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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
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):22–29
Klemperer NS, Pickart CM (1989) Arsenite inhibits two steps in the ubiquitin-dependent proteolytic pathway. J Biol Chem 264(32):19245–19252
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):121–130
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), e7757
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):13618–13623
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):32559–32573
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):866–873
Takao K, Samejima K (1999) Arginyl-tRNA-protein transferase activities in crude supernatants of rat tissues. Biol Pharm Bull 22(9):1007–1009
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):373–381
Zubay G (1966) Isolation of transfer RNA. In: Cantoni GL, Davies DR (eds) Procedures in nucleic acid research. Harper and Row, New York, pp 455–460
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 3:540–543
Kato M, Nozawa Y (1984) Complete purification of arginyl-tRNA: protein arginyltransferase from hog kidney and production of its antibody. Anal Biochem 143(2):361–367
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):731–737
Acknowledgments
The author would like to thank Dr. Keijiro Samejima for his help with this manuscript.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2015 Springer Science+Business Media New York
About this protocol
Cite this protocol
Takao, K. (2015). Assay of Arginyltransferase Activity by a Fluorescent HPLC Method. In: Kashina, A. (eds) Protein Arginylation. Methods in Molecular Biology, vol 1337. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2935-1_12
Download citation
DOI: https://doi.org/10.1007/978-1-4939-2935-1_12
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2934-4
Online ISBN: 978-1-4939-2935-1
eBook Packages: Springer Protocols