A novel, inducible, citral lyase purified from spores of Penicillium digitatum.

A novel lyase, combining hydratase and aldolase activity, that converts citral into methylheptenone and acetaldehyde, was purified from spores of Penicillium digitatum. Remarkably, citral lyase activity was induced 118-fold by incubating nongerminating spores with the substrate, citral. This cofactor independent hydratase/aldolase, was purified and found to be a monomeric enzyme of 31 kDa. Citral lyase has a Km of 0.058 mm and a Vmax of 52.6 U.mg-1. Enzyme activity was optimal at 20 degrees C and pH 7.6. The enzyme has a strong preference for the trans isomer of citral (geranial). Citral lyase also converts other alpha,beta-unsaturated aldehydes (farnesal, methyl-crotonaldehyde, decenal and cinnemaldehyde).     

Cytochrome P450 isozymes catalyzing 4-hydroxylation of parkinsonism-related compound 1,2,3,4-tetrahydroisoquinoline in rat liver microsomes.

Microsomal 4-hydroxylase of 1,2,3,4-tetrahydroisoquinoline (TIQ), a possible candidate for causing Parkinson disease, was characterized by using rat hepatic microsomes and purified P450 isozymes. Kinetic analysis revealed that Km and Vmax values (mean +/- SE) for hepatic microsomal TIQ 4-hydroxylase of male Wistar rats were 319.6 +/- 26.8 microM and 12.13 +/- 1.43 pmol.min-1.mg-1 protein, respectively. When TIQ 4-hydroxylase activity was compared in Wistar (an animal model of extensive debrisoquine metabolizers) and Dark Agouti (an animal model of poor debrisoquine metabolizers) rats, significant strain (Wistar greater than Dark Agouti) and sex (male greater than female) differences were observed. The microsomal activity toward TIQ 4-hydroxylation was increased by pretreatment of male Wistar rats with P448 inducers (beta-naphthoflavone and sudan I), but not with phenobarbital. Pretreatment with propranolol, an inhibitor of P450 isozymes belonging to the P450 IID gene subfamily, decreased TIQ 4-hydroxylase activity. P450 BTL, a P450 isozyme belonging to the IID subfamily, showed TIQ 4-hydroxylase activity of 64.1 pmol.min-1.nmol P450(-1), which was 3.2-fold that of microsomes (20.9 pmol.min-1.nmol P450(-1)). Antibody (IgG) against this isozyme suppressed microsomal TIQ 4-hydroxylase activity concentration-dependently. A male-specific P450 ml (P450IIC11) catalyzed this reaction to a much lesser extent (10.0 pmol.min-1.nmol P450(-1)), and its antibody did not affect the microsomal activity. These results suggest that TIQ 4-hydroxylation in hepatic microsomes are catalyzed predominantly by a P450 isozyme (or isozymes) belonging to the IID gene subfamily in non-treated rats and its immunochemically related P450 isozyme (or isozymes), and that a P450 isozyme (or isozymes) belonging to the IA subfamily also participates in TIQ 4-hydroxylation in rats pretreated with P448-inducers.     

Cytotoxic ether phospholipids. Different affinities to lysophosphocholine acyltransferases in sensitive and resistant cells

