HETEROGENEITY IN PORCINE PITUITARY LUTEINIZING-HORMONE - AMINO-ACID AND CARBOHYDRATE ANALYSIS

Pituitary LH from porcine pituitary glands was purified by a buffered ethanol extraction procedure, ion exchange on DEAE- and carboxymethyl- cellulose, and molecular exclusion on Sephacryl S-200. Purity was asse ssed by amino acid composition, N-terminal sequence, and polyacrylamid e gel electrophoresis. Subunits were isolated by countercurrent distri bution and reverse phase HPLC. Four major forms of the alpha-subunit w ere detected: 1-96 (50%), 3-96 (23%), 4-96 (16%), and 7-96 (11%). [The original sequence report described only the 7-96 form, but we have de tected the other forms in our studies of porcine FSH and in this and o ther species of LH.] Comparable N-terminal heterogeneity was not obser ved for the beta-subunit. Additional heterogeneity was observed for bo th subunits, atrributable to heterogeneity in the N-linked oligosaccha ride moieties. The ioslated subunits were submitted to detailed compos tional carbohydrate analysis, using pulsed amperometric detection of t he HPLC-resolved sugar monomers after trifluoroacetic acid hydrolysis. Sialic acid and sulfate esters were estimated on separate hydrolyzate s. The compositional data suggest that the two alpha-subunit N-linked moieties are hybrid complex biantennary structures with sulfated N-ace tylgalactosamine (40-50%). Sixty to 70% of the alpha-subunit oligosacc harides are fucosylated. The beta-subunit of porcine LH has a single g lycosylation site, which contains a mixture of biantennary oligosaccha ride chains (80-90%) ending in N-acetylgalactosamine, half of which ar e sulfated. The balance (10-20%) are hybrid chains ending in sialylate d galactose. The majority of the oligosaccharide on the beta-subunit i s fucosylated.