Secretion of gelatinases and activation of gelatinase A (MMP-2) by human rheumatoid synovial fibroblasts.

In monolayer cultures human rheumatoid synovial fibroblasts (HRSF) secrete gelatinase A (MMP-2) and, unlike other human fibroblasts, to a minor extent also gelatinase B (MMP-9) as inactive proenzymes. In this regard HRSF resemble the fibrosarcoma cell line HT-1080. Unlike HT-1080, however, HRSF do not increase the secretion of MMP-9 in response to phorbol-12-myristate-13-acetate. This indicates that in HRSF the protein kinase C pathway for an enhanced MMP-9 secretion is inactive. None of the substances used in our study increased MMP-9 secretion, but some of them inhibited MMP-9 secretion. The secretion of MMP-2 could not be enhanced either, not even by dbcAMP, which has been reported to be effective in Sertoli and peritubular cells. Activation of MMP-2 in HRSF could be induced by treatment with concanavalin A (ConA) or cytochalasin D, as was shown for other cell types. This activation was not accompanied by a significant change in the amount of secreted TIMP-1 and TIMP-2. In contrast to reports on human skin fibroblasts, however, the activation of MMP-2 could not be induced in HRSF by treatment of the cells with monensin or sodium orthovanadate. Moreover, monensin was shown to act as an inhibitor of ConA- or cytochalasin D-mediated activation. Additionally, and in contrast to a report on a rat fibroblast cell line, MMP-2 activation is not mediated via the MAP kinase pathway in HRSF: PD 98059, a specific inhibitor of MAP kinase kinase, did not inhibit the activation of MMP-2. Similarly ineffective were PD 169316, an inhibitor for p38 MAP kinase, other inhibitors for protein kinases as lavendustin A, Gö 6983, wortmannin, rapamycin, as well as the protein tyrosine kinase inhibitors herbimycin A and genistein. Only staurosporin, a broad spectrum inhibitor of protein kinases, and the ionophores monensin and A 23187 effectively inhibited MMP-2 activation in HRSF. Our results demonstrate that MMP-2 can be activated by quite different pathways, and that different cells, even when belonging to the fibroblast family, do not necessarily use the same activating pathways.

[Establishment of an in vitro model for rheumatoid arthritis as test system for therapeutical substances]. / Etablierung eines in vitro Testsystems für die rheumatoide Arthritis zur Testung therapeutisch relevanter Wirkstoffe.

In our Tissue Engineering group a 3D in vitro model for rheumatoid Arthritis (in vitro pannus) was established with the aim to develop a standardized drug-screening test to analyze the effects of drugs and different biological substances. The advanced model consists of chondrocyte pellet cultures interacting with rheumatoid arthritis (RA) synovial cell cultures. To establish interactive 3D co-cultures defined rheumatoid arthritis synovial cell populations were centrifuged directly on chondrocyte pellet cultures. Histochemical stainings during time of co-culture revealed obvious invasion by RA synovial cell populations into the chondrocyte matrix. Gene expression analysis showed a downregulation of collagen type II expression in chondrocytes within 2 weeks after co-culture with RA synovial cells. Those interactive co-cultures allow the study of single cell populations as well as the cellular interactions in this system under in vitro conditions. Thus, the established co-culture model may be suitable for routine screening tests, which can be useful in supplementing animal experiments in basic research and drug testing.

Activation of progelatinase B by membranes of human polymorphonuclear granulocytes.

