The collagen substrate specificity of rat uterus collagenase

The collagen substrate specificity of rat uterus collagenase was studied as a function of both collagen type and species of substrate origin. For each collagen examined, values for the basic kinetic parameters, Km and Vmax (kcat), were determined on collagen in solution at 25 degrees C. In all cases, Lineweaver-Burk plots were linear and rat uterus collagenase behaved as a normal Michaelis-Menten enzyme. Collagen types I, II, and III of all species tested were degraded by rat uterus collagenase. Collagen types IV and V were resistant to enzymatic attack. Both enzyme-substrate affinity and catalytic rates were very similar for all susceptible collagens (types I-III). Values for Km ranged from 0.9 to 2.5 X 10(-6) M. Values for kcat varied from 10.7 to 28.1 h-1. The homologous rat type I collagen was no better a substrate than the other animal species type I collagens. The ability of rat uterus collagenase to degrade collagen types I, II, and III with essentially the same catalytic efficiency is unlike the action of human skin fibroblast collagenase or any other interstitial collagenase reported to date. The action of rat uterus collagenase on type I collagen was compared to that of human skin fibroblast collagenase, with regard to their capacity to cleave collagen as solution monomers versus insoluble fibrils. Both enzymes had essentially equal values for kcat on monomeric collagen, yet the specific activity of the rat uterus collagenase was 3- to 6-fold greater on collagen fibrils than the skin fibroblast enzyme. Thus, in spite of their similar activity on collagen monomers in solution, the rat uterus collagenase can degrade collagen aggregated into fibrils considerably more readily than can human skin fibroblast collagenase.     

The gelatinolytic activity of rat uterus collagenase

The collagenase produced by rat uterine cells in culture has been examined for its ability to degrade denatured collagen. Acting as a gelatinase, rat uterus collagenase was able to successfully degrade the denatured chains of collagen types I through V. In addition, the enzyme produced multiple cleavages in these chains and displayed values for Km of 4-5 microM, compared to values of 1-2 microM when native collagen was used as substrate. Furthermore, rat uterus collagenase degraded the alpha 2 chain of denatured type I collagen at a significantly faster rate than the alpha 1 chain, as previously observed for human skin fibroblast collagenase. In contrast to the action of human skin collagenase, however, the rat uterus enzyme was found to be a markedly better gelatinase than a collagenase, degrading the alpha chains of denatured type I guinea pig skin collagen at rates some 7-15-fold greater than native collagen. Human skin collagenase degrades the same denatured chains at rates ranging from 13-44% of its rate on native collagen. Rat uterus collagenase, then, is approximately 50 times better a gelatinase than is human skin collagenase. In addition to its ability to cleave denatured collagen chains at greater rates than native collagen, the rat uterus collagenase also attacked a wider spectrum of peptide bonds in gelatin than does human skin collagenase. In addition to cleaving the Gly-Leu and Gly-Ile bonds characteristic of its action on native collagen, rat uterus collagenase readily catalyzed the cleavage of Gly-Phe bonds in gelatin. The rat enzyme was also capable of cleaving Gly-Ala and Gly-Val bonds, although these bonds were somewhat less preferred by the enzyme. The cleavage of peptide bonds other than Gly-Leu and Gly-Ile appears to be a property of the collagenase itself and not a contaminating protease. Thus, it appears that the collagenase responsible for the degradation of collagen during the massive involution of the uterus might also act as a gelatinase to further degrade the initial products of collagenolysis to small peptides suitable for further metabolism.     

A latent form of collagenase in the involuting rat uterus and its activation by a serine proteinase.

1. The involuting rat uterus displays an extremely rapid breakdown of collagen. Collagenase activity can be assayed directly in the insoluble 6000g pellet of uterine homogenates. At 1 day post partum, about 85% of this collagenase activity is in a latent form. 2. This latent form can be activated by trypsin or by a serine proteinase present in the uterine pellets. 3. The activating enzyme of the tissue is inhibited by a wide spectrum of trypsin inhibitors, including Trasylol, soya-bean and lima-bean trypsin inhibitors, snail inhibitor and di-isopropyl phosphoro-fluoridate. Partial inhibition is produced by benzamidine, phenylmethanesulphonyl fluoride, epsilon-aminohexanoate, leupeptin, antipain and alpha1-antitrypsin. Ovomucoid, 7-amino-1-chloro-3-tosylamido-1-heptan-2-one and 1-chloro-4-phenyl-3-(N-benzyloxy-carbonyl)amino-L-butan-2-one are not inhibitory. 4. Extraction of uterine pellets with 0.1 M-CaCl2 at 60 degrees C releases both latent and active collagenase. Exclusion chromatography on Sephadex G-100 gives an apparent molecular weight of approx. 77000 for the latent form and 66000 for the active form. The latent form is suggested to be a zymogen of collagenase.     

