Type X collagen contains two cleavage sites for a vertebrate collagenase

Type X collagen was cleaved at two sites by a purified human skin collagenase. Two experimental approaches were used to identify the location of the cleavage sites. First, native type X collagen was digested with the enzyme, and the rotary-shadowed products were visualized in the electron microscope. The major collagenase fragment of type X contained the epitope recognized by a monoclonal antibody (X-AC9). The antibody was used as a point of reference to locate the position of the cleavage fragment within the native molecule. Second, the digestion of radiolabeled type X collagen substrates was analyzed by gel electrophoresis. The complete cleavage of type X generated three products with 32-, 18-, and 9-kDa chains. The 32-kDa peptides were present in a triple-helical conformation and demonstrated a midpoint denaturation temperature of 43 degrees C in CD experiments. The 18-kDa peptide contained the tyrosine-rich globular domain of the molecule. The 9-kDa peptide was derived from the triple-helical end of the native molecule. Type X collagen was cleaved more rapidly by the vertebrate collagenase than was type II collagen in in vitro solution studies.     

Comparison of vertebrate collagenase and gelatinase using a new fluorogenic substrate peptide

A fluorogenic substrate for vertebrate collagenase and gelatinase, Dnp-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2, was designed using structure-activity data obtained from studies with synthetic inhibitors and other peptide substrates of collagenase. Tryptophan fluorescence was efficiently quenched by the NH2-terminal dinitrophenyl group, presumably through resonance energy transfer. Increased fluorescence accompanied hydrolysis of the peptide by collagenase or gelatinase purified from culture medium of porcine synovial membranes or alkali-treated rabbit corneas. Amino acid analysis of the two product peptides showed that collagenase and gelatinase cleaved at the Gly-Leu bond. The peptide was an efficient substrate for both enzymes, with kcat/Km values of 5.4 microM-1 h-1 and 440 microM-1 h-1 (37 degrees C, pH 7.7) for collagenase and gelatinase, respectively. Under the same conditions, collagenase gave kcat/Km of about 46 microM-1 h-1 for type I collagen from calf skin. Since both enzymes exhibited similar Km values for the synthetic substrate (3 and 7 microM, respectively), the higher catalytic efficiency of gelatinase reflects predominantly an increase in kcat. Both enzymes were inhibited by HSCH2(R,S)CH[CH2CH(CH3)2]CO-L-Phe-L-Ala-NH2 in this assay (50% inhibition at 20 nM and less than 1 nM for collagenase and gelatinase, respectively). Soluble type I collagen was a competitive inhibitor of peptide hydrolysis by collagenase (KI = 0.8 microM) and exhibited mixed inhibition of gelatinase (KI = 0.3 microM).     

A convenient fluorescent assay for vertebrate collagenases

A versatile, convenient assay for vertebrate collagenases has been developed using the fluorescent peptide substrate dansyl-Pro-Gln-Gly-Ile-Ala-Gly-D-Arg. This sequence resembles that of collagen at the site of cleavage but includes modifications designed to eliminate nonspecific hydrolysis by contaminating peptidases. Both human skin fibroblast and bovine corneal cell collagenases cleave the substrate specifically at the Gly-Ile bond. Plasmin, thrombin, trypsin, alpha-chymotrypsin, carboxypeptidase B, and bacterial collagenase do not cleave the substrate. Elastase and angiotensin converting enzyme display 20- and 400-fold less activity than the vertebrate collagenases, respectively, and cleave the peptide at different positions. The assay is performed by incubating a 5- to 25-microliters aliquot of trypsin-activated sample with an equal volume of 2 mM substrate overnight at 33 degrees C and pH 7.5. Thin-layer chromatography then separates the fluorescent product from the substrate in less than 20 min and allows the detection of subnanogram levels of collagenase. The assay is applicable to the screening of large numbers of samples under different conditions of pH and ionic strength and is readily adaptable for use in a variety of collagenase-dependent systems, such as assays for collagenase activating and/or inducing factors.     

