Adenovirus-mediated human paraoxonase1 gene transfer to provide protection against the toxicity of the organophosphorus pesticide toxicant diazoxon.

Human paraoxonase1 (hPON1) is a potential therapeutic against the toxicity of organophosphorus (OP) pesticides and chemical warfare nerve agents. We tested whether PON1 gene transfer using adenovirus provides protection against the toxicity of the OP diazoxon. Using an adenovirus construct containing hPON1 gene, we showed elevated levels of recombinant hPON1 in vitro in 293A cells and in vivo in mice. The recombinant enzyme was secreted by 293A cells into culture medium and into the systemic circulation of mice. Western blotting revealed that the virally expressed hPON1 had the expected molecular weight of 45 kDa. Recombinant hPON1 in mice was in complex with mouse high-density lipoprotein (HDL) and migrated more slowly than endogenous hPON1 in the human HDL complex. Mice injected with adenovirus expressed PON1 at 600-3480 U ml(-1) on day 5 post-treatment, which is 8-50-fold above endogenous. Six mice expressing hPON1 survived 2LD(50) doses of diazoxon. Four of the six mice survived a second dose of diazoxon (for a total of 4LD(50)) administered 24 h later. In contrast, none of the three mice in the control group survived one 2LD(50) dose. These results show that hPON1 in mice functions as a prophylactic and offers significant protection against lethal doses of diazoxon.

Farnesyltransferase inhibitors block the neurofibromatosis type I (NF1) malignant phenotype.

Neurofibromatosis type I (NF1) is a hereditary tumor and developmental disorder whose defective gene was cloned previously. The protein product of the NF1 gene, neurofibromin, contains a domain that shows significant sequence homology to the known catalytic domains of mammalian Ras GTPase-activating proteins (GAP) and the yeast IRA1 and IRA2 proteins. This homologous region of neurofibromin has been shown to exhibit GAP activity toward Ras proteins. Malignant schwannoma cell lines from NF1 patients contain normal levels of GAP and nonmutated Ras proteins but barely detectable levels of neurofibromin, based on genetic mutations in the NF1 gene. Because these cells contain constitutively activated Ras.GTP, it has been proposed that neurofibromin may be the sole negative regulator of Ras in these cells. Overall, these results have implied an important role of the Ras signaling pathway in NF1 malignant schwannomas. Recently, several laboratories have developed small molecule inhibitors of Ras function that inhibit the enzyme farnesyltransferase (FT). FT-mediated post-translational farnesylation of Ras proteins is absolutely necessary for Ras function since this modification is required for the anchoring of Ras proteins to the plasma cell membrane. Although previous studies have shown that FT inhibitors can block the growth of tumor cells carrying mutant Ras proteins, it remained unclear how this class of inhibitors would affect tumor cells such as in NF1, whose malignant growth appears to be mediated by up-regulation of wild-type Ras activity. Thus, in the current study, we investigated whether BMS-186511, a bisubstrate analogue inhibitor of FT, would inhibit the malignant growth properties of a cell line established from malignant schwannoma of an NF1 patient. Our results indicate that the malignant growth properties of ST88-14 cells, the most malignant cell line among several well-characterized NF1 cells, are inhibited by BMS-186511 in a concentration-dependent manner. Following treatment with BMS-186511, ST88-14 cells became flat, nonrefractile, were contact-inhibited, and lost their ability to grow in soft agar. In the drug-exposed cells, Ras proteins were prevented from FT-mediated membrane association. BMS-186511 was found to specifically inhibit FT, but not geranylgeranyltransferase I, a closely related enzyme. Thus, it is conceivable that FT inhibitors may ultimately become the first generation of drugs against the malignant phenotype in NF1 based on rational insights into the mechanism of action of neurofibromin.

Preclinical antitumor activity of BMS-214662, a highly apoptotic and novel farnesyltransferase inhibitor.

