Peter Dehm

Synthesis and extrusion of collagen by freshly isolated cells from chick embryo tendon

Abstract Matrix-free cells were isolated from chick embryo tendons by digesting the tendons with trypsin and bacterial collagenase. Microscopic examination of the isolated cells indicated that they were essentially homogeneous and testing the cells with the Trypan Blue technique indicated that most of the cells were viable. The cells rapidly incorporated [ 14 C]proline into protein for up to 2 h and well over half of the [ 14 C]proline incorporated into protein was in collagen. Centrifuging the samples at low speed at the end of the incubation removed the cells from most of the newly-synthesized extracellular collagen. About 90 % of the [ 14 C]protein in the medium was fully hydroxylated collagen polypeptides which were at least as large as the α-chains of interstitial collagen. Incubating the cells with varying concentrations of α,α′-dipyridyl inhibited the hydroxylation of protocollagen and demonstrated that un-hydroxylated collagen is not extruded at a normal rate. Incubation with cis -hydroxyproline or with azetidine-2-carboxylic acid provided the first quantitative confirmation of earlier suggestions that collagen containing one of several proline analogues is also not extruded at a normal rate.

Time lag in the secretion of collagen by matrix-free tendon cells and inhibition of the secretory process by colchicine and vinblastine☆

Matrix-free cells were isolated from chick embryo tendons and incubated with [14Cproline in order to follow the time course for the synthesis and secretion of [14C]-collagen. The results indicated a lag of 2–3 min between the time at which the incorporation of [14C]proline and the synthesis of peptide-bound [14C]hydroxyproline reached steady-state levels. A lag of about 18 min was observed between the initiation of the synthesis of collagen [14C]hydroxyproline and the secretion of [14C]collagen from the cells into the incubation medium. Colchicine and vinblastine were found to inhibit the rate of secretion of [14C]-collagen by about 70% and with appropriate concentrations of these agents the amount of intracellular [14C]collagen increased about 2-fold in 2 h. The results suggested that the secretion of collagen depends at least in part on the intensity of microtubules. Addition of dibutyryl cyclic AMP to the incubation medium produced a small increase in the fraction of total [14C]collagen which was secreted into the medium by the cells in 1 h.

Incorporation of cis-hydroxyproline into collagen by tendon cells. Failure of the intracellular collagen to assume a triple-helical conformation

Abstract Matrix-free cells from the tendons of chick embryos were incubated with cis-4-hydroxy-L-proline, a proline analogue previously shown to be incorporated into collagen and other proteins. The analogue (200 μg/ml) decreased the secretion of 14C-labeled collagen by the cells by about 60% and produced a small increase in the amount of intracellular 14C-labeled collagen. The small amount of 14C-labeled protein still secreted in the presence of the analogue consisted of small peptides which were at least in part derived from 14C-labeled collagen. Limited pepsin digestion of the 14C-labeled protein from the cell fraction indicated that the incorporation of the proline analogue into collagen interfered with the formation of the triple-helical conformation.

Effect of the proline analogue azetidine-2-carboxylic acid on collagen synthesis in vivo I. Arrest of collagen accumulation in growing chick embryos☆

Abstract Previous studies demonstrated that the proline analogue azetidine-2-carboxylic acid was incorporated in collagen synthesized in vitro and that the collagen containing the analogue was extruded from cells at a decreased rate. In the present study azetidine-2-carboxylic acid was administered for a 5-day period to rapidly growing chick embryos and it was found that the analogue arrested the accumulation of collagen in the embryos. The effect on collagen accumulation was always more marked than any effect on weight or non-collagen proteins and there was no histological evidence of non-specific toxicity. The decrease in collagen content was accompanied by a marked increase in the fragility of the embryos. Although an increase in the rate of collagen degradation was not completely excluded, the results suggested that the decrease in collagen content of the embryos can be explained by the intracellular synthesis of a collagen which contains the analogue and which cannot be extruded at a normal rate. After the cells have accumulated a large pool of analogue-containing collagen, they reach a new steady state in which both the rate of collagen synthesis and extrusion are decreased.

Characterization of the precursor forms of the α1 and α2 chains of collagen from matrix-free tendon cells

Abstract The precursor form of collagen known as “procollagen” or “transport form” was recovered from the incubation medium of matrix-free cells from embryonic tendon. The polypeptide chains of the collagen were separated by polyacrylamide gel electrophoresis in sodium dodecyl sulfate and examined by amino acid analysis. The results indicated that the additional peptides on both the pro- α 1 and pro- α 2 chain contained about 130 residues of amino acids and were similar in composition. Labeling of the polypeptide chains with [ 14 C] cystine indicated that the pro- α 1 and the pro- α 2 chains contained about the same amount of cystein or cystine.