Equistatin, a New Inhibitor of Cysteine Proteinases from Actinia equina, Is Structurally Related to Thyroglobulin Type-1 Domain*

(Received for publication, November 19, 1996, and in revised form, March 23, 1997)

Brigita Lenarcic Dagger , Anka Ritonja , Borut Strukelj , Boris Turk and Vito Turk

From the Department of Biochemistry and Molecular Biology, Jozef Stefan Institute, Jamova 39, 1000 Ljubljana, Slovenia

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

It is well known that the activities of the lysosomal cysteine proteinases are tightly regulated by their endogenous inhibitors, cystatins. Here we report a new inhibitor of cysteine proteinases isolated from sea anemone Actinia equina. The inhibitor, equistatin, is an acidic protein with pI 4.7 and molecular weight of 14,129. It binds tightly and rapidly to cathepsin L (ka = 5.7 × 107 M-1 s-1, Ki = 0.051 nM) and papain (ka = 1.2 × 107 M-1 s-1, Ki = 0.57 nM). The lower affinity for cathepsin B (Ki = 1.4 nM) was shown to be due mainly to a lower second order association rate constant (ka = 0.04 × 106 M-1 s-1). The inhibitor is composed of 128 amino acids forming two repeated domains with 48% identity. Neither of the domains shows any sequence homology to cystatins, but they do show a significant homology to thyroglobulin type-1 domains. A highly conserved consensus sequence motif of Cys-Trp-Cys-Val together with conserved Cys, Pro, and Gly residues is present in major histocompatibility complex class II-associated p41 invariant chain, nidogen, insulin-like growth factor proteins, saxiphilin domain a, pancreatic carcinoma marker proteins (GA733), and chum salmon egg cysteine proteinase inhibitor. In each of the domains of the equistatin, the three residues are similarly conserved, and the sequences Val-Trp-Cys-Val and Cys-Trp-Cys-Val are present in domains a and b, respectively. We suggest that equistatin belongs to a new superfamily of protein inhibitors of cysteine proteinases named thyroglobulin type-1 domain inhibitors. This superfamily currently includes equistatin, major histocompatibility complex class II- associated p41 invariant chain fragment, and chum salmon egg cysteine proteinase inhibitor.


INTRODUCTION

Sea anemones are known to be a rich source of variety of polypeptide neurotoxins (1, 2) and neuropeptides (3), but little is known about the presence of proteolytic enzymes and their inhibitors. A chymotrypsin-like protease was first isolated from Metridium senile and shown to possess the same zymogen activation and active site chemistry as the proteinase from mammalian pancreas (4). Early reports on the existence of proteinase inhibitors in different species of sea anemones (5-8) were followed by the isolation (9) and primary structure determination of an elastase inhibitor from Anemonia sulcata (10). The inhibitor was found to be a nonclassical Kazal-type inhibitor with respect to positioning of the half-cystines. More recently, the structure of a Kunitz-type proteinase inhibitor purified from the Caribbean sea anemone Stichodactyla heliantus has been determined by NMR spectroscopy (11).

Cysteine proteinases are members of one of the four mechanistic classes of proteinases and, together with their endogenous protein inhibitors, cystatins, play an important role in intracellular degradation (12). They have not yet been found in sea anemones.

In this article we describe the isolation of a new inhibitor of papain-like cysteine proteinases from sea anemone, Actinia equina, designated as equistatin, the kinetic properties of its interaction with papain-like cysteine proteinases, and its amino acid sequence.


EXPERIMENTAL PROCEDURES

Enzymes

Papain (2 × crystallized) and clostripain were purchased from Sigma (Germany), and Ep-475,1 a specific inhibitor of cysteine proteinases, was obtained from Peptide Research Foundation (Japan). The Staphylococcus aureus V8 proteinase was obtained from Miles (UK), and glycyl endopeptidase was a gift from Dr. Alan J. Barrett (The Babraham Institute, Cambridge, UK) and was prepared as described (13). Recombinant human cathepsin B and human cathepsin L were prepared as described previously (14, 15).

