Triose phosphate isomerase from the coelacanth. An approach to the rapid determination of an amino acid sequence with small amounts of material.
The preparation and purification of cyanogen bromide fragments from [(14)C]carboxymethylated coelacanth triose phosphate isomerase is presented. The automated sequencing of these fragments, the lysine-blocked tryptic peptides derived from them, and also of the intact protein, is described. Combination with results from manual sequence analysis has given the 247-residue amino acid sequence of coelacanth triose phosphate isomerase in 4 months, by using 100mg of enzyme. (Two small adjacent peptides were placed by homology with the rabbit enzyme.) Comparison of this sequence with that of the rabbit muscle enzyme shows that 207 (84%) of the residues are identical. This slow rate of evolutionary change (corresponding to two amino acid substitutions per 100 residues per 100 million years) is similar to that found for glyceraldehyde 3-phosphate dehydrogenase. The reliability of sequence information obtained by automated methods is discussed.
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D005075:Biological Evolution; D002238:Carbohydrate Epimerases / Q000032:analysis*; D002250:Carbon Radioisotopes; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D003488:Cyanogen Bromide; D004591:Electrophoresis, Polyacrylamide Gel; D005069:Evaluation Studies as Topic; D005399:Fishes; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D007461:Iodoacetates; D008722:Methods; D008832:Microchemistry; D009132:Muscles; D009943:Organophosphorus Compounds; D010446:Peptide Fragments / Q000032:analysis; D011817:Rabbits; D013045:Species Specificity; D013056:Spectrophotometry, Ultraviolet; D013820:Thermolysin; D014306:Trioses; D014357:Trypsin
D000596:Amino Acids; D002250:Carbon Radioisotopes; D007461:Iodoacetates; D009943:Organophosphorus Compounds; D010446:Peptide Fragments; D014306:Trioses; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D014357:Trypsin; D013820:Thermolysin; D002238:Carbohydrate Epimerases; D003488:Cyanogen Bromide