Contributions of the MMP-2 collagen binding domain to gelatin cleavage. Substrate binding via the collagen binding domain is required for hydrolysis of gelatin but not short peptides.

Two matrix metalloproteinases, MMP-2 and MMP-9, contain each three fibronectin type II-like modules, which form their collagen binding domains (CBDs). The contributions of CBD substrate interactions to the catalytic activities of these gelatinases have attracted special interest. Recombinant (r) CBDs retain collagen binding properties and deletions of CBDs in these MMPs reduce activities on collagen and elastin. We have characterized further the requirement of the CBD for MMP-2 cleavage of gelatin. The analyses used intact rMMP-2 and rCBD to eliminate any confounding effects that might result from structural perturbations in rMMP-2 induced by deletion of the approximately 20 kDa internal CBD. In protein-protein binding assays, 2% DMSO disrupted gelatin interactions of both rCBD and rMMP-2. At this concentration, DMSO also reduced the gelatinolytic activity by approximately 70%, pointing to a central role of CBD-substrate interactions during MMP-2 cleavage of gelatin. Subsequently, soluble rCBD was determined to competitively inhibit gelatin binding of unmodified rMMP-2 to gelatin by 73% and to reduce the MMP-2 degradation of gelatin by 70-80%. The residual gelatin cleavage that was not inhibited even by molar excess rCBD could be accounted for by degradation of short substrate molecules. Indeed, rCBD inhibited rMMP-2 cleavage of an 11 amino acid collagen-like peptide substrate (NFF-1) by less than 10%. These observations were confirmed with enzyme extracts from experimental tumors in mice. In the presence of rCBD, approximately 65% of the MMP-derived gelatinolytic activity was eliminated. Together, these results demonstrate that the CBD is absolutely required for MMP-2 cleavage of full-length collagen alpha-chains, but not for short protein fragments such as those generated by hydrolysis of gelatin.     

Human leucocyte neutral proteases, with special reference to collagen metabolism.

Three different types of neutral proteases related to collagen metabolism have been found in the granule fraction of human leucocytes from normal adults, using collagen, gelatin, and synthetic peptides as substrates. These are collagenase, an enzyme showing a potent hydrolytic activity against gelatin but little against native collagen, and one splitting the cross-links region of collagen. Their molecular weights were estimated to be about 75,000 150,000, and 25,000, respectively, by gel chromatography. The former two enzymes were inhibited by a alpha2-macroglobulin and ethylenediaminetetraacetate, but not by alpha1-proteinase inhibitor (alpha1-antitrypsin) or phenylmethylsulfonylfluoride, while the latter enzyme, associated in behavior with an enzyme hydrolyzing succinyl-(l-alanyl)3-p-nitroanilide, was inhibited by alpha1-proteinase inhibitor, alpha2-macroglobulin, and phenylmethylsulfonylfluoride, but not by ethylenediaminetetraacetate. A possible cooperative function of these enzymes in collagen catabolism is discussed.     

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.     

Determination of matrix metalloproteinase activity using biotinylated gelatin

Matrix metalloproteinases (MMPs) and, specifically, MMP-2 (gelatinase A) and MMP-9 (gelatinase B) are strongly associated with malignant progression and matrix remodeling. These enzymes are a subject of intensive studies involving screening of comprehensive chemical libraries of synthetic inhibitors. There is no simple method available for measurement of activity of gelatinases and related MMPs. Here, we report a simple, inexpensive, and highly sensitive assay for MMP activity. The assay performed in a 96-well microtiter plate format employs biotin-labeled gelatin (denatured collagen type I) as a substrate. Following the substrate cleavage, only the proteolytic fragments bearing biotin moieties are captured by streptavidin coated on the plastic surface and the captured fragments with at least two biotin molecules should be revealed by streptavidin conjugated with horseradish peroxidase. The frequency of lysine residues is low in collagen type I relative to the MMP cleavage sequences (PXGX). Accordingly, the majority of the cleavage products must be devoid of biotin or possess only one biotin group. Both of these types of fragments cannot be recognized by the horseradish peroxidase-streptavidin conjugate. Therefore, higher gelatinolytic activity is associated with lower signal in the assay. This 2-h assay allows identification of gelatinolytic activity of MMP-2 in concentrations as low as 0.16 ng/ml. The sensitivity of this ELISA-like assay is comparable to that of gelatin zymography, a method widely used to detect gelatinases. However, in contrast to zymography, the assay directly measures the enzymatic activity of MMP samples. The gelatinolytic activity assay permits efficient analyses and screening of the MMP inhibitor panels and allows quantitation of gelatinolytic activity of various MMPs in solution as well as on cell surfaces.     

