Isolation of multiple forms of metleghemoglobin reductase and leghemoglobin components from lupin nodules

Method for simultaneous obtaining in homogeneous state of two main forms of metleghemoglobin reductase and main leghemoglobin components from lupine nodules was worked out. The method included steps of saturation with ammonium sulphate (40-80%), gel-filtration on Ultrogel AcA 44, isoelectric focusing and repeated isoelectric focusing. As result the forms of metleghemoglobin reductase with molecular weights 62 and 66 kDa were obtained purified 725 and 402 times respectively and obtained in homogeneous state. The total yield of activity was 37%. The form with 62 kDa molecular weight was more active.     

Aldose reductase from human skeletal and heart muscle. Interconvertible forms related by thiol-disulfide exchange

Aldose reductase was purified from human skeletal and heart muscle by a rapid and efficient scheme involving Red Sepharose chromatography, chromatofocusing on Pharmacia PBE 94, and hydroxylapatite high pressure liquid chromatography. The scheme afforded homogeneous enzyme, 65% recovery, in 2 days. All muscle samples express aldose reductase but not the closely related aldehyde reductase. Aldose reductase is isolated in one of two forms that are distinguishable by their kinetic patterns with glyceraldehyde as substrate and which are interconvertible by treatment with dithiothreitol. Both forms are capable of catalyzing the reduction of glucose (Km = 68 mM), and both are highly sensitive to inhibition by aldose reductase inhibitors. The reduction of glucose was shown to be nearly stoichiometric with production of sorbitol (92 +/- 2%). Dialysis of aldose reductase in the absence of thiols or NADP converts it into a form that shows markedly different kinetic properties, including very weak catalytic activity toward glucose and insensitivity to aldose reductase inhibitors. This modified form can be converted back into the native form by dithiothreitol. Thiol titration of the two forms of aldose reductase with Ellman's reagent indicated that two thiol groups were lost when the enzyme was dialyzed in the absence of dithiothreitol or NADP.     

Purification of Squash NADH:Nitrate Reductase by Zinc Chelate Affinity Chromatography

NADH:nitrate reductase (EC 1.6.6.1) was isolated and purified from the green cotyledons of 5-day-old squash seedlings (Cucurbita maxima L.). The 10-hour purification procedure consisted of two steps: direct application of crude enzyme to blue Sepharose and specific elution with NADH followed by direct application of this effluent to a Zn²⁺ column with elution by decreasing the pH of the phosphate buffer from 7.0 to 6.2. The high specific activity (100 micromoles per minute per milligram protein) and high recovery (15-25%) of electrophoretically homogeneous nitrate reductase show that the enzyme was not damaged by exposure to the bound zinc. With this procedure, homogeneous nitrate reductase can be obtained in yields of 0.5 milligram per kilogram cotyledons.     

Calmodulin antagonists inhibit formation of platelet-activating factor in stimulated human neutrophils

Conversion of native, 97-100 kDa rat liver microsomal HMG CoA reductase to membrane-bound 62 kDa and soluble 52-56 kDa catalytically active forms was catalyzed in vitro by the calcium-dependent, leupeptin- and calpastatin-sensitive protease calpain-II purified from rat liver cytosol. Cleavage of the native 97-100 kDa reductase was enhanced by pretreatment (inactivation) of microsomes with ATP(Mg2+) and liver reductase kinase (compared to protein phosphatase-pretreated controls). This was reflected in a loss of the 97-100 kDa species and an increase in the soluble 52-56 kDa species (total enzyme activity and specific immunoblot recovery).     

Regulation of crystalline lens aldose reductase activity. Nonhyperbolic oxidation kinetics of NADPH by glucose

A homogeneous aldose reductase was isolated from bovine eye lens tissue by using affinity chromatography on blue agarose. A kinetic analysis of the initial rates of NADPH oxidation at 0.5-100 mM glucose and at 1.2-10 microM NADPH was carried out. The Line-weaver-Burk plots for glucose concentration were nonlinear at fixed concentrations of NADPH and linear at fixed concentrations of glucose. It was shown that the experimental plots reflect the mechanisms, in which substrate regulation of enzyme activity is effectuated by glucose binding to the regulatory site or is due to the shift of the equilibrium between the isomeric forms of aldose reductase.     

