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R Pietruszko

Publications and source records attributed to R Pietruszko.

At least 55 records · Page 3Linked to original sources
PubMed1989

Human aldehyde dehydrogenase: coenzyme binding studies.

The binding of NADH and NAD+ to the human liver cytoplasmic, E1, and mitochondrial, E2, isozymes at pH 7.0 and 25 degrees C was studied by the NADH fluorescence enhancement technique, the sedimentation technique, and steady-state kinetics. The binding of radiolabeled [14C]NADH and [14C]NAD+ to the E1 isozyme when measured by the sedimentation technique yielded linear Scatchard plots with a dissociation constant of 17.6 microM for NADH and 21.4 microM for NAD+ and a stoichiometry of ca. two coenzyme molecules bound per enzyme tetramer. The dissociation constant, 19.2 microM, for NADH as competitive inhibitor was found from steady-state kinetics. With the mitochondrial E2 isozyme, the NADH fluorescence enhancement technique showed only one, high-affinity binding site (KD = 0.5 microM). When the sedimentation technique and radiolabeled coenzymes were used, the binding studies showed nonlinear Scatchard plots. A minimum of two binding sites with lower affinity was indicated for NADH (KD = 3-6 microM and KD = 25-30 microM) and also for NAD+ (KD = 5-7 microM and KD = 15-30 microM). A fourth binding site with the lowest affinity (KD = 184 microM for NADH and KD = 102 microM for NAD+) was observed from the steady-state kinetics. The dissociation constant for NAD+, determined by the competition with NADH via fluorescence titration, was found to be 116 microM. The number of binding sites found by the fluorescence titration (n = 1 for NADH) differs from that found by the sedimentation technique (n = 1.8-2.2 for NADH and n = 1.2-1.6 for NAD+).(ABSTRACT TRUNCATED AT 250 WORDS)

Aldehyde Dehydrogenase↗
PubMed1989

Human aldehyde dehydrogenase. Purification and characterization of a third isozyme with low Km for gamma-aminobutyraldehyde.

An enzyme which catalyzes dehydrogenation of gamma-aminobutyraldehyde has been purified to homogeneity from human liver and identified as an isozyme of aldehyde dehydrogenase (EC 1.2.1.3); two other isozymes, previously obtained in a homogeneous form, are known as E1 and E2. Affinity chromatography on NAD-agarose (N6 with 8 carbon spacer) yields homogeneous enzyme which migrates as two components on isoelectric focusing with pI = 5.3 and 5.45. These two components, separated by fast protein liquid chromatography on a Mono-P HR 5/20 column, have similar Km values for gamma-aminobutyraldehyde, acetaldehyde, propionaldehyde, and NAD. The Km value for gamma-aminobutyraldehyde is 8.0-14.0 microM versus 760 microM for E1 and 512 microM for E2. The enzyme's molecular weight, subunit molecular weight, and amino acid composition are similar to those of the E1 and E2 isozymes. The enzyme also interacts with anti-E1 and anti-E2 antibodies; it is relatively insensitive to disulfiram inhibition and is neither activated nor inhibited by magnesium. Its absorption spectrum, where the ratio of 280/260 nm is 1.1 and a weak absorption is seen in the 340 nm range (Racker band), suggests the presence of bound coenzyme. gamma-Aminobutyraldehyde dehydrogenase (with Km value of 15 microM for gamma-aminobutyraldehyde) was previously partially purified from Pseudomonas fluorescens (Jakoby, W.B., and Fredericks, J. (1959) J. Biol. Chem. 234, 2145-2150) but never from a mammalian organism.

Aldehyde Dehydrogenase↗
PubMed1989

Human brain glyceraldehyde-3-phosphate dehydrogenase, succinic semialdehyde dehydrogenase and aldehyde dehydrogenase isozymes: substrate specificity and sensitivity to disulfiram.