胰蛋白酶消化法测定牛胶原蛋白三股螺旋结构的含量

本研究建立了一种测定胶原蛋白的三股螺旋结构含量的方法。该方法通过使用柱前衍生高效液相色谱(HPLC)法表征经胰蛋白酶酶解后胶原蛋白羟脯氨酸(Hyp)质量浓度的变化,进而对胶原蛋白的三股螺旋结构进行定量。探讨了不同的酶解时间(0~48h)、酶与底物的比例(1∶100、1∶50和1∶20)和温度(20、25、30、37℃)对明胶降解率的影响。获得了酶解的最佳条件——当胰蛋白酶与底物的比例为1∶50时,25℃酶解3h。使用该方法对明胶胶原蛋白混合液检测,结果表明,该方法能灵敏(RSD<10%)的测定胶原蛋白三股螺旋结构的含量。该方法不仅可用于生物组织研究领域,也可用于胶原蛋白食品、保健品和组织工程产品质量的评价。     

The presence of collagenase in collagen preparations.

The frequently observed instability of neutral salt solutions of native collagen extracted from various sources and partially purified by standard procedures has been studied by disc electrophoresis in polyacrylamide gel and by electron microscopic examination of segment long spacing crystallites. The phenomenon has revealed time and temperature dependency, pH optima near neutrality, and inhibition by sodium EDTA and serummin addition, collagen breakdown has been found to be quantitatively related to the state of aggregation of the substrate, being more marked in reconstituted collagen gels than in collagen in solutionma typical pattern of animal collagenase degradation of native collagen into two fragments designated as TC-A and TC-B has been observed under certain conditions. It is concluded that the degradation of native collagen in neutral salt solution is due to a specific collagenase, and that this enzyme probably remains bound to collagen throughout the process of extraction and partial purification. Experiments with gelatin suggest that, in addition to collagenase, a nonspecific proteolytic activity may also be present in collagen preparations.     

Purification and characterization of a trypsin-like serine proteinase from rat brain slices that degrades laminin and type IV collagen and stimulates protease-activated receptor-2

A trypsin-like serine proteinase was purified from the incubation medium of rat brain slices by gelatin zymography. The purification consisted of ammonium sulfate precipitation, benzamidine-Sepharose 6B affinity chromatography, and carboxymethyl-cellulose and gel filtration chromatographies. The gelatinolytic activity, identified at 22 kDa (P22) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions, was eluted as one active peak throughout the purification, and the final preparation gave a single protein peak on reverse-phase HPLC. Diisopropyl fluorophosphate, benzamidine, p-toluenesulfonyl-L-lysine chloromethyl ketone, and aprotinin completely inhibited the activity of P22, whereas phenanthroline, p-toluene-sulfonyl-L-phenylalanine chloromethyl ketone, and elastinal did not. P22 efficiently digested the extracellular matrix proteins laminin and type IV collagen. P22 produced an increase in intracellular Ca2+ concentration in A172 glioblastoma, which was desensitized through prior stimulation with protease-activated receptor-2 agonist peptide SLIGKV, indicating that P22 can stimulate protease-activated receptor-2. Rat brain penetration injury induced gelatinolytic activity in the lesioned area whose molecular size was consistent with that of P22. These results indicated that on incubation of rat brain slices, a trypsin-like serine proteinase was secreted into the medium that was capable of digesting extracellular matrix and stimulating protease-activated receptor-2. It is suggested that the gelatinolytic activity induced by brain injury might be that of P22.     

