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R Pietruszko

Publications and source records attributed to R Pietruszko.

At least 19 recordsLinked to original sources
PubMed2001

Catalysis of dehydrogenation of 4-trans-(N,N-dimethylamino)cinnamaldehyde by aldehyde dehydrogenase.

4-trans-(N,N-dimethylamino)cinnamaldehyde (DACA) is a chromophoric and fluorogenic substrate of aldehyde dehydrogenase. Fluorescence of DACA is enhanced by binding to aldehyde dehydrogenase in the absence of catalysis both in the presence and absence of the coenzyme analogue 5'AMP. DACA binds to aldehyde dehydrogenase with a dissociation constant of 1-3 microM and stoichiometry of 2 mol mol(-1) enzyme. Incorporation of DACA during catalysis was also investigated and found to be 2 mol DACA mol(-1) enzyme. Effect of pH on the stoichiometry of DACA incorporation during catalysis has shown that DACA incorporation remained constant at 2 mol DACA mol(-1) enzyme, despite a 74-fold velocity enhancement between pH 5.0 and 9.0. Increase of pH increased decomposition of enzyme-acyl intermediate without affecting the rate-limiting step of the reaction. At pH 7.0 the pH stimulated velocity enhancement was 10-fold over that at pH 5.0; further velocity enhancement (11.5-fold that of pH 7.0) was achieved by 150 microM Mg(2+) ions. The velocity at pH 7.0 with Mg(2+) exceeded that of pH 9.0, and that at maximal pH stimulation at pH 9.5. It was observed that level of intermediate decreased to about 1 mol mol(-1) enzyme, indicating that Mg(2+) ions increased the rate of decomposition of the enzyme-acyl intermediate and shifted the rate-limiting step of the reaction to another step in the reaction sequence.

Aldehyde Dehydrogenase↗
PubMed2001

Betaine aldehyde dehydrogenase from rat liver mitochondrial matrix.

An NAD-linked aldehyde dehydrogenase which in addition to aliphatic and aromatic aldehydes, metabolizes aminoaldehydes and betaine aldehyde, has been purified to homogeneity from male Sprague-Dawley rat liver mitochondria. The properties of the rat mitochondrial enzyme are similar to those of a rat liver cytoplasmic betaine aldehyde dehydrognase and the human cytoplasmic E3 isozyme. The primary structure. of four tryptic peptides were also similar; only one difference in primary structure was observed. The close similarity of properties of the cytoplasmic with the mitochondrial form suggest that the cytoplasmic and mitochondrial betaine aldehyde dehydrogenase may be coded for by the same nuclear gene. Investigation of the mitochondrial form by isoelectric focusing resulted in visualization of multiple forms, different from those seen in the cytoplasm suggesting that the enzyme may be processed in the mitochondria.

Aldehyde Oxidoreductases↗
PubMed2001

Human aldehyde dehydrogenase catalytic activity and structural interactions with coenzyme analogs.

K(m) and V(max) values for 10 coenzyme analogs never previously studied with any aldehyde dehydrogenase and NADP(+) were compared with those for NAD(+) for three human aldehyde dehydrogenases (EC 1.2.1.3); the cytoplasmic E1 (the product of the aldh1 gene), the mitochondrial E2 (the product of the aldh2 gene) and the cytoplasmic E3 (the product of the aldh9 gene) isozymes. Structural information on changes in coenzyme-protein interactions were obtained via molecular dynamics (MD) studies with the E2 isozyme and quantum mechanical (QM) calculations were used to study changes in charge distribution of the pyridine ring and relative free energies of solvation of the purine ring in the analogs. E1 showed the broadest substrate specificity and was the only isozyme subject to substrate inhibition, both of which are suggested to be due to the two coenzyme conformations observed previously in the sheep crystal structure. NADP(+) selectivity is indicated to be influenced by Glu195 in E1 and E2. Substitutions in the purine ring affected K(m) but not V(max), with the changes in K(m) being dominated by the hydrophobicity of the purine ring as indicted by the QM calculations. Substitutions in the pyridine ring sometimes rendered the coenzymes inactive, with no consistent pattern observed for the three coenzymes. Structural analysis of the coenzyme analog-E2 MD simulations revealed different structural perturbations of the surrounding active site, though interactions with Asn169 and Glu399 were preserved in all cases.