BMS-214662 is a potent and selective inhibitor of farnesyltransferase (FTI). In rodent fibroblasts transformed by oncogenes, BMS-214662 reversed the H-Ras-transformed phenotype but not that of K-Ras or other oncogenes. In soft agar growth assays, BMS-214662 showed good potency in inhibiting H-ras-transformed rodent cells, A2780 human ovarian carcinoma tumor cells, and HCT-116 human colon carcinoma tumor cells. Inhibition of H-Ras processing in HCT-116 human colon tumor cells was more rapid than in H-Ras-transformed rodent fibroblast tumors. BMS-214662 is the most potent apoptotic FTI known and demonstrated broad spectrum yet robust cell-selective cytotoxic activity against a panel of cell lines with diverse histology. The presence of a mutant ras oncogene was not a prerequisite for sensitivity. Athymic and conventional mice were implanted s.c. with different histological types of human and murine tumors, respectively. BMS-214662 was administered both parenterally and p.o. and was active by all these routes. Curative responses were observed in mice bearing staged human tumor xenografts including HCT-116 and HT-29 colon, MiaPaCa pancreatic, Calu-1 lung, and EJ-1 bladder carcinomas. A subline of HCT-116, HCT-116/VM46, resistant to many standard cytotoxic agents by means of a multiple drug resistance mechanism, remained quite susceptible to BMS-214662, and borderline activity was achieved against N-87 human gastric carcinoma. Two murine tumors, Lewis lung carcinoma and M5076 sarcoma, were insensitive to the FTI. In a study performed using Calu-1 tumor-bearing mice, no obvious schedule dependency of BMS-214662 was observed. The FTI, BMS-214662, demonstrated broad spectrum activity against human tumors, but murine tumors were not as sensitive.

Identification of polyphosphoinositide-specific phospholipase C and its resolution from phosphoinositide-specific phospholipase C from human platelet extract.

Sonicated platelet extract, obtained after high-speed centrifugation, was fractionated by phosphocellulose chromatography, and assayed for phosphodiesteric cleavage of phosphatidylinositol (PI), phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-biphosphate (PIP2) using (i) single bilayer vesicles from total platelet membrane lipids and (ii) single bilayer vesicles from individual pure lipids. When total platelet lipids were used as the substrate, two clear-cut, but incompletely resolved, peaks of phosphoinositide-specific phospholipase C (PI-PLC) were detected; one of these activities hydrolysed PIP alone, whereas the second peak hydrolysed all three phosphoinositides. When pure phosphoinositides were used as the substrate, three distinct peaks of PI-PLC were observed; two of these peak activities correspond to the peaks when assays were conducted with total platelet membrane lipids. The third peak of activity hydrolysed PIP2 and PIP, but not PI. These results clearly show that there exist PIP/PIP2-specific PLC enzymes that do not act on PI, and escaped detection thus far because of the general practice of using PI as the substrate to monitor PI-PLC activities. I propose that these PIP/PIP2-specific PLC enzymes are the real candidates that respond to agonist occupation of receptors on the cell surface to generate the two putative second messengers inositol triphosphate and diacylglycerol.

A novel candidate for receptor-coupled phospholipase C purified from human platelets.

The breakdown of polyphosphoinositides (PPI) but not phosphatidylinositol (PI) has been hypothesized as the primary event following agonist (hormones/growth factors/neurotransmitters) stimulation in a wide variety of systems. This, in turn, predicts the existence of a phospholipase C (PLC) enzyme that shows specificity to PPI. Ideally, this PPI-specific PLC activity should not be absolutely dependent on Ca2+ because of its proposed role in Ca2+ mobilization. I have recently identified two PLC activities that are specific to PPI and have described their resolution from a PLC that acts on all three phosphoinositides (Manne, 1987). In this report, I describe purification to near homogeneity of one of these PLC activities. The enzyme shows maximal activity towards PPI in the presence of physiological Mg2+ concentrations, and does not act on PI under conditions optimal for PPI hydrolysis. However, a weak PI hydrolytic activity, representing about 1/8th to 1/20th of that observed with PPI is detected when 0-100 microM Ca2+ is present in the assay. This weak PI hydrolytic activity is strongly inhibited by mM Ca2+, which is required at mM levels for most of the PLC enzymes described in literature. The size of the native enzyme as determined by gel filtration (high performance liquid chromatography) is 140 kDa. Analysis of the purified enzyme by HPLC on Zorbax GF-250 column showed a single major peak that coincided with the enzyme activity. Under both denaturing and non-denaturing conditions of SDS-polyacrylamide gel electrophoresis, the highly purified enzyme shows two major bands of 38 kDa and 42 kDa, which together represent about 90% of the total stain on the gel.

Bisubstrate inhibitors of farnesyltransferase: a novel class of specific inhibitors of ras transformed cells.