Interaction of pro-matrix metalloproteinase-9/proteoglycan heteromer with gelatin and collagen

Previously we have shown that THP-1 cells synthesize matrix metalloproteinase-9 (MMP-9) where a fraction of the enzyme is strongly linked to a proteoglycan (PG) core protein. In the present work we show that these pro-MMP-9.PG heteromers have different biochemical properties compared with the monomeric form of pro-MMP-9. In these heteromers, the fibronectin II-like domain in the catalytic site of the enzyme is hidden, and the fibronectin II-like-mediated binding to gelatin and collagen is prevented. However, a fraction of the pro-MMP-9.PG heteromers interacted with gelatin and collagen. This interaction was not through the chondroitin sulfate (CS) part of the PG molecule but, rather, through a region in the PG core protein, a new site induced by the interaction of pro-MMP-9 and the PG core protein, or a non-CS glycosaminoglycan part of the PG molecule. The interaction between pro-MMP-9.PG heteromers and gelatin was weaker than the interaction between pro-MMP-9 and gelatin. In contrast, collagen I bound to pro-MMP-9.PG heteromers and pro-MMP-9 with approximately the same affinity. Removal of CS chains from the PG part of the heteromers did not affect the binding to gelatin and collagen. Although the identity of the PG core protein is not known, this does not have any impact on the described biochemical properties of the heteromer or its pro-MMP-9 component. It is also shown that a small fraction of the PG, which is not a part of the pro-MMP-9.PG heteromer, can bind gelatin. As for the pro-MMP-9.PG heteromers, this was independent of the CS chains. The structure that mediates the binding of free PG to gelatin is different from the corresponding structure in the pro-MMP-9.PG heteromer, because they were eluted from gelatin-Sepharose columns under totally different conditions. Although only a small amount of pro-MMP-9.PG heteromer is formed, the heteromer may have fundamental physiological importance, because only catalytic amounts of the enzyme are required to digest physiological targets.     

Characterization of gelatinase from pig polymorphonuclear leucocytes. A metalloproteinase resembling tumour type IV collagenase.

The metalloproteinase 'gelatinase' stored in the granules of pig polymorphonuclear leucocytes has been purified in the latent form. The enzyme is secreted as an Mr 97,000 proenzyme that can be activated in the presence of 4-aminophenylmercuric acetate (APMA) by self-cleavage to generate lower-Mr species, of which an Mr 88,000 form was the most active. Trypsin-initiated activation generated different Mr gelatinases of much lower specific activity. Activation was slowed but not prevented by the presence of the tissue inhibitor of metalloproteinases, TIMP. The activated gelatinase formed a stable complex (Mr 144,000) with TIMP, in a Zn2+- and Ca2+-dependent manner, and complex formation was inhibited by the presence of the substrate gelatin. Similar to the human granulocyte gelatinase, the organomercurial-activated pig enzyme degraded gelatin and TCA and TCB fragments of type I collagen, as well as elastin and types IV and V collagen. The degradation of type IV collagen was shown, both by polyacrylamide-gel electrophoresis and by electron microscopic analysis, to generate 3/4 and 1/4 fragments as described for mouse tumour type IV collagenase. Furthermore, an antiserum raised to mouse type IV collagenase recognized the pig granulocyte gelatinase. An antiserum to the pig polymorphonuclear leucocyte gelatinase recognized other high-Mr gelatinases, including those from human granulocytes, pig monocytes and rabbit connective tissue cells, but not the Mr 72,000 enzyme from connective tissue cells. These data suggest that there are two distinct major forms of gelatinolytic activity that also cause specific cleavage of type IV collagen. These enzymes are associated with a wide variety of normal connective tissue and haemopoietic cells, as well as many tumour cells.     