Adult↗
PubMed2000

Betaine aldehyde, betaine, and choline levels in rat livers during ethanol metabolism.

Betaine aldehyde levels were determined in rat livers following 4 weeks of ethanol feeding, employing the Lieber-De Carli liquid diet. The results showed that the levels of betaine aldehyde are unaffected by alcohol feeding to rats. These levels in both experimental and control animals were found to be quite low, 5.5 nmol/g liver. Betaine aldehyde levels have not been determined previously in mammalian liver because of methodological difficulties. This investigation employed fast atom bombardment-mass spectroscopy to determine the levels of betaine aldehyde, betaine, and choline. The decrease in betaine levels following ethanol administration confirmed the results of other investigators. Choline levels determined during this investigation were lower than previously reported. The reason for starting this investigation was the fact that the enzyme that catalyzes betaine aldehyde dehydrogenation to betaine, which is distributed in both mitochondria and the cytoplasm, was found to also metabolize acetaldehyde with K(m) and V(max) values lower than those for betaine aldehyde. Thus, it appeared likely that the metabolism of acetaldehyde during ethanol metabolism might inhibit betaine aldehyde conversion to betaine and thereby result in decreased betaine levels (Barak et al., Alcohol 13: 395-398, 1996). The fact that betaine aldehyde levels in alcohol-fed animals were similar to those in controls demonstrates that competition between acetaldehyde and betaine aldehyde for the same enzyme does not occur. This complete lack of competition suggests that betaine aldehyde dehydrogenase in the mitochondrial matrix may totally metabolize betaine aldehyde to betaine without any involvement of cytoplasmic betaine aldehyde dehydrogenase.

Acetaldehyde↗
PubMed1999

Metabolism of retinaldehyde and other aldehydes in soluble extracts of human liver and kidney.

Purification and characterization of enzymes metabolizing retinaldehyde, propionaldehyde, and octanaldehyde from four human livers and three kidneys were done to identify enzymes metabolizing retinaldehyde and their relationship to enzymes metabolizing other aldehydes. The tissue fractionation patterns from human liver and kidney were the same, indicating presence of the same enzymes in human liver and kidney. Moreover, in both organs the major NAD(+)-dependent retinaldehyde activity copurified with the propionaldehyde and octanaldehyde activities; in both organs the major NAD(+)-dependent retinaldehyde activity was associated with the E1 isozyme (coded for by aldh1 gene) of human aldehyde dehydrogenase. A small amount of NAD(+)-dependent retinaldehyde activity was associated with the E2 isozyme (product of aldh2 gene) of aldehyde dehydrogenase. Some NAD(+)-independent retinaldehyde activity in both organs was associated with aldehyde oxidase, which could be easily separated from dehydrogenases. Employing cellular retinoid-binding protein (CRBP), purified from human liver, demonstrated that E1 isozyme (but not E2 isozyme) could utilize CRBP-bound retinaldehyde as substrate, a feature thought to be specific to retinaldehyde dehydrogenases. This is the first report of CRBP-bound retinaldehyde functioning as substrate for aldehyde dehydrogenase of broad substrate specificity. Thus, it is concluded that in the human organism, retinaldehyde dehydrogenase (coded for by raldH1 gene) and broad substrate specificity E1 (a member of EC 1. 2.1.3 aldehyde dehydrogenase family) are the same enzyme. These results suggest that the E1 isozyme may be more important to alcoholism than the acetaldehyde-metabolizing enzyme, E2, because competition between acetaldehyde and retinaldehyde could result in abnormalities associated with vitamin A metabolism and alcoholism.

