Purification and characterization of mitochondrial biotin-dependent carboxylases from native tissues

Biotin-dependent carboxylases catalyze the MgATP- and bicarbonate-dependent carboxylation of various acceptor substrates through a two-step carboxylation reaction. Biotin-dependent carboxylases play an essential role in the metabolism of key biomolecules and, therefore, they are the subject of ongoing drug discovery efforts, as well as of studies seeking to better characterize their structure and function. It has been an ongoing challenge to obtain high yields of mammalian biotin-dependent carboxylases for in vitro experimentation; these enzymes have not been successfully purified when recombinantly expressed from a bacterial expression host and only low yields of these recombinant, vertebrate enzymes have been obtained through expression in cell culture systems. Here, we describe a revived protocol to isolate mitochondrial biotin-dependent carboxylases (pyruvate carboxylase, propionyl-CoA carboxylase, and 3-methylcrotonyl-CoA carboxylase) from fresh pig liver. This serves as an inexpensive and effective alternative to using a recombinant expression system. This scalable protocol can be completed in less than 48 h and affords effective separation of mammalian, mitochondrial, biotin-dependent carboxylases from cellular components. The purified, mitochondrial biotin-dependent carboxylases can be used in downstream experiments focused on kinetic and structural characterization, as well as in initial drug discovery experiments.