The presence of collagenase in collagen preparations

The frequently observed instability of neutral salt solutions of native collagen extracted from various sources and partially purified by standard procedures has been studied by disc electrophoresis in polyacrylamide gel and by electron microscopic examination of segment long spacing crystallites. The phenomenon has revealed time and temperature dependency, pH optima near neutrality, and inhibition by sodium EDTA and serum. In addition, collagen breakdown has been found to be quantitatively related to the state of aggregation of the substrate, being more marked in reconstituted collagen gels than in collagen in solution. A typical pattern of animal collagenase degradation of native collagen into two fragments designated as TC^A and TC^B has been observed under certain conditions. It is concluded that the degradation of native collagen in neutral salt solution is due to a specific collagenase, and that this enzyme probably remains bound to collagen throughout the process of extraction and partial purification. Experiments with gelatin suggest that, in addition to collagenase, a nonspecific proteolytic activity may also be present in collagen preparations.     

Purification of an insect derived recombinant human ADAMTS-1 reveals novel gelatin (type I collagen) degrading activities

ADAMTS-1 (A Disintegrin And Metalloprotease with ThromboSpondin repeats) is a member of a family of secreted proteolytic enzymes with a complex modular structure. These enzymes are characterised by an N-terminal metalloproteinase domain, a disintegrin-like domain and a carboxyl terminal region containing variable numbers of a repeat sequence with homology to thrombospondin-1. The expression of the gene for ADAMTS-1 has been associated with inflammation, ovulation, angiogenesis, cellular proliferation and bone formation. ADAMTS-1 can proteolytically process large proteoglycans indicating a potential role in extracellular matrix turnover. In this study, we have tested ADAMTS-1 activity in gelatin zymogram assays. Since previous data demonstrate that ADAMTS-1 is a matrix metalloproteinase (MMP) substrate and is highly unstable in conditioned medium from eukaryotic cell types, we created an insect cell line expressing human ADAMTS-1. We isolated an epitope tagged full-length recombinant ADAMTS-1 from serum free insect cell conditioned medium. The purified protein had aggrecanase activity and appears as two major bands on the silver stained SDS-PAGE corresponding well to a pro-domain on form of 115 kDa and a pro-domain off form of 90 kDa. Using denatured type I collagen in zymographic analysis we demonstrate that ADAMTS-1 has a previously unreported gelatinolytic activity. Also, we notice that processing of its C-terminal region by an apparently autocatalytic process reveals a 27 kDa species with gelatinolytic activity. Furthermore, we show that MMP2 but not MMP13 remove ADAMTS-1 specific gelatin zymopraphic zones.     

Cathepsin B1. A lysosomal enzyme that degrades native collagen

1. Experiments were made to determine whether the purified lysosomal proteinases, cathepsins B1 and D, degrade acid-soluble collagen in solution, reconstituted collagen fibrils, insoluble collagen or gelatin. 2. At acid pH values cathepsin B1 released (14)C-labelled peptides from collagen fibrils reconstituted at neutral pH from soluble collagen. The purified enzyme required activation by cysteine and EDTA and was inhibited by 4-chloromercuribenzoate, by the chloromethyl ketones derived from tosyl-lysine and acetyltetra-alanine and by human alpha(2)-macroglobulin. 3. Cathepsin B1 degraded collagen in solution, the pH optimum being pH4.5-5.0. The initial action was cleavage of the non-helical region containing the cross-link; this was seen as a decrease in viscosity with no change in optical rotation. The enzyme also attacked the helical region of collagen by a mechanism different from that of mammalian neutral collagenase. No discrete intermediate products of a specific size were observed in segment-long-spacing crystalloids (measured as native collagen molecules aligned with N-termini together along the long axis) or as separate peaks on gel filtration chromatography. This suggests that once an alpha-chain was attacked it was rapidly degraded to low-molecular-weight peptides. 4. Cathepsin B1 degraded insoluble collagen with a pH optimum below 4; this value is lower than that found for the soluble substrate, and a possible explanation is given. 5. The lysosomal carboxyl proteinase, cathepsin D, had no action on collagen or gelatin at pH3.0. Neither cathepsin B1 nor D cleaved Pz-Pro-Leu-Gly-Pro-d-Arg. 6. Cathepsin B1 activity was shown to be essential for the degradation of collagen by lysosomal extracts. 7. Cathepsin B1 may provide an alternative route for collagen breakdown in physiological and pathological situations.     