Inhibitor Purification

A. equina specimens were collected on the northern coast of the Adriatic sea. The anemones (3 kg) were frozen, partially thawed, cut into small pieces, and homogenized in 4.5 liters of deionized water. Nonsoluble material was removed by centrifugation at 13,000 × g for 45 min. The supernatant was adjusted to pH 10.5 and incubated at room temperature for 1 h. Neutralization to pH 7.0 was followed by additional centrifugation at 13,000 × g for 45 min. The clear supernatant was applied to a carboxymethyl papain-Sepharose column (6 × 10 cm) previously equilibrated with 0.01 M Tris/HCl buffer, pH 8.0, containing 1 M NaCl and 0.1% Brij. After thorough washing of the column, bound proteins were eluted with 0.01 M NaOH. Fractions (20 ml) were collected and assayed for inhibitory activity toward papain using benzoyl-DL-Arg-beta -naphthylamide as substrate (16). The inhibitory fractions were pooled and concentrated by ultrafiltration (Amicon YM-5). The concentrate was applied to a Sephadex G-50 column (4.5 × 140 cm) equilibrated with 0.01 M Tris/HCl buffer, pH 7.7, containing 0.1 M NaCl, and eluted at a flow rate of 18 ml/h. Inhibitory fractions with molecular weights of about 16,000 were pooled, concentrated (Amicon, YM-5), and dialyzed against 0.01 M Tris/HCl buffer, pH 7.2. The dialyzed sample was then applied to a DEAE-Sephacel column (2 × 25 cm) equilibrated with the same buffer. The column was washed extensively, and bound proteins were eluted with a linear salt gradient (0-0.1 M NaCl in 0.01 M Tris/HCl buffer, pH 7.2) at a flow rate 18 ml/h. Equistatin eluted at 0.07 M NaCl.

SDS-PAGE and Analytical Isoelectric Focusing

SDS-PAGE and isoelectric focusing were performed on a PhastSystem apparatus (Pharmacia Biotech Inc.) following the manufacturer's instructions. The inhibitor and molecular weight markers ranging from Mr 14,400 to 94,000 were run in the presence of 0.5% SDS and 5% 2-mercaptoethanol on an 8-25% gradient polyacrylamide gel. The pI of the inhibitor was determined by calibrating the gel with isoelectric focusing marker proteins with pI values ranging from 3.5 to 8.15.

Protein Sequence Determination

Equistatin was reduced overnight with beta -mercaptoethanol at 37 °C and S-pyridylethylated (17). Pyridylethylated equistatin was hydrolyzed with glycyl endopeptidase as described (18). 4 nmol of pyridylethylated equistatin were fragmented using 2% (w/w) S. aureus V8 proteinase in 0.5 M sodium lactate buffer, pH 4.0, at 37 °C for 20 h. Both enzyme hydrolyses were performed in a final volume of 500 µl. Reactions were stopped by the addition of trifluoroacetic acid. The resulting peptide mixtures were separated by high performance liquid chromatography (Milton Roy Co.) using a reverse phase ChromSpher C18 column equilibrated with 0.1% (v/v) trifluoroacetic acid in water. Elutions was performed using various linear gradients of 80% (v/v) acetonitrile containing 0.1% (v/v) trifluoroacetic acid. The absorbance was monitored at 215 nm. Protein samples were hydrolyzed in 6.0 M HCl at 110 °C for 24 h. Analyses of the peptide hydrolysates were performed on an Applied Biosystems 421A amino acid analyzer with precolumn phenylisothiocyanate derivatization. An applied Biosystems liquid pulse sequencer 475A, connected on line to a phenylthiohydantoin analyzer 120A from the same manufacturer, was used for automated amino acid sequence analyses.

Determination of Protein Concentration

Protein concentration of equistatin was determined by absorption measurements at 280 nm using a molar absorption coefficient of 28,600 M-1 cm-1 determined by the method of Pace et al. (19) from the amino acid sequence or by the method of Lowry et al. (20) using bovine serum albumin as standard. The concentration of papain was determined spectrophotometrically using a molar absorption coefficient of 56,200 M-1 cm-1 (21).