Aldehyde Oxidoreductases↗
PubMed1999

Binding and incorporation of 4-trans-(N,N-dimethylamino) cinnamaldehyde by aldehyde dehydrogenase.

4-trans-(N,N-Dimethylamino)cinnamaldehyde (DACA) is a chromophoric substrate of aldehyde dehydrogenase (EC 1.2.1.3) whose fate can be followed during catalysis. During this investigation we found that DACA also fluoresces and that this fluorescence is enhanced and blue-shifted upon binding to aldehyde dehydrogenase. Binding of DACA to aldehyde dehydrogenase also occurs in the absence of coenzyme. Benzaldehyde (a substrate), acetophenone (a substrate-competitive inhibitor), and the substrate-competitive affinity reagent bromoacetophenone interfere with DACA binding. Thus, DACA binds to the active site and can be employed for titration of active aldehyde dehydrogenase. Both E1 and E2 isozymes, which are homotetramers, bind DACA with dissociation constants of 1-4 microM with a stoichiometry of 2 mol DACA/ mol enzyme. The stoichiometry of enzyme-acyl intermediate was also found to be 2 mol DACA/ mol enzyme for both E1 and E2 isozymes. Thus, both enzymes appear to have only two substrate-binding sites which participate in catalysis. The level of enzyme-acyl intermediate remained constant at different pH values, showing that enhancement of velocity with pH was not due to altered DACA-enzyme levels. When the reaction velocity was increased even further by using 150 microM Mg2+ the intermediate level was decreased, suggesting that both increased pH and Mg2+ promote decomposition of the DACA-enzyme intermediate. Titration with DACA permits study of aldehyde substrate catalysis before central complex interconversion.

Acetophenones↗
PubMed1999

Mechanism of inhibition of aldehyde dehydrogenase by citral, a retinoid antagonist.

Low concentrations of citral (3,7-dimethyl-2,6-octadienal), an inhibitor of retinoic acid biosynthesis, inhibited E1, E2 and E3 isozymes of human aldehyde dehydrogenase (EC1.2.1.3). The inhibition was reversible on dilution and upon long incubation in the presence of NAD+; it occurred with simultaneous formation of NADH and of geranic acid. Thus, citral is an inhibitor and also a substrate. Km values for citral were 4 microM for E1, 1 microM for E2 and 0.1 microM for E3; Vmax values were highest for E1 (73 nmol x min-1 x mg-1), intermediate for E2 (17 nmol x min-1 x mg-1) and lowest (0.07 nmol x min-1 x mg-1) for the E3 isozyme. Citral is a 1 : 2 mixture of isomers: cis isomer neral and trans isomer, geranial; the latter structurally resembles physiologically important retinoids. Both were utilized by all three isozymes; a preference for the trans isomer, geranial, was observed by HPLC and by enzyme kinetics. With the E1 isozyme, both geranial and neral, and with the E2 isozyme, only neral obeyed Michaelis-Menten kinetics. With the E2 isozyme and geranial sigmoidal saturation curves were observed with S0.5 of approximately 50 nM; the n-values of 2-2.5 indicated positive cooperativity. Geranial was a better substrate and a better inhibitor than neral. The low Vmax, which appeared to be controlled by either the slow formation, or decomposition via the hydride transfer, of the thiohemiacetal reaction intermediate, makes citral an excellent inhibitor whose selectivity is enhanced by low Km values. The Vmax for citral with the E1 isozyme was higher than those of the E2 and E3 isozymes which explains its fast recovery following inhibition by citral and suggests that E1 may be the enzyme involved in vivo citral metabolism.

Acyclic Monoterpenes↗
PubMed1999

Evidence for mitochondrial localization of betaine aldehyde dehydrogenase in rat liver: purification, characterization, and comparison with human cytoplasmic E3 isozyme.

