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PubMed1982

Purification and properties of human liver aldehyde reductases.

Two NADPH-linked aldehyde reductases (alcohol:NADP+ oxidoreductase, EC 1.1.1.2), referred to here as aldehyde reductases I and II, have been purified to homogeneity from human liver by using ammonium sulfate precipitation, ion-exchange chromatography, affinity chromatography and gel filtration. Structural studies show that aldehyde reductase II is a monomer of about 32 000 daltons, whereas aldehyde reductase I is a dimer of two nonidentical subunits of molecular weights about 42 000 and 35 000. The isoelectric pH was determined to be 5.40 for aldehyde reductase II and 8.25 for aldehyde reductase I. Substrate specificity studies show that neither aldehyde reductase I nor II uses glucose as substrate but that both are capable of reducing various other aldehydes such as pyridine 3-aldehyde, butyraldehyde and DL-glyceraldehyde. The pH optimums for aldehyde reductases I and II are pH 6.0 and 7.0 respectively. Aldehyde reductase I uses both NADH and NADPH as cofactor, whereas aldehyde reductase II activity is dependent on NADPH. Aldehyde reductase I activity is more susceptible than aldehyde reductase II activity to inhibition by p-hydroxymercuribenzoate, as reflected by IC50 values of 7.5 microM and 40 microM for aldehyde reductases I and II, respectively. The susceptibility of human liver aldehyde reductases I and II to inhibition by the aldose reductase (EC 1.1.1.21) inhibitors 3,3'-tetramethylene glutaric acid, alrestatin, chromone and sorbinil was determined and compared with that of aldose reductase partially purified from bovine lenses. The aldose reductase inhibitors, besides inhibiting aldose reductase, also inhibit human liver aldehyde reductases I and II to varying degrees.

Alcohol Oxidoreductases↗
PubMed1984

Phosphorus-31 nuclear magnetic resonance study on cytoplasmic aspartate aminotransferase from pig heart. A reinvestigation.

Striking differences of the environment of the phosphate group of pyridoxal-P in cytoplasmic and mitochondrial aspartate aminotransferase have been reported. Since most details of the three-dimensional structure of the active sites of these isozymes are identical, it seemed difficult to rationalize the reported differences. Therefore, the cytoplasmic isozyme was reinvestigated using 31P-NMR at 72.86 MHz. The 31P chemical shift of the cofactor of this isozyme was found to be pH-dependent with a pKa of 6.2. In the presence of 100 mM succinate or 100 mM glutarate, the 31P chemical shift of bound pyridoxal-P remains at 4.71 or 4.79 ppm, respectively, in the pH range from 5.0 to 8.0, indicating that the phosphate group of the cofactor appears to be in its dianionic form. Reduction of the internal pyridoxal-P Schiff's base dramatically increases the pKa of the phosphate group of the phosphopyridoxyl moiety of the protein to 8.3. Hence, our results on the cytoplasmic isozyme are similar to those reported for the mitochondrial isozyme.

Animals↗
PubMed1985

Purification of liver glutamate dehydrogenase by affinity precipitation and studies on its denaturation.

In the presence of glutaric acid, N2,N2'-adipodihydrazido-bis(N6-carbonylmethyl-NAD+)(bis-NAD+ ) forms cross-links between molecules of glutamate dehydrogenase, resulting in precipitation. The dependence of this process on bis-NAD+ and enzyme concentration has been investigated. This procedure has been shown to be effective in the purification of glutamate dehydrogenase from rat and ox liver, and a procedure is presented in which this affinity precipitation procedure is used instead of the affinity chromatography used in an earlier method (McCarthy, A.D., Walker, J.M. and Tipton, K.F. (1980) Biochem. J. 191, 605-611). The ox liver enzyme prepared in this way had not suffered the limited proteolysis that occurs during the preparation of the enzyme by other commonly used procedures. After the purified enzyme had been denatured by treatment with urea, guanidine hydrochloride, or low pH, no recovery of activity could be demonstrated following dilution or, in the last case, dialysis.