In vitro phosphorylation of 3-hydroxy-3-methylglutaryl coenzyme a reductase: Analysis of 32P-labeled,inactivated enzyme

Rat liver microsomal 3-hydroxy-3-methylglutaryl-CoA reductase was inactivated with Mg²⁺ and [γ-³²P]ATP, then solubilized and purified to homogeneity. The ³²P radioactivity was precipitated by antibody to homogeneous rat liver reductase and comigrated with nonprecipitated, homogeneous reductase on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Under nondenaturing conditions, ³²P radioactivity comigrated with reductase protein and activity on polyacrylamide gels. These results provide direct support for the concept that the enzyme is covalently phosphorylated during the in vitro incubation of microsomes with Mg²⁺ and ATP.     

Preparation of highly radioactive homogeneous 32P-labeled hydroxymethylglutaryl coenzyme A reductase from rat liver

A procedure for the preparation of highly radioactive homogeneous ³²P-labeled 3-hydroxy-3-methylglutaryl coenzyme A reductase from rat liver microsomes has been developed. The enzymatic preparation obtained by this procedure has a specific radioactivity 50-fold higher than that reported in previous literature. The purified enzyme was judged to be homogeneous on the basis of comigration of enzyme activity with a single band of protein and ³²P radioactivity on polyacrylamide gels. The ³²P covalently bound to the reductase was removed upon incubation with purified hydroxymethylglutaryl coenzyme A reductase phosphatase from rat liver.     

Purification, structure and properties of the respiratory nitrate reductase of Klebsiella aerogenes.

1. The respiratory nitrate reductase of Klebsiella aerogenes was solubilized from the bacterial membranes by deoxycholate and purified further by means of gel chromatography in the presence of deoxycholate, and anion-exchange chromatography. 2. Dependent on the isolation procedure two different homogeneous forms of the enzyme, having different subunit compositions, can be obtained. These forms are designated nitrate reductase I and nitrate reductase II. Both enzyme preparations are isolated as tetramers having sedimentation constants (s20,w) of 22.1 S and 21.7 S for nitrate reductase I and II, respectively. The nitrate reductase I tetramer has a molecular weight of about 106. 3. In the presence of deoxycholate both enzyme preparations dissociate reversibly into their respective monomeric forms. The monomeric form of nitrate reductase I has a molecular weight of about 260 000 and a sedimentation constant of 9.8 S. For nitrate reductase II these values are 180 000 and 8.5 S, respectively. 4. Nitrate reductase I consists of three different subunits, having molecular weights of 117 000; 57 000 and 52 000, which are present in a 1:1:2 molar ratio, respectively. Nitrate reductase II contains only the subunits with a molecular weight of 117 000 and 57 000 in a equimolar ratio. 5. Treatment at pH 9.5 in the presence of deoxycholate and 0.05 M NaCl or ageing removes the 52 000 Mr subunit from nitrate reductase I. This smallest subunit, in contrast to the other subunits, is a basic protein. 6. The 52 000 Mr subunit has no catalytic function in the intramolecular electron transfer from reduced benzylviologen to nitrate. However, it appears to have a structural function since nitrate reductase II, which lacks this subunit, is much more labile than nitrate reductase I. Inactivation of nitrate reductase II can be prevented by the presence of deoxycholate. 7. The spectrum of the enzyme resembles that of iron-sulfur proteins. No cytochromes or contaminating enzyme activities are present in the purified enzyme. Only reduced benzylviologen was found to be capable of acting as an electron donor. 8. p-Chlormercuribenzoate enhances the enzymatic activity at concentrations of 0.1 mM and lower. At higher p-chlormercuribenzoate concentrations the enzymatic activity is inhibited non-competitively with either nitrate or benzylviologen as a substrate. The inhibition is not counteracted by cysteine.     

