Rapid assay for nitric oxide synthase using thin-layer chromatography.
A simple, sensitive, and rapid method to determine the nitric oxide synthase (NOS) activity in crude cell extracts has been developed. The method takes advantage of differential migration of arginine and citrulline on silica gel thin-layer chromatography (TLC) with the specified buffer system. We have shown that products obtained by treating [14C]arginine with crude mouse hippocampal homogenate can be separated by methanol precipitation followed by TLC. The separated products of the enzyme reaction can be quantitated by radiometric scanning of the TLC plate or by counting in a scintillation counter. Inhibition of conversion of l-arginine to l-citrulline by NG-monomethyl-l-arginine acetate, a specific inhibitor of NOS, confirmed the NOS assay described in this investigation. This method is versatile and allows rapid simultaneous assay of several samples in a short period of time. Therefore, this assay is very useful for both qualitative and quantitative estimation of NOS activity.
Kumar
V B
VB
Geriatric Research Education and Clinical Center, St. Louis VA Medical Center, St. Louis, Missouri 63125, USA. kumar@slu.edu
Bernardo
A E
AE
Alshaher
M M
MM
Buddhiraju
M
M
Purushothaman
R
R
Morley
J E
JE
D000818:Animals; D001120:Arginine / Q000378:metabolism; D002250:Carbon Radioisotopes; D002855:Chromatography, Thin Layer / Q000379:methods*; D002956:Citrulline / Q000378:metabolism; D006624:Hippocampus / Q000201:enzymology; D051379:Mice; D019001:Nitric Oxide Synthase / Q000032:analysis*; D013379:Substrate Specificity
D002250:Carbon Radioisotopes; D002956:Citrulline; D001120:Arginine; D019001:Nitric Oxide Synthase