Amino-terminal residues

A two-site immunometric assay of undecapeptide substance P (SP) has been developed. This assay is based on the use of two different antibodies specifically directed against the N- and C-terminal parts of the peptide (95). Affinity-purified polyclonal antibodies raised against the six amino-terminal residues of the molecule were used as capture antibodies. A monoclonal antibody directed against the carboxy terminal part of substance P (SP), covalently coupled to the enzyme acetylcholinesterase, was used as the tracer antibody. The assay is very sensitive, having a detection limit close to 3 pg/mL. The assay is fiiUy specific for SP because cross-reactivity coefficients between 0.01% were observed with other tachykinins, SP derivatives, and SP fragments. The assay can be used to measure the SP content of rat brain extracts. 

Peptides are named for the number of amino acid residues present, and as derivatives of the carboxyl terminal residue. The primary structure of a peptide is its amino acid sequence, starting from the amino-terminal residue. 

Figure 4-6. The Edman reaction. Phenylisothiocyanate derivatizes the amino-terminal residue of a peptide as a phenylthiohydantoic acid. Treatment with acid in a nonhydroxylic solvent releases a phenyithiohydantoin, which is subsequently identified by its chromatographic mobility, and a peptide one residue shorter. The process is then repeated.

FIGURE 8.7 Synthesis of a protected tripeptide containing a 2-hydroxy-4-methoxybenzyl-protected peptide bond.38 (A) Acylation of the carboxy-terminal residue, (B) removal of both protecting groups, (C) O-acylation of the benzyl-protector by the symmetrical anhydride of the amino-terminal residue, and (D) migration of the protected amino-terminal residue from the oxygen atom to the amino group of the dipeptide ester. 

Lakowicz et al.(]7] VB) examined the intensity and anisotropy decays of the tyrosine fluorescence of oxytocin at pH 7 and 25 °C. They found that the fluorescence decay was best fit by a triple exponential having time constants of 80, 359, and 927 ps with respective amplitudes of 0.29, 0.27, and 0.43. It is difficult to compare these results with those of Ross et al,(68) because of the differences in pH (3 vs. 7) and temperature (5° vs. 25 °C). For example, whereas at pH 3 the amino terminus of oxytocin is fully protonated, at pH 7 it is partially ionized, and since the tyrosine is adjacent to the amino terminal residue, the state of ionization could affect the tyrosine emission. The anisotropy decay at 25 °C was well fit by a double exponential with rotational correlation times of 454 and 29 ps. Following the assumptions described previously for the anisotropy decay of enkephalin, the longer correlation time was ascribed to the overall rotational motion of oxytocin, and the shorter correlation time was ascribed to torsional motion of the tyrosine side chain. 

Two types of subnucleosomal particles which retain many, if not all, of the properties of the intact nucleosome have been identified. The first type contains only H3 and H4, either as a tetramer (Bina-Stein and Simpson, 1977) or an octamer (Simon et al., 1978 Stockley and Thomas, 1979), while the second contains all core histones, each lacking up to 30 amino-terminal residues which have been digested away by trypsin (Whitlock and Simpson, 1977). The fact that other subnucleosomal particles have not been isolated does not necessarily mean that they cannot exist it indicates only that the proper reconstitution or dissociation conditions have not been found. Nevertheless, results to date point to H3-H4 on the one hand, and the trypsin-resistant carboxy-terminal regions of all the core histones on the other hand, as playing controlling structural roles in the formation of the nucleosome and the consequent folding of the DNA. 

Nagai and Thergersen found that an alternative approach was required to achieve efficient expression of the -chain subunits of human hemoglobin in E. coli [55]. In this case, P-globin was produced as a fusion protein that possessed the sequence Ile-Glu-Gly-Arg between the 31 amino-terminal residues of X cll... 

Bachmair, a., Finley, D. and Varshavsky, A. In vivo half-life of a protein is a function of its amino-terminal residue. Science, 1986, 234, 179-186. 

FIGURE 3-14 The pentapeptide serylglycyltyrosylalanylleucine, or Ser-Gly-Tyr-Ala-Leu. Peptides are named beginning with the amino-terminal residue, which by convention is placed at the left. The peptide bonds are shaded in yellow the R groups are in red. 

