Binding of nonphysiological protein and peptide substrates to proteases: differences between urokinase-type plasminogen activator and trypsin and contributions to the evolution of regulated proteolysis

Understanding the regulation of physiological processes requires detailed knowledge of the recognition of substrates by enzymes. One of the most productive model systems for the study of enzyme-substrate interactions is the serine protease family; however, most studies of protease action have used small substrates that contain an activated, non-natural scissile bond. Because few kinetic or structural studies have used protein substrates, the physiologically relevant target of most proteases, it seems likely that important mechanisms of substrate recognition and processing by proteases have not yet been fully elucidated. Consistent with this hypothesis, we have observed that K(m) values for protein substrates are reduced as much as 200-15000-fold relative to those of analogous peptide substrates. Here we examine the thermodynamic consequences of interactions between proteases and their substrates using staphylococcal nuclease (SNase) and SNase variants as model protein substrates. We have obtained values for enthalpy, entropy, and K(d) for binding of proteins and peptides by the nonspecific protease trypsin and the highly specific protease urokinase-type plasminogen activator (u-PA). To avoid cleavage of substrates during these measurements, we used inactive variants of trypsin and u-PA whose catalytic serine S195 had been replaced by alanine. Differences in the K(d) values for binding of protein and peptide substrates closely approximate the large differences observed in the corresponding K(m) values. Improved binding of protein substrates is due to decreased enthalpy, and this effect is pronounced for the selective protease u-PA. Fundamental differences in recognition of analogous protein and peptide substrates may have influenced the evolution of protease specificity.     

A comparison of staphostatin B with standard mechanism serine protease inhibitors

Staphostatins are the endogenous, highly specific inhibitors of staphopains, the major secreted cysteine proteases from Staphylococcus aureus. We have previously shown that staphostatins A and B are competitive, active site-directed inhibitors that span the active site clefts of their target proteases in the same orientation as substrates. We now report the crystal structure of staphostatin B in complex with wild-type staphopain B at 1.9 A resolution. In the complex structure, the catalytic residues are found in exactly the positions that would be expected for uncomplexed papain-type proteases. There is robust, continuous density for the staphostatin B binding loop and no indication for cleavage of the peptide bond that comes closest to the active site cysteine of staphopain B. The carbonyl carbon atom C of this peptide bond is 4.1 A away from the active site cysteine sulfur Sgamma atom. The carbonyl oxygen atom O of this peptide bond points away from the putative oxyanion hole and lies almost on a line from the Sgamma atom to the C atom. The arrangement is strikingly similar to the "ionmolecule" arrangement for the complex of papain-type enzymes with their substrates but differs significantly from the arrangement conventionally assumed for the Michaelis complex of papain-type enzymes with their substrates and also from the arrangement that is crystallographically observed for complexes of standard mechanism inhibitors and their target serine proteases.     

Subsite specificities of granzyme M: a study of inhibitors and newly synthesized thiobenzyl ester substrates

Granzyme M is a member of a family of granule serine proteases that participate in target cell death initiated by cytotoxic lymphocytes. The enzyme is almost exclusively expressed in NK cell types. Granzyme M cleaves at the carboxy side of amino acids with long, hydrophobic side chains like Met, Leu, and Nle. To further study the substrate specificity of the enzyme, a series of peptide thiobenzyl esters was synthesized. The hydrolysis of the substrates with murine and human recombinant forms of granzyme M was observed. The results show that the enzyme has a strong preference for Pro at the P2 position and Ala, Ser, or Asp at the P3 position. These results suggest that the protein residues of the S2 and S3 subsites form important binding interactions that aid in the selection of specific natural substrates for granzyme M. A series of inhibitors was also tested with granzyme M. None of the inhibitors were effective inactivators of granzyme M, including the general serine protease inhibitor, 3,4-dichloroisocoumarin, which is usually a potent inactivator of serine proteases. This suggests that inhibition of granzyme M may be difficult. Also reported for the first time is the method utilized to isolate granzyme M used in this and previous publications. The observations in this paper will be valuable in development of new potent inhibitors for granzyme M as well as assist in determining the biological function of the enzyme.     