Alkyllysophospholipids (ALP) which are 1-O-alkyl analogs of the cell membrane component 1-acyl-sn-glycero-3-phosphocholine (1-acyl-GPC) represent a family of new antitumor drugs. Susceptibility of cells to ALP is correlated to a selective inhibition of fatty acid incorporation into 1,2-diacyl-sn-glycero-3-phosphocholine in intact cells. This report examines oleoyl-CoA-1-acyl-GPC acyl-transferase activities in cell-free systems of ALP-sensitive methylcholanthrene-induced fibrosarcoma cells (MethA cells) and ALP-resistant bone marrow-derived murine macrophages (BMM phi). The specific activities for the oleoyl-CoA-1-acyl-GPC acyltransferases were 1.05 +/- 0.06 nmol X mg-1 X min-1 and 2.98 +/- 0.27 nmol X mg-1 X min-1, respectively. The kinetic parameters for 1-palmitoyl-GPC were Km = 16.6 microM, Vmax = 4.3 nmol X mg-1 X min-1 (BMM phi) and Km = 7.6 microM, Vmax = 2.0 nmol X mg-1 X min-1 (MethA cells). In the presence of 1-O-octadecyl-2-O-methyl racemic glycero-3-phosphocholine (ET-18-OCH3), one of the most potent cytotoxic ALP, the acyltransferase was dose dependently inhibited in MethA cells with a 50% inhibition concentration at 40 micrograms/ml. The BMM phi-acyltransferase was not affected up to 80 micrograms of ET-18-OCH3/ml. The kinetic parameters (Km' = 15.4 microM, Vmax' = 2.2 nmol X mg-1 X min-1) suggest that ET-18-OCH3 is a competitive inhibitor in MethA cells. Inhibitor constants for ET-18-OCH3, calculated from Dixon plots, were found to be 423 microM (BMM phi) and 13 microM (MethA cells) indicating a 33-fold larger affinity of ET-18-OCH3 to the MethA cells than to the BMM phi acyltransferase. From these data we assume that the inhibition of oleic acid incorporation into cellular phosphocholine during the antineoplastic action of ALP may be due to different affinities of the inhibitor to the 1-acyl-GPC acyltransferases in different cell types.     

Human T lymphocyte cAMP-dependent protein kinase: subcellular distributions and activity ranges of type I and type II isozymes.

The role of the type I and type II protein kinase A isozymes in the regulation of human T lymphocyte immune effector functions has not been ascertained. To approach this question, we first characterized the distribution and enzyme activities of the type I and type II protein kinase A (PKA) isozymes in normal, human T lymphocytes. T cells possess both type I and type II isozymes with an activity ratio of 5.0:1 +/- 0.71 (mean +/- SD). The type I isozyme associates predominately with the plasma membrane whereas the type II isozyme localizes primarily to the cytosol. Analyses of isozyme activities demonstrated that T cells from approximately one-third of 16 healthy donors exhibited significantly higher type II isozyme activities (higher type II, type IIH) than the remaining donors (lower type II, type IIL) (mean = 605 +/- 75 pmol.min-1.mg protein-1, P less than 0.001). Scatchard analyses of [3H]cAMP binding in the cytosolic fraction demonstrated similar Kd values (type IIH, 1.1 x 10(-7) M; type IIL, 9.0 x 10(-8) M); however, the Bmax (maximal binding) of the type IIH was 400 fmol/mg protein compared to the Bmax of the type IIL of 126 fmol/mg protein. Scatchard analysis of [3H]cAMP binding to the type I isozyme associated with membrane fragments had a Kd of 5.6 x 10(-8) M and a Bmax of 283 fmol/mg protein. Eadie-Hofstee plots of type IIH and type IIL gave a Km and Vmax of 2.3 mg/ml and 1.5 nmol.mg-1.min-1, and 2.1 mg/ml and 1.6 nmol.mg-1.min-1, respectively. The 3.2-fold higher maximal binding of the type II isozyme in one-third of healthy donors may reflect a greater amount of isozyme protein. The compartmentalization of type I PKA isozyme to the plasma membrane and type II PKA isozyme to the cytosol may serve to localize the isozymes to their respective substrates in T lymphocytes.     

Leukotriene C4 formation catalyzed by three distinct forms of human cytosolic glutathione transferase

The ability of three distinct types of human cytosolic glutathione transferase to catalyze the formation of leukotriene C4 from glutathione and leukotriene A4 has been demonstrated. The near-neutral transferase (mu) was the most efficient enzyme with Vmax= 180 nmol X min-1 X mg-1 and Km= 160 microM. The Vmax and Km values for the basic (alpha-epsilon) and the acidic (pi) transferases were 66 and 24 nmol X min-1 X mg-1 and 130 and 190 microM, respectively. The synthetic methyl ester derivative of leukotriene A4 was somewhat more active as a substrate for all the three forms of the enzyme.     

