Separation of the two most closely related isoenzymes of alkaline phosphatase by two-dimensional electrophoresis.

Seminoma is the most frequent testicular germ cell tumor. While effective curative treatment of the disease is available today, there is to date no tumor marker suited for the diagnosis and follow-up. Several authors have suggested that the germ-cell-specific isoenzyme of alkaline phosphatase (GCAP) might be valuable for this purpose. GCAP shows 98% sequence homology with the placental isoenzyme of alkaline phosphatase (PLAP). Both display a high degree of phenotypic heterogeneity. Until now all attempts to raise an antibody reacting specifically with GCAP have failed. Consequently there is no immunological assay that allows the measurement of GCAP in the presence of PLAP. Two-dimensional electrophoresis with a sigmoid immobilized pH-gradient of 3-10 for the first dimension makes it possible to differentiate clearly between these two closely related isoenzymes. Additionally, it resolves their many phenotypic variants. This is of special interest, since malignant transformation affects the glycosylation patterns of many glycoproteins. For the detection of GCAP and PLAP in two-dimensional electrophoresis it is essential to purify the raw tissue extracts thoroughly. A chromatographic method suited for this purpose is presented.