. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. DNA-DEPENDENT PROTEIN KINASE 151. c » II , mo 10 - •0 0- < a Y1 Y2 Y3 Y4 Y5 Y6 OOCYTE STAGE Figure 3. DNA-dependent protein kinase activity in oocytes at dif- ferent stages. The isolated oocytes were homogenized as described in Materials and Methods (Immunoprecipitation and protein analysis). The tolal protein content of oocytes at each stage was equalized ( 140 ^g) in all the extracts used for DNA-PK activity assay. The specific peptide was used as the substrate. Ku protein detectable in these stages. Induction, using ds
. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. DNA-DEPENDENT PROTEIN KINASE 151. c » II , mo 10 - •0 0- < a Y1 Y2 Y3 Y4 Y5 Y6 OOCYTE STAGE Figure 3. DNA-dependent protein kinase activity in oocytes at dif- ferent stages. The isolated oocytes were homogenized as described in Materials and Methods (Immunoprecipitation and protein analysis). The tolal protein content of oocytes at each stage was equalized ( 140 ^g) in all the extracts used for DNA-PK activity assay. The specific peptide was used as the substrate. Ku protein detectable in these stages. Induction, using dsDNA, of the kinase activity in the oocyte extracts distin- guishes in Xenopus an enzyme (DNA-PK) that is func- tionally similar to the one reported in human cell lines (Carter et 1990). The association of this activity with a Ku-like protein is confirmed with immunodepletion techniques when anti-Ku antibodies remove the activity from the oocyte extracts. Ku protein and DNA-PK cata- lytic subunit (p460) remain as a complex at physiologic salt concentrations, but dissociate at high salt ( M) concentrations (Gottlieb and Jackson, 1993). Therefore, we used anti-Ku antibodies coupled to Protein A-Sepha- rose beads to immunodeplete the holoenzyme from oocyte extracts prepared in low salt buffers. Extracts incubated with these beads lost the DNA-PK activity, whereas other nonspecific antibodies did not affect the activity. These results indicate that the Ku protein is associated with the DNA-PK activity observed in the oocyte extracts. Although considerable evidence indicates that DNA- PK plays a role in DNA repair and recombination (re- viewed by Anderson, 1993), the /'/; vivo targets of the DNA-PK remain largely unknown. Additional studies in clarifying the behavior of the enzyme during early em- bryogenesis may be useful in recognizing specific targets of this enzyme. We are investigating whether the post- fertilization nuclear translocation of the enzyme activity report
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Keywords: ., bookauthorlilliefrankrat, booksubjectbiology, booksubjectzoology