EDTA/gelatin zymography method to identify C1s versus activated MMP‐9 in plasma and immune complexes of patients with systemic lupus erythematosus

Gelatin zymography analysis is a sensitive method and commonly used to characterize and quantify the presence of the gelatinases (MMP‐2 and MMP‐9) in biological samples. In human plasma samples from healthy controls and systemic lupus erythematosus (SLE) patients, we observed a gelatinolytic molecule at 80 kDa, suggestive for activated human MMP‐9. However, by developing and using the EDTA/gelatin zymography method and after purification of the 80 kDa entity, we proved that this molecule was the C1s subunit of the complement system. The zymolytic capacity of C1s was validated and found to be enhanced, in the absence of calcium and in the presence of EDTA. Our findings indicate that for correct identification of gelatinolytic proteins in complex biological samples the use of EDTA/gelatin zymography for enzyme development is advised. In addition, by quantification of EDTA/gelatin zymography analysis and ELISA, we observed that the levels of C1s were higher in plasma and immune complexes of SLE patients than of healthy individuals. Therefore, our data imply that C1s may become a marker for the diagnosis of SLE.     

Recombinant collagen for animal product-free dextran microcarriers

Manufacturers of vaccines and other biologicals are under increasing pressure from regulatory agencies to develop production methods that are completely animal-component-free. In order to comply with this demand, alternative cell culture substrates to those now on the market, primarily collagen or gelatin, must be found. In this paper, we have tested a number of possible substitutes including recombinant collagen, a 100-kDa recombinant gelatin fragment and a peptide derived from a cell-binding region of type I collagen. The small 15-amino acid peptide did not support attachment of human fibroblasts in monolayer culture. The 100-kDa gelatin fragment supported cell attachment in monolayer culture, but was significantly less active than intact porcine gelatin. Recombinant type I collagen was as successful in promoting cell attachment as native collagen, and both were more effective than porcine gelatin. Based on these data, dextran microspheres were treated with the same attachment proteins—porcine gelatin, native collagen, or recombinant collagen. The same trends were observed as in monolayer culture. Concentrations of the recombinant collagen (as well as native collagen) supported cell attachment on dextran microspheres at concentrations as low as 0.01 μg/cm². Treatment of the dextran with a low level of polyethylenimine, a cationic moiety, further enhanced attachment when used in conjunction with the low concentration of recombinant collagen. Where there was increased cell attachment, increased proliferation followed. We are confident, based on these findings, that a fully recombinant substitute could replace gelatin in current microcarrier preparations without losing the cell growth benefits provided by the native protein.     

Characterization of the distinct collagen binding, helicase and cleavage mechanisms of matrix metalloproteinase 2 and 14 (gelatinase A and MT1-MMP): the differential roles of the MMP hemopexin c domains and the MMP-2 fibronectin type II modules in collagen triple helicase activities

