N-myristoyl-transferase activity in cancer cells. Solubilization, specificity and enzymatic inhibition of a N-myristoyl transferase from L1210 microsomes

The activity catalyzed by N-myristoyl transferase (NMT) is described for the first time in microsome-rich fractions from the murine leukemia cell line L1210, rat brain and mouse liver as biological sources. The enzyme from each source can accommodate various types of proteins (protein kinase A, virus structural gag protein or pp60src) as modelized by the use of their N-terminal derived peptides (GNAAAARR, GQTVTTPL and GSSKSKPKDP, respectively). As for some other types of membrane-bound enzymes, NMT activity can be enhanced by pretreatment with various types of detergents, amongst which Triton 770 and deoxycholate were the most potent. Further experiments on the L1210 microsome-rich fractions demonstrate that these two detergents were able to solubilize the microsomal enzyme, without modifying its substrate specificity. Finally, three compounds described in the literature to be inhibitors of NMT activity from other sources were tested for L1210 microsome-associated activity. None of them show any significant potency in inhibiting this activity. A new compound, myristoylphenylalanine, shows a slightly better inhibitory effect on the L1210 microsomal activity than the reference compounds with a median inhibitory concentration (IC50) of 0.2 mM.     

A fluorescence-based assay for N-myristoyltransferase activity

N-myristoylation is the irreversible attachment of a C(14) fatty acid, myristic acid, to the N-terminal glycine of a protein via formation of an amide bond. This modification is catalyzed by myristoyl-coenzyme A (CoA):protein N-myristoyltransferase (NMT), an enzyme ubiquitous in eukaryotes that is up-regulated in several cancers. Here we report a sensitive fluorescence-based assay to study the enzymatic activity of human NMT1 and NMT2 based on detection of CoA by 7-diethylamino-3-(4-maleimido-phenyl)-4-methylcoumarin. We also describe expression and characterization of NMT1 and NMT2 and assay validation with small molecule inhibitors. This assay should be broadly applicable to NMTs from a range of organisms.     

Development of an enzyme-linked immunosorbent assay for measurement of activity of myristoyl-coenzyme A:protein N-myristoyltransferase

Myristoyl-coenzyme A (CoA):protein N-myristoyltransferase (NMT) catalyzes the covalent attachment of myristate to the N-terminal glycine residue of various proteins. To develop a high-throughput assay for NMT, the principle of enzyme-linked immunosorbent assay (ELISA) is used, in which anti-N-myristoylglycine (anti-N-Myr-Gly) monoclonal antibody is utilized for the detection of the N-myristoylglycine moiety of the product of NMT catalysis. Enzyme-catalyzed reaction was performed using recombinant NMT expressed in Escherichia coli, myristoyl-CoA, and an octapeptide substrate that is biotinylated at its C terminus. The mixture of the products of the reaction was added to immunoplate wells precoated with anti-N-Myr-Gly monoclonal antibody. Then, the N-myristoyl-biotinylated octapeptide product was specifically captured by the antibody and stained with streptavidin-biotinylated peroxidase and tetramethylbenzidine substrate. This was followed by absorbance measurement (lambda(450)-lambda(630)). In this ELISA, the calibration curve showed a strong correlation between the concentration of the synthetic N-myristoyl-biotinylated octapeptide and the absorbance, indicating that this system may be useful for enzyme kinetics studies. Using this ELISA system, we assayed for serinal derivatives to determine their NMT inhibitory activity and found that serinal bisulfite inhibits yeast NMT activity. This is the first report of the measurement of NMT activity by the ELISA system.     

An h.p.l.c. assay for protoporphyrinogen oxidase activity in rat liver.

An h.p.l.c. method is described for the assay of protoporphyrinogen oxidase activity in rat liver. A relatively pure protoporphyrinogen IX substrate was obtained by selectively removing any protoporphyrin IX unreduced by sodium amalgam on a small disposable cartridge packed with a strong anion-exchanger. The protoporphyrin IX formed was extracted with dimethyl sulphoxide/methanol (3:7, v/v) containing mesoporphyrin as the internal standard for separation and quantification by reversed-phase chromatography. The Km for protoporphyrinogen was 9.5 +/- 1.6 microM, and the enzyme activities were 0.59 +/- 0.11 nmol of protoporphyrin IX produced/min per mg of mitochondrial protein and 33.5 +/- 2.7 nmol protoporphyrin IX produced/min per g of liver tissue homogenate. The method is applicable to the determination of enzyme activity in small amounts of human liver biopsy.     

