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Background: Glucose is considered as one of the main sources of cell damage related to aldose reductase (AR) action in hyperglycemic conditions and a worldwide effort is posed in searching for specific inhibitors of the enzyme. This AR substrate has often been reported as generating non-hyperbolic kinetics, mimicking a negative cooperative behavior. This feature was explained by the simultaneous action of two enzyme forms acting on the same substrate., Methods: The reduction of different aldoses and other classical AR substrates was studied using pure preparations of bovine lens and human recombinant AR., Results: The apparent cooperative behavior of AR acting on glucose and other hexoses and pentoses, but not on tethroses, glyceraldehyde, 4-hydroxynonenal and 4-nitrobenzaldehyde, is generated by a partial nonclassical competitive inhibition exerted by the aldose hemiacetal on the reduction of the free aldehyde. A kinetic model is proposed and kinetic parameters are determined for the reduction of l-idose., Conclusions: Due to the unavoidable presence of the hemiacetal, glucose reduction by AR occurs under different conditions with respect to other relevant AR-substrates, such as alkanals and alkenals, coming from membrane lipid peroxidation. This may have implications in searching for AR inhibitors. The emerging kinetic parameters for the aldoses free aldehyde indicate the remarkable ability of the enzyme to interact and reduce highly hydrophilic and bulky substrates., General Significance: The discovery of aldose reductase modulation by hemiacetals offers a new perspective in searching for aldose reductase inhibitors to be developed as drugs counteracting the onset of diabetic complications., (Copyright © 2015. Published by Elsevier B.V.)

Purine nucleoside phosphorylase (purine nucleoside: orthophosphate ribosyltransferase, EC 2.4.2.1) was purified 38,750-fold to apparent electrophoretic homogeneity from bovine ocular lens. The enzyme appears to be a homotrimer with a molecular weight of 97,000, and displays non-linear kinetics with concave downward curvature in double-reciprocal plots with orthophosphate as variable substrate. The analysis of the kinetic parameters of bovine lens purine nucleoside phosphorylase, determined both for the phosphorolytic activity on nucleosides and for ribosylating activity on purine bases, indicates the occurrence of a rapid equilibrium random Bi-Bi mechanism with formation of abortive complexes. The effect of pH on the enzyme activity and on the sensitivity of the enzyme to photoinactivation, as well as the effect of thiol reagents on the enzyme activity and stability, strongly suggest the involvement of histidine and cysteine residues in the active site. From the measurements of the kinetic parameters at different temperatures, heats of formation of the enzyme-substrate complex for guanosine, guanine, orthophosphate and ribose 1-phosphate were determined. Activation energies of 15,250 and 14,650 cal/mol were obtained for phosphorolysis and synthesis of guanosine, respectively.

Aldo-keto reductase has been purified 13,000-fold from the lens of the camel (Camelus dromedarius) to a specific activity of 85 U/mg protein. The enzyme is a monomeric protein, exhibiting a Mr = 40,000 upon polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. Camel lens aldo-keto reductase shows a broad substrate specificity, which is strictly dependent on NADPH, and is insensitive to inhibition by Sorbinil and valproate. Aldoses with a carbon chain with more than four residues, as well as glucuronate, are not reduced by the enzyme. On the basis of substrate specificity and sensitivity to inhibition, camel lens aldo-keto reductase appears to be distinct from the so far described aldose, aldehyde and carbonyl reductases.

1. The kinetics of the hydrolysis of nitrophenylphosphate by nonspecific acid phosphatase (orthophosphoric-monoester phosphohydrolase (acid optimum), EC 3.1.3.2.) from Schizosaccharomices pombe was studied. 2. The kinetic parameters, Km and V, were determined as well as the inhibition constants, K1, for the inhibitors, phosphate and fluoride, as a function of pH. 3. The results, interpreted according to the theories of Dixon and Waley indicated the presence of three ionizable groups on the enzyme itself and one on the enzyme-substrate complex. 4. A model of the hydrolysis of phosphoric acid monoesters by the S. pombe acid phosphatase is proposed based on the ionization state of the reactants and on the results of the inhibition by the competitive inhibitors.