Skip to main content
PMC home page
Nucleic Acids Research logoLink to Nucleic Acids Research
. 1992 Mar 25;20(6):1371–1377. doi: 10.1093/nar/20.6.1371

A pyrimidine-guanine sequence-specific ribonuclease from Rana catesbeiana (bullfrog) oocytes.

Y D Liao 1
PMCID: PMC312185  PMID: 1373237

Abstract

A pyrimidine-guanine sequence-specific ribonuclease (RC-RNase) was purified from Rana catesbeiana (bullfrog) oocytes by sequential phosphocellulose, Sephadex G75, heparin Sepharose CL 6B and CM-Sepharose CL 6B column chromatography. The purified enzyme with molecular weight of 13,000 daltons gave a single band on SDS-polyacrylamide gel. One CNBr-cleaved fragment has a sequence of NVLSTTRFQLNT/TRTSITPR, which is identical to residues 59-79 of a sialic acid binding lectin from R. catesbeiana eggs, and is 71% homologous to residues 60-80 of an RNase from R. catesbeaina liver. The RC-RNase preferentially cleaved RNA at pyrimidine residues with a 3' flanking guanine under various conditions. The sequence specificity of RC-RNase was further confirmed with dinucleotide as substrates, which were analyzed by thin layer chromatography after enzyme digestion. The values of kcat/km for pCpG, pUpG and pUpU were 2.66 x 10(7) M-1s-1, 2.50 x 10(7) M-1s-1 and 2.44 x 10(6) M-1s-1 respectively, however, those for other phosphorylated dinucleotides were less than 2% of pCpG and pUpG. As compared to single strand RNA, double strand RNA was relatively resistant to RC-RNase. Besides poly (A) and poly (G), most of synthetic homo- and heteropolynucleotides were also susceptible to RC-RNase. The RC-RNase was stable in the acidic (pH 2) and alkaline (pH 12) condition, but could be inactivated by heating to 80 degrees C for 15 min. No divalent cation was required for its activity. Furthermore, the enzyme activity could be enhanced by 2 M urea, and inhibited to 50% by 0.12 M NaCl or 0.02% SDS.

