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PubMed1991

Plasminogen activators and inhibitors in the neuromuscular system: III. The serpin protease nexin I is synthesized by muscle and localized at neuromuscular synapses.

Recent studies suggest that the nature of events leading to the formation, maintenance, and elimination of synapses may be regulated by cascade-type, locally expressed proteases and protease inhibitors acting on adhesive extracellular matrix components. We have identified a molecule in conditioned medium of murine skeletal muscle cells that in molecular weight, target protease inhibition, heparin-binding and cross-reactivity with authenic antisera is similar to the human serine proteinase inhibitor, protease nexin I. Protease nexin I is a 43-50 kDa glycoprotein of the serpin superfamily (arg-serpin class). Purified anti-protease nexin I antibody (anti-47 kDa) stains adult mouse skeletal muscle in discrete foci that precisely superimpose on synaptic neuromuscular junctions. Protease nexin I appears in patches on surfaces of cultured mouse skeletal myotubes, but not on myoblasts. These patches co-localize with acetylcholine receptor clusters and acetylcholinesterase staining during cellular maturation in culture. Evidence that protease nexin I is a synaptic, extracellular antigen is particularly intriguing since it has been shown to be identical, in structure and activity, with a factor released by glial cells, called glia-derived nexin that stimulates mouse neuroblastoma cell neurite outgrowth and inhibits granule cell migration. Protease nexin I inhibits both tumor cell and myoblast plasminogen activator-mediated destruction of extracellular matrix. Thus, such observations as presented in this report provide further evidence for involvement of cascade proteolytic systems, and their post-translational regulation by specific serpins, in the remodeling that occurs in synapse formation and elimination.

Amyloid beta-Protein Precursor↗

PubMed1980

Inhibition of cell proliferation and protease activity by cartilage factors and heparin.

Proliferating rat smooth muscle cells and fibroblasts have membrane-associated protease activity. High concentrations of heparin inhibited membrane-associated protease activity and cell proliferation, while low concentration of heparin promoted smooth muscle cell proliferation. The inhibition of protease activity and proliferation was abolished when heparin was treated with protamine sulfate or when acid treated fetal calf serum was used. Heparin required the presence of an acid labile factor(s) in serum for the inhibition of protease activity and proliferation. Heparin and antithrombin III in the presence of acid-treated fetal calf serum did not inhibit cell proliferation or protease activity. Cartilage factors isolated from bovine nasal cartilage containing trypsin inhibitory activity, but not papain inhibitory activity, inhibited rat smooth muscle and fibroblast proliferation and surface associated protease activity. The cartilage factors did not require acid-labile components in the fetal calf serum for the inhibitory activity. The inhibitory activity due to heparin and cartilage factors was not permanent under our experimental condition. Protein synthesis was not inhibited by heparin or the cartilage factors. In rat smooth muscle cells and fibroblasts, the expression of surface-associated protease activity was related to the proliferative state of the cells. Surface protease activity was only present on proliferating cells. When surface protease activity was inhibited by high concentrations of heparin in the presence of an acid-labile serum component(s) or cartilage factors, cell proliferation was also inhibited.

Animals↗

PubMed1980

Properties of a calcium-activated protease in squid axoplasm which selectively degrades neurofilament proteins.

Axoplasm extruded from the giant axon of the squid contains Ca2+-activated proteases. The protease in the 100,000 x g of supernatant of axoplasm is very specific and degrades only the 200,000 MW, neurofilament protein (NF200), whereas the protease(s) in the pellet has a much wider range of substrate specificity. The activation of the supernatant protease is restricted to the Ca2+ ion, and no other divalent cation will substitute. The protease requires Ca2+ at a higher concentration than 0.5 mM for activation, and has a pH optimum of about 7.5. Degradation of the NF200 appears to proceed through a 100,000 MW and possibly a 47,000--50,000-MW intermediate form before degradation to TCA-soluble peptides. Activity of the protease is inhibited by divalent cation chelators, Cu2+ and Fe2+, sulphydryl inhibitors, and leupeptin. This specific Ca2+-activated protease in squid axoplasm has identical properties to Ca2+-activated proteases found in various non-neural tissues. Despite its narrow protein substrate specificity, Ca2+-activated protease purified from human platelets effectively degrades squid NF200, suggesting a possible structural relationship between platelet and muscle actin-binding proteins and neurofilament proteins.

