Jacqueline B. Weiss

Electron microscope studies of the effects of endo- and exopeptidase digestion on tropocollagen. A novel concept of the role of terminal regions in fibrillogenesis.

Abstract 1. 1.|Tropocollagen has been treated with pepsin, carboxypeptidase and leucine aminopeptidase in order to determine whether alterations in the molecule would give rise to modified segment-long-spacing and fibrillar precipitates. 2. 2.|Examination of segment-long-spacing from pepsin-digested tropocollagen clearly revealed that shortening at both ends of the molecule had occurred. Treatment with leucine aminopeptidase resulted in shortening at the A (N-terminal) end of the molecule. Carboxypeptidase digestion revealed a shortening of the B (C-terminal) end of the molecule. 3. 3.|Fibrils from pepsin-digested tropocollagen showed changes in morphology dependent on the length of exposure to pepsin. Short digestion (1–5 h) gave rise to fibrils with a symmetric band pattern. At 20 h tactoidal structures with both polarised and symmetric band patterns were formed. Prolonged digestion (5 days) gave rise to symmetric tactoids predominantly. Digestion of tropocollagen with leucine aminopeptidase gave rise to symmetric fibrils. Digestion. with carboxypeptidase gave rise to polarised tactoids. 4. 4.|A hypothesis suggesting that the A end of the collagen molecule is required for orientation of molecules within a fibril and that the B end is required for a fibril diameter-limiting mechanism is proposed on the basis of these results.

Isolation and characterisation of an unusual collagen from hyaline cartilage and intervertebral disc

The major collagen of hyaline cartilage is type II collagen, consisting of 3 identical cul(ll) chains [1,2]. However, additional minor collagenous components have been observed (but not identified) in chick scleral cartilage [3] and chick embryonic limb cartilage [4], and the aB (or al(V)) chain of type V collagen was reported in epiphyseal cartilage [S]. In [6], 3 collagen o-sized chains from human hyaline cartilage were isolated and partially characterised. Two of these chains designated la and 2cu, were very similar to the cwB (al(V)) and cuA ((u2(V)) chains, respectively, of type V collagen but were shown to be genetically distinct by several physicochemical criteria. The third chain 3a, however, resembled the ol(lI) chain of type II collagen and could be a post-translational modification of this chain. We have studied the collagens of bovine nasal cartilage and human intervertebral disc and have identified the lo, 20 and 3a chains in addition to type II collagen in both tissues. However, we have never observed the cut(V) chain which we believe originates only from contaminating perichondrium. We have also isolated an unusual collagen which is different from any other collagen reported in cartilage or disc. This report describes its isolation and characterisation.

Three new α-chains of collagen from a non-basement membrane source

Abstract Three new collagen α chains have been isolated from synovial membrane, gingiva and skin. Two of these have a similar chromatographic behaviour to the α[A] and α[B] chains described by Burgeson et al . (4) from a foetal basement membrane source but have been separated from another contaminating α chain, α[C]. The α[A] and α[B] chains are present in approximately equal amounts. They contain no detectable 3-hydroxyproline, are highly glycosylated and all sugar residues are present as the disaccharides. The percentage of hydroxylation of the lysine is of the order of 70%. Only a third of these hydroxylysine residues are glycosylated. The significance of these peptides, present in a tissue substantially free of basement membrane, is discussed.

Occurrence of type III collagen in inflamed synovial membranes: a comparison between non rheumatoid, rheumatoid, and normal synovial collagens.

Summary Comparison of normal, rheumatoid and inflamed synovia has shown that abnormal amounts of Type III collagen can be isolated from rheumatoid or inflamed synovia, either after digestion of the tissues with pepsin or as an abnormal polymeric form (F2PC) accounting for 10–15% of total polymeric collagen after EDTA treatment, but not from normal synovia. The amount of Type III collagen appears to be related to the degree of inflammation. Differences in susceptibility to pepsin between the bulk of the polymeric collagen from rheumatoid and non rheumatoid synovia were also noted.

Identification of digalactosylcysteine in a glycopeptide isolated from urine by a new preparative technique

Normal urine contains a complex mixture of lowmolecular weight carbohydrate-protein compounds, approximately half of which are in the MW range l-5000 [l-4] . Hpwever, very few, if any, of such glycopeptides have been obtained in a pure state, perhaps due to the variable and sometimes traumatic methods used for their isolation. Some of these methods have in fact been shown to produce, from amino acids and amino sugars, both free and in peptide form, such artifacts as Amadori rearrangement products [5]. We describe here an original method of concentrating urine, which minimizes chemical modifications of the urinary constituents during the concentration process. Using this method, we.‘have now isolated and characterized a peptide,“containing a novel carbohydrate-protein crosslinkage; digalactosylcysteine.

