Sorbitol Dehydrogenase from Bovine Lens: Purification and Properties

Marini, I; Bucchioni, L; Borella, Paola; Corso, Antonella Del; Mura, Umberto

doi:10.1006/abbi.1997.9882

Cited by 28 publications

(31 citation statements)

References 40 publications

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“…Enzyme Purification and Assay-Sorbitol dehydrogenase was purified to electrophoretic homogeneity as described previously (19). The final enzyme preparation (approximately 1 mg/ml) with a specific activity of 51 units/mg was stored at 4°C in 10 mM sodium phosphate, pH 7 (S-buffer) supplemented with 2 mM dithiothreitol and 0.1 mM NADH.…”

Section: Methodsmentioning

confidence: 99%

“…The assay of enzyme activity was performed at 37°C as described previously (19) by following the decrease in absorbance at 340 nm in a reaction mixture (0.5 ml final volume) containing 0.24 mM NADH and 0.4 M D-fructose in a 100 mM Tris-HCl buffer, pH 7.4. The rate of NADH oxidation measured in a parallel assay in which the substrate was omitted was subtracted as a blank.…”

Section: Methodsmentioning

confidence: 99%

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Complete Protection by α-Crystallin of Lens Sorbitol Dehydrogenase Undergoing Thermal Stress

Marini

Moschini

Corso

et al. 2000

Journal of Biological Chemistry

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Sorbitol dehydrogenase (L-iditol:NAD؉ 2-oxidoreductase, E.C. 1.1.1.14) (SDH) was significantly protected from thermally induced inactivation and aggregation by bovine lens ␣-crystallin. An ␣-crystallin/SDH ratio as low as 1:2 in weight was sufficient to preserve the transparency of the enzyme solution kept for at least 2 h at 55°C. Moreover, an ␣-crystallin/SDH ratio of 5:1 (w/w) was sufficient to preserve the enzyme activity fully at 55°C for at least 40 min. The protection by ␣-crystallin of SDH activity was essentially unaffected by high ionic strength (i.e. 0.5 M NaCl). On the other hand, the transparency of the protein solution was lost at a high salt concentration because of the precipitation of the ␣-crystallin/SDH adduct. Magnesium and calcium ions present at millimolar concentrations antagonized the protective action exerted by ␣-crystallin against the thermally induced inactivation and aggregation of SDH. The lack of protection of ␣-crystallin against the inactivation of SDH induced at 55°C by thiol blocking agents or EDTA together with the additive effect of NADH in stabilizing the enzyme in the presence of ␣-crystallin suggest that functional groups involved in catalysis are freely accessible in SDH while interacting with ␣-crystallin. Two different adducts between ␣-crystallin and SDH were isolated by gel filtration chromatography. One adduct was characterized by a high M r of approximately 800,000 and carried exclusively inactive SDH. A second adduct, carrying active SDH, had a size consistent with an interaction of the enzyme with monomers or low M r aggregates of ␣-crystallin. Even though it had a reduced efficiency with respect to ␣-crystallin, bovine serum albumin was shown to mimic the chaperone-like activity of ␣-crystallin in protecting SDH from thermal denaturation. These findings suggest that the multimeric structural organization of ␣-crystallin may not be a necessary requirement for the stabilization of the enzyme activity.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Complete Protection by α-Crystallin of Lens Sorbitol Dehydrogenase Undergoing Thermal Stress

Marini

Moschini

Corso

et al. 2000

Journal of Biological Chemistry

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“…Several factors affect the accumulation of sorbitol including the amount of the enzyme SDH. SDH catalyses the reversible oxidation of sorbitol and other polyalcohols to the corresponding keto-sugars [8]. There are eight chemical species involved in the reaction: NADH(…”

Section: Scd Model (Scd1)mentioning

confidence: 99%

“…The ranges are bounded and are estimated using realistic concentration values derived from experimental data and Michaelis -Menten constants (Km) defined as the rate of the reaction at half-maximal velocity [8]. Table 1 describes the seven reactions and rates involved in SCD.…”

Section: Scd Model (Scd1)mentioning

confidence: 99%

“…However, many such drugs have off-target or unpredicted effects, which perturb the system in other, often unexpected ways. The chemical reactions and kinetic coefficients for the model have been previously studied [8], but a model incorporating the effect of drug treatment has, to our knowledge, not been created. Understanding the exact conditions that lead to the development of sugar cataracts and how drug treatment will potentially reverse the conditions will help better predict and prevent sugar cataracts from developing.…”

Section: Introductionmentioning

confidence: 99%

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Modelling and analysis of the sugar cataract development process using stochastic hybrid systems

Riley¹,

Koutsoukos²,

Riley³

2009

IET Syst. Biol.

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Modelling and analysis of biochemical systems such as sugar cataract development (SCD) are critical because they can provide new insights into systems, which cannot be easily tested with experiments; however, they are challenging problems due to the highly coupled chemical reactions that are involved. The authors present a stochastic hybrid system (SHS) framework for modelling biochemical systems and demonstrate the approach for the SCD process. A novel feature of the framework is that it allows modelling the effect of drug treatment on the system dynamics. The authors validate the three sugar cataract models by comparing trajectories computed by two simulation algorithms. Further, the authors present a probabilistic verification method for computing the probability of sugar cataract formation for different chemical concentrations using safety and reachability analysis methods for SHSs. The verification method employs dynamic programming based on a discretisation of the state space and therefore suffers from the curse of dimensionality. To analyse the SCD process, a parallel dynamic programming implementation that can handle large, realistic systems was developed. Although scalability is a limiting factor, this work demonstrates that the proposed method is feasible for realistic biochemical systems.

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