We describe the biological properties of a new class of potent farnesyltransferase (FT) inhibitors designed as bisubstrate analog inhibitors. These inhibitors incorporate the structural motifs of both farnesyl pyrophosphate and the CAAX tetrapeptide, the two substrates of the reaction catalyzed by FT. Both the phosphinate inhibitor, BMS-185878, and the phosphonate inhibitor, BMS-184467, exhibited higher in vitro FT selectivity than some of the previously reported CVFM peptidomimentics and benzodiazepine analogs. Xenopus oocyte maturation induced by microinjected oncogenic Ras proteins was blocked by coinjected BMS-184467 and BMS-185878. However, both inhibitors showed poor cell activity presumably because of the doubly charged nature of the compounds. Thus, masking the charge on the carboxylate ion markedly improved the cell permeability of BMS-185878, leading to BMS-186511, the methyl carboxyl ester prodrug. BMS-186511 inhibited FT activity in whole cells as determined by inhibition of p21 Ras protein processing, inhibition of farnesylation of proteins including Ras and the accumulation of unfarnesylated Ras proteins in the cytosolic fraction. While the cellular effects of these bisubstrate analog inhibitors had no significant effect on growth of untransformed NIH3T3 cells, they produced pronounced inhibition of Ras transformed cell growth. Both the anchorage dependent and independent growth of ras transformed cells were severely curtailed by micromolar concentrations of BMS-186511. We also found that both H-ras and K-ras transformed cells are affected by this bisubstrate inhibitor. However, K-ras transformed cells appear to be less sensitive. The inhibition of FT activity in cells and the ensuing inhibition of ras transformed cell growth is further manifested in distinct morphological changes in cells. Cells flattened, became less refractile and grew in contact inhibited monolayer. Moreover, the highly diffused character of the actin cytoskeleton in the ras transformed cells was dramatically reverted to an organized network of stress cables crisscrossing the entire cells upon treatment with BMS-186511. All of these effects of BMS-186511 are limited to ras transformed cells that utilize farnesylated Ras, but are not seen in transformed cells that use geranylgeranyl Ras or myristoyl Ras. Significantly, these FT inhibitors did not produce any signs of gross cytotoxicity in untransformed, ras transformed cells or other oncogene transformed cells.

Contribution of Ras GTPase/MAP kinase and cytochrome P450 metabolites to deoxycorticosterone-salt-induced hypertension.

We recently reported that norepinephrine and angiotensin II activate the Ras/mitogen-activated protein (MAP) kinase pathway through generation of a cytochrome P450 (CYP450) and lipoxygenase metabolites. The purpose of this study was to determine the contribution of Ras/MAP kinase to deoxycorticosterone acetate (DOCA)-salt-induced hypertension in rats. Administration of DOCA and 1% saline drinking water to uninephrectomized rats for 6 weeks significantly elevated mean arterial blood pressure (MABP) (166+/-5 mm Hg, n=19) compared with that of normotensive controls (95+/-5 mm Hg, n=7) (P<0.05). The activity of Ras and MAP kinase measured in the heart was increased in DOCA-salt hypertensive rats. Infusion of the Ras farnesyl transferase inhibitors FPT III (138 ng/min) and BMS-191563 (694 ng/min) significantly (P<0.05) attenuated MABP to 139+/-4 mm Hg (n=14) and 126+/-1 mm Hg (n=4), respectively. Moreover, infusion of MAP kinase kinase inhibitor PD-98059 (694 ng/min) also reduced MABP in hypertensive rats. Morphological studies of the kidney showed that treatment of rats with FPT III, which reduced Ras activity, minimized the hyperplastic occlusive arteriosclerosis and fibrinoid vasculitis observed in untreated hypertensive rats. In addition, the rise in CYP450 activity and MABP in hypertensive rats was prevented by the CYP450 inhibitor aminobenzotriazole (50 mg/kg) and was associated with a decrease in Ras and MAP kinase activity in the heart. These data suggest that the Ras/MAP kinase pathway contributes to DOCA-salt-induced hypertension and associated vascular pathology consequent to activation of CYP450.

Potent, cell active, non-thiol tetrapeptide inhibitors of farnesyltransferase.

