Electrophoretic comparison of cellular and soluble galactosyltransferase (lactose synthetase A protein) using specific antibodies.

Rabbit antisera against soluble human milk galactosyltransferase (GT) having anti-GT activity, as demonstrated by inhibition of enzyme activity were used for a comparative study of the molecular sizes of galactosyltransferase. For this purpose, affinity-purified antibodies were used for the identification of milk, serum and effusion galactosyltransferase from native or partially purified preparations resolved by sodium dodecyl sulfate polyacrylamide electrophoresis (SDS-PAGE) by the immune replica technique. Milk galactosyltransferase migrated as a 55-kilodalton (kD) protein, serum and effusion GT slightly faster. Cross-reactive enzyme forms of 110 kD and 20 kD were detected in milk only. In order to establish a relationship between intracellular and soluble galactosyltransferase, HeLa cells were metabolically labeled by [35S]-methionine, cells lysed, subjected to immunoprecipitation and the precipitate analyzed by SDS-PAGE/fluorography: a single band corresponding to the intracellular form of GT have similar mobility as the milk enzyme was detected. These results indicate a close structural similarity between soluble and cellular galactosyltransferase as judged by immunological cross-reactivity and electrophoretic mobility.