Cleavage of bovine skin type III collagen by proteolytic enzymes. Relative resistance of the fibrillar form

We have studied the susceptibility of fibrils formed from fetal bovine skin type III collagen to proteolytic enzymes known to cleave within the helical portion of the molecule (vertebrate and microbial collagenase, polymorphonuclear elastase, trypsin, thermolysin) and to two general proteases of broad specificity (plasmin, Pronase). Fibrils reconstituted from neutral salt solutions, at 35 degrees C, were highly resistant to nonspecific proteolysis by general proteases such as polymorphonuclear elastase, trypsin, and thermolysin but were rapidly dissolved by bacterial and vertebrate collagenases at rates of 12-45 mol X mol-1 X h-1. In solution, type III collagen was readily cleaved by each of the proteases (with the exception of plasmin), as well as by the true collagenases, although at different rates. Turnover numbers determined by viscometry at 35 degrees C were: human collagenase, approximately equal to 1500 h-1; microbial (clostridial) collagenase, approximately equal to 100 h-1; and general proteases, 23-52 h-1. In addition it was shown that pronase cleaves type III collagen in solution at 22 degrees C by attacking the same Arg-Gly bond in the alpha 1(III) chain as trypsin. However, like other proteases, Pronase was rather ineffective against fibrillar forms of type III collagen. It was also shown that transition of type III collagen as well as type I collagen to the fibrillar form resulted in a significant gain of triple helical thermostability as evidenced by a 6.8 degrees C increase in denaturation temperature (Tm = 40.2 degrees C in solution; Tm = 47.0 degrees C in fibrils).     

Collagenase of human skin basal cell epithelioma.

Collagenase of human basal cell epithelioma was purified by sequential ammonium sulfate precipitation, Sephadex gel filtration and affinity chromatography on collagen-polyacrylamide gel. The collagenase, when partially purified, was found to have an approximate molecular weight of 50,000. The purified enzyme contained no caseinolytic activity. On polyacrylamide gel electrophoresis, the purified enzyme gave a single protein band. The purified collagenase cleaved native acid-soluble guinea pig skin collagen at 37 degrees C with a pH optimum of 8. The enzyme was inhibited by EDTA, cysteine, and human serum but not by soybean trypsin inhibitor. Heparin did not stimulate the enzyme activity. Purified collagenase reduced the specific viscosity of native acid-soluble guinea pig skin collagen to 50 per cent of its original value at 27 degrees C. Polyacrylamide gel disc electrophoresis of the reaction products showed bands corresponding to alphaA, betaA, and alphaB fragments. Electron microscopic examination of SLS aggregates of the reaction products showed that the cleavage site by the enzyme was at a point 75 per cent from the "A" end (TCA75) and 25 per cent from the "B" end (TCB25) of the collagen molecule.     

Detection of collagenase activity in oral bacteria

Collagenolytic activity of 12 species of oral bacteria was assessed using two methods of detection. Except for two species, all bacterial strains tested were capable of degrading at least one general protein substrate. Results of collagenolytic activity in a growth assay indicate that Bacteroides gingivalis is the only bacterium capable of degrading collagen when the substrate is sterilized using ethylene oxide. However, if the substrate is sterilized by autoclaving, in the presence or absence of the growth medium, other bacterial species could be shown to be collagenolytic. Collagenolytic activity was also demonstrated when whole or broken cells were used in a [14C]collagen assay. Results from this assay and from inhibition studies indicate that collagenolytic activity can either be the result of the combined activities of both a specific collagenase and nonspecific proteases (B. gingivalis) or nonspecific proteases only (other strains in this study), although in the latter case, the time taken to hydrolyze collagen can be 10 times longer than with a specific collagenase.     

A rapid assay method of collagenase activity using 14C-labeled soluble collagen as substrate.