All previously reported CAAX-based farnesyltransferase inhibitors contain a thiol functionality. We report that attachment of the 4-imidazolyl group, via 1-, 2-, or 3-carbon alkyl or alkanoyl spacers, to Val-Tic-Met or tLeu-Tic-Gln provides potent FT inhibitors. (R*)-N-[[1,2,3,4-Tetrahydro-2-[N-[2-(1H-imidazol-4-yl)ethyl] -L-valyl]-3-isoquinolinyl]carbonyl]-L-methionine ([imidazol- 4-yl-ethyl]-Val-Tic-Met), with FT IC50 = 0.79 nM, displayed potent cell activity in the absence of prodrug formation (SAG EC50 = 3.8 muM).

Farnesyl diphosphate-based inhibitors of Ras farnesyl protein transferase.

The rational design, synthesis, and biological activity of farnesyl diphosphate (FPP)-based inhibitors of the enzyme Ras farnesyl protein transferase (FPT) is described. Compound 3, wherein a beta-carboxylic phosphonic acid type pyrophosphate (PP) surrogate is connected to the hydrophobic farnesyl group by an amide linker, was found to be a potent (I50(FPT) = 75 nM) and selective inhibitor of FPT, as evidenced by its inferior activity against squalene synthetase (I50(SS) = 516 microM) and mevalonate kinase (I50(MK) = > 200 microM). A systematic structure-activity relationship study involving modifications of the farnesyl group, the amide linker, and the PP surrogate of 3 was undertaken. Both the carboxylic and phosphonic acid groups of the beta-carboxylic phosphonic acid PP surrogate are essential for activity, since deletion of either group results in 50-2600-fold loss in activity (6-9, I50 = 4.6-220 microM). The farnesyl group also displays very stringent requirements and does not tolerate one carbon homologation (12, I50 = 17.7 microM), substitution by a dodecyl fragment (14, I50 = 9 microM), or introduction of an extra methyl group at the allylic position (18, I50 = 55 microM). Modifications around the amide linker group of 3 were more forgiving, as evidenced by the activity of N-methyl analog (21, I50 = 0.53 microM), the one carbon atom shorter farnesoic acid-derived retroamide analog (32, I50 = 250 nM), and the exact retroamide analog (49, I50 = 50 nM). FPP analogs such as 3, 32, and 49 are novel, potent, selective, small-sized, nonpeptidic inhibitors of FPT that may find utility as antitumor agents.

Discovery of (R)-7-cyano-2,3,4, 5-tetrahydro-1-(1H-imidazol-4-ylmethyl)-3- (phenylmethyl)-4-(2-thienylsulfonyl)-1H-1,4-benzodiazepine (BMS-214662), a farnesyltransferase inhibitor with potent preclinical antitumor activity.

Continuing structure-activity studies were performed on the 2,3,4, 5-tetrahydro-1-(imidazol-4-ylalkyl)-1,4-benzodiazepine farnesyltransferase (FT) inhibitors. These studies demonstrated that a 3(R)-phenylmethyl group, a hydrophilic 7-cyano group, and a 4-sulfonyl group bearing a variety of substituents provide low-nanomolar FT inhibitors with cellular activity at concentrations below 100 nM. Maximal in vivo activity in the mutated K-Ras bearing HCT-116 human colon tumor model was achieved with analogues carrying hydrophobic side chains such as propyl, phenyl, or thienyl attached to the N-4 sulfonyl group. Several such compounds achieved curative efficacy when given orally in this model. On the basis of its excellent preclinical antitumor activity and promising pharmacokinetics, compound 20 (BMS-214662, (R)-7-cyano-2,3,4, 5-tetrahydro-1-(1H-imidazol-4-ylmethyl)-3-(phenylmethyl)-4-(2-thie nyl sulfonyl)-1H-1,4-benzodiazepine) has been advanced into human clinical trials.

Development of highly potent inhibitors of Ras farnesyltransferase possessing cellular and in vivo activity.

