an ACT assay FadA had a substrate specificity for a two-carbon acetyl-CoA substrate (K(m) 8.7 +/- 1.4 microM) but was able to use ACPs from both type II fatty acid and polyketide synthases (Streptomyces glaucescens FabC ACP, K(m) 10.7 +/- 1.4 microM; E. coli FabC ACP, K(m) 8.8 +/- 2 microM; FrenN ACP, K(m) 44 +/- 12 microM). In the thiolase assay kinetic analyses revealed similar K(m) values for binding of substrates acetoacetyl-CoA (K(m) 9.8 +/- 0.8 microM) and CoA (K(m) 10.9 +/- 1.8 microM). A Cys92Ser mutant of FadA possessed virtually unchanged K(m) values for acetoacetyl-CoA and CoA but had a greater than 99% decrease in k(cat) for the thiolase activity. No detectable ACT activity was observed for the Cys92Ser mutant, demonstrating that both activities are associated with FadA and likely involve formation of the same covalent acetyl-S-Cys enzyme intermediate. An ACT activity with ACP has not previously been observed for thiolases and in the case of the S. collinus FadA is significantly lower (k(cat) 3 min(-1)) than the thiolase activity of FadA (k(cat) 2170 min(-1)). The ACT activity of FadA is comparable to the KAS activity and significantly higher than the ACT activity, reported for a streptomycete KASIII.     

Functional characterization of D-galacturonic acid reductase, a key enzyme of the ascorbate biosynthesis pathway, from Euglena gracilis

D-Galacturonic acid reductase, a key enzyme in ascorbate biosynthesis, was purified to homogeneity from Euglena gracilis. The enzyme was a monomer with a molecular mass of 38-39 kDa, as judged by SDS-PAGE and gel filtration. Apparently it utilized NADPH with a Km value of 62.5+/-4.5 microM and uronic acids, such as D-galacturonic acid (Km=3.79+/-0.5 mM) and D-glucuronic acid (Km=4.67+/-0.6 mM). It failed to catalyze the reverse reaction with L-galactonic acid and NADP(+). The optimal pH for the reduction of D-galacturonic acid was 7.2. The enzyme was activated 45.6% by 0.1 mM H(2)O(2), suggesting that enzyme activity is regulated by cellular redox status. No feedback regulation of the enzyme activity by L-galactono-1,4-lactone or ascorbate was observed. N-terminal amino acid sequence analysis revealed that the enzyme is closely related to the malate dehydrogenase families.     

Purification, properties, and oligomeric structure of glutathione reductase from the cyanobacterium Spirulina maxima

Glutathione reductase [NAD(P)H:GSSG oxidoreductase EC 1.6.4.2] from cyanobacterium Spirulina maxima was purified 1300-fold to homogeneity by a simple three-step procedure involving ammonium sulfate fractionation, ion exchange chromatography on DEAE-cellulose, and affinity chromatography on 2',5'-ADP-Sepharose 4B. Optimum pH was 7.0 and enzymatic activity was notably increased when the phosphate ion concentration was increased. The enzyme gave an absorption spectrum that was typical for a flavoprotein in that it had three peaks with maximal absorbance at 271, 370, and 460 nm and a E1%271 of 23.3 Km values were 120 +/- 12 microM and 3.5 +/- 0.9 microM for GSSG and NADPH, respectively. Mixed disulfide of CoA and GSH was also reduced by the enzyme under assay conditions, but the enzyme had a very low affinity (Km 3.3 mM) for this substrate. The enzyme was specific for NADPH. The isoelectric point of the native enzyme at 4 degrees C was 4.35 and the amino acid composition was very similar to that previously reported from other sources. The molecular weight of a subunit under denaturing conditions was 47,000 +/- 1200. Analyses of pure enzyme by a variety of techniques for molecular weight determination revealed that, at pH 7.0, the enzyme existed predominantly as a tetrameric species in equilibrium with a minor dimer fraction. Dissociation into dimers was achieved at alkaline pH (9.5) or in 6 M urea. However, the equilibrium at neutral pH was not altered by NADPH or by disulfide reducing reagents. The Mr and S20,w of the oligomeric enzyme were estimated to be 177,000 +/- 14,000 and 8.49 +/- 0.5; for the dimer, 99,800 +/- 7000 and 5.96 +/- 0.4, respectively. Low concentrations of urea increased the enzymatic activity, but this increase was not due to changes in the proportions of both forms.     

