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Atlantic cod trypsin I is a highly active cold-adapted protease. This study aimed at further characterization of this enzyme with respect to kinetic parameters, sites of autolysis and stability. For that purpose, trypsin I was purified by anion exchange chromatography. Its purity and identity was verified by SDS-PAGE analysis and mass spectrometry. Concomitantly, another cod trypsin isozyme, trypsin X, previously only described from its cDNA sequence was detected in a separate peak from the ion exchange chromatogram. There was a stepwise increase in the catalytic efficiency (k(cat)/K(m)) of cod trypsin I obtained with substrates containing one to three amino acid residues. As expected, the activity of trypsin I was maintained for longer periods of time at 15 degrees C than at higher temperatures. The residues of the trypsin I molecule most sensitive to autolysis were identified using Edman degradation. Eleven autolytic cleavage sites were detected within the trypsin I molecule. Unfolding experiments demonstrated that autolysis is a contributing factor in the stability of trypsin I. In addition, the data shows that cod trypsin I is less stable towards thermal unfolding than its mesophilic bovine analogue.

Metal binding and conformational stability characteristics of psychrophilic elastase (ACE) from Atlantic cod (Gadus morhua) has been investigated. Chelation to Ca(2+) was found to be important for maintaining the biologically active conformation and for the thermal stability of the enzyme. However, presence of metal ions such as Zn(2+), Fe(3+) and Cu(2+) was found to inhibit its hydrolytic activity and so did the chelating agent EDTA. Both pH and guanidinium chloride induced denaturation of the enzyme was followed by monitoring the changes in the tryptophan fluorescence. ACE exhibited a simple two-state unfolding pattern in both acidic and basic conditions with the midpoint of transition at pH values 4.08 and 10.29, respectively. Guanidinium chloride and heat induced denaturation of the enzyme was investigated at two pH values, 5.50 and 8.00, wherein the enzyme possesses similar tertiary structure but differ in its hydrolytic activity. Guanidinium chloride induced denaturation indicated that the enzyme unfolds with a C(m) of 1.53 M at pH 8.0 and a DeltaG(H2O) of 6.91 kJ mol(-1) (28.65 J mol(-1) residue(-1)) which is the lowest reported for psychrophilic enzymes investigated till-date. However, at pH 5.50, DeltaG(H2O) value is slightly lowered by 0.65 kJ mol(-1) consistent with the observed increase in the apparent quenching constant obtained with acrylamide. On the other hand, increase in T(m) by 38.45 degrees C was observed for the enzyme at acid pH (5.50) in comparison to the heat induced unfolding at pH 8.0. The increase in the apparent T(m) has been attributed to the possible weak intermolecular association of the enzyme molecules at moderately high temperatures that is favoured by the increase in the accessible surface area / dynamics under acidic conditions. The stability characteristics of ACE have been compared with the available data for mesophilic porcine pancreatic elastase and possible mechanism for the low temperature adaptation of ACE has been proposed.

Atlantic cod trypsin I is a cold-adapted proteolytic enzyme exhibiting approximately 20 times higher catalytic efficiency (kcat/KM) than its mesophilic bovine counterpart for the simple amide substrate BAPNA. In general, cold-adapted proteolytic enzymes are sensitive to autolytic degradation, thermal inactivation as well as molecular aggregation, even at temperatures as low as 18-25 degrees C which may explain the problems observed with their expression, activation, and purification. Prior to the data presented here, there have been no reports in the literature on the expression of psychrophilic or cold-adapted proteolytic enzymes from fish. Nevertheless, numerous cold-adapted proteolytic microbial enzymes have been successfully expressed in bacteria and yeast. This report describes successful expression, activation, and purification of the recombinant cod trypsin I in the His-Patch ThioFusion Escherichia coli expression system. The E. coli pThioHis expression vector used in the study enabled the formation of a fusion protein between a highly soluble fraction of HP-thioredoxin contained in the vector and the N-terminal end of the precursor form of cod trypsin I. The HP-thioredoxin part of the fusion protein binds to a metal-chelating ProBond column, which facilitated its purification. The cod trypsin I part of the purified fusion protein was released by proteolytic cleavage, resulting in concomitant activation of the recombinant enzyme. The recombinant cod trypsin I was purified to homogeneity on a trypsin-specific benzamidine affinity column. The identity of the recombinant enzyme was demonstrated by electrophoresis and chromatography.

