The amino acid sequence around the reactive serine residue in alkaline phosphatase from Escherichia coli

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Abstract
Alkaline phosphatase from E coli was labelled with P[image] of high specific radioactivity by the incorporation of inorganic [P32] phosphate, and radioactive techniques were used to study the sequence around the site of incorporation of phosphate. The P32-labelled phosphatase was subjected to partial hydrolysis with pepsin and with acid. The P32-labelled peptides were purified by paper ionophoresis. The peptides were not resolved in one dimension, and a very complex pattern was obtained in two dimensions. Their interrelationships were studied by subjecting them to a further partial hydrolysis. A dipeptide Ala-SerP was identified and shown to be derived from the inversion of SerP-Ala present in the original protein (SerP refers to serine phosphate). The presence of Asp-SerP dipeptides in three forms was detected. A diagonal ionophoretic technique was used to study the interconversion of the aspartate-containing peptides. On the bases of ionophoretic mobilities and periodate oxidation of the radioactive peptides it was concluded that peptides accounting for the sequence (Thr or Ser) -Asp-SerP-Ala-neutral amino-acid were present in the partial acid hydrolysate. The hydrolysis with dilute acid gave a preferential splitting at the aspartate residue, but phosphate was also produced in high yields. Splitting at sites other than aspartate residues was also detected. The results are discussed in connection with the use of tracer techniques in determinations of amino-acid sequences. The similarity between the sequence described and those of other hydroly-tic and proteolytic enzymes is also discussed.