Glucuronidation of bile acids in human liver, intestine and kidney. An in vitro study on hyodeoxycholic acid

The activities of UDP-glucuronyl transferase(s) in homogenates and microsomal preparations of human liver, kidney and intestine were tested with hyodeoxycholic acid (HDC). The various kinetic parameters of the UDC-glucuronidation were determined from time course experiments. In both liver and kidney preparations, HDC underwent a very active metabolic transformation: liver Km = 78 microM, Vmax = 3.3 nmol . min-1 . mg-1 protein; kidney Km = 186 microM, Vmax = 9.9 nmol . min-1 . mg-1 protein. To our knowledge this is the first observation of both an extensive and comparable bile acid glucuronidation occurring in renal and hepatic tissues.     

Isozyme specificity of rat liver glutathione S-transferases in the formation of PGF2 alpha and PGE2 from PGH2

When prostaglandin H2 (PGH2) was incubated with a mixture of glutathione S-transferases (GSTs) obtained from S-hexylglutathione affinity chromatography, as much as 40% of it was transformed into a prostanoid whose Rf value corresponded to that of the standard PGF2 alpha. The reaction product was identified as PGF2 alpha by cochromatography with a standard on TLC and HPLC. The stereochemistry of the hydroxyl groups on C-9 and C-11 of the cyclopentane ring was confirmed by mass-spectral analysis of the butylboronate derivative of the reaction product. Neither PGE2 nor PGD2 could substitute for PGH2 in the reaction mixture, indicating that the mechanism of formation of PGF2 alpha is a direct two-electron reduction of the endoperoxide moiety and not through a reduction of the keto group on PGE2 or PGD2. Individual GST isozymes exhibited distinct differences in their catalytic rates of formation of PGF2 alpha from PGH2. Among various GSTs, isozyme IV, a homodimer of Ya size subunit showed the highest activity with a Vmax value of approximately 6000 nmol.min-1.mg-1. In general, the isozymes containing Ya and Yc subunits exhibited relatively high activity toward PGH2, indicating that it is the non-selenium-dependent glutathione peroxidase activity associated with the GSTs that might be responsible for the reduction of PGH2 to PGF2 alpha. Interestingly, isozyme IV also exhibited the highest PGE2 forming activity with a Vmax value of approximately 3000 nmol.min-1.mg-1 followed by isozyme I, a homodimer of Yb subunit, which had a Vmax value of 420 nmol.min-1.mg-1. Based on these results, it appears that the GSTs play an important role in the biosynthesis of classical PGs. Therefore, it is conceivable that the tissue-specific formation of PGF2 alpha and PGE2 might, in part, be due to the relative distribution of these enzyme activities in a given tissue. Our results have not only confirmed the previously published reports (E. Christ-Hazelhof et al. (1976) Biochim. Biophys. Acta 450, 450-461), but also have characterized the specificity of GST isozymes in the formation of PGF2 alpha.     

Regiospecific hydroxylation of lauric acid at the (omega-1) position by hepatic and kidney microsomal cytochromes P-450 from rainbow trout

Microsomes from liver or kidney of untreated rainbow trout hydroxylated lauric acid specifically at the (omega-1) position. Turnover numbers for liver (2.72 min-1) and kidney (14.1 min-1) were decreased seven- and twofold, respectively, following treatment with beta-naphthoflavone. Laurate hydroxylation activity from untreated trout hepatic microsomes was sensitive to inhibition by SKF-525A, but was not sensitive to metyrapone and only partially inhibited by alpha-naphthoflavone. The temperature optimum of laurate (omega-1) hydroxylation in trout liver microsomes was 25-30 degrees C. The Km and Vmax for (omega-1)- hydroxylaurate formation was 50 microM and 1.63 nmol min-1 mg-1, respectively, in liver and 20 microM and 3.95 nmol min-1 mg-1, respectively, in kidney from untreated trout microsomes. (omega-1) Hydroxylation of laurate, in both liver and kidney microsomes, was sensitive to an antibody raised against a previously purified cytochrome P-450 isozyme (LM2) of trout liver microsomes, which has been shown to be active towards aflatoxin B1. Antibody to the major isozyme of cytochrome P-450 ( LM4b , active towards benzo(a)pyrene) induced by beta-naphthoflavone did not inhibit (omega-1) hydroxylation of laurate in microsomes from untreated or beta-naphthoflavone-treated trout.     