Matrix metalloproteinase-2 (MMP-2, gelatinase A) and membrane type (MT)1-MMP (MMP-14) are cooperative dynamic components of a cell surface proteolytic axis involved in regulating the cellular signaling environment and pericellular collagen homeostasis. Although MT1-MMP exhibits type I collagenolytic but poor gelatinolytic activities, MMP-2 is a potent gelatinase with weak type I collagenolytic behavior. Recombinant linker/hemopexin C domain (LCD) of MT1-MMP binds native type I collagen, blocks MT1-MMP collagenolytic activity in trans, and by circular dichroism spectroscopy, induces localized structural perturbation in the collagen. These changes were reflected by enhanced cleavage of the MT1-LCD-bound collagen by the collagenases MMP-1 and MMP-8 but not by trypsin or MMP-7. Thus, the MT1-LCD alone can initiate triple helicase activity. In contrast, the native and denatured collagen binding properties of MMP-2 reside in the fibronectin type II modules, accordingly termed the collagen binding domain (CBD). Recombinant CBD (but not the MMP-2 LCD) also changed the circular dichroism spectra leading to increased MMP-1 and -8 cleavage of native collagen. However, recombinant CBD reduced gelatin and collagen cleavage by MMP-2 in trans as did CBD23, which comprises the second and third fibronectin type II modules, but not the CBD23 mutant W316A/W374A, which neither binds gelatin nor collagen. This indicates that MMP-2 and MT1-MMP bind collagen at a different site than MMP-1 and MMP-8. Thus, MMP-2 utilizes the CBD in cis for collagen binding and triple helicase activity, which compensates for the lack of collagen binding by the MMP-2 LCD. Hence, the MMP family has evolved two distinct mechanisms for collagen triple helicase activity using two structurally distinct domains, with triple helicase activity occurring independent of alpha-chain hydrolysis.     

A critical role for the membrane-type 1 matrix metalloproteinase in collagen phagocytosis

Degradation of collagen is important for the physiological remodeling of connective tissues during growth and development as well as in wound healing, inflammatory diseases, and cancer cell invasion. In remodeling adult tissues, degradation of collagen occurs primarily through a phagocytic pathway. However, although various steps in the phagocytic pathway have been characterized, the enzyme required to initially fragment collagen fibrils for subsequent phagocytosis has not been identified. We have used laser confocal microscopy, transmission electron microscopy, and biochemical assays to show that human fibroblasts initiate degradation of collagen through the collagenase activity of the membrane-bound metalloproteinase MT1-MMP. Degradation of natural and reconstituted collagen substrates correlated with the expression of MT1-MMP, which was localized at sites of collagen cleavage at the surface of the cells and also within the cells, whereas collagen degradation was abrogated when MT1-MMP expression was blocked by small interfering RNA treatment. In contrast to MT1-MMP, the gelatinolytic activity of MMP-2 was not required for collagen phagocytosis. These studies demonstrate a pivotal role of catalytically active MT1-MMP in preparing collagen fibrils for phagocytic degradation.     

Collagen-fibronectin interactions in normal and rous sarcoma virus-transformed avian tendon cells: Possible mechanisms for increased extracellular matrix turnover after transformation

Summary Using gelatin, casein, and fibronectin as substrates and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), we have identified protein-degrading enzymes in both normal and Rous sarcoma virus-transformed primary avian tendon cells. Although there are some consistent differences in the profile of the gelatinolytic activities (mainly metalloproteinases) between normal and transformed cells, the amounts of fibronectin-degrading activities seem to be comparable. In vitro studies reported here demonstrate that the degradation of fibronectin is partially and specifically inhibited by gelatin and collagen. We therefore propose that the abundant collagen present in normal tendon cells protects fibronectin against degradation. Conversely, in transformed cells, where collagen levels are drastically reduced, fibronectin may be more accessible to degradation. Thus differences in the steady-state levels of fibronectin on normal and transformed cells may be, at least in part, a consequence of changes in collagen levels. This work was supported by the Office of Health and Environmental Research, Office of Energy Research, U.S. Department of Energy, Washington, D.C., under contracts DE-AC03-76-SF00098 and DE-AC03-76-SF01012.     

