Bovin trypsin

Figure 2.14 shows examples of both cases, an isolated ribbon and a p sheet. The isolated ribbon is illustrated by the structure of bovine trypsin inhibitor (Figure 2.14a), a small, very stable polypeptide of 58 amino acids that inhibits the activity of the digestive protease trypsin. The structure has been determined to 1.0 A resolution in the laboratory of Robert Huber in Munich, Germany, and the folding pathway of this protein is discussed in Chapter 6. Hairpin motifs as parts of a p sheet are exemplified by the structure of a snake venom, erabutoxin (Figure 2.14b), which binds to and inhibits... 

Fehlhammer, H., Bode, W., Huber, R. Crystal structure of bovine trypsinogen at 1.8 A resolution. 11. Crystallographic refinement, refined crystal structure and comparison with bovine trypsin. J. Mol. Biol. 

Ruhlman, A., et al. Structure of the complex formed by bovine trypsin and bovine pancreatic trypsin inhibitor. /. Mol. Biol. 77 417-436, 1973. 

Feldmann, RJ, Bing DH, Furie BC, Furie B. Interactive computer surface graphics approach to study of the active site of bovine trypsin. Proc Natl Acad Sci USA 1978 75 5409-12. 

The three-dimensional structure of trypsin inhibitor 1 from C. maxima was determined in 1989 by X-ray crystallography in complex with bovine trypsin and by in aqueous solution. The three-dimensional... 

The PNA chain was linked to the peptide spacer glutamic acid-(y-tert-butyl ester)-(fi-aminohexanoic acid)-(fi-aminohexanoic acid) (Glu [OtBuj-fiAhx-fiAhx) via an enzymatically cleavable Glu-Lys handle. The Glu [OtBuj-fiAhx-fiAhx spacer was coupled to the amino-functionalized membrane by standard Fmoc-Chemistry. Then the membranes were mounted in an ASP 222 Automated SPOT Robot and a grid of the desired format was dispensed at each position. The free amino groups outside the spotted areas were capped and further chain elongation was performed with Fmoc-protected PNA monomers to synthesize the desired PNA oligomers (18). After completion of the synthesis, the PNA oligomers were cleaved from the solid support by incubation with bovine trypsin solution in ammonium bicarbonate at 37 °C for 3 h. 

Woodard, S.L., Mayor, J.M., Bailey, M.R., Barker, D.K., Love, R.T., Lane, J.R., Delaney, D.E., McComas-Wagner, J.M., MaUubhotla, H.D., Hood, E.E., Dangott, L.J., Tichy, S.E., and Howard, J.A. (2003). Maize (Zea mays)-derived bovine trypsin characterization of the first large-scale, commercial protein product from transgenic plants. Biotechnol. Appl. Biochem. 28(Pt. 2) 123-130. 

Materials and Methods. Fully deuterated phycocyanin and protio phycocyanin from Ph. luridum were used. The method of purifying phycocyanin was identical to that used previously (15, 16). The purity of the phycocyanin preparations, the complete substitution of deuterium for hydrogen in the fully deuterated phycocyanin, and the reversibility of the aggregation phenomenon were ascertained as previously (4, 16). Purified bovine trypsin, soybean trypsin inhibitor, and bovine liver catalase were obtained from the Worthington Biochemical Corp., Freehold, N. J., and used without further purification. Bovine a -casein B was kindly supplied by Chien Ho of the University of Pittsburgh. 

The conformational flexible part found in domain II of SSI is in stark contrast with other protein proteinase inhibitors, such as BPTI. In the case of BPTI, the backbone conformation is found to be nearly identical in both the free and the bovine trypsin complex.28 The conformational rigidity of protein proteinase inhibitors has been considered for a long time to be a necessary condition to inhibit their target enzymes and to protect themselves from attack by other proteinases. It is generally recognized that the substrate-like protein proteinase inhibitors, such as BPTI, STI, and Ovomucoid domain 3,... 

The conformation of M73R SSI in the free state was nearly identical to that of the WT SSI since there was almost no chemical shift difference between these two inhibitors. The carbonyl carbon chemical shift profile of the M73R SSI-subtilisin BPN complex was also very similar to that of the WT SSI-subtilisin BPN complex, showing that the conformations of these complexes are also nearly the same. The shift profile of the M73R SSI-bovine trypsin, however, was quite different from those of the subtilisin complexes. This can be taken as a good indication that the conformational flexible segments are responsible for the wide inhibitory activity of SSI. 

