Immunochemical characterization of human lung epoxide hydrolases

Immunochemical techniques were used to investigate the biochemical properties of human lung epoxide hydrolases. Two epoxide hydrolases with different immunoreactive properties were identified. These two epoxide hydrolases were found in both cytosolic and microsomal cell fractions. Immunotitration of enzyme activity showed that enzymes that catalyze the hydration of benzo(a)pyrene 4,5-oxide react with antiserum to rat microsomal epoxide hydrolase; those that hydrate trans-stilbene oxide do not. Immunotitration and Western blot experiments showed that microsomal and cytosolic benzo(a)pyrene 4,5-oxide hydrolases have significant structural homology. Immunohistochemical staining of human lung benzo(a)pyrene 4,5-oxide hydrolase showed that the enzyme is localized primarily in the bronchial epithelium. No cell type-specific localization was observed. An enzyme-linked immunosorbent assay was developed which allows direct quantitation of benzo(a)pyrene 4,5-oxide hydrolase protein. Levels of enzyme protein detected by this assay correlated well with enzyme levels determined by substrate conversion assays.     

Interconversion between 17 beta-hydroxy-5alpha-androstan-3-one (5alpha-dihydrotestosterone) and 5alpha-androstane-3alpha, 17 beta-diol in rat kidney: heterogeneity of 3alpha-hydroxysteroid oxidoreductases.

3alpha-Hydroxysteroid oxidoreductases catalyzing the interconversion between 17 beta-hydroxy-5alpha-androstan-3-one (5alpha-dihydrotestosterone) and 5alpha-androstane-3alpha, 17 beta-diol (3alpha-androstanediol) have been studied in rat kidney. Three enzymes can be distinguished: a soluble NADPH-dependent oxidoreductase, a microsomal NADPH-dependent enzyme and a microsomal NADH-linked enzyme. Traces of the microsomal enzymes are consistently observed in the 108 000 X g supernatant. Studies on crude preparations reveal that these enzymes differ not only in subcellular localization and co-factor requirement, but also in optimum pH, kinetic characteristics, sensitivity to potential steroidal inhibitors and sensitivity to detergents, ionic strength and temperature. Moreover, salient sex differences exist in the activity of all three kidney enzymes. The soluble NADPH-dependent enzyme is more active in female rats whereas both microsomal enzymes are considerably more active in male animals. The microsomal NADH-dependent oxidoreductase displays favorable characteristics to catalyze the 3alpha-dehydrogenation of 3alpha-androstanediol. Evidence is presented that it is mainly this enzyme that enables the kidney to use 3alpha-androstanediol as an efficient precursor for the local formation of 5alpha-dihydrotestosterone.     

Enzymatic deoxyglucosylation of ceramides by microsomes of BHK-21 cells. The effect of deoxyglucose treatment and herpesvirus infection

The microsomal fractions of cultured hamster fibroblasts (BHK-21 cells) catalyze the incorporation of glucose from UDPglucose or of deoxyglucose from UDPdeoxyglucose into a reaction mixture with liposomes consisting of ceramide and phosphatidylcholine. The microsomal fractions also catalyze the transfer of glucose from UDPglucose to endogenous acceptors. The specific activity of ceramide deoxyglucoside or ceramide glucoside formation was significantly higher when microsomal preparations obtained from deoxyglucose-treated or herpesvirus-infected BHK-21 cells were used as the glucosyltransferase source. Deoxyglucose was incorporated from UDPdeoxyglucose into hydroxy- and nonhydroxy-fatty acid-containing ceramides at approximately the same rate. Competitive inhibition of deoxyglucosylation of ceramides by UDPglucose suggests that both reactions were catalyzed by the same enzyme, viz. UDPglucose:ceramide glucosyltransferase. This inhibition of glycosphingolipid synthesis may account, in part, for the inhibitory effect of deoxyglucose on lipid-containing viruses.     

