Plasma inter-alpha-trypsin inhibitor-related urinary glycoprotein EDC1 inhibits the growth of a Burkitt's lymphoma cell line.

A homogeneous preparation of a urinary glycoprotein has been isolated from urine of patients with malignant melanoma and advanced adenocarcinomas of colon and lung. This molecule, Mr 30 kDa, is homologous to EDC1, a proteinase inhibitor antigenically related to plasma inter-alpha-trypsin inhibitor (IATI) originally isolated from the urine of a leukemic patient, E.D. The newly isolated EDC1 inhibits cellular proliferation of a Burkitt's lymphoma cell line, Raji, growing in serum-free medium supplemented with insulin, transferrin, selenium, and linoleic acid. This concentration-dependent inhibitory effect was monitored in terms of change in cell number and 3H-thymidine incorporation. The growth of cells treated with approximately 3.3 pmol EDC1/ml was 50% that of the control group by both assays. EDC1 was not cytotoxic to the cells because the EDC1-treated cells excluded trypan blue and resumed normal growth after removal of EDC1. In addition, EDC1 treatment of Raji cells prelabeled with 3H-labeled DNA did not release more radioactivity into the conditioned medium than the untreated labeled cells. EDC1 did not affect the growth of Hs2B2, a B-lymphoblast cell line, and Hs294T, a human malignant melanoma cell line. Equimolar and larger quantities of other proteinase inhibitors with inhibitory profiles similar to that of EDC1 (alpha-1 proteinase inhibitor, soybean trypsin inhibitor, lima bean trypsin inhibitor, and turkey ovomucoid) did not affect the growth of Raji cells. Raji cells have an absolute requirement of transferrin as a nutrient and require insulin to modulate the expression of transferrin receptors. The cells also synthesize interleukin-1 as an autocrine growth stimulator. EDC1 did not form a detectable complex with transferrin, insulin, or any autocrine factor synthesized by the cells.     

Purification of a high-molecular-weight inhibitor of the calcium-activated proteinase

An inhibitor of the muscle calcium-activated proteinases has been purified from porcine skeletal muscle by using DEAE-cellulose column chromatography, thermal treatment, Sephacryl S-400 column chromatography in 6 M urea and Sephacryl S-300 column chromatography in 6 M urea. Sodium dodecyl sulfate polyacrylamide slab gel electrophoresis shows that the purified inhibitor is homogeneous and has a subunit molecular weight of 172 000. The inhibitor inactivates both the low- and high-calcium-requiring forms of the calcium-activated proteinase but does not inhibit other proteinases against which it has been tried. It thus appears that the inhibitor is specific for the calcium-activated proteinase. Studies using homogeneous inhibitor and high-calcium-requiring proteinase show that one molecule of the inhibitor can inactivate up to eight molecules of the calcium-activated proteinase. Inactivation of the calcium-activated proteinase by the inhibitor cannot be reversed by calcium concentrations as high as 25 mM, thus eliminating the possibility that the inhibitor functions by chelating calcium. The inhibitory peptide appears to be extremely susceptible to proteolysis during its isolation. Even in the presence of synthetic proteinase inhibitors different inhibitor preparations yield homogeneous inhibitory peptides ranging in molecular weight from 145 000 to 172 000. Preparative electrophoresis and column chromatography have been used to isolate putative proteolytic breakdown products of the 172 kDa peptide at 145, 114, 41 and 29 kDa.     

Trypsin Inhibitor from Poecilanthe parviflora Seeds: Purification, Characterization, and Activity Against Pest Proteases

Plants synthesize a variety of molecules, including proteinaceous proteinase inhibitors, to defend themselves of being attacked by insects. In this work, a novel trypsin inhibitor (PPTI) was purified from the seeds of the native Brazilian tree Poecilanthe parviflora (Benth) (Papilioinodeae, Leguminosae) by gel filtration chromatography on a Sephadex G-100 followed by Superdex G75 chromatography (FPLC), Sepharose 4B-Trypsin column, and fractionated by reversed-phase HPLC on a C-18 column. SDS-PAGE showed that PPTI consisted of a single polypeptide chain with molecular mass of about 16 kDa. The dissociation constant of 1.0 x 10(-7) M was obtained with bovine trypsin. PPTI was stable over a wide range of temperature and pH and in the presence of DTT. The N-terminal sequence of the PPTI showed a high degree of homology with other Kunitz-type inhibitors. Trypsin-like activity in midguts of larval Diatraea saccharalis, Anagasta kuehniella, Spodoptera frugiperda, and Corcyra cephalonica were substantially inhibited by PPTI.     