A nonradioactive assay method for determination of enzymatic activity of D-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco)

A sensitive and nonradioactive assay method for activity determination of Rubisco is described. The method is based on thin-layer chromatographic separation of 3-phosphoglycerate (3-PGA) and D-ribulose-1,5-bisphosphate (RuBP). This assay method allows the quantitative determination of Rubisco activity. Rates of carbon dioxide fixation on RuBP determined by this method were comparable to those obtained independently by other methods. This assay method is reproducible and relatively free from interference.     

The role of the gulose-mannose part of bleomycin in activation of iron-molecular oxygen complexes.

A comparison of the complexing properties of metal ions and O2 activation by bleomycin-A2 (BLM-A2) and deglyco-BLM-A2 is presented. Deglyco-BLM-A2 is obtained from the parent derivative by HF cleavage of the sugar moiety followed by h.p.l.c. purification. Complexing of Cu(II) and Fe(III) is studied by using c.d. and e.s.r. spectroscopy. Spin-trapping experiments in the presence of phenyl N-t-butylnitrone indicated lower production of free radicals by deglyco-BLM-A2. Finally, a proposal is made to explain this discrepancy, focusing on the probable role of the gulose-mannose moiety acting as a protecting pocket, comparable with the pocket and picket-fence porphyrins described for haemoproteins.     

Inhibition by glucosamine of myristoylation in human H9 lymphocytes and rat liver cells

N-Myristoyl transferase (NMT) activity was measured in rat liver and H9 cells using an in vitro assay based on acylation of synthetic peptides. Glucosamine was found to inhibit the NMT activity. Using a synthetic peptide mimicking the N-terminus of HIV p27nef a Km value of 2.4 microM and a Vmax of 240 pmol/mg per h was found. In the presence of glucosamine the Vmax was lowered indicating that glucosamine acted as a non-competitive inhibitor. Glucosamine also inhibited incorporation of radiolabelled myristic acid into H9 cell proteins in vivo. In liver cells using a peptide from the N-terminus of p60 SRC only the Vmax was affected.     

Mechanisms of action of NIP71 on N-myristoyltransferase activity

N-Myristoyl-CoA:protein N-myristoyltransferase (NMT) is the enzyme that catalyses the transfer of myristate from myristoyl-CoA to the N-terminal glycine of protein substrates. NMT was highly purified from bovine brain by procedures involving sequential column chromatography on DEAE-Sepharose CL-6B, phosphocellulose, hydroxylapatite, and mono S and mono Q f.p.l.c.. The highly purified NMT (termed NMT·II) possessed high specific activity with peptide substrates derived from the N-terminal sequences of the cAMP-dependent protein kinase and pp60^src (29,800 and 47,600 pmol N-myristoylpeptide formed/min/mg, respectively), intermediate activity with a peptide based on the N-terminal sequence of a viral structural protein (l) (M2; 17,300 pmol N-myristoylpeptide formed/min/mg) and very low activity with a peptide derived from the N-terminal sequence ofmyristoylatedalanine-richC-kinasesubstrate (MARCKS; 1500 pmol myristoylpeptide formed/min/mg). An NMT protein inhibitor (NIP₇₁) isolated from the particulate fraction of bovine brain (King MJ and Sharma RK: Biochem J 291635-639, 1993) potently inhibited highly purified NMT activity (IC₅₀ 23.7 nM). A minor NMT activity (NMT·PU; 30% total NMT activity), which failed to bind to phosphocellulose, was insensitive to NIP₇₁ inhibition. Inhibition of NMT was observed to be via mixed inhibition with respect to both the myristoyl-CoA and peptide substrates with NIP₇₁ having an apparent higher affinity for NMT than the NMT·myristoyl·CoA complex. Inhibition by NIP₇₁ at subsaturating concentrations of myristolyl-CoA and peptide resulted in a sigmoidal pattern of inhibition indicating that bovine brain possesses a potent and delicate on/off switch to control NMT activity.Abbreviations NMT N-myristoyl-CoA:protein N-myristoyltransferase - NMT·I mono Q N-myristoyl-CoA:protein N-myristoyltransferase peak I - NMT·II mono Q N-myristoyl-CoA:protein N-myristoyltransferase peak II - NMT·III mono Q N-myristoyl-CoA:protein N-myristoyltransferase peak III - NIP₇₁ 71 kDa heat-stable N-myristoyltransferase inhibitor protein     