A rapid assay method for vertebrate collagenase (EC 3.4.24.3) activity has been developed using 14C-labeled soluble collagen as substrate. The method is based on the incubation of collagen with enzyme in the presence of glucose to prevent collagen fibril formation followed by selective extraction of the enzyme digestion products into dioxane at a final concentration of 50%. The rate of reaction was about 10 times higher than that obtained by the conventional method using reconstituted collagen fibrils as substrate and the relationship between enzyme activity and concentration was linear over a wider range. When the method was applied to the assay of human granulocyte collagenase, the results showed good correlation with those obtained by the conventional gel method.     

Purification and characterization of a collagenase extracted from rabbit tumours.

A collagenase was purified from homogenates of V2 ascites-cell carcinoma growing in rabbit muscle. (NH4)2SO4 precipitation, ion-exchange and gel-filtration chromatography, and affinity chromatography (by using the CB7 CNBr) cleavage fragment of alpha 1(I) collagen linked to agarose) gave a 268000-fold purification and a sevenfold increase in total enzyme units recovered. The specific activity, defined as mumol of collagen in solution cleaved/h per mg of enzyme at 35 degrees C, WAS 1.74.2. The collagenase had a broad pH optimum from pH7.0 to 9.5, and a mol.wt. of between 33000 and 35000. It was inhibited by dithiothreitol, L-cysteine, D-penicillamine, EDTA and 1,10-phenanthroline, and by both rabbit and human serum. 3. Removal of cations by a chelating resin (Chelex 100) produced as inactive enzyme that could be reactiviated by the addition of Ca2+ ions at concentrations as low as 1muM. Other bivalent cations were not effective. 4. The purified collagenase cleaved peptides alpha2 and alpha1-CB7 (denatured polypeptides of collagen) at 37 degrees C at one site only. [alpha1 (I)]2alpha2 and [alpha1(III)]3 collagens in solution were cleaved at the same site approximately five times more rapidly than [alpha1 (II)]3. 5. An inhibitor of the enzyme in the tumour extracts, which was dissociable from the enzyme at the (NH4) 2SO4 precipitation step of purification, had a mol. wt. of between 40000 and 50000 but was distinct from the alpha1 trypsin inhibitor. 6. Studies with zonal density-gradient centrifugation suggested that the enzyme was bound to fibrillar substrate (collagen) extracellularly, but that it was not associated with enzymes originating in cell mitochondria, microsomal preparations or lysosomes.     

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.     

Purification and properties of a specific collagenase from rabbit synovial fibroblasts.

1. A specific collagenase from the culture medium of rabbit synovial fibroblasts was purified by gel filtration and ion-exchange chromatography. 2. The enzyme was homogenous on polyacrylamide-gel electrophoresis and showed only traces of contaminants when tested in gels with a non-specific antiserum. 3. The rabbit fibroblast collagenase could hydrolyse collagen both in solution and in fibrillar form. Viscometry showed that at 35 degrees C the purified enzyme could hydrolyse greater than 50 nmol of collagen/min per mg of enzyme. 4. The purified collagenase cleaved collagen in solution at either 24 degrees or 35 degrees C into the characteristic 1/4 and 3/4-length fragments. However, as compared with the impure enzyme, the purified enzyme at 35 degrees C had a much decreased capacity to further degrade the initial specific cleavage products. 5. The specific rabbit collagenase had a mol. wt. of approx. 32000 as estimated by sodium dodecyl sulphate-polyacrylamide-gel electrophoresis, and 35000 by gel filtration.     

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.     

