Peter F. Davison

The contribution of labile crosslinks to the tensile behavior of tendons.

The tensile strength of rat tail tendons, measured by the load that ruptures the tendon, varies with the weight of the animal, the vertebra to which the tendon was attached, the pH of the tendon, and the chemical treatment. Reduction with sodium borohydride multiplies the tensile strength by a factor up to 4.5 (the factor diminishing with age) and reduces creep in the strained tendon. It is concluded that strain catalyzes the hydrolysis of aldimine intermolecular crosslinks that contribute significantly to the tensile strength under physiological conditions, and that the crosslinks are stabilized by reduction or with aging. It is postulated that creep is the result of slippage between polymeric assemblies of stably crosslinked molecules. This postulate leads to a plausible description of elongation of connective tissue fibrils in growing animals.

The Effects of Acetic Acid on Collagen Cross-Links

Native, acid-soluble and acid-insoluble collagen from the tail tendons of rats and calves have been reduced by 3H-sodium borohydride. The reduced cross-link derivatives were separated by ion exchange chromatography. The destruction of the cross-links under the conditions of hydrolysis was measured. By applying the appropriate correction factors, the approximate levels of the respective cross-link structures in the variously treated tissues were assessed. We conclude that treatment of collagen by 0.05 M acetic acid for 1 hour or less imposes a profound rearrangement of intermolecular bonds in both the soluble and insoluble preparations. The nature of the intermolecular bonding in native collagen therefore cannot be meaningfully assessed if the preparation is previously exposed to acid.

Tissue collagenase: a simplified, semiquantitative enzyme assay.

Abstract Tissue collagenase activity from the ulcerating rabbit cornea has been quantitated in a sensitive capillary tube assay system with an unlabeled, native collagen substrate. In this assay system, initial rates of gel lysis are proportional to enzyme concentration over a defined range of enzyme concentrations. Increased sensitivity to enzyme with an unlabeled substrate has been achieved by restricting diffusion of enzyme to one dimension, in a capillary gel. Corneal collagenase activity has been measured at concentrations down to 0.1 μg/μl. In addition to its high sensitivity to enzyme, the precision and simplicity of the assay and minimal equipment requirements all recommend its use for routine screening of biological fluids for collagenase activity and in the investigation of the effects of inhibitors and stimulators of collagenase activity.

Cross-linking and aging in rat tendon collagen

Abstract The solubility of native and borohydride-reduced tail tendons from rats of various ages was studied. Even in the youngest animals examined (one month old) almost all the collagen molecules are interlinked but many of the bonds between the chains are labile in strong salt solutions or in dilute acids. These bonds are stabilized by borohydride reduction. Under conditions that maintain the native configuration of the collagen molecules the reduced tendons are insoluble but denaturing reagents can dissolve a considerable fraction from the tendon collagen of young animals. It is deduced that in the young animals all molecules but not all polypeptide chains are inter-linked. In the older animals the level of cross-linking is increased with the result that most of the polypeptides are linked within and between the molecules as is demonstrated by the decreased solubility in denaturing reagents and the increase in polymeric chains present in the material that can be extracted. The cross-linking compounds in the collagen fibrils that can be discriminated by chromatography of an alkali-hydrolyzed, reduced (and 3 H-labeled) tendon were compared for animals of various ages. The proportions of the various reduced cross-link compounds varied with age but no marked increase in the level of incorporated tritium was noted in the old animals. It appears likely that the increased cross-linking indicated by the solubility studies reflects an accumulation of non-reducible cross-links or a more effective redistribution of pre-existing ones.

Heterogeneity of collagens from basement membranes of lens and cornea.