The P-III class of venom metalloproteinases has, in addition to the proteinase domain, a disintegrin-like domain and a cysteine-rich domain. Recent evidence has shown that the nonproteinase domains of the P-III class of hemorrhagic metalloproteinases function in the inhibition of platelet aggregation by blocking essential procoagulant integrins on platelets. A specific role for the highly conserved cysteine-rich domain has yet to be described. In this study, we expressed the cysteine-rich domain from the hemorrhagic metalloproteinase atrolysin A and demonstrated its ability to inhibit collagen-stimulated platelet aggregation. Additionally, the cysteine-rich domain was shown to interact with MG-63 cells to inhibit adhesion to collagen I. These data suggest a functional role for the cysteine-rich domain of the P-III toxins in the observed coagulopathy by targeting the toxin to platelets and inhibiting collagen-stimulated platelet aggregation. These characteristics may function to synergistically increase the hemorrhagic effect of the toxins., (Copyright 2000 Academic Press.)

Snake venom hemorrhagic metalloproteinase toxins that have metalloproteinase, disintegrin-like and cysteine-rich domains are significantly more potent than toxins with only a metalloproteinase domain. The disintegrin-like domains of these toxins differ from the disintegrin peptides found in crotalid and viperid venoms by the nature of their different disulfide bond structure and, in lieu of the disintegrins' signature Arg-Gly-Asp (RGD) integrin binding sequence, there is an XXCD disulfide-bonded cysteinyl sequence in that region. Due to these apparent differences, the contribution to the overall function of the hemorrhagic metalloproteinases by the disintegrin-like domain has been unknown. In this investigation we have expressed in insect cells the disintegrin-like/cysteine-rich (DC) domains of the Crotalus atrox hemorrhagic metalloproteinase atrolysin A and demonstrated that the recombinant protein (A/DC) can inhibit collagen- and ADP-stimulated platelet aggregation. Using synthetic peptides, we have evidence that the region of the disintegrin-like domain that is positionally analogous to the RGD loop of the disintegrins is the site responsible for inhibition of platelet aggregation. For these synthetic peptides to have significant inhibitory activity, the -RSECD- cysteinyl residue must be constrained by participation in a disulfide bond with another cysteinyl residue. The two acidic amino acids adjacent to the middle cysteinyl residue in these peptides are also important for biological activity. These studies emphasize a functional role for the disintegrin-like domain in toxins and suggest structural possibilities for the design of antagonists of platelet aggregation.

A large number of zinc metalloproteinases of varying mol. wts and biological functions has been isolated from crotalid and viperid venoms. Over the past few years, structural studies on these proteinases have suggested their organization into four classes, P-I to P-IV. These proteinases are synthesized in the venom gland as zymogens which are subsequently processed to the active form. The signal and pro-sequences of the proteins are highly conserved. Within the pro-domain lies a consensus sequence which probably functions in a manner similar to the cysteine switch in mammalian collagenases. The proteinase domain is represented by two forms: a two-disulfide and a three-disulfide structure. Crystallographic and modeling studies suggest that the two forms share very similar tertiary structures. The larger venom metalloproteinases (P-II, III and IV) have additional domains on the carboxy side of the proteinase domain. The additional domains that have been identified include disintegrin and disintegrin-like domains, a high-cysteine domain and a lectin-binding domain. It appears that these non-enzymatic domains function to modulate the biological properties of the proteinases. Recently, a family of homologues of the venom zinc metalloproteinases has been described from a variety of organisms including mammals, reptiles and invertebrates. This family of proteins has been termed the ADAMs, for A Disintegrin-like And Metalloproteinase-containing protein. They differ from the venom proteinases in that some of them may not have proteolytic activity. In addition to the domain structure described for the P-III class of venom proteins, the ADAMs have an epidermal growth factor-like domain, a transmembrane domain and a cytoplasmic domain. A description of venom metalloproteinase structure will be outlined in this review, along with the similarities and differences among the venom proteins and the ADAMs family of proteins.

The cDNAs encoding two different Atlantic cod elastases have been isolated and sequenced. The predicted amino acid sequences revealed two preproelastases, consisting of a signal peptide, an activation peptide and a mature enzyme of 242 and 239 amino acids. Amino acid sequence identity between the two cod elastases was 60.1% and identity with mammalian elastases ranged from 50-64%. The two cod elastases contain all the major structural features common to serine proteases, such as the catalytic triad His57, Asp102 and Ser195. Both cod elastases have a high content of methionine, consistent with previous findings in psychrophilic fish enzymes.

