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We report a simple three-step method of generating a homogeneous toxic fragment (toxin) in high yield from B. thuringiensis var. kurstaki. Purified crystals were digested with trypsin at pH 10.5, followed by (NH4)2SO4 precipitation and dialysis. For the HD73 strain the preparation is toxic to eastern-spruce-budworm (Choristoneura fuminiferana) larvae. It(More)
Bacillus thuringiensis produces a 130-140 kDa insecticidal protein in the form of a bipyramidal crystal. The protein in the crystals from the subspecies kurstaki HD-1 and entomocidus was found to contain 16-18 cysteine residues per molecule, present primarily in the disulphide form as cystine. Evidence that all the cysteine residues form symmetrical(More)
Trypsin is shown to generate an insecticidal toxin from the 130-kDa protoxin of Bacillus thuringiensis subsp. kurstaki HD-73 by an unusual proteolytic process. Seven specific cleavages are shown to occur in an ordered sequence starting at the C-terminus of the protoxin and proceeding toward the N-terminal region. At each step, C-terminal fragments of(More)
The kinetic behaviour of human thrombin has been studied with 26 tripeptidyl-p-nitroanilide substrates protected at the N terminus and with 9 unprotected ones. By the regression analysis of experimentally determined 1/Km, kcat and kcat/Km values the individual contribution of each side chain of the various substrates to the kinetic parameters was(More)
The inhibitory constants of a series of synthetic N-carboxymethyl peptide inhibitors and the kinetic parameters (Km, kcat, and kcat/Km) of a series of model synthetic substrates were determined for the membrane-bound kidney metalloendopeptidase isolated from rabbit kidney and compared with those of bacterial thermolysin. The two enzymes show striking(More)
1. The interaction of heparin with human plasmin was investigated measuring plasmin activity and enzyme inactivation in the presence of heparin. Hydrolysis of synthetic substrates (H-D-Val-Leu-Lys-pNA, H-D-Val-Phe-Lys-pNA and H-D-Pro-Phe-Lys-pNA) by plasmin was enhanced by heparin through an increase in kcat values. 2. This effect was the consequence of a(More)
A procedure for the selective isolation of the C-terminal peptides from enzymatic digests of proteins is described. The methodology is based on the diagonal electrophoretic procedure described by R. G. Duggleby and H. Kaplan (1975) Anal. Biochem. 65, 346-354). The carboxyl groups in the protein are amidated with [14C]-methylamine followed by enzymatic(More)
1. The kinetic properties of pancreatic elastase (E.C. were investigated with 33 tripeptidyl-p-nitroanilide substrates, and the Km, kcat and kcat/Km values were determined. 2. The individual contributions of the substrate side chains to the kinetic constants were evaluated by regression analysis. As a result of the additivity of the(More)
The kinetic parameters of the tryptic hydrolysis of tripeptidyl-p-nitroanilide substrates were determined and the data were studied by regression analysis. The sequence of substrates optimal from the viewpoint of kinetic constants 1/Km, kcat and kcat/Km was established and the influence of amino acid side chains on the binding and reactivity of substrates(More)