Affinity Labels↗
PubMed1992

Expression of a hyperthermophilic aspartate aminotransferase in Escherichia coli.

The gene for an archaebacterial hyperthermophilic enzyme, aspartate aminotransferase from Sulfolobus solfataricus (AspATSs), was expressed in Escherichia coli and the enzyme purified to homogeneity. A suitable expression vector and host strain were selected and culture conditions were optimized so that 6-7 mg of pure enzyme per litre of culture were obtained repeatedly. The recombinant enzyme and the authentic AspATSs are indistinguishable: in fact, they have the same molecular weight, estimated by means of SDS-PAGE and gel filtration, the same Km values for 2-oxo-glutarate and cysteine sulphinate and the same UV-visible spectra. Moreover, recombinant AspATSs is thermophilic and thermostable just as the enzyme extracted from Sulfolobus solfataricus. The protocol described may be used to produce thermostable arachaebacterial enzymes in mesophilic hosts.

Aspartate Aminotransferases↗
PubMed1990

Radioisotopic measurement of femtomolar amounts of NAD(P)H in the assay of enzymatic activity at a single cell level.

A radioisotopic method for the assay of reduced or oxidized pyridine nucleotides, based on the interconversion of 2-[U-14C]ketoglutarate or 2-keto[3,4-3H]glutarate and labelled L-glutamate in the reaction catalyzed by glutamate dehydrogenase, was applied to the measurement of lactate dehydrogenase activity in rat pancreatic islet homogenates. Using the tritiated tracer, the limit of sensitivity of the procedure for NAD(P)H assay was close to 1.0 fmol/sample, and lactate dehydrogenase activity could be measured in as little as 0.0005 islet/sample i.e., at a single cell level. This radioisotopic procedure, which can be used for the assay of various metabolites and enzymic activities, thus provides a tool for investigating the heterogeneity in metabolic behaviour of individual cells.

Animals↗
PubMed1991

Glycine transport by intestinal brush border vesicles of the amphibian Discoglossus pictus.

1. In order to determine the different components of glycine uptake by the intestine of the frog, Discoglossus pictus, we have used brush border membrane vesicles isolated by a classical precipitation technique. 2. Enzymatic tests showed that a good purification was obtained. The concentration ratio of alkaline phosphatase was 14.8. 3. Glycine entry in vesicles as a function of time, in presence or absence of sodium, indicated an overshoot which decreased when incubation time was prolonged. The overshoot was dependent on the presence of sodium. 4. The nature of the anion associated to sodium had little effect on glycine uptake. Nevertheless, chloride and thiocyanate appeared more efficient than glutarate. 5. The effect of transmembrane potential was studied by using valinomycin associated with a potassium gradient. The addition of this substance stimulated glycine transport by 43%. 6. The transport at different glycine concentrations showed two components: one non-saturable with weak affinity and the other saturable with strong affinity (Kt = 0.338 mM). 7. In conclusion, glycine transport by the brush border of D. pictus intestine presents a saturable component depending on sodium and on transmembrane electrical potential.

Amino Acids↗
PubMed1981

The effect of pyrazines on the metabolism of tryptophan and nicotinamide adenine dinucleotide in the rat. Evidence of the formation of a potent inhibitor of aminocarboxy-muconate-semialdehyde decarboxylase from pyrazinamide.

