Carboxyl terminal analysis

Loomis, C.R. Walsh, J.P. Bell, R.M. sn-l,2-Diacylglycerol kinase of Escherichia coli. Purification, reconstitution, and partial amino- and carboxyl-terminal analysis. J. Biol. Chem., 260, 4091-4097 (1985)... 

Amino acid analysis itself does not directly give the number of residues of each amino acid in a polypeptide, but it does give amounts from which the percentages or ratios of the various amino acids can be obtained (Table 5.2). If the molecular weight and the exact amount of the protein analyzed are known (or the number of amino acid residues per molecule is known), the molar ratios of amino acids in the protein can be calculated. Amino acid analysis provides no information on the order or sequence of amino acid residues in the polypeptide chain. Because the polypeptide chain is unbranched, it has only two ends, an amino-terminal or N-terminal end and a carboxyl-terminal or C-termuial end. 

Figure 39-15. The leucine zipper motif. A shows a helical wheel analysis of a carboxyl terminal portion of the DNA binding protein C/EBP. The amino acid sequence is displayed end-to-end down the axis of a schematic a-helix. The helical wheel consists of seven spokes that correspond to the seven amino acids that comprise every two turns of the a-helix. Note that leucine residues (L) occur at every seventh position. Other proteins with "leucine zippers" have a similar helical wheel pattern. B is a schematic model of the DNA binding domain of C/EBP. Two identical C/EBP polypeptide chains are held in dimer formation by the leucine zipper domain of each polypeptide (denoted by the rectangles and attached ovals). This association is apparently required to hold the DNA binding domains of each polypeptide (the shaded rectangles) in the proper conformation for DNA binding. (Courtesy ofS McKnight)...

The effects of the feed ratio in the lipase CA-catalyzed polymerization of adipic acid and 1,6-hexanediol were examined by using NMR and MALDI-TOF mass spectroscopies. NMR analysis showed that the hydroxyl terminated product was preferentially formed at the early stage of the polymerization in the stoichiometric substrates. As the reaction proceeded, the carboxyl-terminated product was mainly formed. Even in the use of an excess of the dicarboxylic acid monomer, the hydroxy-terminated polymer was predominantly formed at the early reaction stage, which is a specific polymerization behavior due to the unique enzyme catalysis. 

Volkmann S, Wohrl BM, Tisdale M, Moelling K. Enzymatic analysis of two HIV-1 reverse transcriptase mutants with mutations in carboxyl-terminal amino acid residues conserved among retroviral ribonucleoside H. JBiol Chem 1993 268 2674-2683. 

The COOH-terminal amino acid of a peptide or protein may be analyzed by either chemical or enzymatic methods. The chemical methods are similar to the procedures for NH2-terminal analysis. COOH-terminal amino acids are identified by hydrazinolysis or are reduced to amino alcohols by lithium borohydride. The modified amino acids are released by acid hydrolysis and identified by chromatography. Both of these chemical methods are difficult, and clear-cut results are not readily obtained. The method of choice is peptide hydrolysis catalyzed by carboxypeptidases A and B. These two enzymes catalyze the hydrolysis of amide bonds at the COOH-terminal end of a peptide (Equation E2.3), since carboxypeptidase action requires the presence of a free a-carboxyl group in the substrate. 

Polypeptide chain end-group analysis, (a) Amino-terminal group identification. A more sensitive method, the dansyl chloride method, is described in Methods of Biochemical Analysis 3B. (b) Carboxyl-terminal group identification. Identification of this amino acid is considerably more difficult. 

A particular eukaryotic DNA virus is found to code for two mRNA transcripts, one shorter than the other, from the same region on the DNA. Analysis of the translation products reveals that the two polypeptides share the same amino acid sequence at their amino-terminal ends but are different at their carboxyl-terminal ends. The longer polypeptide is coded by the shorter mRNA Suggest an explanation. 

Denker, B. M., Boutin, P. M., and Neer, E. J. (1995). Interactions between the amino-and carboxyl-terminal regions of Ga subunits Analysis of mutated Ga0/Gaj2 chimeras. Biochemistry 34, 5544-5553. 

This technique gives information about the protein s primary structure, which may include its amino and/or carboxyl terminal groups (Edman, 1950). For recombinant DNA-derived proteins, this analysis serves to confirm the amino acid sequence predicted by the DNA sequence. The analysis can also be useful to determine the protein s homogeneity. 

Figure 1. Summary of sequencing of Bombyx PITH. Bars show the peptide sequenced, in which the designations and numbers correspond to the type of derivatization or enzyme used in digestion CAM, carboxamidomethylation C, chymotrypsin E, V8 protease. Arrows indicate the sequence whose carboxyl-terminals are not determined. Small letters show the residues deduced solely from cDNA analysis. Asterisk indicates the site of probable glycosylation.

Nonapeptides with the sequence determined for F1/F2 were synthesized using manual solid-phase methodologies as both the carboxyl-terminal amidated and free acid forms. On reversed-phase LC analysis, RCM-Fl and RCM-F2 were shown to coelute with the synthetic RCM-amide rather than the RCM-acid. The intact, synthetic nonapeptide Cys-Leu-Ile-Thr-Asn-Cys-Pro-Arg-Gly-NH2 was shown by LC to have the same retention time as native FI rather than F2. The factors were next analyzed using size-exclusion LC with RIA monitoring. The retention times of native FI and F2 corresponded to... 

Climent, L, Sj berg, B.-M., and Huang, C. Y., 1991, Carboxyl-terminal peptides as probes for Escherichia coli ribonucleotide reductase subunit interaction Kinetic analysis of inhibition studies. Biochemistry 30 5164n5171. 

The results of this analysis indicate that major determinants for lipoprotein association are located at the extreme carboxyl terminus of apoE, the G helix, and the carboxyl-terminal half of the Class A helix spanning residues 225-266. Elements that contribute to VLDL association appear to reside in the 245-266 region. It is interesting that although the 244 and 223 variants contain long stretches of putative a-helical structure with class A potential, these fragments do not associate significantly with lipoprotein particles. 

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