Trypsin-like Proteinases

Trypsin-like proteinases are serine proteinases that recognized peptide residues with positively charged side chains (arginyl or lysyl residues) and that effect... 

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. 

The exact form in which non-crosslinked elastin is secreted from smooth muscle cells is yet to be clearly defined. Foster et al. (36) have suggested that a non-cross linked elastin (pro-elastin) is secreted from smooth muscle cells in a form that is approximately 120,000 to 140,000 daltons. They have suggested that proelastin is cleaved to smaller molecular weight forms of non-crosslinked elastin. It should be noted, however, that this view is not entirely supported by data from other laboratories. There are two reports on the use of isolated mRNA from chick aorta suggesting only a 70,000 dalton non-cross linked elastin is the major product of translation (37,38). There is also a recent report suggesting that aortic mRMA translates a 200,000 dalton putative elastin product (39). We have recently isolated a non-crosslinked elastin from the aortas of copper deficient chicks that appears to be 100,000 daltons (27). Its amino acid composition is similar to that for tropoelastin (Table III). A major problem in resolving these points is that the trypsin-like proteinase associated with elastin is not easily denatured or separated from the non-crosslinked forms of elastin. The proteinase is also not readily inhibited by commonly used inhibitors for trypsin-like proteinases (26). 

In the works of A. Konarev (Konarev et al. 1999 Konarev et al., 2004) shows in detail the variability of inhibitors of trypsin-like proteinases in cereals due to resistance to various grain pests. So in wheat trypsin inhibitors are represented by several genetically independent systems of proteins controlled by the genome and B chromosomes ID (endosperm), 3Dp (aleurone layer), IDS and 3Ap (leaf). Trypsin inhibitors of rye are controlled chromosome 3R and barley 3H. The most complex structure of inhibitors was wheat leaves, with the genomic formula AABBDD. In general, it is the sum of the spectra of trypsin inhibitors from several tetraploid (T. turgidum) (AABB) and (Aegilops tauschii Coss.) (DD) (Konarev, 1986 Konarev et al., 2004). 

Total protein, albumin, urea (standard methods) and middle molecules (MM) were determined in citrated plasma [6]. The trypsin-like activity (TLA) of plasma was measured using the chromogenic peptide substrate (Z-glycyl-glycyl-L-arginine-4-nitroanilide) [7]. Evaluation of anti-enzymatic potential in plasma was based on concentrations of the main protease inhibitors -proteinase inhibitor (ttj-PI) and aj-macroglobulin (a -M). Student s t-test was used for statistical analysis. 

Figure B3.1.1 A 15% SDS-polyacrylamide gel stained with Coomassie brilliant blue. Protein samples were assayed for the purification of a proteinase, cathepsin L, from fish muscle according to the method of Seymour et al. (1994). Lane 1, purified cathepsin L after butyl-Sepharose chromatography. Lane 2, cathepsin L complex with a cystatin-like proteinase inhibitor after butyl-Sepharose chromatography. Lane 3, sarcoplasmic fish muscle extract after heat treatment and ammonium sulfate precipitation. Lane 4, sarcoplasmic fish muscle extract. Lanes M, low-molecular-weight standards aprotinin (Mr 6,500), a-lactalbumin (Mr 14,200), trypsin inhibitor (Mr 20,000), trypsinogen (Mr 24,000), carbonic anhydrase (Mr 29,000), gylceraldehyde-3-phosphate dehydrogenase (Mr 36,000), ovalbumin (Mr 45,000), and albumin (Mr 66,000) in order shown from bottom of gel. Lane 1 contains 4 pg protein lanes 2 to 4 each contain 7 pg protein.

C-Methyl-/3-Casein. 14C-Labeled proteins prepared by reductive methylation have potential as substrates in the study of proteolytic enzymes. A serious limitation is that complete methylation of lysine residues results in inhibition of proteolysis by enzymes with trypsin-like specificity (13). It was interesting to determine whether this problem could be overcome by incomplete methylation which left unaltered most of the lysine residues in more or less random distribution throughout the protein. /3-Casein was selected as a suitable protein for this study since it is cleaved by trypsin-like enzymes to well characterized fragments, the y-caseins, in addition to less well characterized fragments, the proteose peptones. We anticipated that this type of study could provide a basis for a general investigation of milk protein transformation by the native milk proteinase which has a specificity similar to trypsin (14). 

The results on the hydrolysis of partially methylated /3-casein by plasmin indicate that proteins radiomethylated to a low level can serve as substrates for trypsin-like enzymes and probably for proteinases in general. Because it is likely that methylation will interfere with enzymatic attack at lysine residues, the complete hydrolysis of /3-casein probably would not be possible. Studies on mastitic milk demonstrate the usefulness of 14C-methyl proteins for qualitative examination of protein hydrolysis in complex multiprotein systems where resolution and characterization of individual protein fragments is difficult. The requirements in such studies are the availability of pure samples of the proteins under investigation and a suitable technique for separating the radio-labeled protein from hydrolytic products. 

The ratio of microfibrillar protein to elastin, however, appears to decrease upon maturation (2). Other proteins are also secreted with microfibrillar protein and elastin. It is now clear that bound to elastin in its non-crosslink form(s) is a trypsin-like neutral proteinase (26). This proteinase effects... 

Love, R. A., Parge, H. E., Wickersham, J. A., Hostomsky, Z., Habuka, N., Moomaw, E. W., Adachi, T., and Hostomska, Z. (1996) The crystal structure of hepatitis C virus NS 3 proteinase reveals a trypsin-like fold and a structural zinc binding site. Cell 87, 331-342. 

The catalytic domains of trypsin-like serine proteinases consist of two six-stranded antiparallel yS-sheets yielding y9-barrels in which the strands are arranged in a so-called Greek key motif (y91-4) followed by an antiparallel hairpin loop (y95-6) [10]. At the intersection of the two barrels reside the residues of the catalytic triad, His57, Aspl02 and Serl95 [14]. The back-... 

Cathepsin G is an enzyme with dual chymotrypsin and trypsin-like specificity and as a leukocyte proteinase it is involved in the early stages of the immune response. Synthesis and inhibitory activity of a-aminoalkylpho-sphonates diphenyl esters as irreversible cathepsin G inhibitors has been described by Lesner et al. The phosphonates (244) and (245) turned out to be especially active. " ... 

Kazal family inhibitors - the type of trypsin inhibitor-like proteinase, commonly found in animals, including invertebrates. Molecules consist of one or more domains, and three disulfide bridges. The sequence of many often conservative, but the site of binding to the enzyme can be variable (Li et al., 2009 Rimphanitchayakit Tassanakajon, 2010) Kazal inhibitors are involved in various pathologies of the pancreas. In particular inhibitor Kazal SPINKl is expressed not only in normal cells of the pancreas, but also transformed. It is... 

---