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Biomedical subjects

C Walsh

Publications and source records attributed to C Walsh.

At least 199 records · Page 11Linked to original sources
PubMed1981

Mechanism-based inactivation of serine transhydroxymethylases by D-fluoroalanine and related amino acids.

Serine transhydroxymethylase, from lamb or rabbit liver, is known to catalyze slow transamination of D-alanine, but not of L-amino acids, in a tetrahydrofolate-independent reaction. Both enzymes will process the D-isomer of beta-fluoroalanine for alpha, beta-elimination of HF to yield an aminoacrylate-pyridoxal-P-enzyme intermediate. This intermediate partitions between harmless hydrolysis to pyruvate, NH4+, and active enzyme-pyridoxal-P (catalytic turnover) and suicidal enzyme alkylation by covalent modification with an average partition ratio of 40-60 turnovers/inactivation event/monomer unit of this tetrameric enzyme. Enzyme inactivation occurs with stoichiometric incorporation of radioactive label from D-[1,2-14C]fluoroalanine. Titration of enzymic cysteinyl --SH groups with 5,5'-dithiobis(2-nitrobenzoate) indicates loss of 1 --SH group on inactivation. Acid hydrolysis of radioactive-inactive enzyme confirms cysteine residue modification. Treatment of inactive enzyme with 6 M urea, then KBH4, followed by acid hydrolysis yields two radioactive compounds, lanthionine and S-carboxyhydroxyethylcysteine, in about equal amounts. The addition of tetrahydrofolate stimulates both pyruvate production and inactivation to equal extents with about a 200-fold rate acceleration at 0.5 mM tetrahydrofolate to turnover numbers of approximately 120 min-1. The Km for D-fluoroalanine is high, 10-60 mM, and this low substrate affinity suggests D-fluoroalanine will not be a useful in vivo agent for selective inactivation of liver cell serine transhydroxymethylases.

Alanine↗
PubMed1980

Studies on the autoinactivation behavior of pure, reconstituted phenobarbital-induced cytochrome P-450 isozyme from rat liver.

NADPH-dependent turnover of O2, uncoupled from cosubstrate hydroxylation, in a reconstituted phospholipid system containing the cytochrome P-450 isozyme purified from the liver microsomes of phenobarbital-induced rats results in autocatalytic inactivation of this P-450 isozyme. At NADPH concentrations of 1.0 mM, half-times for inactivation are 7 to 9 min and reflect processing of 800 molecules of NADPH/enzyme molecule before an inactivation event occurs. Addition of saturating amounts of N,N-dimethylaniline leads to a 3-fold increase of Vmax of NADPH utilization with 40% of these extra molecules providing electrons for the hydroxylation/N-demethylation sequence. About 2300 turnovers occur over the time period of inactivation, with about 425 product formaldehyde molecules being generated in this interval. This inactivation does not occur in intact microsomes from the livers of phenobarbital-treated rats; however, solubilization of the microsomes or addition of exogenous pure reductase renders the P-450 susceptible to the same autoinactivation.

Allylisopropylacetamide↗
PubMed1980

Stereochemical studies of 8-hydroxy-5-deazaflavin-dependent NADP+ reductase from Methanococcus vannielii.

The purified 8-hydroxy-5-deazaflavin-dependent NADP+ reductase from Methanococcus vannielii catalyzes an oxidation-reduction reaction between a novel 8-hydroxy-5-deazaflavin cofactor and nicotinamide adenine dinucleotide phosphate. The reaction was shown to be a direct hydride transfer process. Using stereospecifically 3H-labeled substrates, the steric course of this process was established to be S-specific with respect to the nicotinamide nucleotide. The 8-hydroxy-5-deazaflavin-dependent NADP+ reductase from M. vannielii and the hydrogenase system in the cell-free extracts of Methanobacterium thermoautotrophicum recognize the same side, designated as A side, with respect to the prochiral center at C-5 of the dihydro-8-hydroxy-5-deazaflavin cofactor.

Euryarchaeota↗
PubMed1980

Resonance Raman and coherent anti-stokes Raman scattering spectra of flavin derivatives. Vibrational assignments and the zwitterionic structure of 8-methylamino-riboflavin.

Resonance Raman and coherent anti-stokes Raman scattering spectra are reported for flavins modified by deprotonation at N3 and by CH substitution for N1, N3 and N5, using laser excitation in resonance with the visible electronic transition. Vibrational shifts are used to make qualitative assignments of the observed vibrational modes. Both 8-CH3NH- and ionized 8-OH-riboflavin were found to give markedly different spectra patterns form flavin itself; this observation supports a partial zwitterionic structure for the 8-CH3NH derivatives.

Chemical Phenomena↗
PubMed1980

Flavin analogs as mechanistic probes of adrenodoxin reductase-dependent electron transfer to the cholesterol side chain cleavage cytochrome P-450 of the adrenal cortex.