A collagenolytic proteinase was purified from the intestines of Atlantic cod by (NH4)2SO4 fractionation, hydrophobic interaction chromatography (phenyl-Sepharose) and ion-exchange chromatography (DEAE-Sepharose). The proteinase has an estimated molecular weight of 24.1 (+/- 0.5) kDa as determined by SDS-PAGE and belongs to the chymotrypsin family of serine proteinases. The enzyme cleaves native collagen types I, III, IV and V, and also readily hydrolyzes succinyl-L-Ala-L-Ala-L-Pro-L-Phe-p-nitroanilide (sAAPFpna), an amide substrate of chymotrypsin, as well as succinyl-L-Ala-L-Ala-L-Pro-L-Leu-p-nitroanilide, a reported elastase substrate, but had no detectable activity towards several other substrates of these proteinases or of trypsin. The pH optimum of the enzyme was between pH 8.0 and 9.5 and it was unstable at pH values below 7. Maximal activity of the enzyme when assayed against sAAPFpna was centered between 45 and 50 degrees C. Calcium binding stabilized the cod collagenase against thermal inactivation, but even in the presence of calcium, the enzyme was unstable at temperatures above 30 degrees C.

A cDNA clone encoding a psychrophilic cod chymotrypsinogen has been isolated and characterized. The predicted amino acid sequence reveals a preproenzyme of 263 amino acids containing a unique 18 residue signal sequence. Amino acid sequence identity between the cod and mammalian chymotrypsinogens is 64-68%. Two highly conserved proline residues are substituted in cod chymotrypsin.

Presented here are four new cDNA sequences for hemorrhagic metalloproteinases from Crotalus atrox venom, hemorrhagic toxins a, b, c, and d. Comparison of the translated open reading frames to the mature protein sequences gives evidence for post-translational processing at both the amino and carboxyl termini. This comparison is also the basis for a new classification system for these precursors, based on their different sizes. Protein sequences in the zymogen region support the hypothesis of a cysteine-switch type mechanism of maintaining latency. The coordination geometry around the active site zinc ion is discussed. The relationship between these venom metalloproteinases and a family of mammalian reproductive proteins is also supported by these sequences. The cysteine pattern of the carboxyl-terminal domain of the largest proteinase, hemorrhagic toxin a, is compared to other venom proteinases and to the mammalian proteins, showing both striking similarities and subtle differences. It would appear that these hemorrhagic toxins have resulted from deletions and subsequent divergence from a larger ancestor, one they may have shared with the aforementioned mammalian reproductive proteins.

One of the more significant consequences of crotalid envenomation is hemorrhage. Over the past 50 years of investigation, it is clear that the primary factors responsible for hemorrhage are metalloproteinases present in the venom of these snakes. The biochemical basis for their activity is the proteolytic destruction of basement membrane and extracellular matrix surrounding capillaries and small vessels. These proteinase toxins may also interfere with coagulation, thus complementing loss of blood from the vasculature. Structural studies have shown that these proteinases are synthesized as zymogens and are processed at both the amino and carboxy termini to give the mature protein. The variety of hemorrhagic toxins found in snake venoms is due to the presence of structurally related proteins composed of various domains. The type of domains found in each toxin plays an important role in the hemorrhagic potency of the protein. Recently, structural homologs to the venom hemorrhagic metalloproteinases have been identified in several mammalian reproductive systems. The functional significance of the reproductive proteins is not clear, but in light of the presence of similar domains shared with the venom metalloproteinases, their basic biochemical activities may be similar but with very different consequences. This review discusses the history of hemorrhagic toxin research with emphasis on the Crotalus atrox proteinases. The structural similarities observed among the hemorrhagic toxins are outlined, and the structural relationships of the toxins to the mammalian reproductive proteins are described.

The cDNAs encoding two different anionic forms of Atlantic cod trypsinogen have been isolated and sequenced. The nucleotide sequences include the 5'-noncoding and 3'-noncoding regions in addition to preproenzymes of 241 amino acids. These consist of hydrophobic signal peptides, activation hexapeptides and trypsins of 222 amino acid residues. The cod trypsins contain all the major structural features common to trypsins such as the catalytic triad His57, Asp102 and Ser195. Furthermore, the obligatory Asp189 and the six disulphide bonds are conserved. Eight amino acid residues are different between the isozymes, leading to a difference of four charges. Both cod trypsins are one-amino-acid-residue shorter than most mammalian trypsins as a result of deletion of proline at position 152, and have a high methionine content. In addition, the cod preproenzyme signal and activation peptides differ markedly from their mammalian analogues. The amino acid identity between the cod and bovine trypsins is approximately 60%.