Isolated human granulocyte plasma membranes contain progelatinase B. The binding of progelatinase B to the membrane, however, is relatively weak, and a considerable part of progelatinase B can be removed by simply washing the membrane with buffer. This detachment does not depend on the ionic strength of the buffer, indicating that electrostatic forces do not play an important role in the binding of progelatinase B to the membrane. A complete removal of progelatinase B is achieved by chromatography of neutrophil membranes on gelatin-agarose. The plasma membrane of human granulocytes activates added progelatinase B. This activation is inhibited by soybean trypsin inhibitor and is thus performed by membrane bound serine proteinases. In contrast to other reports that claimed an important role of elastase in activating progelatinase B, we found that this activation is mostly inhibited by chymostatin and not by elastatinal and is thus primarily due to cathepsin G. Proteinase 3 was shown to activate progelatinase B as efficient as neutrophil elastase, i. e. much weaker than cathepsin G. Binding of cathepsin G and elastase to the neutrophil membrane does not change their ability to activate progelatinase B. However, cathepsin G, the most potent activator of the three neutrophil serine proteinases, is only a weak activator, when compared to stromelysin-1. This, as well as only a weak binding of progelatinase B, make it doubtful that activation of membrane-bound progelatinase B by membrane-bound serine proteinases is of significant physiological importance.

Biochemical characterization of the catalytic domain of membrane-type 4 matrix metalloproteinase.

A C-terminal truncated form of membrane-type 4 matrix metalloproteinase (MT4-MMP; MMP 17), lacking the hemopexin-like and transmembrane domain, was expressed in Escherichia coli. The catalytic domain was produced by tryptic activation of the recombinant proenzyme and proved to be catalytically active towards the fluorogenic substrate for matrix metalloproteinases (7-methoxycoumarin-4-yl) acetyl-Pro-Leu-Gly-Leu(3-(2,4-dinitrophenyl)-L-2,3-diaminopro-p ionyl)-Ala-Arg-NH2. In contrast to the other three MT-MMPs (MT1-, MT2-, and MT3-MMP), the catalytic domain of MT4-MMP does not activate progelatinase A, nor does it hydrolyze one of the offered extracellular matrix (ECM) proteins, such as collagen types I, II, III, IV, and V, gelatin, fibronectin, laminin or decorin. TIMP-1, a poor inhibitor of MT1-, MT2- and MT3-MMP, suppresses MT4-MMP activity effectively. The progelatinase A/TIMP-2 complex that usually reacts like TIMP-2 also inhibits MT4-MMP. TIMP-2, a strong inhibitor of other MT-MMPS, inhibits MT4-MMP at low concentrations. With increasing TIMP-2 concentration, however, activity passes through a minimum and then increases until at high TIMP-2 concentration the activity is the same as in the absence of TIMP-2. TIMP-1 or the progelatinase A/TIMP-2 complex do not prevent reactivation of MT4-MMP catalytic domain at high TIMP-2 concentrations.

The cleavage of pro-urokinase type plasminogen activator by stromelysin-1.

Membrane binding of urokinase type plasminogen activator (u-PA) is thought to play a pivotal role in connective tissue remodeling and invasive processes. We compare the ability of different matrix-metalloproteinases involved in connective tissue turnover to cleave pro-urokinase type plasminogen activator between the catalytic domain and the receptor binding part to investigate a potential role for matrix-metalloproteinases in the regulation of membrane-associated proteolytic activity. We employed several forms of human stromelysin-1 (full length, C-truncated, and recombinant catalytic domain), rabbit C-truncated stromelysin-1, the human gelatinases A and B and the human catalytic domain of neutrophil collagenase. The gelatinases and the collagenase did not separate the receptor binding domain of pro-urokinase type plasminogen activator from the catalytic domain, whereas all stromelysin-1 forms cleaved the glutamic acid 143-leucine 144 bond of pro-urokinase type plasminogen activator. This reaction could be inhibited by specific inhibitors of matrix metalloproteinases and was not affected by inhibitors of serine proteinases. The M(r) 31000 cleavage product with leucine 144 as N-terminus displayed no proteolytic activity towards the pro-urokinase type plasminogen activator substrate pyroGlu-Gly-Arg-pNA-HCI (S2444), but it could be activated by an additional treatment with plasmin. Comparison between full length stromelysin-1 and its C-truncated forms, showed that both exhibited the same cleavage properties towards pro-urokinase type plasminogen activator. Thus, the cleavage of pro-urokinase type plasminogen activator by stromelysin-1 is not influenced by the presence or absence of the C-terminal domain. The recombinant catalytic domain of MMP-3 generated pro-urokinase type plasminogen activator, whereas incubation of pro-urokinase type plasminogen activator with the native forms of human or rabbit stromelysin-1 led to a moderate activation of pro-uPA due to an additional cleavage that is catalyzed by a serine proteinase.

