T. Geoffrey Flynn

Human aldose reductase and human small intestine aldose reductase are efficient retinal reductases: consequences for retinoid metabolism

Aldo-keto reductases (AKRs) are NAD(P)H-dependent oxidoreductases that catalyse the reduction of a variety of carbonyl compounds, such as carbohydrates, aliphatic and aromatic aldehydes and steroids. We have studied the retinal reductase activity of human aldose reductase (AR), human small-intestine (HSI) AR and pig aldehyde reductase. Human AR and HSI AR were very efficient in the reduction of all- trans -, 9- cis - and 13- cis -retinal ( k (cat)/ K (m)=1100-10300 mM(-1).min(-1)), constituting the first cytosolic NADP(H)-dependent retinal reductases described in humans. Aldehyde reductase showed no activity with these retinal isomers. Glucose was a poor inhibitor ( K (i)=80 mM) of retinal reductase activity of human AR, whereas tolrestat, a classical AKR inhibitor used pharmacologically to treat diabetes, inhibited retinal reduction by human AR and HSI AR. All- trans -retinoic acid failed to inhibit both enzymes. In this paper we present the AKRs as an emergent superfamily of retinal-active enzymes, putatively involved in the regulation of retinoid biological activity through the assimilation of retinoids from beta-carotene and the control of retinal bioavailability.

Secretion of atrial natriuretic peptide and vasopressin by small cell lung cancer

Background. Hyponatremia in patients with small cell lung cancer (SCLC) is a common clinical problem usually attributed to tumor secretion of arginine vasopressin (AVP). It recently was shown that some SCLC cell lines produce atrial natriuretic peptide (ANP). The purpose of this investigation was to determine the frequency and clinical consequences of secretion of ANP by SCLC and the relative contribution of ANP and AVP to the hyponatremia associated with this disease.

Isolation and characterization of iso-rANP, a new natriuretic peptide from rat atria

Using a specific radioimmunoassay we have isolated and sequenced a new 45-amino acid peptide from rat atria which exhibits similar physiological and pharmacological properties to rat atrial natriuretic peptide (rANP). We have termed the new peptide iso-rANP, because of its functional and structural similarities to rANP. Amino acid sequence differences show that iso-rANP is genetically distinct from rANP. Iso-rANP has a single disulfide bond between residues 23-39 and this portion of the peptide shows substantial homology to rANP and to porcine brain natriuretic peptide (BNP). Little homology is evident at the N- and C-termini of iso-rANP and ANP. Iso-rANP is equipotent with rANP in eliciting diuresis, natriuresis and hypotension in the bioassay rat.

Identification of Pig Brain Aldehyde Reductases with the High-Km Aldehyde Reductase, the Low-Km Aldehyde Reductase and Aldose Reductase, Carbonyl Reductase, and Succinic Semialdehyde Reductase

Abstract: Four NADPH‐dependent aldehyde reductases (ALRs) isolated from pig brain have been characterized with respect to substrate specificity, inhibition by drugs, and immunological criteria. The major enzyme, ALR1, is identical in these respects with the high‐Km aldehyde reductase, glucuronate reductase, and tissue‐specific, e.g., pig kidney aldehyde reductase. A second enzyme, ALR2, is identical with the low‐Km aldehyde reductase and aldose reductase. The third enzyme, ALR3, is carbonyl reductase and has several features in common with prostaglandin‐9‐ketoreductase and xenobiotic ketoreductase. The fourth enzyme, unlike the other three which are monomeric, is a dimeric succinic semialdehyde reductase. All four of these enzymes are capable of reducing aldehydes derived from the biogenic amines. However, from a consideration of their substrate specificities and the relevant Km and Vmax values, it is likely that it is ALR2 which plays a primary role in biogenic aldehyde metabolism. Both ALR1 and ALR2 may be involved in the reduction of isocorticosteroids. Despite its capacity to reduce ketones, ALR3 is primarily an aldehyde reductase, but clues as to its physiological role in brain cannot be discerned from its substrate specificity. The capacity of succinic semialdehyde reductase to reduce succinic semialdehyde better than any other substrate shows that this reductase is aptly named and suggests that its primary role is the maintenance in brain of physiological levels of γ‐hydroxybutyrate.