Active Site Titration

The following buffers were used in all kinetic and equilibrium studies: 0.1 M phosphate buffer, pH 6.0, containing 5 mM dithiothreitol and 1 mM EDTA (for papain and cathepsin B) or 0.34 M sodium acetate buffer, pH 5.5, containing 5 mM dithiothreitol and 1 mM EDTA (for cathepsin L). Active site titrations of cathepsins B and L were performed using cysteine proteinase inhibitor Ep-475 as described previously (22). Papain, further purified by affinity chromatography (23), had a thiol content of 0.92 ± 0.05 mol/mol of enzyme as determined by reaction with 5,5'-dithiobis(2-nitrobenzoic acid).

Active site-titrated papain was used to titrate equistatin as follows. Papain (0.1 µM final concentration) was incubated with increasing amounts of equistatin (0-0.2 µM final concentration) in 200 µl of 0.1 M phosphate buffer, pH 6.0, containing 5 mM dithiothreitol and 1 mM EDTA at 25 °C. After 15 min of incubation, 1800 µl of 100 µM Z-Phe-Arg p-nitroanilide was added, and the residual activity of papain was monitored as described previously at 410 nm with a Perkin-Elmer Lambda 18 spectrophotometer (22). The data were analyzed by computer fitting to the theoretical binding equation (24).

Kinetics of Inhibition of Papain and Cathepsins B and L by Equistatin

The kinetics of the reaction between equistatin and papain, cathepsin B, and cathepsin L were analyzed by continuous measurements of the loss of enzymatic activity in the presence of substrate under pseudo first-order conditions with at least a 10-fold molar excess of inhibitor. Equistatin in increasing concentrations and the fluorogenic substrate (10 µM Z-Phe-Arg 4-methyl-7-coumarylamide) were mixed in a cuvette with buffer (see above) to a final volume of 1.97 ml. The enzyme (30 µl) was added, and the release of product was monitored continuously at excitation and emission wavelengths of 370 and 460 nm, respectively, by a Perkin-Elmer LS50 spectrofluorimeter. The biphasic progress curves were recorded and analyzed according to the model of slow tight binding kinetics using the equation of Morrison (25): [P] = vst + (vz - vs)(1 - e-kt)/k, where [P] is the product concentration, vz and vs are the initial and the steady-state velocities, respectively, t is time, and k is the observed pseudo first-order rate constant for the establishment of equilibrium between enzyme and inhibitor. ka (association rate constant) and kd (dissociation rate constant) values were obtained from the dependence of k on [I] according to the equations k = ka·[I]/(1 + S/Km) + kd and kd = k·vi/vz. The ka values were corrected for substrate competition using the Km values of 65 µM for papain (26), 2 µM for cathepsin L (27), and 150 µM for cathepsin B (16). Ki values were then determined from both individual rate constants (=kd/ka). Less than 3% of the substrate was hydrolyzed during the experiments throughout.


RESULTS AND DISCUSSION

Purification of Equistatin

Equistatin was purified from A. equina by a procedure similar to that used for the isolation of cysteine proteinase inhibitors of human origin (28). Initially, the supernatant was exposed to alkaline pH to dissociate the complexes between the inhibitor and other proteins. The most selective purification step, affinity chromatography on carboxymethyl papain-Sepharose, then allowed separation of papain-inhibiting proteins from the majority of noninhibitory proteins. This was followed by gel filtration on Sephadex G-50 (Fig. 1A), where the low molecular weight inhibitor (equistatin) was separated from high Mr inhibitor(s) of cysteine proteinases, which were not further characterized. Final purification was achieved by DEAE-Sephacel chromatography, from which the inhibitor eluted as a single peak at 0.07 M NaCl (Fig. 1B). About 5 mg of pure equistatin was obtained from 3 kg (fresh weight) of sea anemones.