Specificity of an extracellular proteinase from Conidiobolus coronatus and its inhibition by an inhibitor from insect hemolymph

The relatively little-investigated entomopathogen Conidiobolus coronatus secretes several proteinases into culture broth. Using a combination of ion-exchange and size-exclusion chromatography, we purified to homogeneity a serine proteinase of Mr 30,000-32,000, as ascertained by SDS-PAGE. The purified enzyme showed subtilisin-like activity. It very effectively hydrolyzed N-Suc-Ala(2)-Pro-Phe-pNa with a Km-1.36 x 10(-4) M and Kcat-24 s(-1), and N-Suc-Ala(2)-Pro-Leu-pNa with Km-6.65 x 10(-4) M and Kcat-11 s(-1). The specificity index k(cat)/K(m) for the tested substrates was calculated to be 176,340 s(-1) M(-1) and 17,030 s(-1) M(-1), respectively. Using oxidized insulin B chain as a substrate, the purified proteinase exhibited specificity to aromatic and hydrophobic amino-acid residues, such as Phe, Leu, and Gly at the P1 position, splitting primarily the peptide bonds: Phe(1)-Val(2), Leu(15)-Tyr(16), and Gly(23)-Phe(24). The proteinase appeared to be sensitive to the specific synthetic inhibitors of the serine proteinases DFP (diisopropyl flourophosphate) and PMSF (phenyl-methylsulfonyl fluoride) as well as to some naturally occurring protein inhibitors of chymotrypsin. It is worth noting that the enzyme exhibited the highest sensitivity to inhibition by AMCI-1 (with an association constant of 3 x 10(10) M(-1)), an inhibitor of cathepsin G/chymotrypsin from the larval hemolymph of Apis mellifera, reinforcing the possibility of involvement of inhibitors from hemolymph in insect innate immunity. The substrate specificity and proteinase inhibitor effects indicate that the purified proteinase from the fermentation broth of Conidiobolus coronatus is a subtilisin-like serine proteinase.     

Proteinase inhibitors in Nauphoeta cinerea midgut.

Proteinase inhibitors were studied in the midgut of Nauphoeta cinerea Oliv. (Blattoptera: Blaberidae) in experimental conditions, excluding their nutritional origin. One trypsin inhibitor (TI) with M(r) 8,000 and two subtilisin inhibitors (SI1 and SI2) with M(r) 13,000 and 8,000 were detected after fractionation of total protein preparation on Sephadex G-50. Ninety-four percent of both types of inhibitors was located in anterior midgut (AM). TI was 120-fold purified by FPLC-chromatography on Mono Q. Its isoelectric point was 4.3. TI lost a large part of activity in acidic and especially in alkaline medium. TI, SI1, and SI2 effectively inhibited activities of endogenous proteinases from posterior midgut (PM) of the cockroach. A search for inhibitor of endogenous unusual SH-dependent proteinase from AM revealed in AM a new inhibitor with M(r) 18,000. It was also inactivated in alkaline medium and was effective against proteinases from PM along with unusual SH-dependent proteinase from AM. A mechanism of regulation of activity of midgut proteinases is proposed based on pH-stability of inhibitors.     

A Kunitz proteinase inhibitor from Archidendron ellipticum seeds: purification, characterization, and kinetic properties

