Purification and characterization of a novel pyrazole-sensitive carbonyl reductase in guinea pig lung

Mouse Ehrlich ascites tumor cells incubated with the creatine analog, N-ethylguanidinoacetate (N-amidino-N-ethylglycine), accumulated up to 8 μmol/g packed cells of the creatine-P analog, N-ethylguanidinoacetate-P. Evaluation of N-ethylguanidinoacetate-P as a synthetic phosphagen under in vivo conditions was performed with Ehrlich cells loaded with equimolar amounts of a common reference phosphagen, cyclocreatine-P (1-carboxymethyl-2-imino-3-phosphonoimidazolidine) plus either N-ethylguanidinoacetate-P or creatine-P. It was concluded that N-ethylguanidinoacetate-P has a Gibbs free energy of hydrolysis equal to that of creatine-P and 2 kcal/mol greater than that of cyclocreatine-P. The relative rates of utilization of intracellular phosphagens by Ehrlich cells when their ATP pools were depleted with 2-deoxyglucose were in the order: creatine-P > N-ethylguanidinoacetate-P > cyclocreatine-P. Dietary N-ethylguanidinoacetate was nontoxic even at very high levels to all animal systems tested. Feeding of 2% N-ethylguanidinoacetate to mice or chicks resulted in equimolar replacement of natural by synthetic phosphagen to the following extents: heart, 75%; leg muscle, 50%; and brain 10–25%. N-Ethylguanidinoacetate-P is the most active synthetic phosphagen thus far found to be accumulated by animal tissues.     

Purification and characterization of two forms of microsomal carbonyl reductase in guinea pig liver.

Glycosaminoglycan oligosaccharides generated by treatment of biosynthetically radiolabelled dermatan sulphate and hyaluronic acid with chondroitin AC lyase or testicular hyaluronidase may be resolved into a series of discrete bands by polyacrylamide-gel electrophoresis. Bands were identified by fixation in glacial acetic acid containing 20% (w/v) 2,5-diphenyloxazole followed by fluorography. The bands represented glycans which differed in size by one disaccharide unit. For the larger oligosaccharides (decasaccharides and above) of similar charge: mass ratio, there was a linear relationship between electrophoretic mobility and log Mr. However, the smaller species showed anomalous migration patterns. Consideration of the structures of the fragments produced by the different enzyme treatments suggests that copolymeric and homopolymeric oligosaccharides may be separated by polyacrylamide-gel electrophoresis. There are many potential applications of this technique, foremost amongst them being studies on the molecular size heterogeneity and patterns of enzyme-mediated depolymerization of native glycosaminoglycan chains and investigations into rates of polymer chain elongation and post-polymerization modification reactions so essential to glycosaminoglycan function.     

Activation of carbonyl reductase from pig lung by fatty acids.

The NADPH-linked reductase activity of pig lung carbonyl reductase was activated two- to fivefold by fatty acids with a carbon chain length greater than nine at pH 7.0. cis-Unsaturated fatty acids of C:18 and C:20 were potent activators, showing Ka values of 2-14 microM which were lower than the values of 21-125 microM for saturated fatty acids (C:9 to C:16). Of the fatty acids arachidonic acid (C20:4) gave the highest activation. No significant stimulatory effect was observed with acyl CoAs, fatty alcohols, phospholipids, and nonionic detergents. Anionic detergents (sodium dodecyl sulfate and sarkosyl) stimulated the enzyme activity more than ninefold, but the Ka values for them were much higher than those for the cis-unsaturated fatty acids. Although no change in molecular weight or in subunit composition was observed in the enzyme activated by C20:4, the activation led to a decrease in thermal stability of the enzyme. The binding of C20:4 to the enzyme was instantaneous and reversible, shifted the pH optimum of the activity from 5.8 to 6.5, and changed the inhibitor sensitivity. In addition, C20:4 acted as an allosteric effector abolishing the negative interaction of the enzyme with carbonyl substrates which was seen without the fatty acid, but the activation increased both Vmax and [S]0.5 values for the substrates. Kinetic analysis with respect to NADPH concentration, in which no cooperativity was detected with or without C20:4, indicated that C20:4 was a nonessential activator of mixed type showing a binding constant of 10 microM. These results suggest that cis-unsaturated fatty acids may be potential modulators of pulmonary carbonyl reductase.     