Collagenase production by smooth muscle: correlation of immunoreactive with functional enzyme in the myometrium

A monospecific antibody to rat uterine collagenase has been produced and employed to study the cell of origin and the time course of production of this enzyme in the involuting rat uterus. The specificity of the anti-collagenase antibody was confirmed by immunoprecipitation, Western analysis, and by its ability to inhibit the activity of collagenase. Parallel measurements of functional enzyme, both latent and active, bound to tissue collagen were also made in nonpregnant, gravid, and postpartum rat uteri. Immunohistochemical staining of collagenase in sections of rat uterus showed the enzyme to be present in the perinuclear region of the smooth muscle cells only of the involuting myometrium. No detectable collagenase was present in the prepartum or nonpregnant uterus. Identity of the smooth muscle cells was confirmed using an anti-smooth muscle actin antibody. In addition, the cultured uterine cells from which the immunizing antigen was obtained were also identified as smooth muscle cells. Specificity of the tissue staining was confirmed by the ability of pure rat uterine collagenase to block the reaction of the antibody with the tissue. These observations indicate that smooth muscle cells are capable of producing collagenase and are consistent with the hypothesis that this enzyme presides over the massive collagen degradation seen in postpartum uterine involution. Furthermore, measurement of collagenase bound to uterine collagen revealed that collagenase activity could be detected only at the time that the cells could be seen to be producing the enzyme by immunolocalization. These findings support the concept that collagenase is produced only as needed and not stored, either intra- or extra-cellularly.     

Clofibrate-induced increase in coenzyme A concentration in rat tissues

1. Total, active and latent collagenase activities were determined by direct assay of tissue homogenates. 2. The rate of collagen breakdown during post-partum involution of the rat uterus is correlated with the total activity of collagenase. Both are low at parturition, reach a maximum within 24h and fall slowly to low values of 5 days post partum. This temporal correlation strongly supports the hypothesis that collagenase participates in collagen breakdown in vivo. 3. Further support for this hypothesis is provided by the finding that oestradiol-17 beta (100 micrograms/day, intraperitoneally injected), which inhibits the breakdown of collagen by 36% during the first 4 days of involution, produces a closely corresponding decrease in total collagenase activity. 3. The effect of oestradiol in lowering collagenase activity is not due to alterations in collagen substrate, collagenase kinetic behaviour or latent-to-active enzyme conversion. 4. Of the total assayable collagenase, about 35% is fully active and 65% is in a latent form. 5. About 70% of this latent form can be activated by a serine proteinase found, together with collagenase, in the insoluble fraction of uterine homogenates.     

Hormonal regulation of collagen degradation in the uterus: Inhibition of collagenase expression by progesterone and cyclic AMP

Dibutyryl cyclic AMP markedly increases the ability of progesterone to prevent the expression of collagenase activity in cultures of post-partum rat uterus. Dibutyryl cyclic AMP itself and, to a lesser extent, native cyclic AMP, are capable of producing a partial decrease in enzyme activity, but complete abolition is not observed at high cyclic nucleotide concentrations (5 mM) in the culture medium. Theophylline, when added to cultures, mimics the effect of dibutyryl cyclic AMP. Other cyclic nucleotides were without effect on levels of collagenase activity in the uterine cultures.When non-inhibitory concentrations of either dibutyryl cyclic AMP (1 · 10^?4 M) or theophylline (1 · 10^?4 M) are added to cultures together with a non-inhibitory concentration of either progesterone (5 · 10^?6 M) or the potent progesterone analogue Provera (1 · 10^?8 M) the ability of the tissue to produce collagenase is decreased by 40–70%. Collagenase activity is consistently diminished more than additively by combinations of steroid and cyclic nucleotide. Theophylline mimics the effect of dibutyryl cyclic AMP on steroid activity in culture. In the presence of dibutyryl cyclic AMP, diminution of collagenase activity can be observed at concentrations of steroid more than two orders of magnitude lower than the normal minimally inhibitory dose. Reduction of collagenase activity is reflected in all experiments by a concomitant decrease in the normal proteolytic degradation of collagen in the tissue ex-plants. The possibility that progesterone acts in the uterus to raise cyclic AMP levels is suggested by the fact that uterine tissue, when cultured in the presence of progesterone, contains reduced levels of cyclic nucleotide phosphodiesterase.These data suggest that, in some way a cyclic AMP-mediated system is critically involved in the control of collagenase activity by progesterone in the rat uterus.     

