P-Trypsin

To obtain insight into the molecular basis of action of the cyclotheonamides and to explain the high affinity as a serine protease inhibitor, complexes of CtA with a-thrombin [41a,c] and P-trypsin [41b,c], respectively, were studied by X-ray crystallography. 

Kylanpaa-Back ML, Kemppainen E, Puolakkainen P. Trypsin-based laboratory methods and carboxypepti-dase activation peptide in acute pancreatitis. JOP J Pancreas (Online) 2002 3 34-48. 

Figure 8. Stabilization of proteinases (aT ) on association with their protein inhibitors as a function of the logarithms of the equilibrium association constants (log K ssoc). Horizontal bar lines are used to indicate the range of log Kassoc values. Key to inhibitors (inside Figure, at lower right) OM, chicken ovomucoid for other abbreviations, see Table II. Key to proteinases BT, bovine p-trypsin PT, porcine 6-trypsin ...

Serine endopeptidase Serine in the active site Chymotrypsins A, B and C, peptidase B alkaline, pro-teinases a-and P-trypsin,... 

Fig. 1. Schematic drawing of precursors for selected brain oligopeptides. Shaded areas represent the location of sequences of active peptide products which are normally cleaved by trypsin-like enzymes acting on double-basic amino acid residues. Precursors are not necessarily drawn to scale, (a) CRF precursor (b) proopiomelanocortin (POMC) (c) P-protachykinin (d) proenkephalin A (e) CGRP precursor (f) preprodynorphin, ie, preproenkephalin B. Terms are...

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... 

Weissman, J.S., Kim, P.S. Kinetic role of non-native species in the folding of bovine pancreatic trypsin inhibitor. Proc. Natl. Acad. Sci. USA 89 9900-9904, 1992. 

FIGURE l.l Hydrophobic interaction and reversed-phase chromatography (HIC-RPC). Two-dimensional separation of proteins and alkylbenzenes in consecutive HIC and RPC modes. Column 100 X 8 mm i.d. HIC mobile phase, gradient decreasing from 1.7 to 0 mol/liter ammonium sulfate in 0.02 mol/liter phosphate buffer solution (pH 7) in 15 min. RPC mobile phase, 0.02 mol/liter phosphate buffer solution (pH 7) acetonitrile (65 35 vol/vol) flow rate, I ml/min UV detection 254 nm. Peaks (I) cytochrome c, (2) ribonuclease A, (3) conalbumin, (4) lysozyme, (5) soybean trypsin inhibitor, (6) benzene, (7) toluene, (8) ethylbenzene, (9) propylbenzene, (10) butylbenzene, and (II) amylbenzene. [Reprinted from J. M. J. Frechet (1996). Pore-size specific modification as an approach to a separation media for single-column, two-dimensional HPLC, Am. Lab. 28, 18, p. 31. Copyright 1996 by International Scientific Communications, Inc.. Shelton, CT.]... 

The T2 site also became protected from tryptic hydrolysis after phosphorylation of the native or solubilized sarcoplasmic reticulum vesicles with inorganic phosphate in a calcium free medium in the presence of dimethylsulfoxide or glycerol [121,252]. Under these conditions the Ca -ATPase is converted into a covalent E2-P intermediate, that is analogous in conformation to the E2V intermediate formed in the presence of vanadate. In contrast to this, the T2 site in the stable phosphorylated Ca2E P intermediate generated by the reaction of the Ca -ATPase with chro-mium-ATP in the presence of Ca [178,253] was fully exposed to trypsin, just as it was in the nonphosphorylated Ca2Ei form. Therefore the phosphorylated intermediates show the same sensitivity to trypsin at the T2 site as the corresponding nonphosphorylated enzyme forms. 

The intramolecular distances measured at room temperature with the AEDANS FITC pair were similar in the Ca2Ei and E2V states [297]. Ca and lanthanides are expected to stabilize the Ej conformation of the Ca -ATPase, since they induce a similar crystal form of Ca -ATPase [119,157] and have similar effects on the tryptophan fluorescence [151] and on the trypsin sensitivity of Ca -ATPase [119,120]. It is also likely that the vanadate-stabilized E2V state is similar to the p2 P state stabilized by Pi [418]. Therefore the absence of significant difference in the resonance energy transfer distances between the two states implies that the structural differences between the two conformations at sites recorded by currently available probes, fall within the considerable error of resonance energy transfer measurements. Even if these distances would vary by as much as 5 A the difference between the two conformations could not be established reliably. 

Protein isolation with affinity precipitation has been discussed in detail by Mattiasson and co-workers (see, e.g. Galaev and Mattiassion, 1997) and they have provided an exhaustive tabulation. Polymers varied from alginate.s/chitosan to dextran to NIPAM. Examples of the used proteins are from trypsin, p-glucosidase, xylanase, alkaline protease, etc. It is remarkable that affinity precipitation can sometimes give results comparable to affinity chromatography. 

Wakselman, M. Xie, J. Mazaleyrat, J.-P. Boggetto, N. Vilain, A. C. Montagne, J.-J. Reboud-Ravaux, M. New mechanism-based inactivators of trypsin-like proteinases. Selective inactivation of urokinase by functionalized cyclopeptides incorporating a sulfoniomethyl-substituted mera-aminobenzoic acid residue. J. Med. Chem. 1993, 36, 1539-1547. 

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