Analogs of CVFM (a known nonsubstrate farnesyltransferase (FT) inhibitor derived from a CA1A2X sequence where C is cysteine, A is an aliphatic residue, and X is any residue) were prepared where phenylalanine was replaced by (Z)-dehydrophenylalanine, 2-aminoindan-2-carboxylate, 1,2,3,4-tetrahydroisoquinoline-3-carboxylate (Tic), and indoline-2-carboxylate. The greatest improvement in FT inhibitory potency was observed for the Tic derivative (IC50 = 1 nM); however, this compound was ineffective in blocking oncogenic Ras-induced transformation of NIH-3T3 fibroblast cells. A compound was prepared in which both the Cys-Val methyleneamine isostere and the Tic replacement were incorporated. This derivative inhibited FT with an IC50 of 0.6 nM and inhibited anchorage-independent growth of stably transformed NIH-3T3 fibroblast cells by 50% at 5 microM. Replacing the A1 side chain of this derivative with a tert-butyl group and replacing the X position with glutamine led to a derivative with an IC50 of 2.8 nM and an EC50 of 0.19 microM, a 26-fold improvement over (S*,R*)-N-[[2-[N-(2-amino-3-mercaptopropyl)-L-valyl]-1,2,3,4- tetrahydro-3-isoquinolinyl]carbonyl]-L-methionine. This derivative, (S*,R*)-N-[[2-[N-(2-amino-3-mercaptopropyl)-L-tert-leucyl]-1,2,3,4 - tetrahydro-3-isoquinolinyl]-carbonyl]-L-glutamine, was evaluated in vivo along with (S*,R*)-N-[[2-[N-(2-amino-3- mercaptopropyl)-L-tert-leucyl]-1,2,3,4-tetrahydro-3- isoquinolinyl]carbonyl]-L-methionine methyl ester for antitumor activity in an athymic mouse model implanted ip with H-ras-transformed rat-1 tumor cells. When administered by injection twice a day at 45 mg/kg for 11 consecutive days, both compounds showed prolonged survival time (T/C = 142-145%), thus demonstrating efficacy against ras oncogene-containing tumors in vivo.

Discovery and structure-activity relationships of imidazole-containing tetrahydrobenzodiazepine inhibitors of farnesyltransferase.

2,3,4,5-Tetrahydro-1-(imidazol-4-ylalkyl)-1,4-benzodiazepines were found to be potent inhibitors of farnesyltransferase (FT). A hydrophobic substituent at the 4-position of the benzodiazepine, linked via a hydrogen bond acceptor, was important to enzyme inhibitory activity. An aryl ring at position 7 or a hydrophobic group linked to the 8-position through an amide, carbamate, or urea linkage was also important for potent inhibition. 2,3,4, 5-Tetrahydro-1-(1H-imidazol-4-ylmethyl)-7-(4-pyridinyl)-4-[2-(t rifluo romethoxy)benzoyl]-1H-1,4-benzodiazepine (36), with an FT IC(50) value of 24 nM, produced 85% phenotypic reversion of Ras transformed NIH 3T3 cells at 1.25 microM and had an EC(50) of 160 nM for inhibition of anchorage-independent growth in soft agar of H-Ras transformed Rat-1 cells. Selected analogues demonstrated ip antitumor activity against an ip Rat-1 tumor in mice.

10'-Desmethoxystreptonigrin, a novel analog of streptonigrin.

10'-Desmethoxystreptonigrin, a novel analog of streptonigrin produced by Streptomyces albus, was discovered in a screen for inhibitors of farnesylation of RAS p21 protein. The compound was isolated from the fermentation broth and its structure determined. It is markedly cytotoxic to several human tumor cell lines and also exhibits potent broad-spectrum antibacterial activity.

Characterization of phosphoinositide-specific phospholipase C from human platelets.

Phosphoinositide-specific phospholipase C (PI-PLC) from human platelet cytosol was purified 190-fold to a specific activity of 0.68 mumol of phosphatidylinositol (PI) cleaved/min per mg of protein. It hydrolyses PI and phosphatidylinositol 4,5-bisphosphate (PIP2), but not phosphatidylcholine, phosphatidylserine or phosphatidylethanolamine. The enzyme exhibits an acid pH optimum of 5.5 and has a molecular mass of 98 kDa as determined by Sephacryl S-200 gel filtration. It required millimolar concentrations of Ca2+ for PI hydrolysis, whereas micromolar concentrations are optimal for PIP2 hydrolysis. Mg2+ could substitute for Ca2+ when PIP2, but not PI, was used as the substrate. EDTA was more effective than EGTA in inhibiting the basal PI-PLC activity towards PIP2. Sodium deoxycholate strongly inhibits the purified PI-PLC activity with either PI or PIP2 as substrate. Ras proteins, either alone or in the form of liposomes, have no effect on PI-PLC activity.