An intestinal elastase was purified from Atlantic cod (Gadus morhua) of apparent molecular mass 24.8 kDa as determined by SDS-PAGE and with isoelectric point above pI 9.3. Heat stability and stability towards acidic pH was reduced in the cod enzyme as compared with porcine intestinal elastase. N-terminal amino-acid sequence analysis of cod elastase showed considerable similarity with porcine elastase. The cod enzyme was less sensitive to phenylmethylsulfonyl fluoride inhibition than porcine elastase, but sensitivity towards other inhibitors was similar. Kinetic properties were examined using the substrate Suc-Ala-Ala-Ala-p-nitroanilide and the cod enzyme was found to have more than 2-times turnover rate (kcat) as compared with the porcine enzyme, and slightly higher Km values. Thus, the catalytic efficiency (kcat/Km) of Atlantic cod elastase was about 2-times higher than observed with porcine elastase, which indicates an adaptive response towards the low temperature environmental in which the cod lives. Substrate specificity was studied by digestion of oxidized B-chain of insulin and by using synthetic substrates. Digestion was most rapid at the carbonyl side of alanine residues, but also occurred at valine and leucine residues.

The sequence of two overlapping cDNA clones for the zinc metalloproteinase hemorrhagic toxin e (also known as atrolysin e, EC 3.4.24.44) from the venom gland of Crotalus atrox, the Western diamondback rattlesnake, is presented. The assembled cDNA sequence is 1975 nucleotides in length and encodes an open reading frame of 478 amino acids. The mature hemorrhagic toxin e protein as isolated from the crude venom has a molecular weight of approximately 24,000 and thus represents the processed product of this open reading frame. From the deduced amino acid sequence, it can be hypothesized that the enzyme is translated with a signal sequence of 18 amino acids, an amino-terminal propeptide of 169 amino acids, a central hemorrhagic proteinase domain of 202 amino acids, and a carboxy-terminal sequence of 89 amino acids. The propeptide has a short region similar to the region involved in the activation of matrix metalloproteinase zymogens. The proteinase domain is similar to other snake venom metalloproteinases, with over 57% identity to the low molecular weight proteinases HR2a and H2-proteinase from the Habu snake Trimeresurus flavoviridis. The carboxy-terminal region, which is not observed in the mature protein, strongly resembles the protein sequence immediately following the proteinase domain of HR1B (a high molecular weight hemorrhagic proteinase from the venom of T. flavoviridis) and the members of a different family of snake venom polypeptides known for their platelet aggregation inhibitory activity, the disintegrins. The cDNA sequence bears striking similarity to a previously reported sequence for a disintegrin cDNA. This report is evidence that this subfamily of venom metalloproteinases is synthesized in a proenzyme form which must be proteolytically activated.(ABSTRACT TRUNCATED AT 250 WORDS)

Recently, the complete amino acid sequences have been determined for several snake venom metalloproteinases from the genera Crotalus, Trimeresurus and Lachesis of the Crotalidae family. Among these are both hemorrhagic and nonhemorrhagic metalloproteinases. Despite differences in their molecular weights and activities, they all appear to be related through a single ancestral gene, as observed by the comparison of their amino acid sequences. None of these proteins bear significant similarity to any other known protein, with the exception of the zinc binding site demonstrated in thermolysin and other metalloproteinases. Thus we propose that these proteins define a new family of zinc metalloproteinases which is likely to consist of (but is not necessarily limited to) many of the zinc metalloproteinases found in snake venoms.

1. Two chymotrypsins with isoelectric points pI 6.2 and 5.8 were purified from the pyloric caeca of Atlantic cod using a phenyl-Sepharose column and chromatofocusing chromatography. The apparent molecular weight was 26,000 as judged by SDS-polyacrylamide gel electrophoresis and gel filtration. 2. The cod enzymes differed from bovine chymotrypsin in having a slightly higher molecular weight and more acidic pI points. N-terminal amino acid sequence analysis of cod chymotrypsin B showed considerable similarity with bovine chymotrypsin. 3. Heat stability and stability towards acidic pH were reduced in the cod enzymes. Generally, the cod and bovine chymotrypsins responded similarly to various protease inhibitors. However, the cod chymotrypsins were less sensitive to aprotinin inhibition but more sensitive towards soybean trypsin inhibitor and cysteine. 4. Kinetic properties were examined and the cod enzymes found to be more active towards both ester (N-benzoyl-tyrosine ethyl ester) and amide (N-benzoyl-tyrosine-p-nitroanilide) substrates. The observed differences in kinetic properties are indicative of an adaptive response towards the low temperature environment in which the cod lives.

The venoms of most Crotalidae snakes contain metalloproteinases which are the agents responsible for the production of venom-induced hemorrhage via proteolytic destruction of capillary basement membranes. Prevention of hemorrhage by administration of antiserum is generally not totally effective against damage at the site of envenomation. Therefore, we have investigated alternate methods for the alleviation of hemorrhage by inhibition of the proteolytic activity of the hemorrhagic toxins. The first approach involves the synthesis of carboxyalkyl peptide inhibitors in which the peptide moiety is modeled on the substrate specificity of the toxins. With this approach we have determined that the carboxypentyl group for interaction with the active site Zn+ +ion is most effective. Also, longer peptide moieties enhance the inhibitors' activity giving Ki's in a range of 10(-6) M. Our second approach to hemorrhagic toxin inhibition was to search for the presence of endogenous inhibitors against the toxins in the venom. From the crude venom we have isolated several pyro-glutamate containing peptides, two of which are relatively good inhibitors of the toxins. The isolation and characterization of the endogenous toxin inhibitors as well as the synthetic inhibitors may ultimately serve as a basis for new, effective treatments against venom-induced hemorrhage.

