Advertisement

Assay of Arginyltransferase Activity by a Fluorescent HPLC Method

  • Koichi TakaoEmail author
Protocol
  • 1.5k Downloads
Part of the Methods in Molecular Biology book series (MIMB, volume 1337)

Abstract

Syntheses of fluorescent substrate and product for arginyltransferase, N-aspartyl-N′-dansylamido-1,4-butanediamine (Asp-4DNS), and N-arginylaspartyl-N′-dansylamido-1,4-butanediamine (ArgAsp-4DNS), respectively, including their precursor 4-dansylamidobutylamine (4DNS), are described. Then, HPLC conditions are summarized for a baseline separation of the three compounds in 10 min. The present method, which permits the simultaneous determination of Asp-4DNS, 4DNS, and ArgAsp-4DNS (in eluting order), is advantageous in measuring arginyltransferase activity and detecting the unfavorable enzyme(s) in 105,000 × g supernatant of tissues to ensure accurate determination.

Key words

Arginyltransferase assay Fluorescent HPLC N-Aspartyl-4-dansylamidobutylamine N-Arginylaspartyl-4-dansylamidobutylamine 4-Dansylamidobutylamine 

Notes

Acknowledgments

The author would like to thank Dr. Keijiro Samejima for his help with this manuscript.

References

  1. 1.
    Horinishi H, Kato M, Takahashi T (1976) A sensitive and reproducible procedure for the assay of arginyl-tRNA: protein arginyl transferase. Anal Biochem 75(1):22–29CrossRefPubMedGoogle Scholar
  2. 2.
    Klemperer NS, Pickart CM (1989) Arsenite inhibits two steps in the ubiquitin-dependent proteolytic pathway. J Biol Chem 264(32):19245–19252PubMedGoogle Scholar
  3. 3.
    Wang J, Han X, Saha S, Xu T, Rai R, Zhang F, Wolf YI, Wolfson A, Yates JR III, Kashina A (2011) Arginyltransferase is an ATP-independent self-regulating enzyme that forms distinct functional complexes in vivo. Chem Biol 18(1):121–130PubMedCentralCrossRefPubMedGoogle Scholar
  4. 4.
    Brower CS, Varshavsky A (2009) Ablation of arginylation in the mouse N-end rule pathway: loss of fat, higher metabolic rate, damaged spermatogenesis, and neurological perturbations. PLoS One 4(11), e7757PubMedCentralCrossRefPubMedGoogle Scholar
  5. 5.
    Graciet E, Walter F, Maoileidigh DO, Pollmann S, Meyerowitz EM, Varshavsky A, Wellmer F (2009) The N-end rule pathway controls multiple functions during Arabidopsis shoot and leaf development. Proc Natl Acad Sci U S A 106(32):13618–13623PubMedCentralCrossRefPubMedGoogle Scholar
  6. 6.
    Hu RG, Brower CS, Wang H, Davydov IV, Sheng J, Zhou J, Kwon YT, Varshavsky A (2006) Arginyltransferase, its specificity, putative substrates, bidirectional promoter, and splicing-derived isoforms. J Biol Chem 281(43):32559–32573CrossRefPubMedGoogle Scholar
  7. 7.
    Saha S, Wang J, Buckley B, Wang Q, Lilly B, Chernov M, Kashina A (2012) Small molecule inhibitors of arginyltransferase regulate arginylation-dependent protein degradation, cell motility, and angiogenesis. Biochem Pharmacol 83(7):866–873PubMedCentralCrossRefPubMedGoogle Scholar
  8. 8.
    Takao K, Samejima K (1999) Arginyl-tRNA-protein transferase activities in crude supernatants of rat tissues. Biol Pharm Bull 22(9):1007–1009CrossRefPubMedGoogle Scholar
  9. 9.
    Takao K, Xu YJ, Samejima K, Shirahata A, Nitsu M (1999) Preparation and usefulness of some fluorogenic substrates for assay of arginyl-tRNA-protein transferase by HPLC. Anal Biochem 267(2):373–381CrossRefPubMedGoogle Scholar
  10. 10.
    Zubay G (1966) Isolation of transfer RNA. In: Cantoni GL, Davies DR (eds) Procedures in nucleic acid research. Harper and Row, New York, pp 455–460Google Scholar
  11. 11.
    Mans RJ, Novelli GD (1960) A convenient, rapid and sensitive method for measuring the incorporation of radioactive amino acids into protein. Biochem Biophys Res Commun 3:540–543CrossRefPubMedGoogle Scholar
  12. 12.
    Kato M, Nozawa Y (1984) Complete purification of arginyl-tRNA: protein arginyltransferase from hog kidney and production of its antibody. Anal Biochem 143(2):361–367CrossRefPubMedGoogle Scholar
  13. 13.
    Soffer RL (1970) Enzymatic modification of proteins. II. Purification and properties of the arginyl transfer ribonucleic acid-protein transferase from rabbit liver cytoplasm. J Biol Chem 245(4):731–737PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2015

Authors and Affiliations

  1. 1.Faculty of Pharmaceutical SciencesJosai UniversitySakadoJapan

Personalised recommendations