Neutralizing antibodies to nucleoside diphosphate kinase inhibit the enzyme in vitro and in vivo: evidence for two distinct mechanisms of activation of atrial currents by ATPγS

Abstract

Nucleoside diphosphate kinase (NDPK) participates in multiple cellular functions, yet the molecular mechanisms of its involvement are often unknown, given that there are no specific inhibitors for the enzyme from vertebrates. We developed antibodies against NDPK by immunization of rabbits with the enzyme from bullfrog skeletal muscle. The antibodies specifically recognized the enzyme from frog tissues, and cross-reacted with NDPK from Xenopus. In contrast to mammalian NDPK, the amphibian enzyme elicited antibodies that inhibit potently its catalytic function. We utilized the inhibitory properties of these immunoglobulins to examine the role of NDPK on the ATPgammaS-induced stimulation of Ca2+ and K+ currents of cardiac myocytes. Injection of NDPK-neutralizing Fab fragments into atrial cells reduced considerably the effect of ATPgammaS on muscarinic K+ currents, but not on Ca2+ currents. Therefore, ATPgammaS increases calcium and potassium currents of atrial cells by two distinct mechanisms. NDPK is essential for the conversion of ATPgammaS into GTPgammaS which leads to muscarinic K+ channel activation but not for the stimulation of Ca2+ currents by ATPgammaS. The results demonstrate that antibodies to frog NDPK block the activity of the enzyme in vivo and in vitro, and can be used to determine the relevance of NDPK and its catalytic activity to the function of vertebrate cells.

NDP kinase moves into developing primary cilia

“…), specific for NDP kinase A and B [Gallagher et al, 2003], and two distinct polyclonal antibodies to frog NDP kinase, M1 and F1 [Yi et al, 1996;Otero, 1997]. Acetylated α-tubulin was detected with a monoclonal antibody (clone 6-11B-1, Piperno and Fuller [1985]) from Zymed.…”

“…Protein concentration was determined by the BCA assay (Pierce). Enzyme activity was determined in triplicate by the coupled assay as described [Yi et al, 1996].…”

Copurification of Vimentin, Energy Metabolism Enzymes, and a MER5 Homolog with Nucleoside Diphosphate Kinase

“…Goat anti-human vimentin, goat anti-rabbit muscle PK, goat anti-CK (MM), and monoclonal antibody to GAPDH were from Chemicon. Other sources were as described (12).…”

Association of Nucleoside Diphosphate Kinase with Microtubule-Based Structures

“…Soluble NDPK was purified from human erythrocytes using a modification of an earlier protocol (Yi et al, 1995). Cells were lysed in buffer A (25 mM MOPS, pH 7.5, 1 mM EDTA, 1 mM PMSF) on ice, and centrifuged at 17,600 xg for 1 hour.…”

“…For NDPK detection in NIH-3T3 cells, we utilized a monoclonal antibody specific for NDPK A (NM301) from Santa Cruz Biotechnology, and a polyclonal antibody, Ab-1 (Labvision) that recognizes human NDPKs A and B (amino acids 86-102). Polyclonal antibodies to frog NDPK were used to detect NDPK in A6 cells (Yi et al, 1995;. Secondary antibodies (multiple labeling grade) used for immunofluorescence were from Jackson Immunoresearch Laboratories (West Grove, PA;…”