FURTHER CHARACTERIZATION OF RAT 26,000 PROLACTIN AS A GLYCOPROTEIN WITH ESSENTIALLY O-LINKED CARBOHYDRATE CHAINS

In the rat two major molecular variants of prolactin are recorded i.e. 23,000 M(r) and glycosylated 26,000 M(r) In order to further characterize the glycosylated 26,000 rat prolactin molecular variant, rat pituitary cell lysates were digested with several glycoenzymes and the digestion products submitted to sodium dodecyl sulphate polyacrylamide gel electrophoresis and subsequent immunoblotting. The results were as follows: treatment with 1) neuraminidase, specific for sialic acid, yielded an M(r) decrease of the glycosidic variant from 26,000 to 24,500, 23,800, 23,000 and 22,000; 2) endo-alpha-N-acetylgalactosaminidase, which releases the disaccharide Gal (beta-1-3) GalNac from O-glycans, split 26,000 rat prolactin into a doublet of M(r) 26,000 to 25,500; and 3) mixed exoglycosidases from Turbo cornutus caused a gradual M(r) shift from 26,000 to 23,000. Affinity chromatography on wheat germ agglutinin Sepharose 6MB and soybean agglutinin agarose of rat pituitary homogenates and competitive inhibition tests showed that glycosylated rat prolactin has distinct affinity for these lectins. From the experimental data it is proposed that glycosylated rat prolactin is O-linked through threonine by the disaccharide Gal (beta-1-3) GalNac and possesses at least GalNac, and/or Gal and sialyl residues.