Regulation of human skin collagenase activity by hydrocortisone and dexamethasone in organ culture

Hydrocortisone and dexamethasone (9α-fluoro, 16α-methyl prednisolone) prevent the appearance of collagenase in cultures of normal human skin, human rheumatoid synovium and rat uterus. Hydrocortisone is maximally inhibiting at 10^?7M and dexamethasone at 10^?8M in culture medium. Neither steroid is an inhibitor of enzyme activity. The loss of collagenase activity in cultured tissue is not accompanied by detectable inhibition of protein synthesis. Reduction of enzyme activity in culture medium is concomitant with a parallel cessation of tissue collagen degradation, indicating that the tissue fails to produce active collagenase in the presence of physiologic levels of glucocorticoids.     

Effect of 1,25-dihydroxyvitamin D-3 on phosphate uptake into chick intestinal brush border membrane vesicles

The production of collagenase by human skin explants in culture is prevented by 10(-8) M dexamethasone, 5 . 10(-4) M dibutyryl cyclic AMP, or 2.5 . 10(-3) M theophylline. Decreases in collagenase activity are paralleled by reductions in the degradation of explant collagen during the culture period. Progesterone, which effectively inhibits collagenase production in rat uterine explant cultures, has no effect on human skin explants. The inhibition by cyclic AMP is nucleotide specific. When partially inhibitory concentrations of dexamethasone and dibutyryl cyclic AMP, or dexamethasone and theophylline, are added to culture medium together, the resultant inhibition is that predicted by additivity. Synergistic inhibition, as observed in rat uterus between progesterone and dibutyryl cyclic AMP, fails to occur. Dexamethasone inhibits the production of collagenase by cultured explants of rat uterus, with complete inhibition occurring at 10(-7) M steroid. Synergism between glucocorticoids and dibutyryl cyclic AMP or between dexamethasone and progesterone could not be demonstrated in the uterine culture system. These results suggest the existence of three regulatory systems for the control of collagenase production in mammalian tissues, and that cooperativity between systems may occur on a tissue-specific basis.     

Extraction of collagenase from the 6000 times g sediment of uterine and skin tissues of mice. A comparative study.

An enzyme capable of digesting native collagen in solution at neutral pH was extracted from the 6 000 times g sediment of the involuting uterus of the mouse and of the back skins of mice and rats. The collagenase could be dissociated at cold-room temperature from the sediment in about equal amounts when neutral Tris buffer containing 1.0M NaCl or 5M urea was used for the extraction step. The enzyme has been concentrated by ammonium sulfate precipitation and the activity was measured by using [14C]collagen in solution at pH 7.5. Collagen breakdown products were identified by disc electrophoresis. The amount of enzyme extracted was a function of temperature and salt concentration. As 5M urea extracted collagenase from the sediment in a relatively short time, this method of extraction seems to be a useful tool for serial experiments in the study of collagenase activity in collagen-rich tissues.     

Serine proteinase activation of latent human skin collagenase

Latent and active collagenase were demonstrated following direct extraction from normal skin homogenates with 0.1M calcium chloride at 60 degrees C. 83% of the collagenase activity was in latent form and could be maximally activated with trypsin. Partial activation of the latent enzyme could also be demonstrated by incubation of the skin extract without added trypsin. This endogenous activation was inhibited by the addition of soya bean trypsin inhibitor, trasylol, di-isopropylphosphofluoridate and phenylmethanesulphonylfluoride, none of which inhibited collagenase directly. This suggests that the skin extracts contain a collagenase activating enzyme with the inhibition profile of a serine proteinase. A chymotryptic proteinase with a similar inhibition profile was extracted from normal human skin and partially purified. This enzyme activated fibroblast procollagenase derived from tissue culture of normal skin. The procollagenase was also partially activated by plasmin and chymotrypsin. This is the first demonstration of a collagenase activating enzyme in human skin and raises the possibility that collagenase activation by this mechanism may be responsible for collagen degradation in some disease processes.     

