Kunitz inhibitors

Some of the best investigated anti-nutrients are the enzyme inhibitors present in legumes and other plants. The Bowman-Birk and the Kunitz inhibitors of trypsin and other proteases are among the best characterized. In contrast to the non-specific and widespread influences of tannins and lectins (Carmona, 1996), the Bowman-Birk, Kunitz and other such inhibitors target specific enzymes. Corresponding with this, proteases and other digestive enzymes vary in sensitivity to the different inhibitors. 

Honisberger, M., and Tacchini-Vonlanthen, M. (1983) Ultrastructural localization of Kunitz inhibitor on thin sections of Glycine max (soybean) cv. Maple Arrow by the gold method. Histochemistry 77, 37-50. 

It has been known for some time that many examples of naturally occurring Kunitz inhibitors exist, both isolated and as domains in larger proteins, which inhibit a variety of serine proteases [47]. This strongly suggests that this molecular framework is compatible with inhibition of this general class. The contact region between these inhibitors and their protease targets is known from a... 

Murzin AG, Lesk AM, Chothia C. b- Trefoil Fold patterns of structure and sequence in the kunitz inhibitors interleukins-1J3 and la and fibroblast growth factors. J Mol Biol 1992 223 531-543. 

In cells tested, the Bowman-Birk inhibitor is more potent than the Kunitz inhibitor in decreasing cell growth. Thus, it appears that the antichymotrypsin activity is modulating growth of these cells. 

Pando, S.C., Oliva, M.L., Sampaio, C.A. et al. 2001. Primary sequence determination of a Kunitz inhibitor isolated from Delonix regia seeds. Phytochemistry 57 625-631. 

Three proteins, for which I did not have structural data, should also be mentioned, namely the so-called minibody [147-149] a Kunitz inhibitor domain of the human lipoprotein associated coagulation inhibitor (LACI-D1 Refs. 89 and 90) which resembles BPTI and CP-1, a designed scaffold zinc-finger [150], The minibody is a 61 aa designed... 

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. 

If thrombin and factor Xa, the major activated blood coagulation factors (Fig. 11.6), escape into healthy blood vessels, blood clots will develop and occlude capillaries throughout the body. Direct inhibition of these activated enzymes in the blood flow utilizes serine protease inhibitors, of which there are two common types a Kunitz inhibitor and a serpin. The former possess a Kunitz domain, a convex antiparallel (1-sheet that exactly fits into the concave active site of a serine protease, directly blocking it (lock and key mechanism). By contrast, serpins undergo complex interactions with other proteins to cause conformational changes that bait and block the catalytic action (Fig. 11.12 shows the bait). Table 11.3 fists the major coagulation inhibitors and cofactors, their targets and mechanisms of action. 

The Bowman Birk trypsin inhibitor is the smaller trypsin inhibitor with MW -S.OkD and binding sites for trypsin and chymotrypsin (Birk, 1985). BBI is more heat-stable than the Kunitz inhibitor probably resulting from its great proportion of disulfide cross-linking at 7 per mole BBI. Although BBI is present in much lower amounts in raw soybeans, its relative heat stability may be the main reason for the residual STI activity in moist-heated soybean protein products. Friedman and Brandon (2001) reported about 7 pg/mL BBI in soy-based infant formula as measured by ELISA while Dipietro and Liener (1989) reported <0.1 pg/mg (their limit of detection by ELISA) for SIR... 

The effect of purified soybean Kunitz and BBI trypsin inhibitors as dietary supplements (5, 15, or 50 g/kg) on spontaneous pulmonary metastasis of lung carcinoma 3LL cells as well as human ovarian cancer HRA cells was investigated in mice (Ko-bayashi et al., 2004a). Only Kunitz inhibitor inhibited the formation of lung metastasis in a dose-dependent manner. These results suggest that dietary supplementation of Kunitz inhibitor could more efficiently regulate cell metastatic processes than BBI,... 

Protease inhibitors in soybeans, known as trypsin inhibitors (TIs), play important roles in nutritional properties of soybeans and soy protein products. Two types of TI are the Kunitz inhibitor and the Bowman-Birk inhibitor. The Kunitz inhibitor has a MW of 21,500 with two disulfide bonds, while the Bowman-Birk inhibitor has a MW of 7,900 with seven disulfide bonds (Wolf, 1977). The large ratio of disulfide bonds to MW in the Bowman-Birk inhibitor stabilizes protein conformation and makes the Bowman-Birk inhibitor highly resistant to heat denaturation and inactivation. The Kunitz inhibitor inhibits trypsin, while the Bowman-Birk inhibitor inhibits both trypsin and chymotrypsin. The kinetics of TI inactivation when heating at high water activity were determined by Johnson et al. (1980) they estimated that 83-91% of the TI activity in soybeans is due to the Kunitz inhibitor. Recently, the Bowman-Birk inhibitor was attributed cancer-protecting qualities and also interest exists in using purified soy TI to treat AIDS patients (Kennedy, 1995, 1998 Kennedy Szuhaj, 1994). 

