Abstract
Mammals rely on an elaborate intracellular trafficking pathway for processing and delivering vitamin B(12) to two client enzymes. CblC (also known as MMACHC) is postulated to receive the cofactor as it enters the cytoplasm and converts varied B(12) derivatives to a common cob(II)alamin intermediate. CblD (or MMADHC) reacts with CblC-bound cob(II)alamin forming an interprotein thiolato-cobalt coordination complex and, by a mechanism that remains to be elucidated, transfers the cofactor to methionine synthase. In the mitochondrion, CblB (also known as MMAB or adenosyltransferase) synthesizes AdoCbl from cob(II)alamin and ATP in the presence of an electron donor. CblA (or MMAA), a GTPase, gates cofactor loading from CblB to methylmalonyl-CoA mutase and off-loading of cob(II)alamin in the reverse direction. This chapter focuses on assays for measuring the activities of the four B(12) chaperones CblA-D.