Various procedures are used to analyze protein primary structure. Several protocols are available to label and identify the amino-terminal amino acid residue (Fig. 3-25a). Sanger developed the reagent l-fluoro-2,4-dinitrobenzene (FDNB) for this purpose other reagents used to label the amino-terminal residue, dansyl chloride and dabsyl chloride, yield derivatives that are more easily detectable than the dinitrophenyl derivatives. After the amino-terminal residue is labeled with one of these reagents, the polypeptide is hydrolyzed to its constituent amino acids and the labeled amino acid is identified. Because the hydrolysis stage destroys the polypeptide, this procedure cannot be used to sequence a polypeptide beyond its amino-terminal residue. However, it can help determine the number of chemically distinct polypeptides in a protein, provided each has a different amino-terminal residue. For example, two residues—Phe and Gly—would be labeled if insulin (Fig. 3-24) were subjected to this procedure. 

Identify amino-terminal residue purify and recycle remaining peptide fragment through Edman process. 

FIGURE 3-25 Steps in sequencing a polypeptide, (a) Identification of the amino-terminal residue can be the first step in sequencing a polypeptide. Sanger s method for identifying the amino-terminal residue is shown here, (b) The Edman degradation procedure reveals... 

To sequence an entire polypeptide, a chemical method devised by Pehr Edman is usually employed. The Edman degradation procedure labels and removes only the amino-terminal residue from a peptide, leaving all other peptide bonds intact (Fig. 3-25b). The peptide is reacted with phenylisothiocyanate under mildly alkaline conditions, which converts the amino-terminal amino acid to a phenylthiocarbamoyl (PTC) adduct. The peptide bond next to the PTC adduct is then cleaved in a step carried out in anhydrous trifluo-roacetic acid, with removal of the amino-terminal amino acid as an anilinothiazolinone derivative. The deriva-tized amino acid is extracted with organic solvents, converted to the more stable phenylthiohydantoin derivative by treatment with aqueous acid, and then identified. The use of sequential reactions carried out under first basic and then acidic conditions provides control over... 

FIGURE 3-27 Cleaving proteins and sequencing and ordering the peptide fragments. First, the amino acid composition and amino-terminal residue of an intact sample are determined. Then any disulfide bonds are broken before fragmenting so that sequencing can proceed efficiently. In this example, there are only two Cys (C) residues and... 

Amino-Terminal and Carboxyl-Terminal Modifications The first residue inserted in all polypeptides is Al-formylmethio-nine (in bacteria) or methionine (in eukaryotes). However, the formyl group, the amino-terminal Met residue, and often additional amino-terminal (and, in some cases, carboxyl-terminal) residues may be removed enzymatically in formation of the final functional protein. In as many as 50% of eukaryotic proteins, the amino group of the amino-terminal residue is Al-acetylated after translation. Carboxyl-terminal residues are also sometimes modified. 

Half-lives were measured in yeast for the j8-galactosidase protein modified so that in each experiment it had a different amino-terminal residue. (See Chapter 9 for a discussion of techniques used to engineer proteins with altered amino acid sequences.) Half-lives may vary for different proteins and in different organisms, but this general pattern appears to hold for all organisms. 

Thus the large-scale preparation of pyridoxylated hemoglobin seems to result in mixtures of reaction products. These probably represent modifications at either or both CC- and P-amino-terminal residues as well as surface lysines. A partial characterization of the mixture has been carried out... 

Determination of the amino-terminal residue of a polypeptide by Edman degradation. 

Differential reactivity of a C—H bonds has also been reported for Ni11 complexes of peptides and peptide Schiff bases115 in which it is the carboxyl-terminal bond that is the more reactive. However, jV-salicylideneglycyl-L-valinatocopper is found to react with aqueous formaldehyde to give serine-L-valine containing highly optically pure serine. In this case the reaction obviously occurs stereoselec-tively at the amino-terminal residue.115... 

A second structural feature that has been correlated with protein degradative rates is the N-terminal residue of the mature form of cytoplasmic proteins. This relationship, summarized in table 29.7, has become known as the N-terminal rule. Here the presumption is that the amino-terminal residue is at least partly responsible for recognition by the degradative machinery. Proteins that are degraded according to the N-terminal rule are believed to be recognized by the ubiquitin-ATP-dependent pathway, which we describe shortly. 

The light chain is encoded by three distinct DNA elements—a variable (yL), a joining (/L), and a constant (CL) element (fig. S3.6). During the differentiation of a B cell, a V, element (encoding the first 95 amino-terminal residues)... 

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