Several enzymes active in the presence of NAD with acetaldehyde and propionaldehyde have been purified from human brain and characterized. The enzymes have been identified as aldehyde dehydrogenase (EC 1.2.1.3), NAD-linked succinic semialdehyde dehydrogenase (EC 1.2.1.24), and glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12). Glyceraldehyde-3-phosphate dehydrogenase is extremely heterogeneous with some isozymes active with acetaldehyde, others inactive. The cytoplasmic enzyme, which is the classical glyceraldehyde-3-phosphate dehydrogenase, is inactive with acetaldehyde as substrate; the isozymes that are active with short chain aliphatic aldehydes are localized in the mitochondrial fraction. Properties of glyceraldehyde-3-phosphate dehydrogenase isozymes with respect to short chain aliphatic aldehydes and inhibition by disulfiram are described. Their Km values for acetaldehyde range from 300 to 2000 microM. All glyceraldehyde-3-phosphate dehydrogenases that are active with acetaldehyde are easily inactivated by low concentrations of disulfiram. In all cases activity regain can be obtained with 2-mercaptoethanol; in the case of two glyceraldehyde-3-phosphate isozymes (E8.5 and 9.0), activity can also be regained with cysteine and with glutathione; activity of E6.6 and E6.8 glyceraldehyde-3-phosphate dehydrogenases could not be regained with 33 microM cysteine or glutathione. Succinic semialdehyde dehydrogenase and aldehyde dehydrogenase (EC 1.2.1.3) were also inhibited by disulfiram; their activity could be regained with 2-mercaptoethanol but not with 33 microM cysteine or glutathione. Comparison of human brain succinic semialdehyde dehydrogenase and aldehyde dehydrogenase with glyceraldehyde-3-phosphate dehydrogenase shows that the activity with short chain aldehydes is not unique to aldehyde dehydrogenase; neither is sensitivity to disulfiram; activity with 3,4-dihydroxyphenylacetaldehyde appears to be a unique property of aldehyde dehydrogenase (EC 1.2.1.3).

Aldehyde Dehydrogenase↗
PubMed1988

Human brain "high Km" aldehyde dehydrogenase: purification, characterization, and identification as NAD+ -dependent succinic semialdehyde dehydrogenase.

NAD-dependent succinic semialdehyde dehydrogenase (EC 1.2.1.24) has been purified to homogeneity from human brain via ion-exchange chromatography and affinity chromatography employing Blue Sepharose and 5'-AMP Sepharose. Succinic semialdehyde dehydrogenase was never previously purified to homogeneity from any species; this preparation therefore allows the determination of its molecular weight, subunit molecular weight, subunit composition, isoelectric points, and substrate specificity for the first time. The enzyme is a tetramer of Mr230,000 to 245,000 and consists of weight-nonidentical subunits (Mr 61,000 and 63,000). On isoelectric focusing the enzyme separates into five bands with the following isoelectric points: 6.3, 6.6, 6.8, 6.95, and 7.15. Its substrates include glutaric semialdehyde, nitrobenzaldehyde, and short chain aliphatic aldehydes in addition to succinic semialdehyde which is the best substrate. The Km values for succinic semialdehyde, acetaldehyde, and propionaldehyde are 1,875, and 580 microM, respectively. The enzyme is inactive with 3,4-dihydroxyphenylacetaldehyde and indole-3-acetaldehyde as substrates. Its subcellular localization is in the mitochondrial fraction. Succinic semialdehyde dehydrogenase is sensitive to inhibition by disulfiram (a drug used therapeutically to produce alcohol aversion) resembling, in this respect, aldehyde dehydrogenase (EC 1.2.1.3). It does not, however, interact with the antibody developed in the rabbit vs aldehyde dehydrogenase, suggesting that the two enzymes are structurally distinct.

Aldehyde Dehydrogenase↗
PubMed1988

Heterogeneity of glyceraldehyde-3-phosphate dehydrogenase from human brain.

In an attempt to characterize enzymes from human brain capable of dehydrogenating short chain aliphatic aldehydes, four groups of enzymes which catalyze inorganic phosphate-dependent reversible dehydrogenation of glyceraldehyde 3-phosphate as well as short chain aldehydes have been purified and characterized. Three enzyme groups are visualized as multiple bands on isoelectric focusing: E6.6 (pI 6.65, 6.75, 6.85); E6.8 (pI 6.8, 6.9); E8.5 (pI 8.5, 8.6); one enzyme, E9.0, is seen as a single band pI 9.0. The subcellular localization of E6.8, E8.5 and E9.0 appears to be mitochondrial. The mitochondrial enzymes differ slightly in molecular weight: E6.8 is 142,000 with subunits of 36,000 and 38,000; E8.5 is 120,000 with a subunit weight of 29,500; E9.0 is 133,000 with a subunit of 33,000. The E8.5 and E9.0 enzymes also appear to contain Zr as part of their molecular structure. E6.6 (subcellular localization uncertain) is a dimer with a molecular weight of 98,000 and two subunits of 58,000 and 61,000. The specific activity with glyceraldehyde-3-phosphate is: E6.6, 8.6 IU/mg; E6.8, 13 IU/mg; E8.5, 158 IU/mg; E9.0, 620 IU/mg. With glyceraldehyde 3-phosphate and 1,3-diphosphoglyceric acid and Km values of all the enzymes are similar (10-40 microM), except for E6.8 whose Km for glyceraldehyde 3-phosphate is very sensitive to pH and is extremely low at pH 7.0 (2 microM), while being considerably higher than that for the other enzymes at pH 9.0 (170 microM). The molecular properties, Km values as well as high specific activity with glyceraldehyde 3-phosphate identify E6.8, E8.5 and E9.0 as glyceraldehyde-3-phosphate dehydrogenases (EC 1.2.1.12). The catalytic properties of E6.6 are similar to those of E6.8, E8.5 and E9.0, but its molecular properties are different, precluding definite identification.

