Summary
In this study, we purified insulin-like substance (ILS) in the human pancreatic juice
by the combined use of affinity chromatography and radioimmunoassay (RIA). The amino
acid sequence of ILS in the N-terminal region is the same as that of human insulin.
The influence of the enzymes present in the pancreatic juice on the RIA procedure,
was examined. Trypsin, chymotrypsin and amylase showed steep influences on radioactivity.
The addition of enzyme inhibitors could not reduce pseudo-activity, but the elimination
of enzymes in the pancreatic juice by ultrafiltration with the Mole-Cut (Millipore,
Japan) resulted in a lowering of the pseudo-insulin activity.
Affinity chromatography on Sepharose 4B coupled with anti-porcine insulin was used
to capture ILS. ILS was eluted by 1 M acetic acid from the affinity column monitoring
pH and the insulin activity by RIA.
The amino acid sequences of two components of ILS in amino terminal region were Phe-Val
and Gly-Ile-Val. This indicates that ILS obtained from human pancreatic juice was
the insulin derived from endocrine secretion of pancreas.
Key-Words
Human Pancreatic Juice
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Insulin
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RIA
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Affinity Chromatography