Subcellular distribution and properties of carbonyl reductase in guinea pig lung

On subcellular fractionation, carbonyl reductase (EC 1.1.1.184) activity in guinea pig lung was found in the mitochondrial, microsomal, and cytosolic fractions; the specific activity in the mitochondrial fraction was more than five times higher than those in the microsomal and cytosolic fractions. Further separation of the mitochondrial fraction on a sucrose gradient revealed that about half of the reductase activity is localized in mitochondria and one-third in a peroxidase-rich fraction. Although carbonyl reductase in both the mitochondrial and microsomal fractions was solubilized effectively by mixing with 1% Triton X-100 and 1 M KCl, the enzyme activity in the mitochondrial fraction was more highly enhanced by the solubilization than was that in the microsomal fraction. Carbonyl reductases were purified to homogeneity from the mitochondrial, microsomal, and cytosolic fractions. The three enzymes were almost identical in catalytic, structural, and immunological properties. Carbonyl reductase, synthesized in a rabbit reticulocyte lysate cell-free system, was apparently the same in molecular size as the subunit of the mature enzyme purified from cytosol. These results indicate that the same enzyme species is localized in the three different subcellular compartments of lung.     

Purification and characterization of carbonyl reductase from Geotrichum candidum

Geotrichum candidum is well known for the reduction of prochiral ketones to chiral alcohol with high yield and excellent enantioselectivity. Carbonyl reductase from G. candidum was purified by ammonium sulphate precipitation, anion exchange and hydrophobic interaction chromatographies. Gel filtration chromatography together with SDS-PAGE revealed this protein to be a dimer of 60 kDa subunits. Maximum enzyme activity was found in acetate buffer at pH 5.4 with t_1/2 of 7.13 h at 30 °C and t_1/2 of 2.8 h at 65 °C. The enzyme was inhibited by p-hydroxymercuribenzoate and hydroxylamine indicating the involvement of thiol and carbonyl groups in the reduction reaction catalyzed by the enzyme. Chelating agents also reduced the enzyme activity indicating the requirement of metal ions as cofactors. The purified carbonyl reductase was found to be highly selective for ketones containing naphthyl ring, whereas aryl or hetero-aryl ketones showed very less or no activity at all.     

Purification and characterization of zeta-crystallin/quinone reductase from guinea pig liver.

zeta-Crystallin, a major lens protein of certain mammalian species, has recently been characterized as a novel and active NADPH:quinone oxidoreductase. Here we report the purification of this protein from guinea pig liver by utilizing sequentially: ammonium sulphate precipitation, Blue Sepharose affinity, cation exchange and hydrophobic chromatography steps. This four-step isolation procedure yielded 118-fold purification and a specific activity of 6 U/mg protein when assayed in the presence of 9,10-phenanthrenequinone. Kinetic, immunological and physical properties of this protein have been found to be identical with those of guinea pig lens zeta-crystallin. Western blot analysis using antibodies raised against zeta-crystallin peptides demonstrated the presence of substantial amounts of this protein in human liver homogenates.     

Purification and properties of prostaglandin 9-ketoreductase from pig and human kidney. Identity with human carbonyl reductase.

Prostaglandin 9-ketoreductase (PG-9-KR) was purified from pig kidney to homogeneity, as judged by SDS/PAGE using an improved procedure. The enzyme is pro-S stereoselective with regard to hydrogen transfer from NADPH with prostaglandin E2 as substrate and reduces its 9-keto group with approximately 90% stereoselectivity to form prostaglandin F2 alpha. Approximately 8% of the prostaglandin F formed has the beta-configuration. In addition to catalyzing the interconversion of prostaglandin E2 to F2 alpha, PG-9-KR also oxidizes prostaglandin E2, F2 alpha and D2 to their corresponding, biologically inactive, 15-keto metabolites. Incubation of PG-9-KR with prostaglandin F2 alpha and NAD+ leads to the preferential formation of 15-keto prostaglandin F2 alpha rather than prostaglandin E2. This suggests that the prostaglandin E2/prostaglandin F2 alpha ratio is not determined by the NADP+/NADPH redox couple. The enzyme also reduces various other carbonyl compounds (e.g. 9,10-phenanthrenequinone) with high efficiency. The catalytic properties measured for PG-9-KR suggest that its in vivo function is unlikely to be to catalyze formation of prostaglandin F2 alpha. The monomeric enzyme has a molecular mass of 32 kDa and exists as four isoforms, as judged by isoelectric focusing. PG-9-KR contains 1.9 mol Zn2+/mol enzyme and no other cofactors. Human kidney PG-9-KR was also purified to homogeneity. The human enzyme has a molecular mass of 34 kDa and also exists as four isoforms. Polyclonal antibodies raised against pig kidney PG-9-KR cross-react with human kidney PG-9-KR and also with human brain carbonyl reductase, as demonstrated by Western blot analysis. Sequence data of tryptic peptides from pig kidney PG-9-KR show greater than 90% identity with human placenta carbonyl reductase. From comparison of several properties (catalytical, structural and immunological properties), it is concluded that PG-9-KR and carbonyl reductase are identical enzymes.     