The intraperitoneal or oral administration of pyrazinamide and pyrazinoic acid (pyrazine 2-carboxylic acid) resulted in a marked increase of the NAD content in rat liver. The injections of pyrazine and pyrazine 2,3-dicarboxylic acid exhibited no significant effect on the hepatic NAD content. The boiled extract obtained from liver and kidney of rat injected with either pyrazinamide or pyrazinoic acid exhibited a potent inhibitory effect on the aminocarboxymuconate-semialdehyde decarboxylase (EC 4.1.1.45) activity in either lier or kidney, although pyrazinamide or pyrazinoic acid per se did not inhibit the enzyme activity. The unknown inhibitor of aminocarboxymuconate-semialdehyde decarboxylase was dialysable and heat-stable, and mostly excreted in urine by 6 and 12 h after injected of pyrazinoic acid and pyrazinamide, respectively. Pyrazine 2,3-dicarboxylic acid, pyrazine, nicotinamide, nicotinic acid, tryptophan, anthranilic acid, 5-hydroxyanthranilic acid and quinolinic acid exhibited no significant effect on the aminocarboxymuconate-semialdehyde decarboxylase activity in liver and kidney at the concentration of 1 mM in the reaction mixture. The expired 14CO2 from L-[benzen ring-U-14C]tryptophan was markedly decreased by the pyrazinamide injection, while the urinary excretion of 14C-labeled metabolites from L-tryptophan, mainly quinolinic acid, was markedly increased. These results suggest that the glutarate pathway of L-tryptophan was strongly inhibited by the inhibitor produced after the administration of pyrazinoic acid and pyrazinamide. Pyrazinamide but not pyrazinoic acid also exhibited a significant inhibition of the nuclear enzyme poly(ADP-ribose) synthetase in rat liver.

Animals↗
PubMed1981

Stabilization of the colchicine-binding activity of tubulin by organic acids.

A number of carboxylic acids and organic phosphates were found to be highly effective in stabilizing the colchicine-binding activity of calf brain tubulin. The most active were glutamate, glutarate, delta-aminovalerate, glucose 1-phosphate, glucose 6-phosphate, fructose 1,6-(bis)phosphate, creatine phosphate and 6-phosphogluconate Maximum effects occurred at high concentrations. Combinations of agents were also examined, and the most effective mixture for stabilizing tubulin found thus far was the combination of 1.0 M glutamate, 100 mM glucose 1-phosphate, 1 mM GTP and 0.5 mg/ml of albumin. No loss of activity occurred over 48 h at 37 degrees C with tubulin was present at a concentration of 100 microgram/ml.

Animals↗
PubMed1996

NAD-glutamate dehydrogenase from Halobacterium halobium: inhibition and activation by TCA intermediates and amino acids.

A variety of metabolites have been found to elicit a form of inhibition or activation on an NAD-specific glutamate dehydrogenase (NAD-GDH, EC 1.4.1.2) from Halobacterium halobium. The purified halophilic enzyme was tested with several compounds known to be allosteric modifiers of mammalian glutamate dehydrogenases to determine their effects on enzyme activity. GTP, ATP, ADP and AMP did not affect the enzyme, so these effectors of bovine glutamate dehydrogenase do not play a role in the regulation of the halophilic enzyme. However, the halophilic enzyme was subject to strong inhibition by TCA intermediates. When measuring the initial rate of the reaction, the oxidative deamination of L-glutamate was inhibited by TCA metabolites such as: fumarate, oxalacetate, succinate and malate; by substrate analogues such as: NADP+, D-glutamate and glutarate; and by dicarboxylic compounds such as adipate. On the other hand, all the amino acids tested were activators of this enzyme, except the D-isomer of the substrate L-glutamate that acted as an inhibitor. The relative effectiveness of each inhibitor or activator (Ki or Ka values) was correlated with the dipole moment (mu), HOMO and LUMO molecular orbital energies, optimal distance between two carboxyl groups, and hydrophobicity. Compounds with high dipole moment acted as good activators while compounds with low dipole moment were inhibitors. We have also found that the best activators were amino acids with no polar lateral chain.

Amino Acids↗
PubMed1991

Automated screening of urine samples for carbohydrates, organic and amino acids after treatment with urease.

Eighty-five clinical urine samples and nineteen urine samples previously found by other laboratories to suggest genetic metabolic defects were prepared for trimethylsilylation by treatment with urease, followed by azeotropic dehydration. The "Target Analyte Search" program provided with the VG Trio 2 gas chromatograph-mass spectrometer required 6 min to quantify 103 compounds relative to endogenous urinary creatinine. This technique has been used to confirm diagnoses including cystinuria, lysinuria, medium-chain acyldehydrogenase deficiency, ornithine transcarbamylase deficiency, aspartylglucosaminuria, methylmalonic, propionic and glutaric acidurias.