Aldehydes↗
PubMed1987

Active site of human liver aldehyde dehydrogenase.

Bromoacetophenone (2-bromo-1-phenylethanone) functions as an affinity reagent for human aldehyde dehydrogenase (EC 1.2.1.3) and has been found specifically to label a unique tryptic peptide in the enzyme. Amino-terminal sequence analysis of the labeled peptide after purification by two different procedures revealed the following sequence: Val-Thr-Leu-Glu-Leu-Gly-Gly-Lys. Radioactivity was found to be associated with the glutamate residue, which was identified as Glu-268 by reference to the known amino acid sequence. This paper constitutes the first identification of an active site of aldehyde dehydrogenase.

Acetophenones↗
PubMed1987

Chemical modification of human aldehyde dehydrogenase by physiological substrate.

Employing 3,4-dihydroxyphenylacetaldehyde (dopal) as a substrate for human aldehyde dehydrogenase (aldehyde:NAD+ oxidoreductase, EC 1.2.1.3) in anaerobic conditions, inactivation of both cytoplasmic E1 and mitochondrial E2 isozymes during catalysis has been observed. Incorporation of 14C-labelled dopal has been demonstrated by retention of label following denaturation and exhaustive dialysis and by peptide mapping following tryptic digestion. Incorporation of label gave linear plots vs. activity remaining with up to two molecules incorporated per molecule of enzyme and 30% activity remaining. Further incorporation (up to 16 molecules) occurred, but was non-linear when plotted vs. activity remaining. Protection against activity loss during incorporation of the first two molecules was afforded by NAD, NADH, chloral, and by chloral and NAD together, the last being the most effective. Saturation kinetics gave y-axis intercepts, suggesting interaction at a specific point on the enzyme surface. The Ki value from saturation kinetics was the same as that from the slope replot in catalytic reaction. Peptide mapping of tryptic digests showed that a single peptide was labelled, confirming specificity of interaction. Even in the absence of complete inactivation, the results suggest that reaction with the first two molecules occurs at some point on the enzyme surface important for enzyme activity. The possibility of such a reaction occurring in vivo is discussed.

3,4-Dihydroxyphenylacetic Acid↗
PubMed1987

Purification and characterization of aldehyde dehydrogenase from human brain.

Aldehyde dehydrogenase (EC 1.2.1.3) has been purified from human brain; this constitutes the first purification to homogeneity from the brain of any mammalian species. Of the three isozymes purified two are mitochondrial in origin (Peak I and Peak II) and one is cytoplasmic (Peak III). By comparison of properties, the cytoplasmic Peak III enzyme could be identified as the same as the liver cytoplasmic E1 isozyme (N.J. Greenfield and R. Pietruszko (1977) Biochim. Biophys. Acta 483, 35-45). The Peak I and Peak II enzymes resemble the liver mitochondrial E2 isozyme, but both have properties that differ from those of the liver enzyme. The Peak I enzyme is extremely sensitive to disulfiram while the Peak II enzyme is totally insensitive; liver mitochondrial E2 isozyme is partially sensitive to disulfiram. The specific activity is 0.3 mumol/mg/min for the Peak I and 3.0 mumol/mg/min for the Peak II enzyme; the specific activity of the liver mitochondrial E2 isozyme is 1.6 mumol/min/mg under the same conditions. The Peak I enzyme is also inhibited by acetaldehyde at low concentrations, while the Peak II enzyme and the liver mitochondrial E2 isozyme are not inhibited under the same conditions. The precise relationship of brain Peak I and II enzymes to the liver E2 isozyme is not clear but it cannot be excluded at the present time that the two brain mitochondrial enzymes are brain specific.

Aldehyde Dehydrogenase↗
PubMed1987

Human aldehyde dehydrogenase: metabolism of putrescine and histamine.