In-cell selectivity profiling of serine protease inhibitors by activity-based proteomics

Activity-based proteomics is a methodology that is used to quantify the catalytically active subfraction of enzymes present in complex mixtures such as lysates or living cells. To apply this approach for in-cell selectivity profiling of inhibitors of serine proteases, we designed a novel activity-based probe (ABP). This ABP consists of (i) a fluorophosphonate-reactive group, directing the probe toward serine hydrolases or proteases and (ii) an alkyne functionality that can be specifically detected at a later stage with an azide-functionalized reporter group through a Cu(I)-catalyzed coupling reaction ("click chemistry"). This novel ABP was shown to label the active site of several serine proteases with greater efficiency than a previously reported fluorophosphonate probe. More importantly, our probe was cell-permeable and achieved labeling of enzymes within living cells with efficiency similar to that observed for the corresponding lysate fraction. Several endogenous serine hydrolases whose activities were detected upon in-cell labeling were identified by two-dimensional gel and MS analyses. As a proof of principle, cell-permeable inhibitors of an endogenous serine protease (prolyl endopeptidase) were assessed for their potency and specificity in competing for the in situ labeling of the selected enzyme. Altogether these results open new perspectives for safety profiling studies in uncovering potential cellular "side effects" of drugs (unanticipated off-target inhibition or activation) that may be overlooked by standard selectivity profiling methods.     

A lead discovery strategy driven by a comprehensive analysis of proteases in the peptide substrate space

We present here a comprehensive analysis of proteases in the peptide substrate space and demonstrate its applicability for lead discovery. Aligned octapeptide substrates of 498 proteases taken from the MEROPS peptidase database were used for the in silico analysis. A multiple‐category naïve Bayes model, trained on the two‐dimensional chemical features of the substrates, was able to classify the substrates of 365 (73%) proteases and elucidate statistically significant chemical features for each of their specific substrate positions. The positional awareness of the method allows us to identify the most similar substrate positions between proteases. Our analysis reveals that proteases from different families, based on the traditional classification (aspartic, cysteine, serine, and metallo), could have substrates that differ at the cleavage site (P1–P1′) but are similar away from it. Caspase‐3 (cysteine protease) and granzyme B (serine protease) are previously known examples of cross‐family neighbors identified by this method. To assess whether peptide substrate similarity between unrelated proteases could reliably translate into the discovery of low molecular weight synthetic inhibitors, a lead discovery strategy was tested on two other cross‐family neighbors—namely cathepsin L2 and matrix metallo proteinase 9, and calpain 1 and pepsin A. For both these pairs, a naïve Bayes classifier model trained on inhibitors of one protease could successfully enrich those of its neighbor from a different family and vice versa, indicating that this approach could be prospectively applied to lead discovery for a novel protease target with no known synthetic inhibitors.     

Structural organization in the serine proteases. I. Macromolecular specificity in limited proteolysis

We have been developing computational approaches to increase our ability to analyze the growing body of three-dimensional structural data with applications centered about the serine proteases. The emphasis of these approaches is to compare and contrast macromolecules at the separate levels of secondary, tertiary, and quaternary structure. Our assumption is that in functionally related molecules, regions of structural and/or physicochemical similarity will exhibit functional similarity; regions that are different in structure and/or physicochemical properties will function differently and, therefore, be the source of specificity. Based on this assumption, the independent observations from studies of these enzymes in solution and in biological systems are combined with the structural observations from X-ray crystallographic analysis. A goal of the present research effort is to probe the biomolecular architecture of the serine proteases to evaluate the role of the three-dimensional structure beyond that of the active site in determining recognition and reactivity determinants for these enzymes, and to determine those principles that might be applied successfully to other enzyme systems as well. Of particular note has been our observation of a macromolecular recognition surface which occurs as a topographical feature outside of the active site and under evolutionary control to produce specificity towards macromolecular substrates and inhibitors. In addition we have established the important role of conformational changes that occur beyond the active site of an enzyme and differentiate between natural and synthetic inhibitor-enzyme interactions. This suggests that the specificity and reactivity determinants of a macromolecule are derived from its architecture and structural organization.     

