Application of a copper blotting method to the study of Menkes disease.
Menkes disease is an X-linked recessive disorder of copper metabolism. Deficient quantity or functional activity of a molecule involved in intracellular copper transport is believed to represent the basic defect. We applied an in vitro copper binding assay (copper blotting) to tissue proteins from Menkes patients and controls to evaluate differences in copper-binding. Proteins were separated by SDS-PAGE, electrotransferred to nitrocellulose, and probed with 67CuCl2. Copper-binding polypeptides were visualized by autoradiography. No major differences were observed between a Menkes patient and control subjects in copper blots of post-mortem liver, kidney, or brain--tissues affected clinically by the disturbance of copper metabolism in Menkes disease. We also applied the copper blotting technique to fibroblast proteins from an affected female in whom the gene responsible for Menkes disease is interrupted by a chromosomal translocation, and detected no differences in copper-binding proteins relative to normal controls. These experiments suggest that the gene product defective in Menkes disease is not detectable in copper blots, either because normal tissue levels are below the limits of detection of this method, or because the molecule involved does not bind copper under these conditions.
Kaler
S G
SG
Section on Human Biochemical Genetics, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD.
Gahl
W A
WA
D001345:Autoradiography; D015153:Blotting, Western; D001923:Brain Chemistry; D003300:Copper / Q000302:isolation & purification* / Q000378:metabolism; D003301:Copper Radioisotopes; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D006801:Humans; D007223:Infant; D007668:Kidney / Q000737:chemistry; D008099:Liver / Q000737:chemistry; D008297:Male; D007706:Menkes Kinky Hair Syndrome / Q000378:metabolism*; D010455:Peptides / Q000378:metabolism; D011485:Protein Binding
D003301:Copper Radioisotopes; D010455:Peptides; D003300:Copper