Rapid report
Use of ribonuclease VI from Artemia for the determination of cytidine
2P-phosphate
Antonio R. D|
¨
az, Claudio F. Heredia *
Instituto de Investigaciones Biome
¨
dicas `Alberto Sols', C.S.I.C., Facultad de Medicina U.A.M., Arzobispo Morcillo 4, E-28029 Madrid,
Spain
Received 31 May 1999; received in revised form 21 June 1999; accepted 23 June 1999
Abstract
An enzymatic method has been developed for the quantitative measurement of cytidine 2P-phosphate. Concentrations in
the micromolar range can be measured even in the presence of at least five times greater concentrations of a variety of related
nucleotides. The method is also suitable for detection of the 2P,3P-cyclic nucleotide 3P-phosphodiesterase (EC 3.1.4.37) and its
discrimination from the 2P,3P-cyclic nucleotide 2P-phosphodiesterase (EC 3.1.4.16). ß 1999 Elsevier Science B.V. All rights
reserved.
Keywords: Cytidine 2P-phosphate; Cyclic nucleotide ; Ribonuclease; 2P,3P-Cyclic nucleotide phosphodiesterase; (Artemia)
The quantitative detection of a particular nucleo-
tide present in a mixture with other related mole-
cules, can be achieved with high e¤ciency by high-
performance liquid chromatography, but this re-
quires the development of separation methods and
the assignment of peaks in the resulting chromato-
gram [1]. These problems can be circumvented by
using, as analytical tools, enzymes with a narrow
speci¢city and a very high sensitivity towards the
compound under consideration, which may act either
as a substrate or as an e¡ector. The endoribonu-
clease VI from Artemia ful¢ls these characteristics
with respect to cytidine 2P-phosphate: it is strongly
inhibited by micromolar concentrations of this nu-
cleotide and its activity is not a¡ected by much high-
er concentrations of a wide variety of related nucleo-
tides [2,3]. Therefore it can be used for the
quantitative determination of cytidine 2P-phosphate.
Nucleosides 2P-phosphate are reaction products of
2P,3P-cyclic nucleotide 3P-phosphodiesterase (EC
3.1.4.37) [4^7] and some of them (cytidine 2P-phos-
phate, guanosine 2P-phosphate) are strong modula-
tors of certain ribonucleases at concentrations in
the micromolar range [2,3,8^10]. Hence, the conven-
ience for developing sensitive methods for the quan-
ti¢cation of these compounds and for the character-
ization of the enzymes responsible for their synthesis.
The high sensitivity of Artemia ribonuclease VI to
cytidine 2P-phosphate will facilitate both the determi-
nation of cytidine 2P-phosphate and the detection of
2P,3P-cyclic nucleotide 3P-phosphodiesterase activity
(using as substrate 2P,3P cyclic cytidine phosphate)
and its discrimination from the 2P,3P-cyclic nucleotide
2P-phosphodiesterase (EC 3.1.4.16) whose reaction
product, the 3P-phosphate isomer [11], is not an in-
hibitor of the Artemia ribonuclease. The method de-
0304-4165 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved.
PII: S0304-4165(99)00112-9
* Corresponding author. Fax: +91-397-5353 ;
E-mail: cfheredia@iib.uam.es
BBAGEN 20356 1-10-99
Biochimica et Biophysica Acta 1472 (1999) 404^407
www.elsevier.com/locate/bba