Reaction of serine proteases with substituted isocoumarins: discovery of 3,4-dichloroisocoumarin, a new general mechanism based serine protease inhibitor

The mechanism-based inactivations of a number of serine proteases, including human leukocyte (HL) elastase, cathepsin G, rat mast cell proteases I and II, several human and bovine blood coagulation proteases, and human factor D by substituted isocoumarins and phthalides which contain masked acyl chloride or anhydride moieties, are reported. 3,4-Dichloroisocoumarin, the most potent inhibitor investigated here, inactivated all the serine proteases tested but did not inhibit papain, leucine aminopeptidase, or beta-lactamase. 3,4-Dichloroisocoumarin was fairly selective toward HL elastase (kobsd/[I] = 8920 M-1 s-1); the inhibited enzyme was quite stable to reactivation (kdeacyl = 2 X 10(-5) s-1), while enzymes inhibited by 3-acetoxyisocoumarin and 3,3-dichlorophthalide regained full activity upon standing. The rate of inactivation was decreased dramatically in the presence of reversible inhibitors or substrates, and ultraviolet spectral measurements indicate that the isocoumarin ring structure is lost upon inactivation. Chymotrypsin A gamma is totally inactivated by 1.2 equiv of 3-chloroisocoumarin or 3,4-dichloroisocoumarin, and approximately 1 equiv of protons is released upon inactivation. These results indicate that these compounds react with serine proteases to release a reactive acyl chloride moiety which can acylate another active site residue. These are the first mechanism-based inhibitors reported for many of the enzymes tested, and 3,4-dichloroisocoumarin should find wide applicability as a general serine protease inhibitor.     

Selective and reversible thiol-pegylation, an effective approach for purification and characterization of five fully active ficin (iso)forms from Ficus carica latex

The latex of Ficus carica constitutes an important source of many proteolytic components known under the general term of ficin (EC 3.4.22.3) which belongs to the cysteine proteases of the papain family. So far, no data on the purification and characterization of individual forms of these proteases are available. An effective strategy was used to fractionate and purify to homogeneity five ficin forms, designated A, B, C, D1 and D2 according to their sequence of elution from a cation-exchange chromatographic support. Following rapid fractionation on a SP-Sepharose Fast Flow column, the different ficin forms were chemically modified by a specific and reversible monomethoxypolyethylene glycol (mPEG) reagent. In comparison with their un-derivatized counterparts, the mPEG-protein derivatives behaved differently on the ion-exchanger, allowing us for the first time to obtain five highly purified ficin molecular species titrating 1mol of thiol group per mole of enzyme. The purified ficins were characterized by de novo peptide sequencing and peptide mass fingerprinting analyzes, using mass spectrometry. Circular dichroism measurements indicated that all five ficins were highly structured, both in term of secondary and tertiary structure. Furthermore, analysis of far-UV CD spectra allowed calculation of their secondary structural content. Both these data and the molecular masses determined by MS reinforce the view that the enzymes belong to the family of papain-like proteases. The five ficin forms also displayed different specific amidase activities against small synthetic substrates like dl-BAPNA and Boc-Ala-Ala-Gly-pNA, suggesting some differences in their active site organization. Enzymatic activity of the five ficin forms was completely inhibited by specific cysteine and cysteine/serine proteases inhibitors but was unaffected by specific serine, aspartic and metallo proteases inhibitors.     