A sensitive, specific assay for tissue collagenase using telopeptide-free [3H]acetylated collagen

Collagenase is assayed by incubation with soluble, telopeptide-free collagen extracted from rat skin and labeled with [2-3H]acetic anhydride. Collagen is cleaved by collagenase and the resulting fragments are digested with trypsin and chymotrypsin. Undigested collagen is recovered by precipitation with trichloroacetic acid, collected on glass-fiber filters, and quantitated by liquid scintillation spectrometry. This procedure combines features of the Cawston and Barrett (T.E. Cawston and A.J. Barrett, 1979, Anal. Biochem. 99, 340-345) and the Ryh?nen et al. (L. Ryh?nen et al., 1982, Collagen Rel. Res. 2, 117-130) methods. The first method provides a simple way to prepare large quantities of uniform substrate, while the second increases the specificity of the assay by removal of the labeled telopeptides. The assay is reproducible and linear with time and enzyme concentration. It is approximately 10X more sensitive than the Cawston and Barrett method and can readily detect 1-8 mU collagenase (1 unit equals 1 microgram collagen cleaved/min at 30 degrees C). The substrate is resistant to elastase, trypsin, and chymotrypsin and is completely degraded by bacterial collagenase. Collagenase is the only tissue metalloprotease found, to date, that cleaves the substrate.     

A sensitive method for the separation and quantitation of (14C)proline and (14C)hydroxyproline from the collagen of rat uterus

A specific and sensitive method is described for the isolation and quantitation of [¹⁴C]proline and [¹⁴C]hydroxyproline from uterine collagen of the immature rat. Selectivity is achieved in this isolation by using a protease-free bacterial collagenase. There is complete release of hydroxyproline from uterine protein if the latter is suspended by sonication prior to treatment with collagenase. There is a consistent recovery of [¹⁴C]proline and [¹⁴C]hydroxyproline when they are added to protein hydrolysates of uterus and then subjected to the procedures required for their isolation and quantitation. It is possible using this method to determine the incorporation of [¹⁴C]proline into collagen of the rat uterus and to quantitate its conversion to [¹⁴C]hydroxyproline. Coupled with the colorimetric methods for proline and hydroxyproline, it is also possible to determine their specific activity.     

Preparation, properties, and uses of two fluorogenic substrates for the detection of 5'-(venom) and 3'-(spleen) nucleotide phosphodiesterases

A new method for ³H-labeling of native collagen and a specific microassay for collagenase activity are presented. Acid-soluble type I collagen derived from rat tail tendons was reacted with pyridoxal phosphate and then reduced with NaB³H₄ to yield [³H]collagen with a specific activity of more than 10 μCi/mg. With respect to rate of hydrolysis, trypsin susceptibility, and gelling properties this collagen compares favorably with biosynthetically labeled preparations. It was shown that chemical labeling procedures such as this, or N-acetylation with acetic anhydride, do not adversely affect properties of collagen which are important for its use as substrate in specific assays. The microassay employs 50-μl [³H]collagen gels (1 mg/ml) dispensed in microtest plates. At 36°C this assay combines rapid rate of hydrolysis with low trypsin susceptibility. As little as 1 ng of clostridial collagenase activity can be measured reproducibly. The high specific activity of the [³H]collagen allowed us to explore microassay conditions employing minute quantities of substrate in solution. These studies indicated that native type I collagen whether labeled or not, is cleaved in the helical region by trypsin at subdenaturation temperatures. It was concluded that, in order to remain specific, collagenase assays with collagen in solution as with collagen in fibrils must be performed at 10–12°C below the denaturation temperature, i.e., at 35–37°C with collagen gels and 27–29°C with collagen in solution.     

