Alkaline phosphatase isoenzymes of liver and bone origin are incompletely resolved by wheat-germ-lectin affinity chromatography.
We used wheat-germ-lectin affinity chromatography as a tool to investigate the structure of alkaline phosphatase (ALP, EC 3.1.3.1) and to obtain fractions enriched in either bone or liver ALP activity. Liver and bone isoenzymes in serum samples were incompletely resolved except that the activity in the nonretained fraction (fraction 1) always represented pure liver isoenzyme and constituted a larger percentage of total activity in pooled sera with increased liver ALP activity than in pooled sera with increased bone activity. In contrast, a more avidly retained ALP activity, presumably with high glycosylation, was found in human serum with high activity of bone ALP. Using a solid-phase immunoassay, we examined the fractions obtained from the wheat-germ-lectin-Sepharose 4B column to determine whether the isoenzyme preference of the monoclonal antibody was markedly influenced by the degree of glycosylation. Whether samples contained high proportions of liver or of bone isoenzyme activity, the nonretained fraction contained a higher percentage of liver ALP, whereas the more strongly bound fraction contained a higher percentage of bone ALP. Except for eluted fractions that either contained no detectable N-acetylglucosamine or the highest percentage of it, the avidity of the liver-isoenzyme-specific monoclonal antibody for ALP seemed to be independent of the degree of glycosylation, suggesting that the epitope for monoclonal antibody may be expressed in some structure other than the carbohydrate moieties.
Gonchoroff
D G
DG
Endocrine Research Unit, Mayo Clinic, Rochester, MN 55905.
Branum
E L
EL
O'Brien
J F
JF
D000117:Acetylglucosamine / Q000032:analysis; D000469:Alkaline Phosphatase / Q000097:blood* / Q000276:immunology; D000911:Antibodies, Monoclonal; D000915:Antibody Affinity; D001842:Bone and Bones / Q000201:enzymology*; D002846:Chromatography, Affinity; D004586:Electrophoresis; D006031:Glycosylation; D006801:Humans; D007118:Immunoassay; D007527:Isoenzymes / Q000097:blood* / Q000276:immunology; D008099:Liver / Q000201:enzymology*; D014909:Wheat Germ Agglutinins
D000911:Antibodies, Monoclonal; D007527:Isoenzymes; D014909:Wheat Germ Agglutinins; D000469:Alkaline Phosphatase; D000117:Acetylglucosamine