Hyuntae Kim

Strategies and reagents for photoaffinity labeling of mechanochemical proteins

Publisher Summary This chapter presents some of the uses of nucleotide photoaffinity probes to study various aspects of the interaction of nucleotides with mechanochemical proteins. These applications of photoaffinity probes range from detecting the subunits involved in nucleotide binding to the isolation and sequencing of the active site peptides that are photolabeled. In general, photoaffinity probes have the distinct advantage over classic chemical probes in that the photoactive group is not chemically reactive without exposure to the proper wavelength of light. Several nucleotide photoattinity probes have proved to be excellent tools for identifying the protein subunits that bind specific nucleotides even when studying crude homogenates. They are also quite effective at determining the size and approximate location of the peptides within the binding site under conditions where the peptides are large enough to be separated by SDS-PAGE. However, a number of problems have been encountered, which are particularly serious when attempting to define the active site region at higher resolution. To resolve the problems involved with the HPLC analysis of photolabeled peptides, an alternative approaches to purifying the photolabeled peptide, using HPLC only to confirm the purity of the peptide isolated is resorted to. A good general technique using anion-exclusion chromatography, along with the approach to validate active site labeling, is presented in the chapter.

NAD Glycohydrolases: A possible function in calcium homeostasis

NAD glycohydrolases are the longest known enzymes that catalyze ADP-ribose transfer. The function of these ubiquitous, membrane-bound enzymes has been a long standing puzzle. The NAD glycohydrolase are briefly reviewed in light of the discovery by our laboratory that NAD glycohydrolases are bifunctional enzymes that can catalyze both the synthesis and hydrolysis of cyclic ADP-ribose, a putative second messenger of calcium homeostasis.

PREPARATION OF CYCLIC ADP-RIBOSE, 2'-PHOSPHO-CYCLIC ADP-RIBOSE, AND NICOTINATE ADENINE DINUCLEOTIDE PHOSPHATE : POSSIBLE SECOND MESSENGERS OF CALCIUM SIGNALING

Publisher Summary The metabolism of cyclic ADP-ribose (cADPR) and 2-phospho-cADPR (P-cADPR) in mammals is apparently achieved by enzymes known as nicotinate adenine dinucleotide phosphate (NADP) glycohydrolases. NAD(P)ases catalyze the release of nicotinamide from NAD or NADP with the formation of an enzyme-bound oxocarbenium ion intermediate. This intermediate has three possible fates—(1) it can react via an intermolecular reaction with nicotinamide to regenerate the original dinucleotide or with a variety of other nucleophiles with retention of configuration to form analogs of either NAD or NADP; (2) it can undergo an intramolecular reaction to form cADPR or P-cADPR; or (3) it can react with water to form ADPR or P-ADPR. The methodology described in the chapter uses an NAD(P)ase from the marine mollusk Aplysia californica . The strategy described in the chapter achieves the synthesis of cyclic nucleotides by simple passage of NAD, NADP, or a structural analog through a column of immobilized ADPR cyclase. Conversely, to prepare NAD or NADP analogs, cADPR or P-cADPR can be mixed with the nucleophile of choice and passed through a column of immobilized enzyme. Because the approach involves few reactants and products, purification of the products can be achieved easily.