Leguminous plants in the tropical rainforests are a rich source of proteinase inhibitors and this work illustrates isolation of a serine proteinase inhibitor from the seeds of Archidendron ellipticum (AeTI), inhabiting Great Nicobar Island, India. AeTI was purified to homogeneity by acetone and ammonium sulfate fractionation, and ion exchange, size exclusion and reverse phase chromatography (HPLC). SDS-PAGE of AeTI revealed that it is constituted by two polypeptide chains (alpha-chain, M(r) 15,000 and beta-chain, M(r) 5000), the molecular weight being approximately 20 kDa. N-terminal sequence showed high homology with other serine proteinase inhibitors belonging to the Mimosoideae subfamily. Both Native-PAGE as well as isoelectric focussing showed four isoinhibitors (pI values of 4.1, 4.55, 5.27 and 5.65). Inhibitory activity of AeTI remained unchanged over a wide range of temperatures (0-60 degrees C) and pH (1-10). The protein inhibited trypsin in the stoichiometric ratio of 1:1, but lacked similar stoichiometry against chymotrypsin. Also, AeTI-trypsin complex was stable to SDS unlike the SDS unstable AeTI-chymotrypsin complex. AeTI, which possessed inhibition constants (K(i)) of 2.46 x 10(-10) and 0.5 x 10(-10)M against trypsin and chymotrypsin activity, respectively, retained over 70% of inhibitory activity after being stored at -20 degrees C for more than a year. Initial studies on the insecticidal properties of AeTI indicate it to be a very potent insect anti-feedant.     

Biochemical characterization of Acacia schweinfurthii serine proteinase inhibitor

Abstract

One of the many control mechanisms of serine proteinases is their specific inhibition by protein proteinase inhibitors. An extract of Acacia schweinfurthii was screened for potential serine proteinase inhibition. It was successfully purified to homogeneity by precipitating with 80% (v/v) acetone and sequential chromatographic steps, including ion-exchange, affinity purification and reversed-phase high performance liquid chromatography. Reducing sodium dodecyl sulphate polyacrylamide gel electrophoresis conditions revealed an inhibitor (ASTI) consisting of two polypeptide chains A and B of approximate molecular weights of 16 and 10?kDa, respectively, and under non-reducing conditions, 26?kDa was observed. The inhibitor was shown to inhibit bovine trypsin (K_i of 3.45?nM) at an approximate molar ratio of inhibitor:trypsin (1:1). The A- and B-chains revealed complete sequences of 140 and 40 amino acid residues, respectively. Sequence similarity (70%) was reported between ASTI A-chain and ACTI A-chain (Acacia confusa) using ClustalW. The B-chain produced a 76% sequence similarity between ASTI and Leucaena leucocephala trypsin inhibitor.     

Purification and characterization of a trypsin inhibitor from Putranjiva roxburghii seeds

A highly stable and potent trypsin inhibitor was purified to homogeneity from the seeds of Putranjiva roxburghii belonging to Euphorbiaceae family by acid precipitation, cation-exchange and anion-exchange chromatography. SDS-PAGE analysis, under reducing condition, showed that protein consists of a single polypeptide chain with molecular mass of approximately 34 kDa. The purified inhibitor inhibited bovine trypsin in 1:1 molar ratio. Kinetic studies showed that the protein is a competitive inhibitor with an equilibrium dissociation constant of 1.4x10(-11) M. The inhibitor retained the inhibitory activity over a broad range of pH (pH 2-12), temperature (20-80 degrees C) and in DTT (up to100 mM). The complete loss of inhibitory activity was observed above 90 degrees C. CD studies, at increasing temperatures, demonstrated the structural stability of inhibitor at high temperatures. The polypeptide backbone folding was retained up to 80 degrees C. The CD spectra of inhibitor at room temperature exhibited an alpha, beta pattern. N-terminal amino acid sequence of 10 residues did not show any similarities to known serine proteinase inhibitors, however, two peptides obtained by internal partial sequencing showed significant resemblance to Kunitz-type inhibitors.     

Crystal structure of the bovine α-chymotrypsin:kunitz inhibitor complex. An example of multiple protein:protein recognition sites

The crystal structure of bovine α-chymotrypsin (α-CHT) in complex with the bovine basic pancreatic trypsin inhibitor (BPTI) has been solved and refined at 2.8 Å resolution (R-factor=0.18). The proteinase:inhibitor complex forms a compact dimer (two α-CHT and two BPTI molecules), which may be stabilized by surface-bound sulphate ions, in the crystalline state. Each BPTI molecule, at opposite ends, is contacting both proteinase molecules in the dimer, through the reactive site loop and through residues next to the inhibitor's C-terminal region. Specific recognition between α-CHT and BPTI occurs at the (re)active site interface according to structural rules inferred from the analysis of homologous serine proteinase:inhibitor complexes. Lys15, the P₁ residue of BPTI, however, does not occupy the α-CHT S₁ specificity pocket, being hydrogen bonded to backbone atoms of the enzyme surface residues Gly216 and Ser217. © 1997 John Wiley & Sons, Ltd.     

