Inhibition, trypsin digestion

Ricin is a potent cellular protein toxin contained in the beans of the castor been plant (Ricinus communis), which is extensively cultivated for oil production and is also a common ornamental garden plant. Ricin is able to inhibit ribosomal protein synthesis eventually causing cell death, and owing to these properties it has been allegedly used in terrorist and criminal activities. After trypsin digestion of castor bean crude extracts, Ostin et al. [105] were able to unambiguously... 

Compounds that inhibit oligosaccharide digestion not only impair herbivore nutrition but can also act as antifeedants. A variety of plant-derived secondary metabolites inhibit glycosidases (Table 13.1). Numerous plant protein glycosidase inhibitors have been isolated and some of these have dual functions as both a-amylase and trypsin inhibitors (Table 13.2). 

In the case of sarin-inhibited ChE, the phos-phyl moiety has also been displaced as isopropyl methylphosphonic acid using trypsin digestion and alkaline phosphatase (Nagao et al., 1997 Matsuda et al., 1998). 

Another example of the use of mass spectrometry to delect toxins is to identify ricin, a highly toxic protein that inhibits cell protein synthesis. Ricin is produced from the seeds of Ricinus communis plants (known conunonly as castor beans) [73]. Structurally, ricin is made of A- and B-chains linked by a disulfide bridge. The toxicity of ricin is due primarily to the A-chain, which acts as an RNA A-glycosidase, which leads to ribosome incapacitation and ultimately to cell death. Ricin was identified from crude castor bean extracts using LC-MS/MS. The extract was denatured, reduced, and alkylated prior to trypsin digestion. Ricin identification was based on the detection of marker peptides in the digest. These markers include T5, T7, Til, T12, and T13 from the A-chain and T3, T5, T14, T19, and T20 from the B-chain. MS/MS can provide the amount and sequence of each marker for irrefutable evidence. For quick screening of ricin in crude extracts, MALDI-MS can be used to provide the molecular mass profile of the marker peptides. 

Shielding of Cytochrome b at the Stromal Surface. CpA has no effect on cyt b (9), an expected result since the Pro residue at the penultimate position should block CpA (10). However, the efficacy of trypsin digestion was increased if the membranes were treated first with CpA. o-phenanthroline was added to inhibit CpA before trypsin treatment so that the two proteases did not act simultaneously. Reversing the order of protease addition had no effect, showing that the access of trypsin was increased by prior treatment with CpA. Similar results were obtained using antibodies (i) and (iii) to the NHj- and COOH-termini (unpubl.). Thus, cyt b on the stromal membrane surface is shielded by the COOH-termini of other thylakoid proteins. 

The capacity of Sp to reactivate Me is inhibited by trypsin digestion showing that a protein structure is necessary in Sp to reactivate Me. Besides, heated or boiled Sp are also inactives. Different diets modify Me activity but have no effect on Sp. (Nervi et al, 1975). 

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... 

There is evidence that protease inhibitors selectively regulate the activity of specific digestive enzymes at the level of gene expression (Rosewicz et al., 1989). Specifically, soybean trypsin inhibitor increases secretion of proteases, including a form of trypsin that is resistant to inhibition but does not cause an increase in amylase secretion. Although the relationships between protease inhibitors and exocrine pancreatic secretion have received the most attention, pancreatic secretion is increased when potato fiber is added to the diet (Jacob et al., 2000), although the mechanism and signaling pathway have not been elucidated. 

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