Function and application of a non-ester-hydrolyzing carboxylesterase discovered in tulip

Plants have evolved secondary metabolite biosynthetic pathways of immense rich diversity. The genes encoding enzymes for secondary metabolite biosynthesis have evolved through gene duplication followed by neofunctionalization, thereby generating functional diversity. Emerging evidence demonstrates that some of those enzymes catalyze reactions entirely different from those usually catalyzed by other members of the same family; e.g. transacylation catalyzed by an enzyme similar to a hydrolytic enzyme. Tuliposide-converting enzyme (TCE), which we recently discovered from tulip, catalyzes the conversion of major defensive secondary metabolites, tuliposides, to antimicrobial tulipalins. The TCEs belong to the carboxylesterase family in the α/β-hydrolase fold superfamily, and specifically catalyze intramolecular transesterification, but not hydrolysis. This non-ester-hydrolyzing carboxylesterase is an example of an enzyme showing catalytic properties that are unpredictable from its primary structure. This review describes the biochemical and physiological aspects of tulipalin biogenesis, and the diverse functions of plant carboxylesterases in the α/β-hydrolase fold superfamily.     

Properties of partially purified saccharopine dehydrogenase from human placenta.

Saccharopine dehydrogenase was previously purified 380-fold from human placenta. The enzyme was shown to catalyze the formation of α-aminoadipic-δ-semialdehyde and glutamate from saccharopine, to have a molecular weight of 480,000 on gel filtration, and not to be separable from l-lysine-α-ketoglutarate reductase. Additional properties of the saccharopine dehydrogenase are now described. The pH optimum for the conversion of saccharopine to glutamate and α-aminoadipic-δ-semialdehyde is 8.5 in Tris-HCl buffer and 8.9 in 2-amino-2-methyl-1,3-propanediol buffer. The specificity of the enzyme for Saccharopine and NAD and the inhibition by glutamate and product analogs were tested. It was found the NADP was the only cofactor that could replace NAD in the enzyme reaction and that several NAD analogs were reaction inhibitors. Glutamate was found to be only moderately effective as an inhibitor. Initial velocity studies revealed that the enzyme has an ordered reaction mechanism. The true K_m values for saccharopine and NAD are 1.15 mm and 0.0645 mm, respectively.     

Intermolecular transglycosylating reaction of cyclodextrin glucanotransferase immobilized on capillary membrane

The intermolecular transglycosylating reaction of cyclodextrin glucanotransferase ([EC 2.4.1.19]; CGTase) immobilized on a capillary membrane was investigated using low molecular weight substrates such as cyclodextrin (CD), maltooligosaccharide (MOS), and a CD-MOS mixture. The immobilized CGTase catalyzed the conversion reaction of α-CD to β-CD and MOS or β-CD to α-CD and MOS within a short residence time. The conversion ratio increased as the amount of immobilized CGTase increased. The addition of glucose, maltose, and sucrose as acceptors in the substrate solution containing CD resulted in the acceleration of CD degradation compared with only CD substrate. Furthermore, the MOS substrate (degree of polymerization =2–6) was disproportionated with a conversion ratio exceeding 70% by the immobilized CGTase. These data demonstrate that immobilized CGTase can catalyze intermolecular transglycosylation between low molecular substrates in a few minutes by regulating the amount of immobilized enzyme and the residence time. This might contribute to our comprehension of CGTase-immobilized bioreactors for CD production as well as to the development of new glycosides through its excellent transglycosylation ability.     

