H. Hilbi et al., THE MALONATE DECARBOXYLASE ENZYME-SYSTEM OF MALONOMONAS-RUBRA - EVIDENCE FOR THE CYTOPLASMIC LOCATION OF THE BIOTIN-CONTAINING COMPONENT, Archives of microbiology, 160(2), 1993, pp. 126-131
Malonate decarboxylase of Malonomonas rubra is a complex enzyme system
involving cytoplasmic and membrane-bound components. One of these is
a biotin-containing protein of M(r) 120'000, the location of which in
the cytoplasm was deduced from the following criteria: (i) If the cyto
plasm was incubated with avidin and the malonate decarboxylase subsequ
ently completed with the membrane fraction the decarboxylase activity
was abolished. The corresponding incubation of the membrane with avidi
n, however, was without effect. (ii) Western blot analysis identified
the single biotin-containing polypeptide of M(r) 120'000 within the cy
toplasm. (iii) Transmission electron micrographs of immuno-gold labele
d M. rubra cells clearly showed the location of the biotinyl protein w
ithin the cytoplasm, whereas the same procedure with Propionigenium mo
destum cells indicated the location of the biotin enzyme methylmalonyl
-CoA decarboxylase in the cell membrane. The biotin-containing protein
of the M. rubra malonate decarboxylase enzyme system was not retained
by monomeric avidin-Sepharose columns but could be isolated with this
column in a catalytically inactive form in the presence of detergents
. If the high binding affinity of tetrameric avidin towards biotin was
reduced by destructing part of the tryptophan residues by irradiation
or oxidation with periodate, the inhibition of malonate decarboxylase
by the modified avidin was partially reversed with an excess of bioti
n. Attempts to purify the biotin protein in its catalytically active s
tate using modified avidin columns were without success.