Endogenous inhibitor of calcium activated neutral proteinase from human placenta: Purification and possible mechanism of proteinase regulation

An endogenous inhibitor of calcium activated neutral proteinase has been purified from human placenta. The procedure included chromatography on DEAE cellulose, Ultrogel AcA 22 and milli calcium activated neutral proteinase-sepharose in succession. Endogenous calcium activated neutral proteinase inhibitor was a tetramer with identical subunits of molecular weight 68 kDa. It was specific for milli calcium activated neutral proteinase (Calpain II) which is inhibited by the formation of an inactive enzyme-inhibitor complex and not by sequestering Ca₂₊ from the medium. Although micro calcium activated neutral proteinase (Calpain I) was not inhibited by endogenous calcium activated neutral proteinase inhibitor, it was protected from autolysis in the presence of the inhibitor. The placental endogenous calcium activated neutral proteinase inhibitor thus regulates Ca₂₊ activated proteolysis by ensuring micro calcium activated neutral proteinase activity, while inhibiting milli calcium activated neutral proteinase.     

A new type of low-molecular mass cysteine proteinase inhibitor from pig leukocytes

A new low-molecular mass cysteine proteinase inhibitor (CPI) was purified from the cytosol of peripheral pig leukocytes. The isolation procedure included DEAE chromatography, Sephadex G-100 gel filtration and fast-protein liquid chromatography on Mono Q. The procedure resulted in the isolation of a homogeneous protein with a molecular mass of approximately 12 kDa and a pI of 4.8. The amino terminus is blocked. The amino-acid composition and the sequence of the C-terminal part of the molecule are suggestive of a new family of cystatins. The CPI was found to be a tight-binding inhibitor of both papain and cathepsin L, with Ki values of 0.1 nM and 1 nM, respectively.     

Subtilisin Protein Inhibitor from Potato Tubers

A protein with molecular weight of 21 kD denoted as PKSI has been isolated from potato tubers (Solanum tuberosum L., cv. Istrinskii). The isolation procedure includes precipitation with (NH4)2SO4, gel chromatography on Sephadex G-75, and ion-exchange chromatography on CM-Sepharose CL-6B. The protein effectively inhibits the activity of subtilisin Carlsberg (Ki = 1.67 +/- 0.2 nM) by stoichiometric complexing with the enzyme at the molar ratio of 1 : 1. The inhibitor has no effect on trypsin, chymotrypsin, and the cysteine proteinase papain. The N-terminal sequence of the protein consists of 19 amino acid residues and is highly homologous to sequences of the known inhibitors from group C of the subfamily of potato Kunitz-type proteinase inhibitors (PKPIs-C). By cloning PCR products from the genomic DNA of potato, a gene denoted as PKPI-C2 was isolated and sequenced. The N-terminal sequence (residues from 15 to 33) of the protein encoded by the PKPI-C2 gene is identical to the N-terminal sequence (residues from 1 to 19) of the isolated protein PKSI. Thus, the inhibitor PKSI is very likely encoded by this gene.     

Two apparent human endothelial cell growth factors from human hepatoma cells are tumor-associated proteinase inhibitors

Two polypeptides from secretory products of human hepatoma cells were isolated and characterized on the basis of their stimulation of maintenance and growth of human endothelial cells in serum-free cell culture. Both factors were purified to homogeneity by a combination of reverse-phase, ion exchange, and molecular filtration high performance liquid chromatography. One factor (endothelial cell growth factor (ECGF-2a) had Mr approximately 6,500 and pI near 6. The second (ECGF-2b) had Mr = 27,000 and a pI below 4.0. Both ECGF-2a and ECGF-2b exhibited single NH2-terminal sequences. The first 25 NH2-terminal residues of ECGF-2a and the first 49 residues of ECGF-2b were determined by gas-phase microsequencing. All clearly determined residues of ECGF-2a were identical with human pancreatic secretory trypsin inhibitor. All assignable residues of ECGF-2b were identical with urinary glycoprotein proteinase inhibitor (HI-30/EDC1). Both proteins are absent or at low levels in normal plasma and urine, but appear during acute inflammatory disease and cancer. Amino acid composition of ECGF-2a and ECGF-2b was also similar to human pancreatic secretory inhibitor and HI-30/EDC1, respectively. Both ECGF-2a and ECGF-2b inhibited bovine pancreatic trypsin (2 micrograms/ml) by 50% at 750 ng/ml. ECGF-2a and ECGF-2b stimulated endothelial cell number at a half-maximal dose of 50 ng/ml (8 nM) and 80 to 130 ng/ml (5 to 9 nM) protein, respectively. When assayed under identical conditions, no effect of either factor on human smooth muscle cells, human hepatoma cells, or human, rat, and mouse fibroblasts could be detected.     

