May 2006
Volume 47, Issue 13
Free
ARVO Annual Meeting Abstract  |   May 2006
Endonuclease Activity in Human Tears
Author Affiliations & Notes
  • B.J. Glasgow
    Pathology & Ophthalmology, Jules Stein Eye Institute/UCLA, Los Angeles, CA
  • T.N. Yusifov
    Pathology & Ophthalmology, Jules Stein Eye Institute/UCLA, Los Angeles, CA
  • A.R. Abduragimov
    Pathology & Ophthalmology, Jules Stein Eye Institute/UCLA, Los Angeles, CA
  • O.K. Gasymov
    Pathology & Ophthalmology, Jules Stein Eye Institute/UCLA, Los Angeles, CA
  • Footnotes
    Commercial Relationships  B.J. Glasgow, None; T.N. Yusifov, None; A.R. Abduragimov, None; O.K. Gasymov, None.
  • Footnotes
    Support  NIH Grant EY11224
Investigative Ophthalmology & Visual Science May 2006, Vol.47, 3534. doi:
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      B.J. Glasgow, T.N. Yusifov, A.R. Abduragimov, O.K. Gasymov; Endonuclease Activity in Human Tears . Invest. Ophthalmol. Vis. Sci. 2006;47(13):3534.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: : Human endonucleases play a role in apoptosis in which potentially harmful cells are eliminated. Although endonucleases in tears have not been extensively studied, tear lipocalin (TL) has some activity in vitro related to a conserved sequence common to many lipocalins. Because the specific activity is lower for TL than for DNAse I, the significance of this activity in tears is unclear. Here, the sources of endonuclease activity in tears and their relative activity are determined.

Methods: : Human tears were collected from healthy donors as previously described. Gel filtration (Sephadex G–100) was performed on whole tears. Fractions with endonuclease activity were further isolated using a combination of affinity and anion exchange chromatographies. DNA hydrolyzing activity was determined by the conversion of 0.1 µg sc pUC19 plasmid DNA to its circular form and quantitated by densitometry of images of ethidium stained agarose gels. The masses of the DNA hydrolyzing proteins were estimated in zymograms and by calibration of size exclusion chromatography . DNase activities were characterized by the effect of inhibitors including NiCl2, ZnCl2, G–actin and aurintricarboxylic acid (ATA). To determine the mode of hydrolysis, the cleaved ends of the DNA digested by tear DNAses were analyzed by 3' and 5' end labeling with or without pretreatment with alkaline phosphatase.

Results: : Whole tears have endonuclease activity and the majority of the activity, about 80%, can be attributed to TL. A second protein accounts for about 20% of the relative activity; zymograms revealed an apparent molecular weight of about 35kDa. Both tear endonucleases are Mg+2 dependent and the effect is enhanced in the presence of Ca+2. While activity of TL is minimally affected by Mn+2 the activity of the minor endonuclease is enhanced. Both are inhibited by ATA but not by G actin. The activity of the minor endonuclease was reduced by NiCl2. Both endonucleases are active at physiologic pH. In DNA fragments generated by both endonucleases, the 3' ends were labeled regardless of alkaline phosphatase treatment; the 5' ends could be labeled only after removal of phosphoryl groups. In addition the sizes of the cleaved fragments were greater with the minor endonuclease than TL.

Conclusions: : Two Mg+2 dependent endonucleases were identified in tears; both can be classified as DNases catalyzing hydrolysis of DNA to produce 3'–OH/5'P ends. TL accounts for the majority of endonuclease activity in tears. The minor endonuclease shows enzymatic characteristics that are different from TL. The physiologic roles of endonucleases in tears remain to be elucidated.

Keywords: cornea: tears/tear film/dry eye • protein structure/function • apoptosis/cell death 
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