Animals↗

PubMed1998

Active-site mobility in human immunodeficiency virus, type 1, protease as demonstrated by crystal structure of A28S mutant.

The mutation Ala28 to serine in human immunodeficiency virus, type 1, (HIV-1) protease introduces putative hydrogen bonds to each active-site carboxyl group. These hydrogen bonds are ubiquitous in pepsin-like eukaryotic aspartic proteases. In order to understand the significance of this difference between HIV-1 protease and homologous, eukaryotic aspartic proteases, we solved the three-dimensional structure of A28S mutant HIV-1 protease in complex with a peptidic inhibitor U-89360E. The structure has been determined to 2.0 A resolution with an R factor of 0.194. Comparison of the mutant enzyme structure with that of the wild-type HIV-1 protease bound to the same inhibitor (Hong L, Treharne A, Hartsuck JA, Foundling S, Tang J, 1996, Biochemistry 35:10627-10633) revealed double occupancy for the Ser28 hydroxyl group, which forms a hydrogen bond either to one of the oxygen atoms of the active-site carboxyl or to the carbonyl oxygen of Asp30. We also observed marked changes in orientation of the Asp25 catalytic carboxyl groups, presumably caused by the new hydrogen bonds. These observations suggest that catalytic aspartyl groups of HIV-1 protease have significant conformational flexibility unseen in eukaryotic aspartic proteases. This difference may provide an explanation for some unique catalytic properties of HIV-1 protease.

Binding Sites↗

PubMed1997

Evaluation of the susceptibility of the 3C proteases of hepatitis A virus and poliovirus to degradation by the ubiquitin-mediated proteolytic system.

The picornavirus 3C proteases are required for the processing of viral polyproteins during infections of host cells. Here we report that the 3C protease of the hepatitis A virus, like that of the encephalomyocarditis virus, is a substrate for rapid, ubiquitin-mediated degradation in vitro. Ubiquitin was shown to stimulate the turnover of the hepatitis virus 3C protease, and labeled protease was found to become incorporated into a mixture of high molecular weight species, which is characteristic of conjugation with polyubiquitin chains. In the presence of methylated ubiquitin, a new 33 kDa species formed, consistent with the generation of a monoubiquitin-3C protease conjugate. The rate of degradation of the 3C protease was reduced by inhibitors of the 26S proteasome. A similar evaluation of the 3C protease of poliovirus revealed that it is stable protein and is not conjugated with ubiquitin. It was also determined that the hepatitis A and encephalomyocarditis virus 3C proteases compete with each other for conjugation with ubiquitin and for degradation. This suggests that the two 3C proteases are both recognized by the same ubiquitin system enzyme, or enzymes, responsible for selecting them as targets for destruction.

3C Viral Proteases↗

PubMed1998

The structural stability of the HIV-1 protease.

The most common strategy in the development of HIV-1 protease inhibitors has been the design of high affinity transition state analogs that effectively compete with natural substrates for the active site. A second approach has been the development of compounds that inactivate the protease by destabilizing its quaternary or tertiary structure. A successful optimization of these strategies requires an accurate knowledge of the energetics of structural stabilization and binding, and the identification of those regions in the protease molecule that are critical to stability and function. Here the energetics of stabilization of the HIV-1 protease has been measured for the first time by high sensitivity differential scanning calorimetry. These studies have permitted the evaluation of the different components of the Gibbs energy of stabilization (the enthalpy, entropy and heat capacity changes). The stability of the protease is pH-dependent and due to its dimeric nature is also concentration-dependent. At pH 3.4 the Gibbs energy of stabilization is close to 10 kcal/mol at 25 degreesC, consistent with a dissociation constant of 5x10(-8) M. The stability of the protease increases at higher pH values. At pH 5, the Gibbs energy of stabilization is 14.5 kcal/mol at 25 degreesC, consistent with a dissociation constant of 2.3x10(-11) M. The pH dependence of the Gibbs energy of stabilization indicates that between pH 3.4 and pH 5 an average of 3-4 ionizable groups per dimer become protonated upon unfolding. A structure-based thermodynamic analysis of the protease molecule indicates that most of the Gibbs energy of stabilization is provided by the dimerization interface and that the isolated subunits are intrinsically unstable. The Gibbs energy, however, is not uniformly distributed along the dimerization interface. The dimer interface is characterized by the presence of clusters of residues (hot spots) that contribute significantly and other regions that contribute very little to subunit association. At the dimerization interface, residues located at the carboxy and amino termini contribute close to 75% of the total Gibbs energy (Cys95, Thr96, Leu97, Asn98 and Phe99 and Pro1, Ile3, Leu5). Residues Thr26, Gly27 and Asp29 located at the base of the active site are also important, and to a lesser extent Gly49, Ile50, Gly51 located at the tip of the flap region. The structure-based thermodynamic analysis also predicts the existence of regions of the protease with only marginal stability and a high propensity to undergo independent local unfolding. In particular, the flap region occupies a very shallow energy minimum and its conformation can easily be affected by relatively small perturbations. This property of the protease can be related to the ability of some mutations to elicit resistance towards certain inhibitors.