Profiling serine hydrolase activities in complex proteomes

Serine hydrolases represent one of the largest and most diverse families of enzymes in higher eukaryotes, comprising numerous proteases, lipases, esterases, and amidases. The activities of many serine hydrolases are tightly regulated by posttranslational mechanisms, limiting the suitability of standard genomics and proteomics methods for the functional characterization of these enzymes. To facilitate the global analysis of serine hydrolase activities in complex proteomes, a biotinylated fluorophosphonate (FP-biotin) was recently synthesized and shown to serve as an activity-based probe for several members of this enzyme family. However, the extent to which FP-biotin reacts with the complete repertoire of active serine hydrolases present in a given proteome remains largely unexplored. Herein, we describe the synthesis and utility of a variant of FP-biotin in which the agent's hydrophobic alkyl chain linker was replaced by a more hydrophilic poly(ethylene glycol) moiety (FP-peg-biotin). When incubated with both soluble and membrane proteomes for extended reaction times, FP-biotin and FP-peg-biotin generated similar "maximal coverage" serine hydrolase activity profiles. However, kinetic analyses revealed that several serine hydrolases reacted at different rates with each FP agent. These rate differences were exploited in studies that used the biotinylated FPs to examine the target selectivity of reversible serine hydrolase inhibitors directly in complex proteomes. Finally, a general method for the avidin-based affinity isolation of FP-biotinylated proteins was developed, permitting the rapid and simultaneous identification of multiple serine peptidases, lipases, and esterases. Collectively, these studies demonstrate that chemical probes such as the biotinylated FPs can greatly accelerate both the functional characterization and molecular identification of active enzymes in complex proteomes.     

Inhibitor binding induces active site stabilization of the HCV NS3 protein serine protease domain

Few structures of viral serine proteases, those encoded by the Sindbis and Semliki Forest viruses, hepatitis C virus (HCV) and cytomegalovirus, have been reported. In the life cycle of HCV a crucial role is played by a chymotrypsin-like serine protease encoded at the N-terminus of the viral NS3 protein, the solution structure of which we present here complexed with a covalently bound reversible inhibitor. Unexpectedly, the residue in the P2 position of the inhibitor induces an effective stabilization of the catalytic His-Asp hydrogen bond, by shielding that region of the protease from the solvent. This interaction appears crucial in the activation of the enzyme catalytic machinery and represents an unprecedented observation for this family of enzymes. Our data suggest that natural substrates of this serine protease could contribute to the enzyme activation by a similar induced-fit mechanism. The high degree of similarity at the His-Asp catalytic site region between HCV NS3 and other viral serine proteases suggests that this behaviour could be a more general feature for this category of viral enzymes.     

A comparative study on innate immune parameters in the epidermal mucus of various fish species

Fish epidermal mucus and its components provide the first line of defense against pathogens. Little is known about the role of epidermal mucus enzymes in the innate immune system of fish species such as Arctic char (Salvelinus alpinus), brook trout (S. fontinalis), koi carp(Cyprinus carpio), striped bass (Morone saxatilis), haddock, (Melanogrammus aeglefinus), Atlantic cod (Gadus morhua) and hagfish (Myxine glutinosa). The epidermal mucus samples from these fish were analysed for the specific activities of various hydrolytic enzymes including lysozyme, alkaline phosphatase, cathepsin B and proteases and the enzyme levels were compared among the fish species. Of all the species hagfish mucus showed a high activity for lysozyme and proteases and koi carp mucus had the highest levels of alkaline phosphatase and cathepsin B. A wide variation in enzyme activities was observed among the seven species and also between species of same family such as Arctic char and brook trout (salmonidae), haddock and cod (gadidae). Only lysozyme levels showed a marked variation with salinity where seawater fish showed approximately two times higher lysozyme activity than freshwater-reared fish species. Characterization of proteases with specific inhibitors showed Arctic char, brook trout, haddock and cod having higher levels of serine over metalloproteases whereas koi carp and striped bass had higher levels of metalloproteases over serine proteases. In contrast, hagfish had almost equal proportion of both serine and metalloproteases. This study demonstrates variation in the level of hydrolytic enzymes in the epidermal mucus of fish. These results provide preliminary information for a better understanding of the role of epidermal mucus and its components in the fish innate immune system.     

