tretinoin and 13-cis-retinal

Retinal dehydrogenase (RALDH) isozymes catalyze the terminal oxidation of retinol into retinoic acid (RA) that is essential for embryogenesis and tissue differentiation. To understand the role of mouse type 2 RALDH in synthesizing the ligands (all-trans and 9-cis RA) needed to bind and activate nuclear RA receptors, we determined the detailed kinetic properties of RALDH2 for various retinal substrates. Purified recombinant RALDH2 showed a pH optimum of 9.0 for all-trans retinal oxidation. The activity of the enzyme was lower at 37 degrees C compared to 25 degrees C. The efficiency of conversion of all-trans retinal to RA was 2- and 5-fold higher than 13-cis and 9-cis retinal, respectively. The K(m) for all-trans and 13-cis retinal were similar (0.66 and 0.62 microM, respectively). However, the K(m) of RALDH2 for 9-cis retinal substrate (2.25 microM) was 3-fold higher compared to all-trans and 13-cis retinal substrates. Among several reagents tested for their ability to either inhibit or activate RALDH2, citral and para-hydroxymercuribenzoic acid (p-HMB) inhibited and MgCl(2) activated the reaction. Comparison of the kinetic properties of RALDH2 for retinal substrates and its activity towards various reagents with those of previously reported rat kidney RALDH1 and human liver aldehyde dehydrogenase-1 showed distinct differences. Since RALDH2 has low K(m) and high catalytic efficiency for all-trans retinal, it may likely be involved in the production of all-trans RA in vivo.

Topics: Aldehyde Oxidoreductases; Alitretinoin; Animals; Catalysis; Cloning, Molecular; Diterpenes; Gene Expression Regulation, Enzymologic; Hydrogen-Ion Concentration; Isotretinoin; Kinetics; Mice; Recombinant Proteins; Retinal Dehydrogenase; Retinaldehyde; Temperature; Tretinoin; Vitamin A

Lipocalin-type prostaglandin D synthase is responsible for the biosynthesis of prostaglandin D2 in the central nervous system and the genital organs and is secreted into the cerebrospinal fluid and the seminal plasma as beta-trace. Here we analyzed retinoids binding of the enzyme by monitoring the fluorescence quenching of an intrinsic tryptophan residue, and appearance of circular dichroism around 330 nm, and a red shift of the UV absorption spectra of retinoids. We found that the enzyme binds all-trans- or 9-cis-retinoic acid and all-trans- or 13-cis-retinal, but not all-trans-retinol, with affinities (Kd of 70-80 nM) sufficient for function as a retinoid transporter. All-trans-retinoic acid inhibited the enzyme activity in a noncompetitive manner, suggesting that it binds to the same hydrophobic pocket as prostaglandin H2, the substrate for prostaglandin D synthase, but at a different site in this pocket. It is likely that this enzyme is a bifunctional protein that acts as both retinoid transporter and prostaglandin D2-producing enzyme.

Topics: Alanine; Alitretinoin; Animals; Biological Transport, Active; Circular Dichroism; Cystine; Diterpenes; Intramolecular Oxidoreductases; Isomerases; Isomerism; Kinetics; Lipocalins; Models, Molecular; Protein Conformation; Rats; Recombinant Proteins; Retinaldehyde; Retinoids; Retinol-Binding Proteins; Retinol-Binding Proteins, Plasma; Spectrometry, Fluorescence; Spectrophotometry, Ultraviolet; Tretinoin