Hormonal interactions in mammalian collagenase regulation: Comparative studies in human skin and rat uterus

The production of collagenase by human skin explants in culture is prevented by 10^?8 M dexamethasone, 5·10^?4 M dibutyryl cyclic AMP, or 2.5· 10^?3 M theophylline. Decreases in collagenase activity are paralleled by reductions in the degradation of explant collagen during the culture period. Progesterone, which effectively inhibits collagenase production in rat uterine explant cultures, has no effect on human skin explants. The inhibition by cyclic AMP is nucleotide specific. When partially inhibitory concentrations of dexamethasone and dibutyryl cyclic AMP, or dexamethasone and theophylline, are added to culture medium together, the resultant inhibition is that predicted by additivity. Synergistic inhibition, as observed in rat uterus between progesterone and dibutyryl cyclic AMP, fails to occur.Dexamethasone inhibits the production of collagenase by cultured explants of rat uterus, with complete inhibition occurring at 10^?7 M steroid. Synergism between glucocorticoids and dibutyryl cyclic AMP or between dexamethasone and progesterone could not be demonstrated in the uterine culture system. These results suggest the existence of three regulatory systems for the control of collagenase production in mammalian tissues, and that cooperativity between systems may occur on a tissue-specific basis.     

The distribution of collagenase in normal rat tissues.

A rabbit monospecific anti-rat uterus collagenase antibody has been used to study the distribution of collagenase in normal rat tissues by immunohistochemical methods. Indirect staining was performed with fluorescein-conjugated goat anti-rabbit immunoglobulin G antibody. The organs studied were brain, lung, myocardium, liver, spleen, kidney, adrenal, testes, uterus, xiphoid cartilage, tail tendon, skeletal (triceps) muscle and skin. Collagenase is widely present throughout the connective tissue structures in all organs examined. The enzyme is apparently bound to collagen fibers, reticulum fibers and basement membranes. The results suggest that control of collagenase activity depends on factors other than the presence of the enzyme in tissues.     

Purification and properties of bovine nasal hyaline cartilage collagenase

1. Collagenase from bovine nasal hyaline cartilage was extracted with 1 and 3 M NaCl in Tris-CaCl2 buffer. 2. Two peaks of collagenase activity were revealed on DE52 ion exchange column, collagenase 1 and collagenase 2. 3. The apparent mol. wt of collagenase 1 and 2 as determined by SDS-PAGE were 68 and 43 kDa, respectively. 4. Both enzymes degrade native collagen type II into two characteristic products, TCA(3/4) and TCB(1/4), at 25 degrees C and pH 7.6. 5. Trypsin and aminophenylmercuric acetate were capable of increasing the collagenase 1 activity. 6. The two enzymes can be characterized as metalloproteinases since they were inhibited by EGTA and 1,10-phenanthroline. The use of proteinase inhibitors (N-ethylmaleimide, iodoacetic acid, phenylmethylsulphonyl fluoride, soybean trypsin inhibitor, pepstatin, dithiothreitol) showed that the enzymes do not contain serine, cysteine or aspartic acid in their active sites.     

Differential susceptibility of type X collagen to cleavage by two mammalian interstitial collagenases and 72-kDa type IV collagenase

We have studied the degradation of type X collagen by human skin fibroblast and rat uterus interstitial collagenases and human 72-kDa type IV collagenase. The interstitial collagenases attacked the native type X helix at two loci, cleaving residues Gly92-Leu93 and Gly420-Ile421, both scissions involving Gly-X bonds of Gly-X-Y-Z-A sequences. However, the human and rat interstitial enzymes displayed an opposite and substantial selectivity for each of these potential sites, with the uterine enzyme catalyzing the Gly420-Ile421 cleavage almost 20-fold faster than the Gly92-Leu93 locus. Values for enzyme-substrate affinity were approximately 1 microM indistinguishable from the corresponding Km values against type I collagen. Interestingly, in attacking type X collagen, both enzymes manifested kinetic properties intermediate between those characterizing the degradation of native and denatured collagen substrates. Thus, energy dependence of reaction velocity revealed a value of EA of 45 kcal, typical of native interstitial collagen substrates. However, the substitution of D2O for H2O in solvent buffer failed to slow type X collagenolysis significantly (kH/kD = 1.1), in contrast to the 50-70% slowing (kH/kD = 2-3) observed with native interstitial collagens. Since this lack of deuterium isotope effect is characteristic of interstitial collagenase cleavage of denatured collagens, we investigated the capacity of another metalloproteinase with substantial gelatinolytic activity, 72-kDa type IV collagenase, to degrade type X collagen. The 72-kDa type IV collagenase cleaved type X collagen at both 25 and 37 degrees C, and at loci in close proximity to those attacked by the interstitial enzymes. No further cleavages were observed at either temperature with type IV collagenase, and although values for kcat were not determined (due to associated tissue inhibitor of metalloproteinases-2), catalytic rates appeared to be substantial in comparison to the interstitial enzymes. In contrast, type X collagen was completely resistant to proteolysis by stromelysin. Type X collagen thus appears to be highly unusual in its susceptibility to degradation by both interstitial collagenase and another member of the metalloproteinase gene family.     