Activation of the microsomal glutathione S-transferase by metabolites of alpha-methyldopa

Rat liver microsomes contain a membrane-bound GSH S-transferase (GSH-tr), an enzyme that is involved in the detoxication of xenobiotics. Also located on rat liver microsomes is the cytochrome P450 system, an enzyme complex that catalyzes the conversion of several xenobiotics into reactive intermediates. In this study, it was demonstrated that reactive products from alpha-methyldopa formed by the cytochrome P450 system are able to stimulate microsomal GSH-tr. Also, products formed from alpha-methyldopa that are generated by H2O2-horseradish peroxidase and tyrosinase are able to stimulate the activity of microsomal GSH-tr. GSH was able to prevent the activation of microsomal GSH-tr. Our results indicate that the ortho-quinone or semi-ortho-quinone radical of alpha-methyldopa is responsible for the stimulation of microsomal GSH-tr, probably via arylation of the free sulfhydryl group of microsomal GSH-tr. This conclusion was supported by the observation that 4-methyl-ortho-quinone itself was able to stimulate microsomal GSH-tr via sulfhydryl arylation. Our results are in conformity with the hypothesis that reactive products formed by the cytochrome P450 complex are able to stimulate microsomal GSH-tr and possibly in this way enhance their detoxication.     

A continuous automated assay of lipolysis during perifusion of isolated fat cells

Cysteine sulfinate transaminase (E.C. 2.6.1,l-cysteine sulfinate:2 oxoglutarate aminotransferase) catalyzes the conversion of cysteine sulfinate and α-ketoglutarate to 3-sulfonyl pyruvate and glutamate. A simple two-step assay has been developed to measure the enzyme activity in the high speed supernatant of whole brain homogenate. In the first step, the supernatant is incubated in the presence of exogenous substrate, then glutamate dehydrogenase is added to catalyze the conversion of glutamate to α-ketoglutarate, and the concomitant production of NADH is fluorimetrically monitored. The apparent K_m values of cysteine sulfinate transaminase for cysteine sulfinate and α-ketoglutarate are 1.24 and 0.22 mm, respectively. This assay is extremely rapid and has a high sensitivity, samples containing as low as 30 ng of protein may be accurately assayed.     

Is L-gulonolactone-oxidase the only enzyme missing in animals subject to scurvy?

Evidence has recently appeared implicating an unusual microsomal D-glucuronolactone reductase, which requires carbonyl reagents for activity, in the biosynthesis of ascorbic acid. It was also shown that this microsomal enzyme activity was missing in guinea pigs and primates suggesting that L-gulonolactone oxidase deficiency was not the only defect in animals subject to scurvy. However, we have shown that highly purified L-glulonolactone oxidase catalyzes the conversion of the oxime and semicarbazone of D-glucuronolactone to the corresponding ascorbic acid derivative. There is, therefore, no need to propose a second pathway to ascorbic acid, nor is there evidence for more than the one enzyme defect in scurvy-prone animals.     

Immobilization of liver microsomal cytochrome P-450

Rabbit liver microsomal cytochrome P-450 was immobilized by entrapment in calcium alginate gel. Aminopyrine demethylation experiments showed that the immobilized enzyme system is highly active and exhibits an unimpaired functional stability as compared with crude microsomes. The alginate entrapped microsomes were employed in a fixed bed recirculation reactor, where aminopyrine was continuously demethylated. Such model enzyme reactor can be a useful tool for studying extracorporeal drug detoxification or preparative substrate conversion with microsomal enzyme systems.     

Individual reaction requirements of two enzyme activities, isolated from Aspergillus parasiticus, which together catalyze conversion of sterigmatocystin to aflatoxin B1

Individual reaction requirements were determined for each of two enzyme activities present in Aspergillus parasiticus mycelia which together catalyze conversion of sterigmatocystin (ST) to aflatoxin B1 (AFB1). A postmicrosomal activity (PMA) catalyzed conversion of ST to O-methylsterigmatocystin (OMST) and a microsomal activity (MA) catalyzed conversion of OMST to AFB1. PMA was stimulated two- to three-fold in the presence of S-adenosylmethionine. Addition of NADPH promoted the maximum MA; this activity was not detected when FAD, FMN, NAD, or NADH were utilized individually as cofactors in reaction mixtures. A substantial amount (62%) of MA was lost during isolation of the microsomal fraction, but the activity was completely restored by reconstitution with a heat-treated (100 degrees C) postmicrosomal fraction. The reaction catalyzed by MA was optimum at pH 7.0 and at 17-23 degrees C, whereas the PMA reaction was optimum at pH 8.0-8.5 and at 35-40 degrees C. Apparent Km values of approximately 2.6 X 10(-6) M (for ST) and 6.6 X 10(-7) M (for OMST) were determined for PMA and MA, respectively.     