Factors involved in capillary growth in the heart

Growth of capillaries in the heart occurs under physiological circumstances during endurance exercise training, exposure to high altitude and/or cold, and changes in cardiac metabolism or heart rate elicited by modification of thyroid hormone levels. Capillary growth in all these conditions can be linked with increased coronary blood flow, decreased heart rate, or both. This paper brings evidence that, although increased blood flow due to long-term administration of coronary vasodilators results in capillary growth, a long-term decrease in heart rate induced by electrical bradycardial pacing in rabbits and pigs, or by chronic administration of a bradycardic drug, alinidine, in rats, stimulates capillary growth with little or no change in coronary blood flow. Decreased heart rate results in increased capillary wall tension, increased end-diastolic volume and increased force of contraction, and thus stretch of the capillary wall. This could lead to release of various growth factors possibly stored in the capillary basement membrane. Correlation was found between capillary density (CD) and the levels of low molecular endothelial cell stimulating angiogenic factor (ESAF) both in rabbit and pig hearts with CD increased by pacing. There was no relation between expression of mRNA for basic fibroblast growth factor and CD in sham-operated and paced rabbit hearts. In contrast, mRNA for TGFß was increased in paced hearts, and the possible role of this factor in the regulation of capillary growth induced by bradycardia is discussed.

Characterisation of another short-chain disulphide-bonded collagen from cartilage, vitreous and intervertebral disc

Cartilage is an avascular gel-like tissue having a collagenous meshwork made up mainly of a type of collagen (type II) present only in tissues of a similar nature such as vitreous and intervertebral disc [l-3]. Minor collagens la2a3cr [4] and disulphide-bonded phosphate-soluble collagens [S-7] have been demonstrated in cartilage and intervertebral disc which are not present in other dissimilar tissues. At present, the configuration of the phosphate soluble collagens is not clearly established. Here we describe the isolation and character&ration of a second such collagen from cartihage, vitreous and intervertebral discs.

Isolation and native characterization of cysteine-rich collagens from bovine placental tissues and uterus and their relationship to types IV and V collagens.

A simplified procedure for the fractionation and purification of different collagen types from various tissues is described which is particularly efficient in separating type-V from type-IV collagen, and highmol.-wt. (HMW) aggregates from 7 S collagen. Uterus and maternal villi contain 2 forms of type-V collagen -{α1(V)}2α2(V) and {α1(V)2α2(V)α3(V)}—which have been separated on DEAE-cellulose. Uterus however appears to be the richest source of both HMW aggregates and the {α1(V)2α2(V)α3(V)} collagen, and a probable relationship between these collagens is discussed.

Partial purification of a 5.7K glycoprotein from bovine vitreous which inhibits both angiogenesis and collagenase activity.

An inhibitor of angiogenic activity similar to that described by Raymond and Jacobson (1) has been partially purified and shown to be a glycoprotein of molecular mass 5,700. This inhibitor gave rise to an avascular zone on the chick vitelline plexus and also negated the action of a low Mr angiogenic factor. When the angiogenic inhibitor was incubated with mammalian collagenase it inhibited the enzyme activity by nearly 70%. The relevance of these findings to the role of collagenase in angiogenesis is discussed.

1 Alpha 2 alpha 3 alpha collagen is arthritogenic.

Native 1 alpha 2 alpha 3 alpha collagen (500 micrograms per rat) was both immunogenic and arthritogenic in Alderley Park rats (46% developed arthritis) but only immunogenic in Sprague-Dawley rats. Conversely, native type II collagen (500 micrograms per rat) was immunogenic and arthritogenic in both strains (64% arthritic in Alderley Park strain, 57% arthritic in Sprague-Dawley strain). The inflammatory polyarthritis induced by 1 alpha 2 alpha 3 alpha collagen was similar to that produced by native type II collagen in clinical appearance, time of onset, and histology. Antibodies raised to native bovine type II collagen cross-reacted with native 1 alpha 2 alpha 3 alpha collagen and vice versa. Thus the minor collagen component of cartilage, the 1 alpha 2 alpha 3 alpha collagen, as well as the major collagen component, type II collagen, are immunogenic and arthritogenic in the rat, with strain differences.