Mechanism-based isocoumarin inhibitors for serine proteases: use of active site structure and substrate specificity in inhibitor design

Isocoumarins are potent mechanism-based heterocyclic irreversible inhibitors for a variety of serine proteases. Most serine proteases are inhibited by the general serine protease inhibitor 3,4-dichloroisocoumarin, whereas isocoumarins containing hydrophobic 7-acylamino groups are potent inhibitors for human leukocyte elastase and those containing 7-alkylureidogroups are inhibitors for procine pancreatic elastase. Isocoumarins containing basic side chains that resemble arginine are potent inhibitors for trypsin-like enzymes. A number of 3-alkoxy-4-chloro-7-guanidinoisocoumarins are potent inhibitors of bovine thrombin, human factor Xa, human factor XIa, human factor XIIa, human plasma kallikrein, porcine pancreatic kallikrein, and bovine trypsin. Another cathionic derivative, 4-chloro-3-(2-isothiureidoethoxy) isocoumarin, is less reactive toward many of these enzymes but is an extremely potent inhibitor of human plasma kallikrein. Several guanidinoisocoumarins have been tested as anticoagulants in human plasma and are effective at prolonging the prothrombin time. The mechanism of inhibition by this class of heterocyclic inactivators involves formation of an acyl enzyme by reaction of the active site serine with the isocoumarin carbonyl group. Isocoumarins with 7-amino or 7-guanidino groups will then decompose further to quinone imine methide intermediates, which react further with an active site residue (probably His-57) to form stable inhibited enzyme derivatives. Isocoumarins should be useful in further investigations of the physiological function of serine proteases and may have future therapeutic utility for the treatment of emphysema and coagulation disorders.     

Highly stable glycosylated serine protease from the medicinal plant Euphorbia milii

A serine protease, named as "Milin" was purified to homogeneity from the latex of Euphorbia milii, a medicinal plant of Euphorbiaceae family. The molecular mass (SDS-PAGE), optimum pH and temperature of the enzyme were 51kDa, pH 8.0 and 60 degrees C, respectively. Milin retains full proteolytic activity over a wide range of pH (5.5-12) and temperature (up to 65 degrees C) with casein and azoalbumin as substrates. The activity of milin is inhibited by serine proteases inhibitors like PMSF, APMSF and DFP, but not by any other protease inhibitors such as E-64 and PCMB. Like the other serine proteases from the genus Euphorbia, the activity of milin was not inhibited by the proteinaceous inhibitor soyabean trypsin inhibitor (SBTI) even at very high concentrations that is naturally present in plants. The specific extinction coefficient (epsilon(280 nm)(1%)), molar extinction coefficient (a(m)) and isoelectric point of the enzyme were found to be 29, 152,500 M(-1) cm(-1) and pH 7.2, respectively. The enzyme is a glycoprotein with detectable carbohydrate moiety (7-8%) in its constitution, which is essential for the activity. The numbers of tryptophan, tyrosine and cysteine residues in the sequence of milin were estimated chemically and are 23, 14 and 14, respectively. Of the 14-cysteine residues, 12 constituted 6-disulfide linkages while two are free cysteines. The N-terminal sequence (first 12 amino acid residues) was determined and does not match with any sequence of known plant serine proteases. Perturbation studies by temperature, pH and chaotropes of the enzyme also reveal its high stability as seen by CD, fluorescence and proteolytic activity. Thus, this serine protease may have potential applications in food industry.     

Arresting tissue invasion of a parasite by protease inhibitors chosen with the aid of computer modeling

Computer modeling of the three-dimensional structure of an enzyme, based upon its primary sequence alone, is a potentially powerful tool to elucidate the function of enzymes as well as design specific inhibitors. The cercarial (larval) protease from the blood fluke Schistosoma mansoni is a serine protease hypothesized to assist the schistosome parasite in invading the human circulatory system via the skin. A three-dimensional model of the protease was built, taking advantage of the similarity of the sequence of the cercarial enzyme to the trypsin-like class of serine proteases. A large hydrophobic S-1 binding pocket, suspected from previous kinetic studies, was located in the model and confirmed by new kinetic studies with both synthetic peptide substrates and inhibitors. Unexpected structural characteristics of the enzyme were also predicted by the model, including a large S-4 binding pocket, again confirmed by assays with synthetic peptides. The model was then used to design a peptide inhibitor with 4-fold increased solubility, and a series of synthetic inhibitors were tested against live cercariae invading human skin to confirm that predictions of the model were also applicable in a biologic assay.     

