Purification and characterization of a-amylase from rat pancreatfic acinar carcinoma Comparison with pancreatic a-amylase

a-Amylase was purified to apparent homogeneity from normal pancreas and a transplantable pancreatic acinar carcinoma of the rat by affinity chromatography on a-glucohydrolase inhibitor (a-GHI) bound to aminohexyl-Sepharose 4B. Recovery was 95-100% for both pancreas and tumour a-amylases. They were monomeric proteins, with Mr approx. 54000 on SDS/polyacrylamide-gel electrophoresis. Isoelectric focusing of both normal and tumour a-amylases resolved each into two major isoenzymes, with pI 8.3 and 8.7. Tumour-derived a-amylase contained two additional minor isoenzymes, with pl 7.6 and 6.95 respectively. All four tumour isoenzymes demonstrated amylolytic activity when isoelectric-focused gels were treated with starch and stained with iodine. Two-dimensional electrophoresis, on SDS/10-20% -polyacrylamide-gradient gels after isoelectric focusing, separated each major isoenzyme into doublets of similar Mr values. Pancreatic and tumour-derived a-amylases had similar Km and Ki (a-GHI) values, but the specific activity of the tumour a-amylase was approximately two-thirds that of the normal a-amylase. Although amino acid analysis and peptide mapping with the use of CNBr, N-chlorosuccinimide or Staphylococcus aureus V8 proteinase gave comparable profiles for the two a-amylases, tryptic-digest fingerprint patterns were different. Antibodies raised against the purified pancreatic a-amylase and tumour a-amylase respectively showed only one positive band on immunoblotting after gel electrophoresis of crude extracts of rat pancreas and carcinoma, at the same position as that of the purified enzyme. More than 950 of the a-amylase activity in the pancreas and in the tumour was absorbed by an excess amount of either antibody, indicating that normal and tumour a-amylases are immunologically identical. The presence of additional isoenzymes in the carcinoma, and dissimilarity of tryptic-digest patterns, may reflect an alteration in gene expression or in the post-translational modification of this protein in this heterogeneously differentiated transplantable pancreatic acinar carcinoma.