In:
Biochemical Journal, Portland Press Ltd., Vol. 189, No. 3 ( 1980-09-01), p. 491-499
Abstract:
Embryonic-chick tendon cells were incubated in suspension for 4h with 14C-labelled amino acids, cell extracts were subjected to gel filtration, and the effluent was examined by rocket immunoelectrophoresis by using antibodies specific for the β-subunit of chick prolyl hydroxylase. Two peaks of immunoreactive protein were found. The first peak contained 40% of the immunoreactive protein eluted from the column and 100% of the enzyme activity. Polyacrylamide-slab-gel electrophoresis in sodium dodecyl sulphate of an immunoprecipitate of this peak demonstrated that it consisted of the tetrameric form of prolyl hydroxylase, subunit composition α2β2 where α and β are non-identical subunits. Only the α-subunits were labelled, indicating that they were synthesized during the 4h labelling period. The β-subunits were unlabelled, indicating that they had been synthesized before the labelling period. The second peak eluted from the gel-filtration column contained 60% of the immunoreactive protein eluted from the column and was enzymically inactive. Polyacrylamide-slab-gel electrophoresis of an immunoprecipitate of this peak indicated that it consisted of a single labelled polypeptide chain, identified as cross-reacting protein, which was related to, but not identical with, the β-subunit of prolyl hydroxylase. Pulse–chase experiments were performed on cultured chick tendon cells to demonstrate that α-subunits and cross-reacting protein had half-lives of about 60h. The half-life of β-subunits was considerably longer, and the kinetic pattern was consistent with their being derived from a labelled precursor such as cross-reacting protein. The data presented here indicate that the active tetrameric form of prolyl hydroxylase in cells is assembled from α-subunits which are newly synthesized, and from β-subunits which are derived from cross-reacting protein.
Type of Medium:
Online Resource
ISSN:
0264-6021
Language:
English
Publisher:
Portland Press Ltd.
Publication Date:
1980
detail.hit.zdb_id:
1473095-9
SSG:
12
