LOCALIZATION OF OXALYL-COENZYME-A DECARBOXYLASE, AND FORMYL-COENZYME-A TRANSFERASE IN OXALOBACTER-FORMIGENES CELLS

Knowledge about the cellular location of enzymes concerned with oxalate metabolism in Oxalobacter formigenes is important for the development of concepts about the energy metabolism of this oxalate-dependent anaerobe. The main question asked in this study was whether or not major enzymes of oxalate metabolism (oxalyl-CoA-decarboxylase and formyl-CoA-transferase) were membrane bound. Spheroplasts of the gram-negative cells were prepared using lysozyme, and after osmotic lysis, the membrane fraction was obtained by centrifugation at 280,000 x g. Most (> 90%) of the oxalyl-CoA-decarboxylase and the formyl-CoA-transferase activitites that were recovered were in the soluble cytoplasmic fraction. However, the cytoplasmic fraction also appeared to contain membrane proteins. Of several enzymes that were tested, acid phosphatase appeared to be the best marker of membrane-linked enzymes (39% of the acid phosphatase activity was found in the membrane fraction). This is in contrast to finding only 2.4 and 0.4% of the activities oxalyl-CoA-decarboxylase and formly-CoA-transferase, respectively, in the membrane fraction. Results of immunogold labeling studies also support the concept that oxalyl-CoA-decarboxylase is not a membrane-bound or localized enzyme.