Amino Acids↗
PubMed1994

Ethylmalonic aciduria: an organic acidemia with CNS involvement and vasculopathy.

Five infants from 3 families, one Egyptian, two Yemeni, are described with a progressive encephalopathy, four of whom have been studied in detail. All patients showed vascular lesions of the skin, characterized by waxing and waning petechiae and ecchymoses. Acrocyanosis was present in three patients. All patients showed retinal lesions characterized by tortuous veins. Protracted diarrhea was not a consistent finding, although they had metabolic crisis in association with diarrhea. They did not show failure to thrive. The neurologic symptoms were indicative of a progressive pyramidal tract disease. Three patients died following sudden emergence of severe basal ganglia, putaminal and head of caudate lesions. In one patient the CT changes in brain were suggestive of infarction. The patients who died manifested pulmonary congestion, or wet lung, and respiratory difficulties during the terminal stage of the disease. In all patients before and during the terminal event, mild-to-moderate hematuria, and in two RBC in CSF, was observed. In one patient there was mild hemoperitoneum at the terminal event. The urine organic acids indicated increased excretion of ethylmalonic, methylsuccinic, glutaric, and adipic acids. The patients invariably showed lactic acidosis, but no ketosis, during and in between the acidotic attacks of the disease. The acylcarnitine profile in blood of two patients showed a pronounced increase in C4 and C5 carnitine esters. In three patients, biopsies from petechiae indicated absence of an immune event, showing only fresh hemorrhage. An immunologic study in one patient was normal for the suppressor:cytotoxic lymphocyte ratio and concentration of interleukin-2 receptor during and in between hemorrhagic attacks. The cytochrome c oxidase activity in fibroblasts was normal. The rate of oxidation of glucose, leucine, isoleucine, valine, propionate and butyrate by fibroblasts was normal. The disease is not responsive to treatment with riboflavin, ascorbic acid, vitamin E, glycine, or carnitine. One patient remained stable on prolonged large doses of methylprednisolone. The biochemical defect leading to ethylmalonic aciduria in this disease remains unknown.

Acidosis↗
PubMed1994

CT and MR of the brain in the diagnosis of organic acidemias. Experiences from 107 patients.

The results of CT and/or MRI of the brain in 107 patients with different types of organic acidemia are presented. The CSF spaces were wide in more than two-thirds of the patients, in 46 slightly-to-moderately and in 26 markedly-to-severely dilated. Marked widening of the operculae was found in all 5 patients with glutaric acidemia type 1, but open opercula was also found in other organic acidemias. White matter changes were found in about half the patients, in 28 mildly-to-moderately pronounced, in another 28 marked or severe. Basal ganglia or central pathway pathology was seen in a total of 34 patients, i.e. 32%. These changes in 25 patients involved the caudate and/or lentiform nuclei: in 14 cases the T2 signal was increased and volume loss was present, in 9 cases increased T2 signal with preserved volume was found (in one of these the changes were transient). In 2 patients, both with ethylmalonic aciduria (cause unknown), only small high T2 spots were seen in the caudate heads and the putamina. In 4 patients, all suffering from methylmalonic acidemia, only the globus pallidus was affected. In 3 patients, all with beta-ketothiolase deficiency, high T2 intensity changes were seen only in the postero-lateral putamina. The remaining 8 patients represent a variety of different locations of lesions. The CT or MRI findings in many patients with organic acidemias should alert the radiologist that a neurometabolic disorder may be present; in some cases the location and appearance of the lesions may even suggest the correct diagnosis.

Acidosis↗
PubMed1991

Synthesis and biological activity of 17 alpha-alkynylamide derivatives of estradiol.