Substance P induces the secretion of gelatinase A from human synovial fibroblasts.

We investigated the secretion of the matrix metalloproteinases, interstitial collagenase (matrix metalloproteinase-1), gelatinase A (matrix metalloproteinase-2) and stromelysin-1 (matrix metalloproteinase-3) in human synovial fibroblasts after stimulation with the neuropeptide substance P. Human synovial fibroblasts were stimulated with substance P or interleukin-1 beta (IL-1 beta). In the cell culture media gelatinase A, interstitial collagenase and stromelysin-1 were identified and their activities towards different substrates were determined. Substance P in synovial fibroblasts induced an increase in the overall matrix metalloproteinase activity towards the dinitrophenyl-labelled peptide by 85%, against an increase of 124% after stimulation with IL-1 beta. In case of substance P stimulation, the increase in activity reflects a significantly enhanced secretion of gelatinase A, whereas no significant increase of stromelysin-1 and collagenase secretion could be observed. The matrix metalloproteinase pattern showing the highest gelatinase A secretion was obtained after stimulation with substance P. This pattern was very pronounced and differed very clearly from the pattern seen after IL-1 beta stimulation which caused a significant rise in collagenase and stromelysin-1 activity. We assume that distinct stimulation pathways are involved and that the neuropeptide (substance P), which is always present in the inflamed joint, plays its own and separate role in proliferative processes leading to the cartilage destruction.

Activation of progelatinase A and progelatinase A/TIMP-2 complex by membrane type 2-matrix metalloproteinase.

C-terminal truncated membrane-type 2 matrix metalloproteinase (MT2-MMP1-269), comprising prodomain and catalytic domain, was expressed as a soluble protein in Escherichia coli. Unlike the corresponding form of MT1-MMP, which can be isolated as a 31 kDa protein, MT2-MMP1-269 proved to be comparatively instable, and already the freshly isolated preparation displayed several proteins in SDS-PAGE representing MT2-MMP1-269 (33 kDa) and four N-truncated forms with N-termini methionine32 (30 kDa), isoleucine37 (30 kDa), leucine84 (24 kDa), and leucine93 (22 kDa), the catalytic domain. After thawing of frozen preparations the 33 and the 30 kDa proforms were no longer detectable in SDS-PAGE, and only the 24 and 22 kDa forms remained. The catalytic domain of MT2-MMP activated progelatinase A as well as the progelatinase A/TMP-2 complex by cleaving the 72 kDa progelatinase A to yield 67 kDa gelatinase A, which is then transformed into 62 kDa gelatinase A. The 62 kDa form is about twice as active as the 67 kDa form towards the synthetic substrate N-(2,4)-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg. No significant difference in activity was found between free and complexed gelatinase A forms. the activation of the progelatinase A/TIMP-2 complex proceeds in two steps: At first MT2-MMP is inhibited by the progelatinase A/TIMP-2/MT2-MMP, complex, whereby a ternary complex, progelatinase A/TIMP-2/ MT-2MMP is generated. This ternary complex is then activated by excess MT2-MMP. Our results suggest a mechanism for spatially regulated extracellular gelatinase A activity mediated by activation with membrane-type MMPs; Free gelatinase A is released into the extracellular space, while gelatinase A/TIMP-2 bound to MT-MMP remains anchored on the cell surface.

The recombinant catalytic domain of membrane-type matrix metalloproteinase-1 (MT1-MMP) induces activation of progelatinase A and progelatinase A complexed with TIMP-2.

