Trypsin inhibitors of buffalo seminal plasma.

Two trypsin inhibitors from acid-treated buffalo seminal plasma were purified by gel filtration and affinity chromatography. These acid-stable trypsin inhibitors having charge heterogeneity were homogeneous with respect to size as revealed by gel filtration and SDS-PAGE. Gel filtration data suggest molecular weight value of 9,900 Da for inhibitor I and 10,900 Da for inhibitor II. Molecular weight estimated by SDS-PAGE was found to be 10,600 Da and 11,200 Da for inhibitors I and II, respectively. The hydrodynamic properties such as Stokes radii (1.58 nm and 1.62 nm); intrinsic viscosity (2.5725 ml/g and 2.5025 ml/g) and diffusion coefficient (13.499 x 10(-11) m2/sec. and 13.166X10(-11) m2/sec) respectively for inhibitor I and II were determined by analytical gel filtration. These inhibitors were fairly thermostable and could not be stained by PAS reagent. Both the inhibitors were found to inhibit buffalo acrosin but not bovine chymotrypsin.     

Purification and characterization of proteinase inhibitors from adzuki beans (Phaseolus angularis).

Two proteinase inhibitors, designated as inhibitors I and II, were purified from adzuki beans (Phaseolus angularis) by chromatographies on DEAE- and CM-cellulose, and gel filtration on a Sephadex G-100 column. Each inhibitor shows unique inhibitory activities. Inhibitor I was a powerful inhibitor of trypsin [EC 3.4.21.4], but essentially not of chymotrypsin ]EC 3.4.21.1]. On the other hand, inhibitor II inhibited chymotrypsin more strongly than trypsin. The molecular weights estimated from the enzyme inhibition were 3,750 and 9,700 for inhibitors I and II, respectively, assuming that the inhibitions were stoichiometric and in 1 : 1 molar ratio. The amino acid compositions of both inhibitors closely resemble those of low molecular weight inhibitors of other leguminous seeds: they contain large amounts of half-cystine, aspartic acid and serine, and little or no hydrophobic and aromatic amino acids. Inhibitor I lacks both tyrosine and tryptophan residues. The molecular weights were calculated to be 7,894 and 8,620 for inhibitors I and II, respectively. The reliability of these molecular weights was confirmed by the sedimentation equilibrium and 6 M guanidine gel filtration methods. On comparison with the values obtained from enzyme inhibition, it was concluded that inhibitor I and two trypsin inhibitory sites on the molecule, whereas inhibitor II had one chymotrypsin and one trypsin inhibitory sites on the molecule.     

Trypsin inhibitor in the hemolymph of a solitary ascidian, Halocynthia roretzi. Purification and characterization

A purified preparation of trypsin inhibitor was obtained from the hemolymph of a solitary ascidian, Halocynthia roretzi, by a procedure including trypsin-Sepharose chromatography, DEAE-cellulose chromatography, and Sephadex G-50 gel filtration. The product was a mixture of two isoinhibitors, inhibitors I and II. They were separated from each other by high-performance liquid chromatography on an anion exchanger column, and showed almost identical amino acid compositions. They were also indistinguishable in terms of apparent specific inhibitory activity against bovine trypsin when the activity was assayed with the inhibitors at rather high concentrations (greater than 50 nM). A large difference was observed between them, however, in the inhibition constants, which correspond to the dissociation constants of the inhibitor-trypsin complexes; the inhibition constant of inhibitor I was 90 pM, whereas that of inhibitor II was 4.7 nM. The molecular weights of inhibitors I and II were estimated to be 6,000 and 4,500, respectively, by SDS-polyacrylamide gel electrophoresis, while an almost identical value, 9,000, was obtained for both of them by gel filtration. The molecular weight calculated from the amino acid compositions was 5,929 for both. The isoelectric points were also identical, that is about 5.0. Both of the inhibitors were heat-stable. Ascidian inhibitor I also inhibited other trypsin-like enzymes of mammalian origin, as well as those of ascidian origin.     

Characterization of the proteinase inhibitors from bull seminal plasma and spermatozoa.

Two acid stable proteinase inhibitors are present in bull seminal plasma and washed ejaculated bull spermatozoa. Inhibitor I with a molecular weight of about 8700 (estimated by gel filtration) is a very strong inhibitor of bull sperm acrosin but also inhibits bovine trypsin and chymotrypsin and porcine plasmin; inhibition of porcine pancreatic and urinary kallikrein was not observed. In this respect inhibitor I resembles the well known cow colostrum trypsin inhibitor. Inhibitor II with a molecular weight near 6800 (estimated by gel filtration) inhibits bovine trypsin and chymotrypsin, porcine plasmin and pancreatic and urinary kallikrein as well as bull acrosin. The inhibition specificity of inhibitor II is thus very similar to that of the basic inhibitor from bovine organs (Kunitz-type). In view of the inhibition strength and other characteristics, however, the acid stable bull seminal inhibitors are not identical with the inhibitor from cow colostrum or bovine lung (organs).     