Hemorrhagic toxin e (Ht-e), a metalloproteinase isolated from the venom of the Western Diamondback rattlesnake Crotalus atrox, digests laminin and nidogen, both in their isolated forms and when present in a purified soluble complex. The only common site of cleavage by Ht-e of isolated nidogen and nidogen when complexed with laminin is at amino acid residue 336 in the amino terminal domain. Additionally, nidogen in complex with laminin is also cleaved at sites 322, 351 and 840 as determined by sequence analysis and site 953 as proposed from the molecular mass of a digestion product. Isolated nidogen, on the other hand, was cleaved at amino acid residues 75, 336, 402, and 920, as determined by sequence determinations and approximately at residues 296, 478, 625 and 702 as proposed from the molecular mass values of the generated polypeptide chains. Products from the proteolytic cleavage of the A and B2 chains of laminin were observed with the sites of cleavage determined to be at position 2666 in the laminin A chain and position 1238 in the laminin B2 chain. The laminin digestion products were identical regardless of whether nidogen was present in a complex with the laminin chains.

Type IV collagen, solubilized from Engelbreth-Holm-Swarm (EHS) tumor basement membranes is digested by a hemorrhagic metalloproteinase, Ht-e, isolated from the crude venom of the Western Diamondback rattlesnake, Crotalus atrox. The major proteolytic products have Mr 141,000, 132,000, 87,000, 71,000, 33,000 and approximately 18,000 as estimated by SDS-gel electrophoresis of pepsinized type IV collagen fragments. Sequence analysis of the digestion products reveal that the Mr 141,000, 71,000 and approximately 18,000 band are derived from the alpha 1(IV) chains and the Mr 132,000, 87,000 and 33,000 bands are derived from the alpha 2(IV) chain. The products are stable over 72-hour incubation periods. The cleavage sites on the alpha 1(IV) and alpha 2(IV) chains are not identical. The alpha 1(IV) chains are cleaved in a pepsin susceptible triplet interruption region of the triple helix at position Ala258-Gln259. The alpha 2(IV) chain is cleaved in the triple helical region near the NC2 domain at the Gly191-Leu192 peptide bond. Isolated hexameric NC1 globular domains of type IV collagen are not digested by Ht-e. The present study demonstrates that the venom hemorrhagic metalloproteinase Ht-e has type IV collagenolytic activity. The triple helix of the type IV collagen molecule is cleaved in a region located immediately carboxyl to the flexible NC2 domain. The degradation by Ht-e of type IV collagen, a major component of basement membranes which forms the scaffold of this extracellular structure, may account in part for the hemorrhagic activity of this toxin.

The interaction between four Crotalus atrox hemorrhagic metalloproteinases and human alpha 2-macroglobulin was investigated. The proteolytic activity of the hemorrhagic toxins Ht-c, -d, and -e against the large molecular weight protein substrates, gelatin type I and collagen type IV, was completely inhibited by alpha 2-macroglobulin. The proteolytic activity of Ht-a against the same substrates was not significantly inhibited. Each mole of alpha 2-macroglobulin bound maximally 2 mol of Ht-e and 1.1 mol of Ht-c and Ht-d. These proteinases interacted with alpha 2-macroglobulin rapidly at 22 degrees C. Rate constants based on intrinsic fluorescence measurements were 0.62 X 10(5) M-1 s-1 for interaction of alpha 2-macroglobulin with Ht-c and -d and 2.3 X 10(5) M-1 s-1 for the interaction of alpha 2-macroglobulin with Ht-e. Ht-a interacted with alpha 2-macroglobulin very slowly at 22 degrees C. Increasing the temperature to 37 degrees C and prolonging the time of interaction with alpha 2-macroglobulin resulted in the formation of Mr 90,000 fragments and high molecular weight complexes (Mr greater than 180,000), in which Ht-a is covalently bound to the carboxy-terminal fragment of alpha 2-M. The identification of the sites of specific proteolysis of alpha 2-macroglobulin shows that the cleavage sites for the four metalloproteinases are within the bait region of alpha 2-macroglobulin. Ht-c and -d cleave only at one site, the Arg696-Leu697 peptide bond, which is also the site of cleavage for plasmin, thrombin, trypsin, and thermolysin. Ht-a cleaves alpha 2-macroglobulin primarily at the same site, but a secondary cleavage site at the His694-Ala695 peptide bond was also identified.(ABSTRACT TRUNCATED AT 250 WORDS)

