Tadeusz Wilusz

The refined 2.0 Å X-ray crystal structure of the complex formed between bovine β-trypsin and CMTI-I, a trypsin inhibitor from squash seeds (Cucurbita maxima) Topological similarity of the squash seed inhibitors with the carboxypeptidase A inhibitor from potatoes

The stoichiometric complex formed between bovine β‐trypsin and the Cucurbita maxima trypsin inhibitor I (CMTI‐I) was crystallized and its X‐ray crystal structure determined using Patterson search techniques. Its structure has been crystallographically refined to a final R value of 0.152 (6.0 — 2.0 Å). CMTI‐I is of ellipsoidal shape; it lacks helices or β‐sheets, but consists of turns and connecting short polypeptide stretches. The disulfide pairing is CYS‐3I‐20I, Cys‐10I‐22I and Cys‐16I‐28I. According to the polypeptide fold and disulfide connectivity its structure resembles that of the carboxypeptidase A inhibitor from potatoes. Thirteen of the 29 inhibitor residues are in direct contact with trypsin; most of them are in the primary binding segment Val‐2I (P4) — Glu‐9I (P4′) which contains the reactive site bond Arg‐5I — Ile‐6I and is in a conformation observed also for other serine proteinase inhibitors.

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.

Nuclear magnetic resonance solution and X-ray structures of squash trypsin inhibitor exhibit the same conformation of the proteinase binding loop.

A comparison of the solution nuclear magnetic resonance (n.m.r.) structures of squash trypsin inhibitor from seeds of the squash Cucurbita maxima with the X-ray structure of a trypsin complex of the inhibitor shows that the n.m.r. and X-ray structures are similar in terms of the global folding and secondary structure. The average atomic root-mean-square difference between the 36 n.m.r. structures on the one hand and the X-ray structure is 0.96 A for the backbone atoms and 1.95 A for all heavy atoms. The n.m.r. and X-ray structures exhibit extremely similar conformations of the primary proteinase binding loop. Despite the overall similarity, there are small differences between the mean computed structure and the X-ray structure. The n.m.r. structures have slightly different positions of the segments from residues 16 to 18, and 24 and 25. The n.m.r. results show that the inclusion of stereospecific assignments and precise distance constraints results in a significant improvement in the definition of the n.m.r. structure, making possible a detailed analysis of the local conformations in the protein.

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.

Purification of two low-molecular-mass serine proteinase inhibitors from chicken liver.

Two serine proteinase inhibitors, designated clTI-1 and clTI-2 were purified from livers of chickens to apparent homogeneity by a combination of ethanol-acetone fractionation, gel filtration and ion-exchange chromatography on CM-cellulose and Mono S columns. The inhibitor clTI-1 is a single polypeptide chain, low-molecular-mass protein (Mr about 6200), very stable to heat and ethanol. It inhibits chicken, porcine and bovine trypsins as well as human plasmin. The second protein, clTI-2 of Mr 17,000 was shown to be a very effective inhibitor of both trypsins and human cathepsin G. Since both inhibitors are sensitive to arginine modification with phenylglyoxal it is assumed that this amino acid residue is present at the P1 position of the reactive site peptide bond. The N-terminal amino acid sequence of 28 residues of clTI-2 (SVDVSKYPSTVSKDGRTLVACPRILSPV) revealed a high homology of this protein to the third domain of the chicken ovoinhibitor, whereas, the clTI-1 (APPAAEKYYSLPPGAPRYYSPVV) has some sequence identity to a fragment of the human inter-alpha-trypsin inhibitor.

Isolation and amino acid sequence of two trypsin isoinhibitors from duck pancreas.

DPTI II and DPTI IV, two trypsin inhibitors from duck pancreas, have been isolated by affinity chromatography on immobilized anhydrotrypsin, anion exchange and RP-HPLC. The complete amino acid sequence of both inhibitors was determined after reductive carboxymethylation and digestion with Staphylococcus aureus V8 protease or trypsin. The inhibitors were each found to be a single polypeptide chain comprised of 69 amino acid residues and their molecular masses were estimated at 7687 Da for DPTI II and 7668 Da for DPTI IV. The only difference in amino acid sequence between the two inhibitors is the replacement of Arg for His residue in the C-terminal position of DPTI IV.

High concentrations of sodium chloride facilitate the use of immobilized chymotrypsin for separating virgin forms of specific trypsin inhibitors

It has been shown that specific trypsin inhibitors exhibit also antichymotrypsin activity in the presence of high NaCl concentrations. Taking advantage of this phenomenon a simple procedure of separation of the virgin forms of trypsin inhibitors from squash seeds and porcine pancreas (Kazal) was elaborated. In a typical experiment the inhibitor sample was loaded onto immobilized chymotrypsin equilibrated with 5 M NaCl at pH 8. After washing out unadsorbed material the virgin forms of inhibitors could be eluted either with water, buffer pH 8.0 or 0.02 M citrate buffer pH 2.6 containing no NaCl.

Kazal-type chymotrypsin inhibitor from duck pancreas.

A chymotrypsin inhibitor of the Kazal-type has been isolated from duck pancreas, by affinity chromatography on immobilized chymotrypsin, gel filtration on Bio-Gel P-10 and reverse phase (RP)-HPLC. It inhibits bovine chymotrypsin Aalpha with an association constant (K(a)) of 2.06x10(7) M(-1). The complete amino acid sequence was determined after digestion of pyridylethylated inhibitor with Staphylococcus aureus V8 protease and chemical cleavage with CNBr. Duck pancreatic chymotrypsin inhibitor (DPCI) was found to be a single polypeptide chain composed of 65 amino acid residues, corresponding to a molecular mass of 7191 Da.

Modifications outside the proteinase binding loop in Cucurbita maxima trypsin inhibitor III (CMTI-III) analogues change the binding energy with bovine β-trypsin

Five 26‐peptide analogues of the trypsin inhibitor [Pro18]CMTI‐III containing Leu or Tyr in position 7 and Val or Tyr in position 27: 1 (Leu7, Tyr27), 2 (Tyr7, Val27), 3 (Tyr7, Tyr27), 4 (Leu7, Val27) and 5 (Leu7, Ala18, Tyr27) were synthesized by the solid‐phase method. Analogues 1–4 displayed K a with bovine β‐trypsin of the same order of magnitude as the wild CMTI‐III inhibitor, whereas for analogue 5, this value was lower by about 3 orders of magnitude. This indicated that for the analogues with Pro (but not with Ala) in position 18, the side‐chain interactions between positions 7 and 27 did not play a critical role for the stabilization of the active structure. In addition, these results also suggest that Tyr7 is involved in an additional aromatic interaction with position 41 of the enzyme.