Solubilization and characterization of the leukotriene C4 synthetase of rat basophil leukemia cells: A novel,participate glutathione S-transferase

Rat basophil leukemia cell homogenates effectively catalyze the conversion of leukotriene A₄ to a mixture of leukotrienes C₄ and D₄ in the presence of glutathione. These homogenates also catalyze the formation of adducts of halogenated nitrobenzene with glutathione, as determined spectrophotometrically. While all the classical glutathione S-transferase activity resides in the soluble fraction of the homogenates, the thiol ether leukotriene-generating activity is found in the particulate fraction. This “leukotriene C synthetase” activity has been solubilized from a crude high-speed particulate fraction by means of the nonionic detergent, Triton X-100. The solubilized enzyme is incapable of converting 2,4-dinitrochlorobenzene to a colored product in the presence of glutathione. Nor will it react with 3,4-dichloronitrobenzene. On the other hand, under optimal conditions, this enzyme preparation is capable of generating about 0.1 nmol leukotriene C mg protein^?1 min^?1 in a reaction which continues in linear fashion for at least 10 min. This dissociation in substrate specificity, as well as differences in the inhibition profile, distinguish the enzyme activity in the particulate fraction from rat basophil leukemia cell homogenates from the microsomal glutathione S-transferase which has been described in rat liver homogenates, suggesting that this “leukotriene C synthetase” is a new and unique enzyme.     

Source of the hepatic microsomal trans-2-enoyl CoA hydratase bifunctional protein: endoplasmic reticulum or peroxisomes

The present study was designed to investigate the hepatic localization of the microsomal bifunctional trans-2-enoyl CoA hydratase. Despite the low activity (less than 10%) of peroxisomal marker enzymes in isolated hepatic microsomes (acyl CoA oxidase (this study), catalase, and urate oxidase (L. Cook, M. N. Nagi, J. Piscatelli, T. Joseph, M. R. Prasad, D. Ghesquier, and D. L. Cinti, 1986, Arch. Biochem. Biophys. 245, 24-26), additional evidence in this study suggests that the microsomal enzyme is derived from peroxisomes. For example, the microsomal hydratase activity was associated with the ribosomal fractions but not with the smooth endoplasmic reticulum. In addition, when an extract of the peroxisomal enzyme was incubated with either free ribosomes or membrane-bound ribosomes, marked binding was observed with each of the fractions. Furthermore, the ease of release of the bifunctional enzyme from both free ribosomes and membrane-bound ribosomes by only KCl suggests that the bound enzyme is not a nascent protein. Labeling of liver tissue from DEHP-treated rats with rabbit immune IgG made to the purified microsomal hydratase followed by gold conjugated goat anti-rabbit IgG suggested a single subcellular site for the bifunctional hydratase--the peroxisomal organelle.     

Studies on Tetrahymena membranes. Palmitoyl-coenzyme a desaturase, a possible key enzyme for temperature adaptation in Tetrahymena microsomes.

