Analysis of nucleotides from Tetrahymena by high-performance liquid chromatography

✍ Scribed by Michael P. Reinhart; Mary J. Koroly

• Publisher: Elsevier Science
• Year: 1982
• Tongue: English
• Weight: 451 KB
• Volume: 119
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(82)90603-0

✦ Synopsis

Procedures for the complete extraction and isocratic high-performance liquid chromatography analysis of cellular nucleoside di-and triphosphates are reported. Maximum nucleotide recovery from cultures of the ciliate Tetrahymenapyriformis is achieved if (a) cells are cooled to 4°C prior to harvest; (b) dicarbonic acid diethyl ester, a protein crosslinking reagent, is added prior to neutralization of the cellular acid extract; and (c) 350 mM tri-n-octylamine in Freon is used as a neutralizing agent. These procedures also extend the useful lifetime of the Waters *Bondapak NH> column used for high-performance liquid chromatography analysis. Nucleoside triphosphates are resolved on this column in under 25 min using I25 mM ammonium phosphate, pH 3.15. Nucleoside diphosphates are separated by reducing the buffer concentration to 75 mM.

A number of cellular processes appear to be closely regulated by unusual nucleotides ( 1) such as guanosine 5'-diphosphate-3'diphosphate (ppGp~),~ guanosine 5'-triphosphate-3'-diphosphate (pppGpp) (2), diadenosine 5',5"-P',P4-tetraphosphate (A,,A) (3) diguanosine 5',5"-P',p-tetra-