1. Three pepsins were purified from the gastric mucosa of Atlantic cod (Gadus morhua). 2. The enzymes, called Pepsin I and Pepsin IIa and b, had isoelectric points 6.9, 4.0 and 4.1, respectively, and digested hemoglobin at a maximal rate at a pH of approximately 3. 3. They resembled bovine cathepsin D in being unable to digest the mammalian pepsin substrate N-acetyl-L-phenylalanyl-3,5-diiodo-L-tyrosine. 4. Specificity constants (kcat/Km) for the cod pepsins were lower than for porcine pepsin, and they expressed higher substrate affinity and physiological efficiency at pH 3.5 than at pH 2. 5. The cod pepsins are glycoproteins, and their amino acid composition resembles that of porcine cathepsin D more than that of porcine pepsin. 6. The N-terminal sequence of Atlantic cod pepsins is substantially different from that of porcine pepsin. This indicates a significant evolutionary gap between fish and mammalian pepsins.

The hemorrhagic toxin Ht-d from venom of the Western diamondback rattlesnake is a metalloproteinase with a molecular weight of 23,234. Peptides were obtained from enzymatic and chemical digestions, separated by reverse-phase chromatography, and sequenced in a gas-phase sequenator. The sequence showed a putative zinc binding site similar to that of thermolysin and other metalloproteinases but no overall significant similarity to the sequences of other metalloproteinases and may represent a new subfamily of metalloproteinases. Ht-d was shown to degrade type IV collagen and gelatin types I, III, and V but not interstitial collagens. The digestion of type IV collagen and other basement membrane proteins may allow this proteinase to disrupt capillary membranes causing hemorrhage in surrounding tissues.

Five previously unknown hemorrhagic proteins, designated hemorrhagic toxins a,b,c,d, and e, were isolated from the venom of the western diamondback rattlesnake (Crotalus atrox). Molecular weights of hemorrhagic toxins a-e were determined to be 68 000, 24 000, 24 000, 24 000, and 25 700, respectively, by sodium dodecyl sulfate-phosphate gel electrophoresis using various polyacrylamide gel concentrations. Amino acid composition showed a total of 636, 200, 213, 214, and 219 amino acids for hemorrhagic toxins a-e, respectively. All the hemorrhagic toxins were found to lose their hemorrhagic activities with the metal chelators ethylenediaminetetraacetic acid and 1, 10-phenanthroline. All the hemorrhagic toxins were found to contain approximately 1 mol of zinc/mol of toxin, and they were all demonstrated to be proteolytic when dimethylcasein and dimethylhemoglobin were used as substrates. When zinc was removed from hemorrhagic toxin e with 1,10-phenanthroline, both both the proteolytic and hemorrhagic activities were equally inhibited. When the apohemorrhagic toxin e thus produced was incubated with zinc, the hemorrhagic and proteolytic activities were regenerated to the same extent. CD, UV, and Raman spectroscopy were used to study the structure of native hemorrhagin toxin e as well as the structural changes caused by zinc removal. From CD spectroscopy the native toxin was estimated to consist of 23% alpha helix, 6% beta structure, and 71% random-coil conformation. When over 90% of the zinc was removed, the alpha-helix content dropped from 23 to 7%.

The symptoms which immediately follow envenomation by many crotalid snakes include hypotension, hypovolemia, hemoconcentration, and shock. We have isolated and characterized two proteases (EI and EII) from the venom of Crotalus atrox which may be involved in the onset of these symptoms. EI and EII have molecular weights of 27,500 and 29,200 and isoelectric points of 4.7 and 4.3, respectively. Specific esterolytic activities of EI and EII on N alpha-p-tosyl-L-arginine methyl ester are 51.5 mumol min-1mg-1 and 48.1 mumol min-1 mg-1, respectively. Both enzymes are rather specific in their substrate requirements in that neither was demonstrated to have any proteolytic activity against either of the oxidized chains of insulin, or glucagon. Neither enzyme was shown to have plasmin or fibrinolytic activity. Both enzymes are able to cleave a kininogen analog to release bradykinin. This proteolytic activity is inhibited by aprotinin and phenylmethanesulfonyl fluoride but not by ethylenediaminetetraacetate. The enzymes are active upon the kallikrein substrates S2666 and S2302. The Km values of the enzymes with these substrates are similar to those reported for kallikrein. Structural similarity between the two enzymes was demonstrated by ultraviolet and circular dichroic spectroscopy, and amino acid analysis. Tryptic peptide mapping of the two native enzymes also suggested a large degree of structural similarity. Furthermore, sequence studies on the NH2-terminal regions of the enzymes indicate that they share a significant degree of sequence homology with porcine kallikrein and crotalase, a kallikrein-like enzyme from Crotalus adamanteus. The main physical difference between the two kallikreins reported here appears to be due to the carbohydrate moieties on the enzymes. At present the in vivo role of venom kallikreins in envenomation pathology is uncertain; however, it is possible that they play an important part in giving rise to the initial symptoms of hypotension and shock.