Purification and primary structure determination of two Bowman-Birk type trypsin isoinhibitors from Cratylia mollis seeds

Two Bowman-Birk type trypsin inhibitors (CmTI(1) and CmTI(2)) were purified from Cratylia mollis seeds by acetone precipitation, ion exchange, gel filtration and reverse-phase chromatography. CmTI(1) and CmTI(2), with 77 and 78 amino acid residues, respectively, were sequenced in their entirety and show a high structural similarity to Bowman-Birk inhibitors from other Leguminosae. The putative reactive sites of CmTI(1) are a lysine residue at position 22 and a tyrosine residue at position 49. Different reactive sites, as identified by their alignment with related inhibitors, were found for CmTI(2): lysine at position 22 and leucine at position 49. The dissociation constant K(i) of the complex with trypsin is 1.4 nM. The apparent molecular mass is 17 kDa without DDT and 11 kDa with reducing agent and heating.     

Characterization of a Kunitz trypsin inhibitor with one disulfide bridge purified from Swartzia pickellii

Swartzia pickellii is a Leguminosae that belongs to the Caesalpinioideae sub-family the Swartzia pickellii Trypsin Inhibitor (SWTI), a serine proteinase inhibitor was isolated from its seeds. SWTI is a single polypeptide chain protein and it's structure has 174 amino acid residues, it homologous to other Kunitz plant inhibitors, however shows some major differences: it contains only one disulfide bridge, instead two which are usually found in plant Kunitz inhibitors, and the SWTI reactive site does not contain the usual Arg or Lys residues at the putative reactive site (position 65). A glycosylation site was detected at Asn38 with 1188 kDa carbohydrate portion. The primary structure micro heterogeneity was found combining the sequence determination and mass spectrometry. Three forms of SWTI were actually defined: two glycosylated forms a 20,204 kDa (Arg 165) and 20,185 kDa (His 165) and one deglycosylated form 19,016 kDa (Arg 165), all of them contain a Met residue at position 130.     

Phage display selection of hairpin loop soyacystatin variants that mediate high affinity inhibition of a cysteine proteinase

Two hairpin-loop domains in cystatin family proteinase inhibitors form an interface surface region that slots into the active site cleft of papain-like cysteine proteinases, and determine binding affinity. The slot region surface architecture of the soybean cysteine proteinase inhibitor (soyacystatin N, scN) was engineered using techniques of in vitro molecular evolution to define residues that facilitate interaction with the proteinase cleft and modulate inhibitor affinity and function. Combinatorial phage display libraries of scN variants that contain mutations in the essential motifs of the first (QVVAG) and second (EW) hairpin-loop regions were constructed. Approximately 1010-1011 phages expressing recombinant scN proteins were subjected to biopanning selection based on binding affinity to immobilized papain. The QVVAG motif in the first hairpin loop was invariant in all functional scN proteins. All selected variants (30) had W79 in the second hairpin-loop motif, but there was diversity for hydrophobic and basic amino acids in residue 78. Kinetic analysis of isolated scN variants identified a novel scN isoform scN(LW) with higher papain affinity than the wild-type molecule. The variant contained an E78L substitution and had a twofold lower Ki (2.1 pM) than parental scN, due to its increased association rate constant (2.6 +/- 0.09 x 107 M-1sec-1). These results define residues in the first and second hairpin-loop regions which are essential for optimal interaction between phytocystatins and papain, a prototypical cysteine proteinase. Furthermore, the isolated variants are a biochemical platform for further integration of mutations to optimize cystatin affinity for specific biological targets.     

