Isolation and identification of a protein with capsaicin-inhibited NADH oxidase activity from culture media conditioned by growth of HeLa cells.
A ca. 33.5-kDa protein has been identified as a soluble NADH oxidase activity of culture media conditioned by growth of HeLa cells. The protein appears to be derived from a 34-kDa protein of the HeLa plasma membrane. Both proteins are characterized by an ability to oxidize NADH in the absence of exogenous electron acceptors. The activity is inhibited by 8-methyl-N-vanillyl-6-noneamide (capsaicin). The soluble and the plasma membrane forms of the activity exhibit a similar EC50 of about 5 nM for inhibition of the activity by capsaicin. The activity was purified from culture media conditioned by growth of HeLa cells using DEAE ion exchange chromatography, G-200 size exclusion chromatography, and preparative SDS-PAGE. Purification was monitored on the basis of the capsaicin-inhibited oxidation of NADH, including the final electrophoretic purification. Activity was restored following SDS-PAGE by reduction with dithiothreitol or reduced glutathione in the presence of NADH followed by the addition of 0.03% hydrogen peroxide and preincubation in the presence of NADH for 5-15 min. For affinity purification, the vanillylamine portion of capsaicin was linked to agarose. The agarose-linked vanillylamine bound a ca. 33.5-kDa protein band with capsaicin-inhibited NADH activity from total defined culture media conditioned by growth of HeLa cells. The NADH oxidase activity of both the soluble and the plasma membrane-associated form of the activity was inhibited by antisera corresponding to the 33.5-kDa protein. The antisera also immunoprecipitated and reacted on Western blots with both the soluble (33.5 kDa) and plasma membrane (34 kDa)-associated forms of the capsaicin-inhibited activity. The results identify the capsaicin-inhibited NADH oxidase of the conditioned media of HeLa cells as being a ca. 33.5-kDa shed form of the previously reported capsaicin-inhibited NADH oxidase of the HeLa cell plasma membrane.
Wilkinson
F
F
Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, Indiana, 47907, USA.
Kim
C
C
Cho
N
N
Chueh
P J
PJ
Leslie
S
S
Moya-Camarena
S
S
Wu
L Y
LY
Morré
D M
DM
Morré
D J
DJ
D002211:Capsaicin / Q000494:pharmacology*; D017077:Culture Media, Conditioned / Q000737:chemistry; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D006367:HeLa Cells; D006801:Humans; D008565:Membrane Proteins / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism
D017077:Culture Media, Conditioned; D004791:Enzyme Inhibitors; D008565:Membrane Proteins; D009097:Multienzyme Complexes; C023107:NADH oxidase; D009247:NADH, NADPH Oxidoreductases; D002211:Capsaicin