The sites and relative rates of peptide bond hydrolysis of the oxidized B chain of bovine insulin by two hemorrhagic proteinases, Ht-a and Ht-b, isolated from the venom of the Western Diamondback Rattlesnake, Crotalus atrox, were investigated. The results were compared with previous results on the digestion of the same substrate by the C. atrox hemorrhagic proteinases Ht-c, d, and e. Both toxins, Ht-a and Ht-b, were found to cleave the His5-Leu6, His10-Leu11, Ala14-Leu15, and Tyr16-Leu17 bonds of the oxidized insulin B chain. In addition, Ht-a cleaves the Asn3-Gln4 bond, whereas Ht-b cleaves the Gly23-Phe24 bond. The cleavage specificity of Ht-b on the insulin B chain is identical to that of the weakly hemorrhagic isoenzymes Ht-c and Ht-d. Hemorrhagic proteinase Ht-a cleaves the Ala14-Leu15 bond most rapidly, having a turnover number of 12.6 min-1 which was approximately twice as fast as the Tyr16-Leu17 bond turnover of cleavage. Hemorrhagic proteinase Ht-b, on the other hand, cleaves the Tyr16-Leu17 bond fastest, having a turnover number of 61.3 min-1. The Ala14-Leu15 bond is the peptide bond cleaved the second fastest by Ht-b, with a turnover number of 48.2 min-1. The five hemorrhagic toxins from Crotalus atrox venom were also examined for their capability to hydrolyse basement membrane preparations, and the identities of the digested proteins comprising the basement membrane were determined. From the results it is concluded that all five hemorrhagic toxins cleave laminin and the component of the basement membrane referred to as band a.(ABSTRACT TRUNCATED AT 250 WORDS)

A serine protease shown to be trypsin was purified from the pyloric caeca of Atlantic cod (Gadus morhua), and resolved into three differently charged species by chromatofocusing (pI 6.6, 6.2 and 5.5). All three trypsins had similar molecular mass of 24.2 kDa. N-terminal amino acid sequence analysis of cod trypsin showed considerable similarity with other known trypsins, particularly with dogfish and some mammalian trypsins. The apparent Km values determined at 25 degrees C for the predominant form of Atlantic cod trypsin towards p-tosyl-L-arginine methyl ester and N-benzoyl-L-arginine p-nitroanilide were 29 microM and 77 microM respectively, which are notably lower values than those determined for bovine trypsin (46 microM and 650 microM respectively). The difference was particularly striking when the amidase activity of the enzymes was compared. Furthermore, the kcat values determined for the Atlantic cold trypsins were consistently higher than the values determined for bovine trypsin. The higher catalytic efficiency (kcat/Km) of Atlantic cod trypsin as compared to bovine trypsin may reflect an evolutionary adaptation of the poikilothermic species to low environmental temperatures.

The proteolytic activity of four hemorrhagic metalloproteinases (Ht-a, c, d, and e) isolated from the venom of the Western diamondback rattlesnake (Crotalus atrox) was investigated using isolated extracellular matrix (ECM) proteins. We determined that all of the proteinases are capable of cleaving fibronectin, laminin, type IV collagen, nidogen (entactin), and gelatins. However, none of the proteinases were proteolytic against the interstitial collagen types I and III or type V collagen. With all of the substrates listed above Ht-c and Ht-d produced identical digestion patterns, as would be expected for these isoenzymes. With fibronectin, Ht-a produces a different ratio of products from Ht-c and Ht-d, while Ht-e produces a unique pattern of digestion. Ht-e and Ht-a produced nonidentical patterns with the laminin/nidogen preparation although some similarity was shared between them as well as with the Ht-c/d digestion pattern. Similar results were also observed for these proteinases with nidogen 150 as the substrate. The type IV collagen digestion patterns by Ht-e and Ht-a were similar to the pattern observed with Ht-c/d but differed by two bands. The digestion patterns of the three gelatins produced by the proteinases show differences between Ht-c and Ht-d when compared to Ht-e and Ht-a. This investigation clearly shows that several of the ECM proteins are efficiently digested by these toxins. The proteinases have some digestion sites in common but show differing specificities. In addition, the range of ECM proteins digested by these hemorrhagic proteinases is nearly identical to that demonstrated by the ECM proteinase stromelysin (MMP-3). From these data, and the knowledge of the roles these ECM proteins have in maintaining basement membrane structural/functional integrity, one can envision that the degradation of these ECM proteins could readily lead to loss of capillary integrity resulting in hemorrhage occurring at those sites.

