Tear lipocalin is the major endonuclease in tears.
OBJECTIVE
Human endonucleases are integral to apoptosis in which unwanted or potentially harmful cells are eliminated. The rapid turnover of ocular surface epithelium and microbial colonization of the eyelids are continual sources of DNA in tears. Here, we determine the principal sources of endonuclease activity in tears.


METHODS
Endonucleases in human tears were identified after Sephadex G100 gel filtration. DNA hydrolyzing activity was measured by the conversion pUC19 plasmid DNA to its circular form in agarose gels. Fractions with endonuclease activity were further isolated using a combination ConA-Sepharose DNA, oligo (dT) cellulose, and anion exchange chromatographies. The molecular weights of the DNA hydrolyzing proteins were estimated in zymograms and by calibration of size exclusion chromatography. DNase activities were characterized for activity at a variety of pH and ion concentrations as well as in the presence of inhibitors including NiCl(2), ZnCl(2), 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 using either terminal deoxynucleotidyl transferase or polynucleotide kinase with or without pretreatment with alkaline phosphatase.


RESULTS
Tear lipocalin (TL) accounts for over 75% of the DNA catalytic activity in tears while a second endonuclease, approximately 34 kDa, is responsible for less than 24% of the activity. Both are Mg(2+) dependent enzyme endonucleases that are enhanced by Ca(2+), active at physiologic pH, inhibited by aurintricarboxylic acid, and catalyze hydrolysis of DNA to produce 3'-OH/5'P ends. However, the two enzymes can be distinguished by the inhibitory effect of NiCl(2) and the sizes of the cleaved DNA fragments.


CONCLUSIONS
Two magnesium dependent extracellular endonucleases were identified in tears that are different from other major human extracellular nucleases. TL is the principal endonuclease in human tear fluid. Tear endonucleases have unique characteristics that differ from other known human endonucleases.
Yusifov
Taleh N
TN
Department of Pathology, Jules Stein Eye Institute, University of California, Los Angeles, CA, USA.
Abduragimov
Adil R
AR
Narsinh
Kiran
K
Gasymov
Oktay K
OK
Glasgow
Ben J
BJ
D002413:Cations, Divalent / Q000494:pharmacology; D002850:Chromatography, Gel; D004247:DNA / Q000378:metabolism; D004720:Endonucleases / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis / Q000187:drug effects; D054835:Lipocalin 1 / Q000378:metabolism*; D012015:Reference Standards; D012965:Sodium Chloride / Q000494:pharmacology; D013666:Tears / Q000187:drug effects / Q000201:enzymology*
D002413:Cations, Divalent; D004791:Enzyme Inhibitors; D054835:Lipocalin 1; D012965:Sodium Chloride; D004247:DNA; D004720:Endonucleases