An internally quenched fluorescent substrate for collagenase

A synthetic collagenase substrate containing the internal peptide sequence--Gly-Gly-Pro-Leu-Gly-Pro-Pro-Gly-Pro--has been synthesized, with an N-terminus 4-((4-(dimethylamino)phenyl)azo)-benzoyl (DABCYL) group and C-terminus 5-[2-(acetamido)ethylamino] naphthalene-1-sulfonic acid (AEDANS) moiety resulting in internal quenching of AEDANS fluorescence. Peptide bond hydrolysis results in a large increase in fluorescence at 490 nm upon excitation at 336 nm. The substrate is cleaved exclusively by Clostridium histolyticum collagenase and is completely resistant to attack by proteases like thermolysin, proteinase K, and trypsin. K(m) and V(max) values for substrate hydrolysis by collagenase have been determined, establishing the peptide as one of the best binding substrates for the enzyme. MALDI mass spectrometry using a derivative of the substrate establishes that the sites of cleavage lie within the collagen like domain. The CD spectrum of an analog peptide lacking the donor and acceptor groups reveals spectral features that are reminiscent of weak polyproline structures.     

The gelatinolytic activity of human skin fibroblast collagenase

The gelatinolytic activity of human skin fibroblast collagenase was examined on denatured collagen types I-V. All denatured substrates were cleaved, including types IV and V, which are resistant to collagenase in native form. Interestingly, the earliest major cleavage in denatured collagen types I-III occurred at a 3/4-1/4 locus, resulting in products electrophoretically identical with TCA and TCB fragments of mammalian collagenase action on these native collagens. However, in the denatured substrates, multiple additional proteolytic cleavages followed. The propensity for cleavage at a 3/4-1/4 site in denatured collagen, where sequence is the major specifier of enzymatic action, would seem to indicate that the most favorable amino acid sequence of gamma chains for catalysis is located in this region. The peptide bond specificity of human fibroblast collagenase on gelatin was examined by amino acid sequencing of extensively cleaved denatured type I collagen. Analysis of the NH2-terminal amino acid residues from the resultant gelatin peptides showed sequences of "-H2N-Ile-Y-Gly" and "H2N-Leu-Y-Gly" only (where Y indicates that any amino acid can be found in that position), indicating that Gly-Ile and Gly-Leu bonds are the only sites of collagenase cleavage in this substrate. Whereas the gamma1 chains of denatured collagen types I-III were cleaved at similar rates, fibroblast collagenase was a much better gamma2-gelatinase than gamm1-gelatinase on denatured type 1 collagen. This preference for the cleavage of gamma2(I) was the result of both a higher kcat (750 versus 230 h-1) and lower Km (3.7 versus 7.0 microM) than for a gamma1(1), resulting in an overall selectivity (kcat/Km) of greater than 6-fold. Compared to such kinetic parameters on native collagen, these values indicate that gelatinolysis is somewhat slower than collagenolysis.     

Substrate specificity of the collagenolytic serine protease from Uca pugilator: studies with collagenous substrates

The collagenolytic protease from Uca pugilator was studied with respect to its catalytic properties on collagen types I-V. The crab protease degraded all five collagen types, producing multiple cleavages in the triple helix of each native collagen at 25 degrees C. The major early cleavage in the alpha 1 polypeptide chain of collagen types I-III occurred at a 3/4:1/4 locus, resulting in fragments electrophoretically similar to the TCA and TCB products of mammalian collagenase action. Interestingly, a propensity toward this same cleavage was observed even following thermal denaturation of the substrates. The ability of the crab protease to degrade all native collagen types and to catalyze cleavages at multiple loci in the triple helix distinguishes its action from that of mammalian collagenases. The collagenolytic activity of the crab protease was also examined on fibrillar collagen and compared to that of human skin fibroblast collagenase. Enzyme concentrations of fibroblast collagenase which resulted in the saturation of available substrate sites failed to show such an effect in the case of the crab protease. Binding studies of the crab protease to fibrillar collagen likewise indicated substantially reduced levels of enzyme binding in comparison to fibroblast collagenase. These data suggest that the affinity of the crab protease for native collagen is considerably less than the affinity of mammalian collagenase for this substrate.     