Novel glutathione conjugates formed from epoxyeicosatrienoic acids (EETs)

The catalysis of glutathione (GSH) conjugation to epoxyeicosatrienoic acids (EETs) by various purified isozymes of glutathione S-transferase was studied. A GSH conjugate of 14,15-EET was isolated by HPLC and TLC; this metabolite contained one molecule of EET and one molecule of GSH. Fast atom bombardment mass spectrometry of the isolated metabolite confirmed the structure as a GSH conjugate of 14,15-EET. Studies designed to determine the isozyme specificity of this reaction demonstrated that two isozymes, 3-3, and 5-5, efficiently catalyzed this conjugation reaction. The Km values for 14,15-EET were approximately 10 microM and the Vmax values ranged from 25 to 60 nmol conjugate formed min-1 mg-1 purified transferase 3-3 and 5-5. The 5,6-, 8,9-, and 11,12-EETs were also substrates for the reaction, albeit at lower rates. These results demonstrate that the EETs can serve as substrates for the cytosolic glutathione S-transferases.     

Delta 5-3 beta-hydroxysteroid dehydrogenase-isomerase activity in canine pancreas

Activity of delta 5-3 beta-hydroxysteroid dehydrogenase coupled with steroid-delta 5-4-isomerase was demonstrated for the first time in the pancreas. The enzyme complex was assayed by measuring the conversion of pregnenolone to progesterone as well as of dehydroepiandrosterone to androstenedione and found to be localized primarily in the mitochondrial fraction of dog pancreas homogenates. The delta 5-3 beta-hydroxysteroid dehydrogenase used either NAD+ or NADP+ as co-substrates, although maximal activity was observed with NAD+. In phosphate buffer, pH 7.0 and 37 degrees C, the apparent Km values of the dehydrogenase were 6.54 +/- 0.7 microM for pregnenolone and 9.61 +/- 0.8 microM for NAD+. The apparent Vmax was determined as 0.82 +/- 0.02 nmol min-1 mg-1. Under the same conditions the Km values for dehydroepiandrosterone and NAD+ were 3.3 +/- 0.2 microM and 9.63 +/- 1.6 microM, respectively, and the apparent Vmax was 0.62 +/- 0.01 nmol min-1 mg-1.     

Ecdysone 3-epimerase from the midgut of Manduca sexta (L.).

Ecdysone 3-epimerase was partially purified by ammonium sulfate fractionation from the 100,000 g supernate of Manduca sexta midguts. The enzyme converts ecdysone and 20-hydroxyecdysone to their respective 3-epimers, requires NADH or NADPH and O2 for this reaction, and has the following kinetic parameters: for ecdysone, Km = 17.0 +/- 1.4 microM, Vmax = 110.6 +/- 14.6 pmol min-1 mg-1; for 20-hydroxyecdysone, Km = 47.3 +/- 7.5 microM, Vmax = 131.0 +/- 3.5 pmol min-1 mg-1: for NADPH, Km = 85.4 +/- 10.6 microM; for NADH, Km = 51.3 +/- 1.3 microM. The reaction is irreversible and can be inhibited by various ecdysteroids.     

