Effect of Protein Infused into Sheep Duodenum on Activities of Pancreatic Proteases in Intestinal Digesta and on the Absorption Site of Amino Acids
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Digestion
Milk protein
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Conjugate
Autolysis (biology)
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Abstract Bovine and porcine pancreatic residue, remaining after the extraction of insulin, has been used to prepare a proteinase powder. This powder was used as a source of trypsin and chymo-trypsin. The individual enzymes were isolated and purified by chromatography on sulfopropyl (SP)-Sephadex C-25 and affinity chromatography on soybean trypsin inhibitor (STI)-Sepharose. The bovine proteinase powder contained a-chymotrypsin, trypsin and chymotrypsin B in the ratio 5:2:1. The porcine powder contained cationic trypsin, anionic trypsin and cationic chymotrypsin in the ratio 5 : 1. 4 : 3. The isolated enzymes were characterized and found to be identical with enzymes isolated from fresh tissue with the exception of porcine chymotrypsin. Porcine cationic chymotrypsin was isolated as two distinct forms, A-l and A-2, which appear to be different activation products of porcine chymotrypsinogen A. Both forms resemble bovine a-chymotrypsin, a three chain structure, rather than porcine chymo-trypsin A, a two chain structure. Furthermore, the B-chain appears to be cleaved, possibly at residues Phe89-Lys90.
Sephadex
Chymotrypsinogen
Sepharose
Aprotinin
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The two main trypsin-chymotrypsin isoinhibitors previously purified from lentils (Lens culinaris Medik.), LCI-1 and LCI-4, inhibited one mol of human trypsin (1.05 and 1.00), more than one mol of bovine trypsin (1.53 and 1.38) and human chymotrypsin (1.70 and 1.43) as well as less than one mol of bovine chymotrypsin (0.62 and 0.54, respectively) per mol of inhibitor. Complex formation, together with chemical and enzymatic modification studies, showed that they were Bowman-Birk inhibitors with two independent reactive sites. One of these sites, mainly reacting with trypsin, contained arginine and bound tightly to bovine trypsin, less tightly to human trypsin and loosely to human chymotrypsin. The other reactive site, preferring chymotrypsin, contained tyrosine and bound tightly to human chymotrypsin, less tightly to bovine chymotrypsin and loosely to bovine trypsin. The amounts of bound enzyme exceeding one mol per mol of inhibitor reacted with the “wrong” sites: bovine trypsin with the chymotrypsin-reactive and human chymotrypsin with the trypsin-reactive one. The much higher inhibition of human chymotrypsin compared to that of bovine chymotrypsin resulted from a combination of two effects: the additional binding of human chymotrypsin at the “wrong” reactive site and the weak binding of the bovine chymotrypsin.
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The presence of two specific trypsin-chymotrypsin inhibitors from head parts of the rhynchobdellid leech Theromyzon tessulatum is reported. Two proteins, anti-trypsin chymotrypsin A (ATCA; 14636.6 +/- 131 Da) and anti-trypsin-chymotrypsin B (ATCB; 14368 +/- 95 Da) were purified by size exclusion and anion-exchange chromatography followed by reversed-phase HPLC. Based on amino-acid composition, N-terminal sequence determination (MELCELGQSCSRD-NPQPSNM), matrix assisted laser desorption-time of flight measurement (MALDI-TOF), trypsin mapping comparison, inhibition constant determination (Ki), and influence on amidolytic activity of different serine proteases, it is demonstrated that ATCA and ATCB are novel and highly potent serine-protease inhibitors of trypsin and chymotrypsin (ATCA: 350fM towards trypsin and chymotrypsin; ATCB: 400 and 75 fM towards trypsin and chymotrypsin, respectively). It is further surmised that ATCA and ATCB are linked, in that ATCB would lead to the formation of ATCA after loss of few amino acid residues.
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Pancreas size, and trypsin and chymotrypsin esterase activities were determined in the pancreas and small intestinal contents (digesta) from 84 pigs killed at 3 or at 5 weeks of age. Pancreases and digesta from 3-week-old pigs had low trypsin activity (averaging 0.33 unit/mg fresh tissue; 4.0 units/g of liquid digesta). At 5 weeks of age, trypsin activity per unit of tissue or digesta was about 3.5 times greater. Chymotrypsin activity was high at 3 weeks (0.84 unit/mg pancreatic tissue; 17.2 units/g digesta), and was only 1 to 2 times greater at 5 weeks. Digesta from three pigs killed at birth averaged 13.1 units chymotrypsin activity/g of digesta, but had no trypsin activity. The trypsin and chymotrypsin activities in the pancreases from these three pigs averaged 0.43 and 0.89 unit/mg of fresh tissue respectively. The "nonparallel" change in the rate of synthesis and secretion of pancreatic enzymes between 3 and 5 weeks of age reduced the proportion of chymotrypsin to trypsin activity (2.64 to 1.31 for pancreas; 3.39 to 1.41 for digesta). Possible mechanisms involved in this phenomenon are discussed. Increases in pancreas size and body weight were nearly proportional, but the rate of enzyme activity increased as much as 4.1 times (pancreatic trypsin) that of body weight increase. The size and enzyme activity of the pancreas were similar in male and female pigs. Three-week-old males had more trypsin activity in the digesta than did females of the same age. Body weight gains to 3 or 5 weeks of age were positively correlated with total trypsin (P < 0.05) and chymotrypsin (P < 0.01) activity in the digesta.
Esterase
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The stability of trypsin and chymotrypsin was determined in human intestinal juice. On a weight per weight basis at the end of one-half hour as much as 63% of chymotrypsin was still enzymatically active while only 8% of the trypsin activity could be detected. In fact, 30% of the chymotrypsin still remained at the end of 2 hours.
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Site-directed mutagenesis
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Ascaridia galli
Fowl
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Photoreactive derivatives of the Bowman-Birk trypsin-chymotrypsin inhibitor (BBI) from soybeans and of CI, the trypsin-chymotrypsin inhibitor from chick peas, were prepared by selective modification of the epsilon-amino groups of lysine residues with 2-nitro-4(5)-azidophenylsulfenyl chlorides (2,4(5)-NAPS-C1). The ultraviolet absorption spectra of the photolabeled inhibitors indicated that three out of the five lysines of BBI and one of the seven lysines of CI were modified. The inhibitory activity of the modified inhibitors towards trypsin and chymotrypsin was not reduced even after photolysis. The specific lysine residues that constitute the trypsin-inhibitory sites of BBI and CI did not react with the photoreactive reagents. Further modification of the photoreactive derivatives of BBI and CI with maleic anhydride, directed towards the trypsin-reactive sites, resulted in almost complete loss of the trypsin-inhibiting activity without reducing the ability to inhibit chymotrypsin. A pronounced potentiation effect (approximately 2x) of the chymotrypsin inhibiting activity was noted for 2,5-NAPS-CI and it was retained even after maleylation followed by photolysis, raising the possibility of exposure of an additional chymotrypsin inhibitory site in CI.
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