F. S. Steven

The effect of chelating agents on collagen interfibrillar matrix interactions in connective tissue

Abstract 1. 1.The interaction of collagen fibrils and the interfibrillar matrix of tendon has been studied. 2. 2.Treatment of tendon with chelating agents at pH 7.5 results in the subsequent dispersion of collagen fibrils in dilute acetic acid. 3. 3.The dispersed collagen fibrils have an amino acid composition which is almost identical with the best preparations of tendon gelatin, indicating the chemical purity of the collagen. 4. 4.The action of chelating agents can be reversed by equilibration of the treated tendon with calcium acetate prior to acetic acid extraction. 5. 5.The interfibrillar matrix can be isolated in a purified form virtually free of collagen. 6. 6.It is suggested that metal ions play a part in the interaction of collagen fibrils and the interfibrillar matrix in tendon.

A neutral protease in rheumatoid synovial fluid capable of attacking the telopeptide regions of polymeric collagen fibrils

Fluorescent-labelled polymeric collagen fibrils have been prepared which contain three fluoresein residues in the telopeptide regions and four fluorescein residues in the helical region of each tropocollagen unit within the polymer. This material has been used as a substrate for the study of enzymes present in the synovial fluid of inflamed rheumatoid joints which are capable of degrading polymeric collagen fibrils. Two enzyme systems were observed, one inhibited by EDTA and having the properties of the known synovial collagenase, the other having the properties of a neutral protease. The neutral protease was found to be present in sonicates of the polymorphonuclear leucocytes in the synovial fluids of inflamed joints. This enzyme attacked the telopeptides of fluorescein-labelled polymeric collagen fibrils and was similar to trypsin in removing two residues of fluorescein-labelled peptides per tropocollagen molecule within the polymeric collagen fibrils but did not depolymerise the polymeric collagen fibrils.

Interaction of tropocollagen with protein-polysaccharide complexes: An analysis of the ionic groups responsible for interaction

Abstract Human intervertebral disc protein-polysaccharide complex (PP-L) has been separated by isoelectric focussing analysis into three main subunits and four minor components. PP-L interacted with tropocollagen in the isoelectric-focussing column to yield a single complex zone, containing both uronic acid and hydroxyproline, with an isoionic point intermediate between those of tropocollagen and PP-L. This complex, formed at 4°, was shown by electron microscopy to be in the form of fibrous long-spacing fibrils, whilst the complex formed at 37° consisted of native-type fibrils. Chemical and enzymic modification of the PP-L demonstrated that the sulphate group was largely responsible for interaction with tropocollagen during isoelectric focussing. The initial interaction was independent of the core protein and independent of the length of the glycosaminoglycan chains. The guanidino groups of arginine and the ϵ-amino groups of lysine were found to be the sites of interaction of tropocollagen with PP-L.

The Isolation of Chemically Pure Elastins in a Form Suitable for Mechanical Testing

Highly purified elastic fibers have been prepared by three different techniques from adult bovine ligamentum nuchae in the form of strips suitable for scanning electron microscopy7 and for mechanical testing. The purity of these ehstins has been controlled by amino acid analyses carried out on samples withdrawn at given time intervals during enzymatic, alkaline and formic acid pretreatments. Mechanical testing of intact ligamentum nuchae revealed two distinct phases during extension; the first phase was a reflection of the extension of elastin fibers while the second phase was a reflection of the extension of collagen fibers. The first phase could be studied independently by using strips of elastin fibers prepared by enzymatic or alkaline pretreatment which removed all the collagen. Although the formic acid treatment also removed the collagen, partial destruction of the desmosine and isodesmosine cross-links and also peptide bonds took place with the resultant loss in stiffness of the elastin fibers compa...

A trypsin-like neutral protease on Ehrlich ascites cell surfaces: its role in the activation of tumour-cell zymogen of collagenase.

