Abstract
Summary
The insulin content of microdissected rat islets when measured by the two-antibody immunoassay method was approximately one-fourth that obtained when the epididymal fat pad method was used. Insulin content of the subcellular goosefish islet fractions after acid-alcohol extraction was approximately 103 U/g when measured by the fat pad method, as compared to 0.063 U/g when measured by the immunoassay method. Thus there is a 1600-fold difference in the values obtained using these two methods.
About 84% of the total insulin contained in goosefish islets (estimated by the immunoassay method, after acid-alcohol extraction) was recovered in the mitochondria plus secretion granule fraction. Using the fat pad, 74% of the total insulin was found in this subcellular fraction. Acid-alcohol extraction of the secretion granule plus mitochondria, produced an eightfold increase in apparent insulin content when measured by the immunoassay method; thus most of the insulin contained in the secretion granule was not available to react with the insulin-antibody until it was extracted or modified by the acid-alcohol extracted and unextracted subcellular fraction.
One microliter of antiserum (to beef-pork insulin) was able to completely neutralize the effect of 500 μU of either bovine or rat (islet) insulin as measured by the fat pad method. However, five times this amount of antiserum was needed to neutralize the same quantity of goosefish insulin. The observed 1600-fold difference in insulin content of goosefish islet tissue when measured by the fat pad and immunoassay methods indicates that goosefish insulin is hardly able to displace beef 131I insulin from its combination with the (beef-pork) antibody, whereas in the absence of a competing insulin only five times the quantity of antiserum is required to neutralize the effects of goosefish insulin.
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