Skip to main content
NCBI home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1994 Apr 25;22(8):1456–1462. doi: 10.1093/nar/22.8.1456

Purification of a calcium dependent ribonuclease from Xenopus laevis.

C W Seidel 1, L J Peck 1
PMCID: PMC308005  PMID: 8190637

Abstract

We have purified a Ca2+ dependent ribonuclease from the oocytes of Xenopus leavis. Two properties of this ribonuclease set it apart from other known nucleases. First, Ca2+ was required for ribonuclease activity, and Mg2+ would not substitute. Second, the enzyme specifically degraded RNA and digestion of double or single stranded DNA was not observed. Ca2+ dependent ribonuclease activity of the purified 36-kDa protein was directly observed after renaturation of the protein following electrophoresis in an SDS-Laemmli gel. In addition, the enzyme was shown to have endoribonuclease activity at numerous sites. The Ca2+ dependence suggests that the ribonuclease activity may be modulated by changes in the level of intracellular Ca2+ and thereby provide a direct link to signal transduction systems.

Full text

PDF
1456

Images in this article

1458Image
on p.1458
1459Image
on p.1459
1460Image
on p.1460
1460Image
on p.1460

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. ALEXANDER M., HEPPEL L. A., HURWITZ J. The purification and properties of micrococcal nuclease. J Biol Chem. 1961 Nov;236:3014–3019. [PubMed] [Google Scholar]
  2. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  3. Busa W. B., Nuccitelli R. An elevated free cytosolic Ca2+ wave follows fertilization in eggs of the frog, Xenopus laevis. J Cell Biol. 1985 Apr;100(4):1325–1329. doi: 10.1083/jcb.100.4.1325. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bürglin T. R., Mattaj I. W., Newmeyer D. D., Zeller R., De Robertis E. M. Cloning of nucleoplasmin from Xenopus laevis oocytes and analysis of its developmental expression. Genes Dev. 1987 Mar;1(1):97–107. doi: 10.1101/gad.1.1.97. [DOI] [PubMed] [Google Scholar]
  5. Deutscher M. P. Ribonuclease multiplicity, diversity, and complexity. J Biol Chem. 1993 Jun 25;268(18):13011–13014. [PubMed] [Google Scholar]
  6. Giebelhaus D. H., Zelus B. D., Henchman S. K., Moon R. T. Changes in the expression of alpha-fodrin during embryonic development of Xenopus laevis. J Cell Biol. 1987 Aug;105(2):843–853. doi: 10.1083/jcb.105.2.843. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Glikin G. C., Ruberti I., Worcel A. Chromatin assembly in Xenopus oocytes: in vitro studies. Cell. 1984 May;37(1):33–41. doi: 10.1016/0092-8674(84)90298-8. [DOI] [PubMed] [Google Scholar]
  8. Kimelman D., Kirschner M. Synergistic induction of mesoderm by FGF and TGF-beta and the identification of an mRNA coding for FGF in the early Xenopus embryo. Cell. 1987 Dec 4;51(5):869–877. doi: 10.1016/0092-8674(87)90110-3. [DOI] [PubMed] [Google Scholar]
  9. Krieg P. A. Improved synthesis of full-length RNA probe at reduced incubation temperatures. Nucleic Acids Res. 1990 Nov 11;18(21):6463–6463. doi: 10.1093/nar/18.21.6463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Krieg P. A., Melton D. A. In vitro RNA synthesis with SP6 RNA polymerase. Methods Enzymol. 1987;155:397–415. doi: 10.1016/0076-6879(87)55027-3. [DOI] [PubMed] [Google Scholar]