Purification and characteristics of an endogenous alpha-amylase inhibitor from barley kernels

An inhibitor of malted barley (Hordeum vulgare cv Conquest) α-amylase II was purified 125-fold from a crude extract of barley kernels by (NH₄)₂SO₄ fractionation, ion exchange chromatography on DEAE-Sephacel, and gel filtration on Bio-Gel P 60. The inhibitor was a protein with an approximate molecular weight of 20,000 daltons and an isoelectric point of 7.3. The protein was homogeneous, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino acid analysis indicated the presence of about 9 half-cystine residues per mole. The neutral isoelectric point of the inhibitor suggested that some of the apparently acidic residues (glutamic and aspartic) existed in the amide form. The first twenty N-terminal amino acids were sequenced. Some homology appeared to exist between the α-amylase II inhibitor and trypsin inhibitor from barley. Complex formation between α-amylase II and the inhibitor was detected by the appearance of a new molecular weight species after gel filtration on Bio-Gel P 100. Enzyme and inhibitor had to be preincubated for 5 min, prior to assaying for enzyme activity before maximum inhibition was attained. Inhibition increased at higher pH values. At pH 5.5, an approximately 1100 molar excess of inhibitor over α-amylase II produced 40% inhibition, whereas, at pH 8.0, a 1:1 molar ratio of inhibitor to enzyme produced the same degree of inhibition.     

Purification, partial characterization, and immunological relationships of multiple low molecular weight protease inhibitors of soybean.

Five protease inhibitors, I--V, in the molecular weight range 7000--8000 were purified from Tracy soybeans by ammonium sulfate precipitation, gel filtration on Sephadex G-100 and G-75, and column chromatography on DEAE-cellulose. In common with previously described trypsin inhibitors from legumes, I--V have a high content of half-cystine and lack tryptophan. By contrast with other legume inhibitors, inhibitor II contains 3 methionine residues. Isoelectric points range from 6.2 to 4.2 in order from inhibitor I to V. Molar ratios (inhibitor/enzyme) for 50% trypsin inhibition are I = 4.76, II = 1.32, III = 3.22, IV = 2.17, V = 0.97. Only V inhibit chymotrypsin significantly (molar ratio = 1.33 for 50% inhibition). The sequence of the first 16 N-terminal amino acid residued of inhibitor V is identical to that of the Bowman-Birk inhibitor; all other observations also indicate that inhibitor V and Bowman-Birk are identical. The first 20 N-terminal amino acid residues of inhibitor II show high homology to those of Bowman-Birk inhibitor, differing by 1 deletion and 5 substitutions. Immunological tests show that inhibitors I through IV are fully cross-reactive with each other but are distinct from inhibitor V.     

Purification and properties of a subtilisin inhibitor and an associated trypsin inhibitor from Dolichos biflorus

A subtilisin inhibitor and an associated trypsin inhibitor from Dolichos biflorus were purified to homogeneity by conventional methods such as chromatography on DEAE-cellulose, gel filtration on Sephadex G-75, PAGE and affinity chromatography. The final preparations were homogeneous on PAGE. Their pI were 7.66 and 7.70, respectively. The dissociation constant of the complex of the inhibitor with subtilisin was 2.69.10(-10) M. Both the inhibitors were stable to heat, TCA and ethanol. The molecular weights of the subtilisin inhibitor and the associated trypsin inhibitor by gel filtration were 7500 and 8200, respectively.     

Isolation, characterization and kinetics of goat cystatins

Two cysteine proteinase inhibitors I and II were purified from goat kidney using alkaline denaturation, ammonium sulphate fractionation, gel filtration on Sephadex G-75 and ion exchange chromatography on DEAE cellulose. The purified inhibitors were homogenous and showed a single band on SDS PAGE under reducing and non-reducing conditions with an apparent molecular mass of 67 kDa. The cystatin forms were stable in the range of pH 3–10 and up to 95 °C. Immunological identity with the sheep LMW kininogen was obtained suggesting that the inhibitor is closely related to kininogens. Spectral studies confirm that the inhibitors have predominantly an α-helical structure and undergo major conformational changes during complex formation with papain. The inhibitors had similar inhibitory activities on cysteine proteinases. Both inhibitors inhibited papain, ficin and bromelain competitively, with maximum affinity for papain. The overall lower affinity of these inhibitors to cysteine proteinases compared to other known cystatins can be attributed to the unusual N-terminal sequence where Leu is substituted by Ile. Furthermore, N-terminal sequence analysis revealed maximum homology to mammalian LMW kininogen.     

