Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1980 Aug 1;189(2):277–284. doi: 10.1042/bj1890277

Use of polynucleotide/polyacrylamide-gel electrophoresis as a sensitive technique for the detection and comparison of ribonuclease activities.

T P Karpetsky, G E Davies, K K Shriver, C C Levy
PMCID: PMC1161998  PMID: 6161609

Abstract

A technique is described in which the incorporation of a polynucleotide substrate into the matrix of a polyacrylamide gel allows the use of electrophoresis for the detection of polycationic ribonuclease activity rather than simply the presence of protein. Because use is made of the catalytic properties of ribonucleases, polynucleotide/polyacrylamide-gel electrophoresis is apparoximately 10(5) times more sensitive for the detection of these enzymes than conventional gel electrophoresis with the use of protein-staining dyes. Initial studies showed that the poor migration, in the gels, of highly charged polycationic ribonucleases in the presence of negatively charged synthetic polynucleotides could be overcome by high concentrations of spermine. The positively charged polyamine, by neutralizing the polyanionic polynucleotide, enabled these basic enzymes to migrate considerable distances in the gel. Electrophoresis of the RNAases under conditions of low pH, and incubation of the gel at neutral pH followed by staining for polynucleotide, resulted in coloured gels containing clear bands that define regions of enzyme activity. Alterations in spermine concentration or substrate identity caused changes in the positions of these bands, suggesting a dynamic interaction among the enzyme, polyamine and polynucleotide. Because of the advantages, in terms of selectivity and sensitivity of polynucleotide/polyacrylamide-gel electrophoresis, this technique was used to demonstrate the nuclease homogenity of three purified bovine muscle enzymes, and to compare these enzymes with each other, as well as with bovine pancreatic ribonuclease A.