The intrinsic isotope effect on the reduction of the FAD-containing dehydrogenase electron transferase, adrenodoxin reductase, by (4S)-[2H]NADPH has been determined to be 7.1 to 7.7. The replacement of FAD by a series of FAD analogs at the active site of adrenodoxin reductase with oxidation-reduction potentials which vary over a range of 212 mV has made it possible to extrapolate to this limiting value from the variation in the observed isotope effect on Vmax with flavin midpoint potential. Stop-flow studies which allow the direct determination of the intrinsic isotope effect on the reductive half-reaction corroborate this result. During the steady state reduction of ferricyanide by the native enzyme under conditions of Vmax, this isotope effect is almost fully expressed (VH/VD = 6.7 to 6.8). In contrast, we observe a dramatic attenuation of the intrinsic isotope effect (due to hydride transfer to flavin) when the oxidative half-reaction is mediated by the natural acceptor protein, the 2Fe/2S ferredoxin, adrenodoxin. In a coupled three-protein system, the adrenodoxin-mediated reductions of both the artificial electron acceptor, cytochrome c, and the physiological electron acceptor, cytochrome P-450scc, by adrenodoxin reductase occur at similar rates and with similar kinetic isotope effects (1.9 to 2.0) when (4S)-[2H]NADPH is the reductant. We infer similar mechanisms for the reduction of both cytochromes. These results are in agreement with previous studies (Lambeth, J.D., and Kamin, H. (1979) J. Biol. Chem. 254, 2766-2774) which show that the reductive half-reaction is not solely rate-determining in adrenodoxin-mediated processes. The observation of a linear free energy relationship between Vmax and the flavin midpoint potential during steady state reduction of ferricyanide confirms that the reductive half-reaction is rate-determining in this assay. The relationship between Vmax and flavin midpoint potential in reactions which require adrenodoxin suggests that the midpoint potential of native adrenodoxin reductase has been optimized. Thus, the apoenzyme of adrenodoxin reductase tailors the midpoint potential of bound FAD in order to balance the activation energies of the reductive and oxidative half-reactions.

Adrenal Cortex↗
PubMed1980

Mechanism-based inactivation of pig heart L-alanine transaminase by L-propargylglycine. Half-site reactivity.

L-Alanine transaminase (EC 2.6.1.2) from pig heart was found to be a dimer, with a subunit molecular weight of 55,000 and one pyridoxal phosphate bound/subunit. Seven free sulfhydryl groups/subunit were detected. Isoelectric focusing revealed three species (native pI values, 5.3 to 5.5). L-Propargylglycine was found to inactivate the enzyme at 37 degrees C with a KI = 3.9 mM and an observed maximal first order rate constant, kinact = 0.26 min-1. Incorporation of 1 [14C]propargylglycine molecule/dimer leads to greater than 97% inactivation, suggesting half-site reactivity, while the unalkylated subunit is still apparently capable of processing L-alanine, L-propargylglycine, and beta-chloro-L-alanine. The minimal stoichiometric ratio necessary for inactivation was determined to be 2.7 L-propargylglycine molecules/enzyme subunit, 2.2 molecules/subunit undergoing transamination before inactivation ensues. A deuterium kinetic isotope effect of 3.5 was observed for inactivation with DL-[2-2H]propargylglycine.

Alanine Transaminase↗
PubMed1980

Appearance of heat shock proteins during the induction of multiple flagella in Naegleria gruberi.

A heat shock to amebae of the amebo-flagellate Naegleria gruberi during differentiation into swimming flagellates results in the induction of heat shock proteins as well as multiple flagella. The principal heat shock proteins migrate on sodium dodecyl sulfate-polyacrylamide gels with apparent molecular weights of 96,000, 77,000, 70,000, and 68,000. These proteins are synthesized preferentially when cells at 25 degrees C are shifted to temperatures above 32 degrees C. The maximal incorporation of methionine into heat shock proteins occurs at 38.2 degrees C, the temperature at which maximal induction of multiple flagella has been reported. Synthesis of heat shock proteins requires new transcription as judged by the ability of actinomycin D to inhibit their synthesis during the first 15 min of heat shock but not thereafter. Although heat shock can induce multiple flagella only when applied during a restricted interval, heat shock proteins are induced at any time cells are shifted to 38.2 degrees C. The response to heat shock of the Naegleria heat shock proteins resembles that of Drosophila heat shock proteins, but the two groups of proteins differ in both size and number. Naegleria heat shock proteins are, however, strikingly similar in size to a group of heat-induced proteins found in chick embryo fibroblast, mouse L, and BHK cells.

Amoeba↗
PubMed1980

Oxygen reactivity of p-hydroxybenzoate hydroxylase containing 1-deaza-FAD.