Human granulocyte elastase is inhibited by the urinary trypsin inhibitor

Two forms of urinary trypsin inhibitor, A and B, were purified from the urine of pregnant women. Form A was the only inhibitor present in fresh urine and inhibitor B arose from degradation of A upon storage of urine. The molecular masses of A and B were about 44 and 20 kDa, respectively, as judged from dodecyl-sulfate polyacrylamide gel electrophoresis, but about 60 kDa and 30 kDa, respectively, as judged from gel filtration analysis. The discrepancy can perhaps be explained by the carbohydrate content amounting to about 10% of each inhibitor. After reduction with mercaptoethanol, inhibitor A and inhibitor B had identical apparent molecular masses of about 20 kDa on dodecyl-sulfate gel electrophoresis. These results and the results of amino acid analysis suggest that one molecule of inhibitor A yields two molecules of inhibitor B. On agarose gel electrophoresis inhibitor A migrated as a rather broad band in the prealbumin region and inhibitor B as 3 well defined bands in the beta-region. Specific antisera were raised against inhibitor A and B. The two inhibitors showed the immunologic reaction of identity with each other and with the plasma inter-alpha-trypsin inhibitor, when using either antiserum. The inhibitors both gave quantitative inhibition of bovine trypsin, the results indicating a 4/1 trypsin/inhibitor molar ratio for A and a 2/1 ratio for B. The two substances also effectively inhibited granulocyte elastase. No inhibition of porcine pancreatic elastase was demonstrable.     

The complete amino acid sequence of barley trypsin inhibitor

The amino acid sequence of barley trypsin inhibitor has been determined. The protein is a single polypeptide consisting of 121 amino acid residues and has Mr = 13,305. No free sulfhydryl groups were detected by Ellman's reagent, which indicates the presence of five disulfide bridges in the molecule. The primary site of interaction with trypsin was tentatively assigned to the arginyl-leucyl residues at positions 33 and 34. On comparison of the sequence of this inhibitor with those of other proteinase inhibitors, we found that the barley trypsin inhibitor could not be classified into any of the established families of proteinase inhibitors (Laskowski, M., Jr., and Kato, I. (1980) Annu. Rev. Biochem. 49, 593-626) and that this inhibitor should represent a new inhibitor family. On the other hand, this trypsin inhibitor showed a considerable similarity to wheat alpha-amylase inhibitor (Kashlan, N., and Richardson, M. (1981) Phytochemistry (Oxf.) 20, 1781-1784) throughout the whole sequence, suggesting a common ancestry for both proteins. This is the first case of a possible evolutionary relationship between two inhibitors directed to totally different enzymes, a proteinase and a glycosidase.     

Isolation, characterization and cDNA sequencing of a Kazal family proteinase inhibitor from seminal plasma of turkey (Meleagris gallopavo)

The turkey reproductive tract and seminal plasma contain a serine proteinase inhibitor that seems to be unique for the reproductive tract. Our experimental objective was to isolate, characterize and cDNA sequence the Kazal family proteinase inhibitor from turkey seminal plasma and testis. Seminal plasma contains two forms of a Kazal family inhibitor: virgin (Ia) represented by an inhibitor of moderate electrophoretic migration rate (present also in the testis) and modified (Ib, a split peptide bond) represented by an inhibitor with a fast migration rate. The inhibitor from the seminal plasma was purified by affinity, ion-exchange and reverse phase chromatography. The testis inhibitor was purified by affinity and ion-exchange chromatography. N-terminal Edman sequencing of the two seminal plasma inhibitors and testis inhibitor were identical. This sequence was used to construct primers and obtain a cDNA sequence from the testis. Analysis of a cDNA sequence indicated that turkey proteinase inhibitor belongs to Kazal family inhibitors (pancreatic secretory trypsin inhibitors, mammalian acrosin inhibitors) and caltrin. The turkey seminal plasma Kazal inhibitor belongs to low molecular mass inhibitors and is characterized by a high value of the equilibrium association constant for inhibitor/trypsin complexes.     

Inactivation of human cystatin C and kininogen by human cathepsin D

A papain inhibitor or 22 kDa was isolated from human placenta and shown to be identical to residues Cys246-Leu373 of the third domain of human kininogen. This kininogen domain and recombinant human cystatin C were inactivated by peptide bond cleavages at hydrophobic amino acid residues due to the action of cathepsin D. These results further support the proposed role cathepsin D in the regulation of cysteine proteinase activity.