Full text

PDF
1371

Images in this article

1372Image
on p.1372
1373Image
on p.1373
1375Image
on p.1375

Selected References

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

  1. Beintema J. J., Broos J., Meulenberg J., Schüller C. The amino acid sequence of snapping turtle (Chelydra serpentina) ribonuclease. Eur J Biochem. 1985 Dec 2;153(2):305–312. doi: 10.1111/j.1432-1033.1985.tb09301.x. [DOI] [PubMed] [Google Scholar]
  2. Blackburn P., Wilson G., Moore S. Ribonuclease inhibitor from human placenta. Purification and properties. J Biol Chem. 1977 Aug 25;252(16):5904–5910. [PubMed] [Google Scholar]
  3. Boguski M. S., Hieter P. A., Levy C. C. Identification of a cytidine-specific ribonuclease from chicken liver. J Biol Chem. 1980 Mar 10;255(5):2160–2163. [PubMed] [Google Scholar]
  4. Bond M. D., Vallee B. L. Isolation of bovine angiogenin using a placental ribonuclease inhibitor binding assay. Biochemistry. 1988 Aug 23;27(17):6282–6287. doi: 10.1021/bi00417a013. [DOI] [PubMed] [Google Scholar]
  5. 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: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  6. Chang L. H., Hsieh J. C., Chen W. L., Tam M. F. Identification of rat liver glutathione S-transferase Yb subunits by partial N-terminal sequencing after electroblotting of proteins onto a polyvinylidene difluoride membrane from an analytical isoelectric focusing gel. Electrophoresis. 1990 Jul;11(7):589–593. doi: 10.1002/elps.1150110710. [DOI] [PubMed] [Google Scholar]
  7. Darnbrough C., Ford P. J. Turnover and processing of poly(A) in full-grown oocytes and during progesterone-induced oocyte maturation in Xenopus laevis. Dev Biol. 1979 Aug;71(2):323–340. doi: 10.1016/0012-1606(79)90173-8. [DOI] [PubMed] [Google Scholar]
  8. Donis-Keller H., Maxam A. M., Gilbert W. Mapping adenines, guanines, and pyrimidines in RNA. Nucleic Acids Res. 1977 Aug;4(8):2527–2538. doi: 10.1093/nar/4.8.2527. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Donis-Keller H. Phy M: an RNase activity specific for U and A residues useful in RNA sequence analysis. Nucleic Acids Res. 1980 Jul 25;8(14):3133–3142. doi: 10.1093/nar/8.14.3133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Feldman M., Kohtz D. S., Kleinberg D. L. Isolation and characterization of monoclonal antibodies against ribonuclease inhibitor. Biochem Biophys Res Commun. 1988 Nov 30;157(1):286–294. doi: 10.1016/s0006-291x(88)80045-7. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Hewick R. M., Hunkapiller M. W., Hood L. E., Dreyer W. J. A gas-liquid solid phase peptide and protein sequenator. J Biol Chem. 1981 Aug 10;256(15):7990–7997. [PubMed] [Google Scholar]
  13. Johnston R. F., Pickett S. C., Barker D. L. Autoradiography using storage phosphor technology. Electrophoresis. 1990 May;11(5):355–360. doi: 10.1002/elps.1150110503. [DOI] [PubMed] [Google Scholar]
  14. Kamiya Y., Oyama F., Oyama R., Sakakibara F., Nitta K., Kawauchi H., Takayanagi Y., Titani K. Amino acid sequence of a lectin from Japanese frog (Rana japonica) eggs. J Biochem. 1990 Jul;108(1):139–143. doi: 10.1093/oxfordjournals.jbchem.a123153. [DOI] [PubMed] [Google Scholar]
  15. Katoh H., Yoshinaga M., Yanagita T., Ohgi K., Irie M., Beintema J. J., Meinsma D. Kinetic studies on turtle pancreatic ribonuclease: a comparative study of the base specificities of the B2 and P0 sites of bovine pancreatic ribonuclease A and turtle pancreatic ribonuclease. Biochim Biophys Acta. 1986 Oct 17;873(3):367–371. doi: 10.1016/0167-4838(86)90085-3. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Lockard R. E., Alzner-Deweerd B., Heckman J. E., MacGee J., Tabor M. W., RajBhandary U. L. Sequence analysis of 5'[32P] labeled mRNA and tRNA using polyacrylamide gel electrophoresis. Nucleic Acids Res. 1978 Jan;5(1):37–56. doi: 10.1093/nar/5.1.37. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Nagano H., Kiuchi H., Abe Y., Shukuya R. Purification and properties of an alkaline ribonuclease from the hepatic cytosol fraction of bullfrog, Rana catesbeiana. J Biochem. 1976 Jul;80(1):19–26. doi: 10.1093/oxfordjournals.jbchem.a131251. [DOI] [PubMed] [Google Scholar]
  19. 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]
  20. Ng S. Y., Parker C. S., Roeder R. G. Transcription of cloned Xenopus 5S RNA genes by X. laevis RNA polymerase III in reconstituted systems. Proc Natl Acad Sci U S A. 1979 Jan;76(1):136–140. doi: 10.1073/pnas.76.1.136. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Nitta R., Katayama N., Okabe Y., Iwama M., Watanabe H., Abe Y., Okazaki T., Ohgi K., Irie M. Primary structure of a ribonuclease from bullfrog (Rana catesbeiana) liver. J Biochem. 1989 Nov;106(5):729–735. doi: 10.1093/oxfordjournals.jbchem.a122924. [DOI] [PubMed] [Google Scholar]
  22. Pao C. I., Lee T. C., Liao Y. D., Wu C. W. An N-terminally fused Xenopus transcription factor IIIA synthesized in Escherichia coli is biologically active. J Biol Chem. 1988 Jul 25;263(21):10295–10299. [PubMed] [Google Scholar]
  23. Sakakibara F., Kawauchi H., Takayanagi G., Ise H. Egg lectin of Rana japonica and its receptor glycoprotein of Ehrlich tumor cells. Cancer Res. 1979 Apr;39(4):1347–1352. [PubMed] [Google Scholar]
  24. Shastry B. S., Ng S. Y., Roeder R. G. Multiple factors involved in the transcription of class III genes in Xenopus laevis. J Biol Chem. 1982 Nov 10;257(21):12979–12986. [PubMed] [Google Scholar]
  25. Sideris D. C., Fragoulis E. G. Purification and characterization of a ribonuclease specific for poly(U) and poly(C) from the larvae of Ceratitis capitata. Eur J Biochem. 1987 Apr 15;164(2):309–315. doi: 10.1111/j.1432-1033.1987.tb11059.x. [DOI] [PubMed] [Google Scholar]
  26. Strydom D. J., Fett J. W., Lobb R. R., Alderman E. M., Bethune J. L., Riordan J. F., Vallee B. L. Amino acid sequence of human tumor derived angiogenin. Biochemistry. 1985 Sep 24;24(20):5486–5494. doi: 10.1021/bi00341a031. [DOI] [PubMed] [Google Scholar]
  27. Titani K., Takio K., Kuwada M., Nitta K., Sakakibara F., Kawauchi H., Takayanagi G., Hakomori S. Amino acid sequence of sialic acid binding lectin from frog (Rana catesbeiana) eggs. Biochemistry. 1987 Apr 21;26(8):2189–2194. doi: 10.1021/bi00382a018. [DOI] [PubMed] [Google Scholar]
  28. Wolffe A. P. Xenopus transcription factors: key molecules in the developmental regulation of differential gene expression. Biochem J. 1991 Sep 1;278(Pt 2):313–324. doi: 10.1042/bj2780313. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES