Limited proteolysis of p-chloromercuribenzoate-dissociated hemoglobin by trypsin.

p-Chloromercuribenzoate-treated hemoglobin was digested by trypsin. The hydrolysate was subjected to gel-filtration on Bio-Gel P-4 and Sephadex G-50 columns, ion-exchange chromatography on CM-Sephadex and DE 52 columns, and paper electrophoresis. Peptides obtained by this procedure were analyzed for amino acid compositions and amino-terminal amino acid sequences. The results showed that p-chloromercuribenzoate-treated hemoglobin was hydrolyzed to a limited extent by trypsin at the bonds involving the carboxyl group of a lysine or arginine residue in planes A--E in the parent hemoglobin, which represent the external region of the parent tetramer. It is concluded therefore that the slight modification of hemoglobin enhances the susceptibility of the protein to proteases and that the hydrolysis of the modified protein is limited.     

Limited proteolysis by trypsin influences activity of maize phosphoenolpyruvate carboxylase

Maize phosphoenolpyruvate carboxylase (PEPC) was rapidly and completely inactivated by very low concentrations of trypsin at 37 degrees C. PEP+Mg2+ and several other effectors of PEP carboxylase offered substantial protection against trypsin inactivation. Inactivation resulted from a fairly specific cleavage of 20 kDa peptide from the enzyme subunit. Limited proteolysis under catalytic condition (in presence of PEP, Mg2+ and HCO3) although yielded a truncated subunit of 90 kDa, did not affect the catalytic function appreciably but desensitized the enzyme to the effectors like glucose-6-phosphate glycine and malate. However, under non-catalytic condition, only malate sensitivity was appreciably affected. Significant protection of the enzyme activity against trypsin during catalytic phase could be either due to a conformational change induced on substrate binding. Several lines of evidence indicate that the inactivation caused by a cleavage at a highly conserved C-terminal end of the subunit.     

Human inter-alpha-trypsin inhibitor. Limited proteolysis by trypsin, plasmin, kallikrein and granulocytic elastase and inhibitory properties of the cleavage products.

The acid-labile inter-alpha-trypsin inhibitor is cleaved enzymatically in vivo, liberating a smaller acid-stable inhibitor. The molar ratio of native inhibitor to this smaller inhibitor in plasma is significantly changed in some severe cases of inflammation and kidney injury. To clarify this observation on a molecular basis, the action of four different types of proteinases (trypsin, plasmin, kallikrein and granulocyte elastase) on the inter-alpha-trypsin inhibitor was studied. The initial rate of cleavage of the inter-alpha-trypsin inhibitor by a 1.3-fold molar excess of proteinase over inhibitor was found to be 4375 nM x min-1 with granulocyte elastase, 860 nM x min-1 with trypsin, 67 nM x min-1 with plasmin, and 0.3 nM X min-1 with kallikrein. Obviously, of the enzymes studied so far, the granulocyte elastase known to be released during severe inflammatory processes is by far the most potent proteinase in the transformation of the inter-alpha-trypsin inhibitor. The inter-alpha-trypsin inhibitor and its cleavage products inhibit bovine trypsin very strongly (Ki = 10(-9)--10(-11) M), porcine plasmin much less strongly, human plasmin very weakly and pancreatic kallikrein practically not at all.     

Studies on simultaneous inhibition of trypsin and chymotrypsin by horsegram Bowman-Birk inhibitor

Bowman-Birk inhibitors (BBI) isolated from plant seeds are small proteins active against trypsin and/or chymotrypsin. These inhibitors have been extensively studied in terms of their structure, interactions, function and evolution. Examination of the known three-dimensional structures of BBIs revealed similarities and subtle differences. The hydrophobic core, deduced from surface accessibility and hydrophobicity plots, corresponding to the two tandem structural domains of the double headed BBI are related by an almost exact two-fold, in contrast to the reactive site loops which depart appreciably from the two-fold symmetry. Also, the orientations of inhibitory loops in soybean and peanut inhibitors were different with respect to the rigid core. Based on the structure of Adzuki bean BBI-trypsin complex, models of trypsin and chymotryspin bound to the monomeric soybean BBI (SBI) were constructed. There were minor short contacts between the two enzymes bound to the inhibitor suggesting near independence of binding. Binding studies revealed that the inhibition of one enzyme in the presence of the other is associated with a minor negative cooperativity. In order to assess the functional significance of the reported oligomeric forms of BBI, binding of proteases to the crystallographic and non-crystallographic dimers as found in the crystal structure of peanut inhibitor were examined. It was found that all the active sites in these oligomers cannot simultaneously participate in inhibition.     

Limited trypsin proteolysis of photoreceptor GTP-binding protein. Light- and GTP-induced conformational changes

The amphibian photoreceptor rod outer segment contains a guanine nucleotide-binding complex which consists of a 39,000-dalton polypeptide that binds guanine nucleotides (G protein), a 36,000-dalton polypeptide (H protein), and an approximately 6,500-dalton polypeptide. Sensitivity to trypsin proteolysis was utilized as a probe of structure-function relationships for these polypeptides. Digestion of the H protein generated fragments of 26,000 and 15,000 daltons whose proteolytic susceptibility was not altered by guanosine triphosphates, light, or membranes. The approximately 6,500-dalton polypeptide was not trypsin sensitive. When the G protein was eluted from illuminated membranes by GTP, trypsin proteolysis cleaved a terminal 1,000-dalton fragment (G1) to yield a 38,000-dalton fragment (G38). With increased digestion time, a 6,000-dalton fragment (G6) was removed from G38 to yield a 32,000-dalton fragment (G32). G32 was subsequently digested to fragments of 23,000 and 12,000 daltons. However, when the G protein was eluted from illuminated membranes by hydrolysis-resistant analogues of GTP, G32 was protected from further digestion. This is consistent with a GTP-induced conformational change in the G protein which is altered by GTP hydrolysis. Proteolysis of the G protein after covalent labeling with a photoaffinity analogue of GTP demonstrated that the analogue is bound to first G38 and then G32, indicating the GTP-binding site is associated with G32. Fragment G6 was cleaved when the G protein was soluble or bound to unilluminated membranes. However, when bound to illuminated membranes, fragments were generated reflecting the loss of 7,500, 9,000, or 11,000 daltons from the G protein. This light-induced alteration in proteolytic susceptibility indicates there is a light-induced conformational change in the G protein. Fragment G1 was not removed from the G protein when it was membrane bound, suggesting G1 is involved in binding to a membrane structure. These data suggest that the light-induced binding of the G protein to illuminated membranes and the reversal of this binding by GTP are mediated through conformational changes in the G protein and that three conformations exist: 1) a basal, inactive conformation; 2) a primed conformation induced by binding to photolyzed rhodopsin, with a high affinity for GTP; and 3) an active conformation, induced by binding of GTP, which activates the catalytic complex of light-activated phosphodiesterase.     

A trypsin and chymotrypsin inhibitor from seeds of Bauhenia purpurea

A trypsin and chymotrypsin inhibitor was partially purified from Bauhenia purpurea seeds and separated from a second inhibitor by Ecteola cellulose chromatography. The factor inhibited bovine trypsin and chymotrypsin as well as pronase trypsin and elastase. It formed a complex with trypsin and with chymotrypsin, but a ternary complex could not be detected. Differences were detected in the effect on trypsin and on chymotrypsin, although one enzyme interfered with the inhibition of the other. The results obtained point to two active centers on the inhibitor for the trypsin and chymotrypsin inhibition such that the one cannot complex with the inhibitor after this inhibitor had complexed with the other.     

A trypsin and chymotrypsin inhibitor from Taenia pisiformis

The somatic extract of mature T. pisiformis has been demonstrated to contain a potent inhibitor capable of inactivating the esterolysis of $\text{N-α-}\text{benzoyl-}\text{L}\text{-arginine ethyl ester}$ and $\text{N-}\text{benzoyl-}\text{L}\text{-tyrosine ethyl ester}$ by trypsin and chymotrypsin, respectively, of bovine, dog and rabbit origin, but not affecting the caseinolytic activity of subtilisin and elastase. The protease inhibitor, partially purified by trichloroacetic acid treatment, Sephadex G-100 column chromatography and affinity chromatography on CNBr-activated Sepharose 4B-bovine chymotrypsin conjugate, was soluble in 5% trichloroacetic acid, stable to heating at 100°C for up to 30 min, tolerated the pH range of 1.5–9.0, and was unaffected by 8 m-urea or 0.2 M-2-mercaptoethanol. The molecular weight of the inhibitor was estimated to be 7000–7200 by Sephadex G-100 chromatography. Activity determinations on crystalline bovine trypsin and chymotrypsin revealed that both inhibitory actions are located on the same or closely adjacent sites of the inhibitor molecule. Complex formation between the inhibitor and mammalian trypsin and chymotrypsin required 3–4 min for completion.     

Three-dimensional structure of soybean trypsin/chymotrypsin Bowman-Birk inhibitor in solution.

