Monoclonal antibodies to bovine UDP-galactosyltransferase. Characterization, cross-reactivity, and utilization as structural probes.
A series of mouse monoclonal antibodies has been developed against a soluble form of bovine UDP-galactose:N-acetylglucosamine galactosyltransferase purified to apparent chemical homogeneity by a combination of affinity and immunoadsorption chromatography. The purified enzyme consists of two molecular mass variants of 42 and 48 kDa. Individual monoclonal antibodies were selected for by their ability to recognize immobilized affinity-purified galactosyltransferase and were not reactive against bovine alpha-lactalbumin and bovine immunoglobulins. Based on competitive binding assays and Western blot analysis with either galactosyltransferase or lactose synthetase (covalently cross-linked alpha-lactalbumin galactosyltransferase), these monoclonal antibodies can be subdivided into four groups. Group A (3 clones) recognize an epitope at or near the alpha-lactalbumin binding site. In addition, this group is cross-reactive with soluble galactosyltransferase from human milk and pleural effusion. Group B (6 clones) and D (1 clone) appear to recognize two different epitopes on the 6-kDa fragment which is released when the 48-kDa galactosyltransferase polypeptide is converted to the 42-kDa form, apparently by proteolysis. Groups A and C (1 clone) recognize epitopes found on both the 48- and 42-kDa polypeptide. Interestingly, immunofluorescence studies indicate that only two monoclonal antibody groups (C and D) are able to decorate membrane-bound galactosyltransferase (Golgi-associated) in formalin-fixed, methanol-, or detergent-permeabilized cells. Thus, these groups of monoclonal antibodies appear to identify four separate structural/functional domains on soluble galactosyltransferase, two of which are not readily accessible for binding in situ.
D000818:Animals; D000911:Antibodies, Monoclonal / Q000302:isolation & purification; D002417:Cattle; D003429:Cross Reactions; D005260:Female; D005700:Galactosyltransferases / Q000032:analysis*; D006056:Golgi Apparatus / Q000201:enzymology; D007124:Immunoenzyme Techniques; D007668:Kidney / Q000201:enzymology; D051379:Mice; D008807:Mice, Inbred BALB C; D008892:Milk / Q000201:enzymology; D006022:beta-N-Acetylglucosaminylglycopeptide beta-1,4-Galactosyltransferase / Q000032:analysis* / Q000276:immunology
D000911:Antibodies, Monoclonal; D005700:Galactosyltransferases; D006022:beta-N-Acetylglucosaminylglycopeptide beta-1,4-Galactosyltransferase