Isolation of the flavodehydrogenase domain of Hansenula anomala flavocytochrome b2 after mild proteolysis by an H. anomala proteinase.
The protomeric chain of Hansenula anomala flavocytochrome b2 was previously shown to be built as the covalent association of two functional domains: an L-lactate dehydrogenase domain and a cytochrome c reductase domain, joined together by a proteolytically sensitive zone. This paper concerns the specific cleavage of this latter zone with a H. anomala proteinase(s) preparation and the purification of the resulting L-lactate dehydrogenase moiety of the molecule with at least 25% recovery, (i.e. one order of magnitude more than for the previously published method). A preliminary characterization of this dehydrogenase domain indicates that it is a tetramer (Mr = 4 x 39000) containing FMN as expected and not heme. It has high L-lactate:ferricyanide oxidoreductase activity (about 70% that of the whole flavocytochrome b2) and the same Km for L(+)-lactate as flavocytochrome b2, but it has no L-lactate:cytochrome c oxidoreductase activity. Its flavin semiquinone is stabilized in the presence of pyruvate as in flavocytochrome b2. The subcellular origin of the H. anomala proteinase in the preparation has not yet been elucidated.
Celerier
J
J
Laboratoire d'Enzymologie Physicochimique, Université Pierre et Marie Curie, Gif-sur-Yvette, France.
Risler
Y
Y
Schwencke
J
J
Janot
J M
JM
Gervais
M
M
D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases; D006868:Hydrolysis; D007700:Kinetics; D007770:L-Lactate Dehydrogenase / Q000032:analysis / Q000302:isolation & purification*; D042844:L-Lactate Dehydrogenase (Cytochrome); D008928:Mitochondria / Q000201:enzymology; D009245:NADH Dehydrogenase / Q000032:analysis; D010446:Peptide Fragments / Q000302:isolation & purification; D010843:Pichia / Q000201:enzymology*; D004718:Saccharomycetales / Q000201:enzymology*; D013347:Subcellular Fractions / Q000201:enzymology
D010446:Peptide Fragments; D007770:L-Lactate Dehydrogenase; D042844:L-Lactate Dehydrogenase (Cytochrome); D009245:NADH Dehydrogenase; D010450:Endopeptidases