Purification and properties of an alpha-amylase inhibitor from wheat.

Four inhibitors of alpha-amylase (EC 3.2.1.1) were separated from an alcohol extract of wheat by ion-exchange chromatography on DE52-cellulose. One inhibitor, which showed the greatest specificity for human salivary amylase relative to human pancreatic amylase, has been purified by the following steps: (a) alcohol fractionation (60--90%) of water extract (b) ion-exchange chromatography on QAE-Sephadex A-50; (c) re-chromatography on DE52-cellulose and (d) gel filtration on Sephadex G-50. The purified inhibitor is 100 times more specific for human salivary amylase than for human pancreatic amylase. It shows an electrophoretic mobility of 0.2 on disc gel electrophoresis and a molecular weight of about 21 000. This inhibitor contributes about 16% to the total salivary amylase inhibiting power of the wheat extract.     

Isolation and characterization of trypsin inhibitors from cowpea (Vigna unguiculata)

The trypsin inhibitor fraction from cowpea (Vigna unguiculata) has been purified and characterized. Although the total trypsin inhibitor as purified by affinity chromatography on immobilised trypsin was shown to be heterogeneous by gel electrophoresis and isoelectric focusing as well as by function, it was relatively homogeneous in MW (ca 17 000) on gel filtration. The total trypsin inhibitor was divided into inhibitors active against trypsin only and active against trypsin and chymotrypsin by affinity chromatography on immobilised chymotrypsin. The ‘trypsin-only’ inhibitor was the major component of the total trypsin inhibitor. It was shown by isoelectric focusing and gel electrophoresis to contain several isoinhibitors. Determination of the combining weight of this inhibitor and investigation of the complexes formed with trypsin by gel filtration indicated the presence of two protease binding sites per inhibitor molecule. The chymotrypsin/trypsin inhibitor was also shown to be composed of several isoinhibitors. On the basis of gel electrophoresis and gel filtration in dissociating and non-dissociating media both inhibitors were considered to be dimeric molecules with the subunits linked by disulphide bonds; this implies that the ‘trypsin-only’ inhibitor has one binding site per subunit.     

Large Scale Purification of Isoinhibitors of Trypsin from Swine Colostrum Using Zinc Chelate Chromatography and Chromatofocusing

Low molecular weight trypsin inhibitors were purified from swine colostrum on a large scale under mild conditions. Ammonium sulfate fractionation and metal chelate chromatography on zinc chelate Sepharose and phenyl Sepharose were used for removal of the bulk of proteins. The inhibitors showed only a weak hydrophobic interaction with phenyl Sepharose even in the presence of 1 M (Nll₄)₂SO4, and advantage was taken of this property to remove the inhibitors from contaminating colostrum proteins which remained tightly adsorbed to phenyl Sepharose under these conditions. The low and high molecular weight inhibitors were then separated by gel filtration on Bio-Gel P-300. The low molecular weight material was eluted in three major inhibitor fractions on DEAE-Sepharose.

Chromatofocusing of these fractions provided greater resolution of the inhibitors, and several previously unreported inhibitor peaks were detected. The six major inhibitors purified by chromatofocusing were homogeneous as judged by polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. These inhibitors were composed of a single polypeptide chain with a molecular weight of 18,000 as determined by Sephacryl S-200 gel filtration and polyacrylamide qel electrophoresis in the presence of sodium dodecyl sulfate and e-mercaptoethanol. The specific activities of the pure inhibitors were approximately 30% higher than those previously reported.     

Isolation and characterization of 5'-nucleotidase inhibitor from rat liver.