Nonspecific interaction of proteoglycans with chromatography media and surfaces: effect of this interaction on the isolation efficiencies

Nonspecific adsorption of proteoglycans to chromatography media and surfaces is demonstrated. This adsorption is highly dependent on the nature of the chromatography media and the precise buffer conditions. For a given buffer the amount of adsorption decreases as the pH of the buffer is increased. It is also highly dependent on buffer concentration and increases as the buffer concentration is increased. The effect of salts such as LiCl, NaCl, KCl, and MgCl2 was generally small and complex so that the presence of the salt both increased and decreased the amount of adsorption depending on the buffer conditions. In contrast, the effect due to the presence of guanidine hydrochloride (Gdn-HCl) was relatively large and complex. At low Gdn-HCl concentrations there generally was a large increase in the amount of adsorption, reaching a maximum at approximately 0.5 M Gdn-HCl and decreasing with further increases in Gdn-HCl concentration. Detergents such as 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps) and sodium dodecylsulfate generally reduced the amount of nonspecific adsorption, although in the presence of both the detergent and Gdn-HCl, the effect due to Gdn-HCl predominated. In commonly used buffers such as 0.5 M sodium acetate (NaOAc), pH 7.0 (buffer F), and 4 M Gdn-HCl in 0.05 M NaOAc, pH 5.8 (buffer D), adsorption to surfaces and chromatography media such as Sepharose CL-2B, cellulose, and controlled pore glass (CPG) is highly significant and it is particularly large for cellulose and CPG.(ABSTRACT TRUNCATED AT 250 WORDS)     

Modulation by ammonium ions of gill microsomal (Na+,K+)-ATPase in the swimming crab Callinectes danae: a possible mechanism for regulation of ammonia excretion

The modulation by Na(+), K(+), NH(4)(+) and ATP of the (Na(+),K(+))-ATPase in a microsomal fraction from Callinectes danae gills was analyzed. ATP was hydrolyzed at high-affinity binding sites at a maximal rate of V=35.4+/-2.1 Umg(-1) and K(0.5)=54.0+/-3.6 nM, obeying cooperative kinetics (n(H)=3.6). At low-affinity sites, the enzyme hydrolyzed ATP obeying Michaelis-Menten kinetics with K(M)=55.0+/-3.0 microM and V=271.5+/-17.2 Umg(-1). This is the first demonstration of a crustacean (Na(+),K(+))-ATPase with two ATP hydrolyzing sites. Stimulation by sodium (K(0.5)=5.80+/-0.30 mM), magnesium (K(0.5)=0.48+/-0.02 mM) and potassium ions (K(0.5)=1.61+/-0.06 mM) exhibited site-site interactions, while that by ammonium ions obeyed Michaelis-Menten kinetics (K(M)=4.61+/-0.27 mM). Ouabain (K(I)=147.2+/-7.microM) and orthovanadate (K(I)=11.2+/-0.6 microM) completely inhibited ATPase activity, indicating the absence of contaminating ATPase and/or neutral phosphatase activities. Ammonium and potassium ions synergistically stimulated the enzyme, increasing specific activities up to 90%, suggesting that these ions bind to different sites on the molecule. The presence of each ion modulates enzyme stimulation by the other. The modulation of (Na(+),K(+))-ATPase activity by ammonium ions, and the excretion of NH(4)(+) in benthic crabs are discussed.