Purification and characterization of 2-alkenal reductase.

The purification of a 2-alkenal reductase to homogeneity from a rat liver 100 000 times g supernatant is described. Its molecular weight has been determined by Sephadex G-100 chromatography and sodium dodecylsulfate polyacrylamide gel electrophoresis before and after reduction with mercaptoethanol and carboxymethylation. The monometric form has a molecular weight of 45 000. It tends to form, to a very small extent, dimeric and trimeric aggregates of molecular weights 90 000 and 135 000. The isoelectric point (IP) was determined to be 6.2 by isoelectric focusing.     

Purification and characterization of an enantioselective carbonyl reductase from Candida viswanathii MTCC 5158

A highly enantioselective carbonyl reductase produced by a new yeast strain Candida viswanathii MTCC 5158, which was isolated using an acetophenone enriched medium, has been purified and characterized. The enzyme has been purified to near homogeneity using ammonium sulfate precipitation, ion exchange and gel filtration chromatography. The molecular properties of the carbonyl reductase suggested the native enzyme to be tetrameric, with an apparent molecular weight of 120 kDa, the monomer being about 29 kDa. Acetyl aryl ketones were found to be the preferred substrates for the enzyme and the best reaction was the enantioselective reduction of acetophenone. The enzyme yielded (S)-alcohol in preference to (R)-alcohol and utilized NADH, but not NADPH as the cofactor. The purified enzyme exhibited maximum enzyme activity at pH 7.0 and 60 °C. The enzyme retained about 80% of its activity after 7 h incubation at 25 °C in sodium phosphate buffer (50 mM, pH 7.0). The addition of reducing agents like dithiothreitol and β-mercaptoethanol enhanced the enzyme activity while organic solvents, detergents and chaotropic agents had deleterious effect on enzyme activity. Metal chelating agents like hydroxyquinoline and o-phenanthroline have significant effect on enzyme activity suggesting that the carbonyl reductase required the presence of a tightly bound metal ion for activity or stability. The maximum reaction rate (V_max) and apparent Michaelis–Menten constant (K_m) for acetophenone and NADH were 59.21 μmol/(min mg) protein and 0.153 mM and 82.64 μmol/(min mg) protein and 0.157 mM at a concentration range of 0.2–2 mM acetophenone (NADH fixed at 0.5 mM) and 0.1–0.5 mM NADH (acetophenone fixed at 2 mM), respectively.     

Characterization of pulmonary carbonyl reductase of mouse and guinea pig

Carbonyl reductases were purified from mouse and guinea pig lung. The mouse enzyme exhibited structural and catalytic similarity to the guinea pig enzyme: tetrameric structure consisting of an identical 23 kDa subunit; basicity (pI of 8.8); low substrate specificity for aliphatic and aromatic carbonyl compounds; dual cofactor specificity for NADPH and NADH; stereospecific transfer of the 4-pro S hydrogen of NADPH; and sensitivity to pyrazole, 2-mercaptoethanol and ferrous ion. Although 3-ketosteroids were extensively reduced by the mouse enzyme but not by the guinea pig enzyme in the forward reaction, the two enzymes similarly oxidized some alicyclic alcohols such as acenaphthenol, cyclohex-2-en-1-ol and benzenedihydrodiol in the presence of NADP+ and NAD+. A partial similarity between the two enzymes was observed immunologically, using antibodies against the purified guinea pig enzyme. The lung enzymes differ in several aspects from other oxidoreductases from extrapulmonary tissues. The immunoreactive protein was detected only in lung of the tissues of the two species.     