Seven estradiol (E2) derivatives with an alkynylamide side chain at the 17 alpha position were synthesized starting from ethynylestradiol (EE2). The main chemical step was the coupling reaction of the acetylide ion of EE2 with carbon dioxide, glutaric anhydride or bromoalkyl ortho ester. The synthesis of these compounds is fast (3-6 steps according to the compound) and is easily achieved with good yield. Five compounds with different side chain lengths were evaluated for uterotrophic and antiuterotrophic activity in the CD-1 mouse. None of the tested compounds shows estrogenic activity in this sensitive in vivo system. At low doses (1 and 3 micrograms), a 14-57% inhibition of E2-induced uterine growth was observed while no additional inhibition was observed at the 10, 20 and 30 micrograms doses. In human breast carcinoma cells in culture, all compounds show estrogenic activity at high concentrations while only compound 39 (N-butyl,N-methyl-8-[3',17' beta-dihydroxy estra-1',3',5'(10')-trien-17' alpha-yl]-7-octynamide) possesses antiproliferative or antiestrogenic effects. No significant correlation could be demonstrated between alkynylamide side chain length and estrogenic or antiestrogenic activity. Among the compounds tested, the derivative of EE2 possessing a five-methylene (CH2) side chain (compound 39) possesses the best antiestrogenic activity (44 +/- 7% in the CD-1 mouse uterus assay at the 3 micrograms dose and 57 +/- 4% at 0.1 nM in human ZR-75-1 cancer cells in culture.

Animals↗
PubMed1995

Bifunctional activity labels for selection of filamentous bacteriophages displaying enzymes.

Two bifunctional activity labels of beta-lactamases or penicillin binding proteins have been prepared. They feature a penicillin sulfone derivative, i.e. a suicide substrate of serine beta-lactamases, or a penicillin derivative connected to a biotin moiety through a spacer containing a disulfide bridge. The biotinyl spacer 4 was prepared by coupling biotin to epsilon-amino-caproic acid, then to cystamine, and purified by transient protection with t-Boc. The penicillin sulfone inhibitor 13 was prepared by chemoselective sulfonylation of methoxymethyl 6-aminopenicillinate with pentafluorophenoxy- or benzyloxy-carbonylmethylsulfonyl chloride (9), followed by permanganate oxidation. Both direct coupling of the activated ester 13b and indirect coupling of the acid 13c obtained by benzyl ester deprotection, afforded the biotinylated sulfone inhibitor 16. The acid 6 resulting from reaction of the biotinyl spacer 4 with glutaric anhydride was activated as pentafluorophenyl-ester 7 and reacted with 6-aminopenicillanic acid to afford the penicillin binding protein label 18. Selection of the most active beta-lactamase displayed on phage from a mixture containing less active enzymes could be accomplished in three rounds of labeling and affinity chromatography using suicide inhibitor 16.

Bacterial Proteins↗
PubMed2004

Protein carboxyl amidation increases the potential extent of protein polyethylene glycol conjugation.

Chemical coupling of polyethylene glycol (PEG) to therapeutic proteins reduces their immunogenicity and prolongs their circulating half-life. The limitation of this approach is the number and distribution of sites on proteins available for PEGylation (the N terminus and the -amino group of lysines). To increase the extent of PEGylation, we have developed a method to increase the number of PEGylation sites in a model protein, recombinant methionine alpha,gamma-lyase (recombinant methioninase; rMETase), an enzyme cancer therapeutic cloned from Pseudomonas putida. rMETase was first PEGylated with methoxypolyethylene glycol succinimidyl glutarate-5000 with a molar ratio of PEG:rMETase of 15:1. The carboxyl groups of the initially PEGylated protein were then conjugated with diaminobutane, resulting in carboxyl amidation. This reaction was catalyzed by 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide, a water-soluble carbodiimide. The steric hindrance provided by the PEG chains already coupled to the protein prevented cross-linking between rMETase molecules during the carboxyl amidation reaction. The carboxyl-amidated PEGylated rMETase was hyper-PEGylated at a molar ratio of PEG to PEG-rMETase of 60:1. Biochemical analysis indicated that 13 PEG chains were coupled to each subunit of rMETase after hyper-PEGylation compared with 6-8 PEG chains attached to the non-carboxyl-amidated PEG-rMETase. Approximately 15-20% of the non-PEGylated rMETase activity was retained in the hyper-PEGylated molecule. Immunogenicity of the hyper-PEG-rMETase was significantly reduced relative to PEG-rMETase and rMETase. Initial results suggest that hyper-PEGylation may become a new strategy for PEGylation of protein biologics.