Biosynthetic and structural properties of endothelial cell type VIII collagen

A highly unusual endothelial cell collagen (Sage, H., Pritzl, P., and Bornstein, P., (1980) Biochemistry 19, 5747-5755) has been characterized in greater detail. Pulse-chase experiments with bovine aortic endothelial cells revealed two nondisulfide-bonded collagens, of apparent chain Mr = 177,000 and 125,000, with an estimated synthesis and secretion time of 75 min. Stepwise, quantitative processing to stable lower molecular weight forms as described for type I procollagen was not observed. Endothelial collagen was secreted over a temperature range of 24-37 degrees C and, prior to heat denaturation, did not display affinity for a gelatin-binding fragment of fibronectin coupled to Sepharose. The presence of a pepsin-resistant domain (Mr = 50,000) in both the soluble and cell layer-associated forms of this protein was shown by ion exchange chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Endothelial collagen was cleaved by vertebrate collagenase into several discrete fragments that differed in molecular weight from the characteristic alpha A and alpha B fragments generated from the interstitial collagens. Nontriple helical domains corresponding to the NH2- and COOH-terminal propeptides of other procollagen types were not found after incubation of endothelial collagen with bacterial collagenase. Additional evidence for the lack of extended noncollagenous sequences was provided by studies with mast cell proteases, which convert native procollagen to collagen but are unreactive toward native interstitial collagens. Endothelial collagen was not cleaved by these enzymes at 37 degrees C, but, as observed for interstitial collagen alpha chains, required prior heating at elevated temperatures for cleavage to occur. In view of this unique set of structural characteristics, and a distribution that is not restricted to the endothelium, we have designated this protein as type VIII collagen.     

Characterization of extracellular alkaline proteases and collagenase induction in Vibrio alginolyticus

The number and approximate molecular weights of extracellular alkaline proteases produced by Vibrio alginolyticus were determined by gelatin-PAGE. Three major bands of protease activity with apparent molecular weights of approximately 28 000, 22 500 and 19 500 (proteases 1, 2 and 3, respectively) and two minor bands of protease activity with apparent molecular weights of approximately 15 500 and 14 500 (proteases 4 and 5, respectively) were obtained after gelatin-PAGE. The activities of the five proteases were inhibited by serine protease inhibitors but their activities were not affected by inhibitors of trypsin-like enzymes. Histidine, which inhibited V. alginolyticus collagenase, did not inhibit the activities of the alkaline serine proteases. The production of protease 1, however, was enhanced by histidine. Protease 1 production was also affected by temperature and production was depressed at 37 degrees C. Gelatin-PAGE of a commercial V. alginolyticus collagenase preparation revealed four bands of activity which were identified as collagenases with apparent molecular weights of approximately 45 000, 38 500, 33 500 and 31 000. The collagenase preparation was contaminated with two serine proteases. The release of [3H]proline from collagen matrices produced by smooth muscle cells was shown to be a sensitive assay for bacterial collagenases and was used to show that V. alginolyticus produced a basal constitutive level of extracellular collagenase. The constitutive levels of collagenase were affected by aeration.     

A rapid sensitive collagenase assay.

A rapid, sensitive collagenase assay has been developed using¹⁴C-acetylated collagen as a substrate. Acid-soluble calfskin collagen was labeled with [1-¹⁴C]acetic anhydride at pH 8. The acetylated collagen had a specific activity of 6.25 × 10⁵ dpm/mg protein. Collagen was not denatured as evidenced by its resistance to nonspecific proteolysis and sensitivity to bacterial collagenase. Polyacrylamide gel electrophoresis of the acetylated protein showed that the radioactivity was present in the three bands corresponding to the α, β, and γ components of collagen. The rate of release of ¹⁴C from labeled collagen by Clostridium histolyticum collagenase was proportional to enzyme and substrate concentration.     