Fig. 1. Purification of equistatin by column chromatography. A, size exclusion chromatography on Sephadex G-50 of inhibitory fractions obtained from carboxymethyl papain-Sepharose. B, ion exchange chromatography on DEAE-Sephacel of inhibitory active fractions (102-125) obtained from A. Bound proteins were eluted with a 0.0-0.1 M NaCl gradient as indicated. Experimental conditions are described under "Experimental Procedures." bullet , A280; open circle , % inhibition of papain; ----, NaCl concentration.
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SDS-PAGE and Analytical Isoelectric Focusing

On SDS-PAGE under reducing conditions, equistain migrates as a single band with Mr of about 16,000 (Fig. 2A). The molecular weight is higher than the molecular weights of either stefins or cystatins (Mr ~ 11,000 and 13,000, respectively) but lower than those of kininogens (Mr ~ 50,000-100,000) (12). The stefins, the cystatins, and the kininogens are proteins with similar sequences and, until recently, were the only known endogenous inhibitors of papain-like cysteine proteinases. On analytical isoelectric focusing, the inhibitor is shown to be an acidic protein with a pI value of 4.7. Very faint bands with pI values of 4.9 and 4.5, probably corresponding to the isoforms of the inhibitor (see below for explanation), could also be seen (Fig. 2B).


Fig. 2. Electrophoretic analyses of equistatin. A, SDS-PAGE of purified inhibitor. Lane 1, equistatin; lane 2, molecular weight standards. Before electrophoresis, the sample was reduced with beta -mercaptoethanol for 10 min at 100 °C. The gel was stained with Coomassie Blue. B, isoelectric focusing of equistatin. Lane 1, pI standards; lane 2, equistatin.
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Amino Acid Sequence of Equistatin

The major and minor N-terminal amino acid sequences, labeled NI-1 and NI-2, respectively, are shown in Fig. 3A. Sequence analyses of the peptides derived from glycyl endopeptidase digestion provided the amino acid sequence of the whole molecule (Fig. 3A). The largest peptide, G-3, spanned the middle part of the inhibitor and overlapped with both NI sequences. The C-terminal sequence was confirmed by peptides G-5 and G-(5+6), which ended with a pyridylethylated Cys residue that is not a glycyl endopeptidase cleavage site. Additional overlapping peptides, designated as E peptides (Fig. 3A) were obtained by S. aureus V8 proteinase digestion. During protein sequence analysis we have observed sequence polymorphism mainly in the middle part of the molecule (Fig. 3A). However, the yield of these residues was lower than 20% when compared with the main sequence. The observed sequence heterogeneity together with the results of isoelectric focusing reveals the presence of at least two closely related isoforms. As the isolation procedure involves the use of many anemone specimens, the difference in amino acid composition could arise from allelic polymorphism.


Fig. 3. Amino acid sequence of equistatin. A, amino acid sequence and strategy of sequence determination of equistatin. The N-terminal amino acid sequences of native inhibitor (NI-1 and NI-2) and peptides were determined by automated Edman degradation. The peptides were generated by the action of glycyl endopeptidase (G peptides) and S. aureus V8 (E peptides) proteinases. The amino acid residues shown under the major sequence were determined as sequence polymorphism. B, alignment of the amino acid sequences of the two equistatin domains. Alignment of fragments 1-64 and 65-128 was determined by using Genetic Computer Group, Inc. program software package version 7.0 (University of Wisconsin, Madison, WI). Identical amino acids are indicated with |, conservative replacements are indicated by :, and less similar residues are indicated by ·.
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The inhibitor comprises 128 amino acid residues including these 11 cysteines and has a molecular weight of 14,129. The inhibitor has no potential glycosylation sites of the Asn-X-(Thr/Ser) type.

