Antoni Polanowski

The squash family of serine proteinase inhibitors. Amino acid sequences and association equilibrium constants of inhibitors from squash, summer squash, zucchini, and cucumber seeds

Six amino acid sequences for trypsin inhibitors isolated from squash, summer squash, zucchini, and cucumber seeds were determined. All these inhibitors along with the two previously sequenced squash inhibitors (1) form the squash inhibitor family. The striking characteristic of the family is that its member inhibitors are very small (29-32 residues, 3 disulfide bridges). The association equilibrium constants with bovine beta trypsin for 6 squash family inhibitors were determined and range from 5.9 X 10(10) to 9.5 X 10(11) M-1.

Purification and partial characterization of the pancreatic proteolytic enzymes trypsin, chymotrypsin, and elastase from the chicken.

The purpose of this work was to isolate, purify and partially sequence trypsin, chymotrypsin and elastase from the chicken pancreas. The extraction of the pancreatic zymogens with 0.5 M CaCl2 at pH 7.5 for 9 h appeared to be most effective in obtaining maximum recovery of the three enzymes. The sequential Cucurbita maxima trypsin inhibitor I/bovine pancreas trypsin inhibitor/soybean trypsin inhibitor affinity chromatography gave the best result for the isolation of trypsin, chymotrypsin and elastase, respectively, from the same extract. For each proteinase, multiple form of enzymatic activity could be observed after gel electrophoresis and each form was further purified on an ion-exchange column. The N-terminal amino acid sequence of trypsin and chymotrypsin showed homologies with the bovine enzymes whereas elastase showed homologies with the porcine enzyme. The molecular mass of trypsin, chymotrypsin and elastase were estimated to be 23,500, 25,700 and 25,000, respectively, which are values close to those in mammalian species. Although some kinetic constants (Km and k(cat)/Km) appeared different from those observed in other species, the pH dependent enzymatic activities were similar to those reported in other animal species.

Aspartic proteinase from Penicillium camemberti : purification, properties and substrate specificity

An acid proteinase from the culture filtrate of Penicillium camemberti was isolated in a two-step purification procedure by cation exchange chromatography and gel filtration. The enzyme is an aspartic proteinase inhibited by pepstatin, DAN, and EPNP, with a molecular mass determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 33.5 kDa. The optimum activity for hydrolysis of denatured hemoglobin is around pH 3.4. The enzyme is highly specific for the aromatic and hydrophobic amino acid residue in insulin B-chain and, like pepsin, selectively splits only one Leu7-Met8 peptide bond in the squash trypsin inhibitor CMTI 1. The hydrolyzed bond can be resynthesized by P. camemberti proteinase at neutral pH.

Production of exocellular proteolytic enzymes by various species of Penicillium

Ten species of fungi belonging to the genus Penicillium were investigated for their ability to produce proteolytic enzymes. The fungi were cultivated on five different media. The synthesis of Penicillium enzymes depends on the kind of medium. The highest activity of hemoglobin-degrading proteinases was found in cultures of P. candidum, P. cyclopium, and P. piscaricum in Mosolov medium. The best producers of proteinases active at pH 7.0 were P. lilacinum and P. chrysogenum, using modified Sanders medium.

On the genetic and structural similarities between the squash seeds polypeptide trypsin inhibitors and wheat germ agglutinin.

SummaryIn this report we propose the disulfide bridges alignment in the squash polypeptide trypsin inhibitors. The prediction is based on the extensive homology in the amino acid sequence between these inhibitors and a portion of the wheat germ agglutinin domains for which the position of the disulfide bridges are known.

SERINE PROTEINASE INHIBITOR PROFILES IN THE HEMOLYMPH OF A WIDE RANGE OF INSECT SPECIES

1. The inhibition of trypsin, chymotrypsin, neutrophil elastase and cathepsin G, and pancreatic elastase by the hemolymph of 14 insect species in six orders has been investigated. 2. All samples showed great diversity in terms of both total proteinase inhibitory capacity and specificity. 3. The highest total inhibitory capacity was found in the larval hemolymph of species in the beetle family Tenebrionidae and the lowest in that of an adult coreid bug, Acanthocephala femorata.

The antifungal properties of chicken egg cystatin against Candida yeast isolates showing different levels of azole resistance

The increasing incidence of fungal infections together with the emergence of strains resistant to currently available antifungal drugs calls for the development of new classes of antimycotics. Naturally occurring antifungal proteins and peptides are of interest because of low toxicity, immunomodulatory potential and mechanisms of action distinct from those of currently available drugs. In this study, the potent antifungal activity of cystatin, affinity‐purified from chicken egg white (CEWC), against the most frequent human fungal pathogens of the genus Candida was identified and characterised. CEWC inhibited the growth of azole‐sensitive Candida albicans isolates with minimal inhibitory concentration (MIC) values ranging from 0.8 to 3.3 μmol l−1, a potency comparable with those of fluconazole and histatin 5, the antimicrobial peptide of the human saliva. Similarly to histatin 5, CEWC activity was not compromised in azole‐resistant isolates overproducing the multidrug efflux transporters Cdr1p and Cdr2p and did not antagonise fluconazole or amphotericin B. CEWC had candidacidal activity, as revealed by the time‐kill assay, and, similarly to histatin 5, completely inhibited the growth at supra‐MIC concentrations. This was in contrast to the fungistatic effect and trailing growth observed with fluconazole. CEWC inhibited the growth of Candida parapsilosis and Candida tropicalis at similar concentrations, whereas Candida glabrata was more resistant to CEWC.