Solubilization, purification, and characterization of a membrane-bound phospholipase A2 from the P388D1 macrophage-like cell line

The release of free arachidonic acid from membrane phospholipids is believed to be the rate-controlling step in the production of the prostaglandins, leukotrienes, and related metabolites in inflammatory cells such as the macrophage. We have previously identified several different phospholipases in the macrophage-like cell line P388D1 potentially capable of controlling arachidonic acid release. Among them, a membrane-bound, alkaline pH optimum, Ca2+-dependent phospholipase A2 is of particular interest because of the likelihood that the regulatory enzyme has these properties. This phospholipase A2 has now been solubilized from the membrane fraction with octyl glucoside and partially purified. The first two steps in this purification are butanol extractions that yield a lyophilized, stable preparation of phospholipase A2 lacking other phospholipase activities. This phospholipase A2 shows considerably more activity when assayed in the presence of glycerol, regardless of whether the substrate, dipalmitoylphosphatidylcholine, is in the form of sonicated vesicles or mixed micelles with the nonionic surfactant Triton X-100. Glycerol (70%) increases both the Vmax and the Km with both substrate forms, giving a Vmax of about 15 nmol min-1 mg-1 and an apparent Km of about 60 microM for vesicles and a Vmax of about 100 nmol min-1 mg-1 and an apparent Km of about 1 mM for mixed micelles. Vmax/Km is slightly greater for vesicles than for mixed micelles. The lyophilized preparation of the enzyme is routinely purified about 60-fold and is suitable for evaluating phospholipase A2 inhibitors such as manoalide analogues. Subsequent steps in the purification are acetonitrile extraction followed by high performance liquid chromatography on an Aquapore BU-300 column and a Superose 12 column. This yields a 2500-fold purification of the membrane-bound phospholipase A2 with a 25% recovery and a specific activity of about 800 nmol min-1 mg-1 toward 100 microM dipalmitoylphosphatidylcholine in mixed micelles. When this material was subjected to analysis on a Superose 12 sizing column, the molecular mass of the active fraction was approximately 18,000 daltons.     

Characteristics of the methylammonium/ammonium transport systems of the nitrogen-fixing cyanobacterium Anabaena 7120 (ATCC 27893)

NH4(+)-transport in Anabaena 7120 was studied using the NH4+ analogue, 14CH3NH3+. At pH 7, two energy-dependent NH4(+)-transport systems were detected in both N2- and NO3(-)-grown cells, but none in NH4(+)-grown cells. Both transport systems showed a low and a high affinity mode of operation depending on the substrate concentration. One of the transport systems showed Km values of 8 microM (Vmax = 1 nmole min-1mg-1protein) and 80 microM (Vmax = 7 nmole min-1mg-1protein), and was insensitive to L-methionine-DL-sulphoximine, a glutamate analogue and irreversible inhibitor of glutamine synthetase. The other transport system showed Km values of 2.5 microM (Vmax = 0.1 nmole min-1mg-1protein) and 70 microM (Vmax = 0.7 nmole min-1mg-1protein), and was sensitive to L-methionine-DL-sulphoximine. Intracellular accumulation of free 14CH3NH3+ showed a biphasic pattern in response to variation in external 14CH3NH3+ concentrations. A maximum intracellular concentration of 2.5 mM and 7.5 mM was reached in the external 14CH3NH3+ concentration range of 1-50 microM and 1-500 microM, respectively. At pH 9, an energy-independent diffusion of 14CH3NH2 leading to a higher intracellular accumulation and assimilation rate, than that at pH 7, was observed.     

Purification and characterization of dimethylallyl tryptophan synthase from Claviceps purpurea.

