Purification and biological activity of a single charge isomer of pituitary-derived chicken growth hormone.
The chicken pituitary gland contains a number of naturally occurring, developmentally regulated forms of GH which have identical molecular weights but differ in their isoelectric points. In order to characterize their biological properties, each must be separated from non-GH proteins and other forms of GH. Chickens GH (cGH) was separated from other pituitary proteins by immunoaffinity chromatography using an anti-GH monoclonal antibody covalently linked to Sepharose 4B. The cGH eluted from this column as a single peak and migrated as a single band during sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), but showed multiple bands on isoelectric focussing. This material was chromatographed on a high-performance cation exchange column, and separation of charge isomers was monitored by a combination of isoelectric focussing and immunoblotting. Chicken GH eluted from this column in two distinct peaks. The minor peak (cGH P1) contained an isomer with an isoelectric point of 6.86 and the major peak (cGH P2) an isomer with an isoelectric point of 7.52. Each isomer migrated as a single band during isoelectric focussing and SDS-PAGE (Mr = 23,500), and as a single peak during high-performance gel permeation chromatography and reverse-phase high-performance liquid chromatography. Analysis of cGH P2 through 30 cycles in a gas-phase microsequencer gave an amino acid sequence identical to that predicted by translation of the GH complementary DNA nucleotide sequence. This single charge isomer increased the rate of lipolysis in chicken adipose tissue explants by about fourfold and was able to displace 125I-labelled cGH from binding sites in liver membranes with a dissociation constant of about 4 nmol/l.(ABSTRACT TRUNCATED AT 250 WORDS)
Houston
B
B
Department of Molecular Endocrinology, AFRC Institute of Animal Physiology and Genetics Research, Edinburgh Research Station, Roslin, Midlothian.
O'Neill
I E
IE
Mitchell
M A
MA
Goddard
C
C
D000273:Adipose Tissue / Q000378:metabolism; D000818:Animals; D002645:Chickens / Q000378:metabolism*; D002850:Chromatography, Gel / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005260:Female; D005990:Glycerol / Q000378:metabolism; D013006:Growth Hormone / Q000302:isolation & purification* / Q000378:metabolism; D007525:Isoelectric Focusing; D007536:Isomerism; D008066:Lipolysis; D008297:Male; D010902:Pituitary Gland / Q000032:analysis*
D013006:Growth Hormone; D005990:Glycerol