The sporulation-specific penicillin-binding protein 5a from Bacillus subtilis is a DD-carboxypeptidase in vitro

A chemiluminescence method for determining acetylcholinesterase activity is described. It is an adaptation of the chemiluminescence assay of acetylcholine described by Israël & Lesbats [(1981) Neurochem. Int. 3, 81-90; (1981) J. Neurochem. 37, 1475-1483]. The acetylcholinesterase activity is measured by monitoring the increase in light emission produced by the accumulation of choline or by determining the amount of choline generated after a short interval. The assay is rapid and sensitive, and uses the natural substrate of the enzyme. Kinetic data obtained with this procedure for acetylcholinesterase from Torpedo and Electrophorus electric organs were comparable with those obtained by using the method of Ellman, Courtney, Andres & Featherstone [(1961) Biochem. Pharmacol. 7, 88-95]. In addition, it was shown that sodium deoxycholate totally inactivated Torpedo acetylcholinesterase but not the Electrophorus enzyme. Competitive inhibitors of acetylcholinesterase protected the enzyme from inactivation.     

A new,robust, and nonradioactive approach for exploring N-myristoylation

Myristoyl-CoA (CoA):protein N-myristoyltransferase (NMT) catalyzes protein modification through covalent attachment of a C14 fatty acid (myristic acid) to the N-terminal glycine of proteins, thus promoting protein-protein and protein-membrane interactions. NMT is essential for the viability of numerous human pathogens and is also up-regulated in several tumors. Here we describe a new, nonradioactive, ELISA-based method for measuring NMT activity. After the NMT-catalyzed reaction between a FLAG-tagged peptide and azido-dodecanoyl-CoA (analog of myristoyl-CoA), the resulting azido-dodecanoyl-peptide-FLAG was coupled to phosphine-biotin by Staudinger ligation, captured by plate-bound anti-FLAG antibodies and detected by streptavidin-peroxidase. The assay was validated with negative controls (including inhibitors), corroborated by HPLC analysis, and demonstrated to function with fresh or frozen tissues. Recombinant murine NMT1 and NMT2 were characterized using this new method. This versatile assay is applicable for exploring recombinant NMTs with regard to their activity, substrate specificity, and possible inhibitors as well as for measuring NMT-activity in tissues.     

A malachite green colorimetric assay for protein phosphatase activity.

A simple and sensitive colorimetric assay for protein phosphatase activity based on the determination of released Pi by an improved malachite green procedure (A. A. Baykov, O. A. Evtushenko, and S.M. Avaeva, 1988, Anal. Biochem. 171, 266-270) is described. Proteins must be removed or stabilized prior to Pi determination with 0.25 N sulfuric acid or 3% (w/w) perchloric acid. Alternatively, to avoid possible acid hydrolysis of phosphate groups from organic compounds during deproteinization, the protein present in the phosphatase assay mixture can be stabilized with sodium dodecyl sulfate. In this case, the excess detergent is subsequently removed by precipitation with KCl because it colors with the malachite green reagent. The above procedure was applied to the determination of phosphorylase phosphatase activity in bovine brain extracts and the results are comparable to those obtained with the radioisotopic phosphatase assay.     

Measurement of 5-aminolaevulinate synthase activity by gas-liquid chromatography with electron-capture detection.

