Isolation of unhydroxylated type I procollagen folding of the protein in vitro.
Isolation of unhydroxylated type I procollagen folding of the protein in vitro.
复制标题
体外分离蛋白质的非羟基化 I 型前胶原折叠。
DOI：
10.1016/0003-9861(81)90411-2
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发表时间：
1981
影响因子：
3.9
通讯作者：
D. Prockop
中科院分区：
文献类型：
--
作者：
C. Fiedler;P. Bruckner;T. Hayashi;D. Prockop
Tendons from 17-day-old chick embryos were incubated with α,α′-dipyridyl so that they synthesized type I protocollagen, the unhydroxylated form of type I procollagen. The incubation medium was cooled to 4 to 15 °C so that the protocollagen secreted into the medium folded into a triple-helical conformation, and the protein was purified by ion-exchange chromatography and velocity sedimentation. The isolated protocollagen had the expected amino acid composition in that it was similar to procollagen but it contained only 1 to 8 residues per 1000 of hydroxyproline and 1 residue per 1000 of hydroxylysine. At 15 °C, the circular dichroism spectrum of the protein was similar to that of triple-helical procollagen or collagen, and the protein underwent a thermal transition with a midpoint of about 25.5 °C. The folding and unfolding of the protein was reversible in that on recooling the denatured protein, essentially all the initial helicity was recovered. The folded form of the protein, however, did not consist of perfectly aligned triple-helical structures. Analysis of the circular dichroism spectrum demonstrated that the specific mean residue ellipticities of the peaks at 221 and 198 nm were less than the expected values of native procollagen. Also, the thermal transition for protocollagen was broader than that observed with procollagen. When examined by zonal centrifugation at 4 °C, most of the protein sedimented with the same apparent sedimentation coefficient as native procollagen but the peak of protocollagen was asymmetric and tailed toward the bottom of the gradient. When incubated with prolyl hydroxylase, some of the prolyl residues were hydroxylated. When incubated with a mixture of trypsin and chymotrypsin, about 40% of the protein was cleaved under conditions in which triple-helical procollagen and collagen resisted digestion. The results demonstrated that although type I protocollagen folds and refolds reversiblyin vitro, about half of the molecules present in the folded form of the protein contain short regions which are not in a triple-helical conformation.
