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Do alpha-crystallins protect catalase against UV damage?

The original finding that lens [Alpha]-crystallin could function as a molecular chaperone, protecting many proteins against heat denaturation, was made by Horwitz (1). Not only did [Alpha]-crystallin protect structural proteins of the lens, but it also protected selected enzyme activities against heat-induced inactivation. That UVA irradiation causes structural and functional damage to the catalase of the lens was discovered by Zigman et al. (2). In this preliminary study, we tested the ability of bovine [Alpha]crystallin to protect the activity of catalase from UVA inhibition.

Sigma 2x crystallized beef liver catalase (0.84mg/ml; 36 units/ml) in 0.1 M, pH 7.2 phosphate buffer was exposed to Woods lamp UVA radiation at [approximately]2.5 mW/[cm.sup.2], for 12 h at 20 [degrees] C, under an atmosphere of 95% air:5% C[O.sub.2]. The optical densities at 280 nm ([O.D..sub.280]) of both catalase and [Alpha]-crystallin solutions were determined spectrophotometrically. The [O.D..sub.280] of both solutions ranged from 0.350 to 0.700 units. Mixtures of catalase and [Alpha]-crystallin solutions having an equivalent [O.D..sub.280], or in which the [Alpha]-crystallin [O.D..sub.280] was twice (molar equivalence) or three times that of the catalase, were prepared and irradiated. One variation of the above protocol was to preincubate catalase for 12 h with a solution of UVA-irradiated tryptophan (UV-trp; 1% in P[O.sub.4] buffer) as a catalase inhibitor. This was done to assess the ability of [Alpha]-crystallin to protect catalase activity from UV-trp. Another variation was to use a 10,000 x g supernatant of dogfish lens-capsule epithelium as the catalase source. Its preparation is described in our companion short report (3). [TABULAR DATA FOR TABLE I OMITTED] Alpha-crystallin was added during UVA exposure to reduce the damaging effect on lens catalase.

Oxygen production (i.e., catalase activity) was measured with an [O.sub.2] electrode and meter (Microelectrodes, Inc.) and displayed as a function of time with a chart recorder. The percent [O.sub.2] produced per minute for treated catalase was compared with the activity of known catalase standards.

Table I shows that [Alpha]-crystallin partially protects purified catalase activity when present at 2x (equimolar) and 3x the [O.D..sub.280] of the catalase. When UVA-exposed 1% trytophan (UV-trp) was added as the perturbant of catalase activity (4), the results in Table I were also obtained: the presence of 2x [O.D..sub.280] (or equimolar) [Alpha]-crystallin protected the catalase activity from UV-trp by about 34%. When homogenized and centrifuged dogfish lens-capsule epithelial-cell catalase activity was used to test [Alpha]crystallin's protection against UVA (Table I), [Alpha]-crystallin was also found to be protective.

In the living lens, most catalase activity is in the epithelium where [Alpha]-crystallin is also present. A higher concentration of alpha is present in the soluble phase, while catalase is mainly in the peroxysomes. However, cellular damage releases catalase into the soluble phase.

The results show that [Alpha]-crystallin can protect against the UVA inhibition of catalase enzymatic activity, as was shown for other enzymes by Borkman et al. (5). The concept that [Alpha]crystallin has a great variety of molecular chaperone activities is supported by this study.

We dedicate this to the fond memory of Arlene Horwitz, who appreciated the time she spent at Woods Hole and the Marine Biological Laboratory. We thank Linlin Ding for providing [Alpha]crystallin (Jules Stein Eye Institute) and Ray Borkman and Grady Knight (Georgia Institute of Technology) for providing the molar extinction coefficient for [Alpha]-crystallin. Supported by: National Eye Institute (NIH) # EY 3897 (Horwitz).

Literature Cited

1. Horwitz, J., 1993. Invest. Ophthal. & Vis. Sci. 34: 10-22.

2. Zigman, S., J. Reddan, J. B. Schultz, and T. McDaniel. 1996. Photochem. Photobiol. 63: 818-824.

3. Zigman, S. 1997. Biol. Bull. 193: 253-254.

4. Zigman, S., T. Yulo, and G. A. Griess. 1976. Mol. Cell. Biochem. 11: 131-135.

5. Borkman, R. F., G. Kuyer, and B. Obi. 1996. Exp. Eye Res. 62: 141-148.
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Title Annotation:ultraviolet radiation protection
Author:Horwitz, Joseph; Zigman, Seymour
Publication:The Biological Bulletin
Date:Oct 1, 1997
Words:677
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