Aminopeptidase I may also be used as a reagent in the assay of endoprotease activities with a synthetic substrate in a two-stage assay. In the first stage, the endoprotease cleaves a peptide, such as Z-Y-X-Leu-p-nitroanilide, with the X, Y, and Z residues being chosen according to the specificity of the endoprotease.
The aminopeptidase is purified about 2000-fold by column chromatography on CM-cellulose, hydroxylapatite and Gly-Pro AH-Sepharose. [Imai et al. J Biochem (Tokyo) 93 431 1983, Schomburg & Schomburg Springer Handbook of Enzymes 2nd Edn vol 6 p 286 2002.] DNA (deoxyribonucleic acids). The essential structures of chromosomes are DNA and contain the genetic “blueprint” in the form of separate genes. They are made up of the four deoxyribonucleic acids (nucleotides): adenylic acid, guanylic acid, cytidylic acid and thymidylic acid (designated A, G, C, T respectively) linked together by their phosphate groups in ester bonds between the 3' and 5' hydroxy groups of the 2'-deoxy-D-ribose moiety of the nucleotides. The chains form a double-stranded spiral (helix) in which the two identical nucleotide sequences run antiparallel with the heterocyclic bases hydrogen bonded (A….T, G….C) forming the “ladder” between the strands. Short sequences of DNA are available commercially, are commercially custom made or synthesised in a DNA synthesiser and purified by HPLC. Their purity can be checked by restriction enzyme cleavage followed by gel electophoresis, or directly by gel electrophoresis or analytical HPLC. Commercial DNAs are usually pure enough for direct use but can be further purified using commercially available kits involving binding to silica or other matrices and eluting with Tris buffers. There are now rapid “throughput” techniques for sequencing DNA which are very accurate.
