Graduate Thesis Or Dissertation
 

Studies of the functional tyrosyl residues of insulin

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  • Acetylimidazole was used to acetylate the tyrosyl residues of insulin. The kinetics of acetylation were studied by termination of the reaction at several time intervals, isolation of acetylated insulin and determination of 0-acetyltyrosyl groups by deacetylation with hydroxylamine. The deacetylation was also studied kinetically by means of the absorbancy change during the reaction. The degree of availability of the tyrosyl groups in native insulin, and of the 0-acetyltyrosyl groups in the acetylated insulin was studied by the above kinetic studies. Two tyrosyl residues were acetylated at a rate sufficiently more fast than the remaining two so that a diacetyl derivative could be isolated. This preparation was fully active. The completely acetylated insulin was only thirty percent as active as native insulin in the mouse convulsion test. Native and acetylated insulin were inactivated by seventy percent on treatment with hydroxylamine. These and other findings lead to the conclusion that one or both of the slowly acetylated tyrosyl residues of insulin are involved in the mechanism of action of the hormone. In order to determine the location of the two acetylated tyrosyl residues, the diacetyl derivative of insulin was oxidized and the A and B chains isolated were assayed for acetyl groups. The diacetyl derivative was also digested separately with trypsin and chymotrypsin, and each core was also assayed for acetyl groups. From the results of these assays it was concluded that the diacetyl derivative of insulin was acetylated to the A19 and B16 tyrosyl residues, which are characterized as reactive, while the other two tyrosyl residues, A14 and B26 are non-reactive or bound. The fact that both tyrosyl B26, and asparaginyl A21 are bound and essential for hormone activity suggests a specific tyrosyl B26-carboxylate A21 interaction essential for maintaining the native conformation and the activity of insulin. The usefulness of the kinetic and structural studies employed here for evaluation of the degree of exposure of tyrosyl residues, selective blocking of tyrosyl groups, and determination of the location of reactive and non-reactive tyrosyl residues of insulin, for the purpose of structure-and-function studies on other problems, is discussed.
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