Abstract
Abstract An enzyme was found in the muscle layers of Ascaris lumbricoides which was similar to the protocollagen proline hydroxylase found in chick embryos and other vertebrates in that it synthesized 14C-hydroxyproline when incubated with 14C-proline-labeled protocollagen from chick cartilage. The Ascaris enzyme was also similar in that it required atmospheric oxygen, α-ketoglutarate, iron, and ascorbate in order to synthesize 14C-hydroxyproline. A further similarity between the Ascaris hydroxylase and the one in chick embryos was that preparations of proline-labeled protocollagen from the cuticle of Ascaris served as substrates only after they were boiled. The Ascaris hydroxylase differed from the chick embryo enzyme in that it was partially inhibited by incubation in normal atmospheric air, and the rate of the reaction increased by about 50% as the oxygen content of the gas phase was reduced to 1%. The Km for α-ketoglutarate for the Ascaris enzyme was about 4 x 10-4 m, or about 100-fold greater than the Km for α-ketoglutarate with the chick embryo enzyme. A further difference was that the Ascaris enzyme was not inhibited by poly-l-proline form II which competitively inhibits the chick embryo enzyme.
Highlights
A further similarity between the Ascaris hydroxylase and the one in chick embryos was that preparations of proline-labeled protocollagen from the cuticle of Ascaris served as substrates only after they were boiled
In order to prepare protocollagen from the muscle layers, the cuticles were detached from the worms, and the soft internal organs were removed by blunt dissection
The presence of protocollagen proline hydroxylase has been est.ablished in a variety of vertebrate tissues (7, 15, 16), and it has been purified from chick embryos (8,11,12) and subsequently from the skin of newborn rats (13)
Summary
Preparation of Protocollagen Hydroxylase from Chick EmbryosProtocollagen proline hydroxylase was prepared from 12-day-old chick embryos with the procedures described previously (8). From 200 to 400 chick embryos were used, and the enzyme was purified through the calcium phosphate gel step. As indicated previously (5, 8, 9), the specific activity of the enzyme preparations obtained by purification through the calcium phosphate gel step was more than 50 times the specific activity of crude extracts of chick embryos, and the various preparations used here had specific activities of 5 to 10 units per mg of enzyme protein. Preparation of Proline-labeled Protocollagen from Chick Embryos and from Ascaris-The 14C-proline-labeled protocollagen from chick embryos was prepared by incubating cartilaginous tibiae from lo-day-old embryos with 14C-proline and 1 mM CX,O(‘dipyridyl (9).
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