Betaine aldehyde dehydrogenase has been purified to homogeneity from rat liver mitochondria. The properties of betaine aldehyde dehydrogenase were similar to those of human cytoplasmic E3 isozyme in substrate specificity and kinetic constants for substrates. The primary structure of four tryptic peptides was also similar; only two substitutions, at most, per peptide were observed. Thus, betaine aldehyde dehydrogenase is not a specific enzyme, as formerly believed; activity with betaine aldehyde is a property of aldehyde dehydrogenase (EC 1.2.1.3), which has broad substrate specificity. Up to the present time the enzyme was thought to be cytoplasmic in mammals. This report establishes, for the first time, mitochondrial subcellular localization for aldehyde dehydrogenase, which dehydrogenates betaine aldehyde, and its colocalization with choline dehydrogenase. Betaine aldehyde dehydrogenation is an important function in the metabolism of choline to betaine, a major osmolyte. Betaine is also important in mammalian organisms as a major methyl group donor and nitrogen source. This is the first purification and characterization of mitochondrial betaine aldehyde dehydrogenase from any mammalian species.

Aldehyde Oxidoreductases↗
PubMed1998

N-tosyl-L-phenylalanine chloromethyl ketone, a serine protease inhibitor, identifies glutamate 398 at the coenzyme-binding site of human aldehyde dehydrogenase. Evidence for a second "naked anion" at the active site.

Human aldehyde dehydrogenase isozymes were inactivated by N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), an inhibitor of chymotrypsin. The inactivation was a first-order process that followed saturation kinetics. NAD and chloral when used together protected against inactivation. In steady-state kinetics, TPCK produced only slope effects versus varied NAD, both slope and intercept effects versus varied glycolaldehyde were produced, indicating that TPCK reacted with the same enzyme form with which NAD reacted. Ki values from steady-state kinetics and saturation kinetics were comparable. Use of [3H]-labeled TPCK showed that inactivation was associated with the incorporation of two molecules of TPCK per molecule of enzyme. The label incorporation occurred into a single tryptic peptide and also into a single chymotryptic peptide of the E1 isozyme. Purification of labeled peptides, followed by sequencing, demonstrated that E398 of aldehyde dehydrogenase was labeled. Reaction of a haloketone, TPCK, with a carboxyl group of E398 indicates that E398 occurs as a "naked anion" within the molecule. This paper constitutes identification of the second (after E268) "naked anion" at the active site of aldehyde dehydrogenase.

Aldehyde Dehydrogenase↗
PubMed1998

Methylglyoxal as substrate and inhibitor of human aldehyde dehydrogenase: comparison of kinetic properties among the three isozymes.

Methylglyoxal was demonstrated to be a substrate for the isozymes E1, E2 and E3 of human aldehyde dehydrogenase. Pyruvate was the product from the oxidation of methylglyoxal by the three isozymes. At pH 7.4 and 25 degrees C, the major and minor components of the E3 isozyme catalyzed the reaction with Vmax of 1.1 and 0.8 mumol NADH min-1 mg-1 protein, respectively, compared to 0.067 and 0.060 mumol NADH min-1 mg-1 protein for the E1 and E2 isozymes, respectively. The E2 isozyme had a K(m) for methylglyoxal of 8.6 microM, the lowest compared to 46 microM for E1 and 586 and 552 microM for the major and minor components of the E3 isozyme, respectively. Both components of the E3 isozyme showed substrate inhibition by methylglyoxal, with Ki values of 2.0 mM for the major component and 12 mM for the minor component at pH 9.0. Substrate inhibition by methylglyoxal was not observed with the E1 and E2 isozymes. Methylglyoxal strongly inhibited the glycolaldehyde activity of the E1 and E2 isozymes. Mixed-type models of inhibition were employed as an approach to calculate the inhibition constants, 44 and 10.6 microM for E1 and E2 isozymes, respectively.