Imidazoleacetaldehyde and gamma-aminobutyraldehyde, metabolites of histamine and putrescine, respectively, have been shown to be substrates of human liver aldehyde dehydrogenase (EC 1.2.1.3) cytoplasmic (E1) and mitochondrial (E2) isozymes. The Km values at pH 7.4 and 500 microM NAD for imidazoleacetaldehyde and gamma-aminobutyraldehyde for the E1 isozyme are 40 and 800 microM, respectively, and for the E2 isozyme are 50 and 500 microM, respectively. The Km values with gamma-aminobutyraldehyde with both isozymes are high relative to Km values with acetaldehyde (50 microM for E1 and 1 microM for E2). Since activity with both imidazoleacetaldehyde and gamma-aminobutyraldehyde in crude liver homogenates paralleled that of aldehyde dehydrogenase (EC 1.2.1.3) during purification it appears likely that in the human liver this enzyme is responsible for metabolism of both compounds. If this is the case, interaction between metabolism of histamine and putrescine and that of alcohol is likely. Both imidazoleacetaldehyde and gamma-aminobutyraldehyde were synthesized in this laboratory and their stability has been investigated. Procedures for assaying aldehyde dehydrogenase employing synthetic metabolites of histamine and putrescine are provided.

Aldehyde Dehydrogenase↗
PubMed1987

Multiplicity and identity of human aldehyde dehydrogenases.

Human "high Km" aldehyde dehydrogenases have been purified to homogeneity and characterized. The "high Km" enzymes are structurally and catalytically distinct from "low Km" aldehyde dehydrogenases; they are not isozymes of the "low Km" enzymes.

1-Pyrroline-5-Carboxylate Dehydrogenase↗
PubMed1986

Bromoacetophenone as an affinity reagent for human liver aldehyde dehydrogenase.

Human liver aldehyde dehydrogenase isozymes E1 and E2 (EC 1.2.1.3) are both completely and irreversibly inactivated by bromoacetophenone (2-bromo-1-phenylethanone). Steady-state kinetics with both acetophenone and chloroacetophenone indicated interaction with the same enzyme form as the aldehyde substrate. Saturation kinetics with chloroacetophenone and bromoacetophenone indicated interaction at a specific site on the enzyme surface and gave a dissociation constant similar to that from steady-state kinetics, suggesting that the same processes were being observed by both methods and that the active site may be involved. Protection against inactivation was afforded by chloral and NAD together. Stoichiometry of inactivation showed the first 2 equiv per tetramer to abolish the majority of catalytic activity; 4 equiv inactivated both isozymes with complete loss of esterase, NAD-stimulated esterase, and dehydrogenase activities. Peptide mapping of enzyme modified with [carbonyl-14C]bromoacetophenone of CNBr digests (E1) and tryptic digests (E1 and E2) showed one peptide to be preferentially labeled. The above results together with the similarity of bromoacetophenone to the substrate benzaldehyde suggest bromoacetophenone may react with a residue in the active site of aldehyde dehydrogenase. Amino acid analysis of the labeled E1 tryptic fragment indicated reaction with a different peptide from that with which iodoacetamide reacts.

Acetophenones↗
PubMed1986

Purification and characterization of human liver "high Km" aldehyde dehydrogenase and its identification as glutamic gamma-semialdehyde dehydrogenase.

The enzyme previously considered as an isozyme (E4, ALDH IV) of human liver aldehyde dehydrogenase (NAD+) (EC 1.2.1.3) has been purified to homogeneity by the use of ion exchange chromatography on CM-Sephadex and affinity chromatography on Blue Sepharose CL-6B and 5'-AMP Sepharose 4B and identified as glutamic gamma-semialdehyde dehydrogenase, or more precisely 1-pyrroline-5-carboxylate dehydrogenase (EC 1.5.1.12). Glutamic gamma-semialdehyde dehydrogenase was never previously purified to homogeneity from any mammalian species. The homogeneous enzyme is seen on isoelectric focusing gels as two fine bands separated by 0.12 pH units: pI = 6.89 and 6.77. In addition, the enzyme also appears as two bands in gradient gels; however, in polyacrylamide gels containing sodium dodecyl sulfate the enzyme migrates as one band, indicating that its subunits are of identical size. Because the enzyme molecule is considerably smaller (Mr approximately 142,000-170,000) than that of aldehyde dehydrogenases (EC 1.2.1.3) (Greenfield, N. J., and Pietruszko, R. (1977) Biochim. Biophys. Acta 483, 35-45; Mr approximately 220,000) and its subunit weight is different (70,600 versus approximately 54,000 for E1 and E2 isozymes), the enzyme is not an isozyme of aldehyde dehydrogenase previously described. The Michaelis constants for glutamic gamma-semialdehyde dehydrogenase with acetaldehyde and propionaldehyde are in the millimolar range. Its substrate specificity within the straight chain aliphatic aldehyde series is essentially confined to that of acetaldehyde and propionaldehyde with butyraldehyde and longer chain length aldehydes being considerably less active. Other substrates include succinic, glutaric, and adipic semialdehydes in addition to glutamic gamma-semialdehyde. The reaction velocity with glutamic gamma-semialdehyde is at least an order of magnitude larger than with carboxylic acid semialdehydes. Aspartic beta-semialdehyde is not a substrate. The reaction catalyzed appears to be irreversible. Although NADP can be used, NAD is the preferred coenzyme. The enzyme also exhibits an unusual property of being subject to substrate inhibition by NAD.