A Nomenclature System for the Aldo-Keto Reductase Superfamily

As new members of the AKR superfamily are identified the need for a systematic and expandable nomenclature has risen, especially since some members of the superfamily have multiple names based on substrate specificity. We have proposed a nomenclature system for the AKR superfamily that is similar to the P450 system but based on amino acid sequence comparisons instead of nucleotide sequence comparisons. Our system uses percent amino acid identities to delineate families and subfamilies within the larger superfamily. Although there are not as many AKRs as P450s, having a flexible nomenclature system will allow for easy incorporation of new proteins into the superfamily.

Enzymology and molecular biology of carbonyl metabolism 6

Aldehyde Dehydrogenase. Crystal Structure of a Class 3 Aldehyde Dehydrogenase at 2.6angstrom Resolution Z-J. Liu, et al. Conserved Residues in the Aldehyde Dehydrogenase Family: Locations in the Class 3 Tertiary Structure J. Hempel, et al. Class 3 Aldehyde Dehydrognease: A View from the Hills R. Lindahl, et al. Human Corneal and Lens Aldehyde Dehydrogenases: Purification and Properties of Human Lens ALDH1 and Differential Expression as Major Soluble Proteins in Human Lens (ALDH1) and Cornea (ALDH3) G. King, R. Holmes. Alcohol Dehydrogenase. Alcohol Dehydrogenase Variability: Evolutionary and Functional Conclusions from Characterization of Further Variants H. Jornvall, et al. Three-Dimensional Structures of Human Alcohol Dehydrogenase Isoenzymes Reveal the Molecular basis for Their Functional Diversity T.D. Hurley, et al. Mammalian Class II Alcohol Dehydrogenase: A Highly Variable Enzyme J.-O. Hoog, S. Svensson. Activity of Liver Alcohol Dehydrogenases on Steroids D.K. Wilson, et al. Aldo/Keto Reductases. Structural Studies of Aldo-Keto Reductase Inhibition D.K. Wilson, et al. Aldehyde Reductase: Catalytic Mechanism and Substrate Recognition O.A. Barski, et al. Study of Non-Covalent Enzyme-Inhibitor Complexes of Aldose Reductase by Electrospray Mass Spectrometry N. Potier, et al. 55 Additional Articles. Index.

Structure and mechanism of aldehyde reductase.

Aldehyde reductase (ALR1, EC 1.1.1.2) and aldose reductase (ALR2, EC 1.1.1.21) catalyze the NADPH-dependent reduction of a wide range of aromatic and aliphatic aldehydes to their corresponding alcohols. Despite a recently expressed opinion that aldose reductase is of little consequence (Harding, 1992) the past few years have seen a great advancement in our knowledge of the structure and mechanism of both enzymes, and of aldose reductase in particular. The three-dimensional structure of pig (Rondeau et al., 1992) and human (Wilson et al., 1992) aldose reductase revealed that as an oxido-reductase the enzyme is unique in that it has a β/α-TIM barrel structure and is the first oxido-reductase known to possess such a structure and to not have a dinucleotide or Rossmann binding fold. Kinetic studies have shown that the enzyme operates by an ordered mechanism with NADPH binding first (Grimshaw et al, 1990; Kubiseski et al., 1992). The binding of coenzyme is very tight (≪lμM, see Grimshaw and Lai in this Proceedings) and following coenzyme binding there is a conformational change in the enzyme. This has been shown by fluorescence spectroscopy (Kubiseski et al., 1992); by a combination of chemical modification and X-ray crystallography (Kubiseski et al., 1994) and from a comparison of the three-dimensional structures of human and porcine aldehyde and aldose reductase (El-Kabbani et al., 1994). Structural studies have also revealed that an aldose reductase inhibitor (ARI), zopolrestat, binds in the active site (Wilson et al., 1993) and not at a site removed from the active site as suggested by the fact that all ARIs are uncompetitive or non-competitive inhibitors in the forward direction of the reaction (Sato & Kador, 1990). Most ARIs are in fact competitive with the alcohol product and binding of ARIs at the active site is, of course, feasible and understandable (Sato & Kador, 1990).

