Subcellular localization, aggregation state, and catalytic activity of microsomal P450 cytochromes modified in the NH2-terminal region and expressed in Escherichia coli.

This laboratory previously expressed cDNAs encoding rabbit liver cytochrome P450 2E1 (the ethanol-inducible isoform) and the corresponding protein lacking amino acids 3-29, a proposed membrane anchor, in Escherichia coli. Unexpectedly, the shortened protein, like the full-length form, was found to be predominantly located in the bacterial inner membrane rather than the cytosol and to have full catalytic activity. Additional proteins with alterations in the NH2-terminal region of P450 2E1 or P450 2B4 (the phenobarbital-inducible isoform) were similarly expressed, and it was concluded that such modifications can change the cytochrome to an increased cytosolic localization and that the first two hydrophobic segments are not uniquely involved in attachment to the bacterial membrane (Pernecky et al., 1993, Proc. Natl. Acad. Sci. USA 90, 2651-2655). In the present study, three chimeric cytochromes were produced to determine the effect on subcellular localization: 2E1:2B4, in which the first 17 residues of 2E1 (delta 3-29) replaced the corresponding 17 residues in 2B4 (delta 2-27), and BM-3:2B4 and BM-3:2E1, in which the first 19 residues of P450BM-3 replaced the first 17 in 2B4 (delta 2-27) and 2E1 (delta 3-29), respectively. Of the total cytochrome expressed, the localization in the E. coli cytosol was about 60, 70, and 80% for the respective chimeras, with 80% being the highest for any P450 we have examined. A plot of the extent of membrane binding versus hydropathy of the NH2-terminal region showed that the terminal sequence strongly influences the subcellular distribution and that a group of 2E1 proteins and a group of 2B4 proteins each have other regions that characteristically determine the extent of membrane attachment. The role of the NH2-terminal region in the high level of aggregation of purified full-length P450 is indicated by the finding that the multimeric state of 2E1 or 2B4 is unaffected by sodium cholate at concentrations that convert 2E1 (delta 3-29) or 2B4 (delta 2-27) to the monomeric state. In contrast to our earlier experience with P450 2E1, purified P450 2B4 (delta 2-27) has on the average only about half the activity of full-length 2B4 with substrates that undergo oxidative dealkylation or oxygenation at a hydroxyl group.

Characterization of the inactivation of nitric oxide synthase by NG-methyl-L-arginine: evidence for heme loss.

The nitric oxide synthases (NOS) are a unique family of P450-type hemoproteins that catalyze the formation of .NO and citrulline from L-arginine, oxygen, and NADPH. NG-Methyl-L-arginine (L-NMA) has been shown to function as a slow, partially uncoupled alternate substrate and mechanism-based inhibitor of the inducible NOS [Olken, N. M., & Marletta, M. A. (1993) Biochemistry 32, 9677-9685]. In this report, the inactivation of NOS by L-NMA has been investigated in detail. Inactivation fails to occur under an argon atmosphere, establishing turnover dependence. The partition ratio, defined as the number of molecules of citrulline formed per NOS monomer inactivated, is 108 +/- 3. By utilizing NG-methyl-L-[2,3-3H2]arginine and NG-[14C]methyl-L-arginine, the stoichiometry of radiolabeling is 0.11 +/- 0.01 equiv of tritium and 0.41 +/- 0.10 equiv of carbon-14 per inactivated NOS monomer. Dialysis under native conditions does not change this stoichiometry. However, dialysis of NOS following denaturation decreases the stoichiometry of radiolabeling to 0.08 +/- 0.04 equiv of tritium and 0.12 +/- 0.04 equiv of carbon-14 per inactivated NOS monomer. Absolute and CO-reduced difference spectroscopy indicates that inactivation of L-NMA is accompanied by a substantial loss of the heme chromophore, which is not prevented by catalase. HPLC analysis of NOS heme following inactivation with L-NMA indicates substantial loss of heme. These findings suggest that multiple mechanisms may contribute to the loss of NOS activity by L-NMA, including heme loss and possibly protein and cofactor modification.

NG-methyl-L-arginine functions as an alternate substrate and mechanism-based inhibitor of nitric oxide synthase.

