| 2249975 |
Dreyer JL: Plasma membrane dehydrogenases in rat brain synaptic membranes. J Bioenerg Biomembr. 1990 Oct;22(5):619-33.
Multiplicity and subunit composition.. Plasma membrane redox enzymes have been investigated in synaptic membranes from rat brain nerve terminals. UV-Vis spectra of intact synaptic plasma membranes are presented and the presence of a b-type cytochrome, detectable at 77 degrees K and sensitive to NADH or NADPH, is shown. The molecular characterization of rat synaptic NADH-dehydrogenases was further performed on solubilized enzymes using a recently developed nondissociating polyacrylamide gel electrophoresis technique. Synaptic plasma membranes were solubilized with 1% sodium cholate or Triton X-114 and centrifuged. The supernatant retained over 60% of the NADH-dehydrogenase activity, tested with either DCIP2 or ferricyanide as substrates, together with NADH. Both enzyme activities were insensitive toward rotenone. This extraction procedure also solubilized about 50% of the proteins. When submitted to polyacrylamide gel electrophoresis under nondenaturing conditions and stained for NADH-dehydrogenase activity, five bands of different mobilities were detected. The multiple NADH-dehydrogenases of synaptic plasma membranes were investigated by means of band excision and the five excised bands each submitted to amino acid analysis and to 2-D electrophoresis. The subunit composition of each band was then deduced, together with the molecular weight and pI of each respective subunit. NADH-dehydrogenases have also been purified by means of FPLC on Mono-P (chromatofocusing) followed by gel filtration on Superose 12. NADH-Dehydrogenase IV and V could be purified in their active forms by this approach. |
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