Full text

PDF
277

Images in this article

280Fig. 2.
on p.280

Selected References

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

  1. Davidson B., Fasman G. D. The single-stranded polyadenylic acid-poly-L-lysine complex. A conformational study and characterization. Biochemistry. 1969 Oct;8(10):4116–4126. doi: 10.1021/bi00838a032. [DOI] [PubMed] [Google Scholar]
  2. Davies G. E., Karpetsky T. P., Levy C. C. The ribonucleases of bovine skeletal muscle. Biochem J. 1980 Aug 1;189(2):263–275. doi: 10.1042/bj1890263. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Faiferman I., Hamilton M. G., Pogo A. O. Nucleoplasmic ribonucleoprotein particles of rat liver. II. Physical properties and action of dissociating agents. Biochim Biophys Acta. 1971 Apr 8;232(4):685–696. [PubMed] [Google Scholar]
  4. Faust C. H., Jr, Diggelmann H., Mach B. Isolation of poly(adenylic acid)-rich ribonucleic acid from mouse myeloma and synthesis of complementary deoxyribonucleic acid. Biochemistry. 1973 Feb 27;12(5):925–931. doi: 10.1021/bi00729a021. [DOI] [PubMed] [Google Scholar]
  5. Frank J. J., Hawk I. A., Levy C. C. Polyamine activation of staphylococcal nuclease. Biochim Biophys Acta. 1975 Apr 16;390(1):117–124. doi: 10.1016/0005-2787(75)90014-3. [DOI] [PubMed] [Google Scholar]
  6. Glasser R., Gabbay E. J. Topography of nucleic acid helices in solutions. 3. Interactions of spermine and spermidine derivatives with polyadenylic-polyuridylic and polyinosinic-polycytidylic acid helices. Biopolymers. 1968;6(2):243–254. doi: 10.1002/bip.1968.360060207. [DOI] [PubMed] [Google Scholar]
  7. Goldstein J. Resistance of RNA to thermal denaturation in the presence of polyamines. Biochim Biophys Acta. 1966 Sep;123(3):620–623. doi: 10.1016/0005-2787(66)90231-0. [DOI] [PubMed] [Google Scholar]
  8. Higuchi S., Tsuboi M. The effects of polyamines on the melting of polyriboadenylic acid plus polyribouridylic acid complexes in a solution. Bull Chem Soc Jpn. 1966 Sep;39(9):1886–1893. doi: 10.1246/bcsj.39.1886. [DOI] [PubMed] [Google Scholar]
  9. Igarashi K., Kumagai H., Watanabe Y., Toyoda N., Hirose S. Change of substrate specificity by polyamines of ribonucleases which hydrolyze ribonucleic acid at linkages attached to pyrimidine nucleotides. Biochem Biophys Res Commun. 1975 Dec 1;67(3):1070–1077. doi: 10.1016/0006-291x(75)90783-4. [DOI] [PubMed] [Google Scholar]
  10. Janik B., Kotick M. P., Kreiser T. H., Reverman L. F., Sommer R. G., Wilson D. P. Synthesis and properties of poly 2'-fluoro-2'-deoxyuridylic acid. Biochem Biophys Res Commun. 1972 Feb 16;46(3):1153–1160. doi: 10.1016/s0006-291x(72)80095-0. [DOI] [PubMed] [Google Scholar]
  11. Karpetsky T. P., Boguski M. S., Levy C. C. Structures, properties, and possible biologic functions of polyadenylic acid. Subcell Biochem. 1979;6:1–116. doi: 10.1007/978-1-4615-7945-8_1. [DOI] [PubMed] [Google Scholar]
  12. Karpetsky T. P., Levy C. C. Thermal inactivation of a Citrobacter ribonuclease: influence of polyamines and ionic strength. Arch Biochem Biophys. 1977 Jul;182(1):203–213. doi: 10.1016/0003-9861(77)90300-9. [DOI] [PubMed] [Google Scholar]
  13. Levy C. C., Mitch W. E., Schmukler M. Effect of polyamines on a ribonuclease which hydrolyzes ribonucleic acid at uridylic acid residues. J Biol Chem. 1973 Aug 25;248(16):5712–5719. [PubMed] [Google Scholar]
  14. Matsuo K., Tsuboi M. The effects of polyamines on the melting of polyriboinosinic acid plus polyribocytidylic acid complex in solution. Bull Chem Soc Jpn. 1966 Feb;39(2):347–352. doi: 10.1246/bcsj.39.347. [DOI] [PubMed] [Google Scholar]
  15. Montenay-Garestier T., Brun F., Hélène C. Interaction of disulfide groups with nucleic acid bases. Photochem Photobiol. 1976 Feb;23(2):87–91. doi: 10.1111/j.1751-1097.1976.tb06778.x. [DOI] [PubMed] [Google Scholar]
  16. Rabin E. Z., Weinberger V. The isolation, purification, and properties of a ribonuclease from normal human urine. Biochem Med. 1975 Sep;14(1):1–11. doi: 10.1016/0006-2944(75)90014-9. [DOI] [PubMed] [Google Scholar]
  17. Randles J. W. Ribonuclease isozymes in Chinese cabbage systemically infected with turnip yellow mosaic virus. Virology. 1968 Dec;36(4):556–563. doi: 10.1016/0042-6822(68)90187-6. [DOI] [PubMed] [Google Scholar]
  18. 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]
  19. Sakai T. T., Cohen S. S. Effects of polyamines on the structure and reactivity of tRNA. Prog Nucleic Acid Res Mol Biol. 1976;17:15–42. doi: 10.1016/s0079-6603(08)60064-1. [DOI] [PubMed] [Google Scholar]
  20. Sakai T. T., Torget R., I J., Freda C. E., Cohen S. S. The binding of polyamines and of ethidium bromide to tRNA. Nucleic Acids Res. 1975 Jul;2(7):1005–1022. doi: 10.1093/nar/2.7.1005. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Schmukler M., Jewett P. B., Levy C. C. The effects of polyamines on a residue-specific human plasma ribonuclease. J Biol Chem. 1975 Mar 25;250(6):2206–2212. [PubMed] [Google Scholar]
  22. Sprecher-Goldberger S. Differences between the structures of poliovirus and Sindbis virus infectious ribonucleic acids. Arch Gesamte Virusforsch. 1967;20(2):225–234. doi: 10.1007/BF01241276. [DOI] [PubMed] [Google Scholar]
  23. Thrierr J. C., Deubel V., Leng M. Interaction between polyribouridylic acid and spermine. Biochimie. 1972;54(9):1115–1119. doi: 10.1016/s0300-9084(72)80015-4. [DOI] [PubMed] [Google Scholar]
  24. Weber K., Osborn M. The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem. 1969 Aug 25;244(16):4406–4412. [PubMed] [Google Scholar]
  25. Wilson C. M. A rapid staining technique for detection of RNase after polyacrylamide gel electrophoresis. Anal Biochem. 1969 Oct 1;31(1):506–511. doi: 10.1016/0003-2697(69)90294-2. [DOI] [PubMed] [Google Scholar]
  26. Wilson C. M. Plant Nucleases: III. Polyacrylamide Gel Electrophoresis of Corn Ribonuclease Isoenzymes. Plant Physiol. 1971 Jul;48(1):64–68. doi: 10.1104/pp.48.1.64. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. van Loon L. C. Polynucleotide-acrylamide gel electrophoresis of soluble nucleases from tobacco leaves. FEBS Lett. 1975 Mar 1;51(1):266–269. doi: 10.1016/0014-5793(75)80903-3. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES