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Tear lipocalin is the major endonuclease in tears.

Yusifov TN, Abduragimov AR, Narsinh K, Gasymov OK, Glasgow BJ - Mol. Vis. (2008)

Bottom Line: However, the two enzymes can be distinguished by the inhibitory effect of NiCl(2) and the sizes of the cleaved DNA fragments.TL is the principal endonuclease in human tear fluid.Tear endonucleases have unique characteristics that differ from other known human endonucleases.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Jules Stein Eye Institute, University of California, Los Angeles, CA, USA.

ABSTRACT

Purpose: 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.

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Related in: MedlinePlus

Endonuclease activity in gel filtration fractions of human tears. A shows the agarose gel of products from hydrolysis of pUC19 human tears (diluted 1:5) Aliquots were removed from the reaction mixture at successive 15-min. intervals (Lanes 1–6); Lane 7 shows sc pUC19 incubated only. The forms of the plasmid are indicated as relaxed (r) and supercoiled (sc). B demonstrates endonuclease activity profile of fractions superimposed on the absorbance elution profile from filtration of human tears (2.5 ml) on Sephadex G-100 column. (□) stands for absorbance; (●) stands for DNA hydrolyzing activity profile calculated by the densitometry of bands in the agarose gel electrophoresis of plasmid DNA sc pUC19. Each point represents the activity resulting from incubation of 5 μl of the various fractions with the plasmid.
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f2: Endonuclease activity in gel filtration fractions of human tears. A shows the agarose gel of products from hydrolysis of pUC19 human tears (diluted 1:5) Aliquots were removed from the reaction mixture at successive 15-min. intervals (Lanes 1–6); Lane 7 shows sc pUC19 incubated only. The forms of the plasmid are indicated as relaxed (r) and supercoiled (sc). B demonstrates endonuclease activity profile of fractions superimposed on the absorbance elution profile from filtration of human tears (2.5 ml) on Sephadex G-100 column. (□) stands for absorbance; (●) stands for DNA hydrolyzing activity profile calculated by the densitometry of bands in the agarose gel electrophoresis of plasmid DNA sc pUC19. Each point represents the activity resulting from incubation of 5 μl of the various fractions with the plasmid.

Mentions: Peaks A and B refer to peaks of nuclease activity observed in Figure 2B. The percentages of total activity are shown in parentheses.


Tear lipocalin is the major endonuclease in tears.

Yusifov TN, Abduragimov AR, Narsinh K, Gasymov OK, Glasgow BJ - Mol. Vis. (2008)

Endonuclease activity in gel filtration fractions of human tears. A shows the agarose gel of products from hydrolysis of pUC19 human tears (diluted 1:5) Aliquots were removed from the reaction mixture at successive 15-min. intervals (Lanes 1–6); Lane 7 shows sc pUC19 incubated only. The forms of the plasmid are indicated as relaxed (r) and supercoiled (sc). B demonstrates endonuclease activity profile of fractions superimposed on the absorbance elution profile from filtration of human tears (2.5 ml) on Sephadex G-100 column. (□) stands for absorbance; (●) stands for DNA hydrolyzing activity profile calculated by the densitometry of bands in the agarose gel electrophoresis of plasmid DNA sc pUC19. Each point represents the activity resulting from incubation of 5 μl of the various fractions with the plasmid.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2254967&req=5

f2: Endonuclease activity in gel filtration fractions of human tears. A shows the agarose gel of products from hydrolysis of pUC19 human tears (diluted 1:5) Aliquots were removed from the reaction mixture at successive 15-min. intervals (Lanes 1–6); Lane 7 shows sc pUC19 incubated only. The forms of the plasmid are indicated as relaxed (r) and supercoiled (sc). B demonstrates endonuclease activity profile of fractions superimposed on the absorbance elution profile from filtration of human tears (2.5 ml) on Sephadex G-100 column. (□) stands for absorbance; (●) stands for DNA hydrolyzing activity profile calculated by the densitometry of bands in the agarose gel electrophoresis of plasmid DNA sc pUC19. Each point represents the activity resulting from incubation of 5 μl of the various fractions with the plasmid.
Mentions: Peaks A and B refer to peaks of nuclease activity observed in Figure 2B. The percentages of total activity are shown in parentheses.

Bottom Line: However, the two enzymes can be distinguished by the inhibitory effect of NiCl(2) and the sizes of the cleaved DNA fragments.TL is the principal endonuclease in human tear fluid.Tear endonucleases have unique characteristics that differ from other known human endonucleases.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Jules Stein Eye Institute, University of California, Los Angeles, CA, USA.

ABSTRACT

Purpose: 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.

Show MeSH
Related in: MedlinePlus