(1) Microsomes from a thermotolerant Tetrahymena NT-1 catalyze the conversion of palmitoyl-CoA to palmitoleate. (2) Palmitoyl-CoA desaturase enzyme requires molecular oxygen and NADH or NADPH as cofactor and its activity is inhibited by cyanide. A pH optimum range 7.0--7.3 is observed. (3) There is a clear break at 30 degrees C and a slight bend around 15 degrees C in the Arrhenius plots of palmitoyl-CoA desaturase activity. (4) After quenching from 39.5 degrees C, at 26 degrees C microsomal membranes show small particle-free areas, when examined by freeze-fracture electron microscopy, indicating the onset of phase separation. Larger smooth areas devoid of membrane-intercalated particles are observed in microsomes at 23 and 15 degrees C. The results support evidence that the thermally induced transition of desaturase enzyme activity in related to the altered membrane properties due to temperature change.     

Sterol carrier protein hypothesis: requirement for three substrate-specific soluble proteins in liver cholesterol biosynthesis.

The 105,000 x g supernatant (S₁₀₅) of liver is required for the conversion of squalene to cholesterol by microsomal membranes. Substantial controversy has existed concerning the properties of what was originally considered to be a single sterol carrier protein present in S₁₀₅ and required for this conversion. We have now resolved this controversy by the discovery that S₁₀₅ contains several sterol carrier proteins. Based upon experiments with three substrates, three substrate-specific soluble proteins (with different properties) have been identified which operate at distinct points in microsomal cholesterol synthesis. These proteins are provisionally designated sterol carrier protein₁ (SCP₁), sterol carrier protein₂ (SCP₂), and sterol carrier protein₃ (SCP₃). SCP₁ is required for the microsomal conversion of squalene to lanosterol, SCP₂ for the microsomal conversion of 4,4-dimethyl-Δ⁸-cholesterol to C₂₇-sterols, and SCP₃ for the microsomal conversion of 7-dehydrocholesterol to cholesterol. Available evidence is consistent with the proposal that a given sterol carrier protein is a soluble constituent of a single microsomal enzyme or enzyme complex, and that it participates both as a carrier for the water-insoluble substrate and as an essential enzyme constituent facilitating catalysis. It may well be that enzymatic transformations of water-insoluble substrates require both microsomal membranes and substrate-specific soluble proteins. This requirement could be a common biological mechanism for water-insoluble substrates.     

Catalytic promiscuity of a bacterial α-N-methyltransferase

The posttranslational methylation of N-terminal α-amino groups (α-N-methylation) is a ubiquitous reaction found in all domains of life. Although this modification usually occurs on protein substrates, recent studies have shown that it also takes place on ribosomally synthesized natural products. Here we report an investigation of the bacterial α-N-methyltransferase CypM involved in the biosynthesis of the peptide antibiotic cypemycin. We demonstrate that CypM has low substrate selectivity and methylates a variety of oligopeptides, cyclic peptides such as nisin and haloduracin, and the ε-amino group of lysine. Hence it may have potential for enzyme engineering and combinatorial biosynthesis. Bayesian phylogenetic inference of bacterial α-N-methyltransferases suggests that they have not evolved as a specific group based on the chemical transformations they catalyze, but that they have been acquired from various other methyltransferase classes during evolution.     

Cytochrome P-450 C21scc: One enzyme with two actions: Hydroxylase and lyase

Testis, adrenal, ovary and placenta contain a microsomal cytochrome P-450 that is capable of converting progesterone to androstenedione and pregnenolone to dehydroepi-androsterone. This conversion requires 17-hydroxylation followed by C_17,20-lyase activity which are both catalyzed by this one protein. Gene cloning and Northern blotting reveal that, at least in man, the same gene is responsible for both testicular and adrenal enzymes. The enzyme was first purified from neonatal pig testis. Both the testicular and adrenal enzymes show a marked preference for the 5-ene substrate (pregnenolone) in keeping with the extensive use of the 5-ene pathway in that species. Affinity alkylation with 17-bromoacetoxyprogesterone reveals a conserved cysteine at the active site of the enzyme and confirms the conclusion that a single enzyme catalyzes both reactions. Under some circumstances the enzyme catalyzes only 17-hydroxylation to permit the formation of the C₂₁ steroid cortisol. The regulation of lyase activity, i.e. the determination of the extent to which the second activity is expressed, results from the availability of P-450 reductase. No doubt the greater concentration of this protein in testicular as opposed to adrenal microsomes (× 3.5) is responsible for the production of androgens in the testis and cortisol in the adrenal. Testicular cytochrome b₅ also specifically stimulates lyase activity and also causes the porcine enzyme to catalyze a new reaction, i.e. Δ¹⁶-synthetase, resulting in synthesis of the important pheromone androsta-4,16-dien-3 one from progesterone.     