A truncated form of the membrane-type matrix metalloproteinase-1 [(Ala21-Ile318)proMT1-MMP] lacking the hemopexin-like and trans-membrane domain was produced in E. coli. We demonstrate that the recombinant proenzyme was autoproteolytically processed to a fully active catalytic domain with N-terminal Ile114. The catalytic domain of MT1-MMP initiated the activation of progelatinase A and progelatinase A complexed with tissue inhibitor of metalloproteinases-2 (TIMP-2). As a typical soluble metalloproteinase it was able to cleave physiologic as well as synthetic substrates. Our kinetic data demonstrate that TIMP-2 is a potent inhibitor for the recombinant enzyme.

Progelatinase B forms from human neutrophils. complex formation of monomer/lipocalin with TIMP-1.

The three forms of neutrophil gelatinase B-monomer, homodimer and monomer/lipocalin complex-, were isolated from phorbolester stimulated neutrophil granulocytes by chromatography on gelatin-Sepharose and heparin-Ultrogel. On average, about 50% of the monomer/lipocalin complex was found to be complexed with TIMP-1. After activation with trypsin monomer, homodimer and monomer/lipocalin complex displayed a specific activity of about 2000 mU/mg towards the substrate N-(2,4)-dinitrophenyl-Pro-Gln-Gly-lle-Ala-Gly-Gln-D-Arg, whereas the monomer/lipocalin/TIMP-1 complex could be activated to a specific activity of only 200 mU/mg. The ternary monomer/lipocalin/TIMP-1 complex behaves like the progelatinase A-TIMP-2 complex and the progelatinase B-TIMP-1 complex in that it is an inhibitor for active metalloproteinases (MMPs) and, after activation, a gelatinase with a pronouncedly reduced activity. When the monomer/lipocalin/TIMP-1 complex inhibits an MMP, a quaternary complex monomer/lipocalin/TIMP-1/MMP is generated which after activation shows a sixfold higher proteolytic activity than the active ternary complex.

Generation and activity of the ternary gelatinase B/TIMP-1/LMW-stromelysin-1 complex.

Incubation of progelatinase B, isolated from human polymorphonuclear leukocytes, with TIMP-1 leads to the formation of the progelatinase B/TIMP-1 complex. This complex behaves like a Janus in a similar manner as we previously described for the progelatinase A/TIMP-2 complex. It shows the properties of TIMP-1 and is a better inhibitor for gelatinase A than for gelatinase B. Treatment with trypsin leads to activation of the binary complex. The activity, however, amounts only to slightly more than 10% of the activity of free gelatinase B, not complexed with TIMP-1. When the progelatinase B/TIMP-1 complex inhibits an active matrix metalloproteinase, a ternary complex is generated that after activation displays a distinct higher proteolytic activity than the active binary complex. The active binary complex cannot be transformed into the active ternary complex.

Synovial and peritoneal macrophages in organoid culture.

Cultivation of macrophages and their progenitors has been very useful for elucidation of function, behaviour and morphology of these cells. The purpose of this contribution is to describe a new in vitro system (organoid, high density or micromass culture) which proved to be convenient for cultivation of macrophages derived from human synovial fluid and tissue and mouse peritoneal fluid. Using this method, highly differentiated and functionally active macrophages of marked purity and long maintenance (up to 2 weeks) could be obtained even after previous cultivation and subcultivation in monolayer culture. The macrophages were identified by electron microscopy and immunomorphology using HLA-DR-DP, CD-68 (markers for human macrophages), anti-human-polymorphonuclear leukocyte-gelatinase and F4/80 (a mouse macrophage surface marker). The significance of this method as a research tool in the study of cartilage degradation by macrophages in co-cultures is stressed.

Activity of ternary gelatinase A-TIMP-2-matrix metallo-proteinase complexes.

