Studies on the Formation of Collagen

Abstract

The discovery of a form of collagen which can be extracted from fresh connective tissue in cold, neutral, and mildly aklaline media (1-5) has suggested that a fraction of the total collagen exists in a dispersed or loosely aggregated state in the extracellular, extrafibrillar matrix of the connective tissues. The process whereby a dispersion of collagen molecules (tropocollagen (6)) might be polymerized in the tissues was suggested by the observation that simple warming of the extract to physiological temperatures results in the precipitation of fibrils with the typical detailed fine structure of native collagen (4, 5).In addition to describing a new procedure for isolating and purifying the extracted collagen, this paper presents some results of an exploration of the properties of crude neutral salt extracts of fresh guinea pig corium as a base line for further studies on the role of the extracted components in the fibrogenesis of collagen and the physiology of connective tissues. Mogerials and General ProcedureGuinea pigs of both sexes ranging in weight from 100 to 500 gin. were fed Purina rabbit chow pellets (containing antibiotics) supplemented with lettuce daily and 25 rag. of vitamin C three times weekly.The hair was removed with an electric clipper and the animals sacrificed by intxaperitoneal administration of nembutal followed by exanguination da intracardiac puncture. The skin of the trunk was removed, the epidermis and subcutaneous tissue dissected away and the corium ground in a hand driven meat grinder, the cutting edges of which were kept cold with

ALTERATIONS IN STATE OF MOLECULAR AGGREGATION OF COLLAGEN INDUCED IN CHICK EMBRYOS BY SS-Aminopropionitrile (LATHYRUS FACTOR)

“…The specific optical rotation, intrinsic viscosity, and sedimentation constant are essentially similar to those for dissolved normal collagen of other species (43)(44)(45)42). The hydroxyproline, proline, and glycine values are within the range for normal collagens.…”

“…Extract viscosity and hydroxyproline content were dependent upon dosage as indicated in Table I. Relative viscosity and hydroxyproline were always determined on all extracts since the former measurement provides confidence that the hydroxyproline is present in the usual highly asymmetric type of collagen molecule (42). Extractible collagen appeared within I hour after administration of the drug and increased steadily with time at least to 72 hours (Text- fig.…”

“…After 24 hours the viscous solution was centrifuged at 100,000 g for 1 hour to remove a very small insoluble residue. The clear solution was then fractionated as described previously (42) for saline extracts of guinea pig skin. The final purified collagen precipitate was redissolved in 0.5 per cent acetic acid to produce a clear viscous solution which could then be stored in the refrigerator for several months.…”

Studies on the Mode of Action of Lathyrogenic Compounds

“…As a partial check on the efficacy of the TCA-ethanol procedure, the purified collagens, dissolved in phosphate buffer, pH 7.6, ionic strength 0.45, were examined in the Spinco analytical ultracentrifuge at 59,800 l~m at 5°C; 1 the presence of a single, hypersharp, slowly moving peak indicated that most of the non-collagenous material had been removed (6).…”

“…The collagen was purified by the TCA-ethanol method (6); wherever possible the purified collagen solution was used immediately, but where temporary storage was essential, collagen was lyophilised and stored at 5°C, solution being effected with 0.5 ~t acetic acid, using a cooled glass homogeniser (6).…”