A new protein inhibitor of trypsin and activated hageman factor from pumpkin (Cucurbita maxima) seeds

A protein inhibitor (CMTI‐V; M r 7106) of trypsin and activated Hageman factor (Factor XIIa), a serine protease involved in blood coagulation, has been isolated for the first time from pumpkin (Cucurbita maxima) seeds by means of trypsin‐affinity chromatography and reverse phase high performance liquid chromatography (HPLC). The dissociation constants of the inhibitor complexes with trypsin and Factor XIIa have been determined to be 1.6 × 10−8 and 4.1 × 10−8 M, respectively. The primary structure of CMTI‐V is reported. The protein has 68 amino acid residues and one disulfide bridge and shows a high level of sequence homology to the Potato I inhibitor family. Furthermore, its amino terminus consists of an N‐acetylates Ser. The reactive site has been established to be the peptide bond between Lys44‐Asp45. The modified inhibitor which has the reactive site peptide bond hydrolyzed inhibits trypsin but not the Hageman factor.

[1]  J. Bieth.  Some Kinetic Consequences of the Tight Binding of Protein-Proteinase-Inhibitors to Proteolytic Enzymes and their Application to the Determination of Dissociation Constants , 1974 .

[2]  G. Abraham,et al.  A technique for the removal of pyroglutamic acid from the amino terminus of proteins using calf liver pyroglutamate amino peptidase. , 1978, Biochemical and biophysical research communications.

[3]  Y. Hojima,et al.  Pumpkin seed inhibitor of human factor XIIa (activated Hageman factor) and bovine trypsin. , 1982, Biochemistry.

[4]  K. Titani,et al.  Wound-induced proteinase inhibitors from tomato leaves. I. The cDNA-deduced primary structure of pre-inhibitor I and its post-translational processing. , 1985, The Journal of biological chemistry.

[5]  B. Wittmann-Liebold,et al.  Micro‐sequence analysis of peptides and proteins using 4‐NN‐dimethylaminoazobenzene 4′‐isothiocyanate/phenylisothiocyanate double coupling method , 1978 .

[6]  R. Wynn,et al.  Inhibition of human β-factor XIIA by squash family serine proteinase inhibitors , 1990 .

[7]  J. Hejgaard,et al.  Subtilisin inhibitor from seeds of broad bean (vicia FABA); purification, amino acid sequence and specificity of inhibition , 1984 .

[8]  G. Reeck,et al.  Combined use of trypsin-agarose affinity chromatography and reversed-phase high-performance liquid chromatography for the purification of single-chain protease inhibitor from corn seeds. , 1986, Journal of chromatography.

[9]  M. Richardson.  Chymotryptic inhibitor I from potatoes. The amino acid sequence of subunit A. , 1974, The Biochemical journal.

[10]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[11]  P. Andrews,et al.  A procedure for in situ alkylation of cystine residues on glass fiber prior to protein microsequence analysis. , 1987, Analytical biochemistry.

[12]  I. Kato,et al.  Protein inhibitors of proteinases. , 1980, Annual review of biochemistry.

[13]  L. Bosch,et al.  Structural studies on the coat protein of alfalfa mosaic virus. The complete primary structure. , 1977, European journal of biochemistry.

[14]  J Otlewski,et al.  The squash family of serine proteinase inhibitors. Amino acid sequences and association equilibrium constants of inhibitors from squash, summer squash, zucchini, and cucumber seeds. , 1985, Biochemical and biophysical research communications.

[15]  S. Moore,et al.  On the determination of cystine as cysteic acid. , 1963 .

[16]  M. Richardson,et al.  Identification of the reactive (inhibitory) sites of chymotryptic inhibitor I from potatoes , 1977 .

[17]  B. Bidlingmeyer,et al.  Rapid analysis of amino acids using pre-column derivatization. , 1984, Journal of chromatography.

[18]  S. Moore,et al.  The preparation and enzymatic hydrolysis of reduced and S-carboxymethylated proteins. , 1963, The Journal of biological chemistry.

[19]  Yu Wang,et al.  The amino acid sequence of a trypsin inhibitor from the seeds of Momordica charantia Linn. Cucurbitaceae , 1988 .

[20]  J. Hejgaard,et al.  Amino acid sequence of serine protease inhibitor CI-1 from barley. Homology with barley inhibitor CI-2, potato inhibitor I, and leech eglin , 1982 .

[21]  J Otlewski,et al.  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 , 1989, FEBS letters.

[22]  A. Pusztai,et al.  The complete amino acid sequence of the major alpha subunit of the lectin from the seeds of Dioclea grandiflora (Mart). , 1984, European journal of biochemistry.

[23]  Y. Hojima,et al.  Plant inhibitors of serine proteinases: Hageman factor fragment, kallikreins, plasmin, thrombin, factor Xa, trypsin, and chymotrypsin. , 1980, Thrombosis Research.