Mechanism of translational control by partial phosphorylation of the alpha subunit of eukaryotic initiation factor 2.

Catalysis of ternary complex formation by the GDP exchange factor (GEF), in the presence of Mg2+, is blocked by phosphorylation of the alpha subunit of the eukaryotic initiation factor 2 (eIF-2). We proposed earlier that this phosphorylation interferes with the interaction between eIF-2 and GEF (then termed ESP). If so, inhibition should be related to the extent of phosphorylation. However, work in other laboratories indicated that in fully inhibited, heme-deficient lysates only 20-40% of the eIF-2 is phosphorylated. To understand the nature of the molecular lesion in eIF-2-alpha phosphorylation we used a system of pure components in which the rate of exchange of eIF-2-bound [3H]GDP with unlabeled GDP (via the reaction eIF-2-GDP + GEF in equilibrium eIF-2-GEF + GDP) was measured by using mixtures of eIF-2(alpha P) X [eH]GDP and eIF-2 X [3H]GDP in different proportions at constant concentration of eIF-2 X GEF. If, for example, the ratio of eIF-2 X GEF to total (phosphorylated and unphosphorylated) eIF-2 X [3H]GDP was 0.25, the exchange was found to be maximally inhibited when the proportion of eIF-2(alpha P) X [3H]GDP in hte mixture reached 25%. This suggests that the reaction stops because the available GEF is trapped in an inactive complex with eIF-2(alpha P). In the absence of free GEF, eIF-2 would not be able to recycle and initiation would come to a standstill when the available eIF-2 is tied up as eIF-2 X GDP. The trapping of GEF by eIF-s(alpha P) is strongly supported by the following observation. Incubation of eIF-2 X GEF with excess [3H]GDP leads to the formation of eIF-2 X [3H] GDP and free GEF and, if eIF-2(alpha 32P) X GDP is also present, all of the GEF is converted to eIF-2(alpha 32P) X GEF. This suggests that, whereas the equilibrium of the reaction eIF-2 X GEF + GDP in equilibrium eIF-2 X GDP + GEF favors the formation of free GEF, the equilibrium of the reaction eIF-2(alpha P) X GDP + GEF in equilibrium eIF-2(alpha P) X GEF + GDP is in favor of the association of GEF to eIF-2(alpha P).

Effect of divalent metal ions and glycerol on the GTPase activity of H-ras proteins.

The product of the protooncogenic ras gene (p21N ras) exhibits a weak GTPase activity. A significant increase in the GTPase activity associated with p21N ras protein was obtained by using glycerol in the assay mixture. Of the several metal ions tested, only Mg++ and Mn++ are effective divalent cations that support the GTPase activity of p21N ras protein. p21N ras protein exhibits higher GTPase activity and yields higher [3H] GDP binding in the presence of MnCl2 than with MgCl2. Optimal GTPase and [3H] GDP binding are obtained at micromolar concentrations of MgCl2 or MnCl2. Concentrations in the millimolar range of either MgCl2 or MnCl2 are inhibitory to the GTPase activity, whereas [3H] GDP binding was not affected.

Purification and properties of synephrinase from Arthrobacter synephrinum.

Synephrinase, an enzyme catalyzing the conversion of (-)-synephrine into p-hydroxyphenylacetaldehyde and methylamine, was purified to apparent homogeneity from the cell-free extracts of Arthrobacter synephrinum grown on (+/-)-synephrine as the sole source of carbon and nitrogen. A 40-fold purification was sufficient to produce synephrinase that is apparently homogeneous as judged by native polyacrylamide gel electrophoresis and has a specific activity of 1.8 mumol product formed/min/mg protein. Thus, the enzyme is a relatively abundant enzyme, perhaps comprising as much as 2.5% of the total protein. The enzyme essentially required a sulfhydryl compound for its activity. Metal ions like Mg2+, Ca2+, and Mn2+ stimulated the enzyme activity. Metal chelating agents, thiol reagents, denaturing agents, and metal ions like Zn2+, Hg2+, Ag1+, and Cu2+ inhibited synephrinase activity. Apart from (-)-synephrine, the enzyme acted upon (+/-)-octopamine and beta-methoxysynephrine. Molecular oxygen was not utilized during the course of the reaction. The molecular mass of the enzyme as determined by Sephadex G-200 chromatography, was around 156,000. The enzyme was made up of four identical subunits with a molecular mass of 42,000.

