Protein disulfide-isomerase retains procollagen prolyl 4-hydroxylase structure in its native conformation.
Protein disulfide-isomerase was isolated as a homogeneous protein from 15-day-old chick embryos. The enzyme has a molecular weight of 56,000 in SDS-polyacrylamide gel electrophoresis. Its Km value for randomly cross-linked ribonuclease, a protein used as a substrate for the enzyme, was 0.3 microM, and the Km value for DTT was 1.0 microM. Its optimum pH was 7.5 and its optimum temperature, 33 degrees C. The maximal velocity of pure protein disulfide-isomerase from chick embryos under optimal conditions was about 29,000 units/g. Protein disulfide-isomerase was able to activate purified prolyl 4-hydroxylase 2- to 3-fold, the activation being higher for enzyme stored for a longer time. This activation is probably due to the repairing of disulfide exchanges occurring in the prolyl 4-hydroxylase structure during purification and storage. Prolyl 4-hydroxylase activity was very stable in microsomes, however, and protein disulfide-isomerase was unable to increase the microsomal prolyl 4-hydroxylase activity, suggesting that prolyl 4-hydroxylase retains its native conformation in microsomes. Protein disulfide-isomerase was able to reactivate prolyl 4-hydroxylase inactivated by mild H2O2 treatment. The activity obtained after this treatment and protein disulfide-isomerase incubation corresponded to the amount of prolyl 4-hydroxylase tetramer found after H2O2 treatment. The data suggest that protein disulfide-isomerase is able to activate only the tetramer part of the enzyme preparation.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D002642:Chick Embryo; D002850:Chromatography, Gel; D007535:Isomerases / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008970:Molecular Weight; D011393:Procollagen-Proline Dioxygenase / Q000302:isolation & purification / Q000378:metabolism*; D011487:Protein Conformation; D019704:Protein Disulfide-Isomerases
D011393:Procollagen-Proline Dioxygenase; D007535:Isomerases; D019704:Protein Disulfide-Isomerases