NG-Methyl-L-arginine (L-NMA) is one of the most commonly used inhibitors of the nitric oxide synthases (NOS). Results reported here demonstrate that L-NMA is an alternate substrate and a mechanism-based inhibitor of the inducible NOS purified from murine macrophages. The irreversible inhibition displays pseudo-first-order kinetics of inactivation with kinact = 0.07 min-1 and KI = 2.7 microM. Inactivation of NOS is enantiospecific for L-NMA, and substrate protection against inactivation is enantiospecific for L-arginine. L-NMA is hydroxylated, producing NG-hydroxy-NG-methyl-L-arginine (L-NHMA), and both compounds are slow, partially uncoupled alternate substrates for NOS. Processing of L-NMA by NOS results in four amino acid products: L-NHMA, NG-hydroxy-L-arginine (L-NHA), L-arginine, and citrulline. Deformylation of L-NMA and L-NHMA precedes the formation of citrulline and nitric oxide (.NO). Partial uncoupling of NADPH oxidation during L-NMA and L-NHMA processing results in hydrogen peroxide formation. The apparent Km values for L-NMA and L-NHMA are 3.1 and 7.4 microM, respectively. Turnover of L-NMA and L-NHMA to .NO and citrulline is slow relative to L-arginine: Vmax(L-arginine/L-NMA) = 20:1; Vmax(L-arginine)/(L-NHMA) = 13:1. NOS contains a functional cytochrome P-450-type heme, and the formation of these products from L-NMA is consistent with cytochrome P-450 monooxygenase chemistry. Other than the NOS reaction intermediate L-NHA, L-NMA and L-NHMA are the first NG-substituted L-arginines identified as substrates for NOS.

Selective localization of a radioiodinated phospholipid ether analog in human tumor xenografts.

Administration of [125I]-rac-1-O-[12-(m-iodophenyl)dodecyl-2-O-methylglycero-3- phosphocholine (NM-294) to athymic mice implanted with human tumors of several histologies, including adenocarcinoma of the ovary and colon, melanoma and small-cell carcinoma of the lung, resulted in excellent images of the tumors by gamma camera scintigraphy. Images of the tumor were obtained at 5 days or more postinjection, by which time nearly all background activity had cleared from the liver and gastrointestinal tract. Tumor-to-blood ratios at this time were quite high and ranged from approximately 8:1 (melanoma) to 30:1 (ovarian carcinoma), which is consistent with the scintigraphic images obtained in all human tumor models. Lipid extraction of the liver and tumor at 13 days postinjection showed that most of the radioactivity in these tissues remained associated with the parent compound, with only a small amount retained by the liver. Appropriately radioiodinated NM-294 has substantial potential as a tumor-avid radiopharmaceutical.

Selective localization of radioiodinated alkylphosphocholine derivatives in tumors.

We have designed and synthesized two radioiodinated analogs of hexadecylphosphocholine in order to evaluate their tumor imaging potential. 12-(m[125I]iodophenyl)dodecyl phosphocholine (NM-324) and hexadecyl-2-[N,N-dimethyl-N-(m[125I]iodobenzyl)-ammonium] ethyl phosphate (NM-326) demonstrated the ability of such compounds to localize in and thereby visualize the Walker 256 tumor in rats. However, the tumor avidity of NM-324 was far superior to NM-326. In addition, NM-324 showed excellent tumor localization in athymic mice bearing subcutaneous human tumors.

NG-allyl- and NG-cyclopropyl-L-arginine: two novel inhibitors of macrophage nitric oxide synthase.

NG-Methyl-L-arginine has recently been shown to inactivate the inducible murine macrophage nitric oxide (.NO) synthase (Olken, N. M.; Rusche, K. M.; Richards, M. K.; Marletta, M. A. Biochem. Biophys. Res. Commun. 1991, 177, 828-833). NG-Allyl-L-arginine and NG-cyclopropyl-L-arginine were synthesized as potential mechanism-based enzyme inhibitors to exploit the chemistry presumed to occur at the active site. NG-Cyclopropyl-L-arginine was found to be a potent reversible inhibitor with a Ki = 7.7 microM. NG-Allyl-L-arginine was found to be both a potent reversible (Ki = 2.1 microM) and irreversible inhibitor of the enzyme. This irreversible inhibition demonstrated pseudo-first-order inactivation kinetics with kinact = 0.026 min-1 and KI = 3.4 microM. Stereospecific protection of the inactivation was afforded by L-arginine, and saturability of the inactivation rate was observed. Our studies indicate that both reversible and irreversible inhibition of the inducible .NO synthase can be achieved with relatively simple modifications of the substrate L-arginine.

Inactivation of macrophage nitric oxide synthase activity by NG-methyl-L-arginine.

.N = O synthase catalyzes the oxidation of one of the two chemically equivalent guanido nitrogens of L-arginine to nitric oxide (.N = O). NG-Methyl-L-arginine has been previously characterized as a potent competitive inhibitor of both major types of .N = O synthases. Initial rate kinetics were performed with a spectrophotometric assay based on the oxidation of oxy- to methemoglobin by .N = O. NG-Methyl-L-arginine was a competitive inhibitor of .N = O synthase activity derived from activated murine macrophages with a Ki of 6.2 microM. When the enzyme was pre-incubated in the presence of the required cofactors NADPH and tetrahydrobiopterin, time- and concentration-dependent irreversible inactivation of the activity was observed. At 37 degrees C the kinact was 0.050 min-1. This inactivation process exhibited substrate protection, saturation kinetics and required the cofactors necessary for enzymatic turnover. These data indicate that NG-methyl-L-arginine acts as a mechanism-based enzyme inactivator of murine macrophage .N = O synthase.