Polypeptide chain initiation in eukaryotes: reversibility of the ternary complex-forming reaction.

In the last step of polypeptide chain initiation in eukaryotes, the interaction of the 40S preinitiation complex eIF-2.GTP.Met-tRNAi.40S [the complex between the 40S ribosomal subunit and the ternary complex containing equimolar amounts of eukaryotic initiation factor 2 (eIF-2), GTP, and eukaryotic initiator methionyl tRNA (Met-tRNAi)] with a 60S ribosomal subunit in the presence of mRNA, cap binding protein (with "capped" messengers), ATP, and the initiation factors eIF-3, eIF-4a, -4b, -4c, and eIF-5, results in the formation of an 80S initiation complex (Met-tRNAi.80S.mRNA) with concomitant hydrolysis of GTP and liberation of eIF-2 for recycling in subsequent initiation events. However, at physiological Mg2+ concentrations, GDP is known to have approximately equal to 100-fold greater affinity than GTP for eIF-2 and eIF-2 is believed to be released in the form of an eIF-2.GDP complex. Previously, we have shown that initiation factor SP (for eIF-2-stimulating protein) promotes the exchange of eIF-2-bound GDP for GTP and catalyzes ternary complex formation in the presence of Met-tRNAi. Binding of GDP by eIF-2 is indeed so tight that, as we now show, homogeneous preparations of eIF-2 contain upward of 0.5 mol of GDP/mol of eIF-2. We further show that, in the presence of Mg2+ and catalytic amounts of SP, ternary complex formation conforms to the overall reversible reaction eIF-2.GDP + GTP + Met-tRNAi in equilibrium eIF-2.GTP.Met-tRNAi + GDP.

Farnesylation of p21 Ras proteins in Xenopus oocytes.

Unprocessed p21 Ras proteins microinjected into Xenopus oocytes were radiolabeled by coinjected [3H]farnesyl pyrophosphate, a direct farnesyl donor substrate for all known mammalian farnesyltransferases. Mevinolin, an inhibitor of HMG CoA reductase which reduces the levels of mevalonate and thus farnesyl pyrophosphate, blocked oncogenic H-Rasva112 induced germinal vesicle breakdown in oocytes. This mevinolin caused block was completely reversed by co-injected farnesyl pyrophosphate. The putative farnesyltransferase in Xenopus oocytes was identified to be similar to those found in mammalian cells in that it requires an intact CAAX box motif in addition to the conserved cysteine residue at the fourth position from the C-terminus of Ras proteins for its farnesylating activity. Peptide inhibitors of farnesyltransferase such as CVIM and TKCVIM were shown to inhibit farnesylation of microinjected Ras proteins thereby blocking its function namely the induction of oocyte maturation. These results demonstrate that Xenopus oocytes process bacterially produced mammalian Ras proteins in a manner similar to, if not identical with that in mammalian cells, thus validating the continued use of the Xenopus oocyte system for unraveling the functions of Ras proteins. Furthermore, our results indicate that the oocyte system may be a useful in vivo model for studying the farnesylation of human Ras proteins, its regulation, and the effects of farnesyltransferase inhibitors.

Ha-ras proteins exhibit GTPase activity: point mutations that activate Ha-ras gene products result in decreased GTPase activity.

Several ras genes have been expressed at high levels in Escherichia coli and the resultant ras proteins were shown to be functional with respect to their well-known specific, high-affinity, GDP/GTP binding. We were able to detect a weak GTPase activity associated with the purified proteins. The normal cellular ras protein (p21N) exhibits approximately equal to 10 times higher GTPase activity than the "activated" proteins. Even though the turnover rate of the reaction is very low (0.02 mol of GTP hydrolyzed per mol of p21N protein per minute), the reaction appears to be catalytic; one molecule of p21N hydrolyzes more than one molecule of GTP. The GTPase and the GDP binding activities both have been recovered from a Mr 23,000 protein eluted following NaDodSO4/polyacrylamide gel electrophoresis, suggesting that these two activities are associated with the same protein. Mg2+ ions and dithiothreitol are required for GTPase activity and the optimal pH is between 7 and 8. Guanidine X HCl, which is required for solubilizing bacterially expressed ras protein, is strongly inhibitory to GTPase activity at concentrations higher than 0.5 M.