I Marini

Specifically Targeted Modification of Human Aldose Reductase by Physiological Disulfides

Aldose reductase is inactivated by physiological disulfides such as GSSG and cystine. To study the mechanism of disulfide-induced enzyme inactivation, we examined the rate and extent of enzyme inactivation using wild-type human aldose reductase and mutants containing cysteine-to-serine substitutions at positions 80 (C80S), 298 (C298S), and 303 (C303S). The wild-type, C80S, and C303S enzymes lost >80% activity following incubation with GSSG, whereas the C298S mutant was not affected. Loss of activity correlated with enzyme thiolation. The binary enzyme-NADP+ complex was less susceptible to enzyme thiolation than the apoenzyme. These results suggest that thiolation of human aldose reductase occurs predominantly at Cys-298. Energy minimization of a hypothetical enzyme complex modified by glutathione at Cys-298 revealed that the glycyl carboxylate of glutathione may participate in a charged interaction with His-110 in a manner strikingly similar to that involving the carboxylate group of the potent aldose reductase inhibitor Zopolrestat. In contrast to what was observed with GSSG and cystine, cystamine inactivated the wild-type enzyme as well as all three cysteine mutants. This suggests that cystamine-induced inactivation of aldose reductase does not involve modification of cysteines exclusively at position 80, 298, or 303.

Chaperone-like features of bovine serum albumin: a comparison with α-crystallin

Abstract.The chaperone behaviour of bovine serum albumin was compared with that of α-crystallin. The chaperone activity was assessed by measuring: (i) the ability to antagonize protein aggregation induced by heat; (ii) the capability to protect the activity of thermally stressed enzymes and (iii) the effectiveness in assisting the functional recovery of chemically denatured sorbitol dehydrogenase. Despite the lack of structural analogies, both proteins show several functional similarities in preventing inactivation of thermally stressed enzymes and in reactivating chemically denatured sorbitol dehydrogenase. As with α-crystallin, the chaperone action of bovine serum albumin appears to be ATP independent. Bovine serum albumin appears significantly less effective than α-crystallin only in preventing thermally induced protein aggregation. A possible relationship between chaperone function and structural organization is proposed. Together, our results indicate that bovine serum albumin acts as a molecular chaperone and that, for its particular distribution, can be included in the extracellular chaperone family.

Complete protection by α-crystallin of lens sorbitol dehydrogenase undergoing thermal stress

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 α-crystallin. An α-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 α-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 α-crystallin of SDH activity was essentially unaffected by high ionic strength (i.e. 0.5m NaCl). On the other hand, the transparency of the protein solution was lost at a high salt concentration because of the precipitation of the α-crystallin/SDH adduct. Magnesium and calcium ions present at millimolar concentrations antagonized the protective action exerted by α-crystallin against the thermally induced inactivation and aggregation of SDH. The lack of protection of α-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 α-crystallin suggest that functional groups involved in catalysis are freely accessible in SDH while interacting with α-crystallin. Two different adducts between α-crystallin and SDH were isolated by gel filtration chromatography. One adduct was characterized by a highM 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 lowM r aggregates of α-crystallin. Even though it had a reduced efficiency with respect to α-crystallin, bovine serum albumin was shown to mimic the chaperone-like activity of α-crystallin in protecting SDH from thermal denaturation. These findings suggest that the multimeric structural organization of α-crystallin may not be a necessary requirement for the stabilization of the enzyme activity.

Alpha-crystallin: an ATP-independent complete molecular chaperone toward sorbitol dehydrogenase

Abstract.α-Crystallin, the major component of the vertebrate lens, is known to interact with proteins undergoing denaturation and to protect them from aggregation phenomena. Bovine lens sorbitol dehydrogenase (SDH) was previously shown to be completely protected by α-crystallin from thermally induced aggregation and inactivation. Here we report that α-crystallin, in the presence of the SDH pyridine cofactor NAD(H), can exert a remarkable chaperone action by favoring the recovery of the enzyme activity from chemically denaturated SDH up to 77%. Indeed, even in the absence of the cofactor, α-crystallin present at a ratio with SDH of 20:1 (w:w) allows a recovery of 35% of the enzyme activity. The effect of ATP in enhancing α-crystallin-promoted SDH renaturation appears to be both nonspecific and to not involve hydrolysis phenomena, thus confirming that the chaperone action of α-crystallin is not dependent on ATP as energy donor.

Discovering an accessible enzyme: Salivary α‐amylase : Prima digestio fit in ore: A didactic approach for high school students

Human salivary α‐amylase is used in this experimental approach to introduce biology high school students to the concept of enzyme activity in a dynamic way. Through a series of five easy, rapid, and inexpensive laboratory experiments students learn what the activity of an enzyme consists of: first in a qualitative then in a semi‐quantitative way. They also learn how some environmental effectors can influence it. The choice of a “human body” enzyme and not an anonymous commercial enzyme is a very attractive one for students. This laboratory approach will be integrated with theoretical knowledge about α‐amylase, starch, their physiological meaning, and biotechnological applications.

Interconversion pathways of aldose reductase induced by thiol compounds.

The occurrence of protein S-thiolation as a consequence of oxidative stress is widely recognized (Thomas et al., 1990; Lou et at, 1990; Schuppe et al., 1992; Giblin, et al., 1995). However, the role and the relevance of this process are still a matter of debate.

From Purines to Basic Biochemical Concepts: Experiments for High School Students.

Many high school biology courses address mainly the molecular and cellular basis of life. The complexity that underlies the most essential processes is often difficult for the students to understand; possibly, in part, because of the inability to see and explore them. Six simple practical experiments on purine catabolism as a part of a biochemistry course for 16–18 years students that require only simple equipment for thin layer chromatography have been developed, tried, and tested. These include detection and measurement of enzyme activities in biological extracts, detection of reversible and irreversible reactions, creating a pathway from consecutive reactions. They focus on purines because of their multifunctionality in biochemical processes. They allow students to grasp specific concepts and to use them as a framework for approaching metabolic complexity.