A collagenolytic serine protease with trypsin-like specificity from the fiddler crab Uca pugilator

A second collagenolytic serine protease has been isolated from the hepatopancreas of the fiddler crab, Uca pugilator. This enzyme cleaves the native triple helix of collagen under physiological conditions of pH, temperature, and ionic strength. In addition to its collagenolytic activity, the enzyme exhibits endopeptidase activity toward other polypeptides and small molecular weight synthetic substrates. The polypeptide bond specificity of this enzyme is similar to that of bovine trypsin as is its interaction with specific protease inhibitors. The amino-terminal sequence of this enzyme displays significant homology with other serine proteases, most notably with that of crayfish trypsin, and demonstrates that this enzyme is a member of the trypsin family of serine endopeptidases. The relatively unique action of this protease with regard to both collagenous and noncollagenous substrates has important implications concerning the specificity and mechanism of collagen degradation.     

Mammalian chymotrypsin-like enzymes. Comparative reactivities of rat mast cell proteases, human and dog skin chymases, and human cathepsin G with peptide 4-nitroanilide substrates and with peptide chloromethyl ketone and sulfonyl fluoride inhibitors

The extended substrate binding sites of several chymotrypsin-like serine proteases, including rat mast cell proteases I and II (RMCP I and II, respectively) and human and dog skin chymases, have been investigated by using peptide 4-nitroanilide substrates. In general, these enzymes preferred a P1 Phe residue and hydrophobic amino acid residues in P2 and P3. A P2 Pro residue was also found to be quite acceptable. The S4 subsites of these enzymes are less restrictive than the other subsites investigated. The substrate specificity of these enzymes was also investigated by using substrates which contain model desmosine residues and peptides with amino acid sequences of the physiologically important substrates angiotensin I and angiotensinogen and alpha 1-antichymotrypsin, the major plasma inhibitor for chymotrypsin-like enzymes. These substrates were less reactive than the most reactive tripeptide reported here, Suc-Val-Pro-Phe-NA. The thiobenzyl ester Suc-Val-Pro-Phe-SBzl was found to be an extremely reactive substrate for the enzymes tested and was 6-171-fold more reactive than the 4-nitroanilide substrate. The four chymotrypsin-like enzymes were inhibited by chymostatin and N-substituted saccharin derivatives which had KI values in the micromolar range. In addition, several potent peptide chloromethyl ketone and substituted benzenesulfonyl fluoride irreversible inhibitors for these enzymes were discovered. The most potent sulfonyl fluoride inhibitor for RMCP I, RMCP II, and human skin chymase, 2-(Z-NHCH2CONH)C6H4SO2F, had kobsd/[I] values of 2500, 270, and 1800 M-1 s-1, respectively. The substrates and inhibitors reported here should be extremely useful in elucidating the physiological roles of these proteases.     

Analysis of the biomacromolecular architecture of eukaryotic and prokaryotic serine proteases

Summary We have been developing computational approaches to increase our ability to analyze the growing body of three-dimensional structural data with applications centered on the serine proteases and their natural inhibitors and substrates. It is essential that these approaches emphasize the comparison of these macromolecules at the separate levels of secondary, tertiary and quaternary structure. We assume in our analysis that in functionally related macromolecules (i.e., a family of evolutionarily related enzymes), regions of structural and/or physicochemical similarity will exhibit functional similarity; regions that are different in structure and/or physicochemical properties will function differently and, therefore, be the source of observed specificity. It is the intent of our research to encapsulate such knowledge in a form which is capable of observing patterns which may serve as generalizable rules for macrostructural analysis (Liebman, M.N. 1986. Enzyme 36: 150–163), and to serve as the essential tools for the rational design of modified serine proteases and/or their natural inhibitors by the methods available through genetic engineering.     

Evolutionary relationships of the prolyl oligopeptidase family enzymes.