Acute ethanol ingestion modifies the circulating plasma levels of atrial natriuretic peptide

Since ethanol ingestion is associated with a disruption of water and electrolyte balance in a variety of species, we sought to evaluate the regulatory control of atrial natriuretic peptide (ANP) in response to acute doses of ethanol. Male Sprague-Dawley rats were administered a 5-g/kg dose of ethanol (40% w/v) via a gastric tube, while control animals received an equivalent volume of water. Expressed as a percentage of control, plasma ANP levels were 39.0%, 28.5%, and 23.6% in the ethanol-treated animals at 30, 60, and 120 min postintubation, respectively. Ethanol-treated animals displayed blood alcohol concentrations of 89.0, 137.6, and 214.1 mg/dl at the same time periods. After 120 min, plasma renin activity was elevated from 8.7 to 20.3 ng/ml/h in conjunction with an increase in the levels of circulating aldosterone from 16.3 to 42.5 ng/dl and an increase in plasma vasopressin from 2.2 to 3.6 pg/ml. Levels of atrial ANP mRNA remained consistent over the time course of the experiment, and no changes in the amount of ventricular ANP transcript were observed. Tissue ANP levels were similar between ethanol-treated and water-loaded control animals. In vitro experiments using cultured cardiac myocytes suggest that ethanol exposure may not directly affect ANP secretion. We propose that acute ethanol treatment may inhibit atrial distension and subsequently modify the control of ANP release under volume loading conditions.

Purification and Characterization of Four NADPH-Dependent Aldehyde Reductases from Pig Brain

Abstract: By a procedure involving ammonium sulfate precipitation, gel filtration, and affinity chromatography, four aldehyde reductases (ALRs) were purified to enzymatic homogeneity from pig brain. These enzymes, designated ALR1, ALR2, ALR3, and succinic semialdehyde reductase were chemically and physically identical with, respectively, the high‐Km aldehyde reductase, the low‐Km aldehyde reductase, carbonyl reductase, and succinic semialdehyde reductase of other tissues and species. The purification procedure allows the purification of these enzymes from the same tissue homogenate in amounts sufficient for characterization and other enzymatic studies. This methodology should be applicable to the simultaneous and rapid purification of aldehyde reductases from other tissues.

Crystal structure of CHO reductase, a member of the aldo-keto reductase superfamily.

Chinese hamster ovary (CHO) reductase is an enzyme belonging to the aldo‐keto reductase (AKR) superfamily that is induced by the aldehyde‐containing protease inhibitor ALLN (Inoue, Sharma, Schimke, et al., J Biol Chem 1993;268:5894). It shows 70% sequence identity to human aldose reductase (Hyndman, Takenoshita, Vera, et al., J Biol Chem 1997;272:13286), which is a target for drug design because of its implication in diabetic complications. We have determined the crystal structure of CHO reductase complexed with nicotinamide adenine dinucleotide phosphate (NADP)+ to 2.4 Å resolution. Similar to aldose reductase and other AKRs, CHO reductase is an α/β TIM barrel enzyme with cofactor bound in an extended conformation. All key residues involved in cofactor binding are conserved with respect to other AKR members. CHO reductase shows a high degree of sequence identity (91%) with another AKR member, FR‐1 (mouse fibroblast growth factor‐regulated protein), especially around the variable C‐terminal end of the protein and has a similar substrate binding pocket that is larger than that of aldose reductase. However, there are distinct differences that can account for differences in substrate specificity. Trp111, which lies horizontal to the substrate pocket in all other AKR members is perpendicular in CHO reductase and is accompanied by movement of Leu300. This coupled with movement of loops A, B, and C away from the active site region accounts for the ability of CHO reductase to bind larger substrates. The position of Trp219 is significantly altered with respect to aldose reductase and appears to release Cys298 from steric constraints. These studies show that AKRs such as CHO reductase are excellent models for examining the effects of subtle changes in amino acid sequence and alignment on binding and catalysis. Proteins 2000;38:41–48. ©2000 Wiley‐Liss, Inc.