Plasma membrane dehydrogenases in rat brain synaptic membranes. 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.
Dreyer
J L
JL
Department of Biochemistry, University of Fribourg, Switzerland.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002462:Cell Membrane / Q000201:enzymology; D008970:Molecular Weight; D009245:NADH Dehydrogenase / Q000378:metabolism; D010088:Oxidoreductases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013570:Synaptic Membranes / Q000201:enzymology*
D000596:Amino Acids; D010088:Oxidoreductases; D009245:NADH Dehydrogenase