Orf and Hymowitz (1979) identified soybean lines without Kunitz TI, which enable soybeans to be utilized without heating in feed and some food applications. The Kunitz inhibitor accounts for about 80-90% of the TI native activity. This modification was achieved through traditional breeding methods, but has not become a commercialized trait. 

Snail epidermis contains at least six trypsin-kallikrein inhibitors with molecular weights ranging from 6431 to 6591 (70-72). The soybean contains two basic types of protease inhibitors, the Kunitz inhibitor of 21,500 daltons (73) and the Bowman-Birk inhibitor of 7975 daltons (74). The two are quite different proteins as shown in Figure 6. The Great Northern bean (Phaseolus vulgaris) has at least three trypsin isoinhibitors ranging in molecular weight from 8086 to 8884 (15). There are four and possibly six isoinhibitors of trypsin in lima bean (Phaseolus lunatus)(75). 

Although pancreatic secretory trypsin inhibitor (Kazal inhibitor) has not been studied by DSC to our knowledge, it is included because it is homologous to the domains of ovomucoids and ovoinhibi-tors (Laskowski and Kato, 1980). Both the porcine and bovine Kazal inhibitors are relatively stable (Menegatti et al., 1981 De Marco et al., 1982), but less so than the Kunitz inhibitor (BPTI). 

Modifications of the active center of an inhibitor result in changes in the properties. For example, Arg (63) of the Kunitz inhibitor from soybeans can be replaced by Lys without changing the inhibitory behavior, while substitution by Trp abolishes the inhibition of trypsin and increases the inhibition of ch)motrypsin. Indeed, He (64) can be replaced by Ala, Leu, or Gly without change in activity, while the insertion of an amino acid residue, e. g., Arg (63)-Glu (63a)-Ile (64), abolishes all inhibition and makes the inhibitor a normal substrate of trypsin. 

Inhibitor activity is normally determined with commercial animal enzymes, e. g., bovine trypsin or bovine chymotrypsin. The evaluation of a potential effect of the inhibitors on human health assumes that the inhibition of human enzymes is known. Present data show that inhibitors from legumes generally inhibit human trypsin to the same extent or a little less than bovine trypsin. On the other hand, human chymotrypsin is inhibited to a much greater extent by most legumes. Ovomucoid and ovoinhibitor from egg white as well as the Kazal inhibitor from bovine pancreas do not inhibit the human enzymes. The Kunitz inhibitor from bovine pancreas inhibits human trypsin but not chymotrypsin. The data obtained greatly depend not only on the substrate used, but also on the enzyme preparation and the reaction conditions, e. g., on the ratio enzyme/inhibitor. The stability of an inhibitor as it passes through the stomach must also be taken into account in the evaluation of a potential effect (cf. Table 16.15). The Kunitz inhibitor of soybeans, for... 

Inhibitors of Bowman-Birk types are widely distributed in plants and significantly different from the Kunitz inhibitor in their amino acid composition and are able to interact not only with trypsin, but with chymotrypsin (Valueva Mosolov, 2002 Rawlings et al., 2004). Inhibitors of Bowman-Birk type characterized by the presence of two reactive centers on a single polypeptide chain rich in cysteine (7 or more residues in one polypeptide), and the lack of amino acid residues tryptophan and tyrosine. The molecular weight of such inhibitors can vary from 8 to 16 kDa. Sometimes there are inhibitors that contain two domains on one polypeptide chain and active only in relation to one type of enzyme (Valueva Mosolov, 2002 Yan et al., 2009 Mosolov Valueva, 2008). 

Inhibitors of proteolytic enzymes may play an important role in plant protection not only from harmful insects, but also other pests nematodes, the nematodes. Many nematodes are parasitic on plants and cause significant damage to agricultural production. In the gut of nematodes contains active cysteine and serine proteases, including chymotrypsin and kallikrein is similar. It is interesting that when infected with worms, among transcripts of tomato were found proteins related to the family of Kunitz inhibitor (SBH) (Valueva Mosolov, 2002 Schroeder et al., 1995). 

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