Purification and characterization of an enzymically active cleavage product of pig kidney aldehyde reductase

During the purification of pig kidney aldehyde reductase by an established procedure [Flynn, Cromlish & Davidson (1982) Methods Enzymol. 89, 501-506] a second enzyme with aldehyde reductase activity may be purified. When the procedure was performed in the presence of 5 mM-EDTA, only traces of the second reductase, pig kidney aldehyde reductase (minor form), were present. By the criterion of sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, pig kidney aldehyde reductase (minor form) had Mr 35 000, in comparison with Mr 40 200 found for pig kidney aldehyde reductase. Amino acid analysis of both enzymes and tryptic-peptide-map comparisons indicated differences in primary structure. The N-terminus of pig kidney aldehyde reductase (minor form) had the sequence Lys-Val-Leu, in contrast with the blocked (acetylated) N-terminus of pig kidney aldehyde reductase. The C-terminal sequence of both enzymes was the same. Both reductases were immunologically identical by double immunodiffusion and rocket immunoelectrophoresis. Pig kidney aldehyde reductase (minor form) had 50% of the specific activity of pig kidney aldehyde reductase when tested with a variety of aldehyde substrates. Michaelis constants of both enzymes for these substrates and for NADPH were similar, but values for kcat. and kcat./Km indicated that catalytically pig kidney aldehyde reductase was the more efficient enzyme. Typical aldehyde reductase inhibitors, such as phenobarbital and sodium valproate, had the same effect on both enzymes. It was concluded that pig kidney aldehyde reductase (minor form) is an enzymically active cleavage product of pig kidney aldehyde reductase which is formed when the latter is purified in the absence of the metalloproteinase inhibitor EDTA.     

Purification and characterization of beef liver dihydrofolate reductase.

Beef liver dihydrofolate reductase has been purified to homogeneity by using a methotrexate affinity column followed by gel filtration to remove several higher molecular weight proteins. Tightly bound dihydrofolate is removed by hydroxylapatite chromatography. The overall purification is 13,000-fold; the specific activity is 26 units·mg^?1, approximately 25 times higher than previously reported. The enzyme has been shown to be homogeneous by the following criteria: (i) discontinuous gel electrophoresis, (ii) sodium dodecyl sulfate-gel electrophoresis, (iii) velocity sedimentation, (iv) equilibrium sedimentation, and (v) methotrexate titration. The amino acid composition has been determined. Notable features include a single cysteine, three tryptophan and three histidine residues. The N-terminal amino acid is leucine. The molecular weight determined by equilibrium sedimentation is 22,500. The s_20,w⁰ is 2.08 × 10^?13 S and D_20,w⁰ = 10.93 cm²·s^?1. A frictional coefficient of 1.04 indicates that the enzyme is essentially spherical. An isoelectrical point of 6.80 was measured.     

Purification and characterization of aldose reductase and aldehyde reductase from human kidney.

Aldose reductase and aldehyde reductases have been purified to homogeneity from human kidney and have molecular weights of 32,000 and 40,000 and isoelectric pH 5.8 and 5.3, respectively. Aldose reductase, beside catalyzing the reduction of various aldehydes, reduces aldo-sugars, whereas aldehyde reductase, does not reduce aldo-sugars. Aldose reductase activity is expressed with either NADH or NADPH as cofactor, whereas aldehyde reductase utilizes only NADPH. Both enzymes are inhibited to varying degrees by aldose reductase inhibitors. Antibodies against bovine lens aldose reductase precipitated aldose reductase but not aldehyde reductase. The sequence of addition of the substrates to aldehyde reductase is ordered and to aldose reductase is random, whereas for both the enzymes the release of product is ordered with NADP released last.     

Ultrastructural localization of carbonyl reductase in mouse lung

Summary The immunocytochemical localization of tetrameric carbonyl reductase in the mouse lung was determined by an electron-microscopical immunogold procedure using monospecific antibodies against the enzyme. The labelling of carbonyl reductase was observed within the mitochondria of the ciliated and non-ciliated cells of the bronchioles and the type II alveolar pneumocytes, and the density of labelling in the non-ciliated cells was higher than those in the other cells. No significant labelling was detected over other compartments of the epithelial cells. The labelling was undetectable in the type I alveolar cells, alveolar macrophages and connective tissue cells of the lung. These results clearly indicate the localization of carbonyl reductase to the mitochondrial matrix of these epithelial cells, of which the non-ciliated bronchiolar cells contained particularly high amounts of the enzyme.     