1-Pyrroline-5-Carboxylate Dehydrogenase↗
PubMed1986

Human aldehyde dehydrogenase: kinetic identification of the isozyme for which biogenic aldehydes and acetaldehyde compete.

Michaelis constants and maximal velocities for phenylacetaldehyde (a metabolite of phenylethylamine), 3,4-dihydroxyphenylacetaldehyde (a metabolite of dopamine), 5-hydroxyindole acetaldehyde (a metabolite of serotonin), and 3,4-dihydroxyphenylglycolaldehyde (a metabolite of epinephrine and norepinephrine) have been determined for both cytoplasmic (E1) and mitochondrial (E2) isozymes of human liver aldehyde dehydrogenase (EC 1.2.1.3). Kinetic constants with biogenic aldehydes have never been previously determined for individual homogeneous isozymes of aldehyde dehydrogenase from any species. Mathematical treatment of these constants suggests that competition with acetaldehyde during alcohol metabolism would severely inhibit dehydrogenation of biogenic aldehydes with the mitochondrial and not the cytoplasmic isozyme of human liver aldehyde dehydrogenase.

Acetaldehyde↗
PubMed1985

Human mitochondrial aldehyde dehydrogenase inhibition by diethyldithiocarbamic acid methanethiol mixed disulfide: a derivative of disulfiram.

A derivative and possible physiological metabolite of disulfiram, diethyldithiocarbamic acid methanethiol mixed disulfide, is shown here for the first time to inactivate the mitochondrial low-Km isozyme of human aldehyde dehydrogenase (EC 1.2.1.3). By comparing inactivating effects of diethyldithiocarbamic acid mixed disulfides with thiols of increasing chain length evidence is provided that steric hindrance is the reason for lack of inhibition of the mitochondrial enzyme by disulfiram in vitro. Since methanethiol is a normal metabolite [(1983) Annu. Rev. Biochem. 52, 187-222] the results also suggest a mechanism by which aldehyde dehydrogenase is inhibited by disulfiram and diethyldithiocarbamic acid in vivo.

Aldehyde Dehydrogenase↗
PubMed1984

Interaction of Mg2+ with human liver aldehyde dehydrogenase. I. Species difference in the mitochondrial isozyme.

The dehydrogenase activity of the mitochondrial isozyme (E2) of human liver aldehyde dehydrogenase was stimulated about 2-fold by the presence of low concentrations (about 120-140 microM) of Mg2+ in the assay at pH 7.0 using propionaldehyde as substrate. The stimulation was totally reversible by treatment with EDTA. Maximum stimulation was dependent on the concentration of NAD+ used in the assay; an increase in Km value of NAD+ was observed to parallel the increase in maximal velocity with increasing Mg2+ concentration, indicating that alterations in the catalytic properties of the E2 isozyme occur in the presence of Mg2+. The presteady state burst of NADH product was observed to decrease in the presence of Mg2+, suggesting that the rate-limiting step of the dehydrogenase reaction is altered by Mg2+. No evidence for Mg2+-induced alterations in the molecular weight properties of the E2 isozyme was observed using gel filtration column chromatography and fluorescence polarization techniques. In addition, no alterations in the inactivating properties of iodoacetamide or disulfiram were produced by Mg2+. These results suggest that the mechanism by which human mitochondrial aldehyde dehydrogenase (E2) is stimulated by Mg2+ is different from that of the horse enzyme, representing a significant species difference.

Aldehyde Dehydrogenase↗