Amino Acid Sequence Comparison

Alignment of equistatin residues 1-64 with 65-128 shows that the inhibitor consists of a tandem repeat with 48% identity and 60% similarity (Fig. 3B). This indicates that equistatin derives from a single ancestral gene that was duplicated and modified during evolution. However, neither domain shows any sequence homology with the members of the cystatin superfamily (12, 29). A Blast (30) search of the Swiss-Prot data bases (31) revealed high sequence similarity with thyroglobulin type-1 domain, a domain of about 65 amino acid residues that repeats 10 times in the N-terminal part of thyroglobulin (32). A number of other proteins containing the thyroglobulin type-1 domain motif were found. These proteins display a variety of physiological functions in different organisms. Major histocompatibility complex class II-associated p41 invariant chain fragment and chum salmon egg cysteine proteinase inhibitor are potent inhibitors of papain-like cysteine proteinases (33, 34), the former being involved in antigen presentation (35). Nidogen is a glycoprotein that probably plays a central role in the supramolecular organization of basement membranes and is tightly associated with laminin (36). Insulin-like growth factor-binding proteins act as inhibitors of insulin-like growth factor (37). Saxiphilin, characterized by a high affinity for a neurotoxin, saxitoxin (38, 39), and a tumor-associated cell surface antigen known also as GA733 are proteins whose functions are not yet elucidated (40). Fig. 4A shows a schematic diagram of the regions of similarity between equistatin and proteins containing the thyroglobulin type-1 domain, and Fig. 4B shows a sequence alignment relative to the thyroglobulin type-1 domains of equistatin. By introducing only a few short gaps in the alignment, the amino acid identities between the thyroglobulin type-1 domains in equistatin and those in all the other proteins listed in Fig. 4B are approximately 40% for the 49 C-terminal amino acids of both domains. The cysteine-rich sequence motif Cys-Trp-Cys-Val and the positions of some other amino acids (Cys-24, Cys-60, Pro-22, Gln-34, Gly-28, and Gly-49; equista in Fig. 4B) were found to be highly conserved among all related repeats, indicating that the proteins are probably evolutionarily related.


Fig. 4. Alignment of thyroglobulin type-1 repeats. A, schematic diagram of thyroglobulin type-1 repeats as found in thyroglobulin (Tg, 10 repeats), ascidian nidogen (Nido asc, 3 repeats) (49), saxiphilin (Sax, 2 repeats), nidogen (Nido), pancreatic carcinoma marker protein (GA733), p41 invariant chain (p41), insulin-like growth factor binding proteins (IGFBP), egg cysteine protease inhibitor (ECI), and equistatin (2 repeats). B, alignment of the equistatin-related proteins relative to the 49 amino acids of both domains of equistatin (equista and equistb). The comparison sequences are human thyroglobulin domain 1.1 (Tg) (32), human invariant chain (p41) (50), chum salmon egg cysteine proteinase inhibitor (ECI) (34), mouse nidogen (nido) (36), two domains of bullfrog saxifilin (saxa and saxb) (39), human pancreatic carcinoma marker protein (GA733-2) (40), and human insulin-like growth factor binding proteins (IGFBP5) (37). Residues in boldface type are present in at least 5 of 10 sequences. The conserved cysteine residues are indicated with an arrowhead.
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Active Site Titration and Kinetics of Inhibition

The sequence data suggest that the two sequentially homologous parts of equistatin (Fig. 3B) may form two potential proteinase binding sites. The binding stoichiometry of papain (active concentration >= 95%) and equistatin was therefore determined by titration monitored by the loss of enzymatic activity. 0.95 ± 0.04 mol of equistatin was needed to saturate 1 mol of papain, indicating that the two proteins formed an equimolar complex (Fig. 5). It could be suggested that binding of one proteinase molecule to equistatin prevents binding of the second proteinase molecule, probably by steric hindrance. However, there are a number of other possibilities. (i) One of the domains is not inhibitory at all, as observed in the kininogens (41). (ii) One of the domains has substantially lower affinity for proteinases, as found for the mucus proteinase inhibitor interaction with various serine proteinases (42). (iii) Both domains bind to the same proteinase molecule but only one of them binds to the active site; the other binds to another site distant from the active site, as reported for rhodiin binding to thrombin (43). Additional spectroscopic and structural studies involving mutant proteins will therefore be needed to clarify which of the above hypotheses is correct.


Fig. 5. Active site titration of equistatin with papain. Inhibition of 0.1 µM papain with increasing concentrations of equistatin. The fitted curve was generated by nonlinear regression analysis according to Bieth (24).
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The kinetics of binding of equistatin to papain and cathepsins B and L were studied under pseudo first-order conditions assuming 1:1 binding stoichiometry (see above). The pseudo first-order rate constants were found to increase linearly with increasing concentrations of inhibitor [I], in agreement with the proposed binding mechanism (25). Values of the second-order rate constants (ka), the dissociation rate constants (kd), and the equilibrium constants (Ki) are presented in Table I. Rapid binding of equistatin to cathepsin L and papain was observed, but the complexes with papain were ~10-fold less stable, with a 5-fold lower association rate constant and a 2-fold higher dissociation rate constant. The rate of complex formation between equistatin and cathepsin B was substantially slower. Its ka value is >30-fold lower than those for cathepsin L and papain, also reflected in the increased Ki value although the overall effect is partially compensated by a lower kd value. Cathepsin B (44) differs from papain (45) and its homologue cathepsin L (46) by having an additional loop of about 20 amino acids, which partially occludes the active site, thus interfering with inhibitor binding (47).