A novel fungal enzyme, NADPH-dependent carbonyl reductase, showing high specificity to conjugated polyketones. Purification and characterization

A novel enzyme which specifically catalyzes the reduction of conjugated polyketones was purified to homogeneity from cells of Mucor ambiguus AKU 3006. The enzyme has a strict requirement for NADPH and irreversibly reduces a number of quinones such as p-benzoquinone, alpha-naphthoquinone and acenaphthenequione. The enzyme also reduces polyketones such as isatin and ketopantoyl lactone, and their derivatives. The apparent Km values for isatin and ketopantoyl lactone are 49.9 microM and 714 microM, respectively. The reduction of ketopantoyl lactone proceeds stereospecifically to yield L-(+)-pantoyl lactone. The pro-S (A) hydrogen at C-4 of NADPH is transferred to the substrate. The enzyme is not a flavoprotein and consists of two polypeptide chains with an identical relative molecular mass of 27,500. Quercetin, dicoumarol and some SH reagents inhibit the enzyme activity. 3-Methyl-1,2-cyclopentanedione and 1,3-cyclohexanedione are uncompetitive inhibitors with Ki values of 80.9 microM and 64.5 microM, respectively, to ketopantoyl lactone.     

Human lung tryptase. Purification and characterization

Human lung tryptase, a mast cell-derived trypsin-like serine protease, has been isolated from whole human lung tissue obtained at autopsy. Increased yields from this purification process have allowed extensive characterization of the enzyme. One of the critical steps in the purification scheme is the use of a linear heparin gradient to elute active material from cellulose phosphate. Gel filtration studies in 1.0 M NaCl yielded an apparent Mr = 135,000, and subsequent electrophoresis on sodium dodecyl sulfate-polyacrylamide gels demonstrated the presence of two active species with apparent Mr = 30,900 and 31,600. Enzymatic activity was sensitive to NaCl concentrations above 0.05 M and was only 50% in 0.15 M NaCl, decreasing to 18% in 0.6 M NaCl. The effects of synthetic and natural inhibitors have also been studied, confirming the enzyme's trypsin-like characteristics and demonstrating that naturally occurring serum inhibitors are incapable of diminishing its activity. A complete amino acid analysis showed a high tryptophan content. Lastly, antisera to human lung tryptase have been generated, and the immunological identity of active fractions has been investigated as well as the localization of the enzyme to the mast cell granule by immunohistochemical staining.     

Purification and characterization of glutathione reductase from Rhodospirillum rubrum.

Glutathione reductase (NAD(P)H:GSSG oxidoreductase EC 1.6.4.2.) was purified 1160-fold to homogeneity from the nonsulfurous purple bacteria Rhodospirillum rubrum (wild type). Specific activity of the pure preparation was 102 U/mg. The enzyme displayed a typical flavoprotein absorption spectrum with maxima at 274,365, and 459 nm and an absorbance ratio A280/A459 of 7.6. The amino acid analysis revealed an unusually high content of glycine and arginine residues. Titration of the enzyme with 5,5'-dithiobis(2-nitrobenzoic acid) showed a total of two free thiol groups per subunit, one of which is made accessible only under denaturing conditions. An isoelectric point of 5.2 was found for the native enzyme. Km values, determined at pH 7.5, were 6.1 and 90 microM for NADPH and GSSG, respectively. NADH was about 2% as active as NADPH as an electron donor. The enzyme's second choice in disulfide substrate was the mixed disulfide of coenzyme A and glutathione, for which the specific activity and Km values were 5.1 U/mg and 3.4 mM, respectively. A native molecular weight of 118,000 was found, while denaturing electrophoresis gave a value of 54,400 per subunit, thus suggesting that R. rubrum glutathione reductase exists as a dimeric protein. Other physicochemical constants of the enzyme, such as Stokes radius (4.2 nm) and sedimentation coefficient (5.71 S), were also consistent with a particle of 110,000.     

Purification and characterization of dihydrofolate reductase from Mycobacterium phlei.

The dihydrofolate reductase from Mycobacterium phlei was purified and characterized; it has an Mr of 15 000 and a pI of 4.8. It is competitively inhibited by both methotrexate and trimethoprim, although the affinity is less than for other bacterial dihydrofolate reductases.