Ehrlich ascites cells in mice have been shown to have a cell-surface trypsin-like neutral protease (TLNP) with proteolytic and beta-naphthylamidase activity. This activity is inhibited by low-mol.-wt inhibitors of trypsin but not by 11 high-mol.-wt inhibitors of trypsin in free solution. We believe that lack of inhibition is due to protection given to the enzyme by the chemical environment of the cell surface. These cells were demonstrated to export a collagenase zymogen which has been shown to be activated by the cell-surface TLNP. When this protease was completely inhibited by low-mol.-wt inhibitors of trypsin, chymotrypsin was used to activate the collagenase zymogen exported by Ehrlich ascites cells. Examination of the products of collagenolysis at 15 degrees C demonstrated the expected 3/4- and 1/4-length alpha-chain fragments derived from monomeric collagen, confirming that collagenase was one of the enzymes responsible for lysis of the collagen fibrils in the test system.

DEPOLYMERISATION OF HUMAN AND BOVINE POLYMERIC COLLAGEN FIBRILS

Bovine and human polymeric collagen fibrils were incubated at 37°C over a range of pH with a crude lysosomal enzyme preparation obtained from rat liver. Electron microscopic analysis demonstrated depolymerisation of the fibrils over the pH range 3.5 to 7.5 with the cleavage of the fibrils into short segments, followed by unwinding of the protofibrils and cleavage of the protofibrils into protofibrillar particles. The depolymerisation of polymeric collagen to soluble products was followed by hydroxyproline analysis; the pH optimum for this process was approxi- mately 4.0. The soluble depolymerisation products of bovine polymeric collagen fibrils digested at pH 4.0 were found to be native tropocollagen-like molecules which rapidly aggregated at physiological pH to form intermolecularly cross-linked native type fibrils. No soluble hydroxyproline containing products were produced by this enzyme system at physiological pH, although the cleavage of polymeric collagen fibrils could be observed at the electronmic...

Proteins of the human intervertebral disc. The association of collagen with a protein fraction having an unusual amino acid composition.

Collagen fibrils have been prepared from human intervertebral discs by use of the crude bacterial α-amylase procedure. Amino acid analysis of these disc collagens suggests that some non-collagenous protein remains firmly attached to these fibrils. This suggestion was confirmed by a study of the gelatin obtained after autoclaving disc preparations. Hydrolysis of the non-collagenous protein fractions of the disc yielded a number of unidentified ninhydrin positive compounds, one of which (called V∗) was present in quantities of up to 100 residues per 1000 total residues.

Evidence for a latent form a collagenase extracted from rabbit tumour cells

Abstract V 2 ascites cells contain a latent from a mammalian collagenase which can be activated by a short incubation with trypsin.

Collagen-protein-polysaccharide interactions in human intervertebral disc

Abstract The collagen-protein-polysaccharide complex present in human intervertebral disc has been prepared. Normal chromatographic separation techniques failed to fractionate this complex into pure collagen (or derived gelatin) and pure protein-polysaccharide complex. Fractionation was achieved by use of cetyltrimethylammonium bromide (CTAB)-cellulose chromatography. Selective binding of certain peptide fragments within the collagen polypeptide chains to the protein-polysaccharide complex was demonstrated by a combination of collagenase digestion, CTAB-cellulose chromatography and peptide fingerprinting studies. It is concluded that the stability of the collagen-protein polysaccharide complex is due to ionic interaction between the positively charged amino groups on the collagen and the negatively charged sulphonic acid groups on the protein polysaccharide complex, rather than to covalent bonds between these two components. The role of the intact protein core of the protein polysaccharide complex is also discussed.

Single-stage partial depolymerisation of collagen fibrils

Abstract Native polymerised collagen fibrils isolated from young, mature and aged achilles tendons were shown to become increasingly resistant to degradation by either proteolytic enzymes or chemical reagents known to destroy cross-linking sites in collagen. At least two chemically distinct types of cross-linkages were found in mature collagens. Cold alkali treatment either destroyed one type of cross-linkage, or cleaved the peptide chain to which this cross-linkage was attached. Hydroxylamine treatment affected another type of cross-linkage in a similar manner. No single degradation step was found to be capable of complete depolymerisation of mature collagen fibrils.