Protection of a Restriction Enzyme from Heat Inactivation by α - Crystallin

Correspondence to: John F. Hess, Department of Cell Biology and Human Anatomy, School of Medicine, University of California, Davis, CA, 95616-8643; Phone: (530) 752-8420; FAX: (530) 7528520; email: [email protected] Predominating the protein profile of the lens are the crystallins, low molecular weight proteins that compose 90% of the mass of the lens [1,2]. Of the three crystallin classes, α, β, and γ, α-crystallins predominate. Amino acid/DNA sequencing has shown that α-crystallins are evolutionarily related to small heat shock proteins, a finding that is intellectually satisfying when viewed in relation to long term protein stability in the lens [3-5]. Additionally, α-crystallins have been demonstrated to have chaperone-like activity in vitro, protecting heterologous proteins from insults [6-14]. The protective effects of α-crystallin toward heterologous proteins was first demonstrated by the heating of alcohol dehydrogenase (ADH) in the presence and absence of α-crystallin [6]. At 48 °C, in the absence of α-crystallin, ADH aggregates and this aggregation can be monitored by light scattering. In the presence of α-crystallin, heat induced aggregation is eliminated. Numerous other metabolic enzymes were also found to be protected from thermally induced aggregation. The enzymatic activities of the enzymes studied were not reported; inactivation was mentioned although data were not presented. Subsequent to the intial report by Horwitz, other investigators found that α-crystallin offered protection from additional insults. Obi and coworkers reported a reduction in UV induced protein aggregation by α-crystallin [9]. Harding and coworkers reported that α-crystallin protects catalase from steroid inactivation and 6-phosphogluconate dehydrogenase from carbamylation [7,8,10]. Harding and coworkers also reported protection of enzymatic activity by α-crystallin, a function not provided by control proteins [7,8,10]. In a separate report that examined aggregation as a measure of protein denaturation, α-crystallin was shown to prevent aggregation while enzymatic activity was lost [14]. Thus, α-crystallin seemed better able to prevent protein aggregation than preserve enzymatic activity. How these two phenomena differ is unknown. From a protein stability standpoint, the chaperone/thermoprotective activity of α-crystallin could prove to be useful. In other research areas, in vitro stabilization of proteins includes: stabilization of thermolabile enzymes by trehalose [15] or the heat shock protein dnaK [16], the interaction between sorbitol and lysozyme [17], and the effects of Ca and sugars on recombinant DNase I [18]. Notable for discussion here is the recognition that some compounds can have appreciable stabilization/survival effects on intact cells and that methods taken to increase intracellular protein stability can have beneficial consequences for higher organisms. For example, recent reports documenting the increased stability of isolated platelets in the presence of antarctic fish antifreeze proteins [19] and the increased survival of heat shock protein expressing cells when ischemically stressed shows how single proteins can perform cellular life saving functions [20,21]. In addition, transgenic plants that produce the protein stabilizing, small molecular weight compound betaine are found to be more resistant to salt and cold temperature stress [22]. Recently, αB crystallin has also been shown to increase survival of E. coli that are exposed to heat shock [23]. We report a series of experiments designed to test the protective effects of α-crystallin with respect to enzymatic activity. We have chosen to use readily available, well characterized restriction enzymes as the enzymes to assay for α-crystallin chaperone activity. Our rationale was that (1) restriction enzymes are completely heterologous to α-crystallin (2) they are commercially available at low cost (3) their enzymatic activities, including heat inactivation properties are well characterized and (4) they are rapidly and easily assayed for activity. With this system, we have found that the restriction © Molecular Vision