Table I. Kinetic data for the interaction of equistatin with cathepsin L, papain, and cathepsin B

The association rate constants, ka, together with their standard errors were calculated from the dependence of the pseudo first-order rate constant on inhibitor concentration. Dissociation rate constants, kd, were calculated for each inhibitor concentration as described under "Experimental Procedures." The equilibrium inhibition constants (Ki) were calculated from ka and kd. The number of measurements is given in parentheses. The association rate constants, ka, together with their standard errors were calculated from the dependence of the pseudo first-order rate constant on inhibitor concentration. Dissociation rate constants, kd, were calculated for each inhibitor concentration as described under "Experimental Procedures." The equilibrium inhibition constants (Ki) were calculated from ka and kd. The number of measurements is given in parentheses.

Enzyme 10-6 × ka 104 × kd Ki

M-1 s-1 s-1 nM
Cathepsin L 57  ± 2.7 (10) 30  ± 1.0 0.051  ± 0.004
Papain 12  ± 0.6 (9) 65  ± 1.5 0.570  ± 0.04
Cathepsin B 0.4  ± 0.02 (7) 5.6  ± 0.6 1.4  ± 0.2

The kinetic and equilibrium constants for the interaction of equistatin with cathepsins L and B and papain are similar to those reported for the interactions of these enzymes with cystatins (12, 22, 47). The Ki values are also in reasonable agreement with those obtained for various forms of chum salmon egg cysteine proteinase inhibitor (34, 48) although they differ significantly from the values for the p41 form of invariant chain fragment. The latter was found to be a stronger inhibitor of cathepsin L (~10-fold) and a weaker inhibitor of papain (~3-fold) but did not inhibit cathepsin B at all (33).

In conclusion, a new protein inhibitor of papain-like cysteine proteinases was isolated from sea anemone A. equina. The inhibitor, equistatin, is distinct from cystatins but shares significant sequence homology with two other chum salmon egg cysteine proteinase inhibitors, p41 invariant chain fragment and cysteine proteinase inhibitor. The three inhibitors were therefore suggested to form a new superfamily of cysteine proteinase inhibitors. The thyroglobulin type-1 domain motif, common to all three inhibitors, has been identified in a variety of other proteins. Whether this highly conserved thyroglobulin type-1 element indeed acts as an inhibitor of cysteine proteinases in these proteins remains to be established as well as the mechanism of binding to cysteine proteinases.


FOOTNOTES

*   This work was supported by the Ministry of Science and Technology of the Republic of Slovenia.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Dagger    To whom correspondence should be addressed. Tel.: 386 61 1773623; Fax: 386 61 273594; E-mail: brigita.lenarcic{at}ijs.si.
1   The abbreviations used are: Ep-475, L-3-carboxy-trans-2,3-epoxypropylleucylamido-(3-guanidino)butane; PAGE, polyacrylamide gel electrophoresis; Z, benzyloxycarbonyl.

ACKNOWLEDGEMENTS

We thank Dr. Aleksander Lucu for providing us with sea anemones and Dr. Iztok Dolenc and Robert Kuhelj for their gifts of cathepsins L and B, respectively. We also thank Dr. Roger H. Pain for critical reading of the manuscript.