The progelatinase A-TIMP-2 complex behaves like a Janus. Like TIMP (tissue inhibitor of metalloproteinases) it inhibits active matrix metalloproteinases, and activation with 4-aminophenylmercury acetate leads to a gelatinolytic activity. This activity, however, amounts only to less than 10% of that of free gelatinase A not complexed with TIMP-2. When the progelatinase A-TIMP-2 complex inhibits an active matrix metalloproteinase, a ternary complex is generated. After activation with 4-aminophenylmercury acetate this ternary complex displays a more than tenfold proteolytic activity compared to activated gelatinase A-TIMP-2 complex, thus reaching the activity of free gelatinase A. The activity of the ternary complex is nearly independent from the bound matrix metalloproteinase. When the progelatinase A-TIMP-2 complex is activated at first with 4-aminophenylmercury acetate the generation of the ternary complex is made impossible and not such a significant enhancement of activity is observed. These results suggest that gelatinase A-TIMP-2 complex may be a matrix metalloproteinase of the 'second step': It starts its proteolytic attack after it has switched off the activity of other matrix metalloproteinases.

Influence of electromagnetic fields on the enzyme activity of rheumatoid synovial fluid cells in vitro.

Since positive clinical effects have been observed in the treatment of rheumatoid arthritis with electromagnetic fields of weak strength and low frequency range (magnetic field strength: 70 microT; frequency: 1.36-14.44 Hz), an attempt was made to analyse the effects of these electromagnetic fields on enzyme activity in monolayer cultures of rheumatoid synovial fluid cells after single irradiation of the cultures for 24 hours. We only investigated the matrix metalloproteinases (collagenase, gelatinase, proteinase 24.11 and aminopeptidases). It was found that electromagnetic fields of such a weak strength and low frequency range do not generally have a uniform effect on the activity of the different proteinases in vitro. While aminopeptidases do not show any great changes in activity, the peptidases hydrolysing N(2,4)-dinitrophenyl-peptide exhibit a distinct increase in activity in the late phase in culture medium without fetal calf serum. In the presence of fetal calf serum this effect is not observed and enzyme activity is diminished. Our experiments do not show whether such a phase-bound increase in the activity of proteinases in vitro is only one finding in a much broader range of effects of electromagnetic fields, or whether it is a specific effect of weak pulsed magnetic fields of 285 +/- 33 nT on enzyme activity after single irradiation. This question requires further elucidation.

Isolation of latent 31-kDa C-truncated stromelysin and 21-kDa stromelysin from rabbit synovial fibroblasts: an alternative activation pathway for stromelysin.

The processing of culture medium of rabbit synovial fibroblasts led to the isolation of three stromelysin-1 (MMP-3) cleavage products: A 31-kDa protein, which represents a C-truncated latent stromelysin-1, an active stromelysin-1 of 21 kDa, that originates from the 31-kDa proform by activation. A third protein had a molecular mass of 25 kDa representing the C-terminal part of prostromelysin-1 and is missing in the C-truncated latent stromelysin-1. The activation process of human prostromelysin-1 in vitro is known to lead to an active stromelysin-1 with a relative molecular mass of 45 kDa by removing the N-terminal prodomain. This active stromelysin-1 is further processed to a lower molecular mass active form of 28 kDa. Our results obtained for the highly homologous rabbit stromelysin-1 indicate that another activation pathway is possible. In a first step prostromelysin-1 is hydrolysed between Met261-Glu generating a C-truncated latent stromelysin-1, which is activated by cleavage of the Thr83-Phe bond to the 21-kDa stromelysin-1. The latent C-truncated stromelysin-1 is slowly converted even at 4 degrees C into the active form. In the presence of 50 microM ZnCl2 this activation was prevented for at least three weeks. The activation rate is largely enhanced by aminophenylmercury acetate and especially by trypsin. The differences of the 21-kDa stromelysin-1 to a 28-kDa stromelysin-1 isolated from human rheumatoid synovial fluids described earlier are discussed.