The in vitro biosynthesis of dhurrin, the cyanogenic glycoside of Sorghum bicolor.

A microsomal fraction from seedlings of Sorghum bicolor (Linn) Moench has been shown to catalyze the conversion of L-tyrosine to p-hydroxymandelonitrile via p-hydroxyphenylacetaldoxime. This transformation is consistent with the general pathway for cyanogenic glycoside biosynthesis proposed on the basis of in vivo experiments. When the microsomal fraction was combined with a protein fraction from the soluble portion of the cell and uridine diphosphate glucose, it was possible to demonstrate the synthesis of the cyanogenic glycoside dhurrin from L-tyrosine.     

Characterization of phytol-phytanate conversion activity in rat liver

The enzymatic conversion of phytol to phytanic acid was investigated in rat liver postnuclear and other subcellular fractions using [1-3H]phytol as the substrate. The assay method involved incubation of the substrate with appropriate cofactors and the enzyme source, followed by subjecting the mixture to Folch partition and measuring the radioactivity in the upper layer. The phytol-phytanate conversion activity was present in mitochondrial and microsomal fractions. Cytosol had no activity. In mitochondrial fraction, investigation of cofactor requirements indicated that only NAD was required for activity. Other pyridine nucleotides supported the activity to a lesser extent when compared with NAD. FAD at 1 mM concentration did not support the activity. Bovine serum albumin (0.4 mg/ml) stimulated the activity. The reaction did not require molecular oxygen. From substrate kinetic studies, an apparent Km of 14.3 and 11.1 microM was calculated for phytol in mitochondrial and microsomal fractions, respectively. The amount of tritiated water produced from incubation increased linearly up to 7-8 min. The activity was linear with the amount of mitochondrial and microsomal protein up to 200 and 40 micrograms, respectively. Among the various rat tissue homogenates tested, liver had the highest activity. Spleen and kidney had 8-9% of the activity of liver. Brain possessed negligible activity. Both ethanol and pyrazole had no inhibitory effect on phytol-phytanate conversion. This observation and the absence of activity in cytosol suggests that alcohol dehydrogenase may not be involved in phytol-phytanate conversion.     

The conformation and thermal stability of soluble cytochrome P-450 and cytochrome P-450 incorporated into liposomal membranes

The α-helix content of the cytochrome P-450 incorporated into the liposomal membranes of either phosphatidylcholine or microsomal phospholipid insignificantly differed from that of the soluble one. The binding of both type I and type II substrates with cytochrome P-450 incorporated into phosphatidylcholine and microsomal phospholipid membranes did not change the conformation of the polypeptide chain. In contrast to this the type II substrates increased the α-helix content of soluble hemoprotein about 3–5%. Dithionite reduction of the cytochrome P-450 haem increased the degree of α spiralization up to 10% for soluble hemoprotein and up to 5% for the membrane-bound enzyme only. The investigation of the thermal stability of the soluble and liposomal forms of cytochrome P-450 showed that the enzyme incorporated into phospholipid vesicles was highly stable. The heating of the enzyme was followed by a slightly pronounced cooperative transition in contrast to the well-pronounced transition for the soluble cytochrome P-450. Hence, the incorporation of the soluble cytochrome P-450 into phospholipid bilayer does not result in significant change of α-helix content, but the increasing of rigidity and thermal stability of the membrane-bound hemoprotein molecule is observed.