The three-dimensional structure of soybean trypsin/chymotrypsin Bowman-Birk inhibitor in solution has been determined by two-dimensional 1H nuclear magnetic resonance spectroscopy and dynamical simulated annealing using the program XPLOR. The structure was defined by 907 NOEs involving intra- and interresidue contacts which served as distance constraints for a protocol of dynamical simulated annealing. In addition, 48 phi angle constraints involving non-proline amino acids, 29 chi angle constraints, six omega angle constraints for the X-Pro peptide bond, and 35 stereoassignments for prochiral centers were incorporated during the course of the calculation. The protein is characterized by two distinct binding domains for serine protease. Each domain is comprised of a beta-hairpin (antiparallel beta-sheet and a cis-proline-containing type VIb reverse turn) with a short segment making a third strand of antiparallel beta-sheet. The structure determination and refinement are described, and the structure is compared to other structures of Bowman-Birk inhibitors as well as other families of serine protease inhibitors.     

Differences between the binding of trypsin and chymotrypsin by alpha 1-proteinase inhibitor

Complexes of alpha 1-proteinase inhibitor with proteases were examined by SDS-PAGE in 7.5% polyacrylamide gel and in a gel gradient. While the inhibitor-chymotrypsin complex was stable under both sets of conditions, the inhibitor-trypsin complex quantitatively dissociated under the second set of conditions, indicating that trypsin, unlike chymotrypsin, is not linked covalently to the inhibitor. Although the inhibitor sustained at least two discrete cleavages by trypsin, its overall recovery after dissociation was 100%. Due to an increased rate of autolytic breakdown in the presence of the inhibitor, the recovery of trypsin after dissociation was appreciably less than 100%. Based on these observations, a new theory of trypsin inhibition by alpha 1-proteinase inhibitor is proposed. This method is suitable for the examination of other inhibition systems as well.     

Glutathione as an inhibitor of trypsin induced proteolysis

Glutathione has been shown to inhibit trypsin induced proteolytic activity. A concentration of 6 mM of glutathione was found to completely inhibit proteolysis of 3H-proline labelled underhydroxylated procollagen as a substrate, whereas a concentration of 2.1 mM of glutathione caused 50% inhibition of proteolysis. When azocoll was used as a substrate for trypsin 50% inhibition of proteolysis was achieved with 1.4 mM of glutathione, though a complete proteolytic inhibition was attained at 4 mM glutathione. The results suggest that glutathione may be playing an important role in protein metabolism in a variety of disease and stress states.     

Entrapment of soy bean trypsin inhibitor and alpha1-antitrypsin by multilamellar liposomes.

The broad-spectrum protease inhibitors, soy bean trypsin inhibitor and α₁-antitrypsin, were entrapped within anionic multilamellar liposomes; the efficiency of entrapment was 12% for α₁-antitrypsin and 14% for soy bean trypsin inhibitor. Entrapment of ³H-labeled d-glucose, a marker for the aqueous compartment, was dependent upon the nature and concentration of coentrapped antiprotease. Rechromatography of the pooled liposome fractions following solubilization with the detergent Triton X-100 demonstrated latency of the entrapped materials. In addition, entrapment of soy bean trypsin inhibitor, as well as ³H-labeled d-glucose, was shown to be proportional to the net anionie surface charge in the lipid bilayers, suggesting sequestration within the aqueous compartments of the liposomes. This localization was also indicated by the minimal adsorption of antiproteases to anionic liposomes, contrasted with the extensive electrostatic binding of the antiproteases to cationic liposomes.     

Properties of a trypsin and chymotrypsin inhibitor secreted by larval Taenia pisiformis

Living metacestodes of Taenia pisiformis maintained in vitro discharge into the surrounding medium a protease inhibitor, which has been purified from the medium by affinity chromatography on bovine α-chymotrypsin immobilized to CNBr-activated Sepharose 4B. The purified inhibitor was shown to inactivate the hydrolysis of $\text{N-α-}\text{benzoyl}\text{-}\text{L}\text{-}\text{arginine}$ ethyl ester and $\text{N-}\text{benzoyl}\text{-}\text{L}\text{-}\text{tyrosine}$ ethyl ester, respectively, by trypsin and chymotrypsin of bovine, rabbit and dog origin, and also the hydrolysis of casein by both bovine trypsin and bovine α and β chymotrypsins, but it did not affect the enzymic activity of subtilisin, elastase, collagenase, pepsin, rennin and papain. The inhibitor withstood heating at 100°C for up to 30 min, was stable in the pH range of 1.5–8.0, was unaffected by 8 M-urea or 0.2 M-2-mercaptoethanol, and had a molecular weight of about 7000 as calculated from its gel chromatographic behaviour. The inhibitor specifically inhibits either trypsin or chymotrypsin with the formation of stable enzyme inhibitor complexes that are not dissociated by 4 M-KCl. Inhibition of trypsin and chymotrypsin is non-competitive and is linear with inhibitor concentration up to 70–80% inhibition. Inhibitory activities toward both enzymes are functions of the same binding site of the inhibitor molecule. Complex formation between the inhibitor and the enzymes is timedependent; it requires 3–4 min for completion.