Immunohistochemical localization of thioredoxin and thioredoxin reductase in adult rats

Rabbit antisera against homogeneous rat liver thioredoxin and thioredoxin reductase (NADPH-oxidized thioredoxin oxidoreductase, E.C. 1.6.4.5) were prepared and used for immunohistochemical analysis in adult rats. Immunoreactive thioredoxin and thioredoxin reductase were widely distributed in tissues and organs, but varied a lot between cell types. Generally, epithelial cells, neuronal cells and secretory cells, both exocrine and endocrine, showed high immunoreactivity whereas mesenchymal cells with exceptions showed low activity. Surface lining epithelial and keratinizing cells showed high activity. The immunofluorescence was localized in the cytoplasm of cells with enrichments at secretory granules, at the plasma membrane or in the subplasma membrane zone. Variations in secretory cells were seen related to feeding and starvation and to metabolic activity. The distribution of thioredoxin and thioredoxin reductase is compatible with function in thiol-disulfide interchange reaction related to protein synthesis, intracellular transport and different forms of secretion.     

Growth of Trypanosoma cruzi in Human Heart Tissue Cells and Effects of Aminonucleoside of Puromycin, Trypacidin and Aminopterin

SYNOPSIS. Development of a suitable tissue cell system for the growth of T. cruzi required the production of infective metacyclic trypanosomes and tissues which these forms could infect. Human heart was selected from a range of serially-cultured tissue cell lines as the most suitable for intracellular growth. Most metacyclic forms occurred when the growth medium had an initial pH of 7.2 and the cultures were gassed with 5% CO₂/95% air before closure and then incubated at 33 C.
During the first 4 days of incubation ∼ 10% of tissue cells in cultures were infected with 1–5 leishmanial forms of T. cruzi. During the next 4 days, the number of infected cells about doubled and many contained 10 or more leishmanias. The effects of drugs were studied on this 2nd 4-day period of incubation. The aminonucleoside of puromycin affected growth of the intracellular but not the extracellular parasites whereas trypacidin had the opposite effect, inhibiting growth of the extracellular but not the intracellular forms. Aminopterin inhibited the growth of both extracellular and intracellular forms. The selective effects of the aminonucleoside of puromycin against intracellular forms might be due to it being metabolized by the host cells to a more active trypanocide.
A method for isolating intracellular forms of T. cruzi from the tissue cells is described. Dihydrofolate reductase activity was found in these preparations and in similar preparations of the culture and blood forms. The reductase was sensitive to inhibition by aminopterin and a 2, 4-diaminopyrimidine.     

NADPH-cytochrome c (P-450) reductase has the activity of NADPH-linked aquacobalamin reductase in rat liver microsomes.

To elucidate the mammalian system for synthesis of cobalamin coenzymes, microsomal NADPH-linked aquacobalamin reductase was purified and characterized. The enzyme was purified about 534-fold over rat liver microsomal fraction in a yield of about 32%. The purified enzyme was homogeneous in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and had a monomeric molecular weight of 79,000. The purified aquacobalamin reductase showed a high specific activity (about 55 mumol/min per mg protein) of NADPH-cytochrome c (P-450) reductase. About 33% of the NADPH-cytochrome c reductase activity found in the microsomal fraction was recovered in the final purified preparation. The activity ratio of NADPH-cytochrome c reductase/NADPH-linked aquacobalamin reductase was about 5.0 through the purification steps, indicating that the rat liver microsomal NADPH-linked aquacobalamin reductase is the NADPH-cytochrome c reductase.     

Rapid,high-yield purification of rat liver 3-hydroxy-3-methyl-glutaryl-coenzyme a reductase

A rapid method for the purification of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase from the livers of cholestyramine-fed rats is reported. The procedure involves a sequence of separations on affinity chromatography columns consisting of Blue Dextran-Sepharose, agarose-CoA, and agarose-HMG-CoA. The advantage of this method is its flexibility in scavenging enzyme that might be lost during purification, resulting in a yield of homogeneous reductase (specific activity approximately 10,000 nmol/min/mg protein) as high as 50%, which is at least twice that previously reported.     

Purification by affinity chromatography of yeast glutathione reductase, the enzyme responsible for the NADPH-dependent reduction of the mixed disulfide of coenzyme A and glutathione.

Glutathione reductase (NAD(P)H : oxidised-glutathione oxidoreductase, EC 1.6.4.2) was purified from baker's yeast by a new procedure involving affinity chromatography on 2',5'-ADP-Sepharose 4B. The yield was 65% of essentially homogeneous enzyme. The activity was assayed with both glutathione disulfide (GSSG) and the mixed disulfide of coenzyme A and glutathione (CoAssg). The two disulfide substrates gave coinciding activity profiles and a constant ratio of the activities in different chromatographic and electrophoretic systems. No evidence was obtained for the existence of a reductase specific for CoASSG distinct from glutathione reductase. It is concluded that normal baker's yeast contains a single reductase active with both GSSG and CoASSG.     