The prolyl oligopeptidase (POP) family of serine proteases includes prolyl oligopeptidase, dipeptidyl peptidase IV, acylaminoacyl peptidase and oligopeptidase B. The enzymes of this family specifically hydrolyze oligopeptides with less than 30 amino acids. Many of the POP family enzymes have evoked pharmaceutical interest as they have roles in the regulation of peptide hormones and are involved in a variety of diseases such as dementia, trypanosomiasis and type 2 diabetes. In this study we have clarified the evolutionary relationships of these four POP family enzymes and analyzed POP sequences from different sources. The phylogenetic trees indicate that the four enzymes were present in the last common ancestor of all life forms and that the beta-propeller domain has been part of the family for billions of years. There are striking differences in the mutation rates between the enzymes and POP was found to be the most conserved enzyme of this family. However, the localization of this enzyme has changed throughout evolution, as three archaeal POPs seem to be membrane bound and one third of the bacterial as well as two eukaryotic POPs were found to be secreted out of the cell. There are also considerable distinctions between the mutation rates of the different substrate binding subsites of POP. This information may help in the development of species-specific POP inhibitors.     

Proteases in egg, miracidium and adult of Fasciola gigantica. Characterization of serine and cysteine proteases from adult

Proteolytic activity of 0-12 day old eggs, miracidium and adult worm of Fasciola gigantica was assessed and proteases were partially purified by DEAE-Sepharose and CM-cellulose columns. Four forms of protease were separated, PIa, PIb, PIc and PII. Purifications were completed for PIc and PII using Sephacryl S-200 chromatography. A number of natural and synthetic proteins were tested as substrates for F. gigantica PIc and PII. The two proteases had moderate activity levels toward azoalbumin and casein compared to azocasein, while gelatin, hemoglobin, albumin and fibrin had very low affinity toward the two enzymes. Amidolytic substrates are more specific to protease activity. PIc had higher affinity toward BAPNA-HCl (N-benzoyl-arginine-p-nitroanilide-HCl) and BTPNA-HCl (N-benzoyl-tyrosine-p-nitroanilide-HCl) at pH 8.0 indicating that the enzyme was a serine protease. However, PII had higher affinity toward BAPNA at pH 6.5 in the presence of sulfhydryl groups (beta-mercaptoethanol) indicating that the enzyme was a cysteine protease. The effect of specific protease inhibitors on these enzymes was studied. The results confirmed that proteases PIc and PII could be serine and cysteine proteases, respectively. The molecular weights of F. gigantica PIc and PII were 60,000 and 25,000, respectively. F. gigantica PIc and PII had pH optima at 7.5 and 5.5 and K(M) of 2 and 5 mg azocasein/mL, respectively. For amidolytic substrates, PIc had K(M) of 0.3 mM BAPNA/mL and 0.5 mM BTPNA/mL at pH 8.0 and PII had K(M) of 0.6 mM BAPNA/mL at pH 6.5 with reducing agent. F. gigantica PIc and PII had the same optimum temperature at 50 degrees C and were stable up to 40 degrees C. All examined metal cations tested had inhibitory effects toward the two enzymes. From substrate specificity and protease inhibitor studies, PIc and PII could be designated as serine PIc and cysteine PII, respectively.     

Purification of rat urinary kallikrein: comparative studies with rat submandibular gland kallikrein-like serine protease.

A 427-fold purification of rat urinary kallikrein (RUK) was achieved in three steps involving chromatography on columns of DEAE-Sepharose CL-6B, gel filtration on Sephadex G-100 and affinity chromatography on a column of benzamidine-Sepharose. Purified enzyme showed a single band on SDS-PAGE with an estimated molecular weight of 43,000. The amino-terminal sequences of the first 25 residues of RUK resemble the reported sequence for true kallikrein and share 80% identity with rat submandibular gland (RSMG) kallikrein-like serine protease. The RUK is highly reactive towards kallikrein substrates Bz-pro-phe-arg-pNA and DL-val-leu-arg-pNA, and plasmin substrate D-val-leu-lys-pNA. RSMG enzyme is more reactive towards Bz-val-gly-arg-pNA and tosyl-gly-pro-arg-pNA, preferential chromogenic substrates for trypsin-like proteases and thrombin, respectively. Both leupeptin and aprotinin inhibit RUK strongly, but soy bean trypsin inhibitor has no effect on this enzyme. RSMG enzyme is poorly inhibited by any of these inhibitors. The data suggest that although both enzymes are members of tissue kallikrein multigene family, urinary enzyme is a true kallikrein and RSMG enzyme is a kallikrein-like serine protease with different substrate specificity.