Calorimetry, Differential Scanning↗

PubMed2001

Functional determinants of the Epstein-Barr virus protease.

Herpesvirus proteases are essential for the production of progeny virus. They cleave the assembly protein that fills the immature capsid in order to make place for the viral DNA. The recombinant protease of the human gamma-herpesvirus Epstein-Barr virus (EBV) was expressed in Escherichia coli and purified. Circular dichroism indicated that the protein was properly folded with a secondary structure content similar to that of other herpesvirus proteases. Gel filtration and sedimentation analysis indicated a fast monomer-dimer equilibrium of the protease with a K(d) of about 60 microM. This value was not influenced by glycerol but was lowered to 1.7 microM in the presence of 0.5 M sodium citrate. We also developed an HPLC-based enzymatic assay using a 20 amino acid residue synthetic peptide substrate derived from one of the viral target sequences for the protease. We found that conditions that stabilised the dimer also led to a higher enzymatic activity. Through sequential deletion of amino acid residues from either side of the cleavage site, the minimal peptide substrate for the protease was determined as P5-P2'. This minimal sequence is shorter than that for other herpesvirus proteases. The implications of our findings are discussed with reference to the viral life-cycle. These results are the first ever published on the EBV protease and represent a first step towards the development of protease inhibitors.

Amino Acid Sequence↗

PubMed2002

Naturally occurring amino acid polymorphisms in human immunodeficiency virus type 1 (HIV-1) Gag p7(NC) and the C-cleavage site impact Gag-Pol processing by HIV-1 protease.

Human immunodeficiency virus type 1 (HIV-1) protease activity is targeted at nine cleavage sites comprising different amino acid sequences in the viral Gag-Pol polyprotein. Amino acid polymorphisms in protease and in regions of Gag, particularly p7(NC) and the C-cleavage site between p2 and p7(NC), occur in natural variants of HIV-1 within infected patients. Studies were designed to examine the role of natural polymorphisms in protease and to identify determinants in Gag that modulate protease processing activity. Closely related Gag-Pol regions from an HIV-1-infected mother and two children were evaluated for processing in an inducible expression system, for protease activity on cleavage-site analogues, and for impact on replication by recombinant viruses. Gag-Pol regions displayed one of three processing phenotypes based on the appearance of Gag intermediates and accumulation of mature p24(CA). Gag-Pol regions that were processed rapidly to produce p24(CA) resulted in high-level replication by recombinant viruses, while slow-processing Gag-Pol variants resulted in recombinant viruses that replicated with reduced kinetics in both T cell lines and peripheral blood mononuclear cells. Direct impact by Gag sequences on processing by protease was assessed by construction of chimeric Gag-Pol regions and by site-directed mutagenesis. Optimal protease activity occurred when Gag and Pol regions were derived from the same gag-pol allele. Heterologous Gag regions generally diminished rates and extent of protease processing. Natural polymorphisms in novel positions in p7(NC) and the C-cleavage site have a dominant effect on protease processing activity. Accumulation of Gag products after processing at the C site appears to delay subsequent cleavage and production of mature p24(CA).

Alleles↗

PubMed1999

ATP-dependent proteases controlling mitochondrial function in the yeast Saccharomyces cerevisiae.

