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R Pietruszko

Publications and source records attributed to R Pietruszko.

At least 73 records · Page 4Linked to original sources
PubMed1984

Interaction of Mg2+ with human liver aldehyde dehydrogenase. II. Mechanism and site of interaction.

The dehydrogenase reaction of the cytoplasmic isozyme (E1) of human aldehyde dehydrogenase (EC 1.2.1.3) was inhibited while that of the mitochondrial isozyme (E2) was stimulated by low levels of Mg2+ (up to about 150 microM). However, with respect to esterase activity, Mg2+ had a similar effect on both isozymes in that only the NADH-stimulated reaction was effected. Comparison of coenzyme dissociation constants from the dehydrogenase reaction with the coenzyme stimulation constants from the esterase reactions provided the detailed mechanism of the action of Mg2+. The data indicate that Mg2+ acts similarly to bring about alterations in the activity of both isozymes. With E1, where NADH release is the rate-determining step, Mg2+ inhibits by causing a tighter binding of NADH to the enzyme. With E2, however, the stimulation results from the increased rate of acid release from the ternary complex (rate-determining step for E2) when Mg2+ is also present. These results suggest that three enzyme conformers participate in the dehydrogenase reaction and provide a unified mechanism for the stimulation of the dehydrogenase reaction catalyzed by the E2 isozyme, inhibition of the same reaction catalyzed by the E1 isozyme, and the effects of Mg2+ on the esterase reactions (nonstimulated and coenzyme stimulated) of both isozymes.

Aldehyde Dehydrogenase↗
PubMed1984

Human aldehyde dehydrogenase: catalytic activity in oriental liver.

Population genetics followed by purification suggested that "null" mutation in the mitochondrial E2 isozyme of human aldehyde dehydrogenase (EC 1.2.1.3) occurred in the Oriental individuals who are sensitive to alcohol. This report demonstrates that the Oriental E2, thought to be a "null" mutant, is catalytically active and except for maximal velocity and isoelectric point, identical with Caucasian E2 isozyme. The data presented are not inconsistent with mutation but preclude active site of the enzyme as the point at which alteration has occurred; they are, however, inconsistent with "null" mutation.

Aldehyde Dehydrogenase↗
PubMed1983

Interaction of human cytoplasmic aldehyde dehydrogenase E1 with disulfiram.

Two equivalents of symmetrically labeled [14C]-disulfiram (tetraethylthiuram disulfide, Antabuse) interact with human liver cytoplasmic aldehyde dehydrogenase (ALDH) E1 (E.C. 1.2.1.3) inhibiting ca. 90% of total catalytic activity. Inhibition occurs without labeling of the enzyme but is associated with disappearance of four SH groups per molecule of enzyme. Inhibition is reversible by treatment with 2-mercaptoethanol suggesting that disulfiram oxidizes vicinal enzyme SH groups to disulfides. The radioactivity from disulfiram is recovered as diethyldithiocarbamate. SDS gel electrophoresis indicates that vicinal SH groups involved in the interaction with disulfiram occur on the same subunits. This is also consistent with the fact that introduction of disulfide bonds by disulfiram is not accompanied by a major conformational change as evidenced by fluorescence polarization or circular dichroism. Experiments with o-iodosobenzoate suggest the presence of a third SH group in the vicinity of the two interacting with disulfiram. Inhibition of aldehyde dehydrogenase by disulfiram is not easily reversible by glutathione which might explain why new protein synthesis is required to regain enzyme activity in vivo.

Aldehyde Dehydrogenase↗
PubMed1982

Identification of a segment containing a reactive cysteine residue in human liver cytoplasmic aldehyde dehydrogenase (isoenzyme E1).

A single cysteine residue is selectively alkylated by iodoacetamide in cytoplasmic human liver aldehyde dehydrogenase (isoenzyme E1). The amino acid sequence of a 35-residue fragment containing this residue is determined, showing two additional cysteine residues and also three histidine residues. The alkylation is selective for Cys-30 of this fragment, with only little alkylation even at an adjacent residue, Cys-29. The region examined is likely to be of significance in the reaction of this isoenzyme with disulfiram since disulfiram blocks the selective alkylation.

Aldehyde Dehydrogenase↗
PubMed1982

Human aldehyde dehydrogenase: mechanism of inhibition of disulfiram.

Disulfiram labeled with carbon-14 reacts specifically with human liver aldehyde dehydrogenase E1 with loss of catalytic activity and no incorporation of label. Carbon-14-labeled diethyldithiocarbamate is formed and the number of enzyme sulfhydryl groups decreases from 34 to 30 during this process. Activity is recovered by-mercaptoethanol but not by glutathione, the physiological reducing agent.

Aldehyde Dehydrogenase↗
PubMed1982

Oxidation of the 17-aldol (20 beta hydroxy-21-aldehyde) intermediate of corticosteroid metabolism to hydroxy acids by homogeneous human liver aldehyde dehydrogenases.