5'-Nucleotidase (EC 3.1.3.5) is widely distributed in nature. However, it could not be detected in rat liver, because of the presence of specific inhibitors. Such inhibitors were also found in other tissues of rat, but at lower concentrations than that in the liver. The inhibitor activity was enriched in the membrane fraction and was also present in the cytosol fraction. It was sensitive to treatment with 6M urea and trypsin, while heating in a boiling water bath for 10 min or dialysis reduced the activity only slightly. Gel filtration or Sephadex G-50 yielded two types of inhibitors. Inhibitor I inhibited brain 5'-nucleotidase while inhibitor II inhibited both the brain and liver enzymes. Inhibitor II on further purification on CM Sephadex C-25 yielded five fractions with inhibitor activity of which inhibitor IIC was electrophoretically homogeneous. It had a molecular weight of 8500 by SDS gel electrophoresis, was rich in basic amino acids and had a high proportion of beta structure. Interaction of the inhibitor with 5'-nucleotidase brought about modifications in the secondary structure of the inhibitor as seen from the circular dichroism spectrum.     

Trypsin inhibitors in summer squash (Cucurbita pepo) seeds. Isolation, purification and partial characterization of three inhibitors

Three trypsin inhibitors were isolated from summer squash (Cucurbita pepo) seeds and purified to homogeneity by fractionation with ammonium sulphate and methanol, ion-exchange chromatography and gel filtration. All three inhibitors have lysine at their active site. Two of them (II, IV) show the same isoelectric point (at pH 5.6), amino acid composition and molecular mass (3259). The third inhibitor (III) of molecular mass of 3654 and isoelectric point at 4.9 has additionally one histidine residue and two glutamic acid residues more per molecule.     

Protease inhibitors from the parasitic worm Parascaris equorum

Two proteic inhibitors (I and II) of serine proteases have been purified from the parasitic worm Parascaris equorum by affinity chromatography on immobilized trypsin followed by preparative electrophoresis. They have an apparent relative molecular mass of 9000 and 7000 as determined by gel filtration, a slightly acid isoelectric point (5.5 and 6.1) and a similar amino acid composition. Both inhibitors lack serine, methionine and tyrosine. They bind bovine trypsin extremely strongly with an association constant, Ka, larger than 10(9) M-1, and form a 1:1 complex with this protease. The Ka values for the binding to bovine chymotrypsin are approximately 3.3 X 10(8) M-1 (inhibitor I) and approximately 2 X 10(6) M-1 (inhibitor II). Inhibitor I interacts also with porcine elastase (Ka approximately 5 X 10(7) M-1), while inhibitor II is inactive towards this enzyme.     

Purification and partial amino acid sequence of a bovine cartilage-derived collagenase inhibitor

An inhibitor of mammalian collagenase from bovine scapular cartilage has been purified to homogeneity. The inhibitor, extracted from cartilage using 2 M NaCl, was applied to an A-1.5m gel filtration column. Inhibitor eluted at an apparent Mr of 28,000. Further purification was achieved by ion exchange chromatography, gel filtration, and reverse-phase high performance liquid chromatography. A purification of greater than 1,000-fold was achieved. The inhibitor was judged homogeneous by the appearance of a single band on a silver-stained 15% sodium dodecyl sulfate-polyacrylamide gel. Reduced inhibitor had an Mr of 27,400, unreduced inhibitor had an Mr of 23,900. NH2-terminal sequence data were obtained for the first 45 residues. The bovine cartilage-derived inhibitor exhibits greater than 65% homology over the first 23 residues with a collagenase inhibitor purified from human skin fibroblasts maintained in cell culture. This is the first demonstration that collagenase inhibitors extracted directly from tissue may be similar to those obtained from culture medium.     

Identification and characterization of trypsin, chymotrypsin and elastase inhibitors in porcine serum.

Characterization of the trypsin-, chymotrypsin- and elastase-inhibiting properties of porcine serum was carried out by gel filtration on Ultrogel, AcA 44, and agarose gel electrophoresis with subsequent processing for protease-inhibiting activity. Moreover, by allowing the fractions obtained from gel filtration to react with antibodies to porcine serum protease inhibitors, the specific inhibiting properties of these inhibitor molecules were identified. At least six protease inhibitors were identified and partially characterized in porcine serum. Two alpha 2 -macroglobulins (alpha 2 Mf and alpha 2 Ms), homologues to human alpha 2 -macroglobulin, with slightly different electrophoretic mobilities, were both found to exhibit trypsin, chymotrypsin and elastase inhibiting activity. Alpha 1 -Protease inhibitor (Mr 51 000), a homologue to human alpha 1 -protease inhibitor (alpha 1 -antitrypsin), also showed trypsin-, chymotrypsin- and elastase-inhibiting properties. Inter-alpha-trypsin inhibitor (Mr 162 000 and 129000), a porcine serum counterpart to human inter-alpha-trypsin inhibitor, showed trypsin- and chymo-trypsin-inhibiting properties. In addition, a specific trypsin inhibitor, alpha 2 -antigrypsin (Mr 58 000), and a specific elastase inhibitor, beta-elastase inhibitor, were characterized in porcine serum, and these seem to have no counterparts in human serum.     