Regulated protein degradation by ATP-dependent proteases plays a fundamental role in the biogenesis of mitochondria. Membrane-bound and soluble ATP-dependent proteases have been identified in various subcompartments of this organelle. Subunits composing these proteases are evolutionarily conserved from yeast to humans and, in support of an endosymbiotic origin of mitochondria, evolved from prokaryotic ancestors: the PIM1/Lon protease is active in the matrix of mitochondria, while the i-AAA protease and the m-AAA protease mediate the turnover of inner membrane proteins. Most of the knowledge concerning the biogenesis and the physiological role of ATP-dependent proteases comes from studies in the yeast Saccharomyces cerevisiae. Proteases were found to be required for mitochondrial stasis, for the maintenance of the morphology of the organelle and for mitochondrial genome integrity. ATP-dependent proteolysis is crucial for the expression of mitochondrially encoded subunits of respiratory chain complexes and for the assembly of these complexes. Hence, mitochondrial ATP-dependent proteases exert multiple roles which are essential for the maintenance of cellular respiratory competence.

ATP-Dependent Proteases↗

PubMed2006

Chymostatin can combine with pepstatin to eliminate extracellular protease activity in cultures of Aspergillus niger NRRL-3.

Aspergillus strains are being considered as potential hosts for recombinant heterologous protein production because of their excellent extracellular enzyme production characteristics. However, Aspergillus proteases are problematic in that they modify and degrade the heterologous proteins in the extracellular medium. In previous studies we observed that media adjustments and maintenance of a filamentous morphology greatly reduced protease activity and that a low concentration of the aspartic protease inhibitor pepstatin inhibited the latter protease activity to the extent of approximately 90%. In this paper we report that when the serine protease inhibitor chymostatin is used in combination with pepstatin 99-100% of total protease activity in Aspergillus cultures is inhibited. In protease assays a concentration of 30 microM chymostatin combined with 0.075 microM pepstatin was required for maximum inhibition. Inhibitor concentrations of chymostatin and pepstatin of 120 and 0.3 microM, respectively, when added to Aspergillus cultures, has no significant effect on biomass production, glucose utilization or culture pH pattern. The potential of using these protease inhibitors in cultures of recombinant Aspergillus strains producing heterologous proteins will now be investigated to determine if the previously observed recombinant protein denaturing effects of Aspergillus proteases can be negated.

Aspartic Acid Endopeptidases↗

PubMedDate not supplied

Protease activity in protein-free NS0 myeloma cell cultures.

Zymography of concentrated conditioned medium (CM) from protein-free NS0 myeloma cell cultures showed that this cell line produced and released/secreted several proteases. Two caseinolytic activities at 45-50 and 90 kDa were identified as aspartic acid proteases, and at least two cathepsins of the papain-like cysteine protease family with molecular masses of 30-35 kDa were found by gelatin zymography. One of these cathepsins was identified as cathepsin L by using an enzyme assay exploiting the substrate Z-Phe-Arg-AMC and the inhibitor Z-Phe-Tyr-t(Bu)-DMK. The aspartic acid and cysteine proteases were active only at acidic pH and are therefore not a potential risk for degrading the product or affecting cell growth during culture. Secreted proforms of cathepsins may, however, possess mitogenic functions, but addition of anti-procathepsin L antibodies to NS0 cultures did not influence proliferation. The recombinant antibody product was not degraded in cell-free CM incubated at pH 7, but when the pH was decreased to 3.5-4, the aspartic acid proteases degraded the product. Gelatin zymography also revealed the presence of several serine proteases in NS0 CM, one at 85 kDa and two at 50 kDa, with pH optima close to culture pH. Addition of the serine protease inhibitor aprotinin significantly increased the specific proliferation rate as compared to the control. In addition to these data, N-terminal amino acid sequencing identified two proteins in NS0 CM as the protease inhibitors secretory leukocyte protease inhibitor and cystatin C.

Amino Acid Sequence↗

PubMed1995

Multiple protease activities in Giardia intestinalis trophozoites.

Azocasein and a wide range of chromogenic and fluorogenic peptidyl substrates, representing 21 different peptide sequences, were degraded by the intracellular proteases present in lysates of trophozoites of Giardia intestinalis (syn lamblia) Portland 1 strain. Total protease activity differed considerably with the substrate with the p-nitroanilide derivatives Bz-pro-phe-arg-pNA and Bz-phe-val-arg-pNA being most rapidly hydrolysed (specific activities 4.2 +/- 0.4 and 1.1 +/- 0.6 U/mg protein, respectively). Activities were increased (16-72%) by addition of 1 mM dithiothreitol and were maximal, for the substrates monitored, in the range pH 5.5-7.0. These data and the inhibitor susceptibilities of the trophozoite proteases confirmed that the activity was predominantly due to cysteine proteases, although the presence of some serine protease, aspartic protease and aminopeptidase activity in the lysates was also indicated. The multiplicity of the protease activities was confirmed by gelatin-polyacrylamide electrophoretic analysis. Eighteen proteolytic activities with Mr values in the range 30,000 to > 211,000 were detected. These gelatinase activities were enhanced by dithiothreitol, were maximal at or close to pH 6, and were inhibited by cysteine proteinase and some serine proteinase inhibitors. Four of the proteases present in the gels exhibited activity towards fluorogenic amidomethylcoumarin peptides, but with different substrate preferences. The results show that G. intestinalis contains multiple proteases, many of which are of the cysteine type.

