Anna Wilimowska-Pelc

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.

A simple method for isolating trypsin from trichloroacetic acid extracts of bovine pancreas

Abstract Trichloroacetic acid was used to isolate trypsin and trypsinogen from bovine pancreas. Trypsinogen, which is at first soluble in trichloroacetic acid, slowly forms a sediment. In alkaline medium and in the presence of calcium chloride, proenzyme is activated to enzyme which has high proteolytic, esterase, and amidase activity and is homogenous in polyacrylamide gel electrophoresis. It is suggested that the gradual reduction of trypsinogen solubility in trichloroacetic acid is associated with the presence of basic pancreatic trypsin inhibitor in trichloroacetic acid extracts from a bovine pancreas.

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.

Metallo-proteinase from the seedlings of kale (Brassica oleracea L. var. sabellica): : Preparation, partial characterization and substrate specificity.

Metallo-proteinase from 8-d-old seedlings of kale was isolated. The enzyme was extracted with 1% NaCl, concentrated by ammonium sulfate and finally purified by high-performance liquid chromatography. The isolated enzyme had a molecular weight of 22.4 kDa and showed a maximum activity at pH 9.0 using casein as a substrate. Proteolytic activity of proteinase was inhibited by chelators. The inhibition by ethylenediaminetetraacetate (EDTA) was abolished by some divalent metals ions, especially by Zn2+. The enzyme showed activity against the synthetic peptides Suc-Ala-Ala-Pro-Leu-pNA and Suc-Ala-Ala-Pro-Phe-pNA, and hydrolized the following peptide bonds in the oxidized insulin B-chain: Leu6-Cya7, Leu15-Tyr16, Leu17—Val18 and Phe25-Tyr26.