A latent collagenase from rheumatoid synovial fluid. Purification and partial characterization

1. A latent collagenase (EC 3.4.24.3) has been isolated from rheumatoid synovial fluids and purified by (NH4)2SO4 precipitation and column chromatography, utilising Sephadex G-150, DEAE Sephadex A-50 and Sephadex G-100 superfine grade. 2. The final preparation activated by trypsin (EC 3.4.21.4) had a specific activity against thermally reconstituted collagen fibrils of 259 micrograms collagen degraded/min per mg enzyme protein, representing a nearly 800-fold increase over that of the original rheumatoid synovial fluid. 3. The latent collagenase preparation can be activated by trypsin and to some extent by HgCl2 but not by 3 M NaSCN, 3.5 M NaCl, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) or p-chloromercuribenzoate. 4. Inhibition studies and the acrylamide gel electrophoretic pattern of collagen degradation products showed that the trypsin-activated enzyme has the essential features of a neutral collagenase. 5. The molecular weights, determined by calibrated gel filtration, were 52 000 and 43 000 for the latent and the activated enzyme, respectively. 6. The nature of the latency of synovial fluid collagenase is discussed.     

Degradation of type I collagen by rat mucosal keratinocytes. Evidence for secretion of a specific epithelial collagenase

Feeder-cell-independent serially propagating keratinocytes from rat oral mucosa (tongue) dissolved reconstituted type I [3H]collagen fibrils, although rather slowly. Analysis of the conditioned medium from such cultures revealed secretion of a Mr = 65,000 collagenase which remained almost entirely latent in the absence of exogenous protease activity. Addition of trypsin (0.1-1.0 microgram/ml) or plasmin (1.0-4.0 micrograms/ml) resulted in substantial acceleration of the collagenolytic process in stimulated secretion of latent collagenase and, at higher concentrations, in conversion of the latent enzyme to the catalytic form. The keratinocyte collagenase was indistinguishable from interstitial, fibroblast-type collagenases by several criteria including: cleavage of native type I collagen in solution at the characteristic collagenase-sensitive locus at 22 degrees C and dissolution of reconstituted type I collagen fibrils at 35 degrees C; activation by trypsin and by organomercurials and inhibition by Zn2+ and Ca2+ chelators; and cross-reaction with antibody to fibroblast-type procollagenase. Expression of collagenolytic activity in keratinocyte cultures was effectively regulated by cell density. The activity (on a per cell basis) was maximal at 10-20% confluence and was more than 95% "contact-inhibited" at subconfluent and early confluent densities (2-4 X 10(5)/cm2). Our findings show that mucosal keratinocytes possess a potent enzymatic apparatus for degradation of interstitial collagen fibrils which includes a classical vertebrate collagenase.     

Collagenase activity in the normal rat myocardium. An immunohistochemical method

Fibrillar collagen in the myocardium provides a supportive framework for myocytes and capillaries. Disruption of this organized framework has been observed in certain pathological states. Collagen degradation is primarily mediated by the specific enzyme collagenase, which has been found to exist in various tissues including the myocardium. In this report we describe a method that detects collagenase activity in sections of cardiac tissue. This method is on the basis of degradation of collagen by collagenase on one hand and the visualization of disrupted collagen fibers by immunofluorescence on the other. Frozen rat heart sections were incubated under optimal conditions for collagenase activity (37 degrees C in the presence of 0.1 M calcium at pH 7.4) for 24 h and 48 h. Subsequently, immunofluorescence staining with antibody to type I collagen was performed and the collagenous structures were visualized by immunofluorescence light microscopy. As control, untreated rat heart sections and sections incubated in the absence of calcium were similarly treated with antibody. After the 24 h of incubation, we found no change in the structural integrity of collagen fibers. Marked disruption of the type I collagen fibers was observed 48 h after incubation. No evidence of collagen fiber disruption was found in control sections. Experiments with exogenous collagenase resulted in similar collagen fiber disruption in the frozen rat heart sections. We conclude that the disruption of collagen type I fibers after 48 h of incubation, under optimal conditions for collagenolytic digestion, is the result of collagen degradation by intrinsic collagenase of the myocardium.