Aldehyde Dehydrogenase↗
PubMed1997

Tissue distribution of human aldehyde dehydrogenase E3 (ALDH9): comparison of enzyme activity with E3 protein and mRNA distribution.

The tissue distribution of the E3 isozyme of human aldehyde dehydrogenase has been investigated by three methods: enzyme activity assay employing betaine aldehyde as substrate, Western blotting employing E3 isozyme-specific antibodies, and Northern blotting using a human liver E3 cDNA as probe. All three methods showed that E3 isozyme was universally distributed among all tissues tested. The highest levels of the E3 isozyme activity were found in liver, adrenal gland, and kidney. These same tissues also showed highest levels of the E3 protein via the Western blot. This distribution is consistent with the possible physiological role of E3 isozyme in the synthesis of the osmolyte, betaine, and the neurotransmitter, GABA. Northern blot analysis, however, differed from that of enzyme assay and the Western blot in that it showed highest mRNA levels in skeletal and heart muscles, which had low enzyme activities and E3 protein levels.

Adrenal Glands↗
PubMed1997

Cimetidine and other H2-receptor antagonists as inhibitors of human E3 aldehyde dehydrogenase.

The histamine H2-receptor antagonists have been identified as inhibitors of human liver aldehyde dehydrogenase (EC 1.2.1.3) isozymes, E1, E2, and E3. Inhibition was strongest with the E3 isozyme, whose substrates include gamma-aminobutyraldehyde, the aldehyde metabolites of polyamines, and betaine aldehyde. Burimamide, metiamide, cimetidine guanidine, cimetidine, and tiotidine were competitive with aldehyde substrates and noncompetitive with the coenzyme, binding to both the free E3 isozyme and the enzyme-coenzyme binary complex. Cimetidine and tiotidine were the best inhibitors, with Ki values of 1.1 +/- 0.2 microM and 1.0 +/- 0.0 microM, respectively; both are the first ever described potent and selective inhibitors of the E3 isozyme. Examination of the H2-receptor antagonist structures for insight into the moieties accounting for E3 isozyme inhibition pointed to the side-chain polar groups as strongly influencing inhibition, with the cyanoguanidine side chain of cimetidine and tiotidine having the strongest influence. The Ki value of the E3 isozyme for cimetidine was the same as the in vitro dissociation constant for the H2-receptor.

Aldehyde Dehydrogenase↗
PubMed1996

Aldehyde dehydrogenase from adult human brain that dehydrogenates gamma-aminobutyraldehyde: purification, characterization, cloning and distribution.

Enzyme purification and characterization, cDNA cloning and Northern blot analysis were the techniques utilized during this investigation to determine the identity and occurrence of the aldehyde dehydrogenase that metabolizes gamma-aminobutyraldehyde in adult human brain. The purification yielded one major protein which was active with gamma-aminobutyraldehyde. It had the physico-chemical and kinetic properties of the human liver E3 isoenzyme of aldehyde dehydrogenase (EC 1.2.1.3), and also interacted with an anti-(liver E3 isoenzyme) antibody. Tryptic peptides derived from the purified brain protein matched the amino acid sequence of the liver E3 isoenzyme. Employing liver E3 cDNA, a human cerebellar cDNA library was screened and a 2.0 kb cDNA fragment was isolated. The cerebellar cDNA yielded a derived primary structure which differed from the liver E3 amino acid sequence by a single serine-to-cysteine substitution at position 88 (position 84 in the liver sequence). Thus the gamma-amino-butyraldehyde-metabolizing enzyme from human brain can be identified as E3', a variant of the E3 isoenzyme. The catalytic properties of the brain variant were indistinguishable from those of E3, and so the functional importance of this variant is at present unknown. The distribution of this enzyme in brain was investigated by Northern blot analysis, which demonstrated the presence of E3' mRNA in all regions of the human brain. mRNA levels were variable in the different brain areas, with the highest levels in the spinal cord and the lowest in the occipital pole.

Adult↗