Tryptic activity

The E2 form is not cleaved by chymotrypsin, but trypsin cleaves at Arg (T1) and subsequently at Lys (T2) and tryptic inactivation of E2K or E2P forms is linear and associated with cleavage at Arg" (Ti) [104,108], Inactivation of K-phosphatase is delayed because cleavage of T1 and T2 in sequence is required for inactivation of K-phosphatase activity [105],... 

NCD-4 is a nonfluorescent carbodiimide derivative that forms a fluorescent adduct with the Ca -ATPase, accompanied by inhibition of ATPase activity and phos-phoenzyme formation [376-378]. Ca protected the enzyme against the inhibition by NCD-4 and reduced the extent of labeling, suggesting that the reaction may involve the Ca " " binding site. The stoichiometry of the Ca -protected labeling was i 2mole/mol ATPase. The fluorescence emission of the modified Ca -ATPase is consistent with the formation of a protein bound A-acylurea adduct in a relatively hydrophobic environment. After tryptic proteolysis of the NCD-4 labeled ATPase the fluorescence was associated with the A2 band of 24 kDa [376,379]. 

Since the discovery of the enkephalins in 1975 [11] a large number of endogenous opioid peptides have been detected in mammals, and at present three distinct families of opioid peptides are known (for a review, See Ref. 12). These are the enkephalins, the endorphins (a-, (J-, and y-), and the dynorphins and neoendorphins. The recently discovered endomor-phins [13] also may represent endogenous opioid peptides. Peptides with opioid activity have also been isolated from tryptic digests of milk casein... 

Mechanism-based inactivation results in formation of a covalent adduct between the active inhibitor and the enzyme, or between the active inhibitor and a substrate or cofactor molecule. If the mechanism involves covalent modification of the enzyme, then one should not be able to demonstrate a recovery of enzymatic activity after dialysis, gel filtration, ultrafiltration, or large dilution, as described in Chapters 5 to 7. Additionally, if the inactivation is covalent, denaturation of the enzyme should fail to release the inhibitory molecule into solution. If a radiolabeled version of the inactivator is available, one should be able to demonstrate irreversible association of radioactivity with the enzyme molecule even after denaturation and separation by gel filtration, and so on. In favorable cases one should likewise be able to demonstrate covalent association of the inhibitor with the enzyme by a combination of tryptic digestion and LC/MS methods. 

The first belief in the possibility of enzyme stabilization on a silica matrix was stated by Dickey in 1955, but he did not give experimental evidence, only mentioning that his experiments were unsuccessful [65]. A sol-gel procedure for enzyme immobilization in silica was first developed by Johnson and Whateley in 1971 [66]. The entrapped trypsin retained about 34 % of its tryptic activity observed in solution before the encapsulation. Furthermore, the enzyme was not released from the silica matrix by washing, demonstrating the increased stability and working pH range. Unfortunately, the article did not attract attention, although their method contained all the details that may be found in the present-day common approach. This was probably due to its publication in a colloid journal that was not read by biochemists. 

Ling V., Guzzetta A.W., Canova-Davis E., Stults J.T., Hancock W.S., Covey T.R., and Shushan B.I. (1991), Characterization of the tryptic map of recombinant DNA derived tissue plasminogen activator by high-performance liquid chromatography-electrospray ionization mass spectrometry, Anal. Chem. 63, 2909-2915. 

Although more limited, sufficient spectroscopic data is available on the HO-2 isoform to confirm that the proximal ligand is also an imidazole. A 28-kDa catalytically active fragment of human HO-2 obtained by tryptic digestion of the enz5mie expressed in E. coli was shown by optical, resonance Raman, and EPR studies to have a neutral (imionized)... 

In systems used to follow the esterolytic activity of trypsin in the presence of BSA ( ), tannins do not ajpear to interact directly with trypsin because of their greater affinity for reaction with BSA (64). In the absence of BSA seme tryptic activity is lost however even in the presence of glycocholate, which prevents any precipitates from forming. This indicates that bile surfactants may act only to... 

A tryptic fragment with three interchain disulfide bonds between four peptide chains was synthesized by repetitive application of this Cys activation/disulfide forming method, and by this procedure the disulfide connectivities of synthetic porcine C5a-anaphylatoxin was confirmed to be identical to those for the natural peptide. 67 ... 

The most obvious effect of a deficiency in vitamin K in animals is delayed blood clotting, which has been traced to a decrease in the activity of prothrombin and of clotting factors VII, IX, and X (Chapter 12, Fig. 12-17). Prothrombin formed by the liver in the absence of vitamin K lacks the ability to chelate calcium ions essential for the binding of prothrombin to phospholipids and to its activation to thrombin. The structural differences between this abnormal protein and the normal prothrombin have been pinpointed at the N terminus of the 560 residue glycoprotein.e f Tryptic peptides from the N termini differed in electrophoretic mobility. As detailed in Chapter 12, ten residues within the first 33, which were identified as glutamate residues by the sequence analysis on normal prothrombin, are actually y-carboxyglutamate (Gla). The same amino acid is present near the N termini of clotting factors VII, IX, and X. 

Some of the serine proteases are stored in the pancreas as inactive precursors that may be activated by proteolysis. Trypsinogen, for example, is converted to trypsin by the removal of the N-terminal hexapeptide on the cleavage of the bond between Lys-6 and Ile-7 by enterokinase. Chymotrypsinogen is activated by the tryptic cleavage of the bond between Arg-15 and He-16. (In this case, further proteolysis by the chymotrypsin that is released during the activation leads to the different forms of the enzyme—Figure 16.5.)... 

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