Ethanol metabolism by HeLa cells transduced with human alcohol dehydrogenase isoenzymes: control of the pathway by acetaldehyde concentration.

Background: Human class I alcohol dehydrogenase 2 isoenzymes (encoded by the ADH1B locus) have large differences in kinetic properties; however, individuals inheriting the alleles for the different isoenzymes exhibit only small differences in alcohol elimination rates. This suggests that other cellular factors must regulate the activity of the isoenzymes.

Methods: The activity of the isoenzymes expressed from ADH1B*1, ADH1B*2, and ADH1B*3 cDNAs was examined in stably transduced HeLa cell lines, including lines which expressed human low K(m) aldehyde dehydrogenase (ALDH2).

Kinetic and cellular characterization of novel inhibitors of S-nitrosoglutathione reductase.

S-Nitrosoglutathione reductase (GSNOR) is an alcohol dehydrogenase involved in the regulation of S-nitrosothiols (SNOs) in vivo. Knock-out studies in mice have shown that GSNOR regulates the smooth muscle tone in airways and the function of beta-adrenergic receptors in lungs and heart. GSNOR has emerged as a target for the development of therapeutic approaches for treating lung and cardiovascular diseases.

Human carboxylesterases: an update on CES1, CES2 and CES3.

Carboxylesterases belong to Phase I group of drug metabolizing enzymes. They hydrolyze a variety of drug esters, amides, carbamates and similar structures. There are five 'carboxylesterase' genes listed in the Human Genome Organization database.

A multiple reaction monitoring method for absolute quantification of the human liver alcohol dehydrogenase ADH1C1 isoenzyme.

Although significant progress has been made in protein quantification using mass spectrometry during recent years, absolute protein quantification in complex biological systems remains a challenging task in proteomics. The use of stable isotope-labeled standard peptide is the most commonly used strategy for absolute quantification, but it might not be suitable in all instances. Here we report an alternative strategy that employs a stable isotope-labeled intact protein as an internal standard to absolutely quantify the alcohol dehydrogenase (ADH) expression level in a human liver sample.

Expression and characterization of a human carboxylesterase 2 splice variant.

CPT-11 [7-ethyl-10-[4-(1-piperidino)-1-piperidino] carbonyloxycamptothecin or Irinotecan] is a carbamate prodrug that is activated in vivo by carboxylesterase (CES)-2 to SN-38 (7-ethyl-10-hydroxycamptothecin), a potent topoisomerase I inhibitor. There is high interindividual variation when CPT-11 is used in the treatment of colorectal cancer. Several splice variants of CES2 are reported in the expressed sequence tag database.

Assay of the pyruvate dehydrogenase complex by coupling with recombinant chicken liver arylamine N-acetyltransferase.

The activity of the pyruvate dehydrogenase complex has long been determined in some laboratories by coupling the production of acetyl-coenzyme A (acetyl-CoA) to the acetylation of 4-aminoazobenzene-4'-sulfonic acid by arylamine N-acetyltransferase. The assay has some advantages, but its use has been limited by the need for large amounts of arylamine N-acetyltransferase. Here we report production of recombinant chicken liver arylamine N-acetyltransferase and optimization of its use in miniaturized assays for the pyruvate dehydrogenase complex and its kinase.

Structure-function relationships in human glutathione-dependent formaldehyde dehydrogenase. Role of Glu-67 and Arg-368 in the catalytic mechanism.

The active-site zinc in human glutathione-dependent formaldehyde dehydrogenase (FDH) undergoes coenzyme-induced displacement and transient coordination to a highly conserved glutamate residue (Glu-67) during the catalytic cycle. The role of this transient coordination of the active-site zinc to Glu-67 in the FDH catalytic cycle and the associated coenzyme interactions were investigated by studying enzymes in which Glu-67 and Arg-368 were substituted with Leu. Structures of FDH.

Structure-function relationships in human Class III alcohol dehydrogenase (formaldehyde dehydrogenase).

Human Class III alcohol dehydrogenase (ADH), also known as glutathione-dependent formaldehyde dehydrogenase plays an important role in the formaldehyde detoxification and reduction of the nitric oxide metabolite s-nitrosoglutathione (GSNO). It follows a random bi bi kinetic mechanism and prefers bulkier substrates like long chain primary alcohols and glutathione adducts like s-hydroxymethylglutathione and GSNO over smaller alcohols like ethanol. The structure of the FDH.

Human glutathione-dependent formaldehyde dehydrogenase. Structural changes associated with ternary complex formation.

Human glutathione-dependent formaldehyde dehydrogenase plays an important role in the metabolism of glutathione adducts such as S-(hydroxymethyl)glutathione and S-nitrosoglutathione. The role of specific active site residues in binding these physiologically important substrates and the structural changes during the catalytic cycle of glutathione-dependent formaldehyde dehydrogenase was examined by determining the crystal structure of a ternary complex with S-(hydroxymethyl)glutathione and the reduced coenzyme to 2.6 A resolution.

Human glutathione-dependent formaldehyde dehydrogenase. Structures of apo, binary, and inhibitory ternary complexes.

The human glutathione-dependent formaldehyde dehydrogenase is unique among the structurally studied members of the alcohol dehydrogenase family in that it follows a random bi bi kinetic mechanism. The structures of an apo form of the enzyme, a binary complex with substrate 12-hydroxydodecanoic acid, and a ternary complex with NAD+ and the inhibitor dodecanoic acid were determined at 2.0, 2.