In human liver, the oxidation of corticosteroids to 20-hydroxy-21-oic acids proceeds via the formation and oxidation of aldol (20-hydroxy-21-aldehyde) intermediates. Human liver aldehyde dehydrogenases E1 and E2, which we have previously purified to homogeneity, catalyzed the oxidation of the aldol isomer of cortisol (isocortisol) or of 11-deoxycorticosterone (isoDOC) by E1 and E2 respectively, were identified by the criteria of chromatographic mobility, derivatization, and reverse isotope dilution of 4-14C labeled acid end products. Both enzymes showed broad substrate specificity and oxidized both 17-hydroxy and 17-deoxy steroids, though at widely varying rates. Kinetic analysis of the course of oxidation of isocortisol and isoDOC by NAD+ gave intersecting initial velocity plots that conform with a sequential mechanism. The inhibition patterns for both enzymes with thionicotinamide adenine dinucleotide or chloral hydrate were consistent with random sequential behavior.

Adrenal Cortex Hormones↗
PubMed1982

Human aldehyde dehydrogenase: improved purification procedure and comparison of homogeneous isoenzymes E1 and E2.

An improved purification procedure of human aldehyde dehydrogenase (EC 1.2.1.3) isoenzymes E1 and E2 is presented. This procedure employs only three chromatographic steps to produce homogeneous E1 and E2 isoenzymes at 60% overall yield. The isoenzymes have been tested for homogeneity by electrophoresis of native and denatured species, specific activity determinations following rechromatography, as well as mapping of tryptic and CNBr fragments. Total SH group analysis has also been done on each isoenzyme. The results show that both isoenzymes are homogeneous. Similarities between E1 and E2 isoenzymes are noted in the mobility of about 40% of tryptic fragments, total SH content, and the mobility of two CNBr fragments. The results also show considerable structural differences between the isoenzymes in that CNBr maps show fragments from E1 and E2 of different molecular weight and about 60% of tryptic fragments migrate to distinct locations. Only one of SH-containing tryptic fragments migrates to the same location in both isoenzymes. E1 and E2 each consist of subunits which migrate as single bands in both sodium dodecyl sulfate (SDS) and urea electrophoresis. While the mobility of E1 and E2 subunits in SDS gels is similar, it is different in urea, showing that subunits of E1 are distinct from those of E2 and that the isoenzymes do not share subunits. Structural similarity between isoenzymes must, therefore, result from sequence similarity within regions of distinct polypeptide chains composing E1 and E2 molecules. The results presented offer a simplified procedure for preparation of the homogeneous isoenzymes; they also suggest that E1 and E2 are products of distinct genes which probably diverged from a common genetic ancestor through gene duplication and compartmentation of the cell.

Aldehyde Dehydrogenase↗
PubMed1981

Brain aldehyde dehydrogenase in human alcoholics and controls.

Human postmortem brain samples from temporal lobe of 23 alcoholics and 19 controls (13 with cancer and six without cancer) were used for determination of low Km (micromolar) and high Km (millimolar) aldehyde dehydrogenase activity. Despite histories of severe alcoholism leading to death through multiple complications, the mean values for either low or high Km activity did not differ significantly from that of controls similarly studied.

Adult↗
PubMed1980

Kinetic mechanism of horse liver alcohol dehydrogenase SS.

The kinetic mechanism of SS isozyme of horse liver alcohol dehydrogenase is shown by initial velocity and product inhibition studies to be asymmetrical, being random for ethanol oxidation and compulsory ordered for acetaldehyde reduction. Enzyme isomerization seems to account for the asymmetry in the mechanism. In its interaction with NADH, the SS isozyme resembles classical alcohol dehydrogenase; consequently, the maximal velocity in the direction from ethanol to acetaldehyde appears to be determined by the rate of NADH dissociation. In the direction from acetaldehyde to ethanol, the enzyme isomerization step appears to limit the maximal velocity.

Acetaldehyde↗
PubMed1980

On the interaction of human liver aldehyde dehydrogenase E1 isoenzyme with disulfiram and iodoacetamide.

The E1 isoenzyme of human liver aldehyde dehydrogenase (Km acetaldehyde = 30uM, pH 7.0), when incubated with disulfiram at a stoichiometry of four moles disulfiram/tetrameric E1, is immediately inhibited to within 10% of control activity. The inhibition is reversed by 0.1% (v/v) mercaptoethanol, indicating disulfide bridge formation. An indirect attempt to locate, on maps, a peptide binding disulfiram has yielded inconsistent results. Iodoacetamide inhibits E1 slowly; inhibition is facilitated in the presence of NAD, resulting in loss of ca. 90% of control activity. Incubation with 14C iodoacetamide labels a 16,000 dalton CNBr peptide, and a ca. 4,000 dalton tryptic cleavage product. These fragments can be equated with those which have been suggested by disulfiram.

Aldehyde Oxidoreductases↗
PubMed1979

Human stomach alcohol dehydrogenase: isoenzyme composition and catalytic properties.

Two isoenzymes of alcohol dehydrogenase have been purified from human stomach and characterized with regard to electrophoretic mobility and kinetic properties with ethanol, hexanol, and acetaldehyde. Both undergo a time-dependent formation of multiple electrophoretic bands; the total amount of alcohol dehydrogenase activity in an average human stomach is only about 0.2% of that of the liver.

Alcohol Oxidoreductases↗