Amino Acid Sequence↗

PubMed1989

Thiol proteases released in vitro by Fasciola hepatica.

Immature Fasciola hepatica release 11 distinct proteases when cultured in vitro for 16 h as revealed by gelatin-substrate sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Six of these proteases are active in the pH range 4.5 to 8.0. Five are acid proteases, being most active in the pH range 3.0 to 4.5. The majority of proteases released in vitro by immature flukes are also released by mature flukes; however, a 40-kDa protease released by immature flukes is a very minor protease released by mature flukes. The activity of all proteases is inhibited by leupeptin, L-trans-epoxysuccinyl-leucylamido(4-guanidino)butane, phenylmethylsulfonyl fluoride and iodoacetamide and enhanced or stabilized by the reducing agents cysteine and dithiothreitol. Therefore, all F. hepatica in vitro-released proteases identified by gelatin-substrate SDS-PAGE are thiol proteases.

Animals↗

PubMed1982

In vitro interaction of porcine serum and colostrum protease inhibitors with pancreatic trypsin, chymotrypsin and elastase.

The partition of 125I-labelled pancreatic trypsin, chymotrypsin and elastase between the inhibitors, alpha 2-macroglobulin f and s, alpha 1-protease inhibitor, alpha 2-antitrypsin, inter-alpha-trypsin inhibitor and the specific sow colostrum protease inhibitor, was studied in vitro by gradually increasing the concentration of these proteases in blood serum from adult and newborn pigs. As revealed by immunoelectrophoresis in combination with autoradiography, differences were noted in the abilities of the various protease inhibitors to interact with and to form complexes with the three proteases, resulting in changes in location, height and numbers of precipitates. Among the serum inhibitors, alpha 2-macroglobulins showed the highest relative affinity to all three proteases, while alpha 1-protease inhibitor showed a high relative affinity only for chymotrypsin. Serum alpha 2-antitrypsin complexed only with trypsin, with a low relative affinity. alpha 2-Antitrypsin also interacted with chymotrypsin and elastase, but without forming complexes. When complexes of sow colostrum protease inhibitor and trypsin were added to the serum from neonatal pigs, these complexes remained stable. The results obtained from these in vitro studies, indicating differences in the relative affinities of the inhibitors to the various proteases, give some information about the role of the inhibitors in vivo, both in adult and in neonatal pigs.

Animals↗

PubMed1993

Fibrinogenolytic proteases from the venoms of juvenile and adult northern Pacific rattlesnakes (Crotalus viridis oreganus).

1. Venoms of Crotalus viridis oreganus show marked ontogenetic variation in protease activity. Adult venoms are approximately five-fold higher in protease (caseinolytic) activity. 2. Of seven potential protease inhibitors, only EDTA and 1,10-phenanthroline caused a significant decrease in protease activity. Responses of juvenile and adult venoms were essentially equivalent, and attempts at recovery of protease activity of EDTA-treated venoms by the addition of Ca2+ or Zn2+ were unsuccessful. 3. Gel filtration resolved two proteases from juvenile and subadult venoms with approximate M(r) of 100,000 and 78,000. Four proteases were resolved from adult venom, and M(r) estimates were 78,000, 61,000, 35,000 and 19,000. 4. Proteases from juvenile and adult venoms showed fibrinogenolytic activity, each producing some unique degradation products. 5. The occurrence of three "new" proteases in adult venom produced the ontogenetic increase in activity seen in the crude venoms.

Aging↗

PubMed2002

The crystal structure of the Epstein-Barr virus protease shows rearrangement of the processed C terminus.