REFERENCES

  1. Manoleras, N., and Norton, R. S. (1994) Biochemistry 33, 11051-11061 [Medline] [Order article via Infotrieve]
  2. Kem, R. W., Parten, B., Pennington, M. W., Price, D. A., and Dunn, B. M. (1989) Biochemistry 28, 3483-3489 [Medline] [Order article via Infotrieve]
  3. Schmutzler, C., Darmer, D., Diekhoff, D., and Grimmelikhuijzen, C. J. P. (1992) J. Biol. Chem. 267, 22534-22541 [Abstract/Free Full Text]
  4. Gibson, D., and Dixon, G. H (1969) Nature 222, 753-756 [Medline] [Order article via Infotrieve]
  5. Fritz, H., Brey, B., and Beress, L. (1972) Hoppe-Seyler's Z. Physiol. Chem. 353, 19-30 [Medline] [Order article via Infotrieve]
  6. Wunderer, G., Beress, L., Machleidt, W., and Fritz, H. (1976) Methods Enzymol. 45, 881-888 [Medline] [Order article via Infotrieve]
  7. Mebs, D., and Gebauer, E. (1980) Toxicon 18, 97-106 [Medline] [Order article via Infotrieve]
  8. Lenarcic, B., Kokalj, M., and Turk, V. (1986) in Cysteine Proteinases and Their Inhibitors (Turk, V., ed), pp. 609-615, Walter de Gruyter & Co., Berlin
  9. Kolkenbrock, H., and Tschesche, H. (1987) Biol. Chem. Hoppe-Seyler 368, 93-99 [Medline] [Order article via Infotrieve]
  10. Tschesche, H., Kolkenbrock, H., and Bode, W. (1987) Biol. Chem. Hoppe-Seyler 386, 1297-1304
  11. Antuch, W., Berndt, K. D., Chavez, M. A., Delfin, J., and Wüthrich, K. (1993) Eur. J. Biochem. 212, 675-684 [Abstract]
  12. Turk, V., and Bode, W. (1991) FEBS Lett. 285, 213-219 [CrossRef][Medline] [Order article via Infotrieve]
  13. Buttle, D. J., Kembhavi, A. A., Sharp, S., Schute, R. E., Rich, D. H., and Barrett, A. J. (1989) Biochem. J. 261, 469-476 [Medline] [Order article via Infotrieve]
  14. Kuhelj, R., Dolinar, M., Pungercar, J., and Turk, V. (1995) Eur. J. Biochem. 229, 533-539 [Abstract]
  15. Turk, B., Dolenc, I., Turk, V., and Bieth, J. G. (1993) Biochemistry 32, 375-380 [Medline] [Order article via Infotrieve]
  16. Barrett, A. J., and Kirschke, H. (1981) Methods Enzymol. 80, 535-561 [Medline] [Order article via Infotrieve]
  17. Henschen, A. (1986) in Advanced Methods in Protein Microsequence Analysis (Wittman-Liebold, B., Salnikov, J., and Erdmann, V. A., eds), pp. 244-255, Springer-Verlag, Berlin
  18. Buttle, D. J., Ritonja, A., Pearl, L. H., Turk, V., and Barrett, A. J. (1990) FEBS Lett. 260, 195-197 [CrossRef][Medline] [Order article via Infotrieve]
  19. Pace, C. N., Vajdos, F., Fee, L., Grimsley, G., and Gray, T. (1995) Protein Sci. 4, 2411-2423 [Abstract/Free Full Text]
  20. Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275 [Free Full Text]
  21. Brocklehurst, K., Carlson, J., Kierstan, M. P. J., and Crook, E. M. (1973) Biochem. J. 133, 573-584 [Medline] [Order article via Infotrieve]
  22. Turk, B., Krizaj, I., Kralj, B., Dolenc, I., Popovic', T., Bieth, J. G., and Turk, V. (1993) J. Biol. Chem. 268, 7323-7329 [Abstract/Free Full Text]
  23. Blumberg, S., Schechter, I., and Berger, A. (1970) Eur. J. Biochem. 15, 97-102 [Medline] [Order article via Infotrieve]
  24. Bieth, J. G. (1984) Biochem. Med. 32, 387-397 [Medline] [Order article via Infotrieve]
  25. Morrison, J. F. (1982) Trends Biochem. Sci. 7, 102-105 [CrossRef]
  26. Zucker, S., Buttle, D. J., Nicklin, J. H., and Barrett, A. J. (1985) Biochim. Biophys. Acta 828, 196-204 [Medline] [Order article via Infotrieve]