Abstract Collagen was solubilized by pepsin digestion of bovine Descemets membrane and anterior lens capsule. From these preparations “segment long-spacing” crystallites were precipitated by ATP. Comparison with bovine types I, II and III collagens by electron microscopy showed that two new types of segments were present. These new crystallites represented only a small fraction of the collagen precipitated on the microscope grids and therefore it is not certain that the collagen producing these segments is representative of the collagen comprising most of the two basement membranes; these new collagens may be derived from minor components of these tissues. Segments identical to one of those from Descemets membrane were also observed in the mixture of collagens solubilized by pepsin from heart valve.

Aging, and crosslinking in mammlian collagen.

The amount and type of borohydride-reducible crosslinks in collagen have been examined as a function of animal age. In a variety of bovine, canine and human tissues the level of redicible crosslinks decreases with time and the ratios of individual compounds change. There is both tissue and species specificity in the extent of these changes. A decrease in the level of reducible crosslinks correlates with the cessation of growth. Loss of reducible crosslinks does not imply a small total number of crosslinks since physical changes with age imply the opposite. We conclude that reducible crosslinks are converted to a stable nonreducible state and the persistence of low levels of reducible crosslinks may be indicative of a low level of turnover in the tissue. Changes in ratios of reducible crosslinks are of doubtful functioal significance and may simply reflect variation in post-translational modification of lysine residues.

Structural homologies in mammalian neurofilament proteins

Antibody has been prepared to purified neurofilament protein extracted from calf brain. This antibody crossreacts with brain and nerve extracts from all mammals tested but fails to crossreact with frog, cod and chicken nerve extracts. Thus the serologic homology of these neurofilament proteins appears to be restricted to the mammals. Structural similarities between the mammalian neurofilament protein are also indicated by the strong resemblance between iodinated peptide maps prepared from the electrophoretically purified neurofilament proteins.

Guest editorial: mechanisms of development and aging

Abstract The view is expressed that an inappropriate emphasis and research effort is currently directed towards simplistic approaches to the problem of aging; it is concluded that an understanding of the biochemical mechanisms underlying the aging process must be sought using a SYSTEMS analysis, at a level of complexity and integration much greater than that encompassed by any theory thus far considered.

Comparison of specific activities of enzymes from young and old dogs and mice

Aldolases A and B (EC 4.1.2.13) and liver cytoplasmic superoxide dismutase (EC 1.15.1.1) have been purified from young and old dogs and mice. The specific activities of these enzymes were measured and show no diminution with age. In addition, dog aldolase B, dog liver superoxide, and mouse liver aldolase were titrated in crude extracts with specific antisera. No accumulation of cross-reacting material with age was detected. The electrophoretic mobilities of these enzymes and the susceptibilities of dog aldolases A and B to trypsin digestion were also unchanged. These results are additional evidence that the accumulation of inactive enzymes is not an invariable concomitant of aging.

Characterization of human corneal collagenase

Collagenolytic activities have been detected in the culture media from an eye bank cornea and in media from corneal tissues biopsied at the time of surgery. The eye bank cornea was not pathological but was not used for corneal grafting because of the donor age (77). The corneal biopsies were from keratoplasty patients with no history of corneal ulceration. Enzymes from both sources cleave tropocollagen at the same site as do tadpole-tail collagenase and rabbit corneal collagenases, and, because of the restricted cleavage, belong to the class of enzymes known as “tissue collagenases”. The human corneal collagenase activities also degrade reconstituted collagen fibrils. The collagenase activity(ies) from human keratoplasty tissues is inhibited by Calcium-EDTA and N-acetyl-l-cysteine, both of which have been found previously to prevent ulceration in the alkali-burned rabbit cornea and to inhibit collagenases produced by the ulcerating rabbit cornea. The serum antiprotease, α2-Macroglobulin, has also been found to inhibit the collagenase(s) from keratoplasty tissues, an observation which supports the ophthalmologists sometime contention that serum inhibits corneal ulceration. It is hoped that the human corneal collagenase(s) produced by the culture of keratoplasty tissues and of eye bank corneas judged unfit for corneal grafting will facilitate the discovery of effective inhibitors of corneal ulceration.