Amides↗
PubMed2006

Ala-504 is a determinant of substrate binding affinity in the mouse Na(+)/dicarboxylate cotransporter.

The Na(+)/dicarboxylate cotransporters from mouse (mNaDC1) and rabbit (rbNaDC1) differ in their ability to handle adipate, a six-carbon terminal dicarboxylic acid. The mNaDC1 and rbNaDC1 amino acid sequences are 75% identical. The rbNaDC1 does not transport adipate and only succinate produced inward currents under two-electrode voltage clamp. In contrast, oocytes expressing mNaDC1 had adipate-dependent inward currents that were about 60% of those induced by succinate. In order to identify domains involved in adipate transport, we examined the functional properties of a series of chimeric transporters made between mouse and rabbit NaDC1. We find that multiple transmembrane helices (TM), particularly TM 8, 9, and 10, are involved in adipate transport. In TM 10 there is only one amino acid difference between the two proteins, corresponding to Ala-504 in mouse and Ser-512 in rabbit NaDC1. The mNaDC1-A504S mutant had decreased adipate-dependent currents relative to succinate-dependent currents and an increase in the K(0.5) for both succinate and glutarate. We conclude that multiple amino acids from TM 8, 9 and 10 contribute to the transport of adipate in NaDC1. Furthermore, Ala-504 in TM 10 is an important determinant of K(0.5) for both adipate and succinate.

Alanine↗
PubMed2006

Layer-by-layer self-assembled multilayer films of carbon nanotubes and platinum nanoparticles with polyelectrolyte for the fabrication of biosensors.

Platinum nanoparticle-doped chitosan (CHIT) solution can be easily prepared by treating the CHIT solution with aqueous H2PtCl6 solution followed by chemical reduction of Pt(IV) with NaBH4. Multiwalled carbon nanotubes (MWCNT) are then dispersed in the nanoparticle-doped solution. The resulting Pt-CNT-CHIT material brings new capabilities for electrochemical devices by using the synergistic action of Pt nanaoparticles and CNT. Positively charged Pt-CNT-CHIT solution and negatively charged poly(sodium-p-styrenesulfonate) salt (PSS) have been employed to fabricate stable ultrathin multilayer films on gold electrode and quartz glass slides in a layer-by-layer fashion. Cyclic voltammetric and UV-vis adsorption spectroscopy confirms the consecutive growth of the multilayer films. The modified gold electrode allows low-potential detection of hydrogen peroxide with high sensitivity and fast response time. With the immobilization of cholesterol oxidase onto the electrode surface using glutaric dialdehyde, a biosensor that responds sensitively to cholesterol has been constructed. In pH 6.98 phosphate buffer, almost interference free determination of cholesterol has been realized at 0.1 V vs. SCE with a linear range from 0.01 to 3mM and response time<30s. With the immobilization of another cholesterol esterase enzyme layer, the biosensor was used to determine total cholesterol in serum samples with satisfactory results.

Biosensing Techniques↗
PubMed2004

Cholesterol amperometric biosensor based on cytochrome P450scc.

A screen-printed enzyme electrode based on flavocytochrome P450scc (RfP450scc) for amperometric determination of cholesterol has been developed. A one-step method for RfP450scc immobilization in the presence of glutaraldehyde or by entrapment of enzyme within a hydrogel of agarose is discussed. The sensitivity of the biosensor based on immobilization procedures of flavocytochrome P450scc by glutaric aldehyde is 13.8 nA microM(-1) and the detection limit is 300 microM with a coefficient of linearity 0.98 for cholesterol in the presence of sodium cholate as detergent. The detection limits and the sensitivity of the agarose-based electrode are 155 microM and 6.9 nA microM(-1) with a linearity coefficient of 0.99. For both types of electrodes, the amperometric response to cholesterol in the presence of detergent was rather quick (1.5-2 min).

Biosensing Techniques↗