The interaction of tumour-localizing porphyrins with collagen, elastin, gelatin, fibrin and fibrinogen

We have already reported in Balb C mouse transplantable mammary carcinoma, that uroporphyrin I and III are superior as tumour localizers when compared to hematoporphyrin derivative and a derivative thereof, photofrin II. This study compares the binding of porphyrins to proteins which may be found in tumour cells or stroma to investigate whether there is a common binding determinant. Coproporphyrin III and deuteroporphyrin IX which are non-tumour localizing porphyrins, were also part of the comparative study. The interaction of these porphyrins with acid soluble collagen and acid insoluble collagen, elastin, and fibrin was evaluated, and the binding of uroporphyrin isomers I and III and deuteroporphyrin IX to gelatin and fibrinogen, was also determined. The results suggest that collagen, especially the acid soluble form, and gelatin preferentially bind the four porphyrins which localize in mammary carcinoma tissue. The well reported observations that malignant epithelial cells, including breast cancer, produce collagen and contain a rate-limiting enzyme in collagen biosynthesis would support the notion that de novo synthesis of this protein may in part govern the tumour uptake and retention of porphyrins. Elastin, fibrinogen and fibrin showed non-discriminant binding to the porphyrins under study.     

Identification of a novel gelatinolytic metalloproteinase (GMP) in the body wall of sea cucumber (Stichopus japonicus) and its involvement in collagen degradation

Body wall that mainly consists of collagen and polysaccharides is the edible part of sea cucumber and is easy to go autolysis, while the proteinase(s) responsible for autolysis remains unclear. In the present study, a gelatinolytic metalloproteinase (GMP) from the body wall of sea cucumber Stichopus japonicus was purified to homogeneity by a combination of ammonium sulfate fractionation and chromatographic steps including DEAE-Sephacel, Sephacryl S-200, Q-Sepharose and Phenyl-Sepharose. The molecular mass of GMP as estimated by SDS-PAGE and gelatin zymography was 45 kDa. The enzyme revealed high activity at a slightly alkaline pH range (8.0–9.0) and the optimal temperature was at 40–45 °C. Metalloproteinase inhibitors, EDTA, EGTA, and 1,10-phenanthroline, almost completely suppressed the activity, whereas other proteinase inhibitors did not show any effect. Peptide mass fingerprinting of the enzyme obtained 3 peptide fragments with a total of 58 amino acid residues, which was 91.4% identical to an alkaline metalloprotease from Pseudomonas fluorescens, strongly suggesting it is a metalloproteinase. Divalent metal ion Ca²⁺ is essential for its activity, indicating it is a calcium-dependent metalloproteinase. Furthermore, GMP hydrolyzed collagen effectively at 37 °C and gradually even at 4 °C, suggesting its involvement in the autolysis of sea cucumber.     

Mg2+ mediates the cell-substratum interaction of Arg-Gly-Asp-dependent HeLa cell collagen receptors

Three HeLa cell surface collagen receptors of apparent molecular mass 102/58, 87, and 38/33 kDa were eluted from gelatin-Sepharose with salt gradients or Arg-Gly-Asp-containing peptides. To understand how the collagen receptors are involved in HeLa cell spreading on collagen we investigated the effects of divalent cations and Arg-Gly-Asp-containing peptides on adhesion to gelatin, since HeLa cells behave similarly on both native type I collagen and gelatin substrata and also whether Arg-Gly-Asp-containing substrata would substitute for gelatin in facilitating cell spreading. Gly-Arg-Gly-Asp-Ser-containing peptides in solution inhibited HeLa cell spreading onto gelatin and promoted only partial HeLa cell spreading when bound to tissue culture plastic. Both partial spreading of HeLa cells on the Gly-Arg-Gly-Asp-Ser substratum and full spreading on gelatin was dependent on Mg2+, but not on Ca2+. Binding of the 102/58-, 87-, and 38/33-kDa collagen receptors to gelatin-Sepharose was increased fourfold in the presence of Mg2+, and subsequent elution of the collagen receptors and a 45-kDa collagen-binding protein not thought to be involved in HeLa cell spreading was achieved with EDTA. In contrast, affinity chromatography on Gly-Arg-Gly-Asp-Ser-Sepharose eluted predominantly the 45-kDa collagen-binding protein and the 38/33-kDa collagen receptor. In summary, the Mg2(+)-dependent interaction of the collagen receptors with the Arg-Gly-Asp sequence in collagen appears to be essential for the initial events in HeLa cell spreading but is not sufficient for full cell spreading.