Epstein-Barr virus (EBV) belongs to the gamma-herpesvirinae subfamily of the Herpesviridae. The protease domain of the assemblin protein of herpesviruses forms a monomer-dimer equilibrium in solution. The protease domain of EBV was expressed in Escherichia coli and its structure was solved by X-ray crystallography to 2.3A resolution after inhibition with diisopropyl-fluorophosphate (DFP). The overall structure confirms the conservation of the homodimer and its structure throughout the alpha, beta, and gamma-herpesvirinae. The substrate recognition could be modelled using information from the DFP binding, from a crystal contact, suggesting that the substrate forms an antiparallel beta-strand extending strand beta5, and from the comparison with the structure of a peptidomimetic inhibitor bound to cytomegalovirus protease. The long insert between beta-strands 1 and 2, which was disordered in the KSHV protease structure, was found to be ordered in the EBV protease and shows the same conformation as observed for proteases in the alpha and beta-herpesvirus families. In contrast to previous structures, the long loop located between beta-strands 5 and 6 is partially ordered, probably due to DFP inhibition and a crystal contact. It also contributes to substrate recognition. The protease shows a specific recognition of its own C terminus in a binding pocket involving residue Phe210 of the other monomer interacting across the dimer interface. This suggests conformational changes of the protease domain after its release from the assemblin precursor followed by burial of the new C terminus and a possible effect onto the monomer-dimer equilibrium. The importance of the processed C terminus was confirmed using a mutant protease carrying a C-terminal extension and a mutated release site, which shows different solution properties and a strongly reduced enzymatic activity.

Amino Acid Sequence↗

PubMed2003

Development and evaluation of a phenotypic assay monitoring resistance formation to protease inhibitors in HIV-1-infected patients.

A novel phenotypic assay, based on recombinant expression of the HIV-1-protease was developed and evaluated; it monitors the formation of resistance to protease inhibitors. The HIV-1 protease-encoding region from the blood sample of patients was amplified, ligated into the expression vector pBD2, and recombinantly expressed in Escherichia coli TG1 cells. The resulting recombinant enzyme was purified by a newly developed one-step acid extraction protocol. The protease activity was determined in presence of five selected HIV protease inhibitors and the 50% inhibitory concentration (IC(50)) to the respective protease inhibitors determined. The degree of resistance was expressed in terms of x-fold increase in IC(50) compared to the IC(50) value of an HIV-1 wild type protease preparation. The established test system showed a reproducible recombinant expression of each individual patients' HIV-1 protease population. Samples of nine clinically well characterised HIV-1-infected patients with varying degrees of resistance were analysed. There was a good correlation between clinical parameters and the results obtained by this phenotypic assay. For the majority of patients a blind genotypic analysis of the patients' protease domain revealed a fair correlation to the results of the phenotypic assay. In a minority of patients our phenotypic results diverged from the genotypic ones. This novel phenotypic assay can be carried out within 8-10 days, and offers a significant advantage in time to the current employed phenotypic tests.

Acquired Immunodeficiency Syndrome↗

PubMed2001

Protease C2, a cysteine endopeptidase involved in the continuing mobilization of soybean beta-conglycinin seed proteins.

The protease that degrades the beta subunit of the soybean (Glycine max (L.) Merrill) storage protein beta-conglycinin was purified from the cotyledons of seedlings grown for 12 days. The enzyme was named protease C2 because it is the second enzyme to cleave the beta-conglycinin storage protein, the first (protease C1) being one that degrades only the alpha' and alpha subunits of the storage protein to products similar in size and sequence to the remaining intact beta subunit. Protease C2 activity is not evident in vivo until 4 days after imbibition of the seed. The 31 kDa enzyme is a cysteine protease with a pH optimum with beta-conglycinin as substrate of 5.5. The action of protease C2 on native beta-conglycinin produces a set of large fragments (52-46 kDa in size) and small fragments (29-25 kDa). This is consistent with cleavage of all beta-conglycinin subunits at the region linking their N- and C-domains. Protease C2 also cleaves phaseolin, the Phaseolus vulgaris vicilin homologous to beta-conglycinin, to fragments in the 25-28 kDa range. N-Terminal sequences of isolated beta-conglycinin and phaseolin products show that protease C2 cleaves at a bond within a very mobile surface loop connecting the two compact structural domains of each subunit. The protease C2 cleavage specificity appears to be dictated by the substrate's three-dimensional structure rather than a specificity for a particular amino acid or sequence.

Amino Acid Sequence↗