Studies of human pituitary LH containing internally cleaved beta subunit.
We have investigated the origin of the internal peptide bond cleavage found in the beta subunit of a proportion of pituitary human LH (hLH) molecules, as well as the effects of this cleavage (or nick) on the interaction between alpha and beta subunits and on binding of hLH to its receptor. The content of cleaved beta subunit, assessed by the intensity of staining of an approximately 10 kDa component on sodium dodecyl sulphate-polyacrylamide gel electrophoresis of reduced hLH, was variable in batches of hLH prepared from pooled acetone-preserved human pituitary glands. There was evidence of a similar cleavage in purified hTSH, but not in the purified hFSH or human chorionic gonadotrophin examined. Although intact hLH was relatively resistant to cleavage in solution, urea dissociation of hormone followed by dialysis resulted in an increased content of nicked beta subunit, which was largely prevented by incorporation of the proteolytic enzyme inhibitor phenylmethylsulphonyl fluoride (PMSF) and the metal-chelating agent EDTA. Hormone that was virtually free of nicked beta subunit was obtained by urea dissociation of hLH subunits in the presence of PMSF and EDTA followed by dialysis to remove urea, reassociation of subunits at 37 degrees C (pH 7) and purification of reassociated hLH dimer by gel filtration on Sephadex G-100 in the presence of 1-anilinonaphthalene-8-sulphonic acid (ANS).(ABSTRACT TRUNCATED AT 250 WORDS)
Stockell Hartree
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A
AFCR Institute of Animal Physiology and Genetics Research, Babraham, Cambridge.
Shownkeen
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RC
D001665:Binding Sites; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007986:Luteinizing Hormone / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010447:Peptide Hydrolases; D010902:Pituitary Gland / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D011974:Receptors, LH / Q000378:metabolism
D010446:Peptide Fragments; D011974:Receptors, LH; D007986:Luteinizing Hormone; D010447:Peptide Hydrolases