A trypsin sensitive stromelysin isolated from rheumatoid synovial fluid is an activator for matrix metalloproteinases.

The processing of synovial fluids of patients suffering from rheumatoid arthritis led to the characterization of a neutral metalloproteinase with polymorphonuclear leukocyte progelatinase and polymorphonuclear leukocyte procollagenase activating properties. The activator exhibits a relative molecular mass of M(r) 27,000 and is an active form of stromelysin. Thus, it reacts specifically with antibodies raised against human stromelysin, splits polymorphonuclear leukocyte progelatinase in a manner characteristic of stromelysin, and is inhibited by EDTA as well as by a tissue inhibitor of metalloproteinases (TIMP-2). The activator shows a high specificity for the matrix metalloproteinases, polymorphonuclear leukocyte progelatinase and polymorphonuclear leukocyte procollagenase. It shows only weak hydrolysis of casein and gelatin, and it does not activate fibroblast M(r) 72,000 progelatinase. Brief treatment with trypsin does not lead to a significant change in the activator's relative molecular mass, but induces a rapid loss of its activating activity for polymorphonuclear leukocyte progelatinase, while its proteolytic activity against the synthetic substrate, N-(2,4)-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg, is increased about 3-fold. The same tryptic treatment does not affect the activator's proteolytic activity towards casein and gelatin.

Characterization of a gelatinase from human rheumatoid synovial fluid cells.

A metalloproteinase with a specificity for gelatin was isolated from serum-free medium of cultures of rheumatoid synovial fluid. The enzyme showed all the properties of a leukocyte gelatinase. In addition to gelatin this proteinase cleaved the synthetic substrate dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg (Dnp-peptide) rapidly, while casein was a much poorer substrate. This proteinase showed no enzymatic activity against collagen type I, was secreted in a latent form and could be activated by trypsin or organomercurial compounds, such as mersalylic acid or 4-aminophenyl-mercury acetate. The latent enzyme had an apparent molecular mass of 130,000-150,000 estimated by gel filtration or 97,000 by electrophoresis on polyacrylamide gel containing sodium dodecyl sulphate. When analysed by immunoblotting the enzyme was recognized by antibodies raised against human polymorphonuclear leukocyte gelatinase. Although we found synovial fibroblasts to be largely present in the cell cultures we could not detect any fibroblast gelatinase activity.

The complex between a tissue inhibitor of metalloproteinases (TIMP-2) and 72-kDa progelatinase is a metalloproteinase inhibitor.

Human rheumatoid synovial cells in culture secrete both 72-kDa progelatinase and a complex consisting of 72-kDa progelatinase and a 24-kDa inhibitor of metalloproteinases, TIMP-2. In addition, the culture medium contains TIMP-1, the classical inhibitor of metalloproteinases, with a molecular mass of 30 kDa. TIMP-1 does not form a complex with free 72-kDa progelatinase. Free progelatinase and progelatinase complexed with TIMP-2 can be activated with the organomercury compound p-aminophenylmercury acetate. The activated complex shows less than 10% the enzyme activity of activated free gelatinase. The progelatinase-TIMP-2 complex could be shown to be an inhibitor for other metalloproteinases, such as gelatinase and collagenase secreted by human rheumatoid synovia fibroblasts, as well as for the corresponding enzymes from human neutrophils.

Isolation and properties of a metal-dependent endopeptidase from human uterus hydrolysing synthetic collagenase substrates.

