Determination of the Intracellular Levels of Cyclic ADP-Ribose in Cultured Human Cells Using a New Highly Sensitive Fluorescent HPLC Method

Gill, Paramjit Kaur
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Determination of the Intracellular Levels of Cyclic ADP-Ribose in Cultured Human Cells Using a New Highly Sensitive Fluorescent HPLC Method Paramjit Kaur Gill*. Department of Molecular Biology and Immunology, University of North Texas Health Science Center at Fort Worth, TX 76107-2699. Cyclic ADP-ribose (cADPR) is a second messenger that mobilizes intracellular stores of calcium in higher eukaryotic cells. The intracellular concentration of cADPR has previously been estimated to be in the femto- to nanomolar range. Therefore, there is a need for a highly sensitive assay to measure the levels of nucleotide in just a few million cells. Here, we have developed a highly, sensitive, specific, and reproducible fluorescent HPLC method to determine the intracellular concentration of cADPR in cultured cells. The procedure involves extraction of the nucleotide pool in 20% (W/V) TCA followed by the purification of soluble molecules containing two or more soluble molecules containing two or more riboses by boronate affinity chromatography. Purified nucleotides are then digested with phosphodiesterase to degrade all non-cyclic molecules, leaving cADPR intact. Contaminating products of phosphohydrolysis are then eliminated by a second boronate step and the pure preparation of cADPR obtained is converted to monomeric ADP-ribose with NADase (isolated from Bungarus fasciatus). After a third boronate purification, ADP-ribose is chemically derivitized to the etheno-adenine fluorescent form with chloroacetylaldehyde at 60°C, and the εADP-ribose formed is quantified by fluorescent-HPLC on a Partisil 10-SAX column. The specificity of our method was monitored by determining the yield at every step of the protocol with {32P]cADPR. Radiolabeled cADPR was synthesized from [32P]β-NAD+ and pure ADP-ribosyl cyclase from Aplysia californica. [32P]cADPR was subsequently purified by HPLC on a Partisil 10-SAX and a C-18 reverse phase column placed in tandem. While the recovery of a known amount of cADPR through each boronate step of the 4-day protocol was approximately 90%, the overall recovery throughout the procedure was between 30-40%. As expected, our mock incubations (negative controls) in the absence of phosphodiesterase or NADase treatment, as well as chloroacetaldehyde, yielded no εADP-ribose peak. Furthermore, spiking of a cell extract with commercially available cADPR resulted in the formation of a bigger fluorescent peak. Finally, our method indicated an intracellular concentration of cADPR in HeLa cells of 980 pmol of cADPR/10^8 cells. Considering HeLa cells have a larger cytoplasm compared to blood cells, our results agree well with those reported by DaSilva et al. who observed that the intracellular concentration of cADPR was 198 pmol/10^8 Jurkat cells, using a less sensitive chromatographic assay. Applications of this assay as a tool for biochemical investigation as to the role of cyclic ADP-ribose in the signal transduction events of rapamycin as an immunosuppressant and CD83 in B-CLL are discussed. *The laboratory study in this thesis were performed entirely in the laboratory of Dr. Rafael Alvarez-Gonzalez, in the department of Molecular Biology and Immunology and were performed entirely under his supervision. Dr. Alvarez served as my mentor from 1995-1998. Both Drs. Rafael Alvarez-Gonzalez and Ronald Goldfarb, Department Chairman, served as co-mentors from August 1999-March of 2000. From April 2001-July 2001, Dr. Goldfarb served as my mentor.