Dimethylallyl tryptophan synthase (DMAT synthase) catalyzes the alkylation of L-tryptophan by dimethylallyl diphosphate to form 4-(gamma,gamma-dimethylallyl)-L-tryptophan. The enzyme from mycelia of Claviceps purpurea was purified approximately 125-fold to apparent homogeneity by chromatography on n-butyl Sepharose, Q Sepharose, phenyl Sepharose, and Protein Pak as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Analysis by gel filtration chromatography and SDS-PAGE indicated that DMAT synthase is an alpha 2 dimer with a molecular mass of 105 kDa. The purified enzyme was active in metal-free buffer containing EDTA. However, activity was enhanced upon addition of divalent calcium or magnesium ions to the buffer. Values for KM and Vmax were determined in the metal-free EDTA buffer (KMDMAPP, 14 microM; KML-tryptophan, 40 microM; Vmax, 215 nmol min-1 mg-1), 4 mM CaCl2 (KMDMAPP, 8.0 microM; KML-tryptophan, 17 microM; Vmax, 504 nmol min-1 mg-1), and 4 mM MgCl2 (KMDMAPP, 8.0 microM; KML-tryptophan, 12 microM; Vmax, 455 nmol min-1 mg-1). The product was isolated and characterized by 1H NMR, uv, and FAB mass spectrometry.     

N omega-hydroxy-L-arginine is an intermediate in the biosynthesis of nitric oxide from L-arginine.

Authentic N omega-hydroxy-L-arginine was synthesized and used to determine whether it is an intermediate in nitric oxide (.NO) synthesis from L-arginine by macrophage .NO synthase. The apparent Km (6.6 microM) and Vmax (99 nmol x min-1 x mg-1) observed with N omega-hydroxy-L-arginine were similar to those observed with L-arginine (Km = 2.3 microM; Vmax = 54 mumol x min-1 x mg-1). N omega-Hydroxy-D-arginine was not a substrate. Stable isotope studies showed that .NO synthase exclusively oxidized the hydroxylated nitrogen of N omega-hydroxy-L-arginine, forming .NO and L-citrulline. As with L-arginine, O2 was the source of the ureido oxygen in L-citrulline from N omega-hydroxy-L-arginine. In the presence of excess N omega-hydroxy-L-arginine, .NO synthase generated a metabolite of L-[14C]arginine that cochromatographed with authentic N omega-hydroxy-L-arginine. The labeled metabolite exhibited identical chromatographic behavior in three solvent systems and generated the same product (L-citrulline) upon alkaline hydrolysis as authentic N omega-hydroxy-L-arginine. Experiments were then run to identify which redox cofactor (NADPH or tetrahydrobiopterin) participated in the enzymatic synthesis of N omega-hydroxy-L-arginine. Both cofactors were required for synthesis of .NO from either N omega-hydroxy-L-arginine or L-arginine. However, with L-arginine, the synthesis of 1 mol of .NO was coupled to the oxidation of 1.52 +/- 0.02 mol of NADPH; whereas with N omega-hydroxy-L-arginine, only 0.53 +/- 0.04 mol of NADPH was oxidized per mol of .NO formed. These results support a mechanism in which N omega-hydroxy-L-arginine is generated as an intermediate in .NO synthesis through an NADPH-dependent hydroxylation of L-arginine.     

Ca2+-dependent ATPases in the basolateral membrane of rat kidney cortex

The basolateral segment of the rat renal tubular plasma membrane possesses Ca2+-dependent ATPase activity which was independent of Mg2+. Two kinetic forms were found: one, was a high affinity (apparent Km for free Ca2+ of 172 nM) low capacity (Vmax of 144 nmol of Pi X min-1 mg-1 protein) type; the other, had low affinity (apparent Km of 25 microM) and high capacity (896 nmol of Pi X min-1 X mg-1 protein). Mg2+ inhibited both Ca2+-ATPases. The high affinity enzyme exhibited positive cooperativity with respect to ATP, with a n value of 1.6. Ca2+-ATPase activity was not affected by calmodulin and was not inhibited by vanadate. On the other hand, both high and low affinity Ca2+-ATPase activities were increased when 1,25-dihydroxycholecalciferol was given to vitamin D-deficient rats. Kinetically, the enhanced activities were due to an increase in the Vmax values; the apparent affinities for free Ca2+ were not changed. The physiological function of the vitamin D-sensitive, Mg+-independent, Ca2+-ATPase activities remains to be established.     

