COOH-terminal sequences

Although the COOH-terminal residues of proteins and peptides may 

Proteins or large polypeptides can be conveniently hydrolyzed with carboxypeptidases inside dialysis tubing. The released amino acids are easily collected by removing the dialysate (or aliquots) at prescribed times. To illustrate how this can be done, the following example is given. 

Hydrolysis with carboxypeptidase C or carboxypeptidase Y can be performed in a similar manner to that described above except that a buffer at pH 5.3-5.S should be used (0.1 M pyridine adjusted to pH with glacial acetic acid has been used). We have generally used 300 units of carboxypeptidase C per 0.1 ml of reaction mixture containing up to 0.1 pmole of peptide. 

Modification of protein side-chains group-specific reagents 

The protein derivatives described in this chapter are in the main used in amino acid sequence analysis, or for the purpose of determining the content of a given residue in a protein. For such purposes, quantitative modification of the residue in question is mandatory. Consequently, some of the methods described below specify the inclusion of high concentrations of denaturing agents in the reaction mixtures. This is not always essential. For certain functional groups, e.g. amino groups, complete modification is frequently attained with the native protein. 

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Better results are obtained for chain C, which corresponds to the chymotrypsinogen COOH-terminal sequence, when aqueous extracts of oxidized a-chymotrypsin (A4) are chromatographed on Sephadex G-50 equilibrated with 0.05 M HCl (79). Figure 8 shows that three well-separated peaks emerge. The first one is an ill-defined mixture containing chains B and C. The second is chain C in an apparently pure form. The third is the small chain A which does not absorb at 280 m/t. Chromatographically purified chain C can be obtained in an over-all yield of 30%. It contains alanine as the single NH2-terminal residue and all amino acids except histidine and phenylalanine. 

Kaneko, K., Vey, M., Scott, M., Pilkuhn, S., Cohen, F.E., and Prusiner, S.B. (1997a). COOH-terminal sequence of the cellular prion protein directs subcellular trafficking and controls conversion into the scrapie isoform. Proc. Natl. Acad. Sci. U.S.A. 94, 2333-2338. 

In the case of smooth muscle TM, these NH2-terminal modifications can be expected to exert similar but not identical effects since, although their NH2-terminal sequences are the same, their COOH-terminal sequences are different (Fig. 4 and Table I). When chicken gizzard TM was assessed for head-to-tail polymerizability in comparison with the skeletal... 

The aspartic acid residue 261 (215 in the pepsin numbering) is the site of esterification by diazoacetyl-norleucinemethyl ester or similar compounds. As might be expected for an active center residue, it is embedded in conservative segments from Ile-259 to Leu-267 where 6 identities are observed in 9 positions. Finally, in the COOH-terminal sequence from Gly-349 to Ala-370 eight residues are in identical positions. In this section Arg-354 and Arg-362 are common to all the gastric enzymes, while penicillopepsin has a lysine residue at position 354, and a serine residue at position 362. 

Fig. 5. Peptide bonds in rabbit muscle and liver aldolases hydrolyzed by cathepsin M. The COOH-terminal sequences of both enzymes are shown. The primary sites of cleavage are shown by the large arrows and the secondary sites by the small arrows. With muscie aldolase, additional peptides derived from the last 20 amino acid residues, but not including proline 342, are recovered (unpublished observation).

Samson, L, K., Rozenski, J., Samyn, B., Van Aerschot A., Van Beeumen, J. and Herdewijn, P., Screening a random pentapeptide library, composed of 14 D-amino acids, against the COOH-terminal sequence of fructose-1,6-bisphosphate aldolase from Trypanosoma brucei, J. Biol. Chem., 272 (1997) 11378-11383. 

Antibody-based detection methods include immuno-cytochemistry, which gives qualitative data but has very good spatial resolution. Radioimmunoassays provide a quantitative measure of release or content. One of the major limitations of all antibody-based methods is the potential for cross-reactivity among the many peptides. For example, some of the most sensitive gastrin antisera also detect CCK, since the peptides share a common COOH-terminal tetrapeptide sequence. Methods for detection of the mRNAs encoding neuropeptides include Northern blots, which provide quantitative data and information on splice variants, but lack fine anatomical resolution. The more commonly used polymerase chain reaction, which can be quantitative but often is used in a more qualitative manner, provides great sensitivity. Alternatively, in situ hybridization preserves anatomical relationships and can be used to obtain both qualitative and quantitative data. 

Nilsson, I., Whitley, P., and von Heijne, G. (1994). The COOH-terminal ends of internal signal and signal-anchor sequences are positioned differently in the ER translocase. 

Cheung HS, Wang EL, Ondetti MA, Sabo EF, Cushman DW. (1980) Binding of peptide substrates and inhibitors of angiotensin-converting enzyme. Importance of the COOH-terminal dipeptide sequence. J Biol Chem 255 401-407. 

This peptide is also referred to as apo Lp-Ser based on its COOH-terminal amino acid residue (H4, Ml), initially believed to be valine (B8, B9). Its NH2-terminal residue is threonine with the amino acid composition listed in Table 9. The sequence of this peptide announced recently (S34) has the noted feature (Fig. 6), also shared by apo LP-Gln II (B5) and apo LP-Ala (B6) of having a number of basic adjacent to acidic residues. 

These two polypeptides have been shown to have identical amino acid composition (Table 9) but to differ from each other in sialic acid content. Da and D4 have 1 and 2 moles of sialic acid per mole of protein, respectively (A5, A6, B9, BIO, E5). A third form without sialic acid has been isolated by preparative isoelectric focusing (A5). Both D3 and D4 have the same NHa-terminal (serine) and COOH-terminal (alanine) amino acid residue and a molecular weight of about 10,000. The complete amino acid sequence has recently been announced (B6) and is reported in Fig. 7. These studies show that the polysaccharide having sialic acid as its terminal sugar, is linked to threonine 74 of the polypeptide chain. 

By lopping off one terminal amino acid at a time from either the free amino end (N-terminal) or the free COOH end (C-terminal), sequencing techniques give the linkage order of the amino acids in the peptide. 

The NH2-terminal residue of the native enzyme, as well as those of the a and /3 subunits are found to be proline. The COOH-terminal amino acid for both the native enzyme and the a subunit is found to be phenylalanine. However, the COOH-terminal residue of the (3 subunit is still unknown. The NH2-terminal sequences of these two subunits already determined are as follows105, a subunits Pro-Ile-Glu-Leu-Leu-Pro-Glu-Thr-Pro-Ser-Glx-Thr-Ala-Gly-/3 subunits Pro-Ala-Gln-Asp-Asn-Ala-Arg-Phe-Val-Ile-Arg-Asx-Arg-Asx-Except for the NH2-terminal residue, the amino acids in each degradation step of the two subunits are different, suggesting that the enzyme is composed of two nonidentical polypeptide chains. These results suggest that the enzyme consists of eight... 

Lebart, M. C., Mejean, C., Boyer, M., Roustan, C., and Benyamin, Y. (1990). Localization of a new alpha-actinin binding site in the COOH-terminal part of actin sequence. Biochem. Biophys. Res. Commun. 173, 120-126. 

Nguyen M, Millar DG, Yong VW, Korsmeyer SJ, Shore GC (1993), Targeting of Bcl-2 to the mitochondrial outer membrane by a COOH-terminal signal anchor sequence, J. Biol. Chem. 268 25265-25268. 

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