The active site structure of trypsin-like enzymes is considered to be very similar to that of bovine trypsin, yet little is known about them. Refinement of these structures is important also for the purpose of designing physiologically active substances. With a view to comparing the spatial requirements of active sites of these enzymes, dissociation constants of the acyl enzyme-ligand complex, K-, which were defined before, were successfully analyzed By taking advantage of inverse substrates which have an unlimited choice of the acyl component, development of stable acyl enzymes could be possible. These transient inhibitors for trypsin-like enzymes could be candidates for drugs. In this respect, the determination of the deacylation rate constants for the plasmin- and thrombin-catalyzed hydrolyses of various esters were undertaken 77). 

The iron complexes of both human serum transferrin and chicken ovo-transferrin were completely resistant to all proteolytic enzymes tested (7). The metal-free proteins, on the other hand, were rapidly hydrolyzed under similar conditions. Fig. 14 compares results with bovine a-chymo-trypsin. Other enzymes used with similar results were bovine trypsin, bacterial proteinase (Nagarse) and ficin (735). The weaker copper complex of chicken ovotransferrin was hydrolyzed, however, at a rate approx-... 

Thermal Stability. Increased catalytic efficiency at low temperatures is associated often with a decrease in thermal stability (41). Proteolytic enzymes from the pyloric caeca of cod, herring, and mackerel are more heat labile than bovine trypsin (42) the thyroid protease of burbot from cold waters had lower thermal stability than that from burbot in a warmer habitat (43). The half-life of myofibrillar ATPase at 37°C averages 1 min for the enzyme from Antarctic Ocean fish species, 70 min for the enzyme from fish species in the Indian Ocean, and 600 min for the enzyme from East African hot spring fish (33). 

FIGURE 13 Plot of the logarithm of the retention volume (In VR) versus the concentration of the displacing salt, ammonium sulphate, in the HP-HIC mode with the proteins I, insulin B-chain 2, bovine trypsin inhibitor 3, bovine trypsinogen 4, insulin A-chain 5, ribonuclease 6, sperm whale myoglobin 7, horse heart cytochrome c. Data from Ref. 42. 

Tryptic activity measured in vitro with bovine trypsin. Tryptic activity and larval growth reported as percent of control where control equals 100%. Soybean inhibitor was Kunitz inhibitor. Percent relative growth determined by weight gain of S. exigua for 11 days on artificial diet containing the specified phytochemicals. 

Chambers JL, Stroud RM (1979) The accuracy of refined protein structures comparison of two independently refined models of bovine trypsin. Acta Cryst B35 1861-1874... 

Bode W, Schwager P (1975) The refined crystal structure of bovine -trypsin at 1.8 A resolution, II. Crystallographic refinement, calcium binding, benzamidine binding site and active site at pH 7.0. J Mol Biol 98 693-717... 

Bovin trypsin has been modified with semicarbazide in the presence of a carbodiimide probably involving carboxyl groups. Also, monoamine derivatives of a, fi or y cyclodex-trins are introduced into trypsin. The thus modified trypsins are more resistant to autolysis and show some increase in esterase activity. Ribonucleases are also modified by attaching aminoethanol, taurin and 1,2-diaminoethane to approximately six to eight of the 11 available carboxylates using EDC. The modified enzymes lose activity and the cytotoxicity is increased. 

Bovine trypsin can also be chromatographed on CM-cellulose at pH 3.2 in an ionic strength gradient (115). In this way, active trypsin begins to separate from inactive proteins present in commercial preparations or formed during trypsinogen activation in the absence of calcium. After... 

Figures 10 and 11 show how porcine trypsinogen can be prepared in high yield by chromatography on CM-cellulose. The amino acid composition of this protein is given in Table III, with two sets of values for the bovine precursor. Analytical results have also been obtained with a commercial sample of bovine trypsin (123).

J. Koepke, U. Ermler, E. Warkentin, G. Wenzl, and P. Flecker. 2000. Crystal structure of cancer chemopreventive Bowman-Birk inhibitor in ternary complex with bovine trypsin at 2.3 A resolution Structural basis of Janus-faced serine protease inhibitor specificity J. Mol. Biol. 298 477-491. (PubMed)... 

An X-ray structure of a benzothiazole ketone derivative (RWJ-56423) complexed with bovine trypsin (1.9A resolution) showed a hemiketal intermediate formed involving Ser-189, and hydrogen bonds with His-57 and Gln-192 <2003JME3865>. 

Angiotensin-I, methionine enkephalin, substance-P, bovine trypsin, horse cytochrome-C, horse myoglobin, bovine insulin and egg-white lysozyme were purchased from Sigma Chemical Co. (St. Louis, MO) and used without further purification. 

Tanaka, T., McRae, B.J., Cho, K. et al. (1983). Mammalian tissue trypsin-like enzymes. Comparative reactivities of human skin tryptase, human lung tryptase, and bovine trypsin with peptide 4-nitroanilide and thioester substrates. J. Biol. Chem. 258, 13552-13557. 

Table 15.6. Bovine Trypsin Autolysis Fragments, Used for mlz Calibration...

---