Enzymes of vitamin B6 degradation. Purification and properties of 4- and 5-pyridoxolactonases

4-Pyridoxolactone and 5-pyridoxolactone, formed by dehydrogenation of pyridoxal or isopyridoxal during the bacterial degradation of vitamin B6 by Pseudomonas MA-1 and Arthrobacter Cr-7, respectively, are hydrolyzed to the corresponding acids by distinct inducible lactonases which were purified to homogeneity. 4-Pyridoxolactonase from Pseudomonas MA-1 has an Mr of 54,000 and contains two probably identical subunits of Mr = 28,600. It has a pH optimum of 7.0, a Km of 5.9 microM, and a Vmax at 25 degrees C of 35.2 mumol X min-1 X mg-1. 5-Pyridoxolactonase from Arthrobacter Cr-7 has an Mr of 65,200 and also contains two probably identical subunits of Mr = 32,800. It has a pH optimum of 7.1-7.7, a Km of 300 microM, and a Vmax at 25 degrees C of 21.5 mumol-1 X min-1 X mg-1. The two lactonases require no added cofactors or metal ions; their activities are inhibited by sulfhydryl reagents but are not affected by metal-chelating reagents. Although the two lactonases are entirely specific for their respective substrates, 4-pyridoxolactone is a competitive inhibitor (KI = 52 microM) for 5-pyridoxolactonase, and 5-pyridoxolactone is a competitive inhibitor (KI = 48 microM) for 4-pyridoxolactonase.     

Demethylation and denitrosation of nitrosamines by cytochrome P-450 isozymes

Metabolism of nitrosamines was studied in a reconstituted monooxygenase system composed of cytochrome P-450 isozymes purified from liver microsomes of ethanol- and phenobarbital-treated rats. The ethanol-induced isozyme (P-450et) was efficient in catalyzing the demethylation of N-nitrosodimethylamine (NDMA), with a Km of 2.4 mM and Vmax of 7.2 nmol min-1 nmol P-450(-1), but less active with N-nitrosomethylbenzylamine and N-nitrosomethylaniline. The phenobarbital-induced form (P-450b) was ineffective in NDMA metabolism but was active in catalyzing the demethylation of N-nitrosomethylaniline, with an estimated Km of 0.08 mM and a Vmax of 7.2 nmol min-1 nmol-1. P-450et also catalyzed the denitrosation of NDMA with a Km of 13.6 mM and a Vmax of 1.36 nmol min-1 nmol-1. With control liver microsomes, multiple Km values were observed for the demethylation and denitrosation of NDMA. Involvement of superoxide radicals in the metabolism of NDMA was suggested by the action of superoxide dismutase, which inhibited the denitrosation by 43 to 73% and the demethylation by 13 to 22% in different monooxygenase systems. The P-450et-dependent NDMA demethylation was strongly inhibited by 2-phenylethylamine and 3-amino-1,2,4-triazole; these compounds were previously believed not to be inhibitors of P-450-dependent reactions but were found to inhibit microsomal NDMA demethylase. The present results establish the role of P-450 in nitrosamine metabolism and help to clarify some of the previous confusion in this area of research.     

Distinction between the two basic mechanisms of cation transport in the cardiac Na(+)-Ca2+ exchange system