  27. Mason, R. W. (1986) Biochem. J. 240, 285-288 [Medline] [Order article via Infotrieve]
  28. Lenarcic, B., Ritonja, A., Sali, A., Kotnik, M., Turk, V., and Machleidt, W. (1986) in Cysteine Proteinases and Their Inhibitors (Turk, V., ed), pp. 473-487, Walter de Gruyter & Co., Berlin
  29. Barrett, A. J., Fritz, H., Grubb, A., Isemura, S., Järvinen, M., Katunuma, N., Machleidt, W., Müller-Esterl, W., Sasaki, M., and Turk, V. (1986) Biochem. J. 236, 312 [Medline] [Order article via Infotrieve]
  30. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. (1990) J. Mol. Biol. 215, 403-410 [CrossRef][Medline] [Order article via Infotrieve]
  31. Bairoch, A., and Boeckmann, B. (1991) Nucleic Acids Res. 19, 2247-2249 [Medline] [Order article via Infotrieve]
  32. Malthiery, Y., and Lissitzky, S. (1987) Eur. J. Biochem. 165, 491-498 [Abstract]
  33. Bevec, T., Stoka, V., Pungercic, G., Dolenc, I., and Turk, V. (1996) J. Exp. Med. 183, 1331-1338 [Abstract]
  34. Yamashita, M., and Konagaya, S. (1996) J. Biol. Chem. 271, 1282-1284 [Abstract/Free Full Text]
  35. Cresswell, P. (1994) Annu. Rev. Immunol. 12, 259-293 [CrossRef][Medline] [Order article via Infotrieve]
  36. Mann, K., Deutzmann, R., Aumailley, M., Timpl, R., Raimondi, L., Yamada, Y., Pan, T., Conway, D., and Chu, M. (1989) EMBO J. 8, 65-72 [Abstract]
  37. Allander, S. V., Larsson, C., Ehrenborg, E., Suwanichkul, A., Weber, G., Morris, S. L., Bajalica, S., Kiefer, M. C., Luthman, H., and Powell, D. R. (1994) J. Biol. Chem. 269, 10891-10898 [Abstract/Free Full Text]
  38. Morabito, M. A., and Moczydlowski, E. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 2478-2482 [Abstract]
  39. Morabito, M. A., and Moczydlowski, E. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 6651 [Free Full Text]
  40. Szala, S., Froehlich, M., Scollon, M., Kasai, Y., Steplewski, Z., Koprowski, H., and Linnenbach, A. J. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 3542-3546 [Abstract]
  41. Salvesen, G., Parkes, C., Abrahamson, M., Grubb, A., and Barrett, A. J. (1986) Biochem. J. 234, 429-434 [Medline] [Order article via Infotrieve]
  42. Boudier, C., and Bieth, J. G. (1992) J. Biol. Chem. 267, 4370-4375 [Abstract/Free Full Text]
  43. van de Locht, A., Lamba, D., Bauer, M., Huber, R., Friedrich, T., Kröger, B., Höffken, W., and Bode, W. (1995) EMBO J. 14, 5149-5157 [Abstract]
  44. Musil, Dj., Zucic, D., Turk, D., Engh, R. A., Mayr, I., Huber, R., Popovic', T., Turk, V., Towatari, T., Katunuma, N., and Bode, W. (1991) EMBO J. 10, 2321-2330 [Abstract]
  45. Kamphuis, I. G., Kalk, K. H., Swarte, M. B. A., and Drenth, J. (1984) J. Mol. Biol. 179, 233-257 [Medline] [Order article via Infotrieve]
  46. Coulombe, R., Grochulski, P., Sivaraman, J., Menard, R., Mort, J. S., and Cygler, M. (1996) EMBO J. 15, 5492-5503 [Abstract]
  47. Lenarcic, B., Krizaj, I., Zunec, P., and Turk, V. (1996) FEBS Lett. 395, 113-118 [CrossRef][Medline] [Order article via Infotrieve]
  48. Yamashita, M., and Konagaya, S. (1991) J. Biochem. (Tokyo) 110, 762-766 [Abstract]
  49. Nakae, H., Sugano, M., Ishimori, Y., Endo, T., and Obinata, T. (1993) Eur. J. Biochem. 213, 11-19 [Abstract]
  50. Strubin, M., Berte, C., and Mach, B. (1986) EMBO J. 5, 3483-3488 [Abstract]

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