Human hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase: evidence for the regulation of enzymic activity by a bicyclic phosphorylation cascade

Microsomal human liver HMG-CoA reductase has been shown to exist in active (dephosphorylated) and inactive (phosphorylated) forms. Microsomal HMG-CoA reductase was inactivated in vitro by ATP-Mg in a time dependent manner; this inactivation was mediated by reductase kinase. Incubation of inactivated enzyme with phosphatase resulted in a time dependent reactivation (dephosphorylation). Polyacrylamide gel electrophoresis of purified HMG-CoA reductase incubated with reductase kinase and radiolabeled ATP revealed that the 32P radioactivity and HMG-CoA reductase enzymic activity were localized in a single electrophoretic position. Partial dephosphorylation of the phosphorylated enzyme was associated with loss of 32P and increase in HMG-CoA reductase activity. Human reductase kinase also exists in active and inactive forms. The active (phosphorylated) form of reductase kinase can be inactivated by incubation with phosphatase. Phosphorylation of inactive reductase kinase with ATP-Mg and a second kinase, reductase kinase kinase, was associated with a parallel increase in the enzymic activity of reductase kinase and the ability to inactivate HMG-CoA reductase. The combined results present initial evidence for the presence of human HMG-CoA reductase and reductase kinase in active and inactive forms, and the in vitro modulation of its enzymic activity by a bicyclic phosphorylation cascade. This bicyclic cascade system may provide a mechanism for short-term regulation of the pathway for cholesterol biosynthesis in man.     

A heme-C-containing enzyme complex that exhibits nitrate and nitrite reductase activity from the dissimilatory iron-reducing bacterium Geobacter metallireducens

Nitrate reduction in the dissimilatory iron-reducing bacterium Geobacter metallireducens was investigated. Nitrate reductase and nitrite reductase activities in nitrate-grown cells were detected only in the membrane fraction. The apparent K _m values for nitrate and nitrite were determined to be 32 and 10 μM, respectively. Growth on nitrate was not inhibited by either tungstate or molybdate at concentrations of 1 mM or less, but was inhibited by both at 10 and 20 mM. Nitrate and nitrite reductase activity in the membrane fraction was not, however, affected by dialysis with 20 mM tungstate. An enzyme complex that exhibited both nitrate and nitrite reductase activity was solubilized from membrane fractions with CHAPS and was partially purified by preparative gel electrophoresis. It was found to be composed of four different polypeptides with molecular masses of 62, 52, 36, and 16 kDa. The 62-kDa polypeptide [a low-midpoint potential (–207 mV), multiheme cytochrome c] exhibited nitrite reductase activity under denaturing conditions. No molybdenum was detected in the complex by plasma-emission mass spectrometry. Received: 26 March 1999 / Accepted: 16 August 1999     

Interaction between cytochrome P-448 and NADP-cytochrome P-450 reductase in reconstituted microsomal membranes

The regularities of changes in the functional activity of the microsomal monooxygenase system reconstituted by self-assembly from intact rat liver microsomes solubilized with 4% sodium cholate were studied at variable levels of NADPH-cytochrome P-450 reductase and the 3-methylcholanthrene-induced form of cytochrome P-450. Using antibodies against cytochrome P-448, the role of cytochrome P-448 in the overall reaction of benzopyrene hydroxylation induced in the microsomal membrane by a set of molecular forms of cytochrome P-450 was investigated. The effect of NADPH-cytochrome P-450 reductase and cytochrome P-448 incorporation into reconstituted microsomal membranes on benzpyrene metabolism suggests that in intact microsomal membranes benzopyrene metabolism induced by different forms of cytochrome P-450, with the exception of P-448, is limited by reductase is not the limiting component; however, cytochrome P-448 reveals its maximum activity at the cytochrome to reductase optimal molar ratio of 5:1; above this level, the catalytic activity of cytochrome P-448 is lowered.     