A specific assay for 5-aminolaevulinate synthase activity is described, with a sensitivity comparable with that of radiochemical assays. It is based on measurement by g.l.c. with electron-capture detection of the pentafluorobenzyl ester of the ethyl acetoacetate pyrrole derivative of 5-aminolaevulinic acid, and of the corresponding compound from 6-amino-5-oxohexanoic acid used as internal standard. Enzyme activity has been measured in homogenates of rat liver, spleen, kidney and brain, and in human lymphocytes.     

A continuous 96-well plate spectrophotometric assay for branched-chain amino acid aminotransferases

A new, continuous 96-well plate spectrophotometric assay for the branched-chain amino acid aminotransferases is described. Transamination of L-leucine with alpha-ketoglutarate results in formation of alpha-ketoisocaproate, which is reductively aminated back to L-leucine by leucine dehydrogenase in the presence of ammonia and NADH. The disappearance of absorbance at 340 nm due to NADH oxidation is measured continuously. The specific activities obtained by this procedure for the highly purified human mitochondrial and cytosolic isoforms of BCAT compare favorably with those obtained by a commonly used radiochemical procedure, which measures transamination between alpha-ketoiso[1-14C]valerate and L-isoleucine. Due to the presence of glutamate dehydrogenase substrates (alpha-ketoglutarate, ammonia, and NADH) and L-leucine (an activator of glutamate dehydrogenase) in the standard assay mixture, interference with the measurement of BCAT activity in tissue homogenates by glutamate dehydrogenase is observed. However, by limiting the amount of ammonia and including the inhibitor GTP in the assay mixture, the interference from the glutamate dehydrogenase reaction is minimized. By comparing the rate of loss of absorbance at 340 nm in the modified spectrophotometric assay mixture containing leucine dehydrogenase to that obtained in the modified spectrophotometric assay mixture lacking leucine dehydrogenase, it is possible to measure BCAT activity in microliter amounts of rat tissue homogenates. The specific activities of BCAT in homogenates of selected rat tissues obtained by this method are comparable to those obtained previously by the radiochemical procedure.     

Direct measurement of NAD(P)H:quinone reductase from cells cultured in microtiter wells: a screening assay for anticarcinogenic enzyme inducers

We describe a rapid and direct assay of NAD(P)H:(quinone-acceptor) oxidoreductase (EC 1.6.99.2) activity in cultured cells suitable for identifying and purifying inducers of this detoxication enzyme. Hepa 1c1c7 murine hepatoma cells are plated in 96-well microtiter plates, grown for 24 h, and exposed to inducing agents for another 24 h. The cells are then lysed and quinone reductase activity is assayed by the addition of a reaction mixture containing an NADPH-generating system, menadione (2-methyl-1,4-naphthoquinone), and MTT [3-(4,-5-dimethylthiazo-2-yl)-2,5-diphenyltetrazolium bromide]. Quinone reductase catalyzes the reduction of menadione to menadiol by NADPH, and MTT is reduced nonenzymatically by menadiol resulting in the formation of a blue color which can be quantitated on a microtiter plate absorbance reader. The reaction is more than 90% dicoumarol inhibitable and menadione dependent. The results are comparable to those obtained by harvesting cells from larger plates, preparing cytosols, and carrying out spectrophotometric measurements.     

A high-performance-liquid-chromatographic method for the assay of coproporphyrinogen oxidase activity in rat liver.

An h.p.l.c. method was developed for the assay of coproporphyrinogen oxidase activity in rat liver. The protoporphyrinogen IX formed is completely oxidized to protoporphyrin IX for separation and quantification by reversed-phase chromatography with mesoporphyrin as the internal standard. The Km of coproporphrinogen oxidase is 1.01 +/- 0.23 microM. The activities are 4.07 +/- 0.40 nmol of protoporphyrin IX/h per mg of mitochondrial protein and 224 +/- 19 nmol of protoporphyrin IX/h per g of liver tissue homogenate. The method is sensitive enough for measuring enzyme activity in small amounts of human tissue from needle biopsy.     

High-performance liquid chromatography of uroporphyrinogen and coproporphyrinogen isomers with amperometric detection.