  11. Kubota H. Y., Yoshimoto Y., Yoneda M., Hiramoto Y. Free calcium wave upon activation in Xenopus eggs. Dev Biol. 1987 Jan;119(1):129–136. doi: 10.1016/0012-1606(87)90214-4. [DOI] [PubMed] [Google Scholar]
  12. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  13. Liu D. K., Liao W. S., Fritz P. J. Ca2+-stimulated ribonucleases from rat mammary gland and R3230AC mammary adenocarcinoma nuclei. Biochemistry. 1977 Jul 26;16(15):3361–3369. doi: 10.1021/bi00634a012. [DOI] [PubMed] [Google Scholar]
  14. Mohun T. J., Garrett N., Taylor M. V. Temporal and tissue-specific expression of the proto-oncogene c-fos during development in Xenopus laevis. Development. 1989 Dec;107(4):835–846. doi: 10.1242/dev.107.4.835. [DOI] [PubMed] [Google Scholar]
  15. Newport J., Kirschner M. A major developmental transition in early Xenopus embryos: I. characterization and timing of cellular changes at the midblastula stage. Cell. 1982 Oct;30(3):675–686. doi: 10.1016/0092-8674(82)90272-0. [DOI] [PubMed] [Google Scholar]
  16. Newport J., Kirschner M. A major developmental transition in early Xenopus embryos: II. Control of the onset of transcription. Cell. 1982 Oct;30(3):687–696. doi: 10.1016/0092-8674(82)90273-2. [DOI] [PubMed] [Google Scholar]
  17. Nuccitelli R., Yim D. L., Smart T. The sperm-induced Ca2+ wave following fertilization of the Xenopus egg requires the production of Ins(1, 4, 5)P3. Dev Biol. 1993 Jul;158(1):200–212. doi: 10.1006/dbio.1993.1179. [DOI] [PubMed] [Google Scholar]
  18. Oakley B. R., Kirsch D. R., Morris N. R. A simplified ultrasensitive silver stain for detecting proteins in polyacrylamide gels. Anal Biochem. 1980 Jul 1;105(2):361–363. doi: 10.1016/0003-2697(80)90470-4. [DOI] [PubMed] [Google Scholar]
  19. Rosenthal A. L., Lacks S. A. Nuclease detection in SDS-polyacrylamide gel electrophoresis. Anal Biochem. 1977 May 15;80(1):76–90. doi: 10.1016/0003-2697(77)90627-3. [DOI] [PubMed] [Google Scholar]
  20. Sachs A. B. Messenger RNA degradation in eukaryotes. Cell. 1993 Aug 13;74(3):413–421. doi: 10.1016/0092-8674(93)80043-e. [DOI] [PubMed] [Google Scholar]
  21. Sagata N., Oskarsson M., Copeland T., Brumbaugh J., Vande Woude G. F. Function of c-mos proto-oncogene product in meiotic maturation in Xenopus oocytes. Nature. 1988 Oct 6;335(6190):519–525. doi: 10.1038/335519a0. [DOI] [PubMed] [Google Scholar]
  22. Strauss F., Varshavsky A. A protein binds to a satellite DNA repeat at three specific sites that would be brought into mutual proximity by DNA folding in the nucleosome. Cell. 1984 Jul;37(3):889–901. doi: 10.1016/0092-8674(84)90424-0. [DOI] [PubMed] [Google Scholar]
  23. Vriz S., Taylor M., Méchali M. Differential expression of two Xenopus c-myc proto-oncogenes during development. EMBO J. 1989 Dec 20;8(13):4091–4097. doi: 10.1002/j.1460-2075.1989.tb08593.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Wickens M. In the beginning is the end: regulation of poly(A) addition and removal during early development. Trends Biochem Sci. 1990 Aug;15(8):320–324. doi: 10.1016/0968-0004(90)90022-4. [DOI] [PubMed] [Google Scholar]
  25. Young L. S., Dunstan H. M., Witte P. R., Smith T. P., Ottonello S., Sprague K. U. A class III transcription factor composed of RNA. Science. 1991 Apr 26;252(5005):542–546. doi: 10.1126/science.1708526. [DOI] [PubMed] [Google Scholar]

Articles from Nucleic Acids Research are provided here courtesy of Oxford University Press

RESOURCES