The flavin prosthetic group (FAD) of p-hydroxybenzoate hydroxylase (EC 1.14.13.2) was replaced by 1-deaza-FAD (carbon substituted for nitrogen at position 1). An improved method for production of apoenzyme by precipitation with acidic ammonium sulfate was developed. The modified enzyme, in the presence of p-hydroxybenzoate, catalyzed the oxidation of NADPH by oxygen, yielding NADP+ and H2O2, but the ability to hydroxylate p-hydroxybenzoate and other substrates was lost. An analysis of the mechanism of NADPH-oxidase catalysis showed a close analogy between the reaction pathways for native and modified enzymes. In the presence of p-hydroxybenzoate, the rate of NADPH consumption catalyzed by the 1-deaza-FAD form was about 11% that of the native enzyme. Both formed a stabilized flavin-C (4a)-OOH intermediate upon reaction of reduced enzyme with oxygen, but the 1-deaza-FAD enzyme could not utilize this peroxide to hydroxylate substrates, and the peroxide decomposed to oxidized enzyme and H2O2.

4-Hydroxybenzoate-3-Monooxygenase↗
PubMed1980

Studies on the kinetics and stoichiometry of inactivation of Pseudomonas omega-amino acid:pyruvate transaminase by gabaculine.

A homogeneous pyruvate-requiring omega-amino acid transaminase from Pseudomonas species F-126 has been examined for its behavior with gamma-aminobutyrate (GABA) as omega-amino acid substrate and for its susceptibility to the cyclic dihydroaromatic GABA analogue, gabaculine, a known suicide substrate for alpha-ketoglutarate-requiring GABA transaminases (Biochemistry 16, 4604-4610, 1977). One isomer of DL-gabaculine serves as a completely efficient titrant (no product molecules released) for this omega-amino acid transaminase by the anticipated mechanism of bound pyridoxal 5'-phosphate (PLP) derivitization. Stoichiometric titration with [2-3H]gabaculine reveals full inactivation at 0.45 labels/enzyme tetramer (see below), consistent with the subsequent demonstration that there is only 0.45 PLP molecule, bound as phenylhydrazine-titratable aldehyde form, per tetramer. Spectroscopic monitoring of inactivation also agrees with formation, on full inactivation, of 0.45 mol of m-anthranilyl-PNP adduct as the species quantitatively responsible for enzyme inactivation. Incubation of enzyme with excess PLP at 60 degrees C allows loading of enzyme with coenzyme to an average level of approximately 1 PLP/subunit, but even in this case activity is only increased up to 1.5-fold, and 1 to 1.5 molecules of gabaculine/tetramer cause complete inactivation. These data may indicate negative cooperativity between subunits.

Cyclohexanecarboxylic Acids↗
PubMed1980

The metabolic activation of benz[a]anthracene in hamster embryo cells: evidence that diol-epoxides react with guanosine, deoxyguanosine and adenosine in nucleic acids.

The principal nucleoside-hydrocarbon adducts present in hydrolysates of RNA and DNA isolated from hamster embryo cells treated with benz[a]anthracene (BA) were examined by chromatography on Sephadex LH 20 and by high pressure liquid chromatography (HPLC) on Spherisorb 5 ODS. The results extend the previous finding that a non-'bay-region' diol-epoxide, anti-BA-8,9-diol 10,11-oxide (r-8,t-9-dihydroxy-t-10,11-oxy-8,9,10,11-tetrahydrobenz[a] anthracene) is involved in the binding of BA to cellular nucleic acids and show that this diol-epoxide most probably reacts with guanosine and adenosine in RNA and with deoxyguanosine in DNA. The results also show that a 'bay-region' diol-epoxide anti-BA-3,4-diol 1,2-oxide (t-3,-4-dihydroxy-t-1,2-oxy-1,2,3,4-tetrahydrobenz[a]anthracene, which is thought to be involved in the binding of benz[a]anthracene, which is thought to be involved in the binding of benz[a]anthracene to DNA in some situations, reacts mainly with deoxyguanosine.

Adenosine↗
PubMed1980

Additional evidence for the involvement of the 3,4-diol 1,2-oxides in the metabolic activation of 7,12-dimethylbenz[a]anthracene in mouse skin.

The role of vicinal diol-epoxides in the metabolic activation of 7,12-dimethylbenz[a]anthracene to intermediates that react with nucleic acids was investigated using Sephadex LH-20 column chromatography and high pressure liquid chromatography. The results show that some of the hydrocarbon-DNA products formed in mouse skin treated in vivo with 7,12-dimethylbenz[a]anthracene arise from the reaction of DNA with 3,4-dihydro-3,4-dihydroxy-7,12-dimethylbenz[a]anthracene 1,2-oxides which, on the basis of this and other evidence, appears to be a biologically-active metabolite of 7,12-dimethylbenz[a]anthracene. However, since other nucleic acid-hydrocarbon adducts were also present that have not been identified as resulting from the reaction of the 3,4-diol 1,2-oxides with DNA, other mechanisms may also be involved in the metabolic activation of 7,12-dimethylbenz[a]anthracene in mouse skin.

9,10-Dimethyl-1,2-benzanthracene↗