Separation of the primary dehydrogenase from the cytochromes of the nicotinamide adenine dinucleotide (reduced form) oxidase of Bacillus megaterium.
A selective extraction procedure was developed for sequentially extracting a fraction containing the primary dehydrogenase and a fraction containing the cytochromes of the nicotinamide adenine dinucleotide (reduced form) (NADH) oxidase of Bacillus megaterium KM membranes. The primary dehydrogenase (NADH-2,6-dichlorophenolindophenol oxidoreductase) activity was extracted from sonically treated membranes with 0.4% sodium deoxycholate for 30 min at 4 C. The insoluble residue was extracted with 0.4% sodium deoxycholate in 1 m KCl for 30 min at 25 C. A combination of the two extracts and dilution in Mg(2+) gave good recovery of the original membrane NADH oxidase activity. The primary dehydrogenase fraction contained 41% of the membrane protein, no cytochromes, flavine adenine dinucleotide as the sole acid-extractable flavine, and most of the membrane ribonucleic acid (RNA). The cytochrome-containing fraction had 16% of the membrane protein, 61% of the membrane cytochrome with the same relative amounts of cytochromes a and b as the original membrane, no acid-extractable flavine, little RNA, and no oxidoreductase activity. The oxidoreductase fraction remained soluble after removal of deoxycholate whereas the cytochrome fraction became insoluble after removal of deoxycholate-KCl, but the precipitated fraction could be redissolved in 0.4% sodium deoxycholate. Treatment of both fractions with ribonuclease to destroy all of the RNA present did not affect the ability of the fractions to recombine into a functional oxidase unit. Treatment of either fraction with phospholipase A prevented restoration of a functional oxidase when the oxidoreductase and cytochrome fractions were treated in solution, but no affect on restoration of oxidase was observed when the phospholipase A treatment was carried out with the soluble oxidoreductase fraction and the insoluble cytochrome fraction.
D001410:Bacillus megaterium / Q000032:analysis / Q000201:enzymology* / Q000254:growth & development; D001426:Bacterial Proteins / Q000032:analysis; D001647:Bile Acids and Salts; D002462:Cell Membrane / Q000032:analysis / Q000201:enzymology; D002474:Cell-Free System; D011232:Chemical Precipitation; D003580:Cytochromes; D003956:Dialysis; D005486:Flavin Mononucleotide / Q000032:analysis; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis; D005470:Fluorometry; D006863:Hydrogen-Ion Concentration; D007215:Indophenol; D008274:Magnesium; D009243:NAD; D010088:Oxidoreductases / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D010740:Phospholipases; D011189:Potassium Chloride; D011523:Protoplasts; D012329:RNA, Bacterial / Q000032:analysis; D012260:Ribonucleases; D012997:Solvents; D013696:Temperature; D013997:Time Factors; D014465:Ultrasonics; D014732:Vibration
D001426:Bacterial Proteins; D001647:Bile Acids and Salts; D003580:Cytochromes; D012329:RNA, Bacterial; D012997:Solvents; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; D007215:Indophenol; D011189:Potassium Chloride; D005486:Flavin Mononucleotide; D010088:Oxidoreductases; D010740:Phospholipases; D012260:Ribonucleases; D008274:Magnesium