Si Qi Liu

Does sorbinil bind to the substrate binding site of aldose reductase

With benzyl alcohol as the varied substrate, sorbinil was found to be a competitive inhibitor of aldose reductase, an enzyme implicated in the etiology of secondary diabetic complications. The K(is sorbinil) and the Vmax/Km (V/K) benzyl alcohol decreased at low pH with a pK of 7.5 and 7.7, respectively. These observations suggest that both sorbinil and benzyl alcohol bind to the same site on the enzyme. Active site inhibition by sorbinil is consistent with non-competitive inhibition patterns of sorbinil with nucleotide coenzyme or aldehyde as the varied substrate in the direction of aldehyde reduction.

A micromethod for the determination of fructose in human erythrocytes and plasma

Fructose makes up approximately one-sixth to one-third of the total carbohydrate intake and is cleared from the portal blood after absorption more rapidly and completely than glucose. In tissues, fructose can also be formed from glucose by the polyol pathway in which glucose is first reduced to sorbitol by aldose reductase with the mediation of NADPH, and subsequently sorbitol is oxidized to fructose by sorbitol dehydrogenase using NAD as the cofactor. Usually the level of fructose and sorbitol in the body fluid is low. However, in hyperglycemia, increased levels of sorbitol and fructose in several tissues have been observed, possibly due to increased flux of glucose through the polyol pathway [l-3]. The methods for the determination of free fructose in biological samples such as gas chromatography (GC), high performance liquid chromatography (HPLC) and enzymatic [4-61 are of little use with samples containing high concentrations of glucose which is the case for tissues from hyperglycemic subjects. The presence of much higher concentrations of glucose as compared to fructose markedly interferes with the resolution of fructose peak in GC and HPLC analysis of sugars because of the close resolution time for glucose and fructose. In addition, analysis of sugars by GC and HPLC is cumbersome and time consuming as compared to enzymatic methods [4,5]. The most commonly used enzymatic method for fructose determination which utilizes hexokinase, phosphoglucoisomerase and glucose-6-P-dehydrogenase is less accurate when the fructose/glucose ratio in the sample is approximately 0.05 or less [6]. D-Fructose dehydrogenase [7] or fructokinase [8] has also been used to determine fructose in biological samples, but these enzymes are not commercially available. In the present communication we have described a specific and sensitive fluorometric method based upon the quantification of NAD formed as a result of reduction of fructose by NADH in the presence of sorbitol dehydrogenase [9].

Structure-activity correlations in human kidney aldehyde reductase-catalyzed reduction of para-substituted benzaldehyde by 3-acetyl pyridine adenine dinucleotide phosphate

Steady-state kinetic parameters of the human kidney aldehyde reductase-catalyzed reduction of para-substituted benzaldehydes by 3-acetyl pyridine dinucleotide phosphate (3-APADPH) were determined. The kcat of aldehyde reduction by 3-APADPH was 2- to 4-fold lower than by NADPH. The dissociation constant of 3-APADPH from the enzyme-coenzyme complex was higher (77 microM) than that of NADPH (5.3 microM). Primary deuterium kinetic isotope effects on both kcat and kcat/Km for para-substituted benzaldehyde reduction by 3-APADPH (with the exception of para-carboxybenzaldehyde) were equal and on average 2.82 +/- 0.21, suggesting that these reactions follow a rapid equilibrium-ordered reaction scheme in which the hydride transfer step is rate-limiting. Multiple regression analysis of the data suggests that benzaldehyde reduction depends upon electronic substituent effects, characterized by a rho value of 0.5. These data are consistent with a transition state in which the charge on the aldehyde carbonyl increases relative to the charge on this group in the ground state. A positive deviation of para-carboxybenzaldehyde from the linear correlation between other benzaldehydes and the substituent constant sigma + suggests a specific interaction of the carboxyl substituent of the substrate with the enzyme.