THE MALONATE DECARBOXYLASE ENZYME-SYSTEM OF MALONOMONAS-RUBRA - EVIDENCE FOR THE CYTOPLASMIC LOCATION OF THE BIOTIN-CONTAINING COMPONENT

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.