In order to distinguish between the Ping-Pong and sequential mechanisms of cation transport in the cardiac Na(+)-Ca2+ exchange system, the initial rates of the Nai-dependent 45Ca uptake (t = 1 s) were measured in reconstituted proteoliposomes, loaded with a Ca chelator. Under "zero-trans" conditions ([Na]o = [Ca]i = 0) at a fixed [Na]i = 10-160 mM with varying [45Ca]o = 2.5-122 microM for each [Na]i, the Km and Vmax values increased from 7.7 to 33.5 microM and from 2.3 to 9.0 nmol.mg-1.s-1, respectively. The Vmax/Km values show a +/- 2-10% deviation from the average value of 0.274 nmol.mg-1.s-1.microM-1 over the whole range of [Na]i. These deviations are within the standard error of Vmax (+/- 3-7%), Km (+/- 11-17%), and Vmax/Km (+/- 11-19%). This suggests that, under conditions in which Vmax and Km are [Na]i dependent and vary 4-5-fold, the Vmax/Km values are constant within the experimental error. In the presence of K(+)-valinomycin the Vmax/Km values are 0.85 +/- 0.17 and 1.08 +/- 0.18 nmol.mg-1.s-1.microM-1 at [Na]i = 20 and 160 mM, respectively, suggesting that under conditions of "short circuit" of the membrane potential the Vmax/Km values still exhibit the [Na]i independence. At a very low fixed [45Ca]o = 1.1 microM with varying [Na]i = 10-160 mM, the initial rates were found to be [Na]i independent. At a high fixed [45Ca]o = 92 microM the initial rates show a sigmoidal dependence on the [Na]i with Vmax = 13.8 nmol.mg-1.s-1, KmNa = 21 mM, and Hill coefficient nH = 1.5. The presented data support a Ping-Pong (consecutive) mechanism of cation transport in the Na(+)-Ca2+ exchanger.     

Redox cycling of anthracyclines by cardiac mitochondria. I. Anthracycline radical formation by NADH dehydrogenase

In the present study we have used beef heart submitochondrial preparations (BH-SMP) to demonstrate that a component of mitochondrial Complex I, probably the NADH dehydrogenase flavin, is the mitochondrial site of anthracycline reduction. During forward electron transport, the anthracyclines doxorubicin (Adriamycin) and daunorubicin acted as one-electron acceptors for BH-SMP (i.e. were reduced to semiquinone radical species) only when NADH was used as substrate; succinate and ascorbate were without effect. Inhibitor experiments (rotenone, amytal, piericidin A) indicated that the anthracycline reduction site lies on the substrate side of ubiquinone. Doxorubicin and daunorubicin semiquinone radicals were readily detected by ESR spectroscopy. Doxorubicin and daunorubicin semiquinone radicals (g congruent to 2.004, signal width congruent to 4.5 G) reacted avidly with molecular oxygen, presumably to produce O2-, to complete the redox cycle. The identification of Complex I as the site of anthracycline reduction was confirmed by studies of ATP-energized reverse electron transport using succinate or ascorbate as substrates, in the presence of antimycin A or KCN respiratory blocks. Doxorubicin and daunorubicin inhibited the reduction of NAD+ to NADH during reverse electron transport. Furthermore, during reverse electron transport in the absence of added NAD+, doxorubicin and daunorubicin addition caused oxygen consumption due to reduction of molecular oxygen (to O2-) by the anthracycline semiquinone radicals. With succinate as electron source both thenoyltrifluoroacetone (an inhibitor of Complex II) and rotenone blocked oxygen consumption, but with ascorbate as electron source only rotenone was an effective inhibitor. NADH oxidation by doxorubicin during BH-SMP forward electron transport had a KM of 99 microM and a Vmax of 30 nmol X min-1 X mg-1 (at pH 7.4 and 23 degrees C); values for daunorubicin were 71 microM and 37 nmol X min-1 X mg-1. Oxygen consumption at pH 7.2 and 37 degrees C exhibited KM values of 65 microM for doxorubicin and 47 microM for daunorubicin, and Vmax values of 116 nmol X min-1 X mg-1 for doxorubicin and 114 nmol X min-1 X mg-1 for daunorubicin. In marked contrast with these results, 5-iminodaunodrubicin (a new anthracycline with diminished cardiotoxic potential) exhibited little or no tendency to undergo reduction, or to redox cycle with BH-SMP. Redox cycling of anthracyclines by mitochondrial NADH dehydrogenase is shown, in the accompanying paper (Doroshow, J. H., and Davies, K. J. A. (1986) J. Biol. Chem. 261, 3068-3074), to generate O2-, H2O2, and OH which may underlie the cardiotoxicity of these antitumor agents.