Modulation in vitro of 3-hydroxy-3-methylglutaryl coenzyme A reductase in brain microsomes: Evidence for the phosphorylation and dephosphorylation associated with inactivation and activation of the enzyme

The activity of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase in brain microsomes was modified in vitro. The inactivation of the enzyme required Mg²⁺ and ATP or ADP, and an inactivator present both in S₁₀₅ and microsomes. Inactivation was dependent on inactivator concentration and time of preincubation. The inactive reductase in brain microsomes could be completely reactivated by a factor present in brain S₁₀₅. Reactivation of the enzyme also depended on incubation time and the activator concentration. Activator activity was inhibited by NaF, a phosphatase inhibitor. Both the inactivator and the activator appear to be proteins. Our data thus suggest that the inactivation and the reactivation of the reductase in brain microsomes occurs via protein-mediated interconversion to phosphorylated and dephosphorylated forms of the enzyme with differing catalytic activity. The HMG-CoA reductase activity increases almost two-fold during isolation of the brain microsomes. This increase in activity is blocked when brain tissue is homogenized in the medium containing NaF. In rat brain about 50% of the reductase exists in an inactive form in both young and adult rats. The low reductase activity in brain of adult animals does not appear to be related to an increase in the proportion of an inactive phosphorylated form of the enzyme. This suggests that developmental change in the reductase activity is not associated with the change in the proportion of phosphorylated and dephosphorylated forms of the enzyme.     

Purification and characterization of a psychrophilic glutathione reductase from Antarctic ice microalgae <Emphasis Type="Italic">Chlamydomonas</Emphasis> sp. Strain ICE-L

A psychrophilic glutathione reductase from Antarctic ice microalgae Chlamydomonas sp. Strain ICE-L was purified by ammonium sulfate fractionation and three steps of chromatography. The yield was up to 25.1% of total glutathione reductase in the crude enzyme extract. The glutathione reductase activity was characterized by the spectrophotometric method under different conditions. Purified glutathione reductase was separated by SDS-PAGE, which furnished a homogeneous band. The native molecular mass of the enzyme was 115 kDa. Apparent Km values for NADPH and NADH (both at 0.5 mmol L⁻¹ oxidized glutathione) were 22.3 and 83.8 μmol L⁻¹, respectively. It was optimally active at pH 7.5, and it was stable from pH 5 to 9. Its optimum temperature was 25°C, with activity at 0°C 23.5% of the maximum. Its optimum ion strength and optimum Mg²⁺ were 50–90 and 7.5 mmol L⁻¹, respectively. Ca²⁺, Mg²⁺, and cysteine substantially increased the activity of the enzyme but chelating agents, heavy metals (Cd²⁺, Pb²⁺, Cu²⁺, Zn²⁺, etc.), NADPH, and ADP had significant inhibitory effects. This glutathione reductase can be used to study the adaptation and mechanism of catalysis of psychrophilic enzymes, and it has a high potential as an environmental biochemical indicator under extreme conditions.     

Purification and properties of rat liver 3-hydroxy-3-methylglutaryl coenzyme A reductase.

3-Hydroxy-3-methylglutaryl coenzyme A reductase has been purified from rat liver microsomes with a recovery of approx. 25%. The enzyme was homogeneous on gel electrophoresis and enzyme activity comigrated with the single protein band. The molecular weight of the reductase determined by gel filtration on Sephadex G-200 was 200,000. SDS-polyacrylamide gel electrophoresis gave a subunit molecular weight of 52,000 +/- 2000, suggesting that the enzyme was a tetramer. The specific activities of the purified enzyme obtained from rats fed diets containing 0% or 5% cholestyramine were 11,303 and 19,584 nmol NADPH oxidized/min per mg protein, respectively. The reductase showed unique binding properties to Cibacron Blue Sepharose; the enzyme was bound to the Cibacron Blue via the binding sites for both substrates, NADPH and (S)-3-hydroxy-3-methylglutaryl coenzyme A. Antibodies prepared against purified reductase inactivated 100% of the soluble and at least 91% of the microsomal enzyme activity. Immunotitrations of solubilized enzyme obtained from normal and cholestyramine-fed rats indicated that cholestyramine feeding both increased the amount of enzyme protein and resulted in enzyme activation. Administration of increasing amounts of mevalonolactone to rats decreased the equivalence point obtained from immunotitration studies with solubilized enzyme. These data indicate that the antibody cross-reacts with the inactive enzyme formed after mevalonolactone treatment.