A metal-dependent peptidase was isolated from the homogenate of human uterus by standard chromatographic techniques and purified to apparent homogeneity. The peptidase hydrolysed the synthetic vertebrate collagenase substrate 2,4-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg (Dnp-peptide), the synthetic bacterial collagenase substrate 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg (PZ-peptide) and gelatinolytic peptides of gelatin, but was inactive against collagen type I, gelatin and casein. The cleavage site for the Dnp-peptide was the Gly-Ile bond. The enzyme was not only inhibited by metal chelators, such as EDTA, 1,10-phenantroline and dithiothreitol but also by thiol reagents, such as mersalylic acid and N-ethylmaleimid. However, E-64, an inhibitor for thiolproteinases, and leupeptin, an inhibitor for thiol- and serine proteases, did not exhibit any inhibitory activity. Pepstatin, an inhibitor for aspartate proteinases, and inhibitors for serine proteinases like phenylmethanesulfonyl fluoride and Trasylol were ineffective as well. The purified peptidase displayed a single band in the SDS-PAGE with an apparent molecular mass of 65 kDa. Employing isoelectric focusing an IP of 5.0 could be determined. The enzyme's properties are discussed in relation to the proteinase EC 3.4.24.11 and to proteinases of the collagenase family as well as the possibility to discriminate these three metalloproteinase classes by employing the Dnp-peptide.

Ultrastructural changes induced by alpha-sarcin in a human pulmonary tumor grown in naked mice.

alpha-Sarcin is a cytotoxic polypeptide produced by Aspergillus giganteus. It suppresses protein synthesis in yeast and wheat germ extracts and has a purine-specific RNase activity. The substance has been tested for its antitumor properties in a series of induced tumor systems in mice such as sarcoma and carcinoma among others. Although some of the in vitro effects of alpha-Sarcin on certain cellular components have been elucidated, the biological effects leading to cellular damage are still obscure. In this work we analysed the morphological changes in tumor cells derived from human pulmonary adenocarcinoma heterotransplanted and grown in naked mice, induced shortly (24 hours) after a single intratumoral injection of alpha-Sarcin (0.4 mg/tumor). The results obtained were: 1) swelling of mitochondria; 2) cell necrosis with partial removal of necrotic cells by phagocytosis; 3) thickening of interlobular connective tissue; 4) hyperplasia of goblet-cell-like clear cells. The mode of action concerning these cellular changes is presently uncertain. In view of the severity of these structural alterations it seems conceivable that alpha-Sarcin may enter the cell undergoing interactions with different intracellular structures. This would require a selective membrane permeabilization, perhaps induced upon formation of complexes with negatively-charged membrane phospholipids.

The covalent structure of the elastase inhibitor from Anemonia sulcata--a "non-classical" Kazal-type protein.

The amino-acid sequence of the proteinase inhibitor specific for elastases from the sea anemone Anemonia sulcata was determined from performic-acid oxidized inhibitor and from three cyanogen bromide fragments of reduced and carboxymethylated inhibitor. The molecule consists of a single polypeptide chain formed from 48 amino-acid residues and is stabilized by three intramolecular disulfide bridges. After cyanogen bromide cleavage of the native protein at methionines 10 and 28 followed by chymotryptic cleavage two fragments each containing a single disulfide bridge were isolated. These indicated the location of three intramolecular disulfide linkages between Cys4 and Cys34 (part of A-loop), Cys8 and Cys27 (B-loop) and Cys16 and Cys48 (C-loop). The sequential homology and the disulfide pattern identified the elastase inhibitor as a Kazal-type inhibitor in which, however, not only the CysI-CysII segment is rather short but interestingly the Cys4-Cys34 disulfide anchoring point (i.e. CysI-CysV) in the C-loop is shifted by one turn in the alpha-helical segment towards the C-terminus. Thus, the elastase inhibitor is a non-classical Kazal-type inhibitor with respect to the positioning of the half-cystines. The inhibitor molecule was modelled based on the known three-dimensional structure of the silver pheasant ovomucoid third domain. The shortened amino-terminal segment was arranged in such a manner to allow disulfide bridge formation between the first cysteine Cys4 and the replaced Cys34 under maintenance of a suitable binding loop conformation. The characteristic ovomucoid scaffold consisting of a central alpha-helix, an adjacent three-stranded beta-sheet and the proteinase-binding loop cross-connected through disulfide bridges CysI-CysV and CysIII-CysVI was conserved.