Purification and properties of a self-associating, 50-kDa copper-binding protein from brindled mouse livers

The brindled mouse is an animal model of Menkes disease, a fatal, X-linked disease of copper metabolism. A self-associating, 50-kDa copper-binding protein (CuBP) was purified from brindled mouse hepatic cytosols, and some of its properties were determined. When ⁶⁴Cu-labeled whole hepatic cytosols were fractionated on Superose, statistically significantly less than normal ⁶⁴Cu binding was detected in both the fraction which contained the tetramer plus dimer (≈26% less) and the fraction containing the monomer of CuBP (≈37% less). CuBP was purified from brindled mouse hepatic cytosols by successive Mono Q, chelating Superose, and phenyl-Superose columns using the same methods used to purify the protein from normal mice. However, CuBP from the brindled mice was somewhat unstable during the purification. Also, CuBP from the brindled mouse eluted abnormally from the phenyl-Superose column. Thus, while the protein from normal mice eluted at ≈20 min after starting the final water elution step, the brindled mouse protein eluted by ≈5 min. This seemed to be due to abnormal self-association in the column buffers. Consistent with the results using whole cytosols, the purified CuBP from the brindled mouse showed decreased copper binding in both the tetramer and monomer fractions from Superose. Moreover, under the same conditions, CuBP from the brindled mice seemed to have relatively less tetramer and more dimer than normal. The results are consistent with a significant role for CuBP in intracellular copper metabolism, and an abnormal structure of CuBP may be the basic defect in the brindled mice and, by inference, Menkes disease.