Glycosylated equine prolactin (G-ePRL) and nonglycosylated ePRL were p
urified to homogeneity from side fractions obtained during isolation o
f LH/FSH from horse pituitaries. Both PRL forms were isolated together
in high yield by the isolation procedure used for glycosylated porcin
e PRL/(G-pPRL) and pPRL, involving acetone extraction/precipitation, N
aCl and isoelectric precipitation, and gel filtration. Purification of
G-ePRL required additional Con A chromatography. The N-terminal amino
acid sequencing for 32 cycles of G-ePRL and ePRL resulted in sequence
s identical to the known primary structure of ePRL. Based on MALDI mas
s spectrometry analysis and SDS-PAGE mobilities, -G-ePRL and ePRL had
estimated molecular weights of 25,000 and 23,000 Da, respectively. G-e
PRL displayed only 60% of the immunoreactivity of ePRL in homologous r
adioimmunoassay, Using the Nb2 lymphoma cell bioassay, ePRL was found
to have about 1/30th the mitogenic activity of bovine PRL; G-ePRL was
approximately 1/10th as active as ePRL. Glycosylation of G-ePRL at Asn
(31) was confirmed by isolation and sequence analysis of an enzymatica
lly derived G-ePRL glycopeptide spanning residues 29-37. Monosaccharid
e compositions of intact G-ePRL and this glycopeptide were very simila
r (Man(3), GlcNAc(2), GalNAc(1), Fuc(0.6), Gal(0.2), NeuAc(0.15)) and
resembled that of G-pPRL. The glycopeptide contained one sulfate resid
ue as determined by ion chromatography after acid hydrolysis, indicati
ng the presence of a sulfated monosaccharide, Comparative carbohydrate
analysis of G-ePRL and other G-PRL preparations suggests that the fun
ctionally significant Asn(31) carbohydrate unit is a fucosylated compl
ex mono- and/or biantennary oligosaccharide terminating with a sulfate
d GalNAc residue and two or three Man residues.