A reversed-phase h.p.l.c. system, with an ODS-Hypersil column with acetonitrile or methanol in ammonium acetate buffer as mobile phase, is described for the separation of uro-and copro-porphyrinogen isomers. The porphyrinogens are detected amperometrically with sensitivity comparable with that of the fluorescent detection of porphyrins. The effects of pH, buffer concentration and organic modifiers on retention and resolution were studied. The method is suitable for both analytical and preparative separation of porphyrinogens.     

Continuous pH-stat titration method for the assay of lipoprotein lipase activity in vitro

A method was described where the hydrolysis of triolein to fatty acids and glycerol by lipoprotein lipase (LpL) was continuously monitored by titration of the liberated protons in a pH-stat automatic titrator. The method gave results comparable to those obtained by a conventional assay, and was also found to be applicable to the study of crude and partially purified enzyme preparations from both cow milk and rat adipose tissue. In agreement with earlier results obtained by other methods, the first product of hydrolysis was identified as 1,2-(2,3-)diglyceride, which was further converted to 2- or 1-monoglyceride. A good comparison between the titration and conventional methods was also obtained when peptides from human serum very low density lipoproteins (VLDL) were tested either as activators or inhibitors of LpL. Thus, the method appears suitable also for a systematic investigation of the role of substrates, activator(s) and inhibitors on LpL activity in vitro.     

Using a Non-Image-Based Medium-Throughput Assay for Screening Compounds Targeting N-myristoylation in Intracellular Leishmania Amastigotes

We have refined a medium-throughput assay to screen hit compounds for activity against N-myristoylation in intracellular amastigotes of Leishmania donovani. Using clinically-relevant stages of wild type parasites and an Alamar blue-based detection method, parasite survival following drug treatment of infected macrophages is monitored after macrophage lysis and transformation of freed amastigotes into replicative extracellular promastigotes. The latter transformation step is essential to amplify the signal for determination of parasite burden, a factor dependent on equivalent proliferation rate between samples. Validation of the assay has been achieved using the anti-leishmanial gold standard drugs, amphotericin B and miltefosine, with EC₅₀ values correlating well with published values. This assay has been used, in parallel with enzyme activity data and direct assay on isolated extracellular amastigotes, to test lead-like and hit-like inhibitors of Leishmania N-myristoyl transferase (NMT). These were derived both from validated in vivo inhibitors of Trypanosoma brucei NMT and a recent high-throughput screen against L. donovani NMT. Despite being a potent inhibitor of L. donovani NMT, the activity of the lead T. brucei NMT inhibitor (DDD85646) against L. donovani amastigotes is relatively poor. Encouragingly, analogues of DDD85646 show improved translation of enzyme to cellular activity. In testing the high-throughput L. donovani hits, we observed macrophage cytotoxicity with compounds from two of the four NMT-selective series identified, while all four series displayed low enzyme to cellular translation, also seen here with the T. brucei NMT inhibitors. Improvements in potency and physicochemical properties will be required to deliver attractive lead-like Leishmania NMT inhibitors.     

Biochemical,immunological and genetic characterization of natural gramine-free variants of Hordeum vulgare L.

Many barley cultivars (e.g. Arimar) contain the indole alkaloid gramine, but some do not. Among seven gramine-free cultivars tested, two phenotypic classes were found: those with a normal level of the N-methyltransferase (NMT) activity that catalyzes the last two steps of gramine synthesis (e.g. Proctor); and those having neither NMT activity nor protein recognized by polyclonal antibodies raised against purified NMT (e.g. Morex).A 3 × 3 diallel cross with reciprocals was made using cultivars Arimar, Proctor and Morex. The pattern of occurrence of gramine and NMT activity among the F₁ hybrids suggested that Proctor and Morex carried defective alleles of the same nuclear gene governing an early step in the indole alkaloid pathway, and that Morex also carried a recessive allele at a nuclear locus encoding NMT activity. However, no non-parental alkaloid phenotypes were found in the F₂ generation from an Arimar × Morex cross and the ratio of progeny with gramine to those with no alkaloids was 3 : 1. One explanation of these results is tight linkage between genes controlling two of the steps in gramine biosynthesis.