Specific cleavage of human type III collagen by human polymorphonuclear leukocyte elastase

Purified polymorphonuclear leukocyte elastase degraded native human liver type III collagen at 27 degrees C by making a cleavage through the triple helix. The enzyme had no effect on human type I collagen. The reaction was inhibited by phenylmethanesulfonyl fluoride (PhCH2SO2F) but not by EDTA. The collagen reaction products were identical with those generated by human rheumatoid synovial collagenase when analyzed by polyacrylamide gel electrophoresis and gel filtration. NH2-trminal sequence analysis indicated that the enzyme cleaved at an isoleucyl-threonyl bond located 4 residues on the carboxyl side of the established cleavage site for animal collagenases. Therefore, it is likely that in pathologic states, type III collagen can be selectively depleted from the matrix by this enzyme.     

Cleavage of type VII collagen by interstitial collagenase and type IV collagenase (gelatinase) derived from human skin

Type VII collagen is the major structural protein of anchoring fibrils, which are believed to be critical for epidermal-dermal adhesion in the basement membrane zone of the skin. To elucidate possible mechanisms for the turnover of this protein, we examined the capacities of two proteases, human skin collagenase, which degrades interstitial collagens, and a protease with gelatinolytic and type IV collagenase activities, to cleave type VII collagen. At temperatures below the denaturation temperature, pepsin cleaves type VII collagen into products of approximately 95 and approximately 75 kDa. Human skin collagenase cleaved type VII collagen into two stable fragments of approximately 83 and approximately 80 kDa, and the type IV collagenase (gelatinase) produced a broad band of approximately 80 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cleavage of type VII collagen was linear with time and enzyme concentration for both enzymes. Although the Km values were similar for both enzymes, the catalytic rate of cleavage by type IV collagenase is much faster than by interstitial collagenase, and shows a greater rate of increase with increasing temperature. Sequence analysis of the cleavage products from both enzymes showed typical collagenous sequences, indicating a relaxation in the helical part of the type VII collagen molecule at physiological temperature which makes it susceptible to gelatinolytic degradation. Interstitial collagenase from both normal skin cells and cells from patients with recessive dystrophic epidermolysis bullosa, a severe hereditary blistering disease in which both an anchoring fibril defect and excessive production of collagenase can be observed, produced identical cleavage products from type VII collagen. These data suggest a pathophysiological link between increased enzyme levels and the observed decrease or absence of anchoring fibrils.     

Cleavage of Type II and III collagens with mammalian collagenase: site of cleavage and primary structure at the NH2-terminal portion of the smaller fragment released from both collagens.

Collagenase cleavage of human Type II and III collagens has been studied using a highly purified preparation of rabbit tumor collagenase. Progress of the reactions in solution was followed by viscometry and the results indicated that under the conditions employed Type III collagen molecules were cleaved at approximately five times the rate of Type II molecules. Cleavage products of the reactions were isolated in denatured form by agarose molecular sieve chromatography. The molecular weights and amino acid compositions of the products demonstrated that Type II and III molecules had been cleaved at the characteristic three-quarter, one-quarter locus, giving rise to a large fragment derived from the NH2-terminal portion of the molecule and a smaller fragment representing the COOH-terminal region. The amino acid sequence at the NH2-terminal portion of the smaller fragment derived from Type II collagen was determined to be Ile-Ala-Gly-Gln-Arg, and the corresponding region from Type III collagen was found to have the sequence Leu-Ala Gly-Leu-Arg. These sequences for alpha1(II) and alpha1(III) chains adjacent to the site of collagenase cleavage along with previous data for alpha1(I) and alpha2 chains indicate that the minimum specific sequence required for collagenase cleavage is Gly-Ile-Ala or Gly-Leu-Ala. Inspection of the available sequence data for collagen alpha chains indicates that the latter sequences are found in at least three additional locations at which collagenase cleavage does not occur. Each of the sequences which are apparently not substrates for collagenase, however, are followed by a Gly-X-Hyp sequence. We suggest, then, that a minimum of five residues in collagen alpha chains COOH-terminal to the cleavage site comprise the substrate recognition site.