The proteolytic specificities of two zinc hemorrhagic toxins (Ht-c and Ht-d), isolated from Crotalus atrox venom, were investigated by using the oxidized B chain of bovine insulin and synthetic peptide substrates. The enzymes cleaved the Ala14-Leu15 bond of the insulin B chain most rapidly and the Tyr16-Leu17 slightly more slowly. The His5-Leu6, His10-Leu11, and Gly23-Phe24 bonds were also cleaved but at considerably slower rates. In order to assess the substrate length preferences of the enzymes, peptide analogs of the B chain about the Ala14-Leu15 bond were synthesized ranging in length from four to seven residues. The heptapeptide NH2-Leu-Val-Glu-Ala-Leu-Tyr-Leu-COOH was the best peptide substrate tested with the other peptides having decreasing kcat/Km values with decreasing length. The tetrapeptide NH2-Ala-Leu-Tyr-Leu-COOH was not cleaved by the enzymes. Furthermore, this peptide was shown to serve as a competitive inhibitor of the toxins. The N-acetylated pentapeptides and hexapeptides, synthesized to probe the active site environment of the enzymes, were significantly better substrates than their unacetylated counterparts. The toxins had the highest kcat/Km values for the acetylated peptide Ac-Val-Ala-Leu-Leu-Ala-COOH. The data suggest that the toxins may indeed have extended substrate-binding sites, which may accommodate at least six amino acid residues. The best substrate examined thus far for the toxins is the fluorogenic peptide analog 2-aminobenzoyl-Ala-Gly-Leu-Ala-4-nitrobenzylamide, suggestive of similarities between the toxins and mammalian collagenases as well as thermolysin. Mechanisms for inhibition of the enzymes were investigated using amino acid hydroxamates, chloromethyl esters, phosphoramidon and the peptide NH2-Ala-Leu-Tyr-Leu-COOH. All of these inhibitors had Ki values in the 10(-4) M range.

The peptide backbone conformation and salient structural details of oxytocin were examined by laser Raman spectroscopy. Spectra were obtained in the solid phase, water, 2H2O, and dimethyl sulfoxide solutions. A distinct Amide I band was obtained at 1663 cm-1 for aqueous and deuterated samples and 1666 cm-1 for the solid sample. A relatively high frequency Amide III band at 1260 cm-1 was obtained. It is concluded that these Amide I and III bands arise from the "beta-turn"-like conformation of oxytocin. The tyrosine side chain, according to the I850 cm-1/I830 cm-1 intensity ratio, is exposed to the solvent. The S-S stretching vibration at 512 cm-1 indicates the conformation of C-C-S-S-C-C in the disulfide bridge of oxytocin in the ring is gauche-gauche-gauche.

Two hemorrhagic proteinases from Crotalus atrox venom, hemorrhagic toxin c (Ht-c) and hemorrhagic toxin d (Ht-d), were characterized and compared to one another. The two toxins are zinc metalloproteinases which both have molecular weights of 24,000. Their isoelectric points are slightly acidic, Ht-c being the more basic of the two with an isoelectric point of 6.2, whereas Ht-d has an isoelectric point of 6.1. Only minor differences were found in the amino acid compositions of the two toxins. The toxins were both demonstrated to be hemorrhagic, using an in vivo assay, and also proteolytic. Prior treatment of the hemorrhagic proteinases with ethylenediaminetetraacetic acid and o-phenanthroline eliminated both the hemorrhagic and the proteolytic activities. Aprotinin and phenylmethylsulfonyl fluoride had no effect upon these activities. The pH optimum of the proteolysis by Ht-c and Ht-d on hide powder azure as the substrate was between pH 8 and pH 9. The circular dichroism spectra for Ht-c and Ht-d appear almost identical with respect to minima positions and elipticities, indicative of very similar solution structures for the two enzymes. Antiserum raised in mice against Ht-c was assayed on double-diffusion Ouchterlony plates for cross-reactivity with other hemorrhagic toxins from C. atrox venom. From this experiment it was concluded that the two hemorrhagic proteinases Ht-c and Ht-d share identical antigenic structures. This was corroborated by tryptic mapping of the two toxins. Only one major difference was observed from the maps. In the case of Ht-c, it was determined that an aspartate was substituted by an alanine when compared to Ht-d. From these characterization studies we conclude that Ht-c and Ht-d are isoenzymes with only very minor differences in their structures.