Proteinase Inhibitors in Germinating Barley Embryos

The activities of trypsin inhibitors and Aspergillus-proteinase inhibitors have been assayed in barley embryos germinating in different conditions. The Aspergillus-proteinase inhibitor activity increased about 20-fold during 6 days at 20°C both in separated embryos germinating on nutrient agar and in the embryos of undissected grains germinating on agar with or without mineral salts. Significant inhibitor activity was also present in root tips at later stages of vegetative growth. Whether any changes occurred in the molecular weight distribution of the inhibitors present in the seedlings was studied by gel filtration on Sephadex G-75; only minor changes were observed. The trypsin inhibitor activity per seedling remained roughly constant in all cultures. In gel filtration experiments all the activity appeared to be due to the inhibitor species present in resting embryos.     

Secretion of metalloproteinases by stimulated capillary endothelial cells. II. Expression of collagenase and stromelysin activities is regulated by endogenous inhibitors

Rabbit brain capillary endothelial cells treated with 12-O-tetradecanoylphorbol-13-acetate produce the metalloproteinases, procollagenase and prostromelysin, as up to 20% of their total secreted protein. However, little or no catalytic activity of these enzymes can be found after treatment with either trypsin or an organomercurial agent, which are able to activate the proenzymes in the medium from stimulated rabbit fibroblasts. We now have shown that enzyme activities of procollagenase and prostromelysin are revealed after conditioned medium is analyzed by gel filtration chromatography or by electrophoresis on sodium dodecyl sulfate-substrate gels. In both systems, the metalloproteinases were separated from metalloproteinase inhibitors. The major inhibitor of Mr = 30,000 from capillary endothelial cells was immunologically identical with the rabbit tissue inhibitor of metalloproteinases. Two additional inhibitors of metalloproteinases at Mr = 22,000 and 19,000 were also observed. Inhibitors were present in the conditioned medium from rabbit fibroblasts in much lower quantities and were also qualitatively different. When gel filtration chromatography was used to remove the tissue inhibitor of metalloproteinases from medium conditioned by stimulated capillary endothelial cells, both activatable procollagenase and prostromelysin were readily demonstrable. These data suggest that endogenous inhibitors regulate the expression of metalloproteinases secreted by endothelial cells.     

Purification and characterization of two serine protease inhibitors from the hemolymph of Mythimna unipuncta

Two serine protease inhibitors, trypsin inhibitor and alpha-chymotrypsin inhibitor, were isolated from the hemolymph of Mythimna unipuncta. Mythimna trypsin and alpha-chymotrypsin inhibitors were purified by gel filtration and anion-exchange chromatography. They displayed molecular masses of 52 kDa and 43 kDa, respectively, as determined by electrophoresis under reducing and non-reducing conditions on denaturing polyacrylamide gels. Their isoelectric points were evaluated by isoelectric focusing and two-dimensional electrophoresis. Their N-terminal sequences have been analyzed as APSDTTIAETLTITEEFFPD and FDESFGFQGPSTYEKTPLGEP, respectively. The role of these inhibitors in the regulation of the defense reaction of the insect is discussed.     

Concurrent isolation of a Kunitz-type trypsin inhibitor with antifungal activity and a novel lectin from Pseudostellaria heterophylla roots

A simple purification protocol, involving ion exchange chromatography on DEAE-cellulose and CM-cellulose and fast protein liquid chromatography-gel filtration on Superdex 75, was employed to isolate a Kunitz-type trypsin inhibitor with antifungal activity and a novel lectin from Pseudostellaria heterophylla roots. Both the trypsin inhibitor and the lectin were unadsorbed on DEAE-cellulose and adsorbed on CM-cellulose. They could be separated from one another by gel filtration on Superdex 75 in which the 36-kDa lectin appeared as the first peak and the 20.5-kDa trypsin inhibitor as the second peak. P. heterophylla trypsin inhibitor exhibited a trypsin inhibitory potency similar to that of soybean trypsin inhibitor. It also demonstrated antifungal activity toward Fusarium oxysporum like aprotinin and Kunitz-type trypsin inhibitors from soybeans and lima beans. P. heterophylla lectin was devoid of antifungal activity and exhibited low thermostability and also lability in the presence of acid and alkali. The novel aspects of the present report include demonstration of antifungal activity in Kunitz-type trypsin inhibitors and isolation of a novel lectin as well as a trypsin inhibitor from roots.