Immunologically active peptides that accompany hen egg yolk immunoglobulin Y: separation and identification.

Abstract The protein mixture of cytokine-inducing activity accompanying chicken immunoglobulin Y, named yolkin, consists of several peptides of molecular weight (MW) ranging from over 1 to 35 kDa. Yolkin and its constituent peptides were found to be efficient inducers of interleukin (IL)-1β, IL-6 and IL-10 secretion. N-terminal amino acid sequences of eight of the electrophoretically purified yolkin constituents revealed that all of them are homological to some fragments of the C-terminal domain of vitellogenin II. The fractions of MW about 4 and 12 kDa are free of carbohydrates and start at position 1732 in the vitellogenin amino acid sequence; whereas the other fractions (MW about 16, 19, 23, 29, 32 and 35 kDa) appeared to be glycoproteins corresponding to the amino acid sequence of vitellogenin starting at position 1572. From these data, it is concluded that yolkin most likely represents vitellogenin-derived peptides that possess cytokine-inducing activity and are, at least partially, responsible for such properties of separated immunoglobulin Y preparation. This finding reveals a new role for vitellogenin as a reservoir of polypeptides that may play an important role in the innate immune system of the developing embryo.

A Kazal-type serine proteinase inhibitor from chicken liver (clTI-1): purification, primary structure, and inhibitory properties.

Low-molecular-mass trypsin inhibitor (clTI-1; chicken liver Trypsin Inhibitor-1) was purified from chicken liver by extraction with perchloric acid, ammonium sulfate precipitation, a combination of ethanol-acetone fractionation followed by gel filtration, ion-exchange chromatography and RP-HPLC on a C18 column. The inhibitor occurs in two isoforms with molecular masses of 5938.56 and 6026.29 Da (determined by MALDI TOFF mass spectrometry). The complete amino acid sequences of both isoforms were determined (UniProtKB/Swiss-Prot P85000; ISK1L_CHICK). The inhibitor shows a high homology to Kazal-type family inhibitors, especially to trypsin/acrosin inhibitors and pancreatic secretory trypsin inhibitors. clTI-1 inhibits both bovine and porcine trypsin (K(a)=1.1 x 10(9) M(-1) and 2.5 x 10(9) M(-1), respectively). Significant differences were shown in the inhibition of the anionic and cationic forms of chicken trypsin (K(a)=4.5 x 10(8) M(-1) and 1.2 x 10(10) M(-1)). Weak interaction with human plasmin (K(a)=1.2 x 10(7) M(-1)) was also revealed.

Pancreatic secretory trypsin inhibitor acts as an effective inhibitor of cysteine proteinases gingipains from Porphyromonas gingivalis.

BACKGROUND AND OBJECTIVE Porphyromonas gingivalis has been implicated as the major pathogen of periodontitis in adults. This organism produces an array of virulence factors, of which cysteine proteinases, referred to as gingipains K and R, are believed to play a crucial role in pathogenicity. The aim of this study was to investigate the susceptibility of gingipains K and R to inhibition by a pancreatic secretory trypsin inhibitor. MATERIAL AND METHODS Enzyme activities were measured spectrophotometrically using chromogenic turnover substrates. To estimate the value of the association constant (Ka), constant amounts of enzyme were reacted with increasing amounts of inhibitor to reach equilibrium. The Ka was calculated by fitting the experimental data to the given equation. RESULTS In this study it was shown that gingipains are susceptible to pancreatic Kazal-type trypsin inhibitors (pancreatic secretory trypsin inhibitors). Bovine pancreatic secretory trypsin inhibitor, having an Arg residue at the P1 position of the reactive site, specifically inhibited the activity of the Arg-specific cysteine proteinase gingipain R, whereas porcine inhibitor, possessing a Lys residue at the P1 position, exhibited activity only against the Lys-specific cysteine proteinase gingipain K. The Ka values for the inhibitor-proteinase interaction were 1.6 x 10(6) m(-1) and 2.0 x 10(4) m(-1) for gingipain R and gingipain K, respectively. CONCLUSION This finding is the first demonstration of the inhibitory potency of the Kazal-type specific trypsin inhibitors against cysteine proteinases. These discoveries open new possibilities for the use of naturally occurring inhibitors, displaying activity across enzyme families, as a model in designing new molecules of therapeutic significance.