Tryptic cleavage

Figure 5. Partial peptidic sequences of peptides resulting fi-om tryptic cleavage PME isoforms.

Fig. 1. Amino acid sequence homology between the neonatal fast-twitch and slow-twitch skeletal muscle forms of the Ca -ATPase. The sequence of the slow Ca -ATPase is shown above the neonatal fast-twitch form, with nonhomologous amino acids indicated by asterisks. The sequence of the slow ATPase is shifted to the right by one residue at residue 505 to allow realignment after the difference in sequence length. Ml-MlO, membrane spanning regions S1-S5, stalk sectors Tl, T2, major tryptic cleavage sites P,...

Fig. 2. The hypothetical structure of Ca -ATPase. The structure consists of three major cytoplasmic domains, a pentahelical stalk region, and an intramembranous domain with ten, presumably helical, transmembrane segments. Ti and T2 mark the tryptic cleavage sites. Inset charged amino acids in and near the transmembrane region that may contribute to a Ca " -channel. Adapted from Brandi et al. [8].

Figure 6.3 The time dependence of the number of peaks found after tryptic cleavage of egg white model mortar...

Ethylenimine may be used to introduce additional sites of tryptic cleavage for protein structural studies. In this case, complete sulfhydryl modification is usually desired. Proteins are treated with ethylenimine under denaturing conditions (6-8 M guanidine hydrochloride) in the presence of a disulfide reductant to reduce any disulfide bonds before modification. Ethylenimine may be added directly to the reducing solution in excess (similar to the procedure for Aminoethyl-8 described previously) to totally modify the —SH groups formed. 

Raftery, M.A., and Cole, R.D. (1963) Tryptic cleavage at cysteinyl peptide bonds. Biochem. Biophys. Res. Comm. 10, 467-472. 

Enkephalins Promoter and terminator of 2S1 albumin gene of A. thaliana Integrated in 2S1 albumin of A. thaliana and flanked by tryptic cleavage sites A. thaliana (seed) Brassica napus (seed) 2.9% ofTSP, (200 nmol g 1) 50 nmol 3... 

This modification of the DMAA program permitted identification of the first 14 residues. Identification of the polar amino acids in positions 15 and 16 could not be achieved whereas glycine in position 17 could again be recognized. The remaining unknown section of the sequence was elucidated by the manual Edman degradation procedure after tryptic cleavage of C3... 

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.)... 

The ability of Ca2+ to crosslink groups and so stabilize proteins against thermal denaturation or hydrolytic attack is well illustrated by thermolysin, which involves four Ca2+ and a catalytic Zn2+. The function of the calcium is to stabilize the protein structure.404 Calcium also protects surface protein of the neural retina from tryptic cleavage.405... 

Phospholipase A2 catalyzes the hydrolysis of the 2-acyl linkage of all phospholipids. It is secreted as a zymogen by the pancreas, and converted to the active enzyme via a specific tryptic cleavage of the Arg-Ala link which removes an N-terminal heptapeptide from the proenzyme. Phospholipase A2 is one example of the way in which the metabolism of phospholipids is sensitive to calcium at several key points. 

FIGURE 8.1 Schematic illustration of Gad c 1. The sites of expected susceptibility to tryptic cleavage are indicated by quadrates. (Modified from Elsayed, S. and Apold, J., Allergy, 38, 451, 1983. With permission.)... 

Andersen, J.P., Vilsen, B Collins, Jit., Jorgensen, P.L. (1986). Localization of ErE2 conformational transitions of sarcoplasmic reticulum Ca-ATPase by tryptic cleavage and hydrophobic labeling. J. Membr. Biol. 93, 85-92. 

The contact surface between the a and /3 subunits is very broad ( 1100 A2) and is mostly hydrophobic in character.7 The a subunit loops following strands 2,3,4, and 5 (Fig. 7.2) make important contacts with the /3 subunit. The 50-residue subdomain of the /3 subunit (residues 260-310) comprised of several long /3 hairpin loops makes several contacts with the a subunit (Fig. 7.5).7 Contacts in this region probably prevent tryptic cleavage of the /3 subunit in the 02/82 complex.34-35 The fact that the sites of tryptic cleavage of the /3 subunit (Lys-272, R-275, and K-283) are close to the a/(3 contact surface may explain why proteolytically cleaved /3 subunit does not associate with the a subunit.59-106 In contrast, cleavage of the /3 subunit by proteinase K at Glu-296, a residue at a position in the subdomain (residues 260-310) that does not make direct contact with the a subunit, yields an enzymatically active nicked f3 subunit that interacts weakly with a subunits.61 ... 

In addition to the Mo center shown in Fig. 16, SO contains an N-terminal domain with a h5-type cytochrome (72) that dominates the visible absorption and RR spectra of the holoenzyme. Hence, RR characterization of the Mo center has been confined to studies of the Mo-domain of recombinant human SO. Resonance Raman spectra of the Mo-domain obtained with 488-nm excitation for samples prepared by tryptic cleavage of the overexpressed and purified K108R variant of the holoenzyme (73) and by overexpression and purification of the His-tagged Mo-domain (74), are compared in Fig. 19. Of particular importance is that the bands at 1006, 1161, and 1532 cm-1 in the Mo-domain samples prepared by tryptic cleavage [Fig. 19(a)] are no longer observed in the... 

Figure 19. Resonance Raman spectra of the oxidized Mo domain of recombinant human SO. (a) The Mo domain prepared by tryptic cleavage of the K108R variant of the holoenzyme. (b) His-tagged Mo-domain. Spectra recorded using 488-nm excitation for samples (0.5-1.0 mM in Mo) in 50-mM tricine buffer, pH 8.0, frozen at 17-25 K. Bands marked an asterisk correspond to lattice modes of ice and bands marked with P correspond to nonresonantly enhanced protein modes. A linear ramp has been subtracted to correct for a sloping fluorescence background.

A MALDI-TOF mass spectrum of human hemoglobin Hb Miyazono. Two p-globins (one normal and one mutant) are detected. B MALDI spectrum of the tryptic cleavage of the (3-globin from Hb Miyazono mutant of human hemoglobin. Mutated peptides T9m and T8+T9m are detected. By tandem mass spectrometry, the mutation is characterized as the substitution 79D-E. Reproduced (modified) from Wada Y., Journal of Chromatography B, 781, 291-301, 2002, with permission. 

Fig. 8. The peptide map for the tryptic cleavage of apolipoprotein B which was obtained on a Waters radially compressed analytical system with a mobile phase of 1% triethylam-monium phosphate, pH 3.2, and a gradient of acetonitrile. The flow rate was 1 ml/min. Adapted from Hancock and Sparrow (1981d).

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