alpha-Amylase was purified to apparent homogeneity from normal pancreas and a transplantable
pancreatic acinar carcinoma of the rat by affinity chromatography on alpha-glucohydrolase inhibitor (alpha-GHI) bound to
aminohexyl-Sepharose 4B. Recovery was 95-100% for both pancreas and tumour
alpha-amylases. They were monomeric
proteins, with Mr approx. 54000 on SDS/
polyacrylamide-gel electrophoresis. Isoelectric focusing of both normal and tumour
alpha-amylases resolved each into two major
isoenzymes, with pI 8.3 and 8.7. Tumour-derived
alpha-amylase contained two additional minor
isoenzymes, with pI 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
alpha-amylases had similar Km and Ki (alpha-GHI) values, but the specific activity of the tumour
alpha-amylase was approximately two-thirds that of the normal
alpha-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
alpha-amylases, tryptic-digest fingerprint patterns were different.
Antibodies raised against the purified
pancreatic alpha-amylase and tumour
alpha-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 95% of the
alpha-amylase activity in the pancreas and in the tumour was absorbed by an excess amount of either antibody, indicating that normal and tumour
alpha-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.