Identification of intracellular degradation intermediates of aldolase B by antiserum to the denatured enzyme.

AUTOR(ES)

Reznick, A Z

RESUMO

A method has been developed that enables us to identify intracellular degradation intermediates of fructose-bisphosphate aldolase B (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13). This method is based on the use of antibody against thoroughly denatured purified aldolase. This antibody has been shown to recognize only denatured molecules, and it did not interact with "native" enzyme. supernatants (24,000 X g for 30 min) of liver and kidney homogenates were incubated with antiserum to denatured enzyme. The antigen-antibody precipitates thus formed were subjected to NaDodSO4/PAGE, followed by electrotransfer to nitrocellulose paper and immunodecoration with antiserum to denatured enzyme and 125I-labeled protein A. Seven peptides with molecular weights ranging from 38,000 (that of the intact subunit) to 18,000, which cross-reacted antigenically with denatured fructose-bisphosphate aldolase, could be identified in liver. The longest three peptides were also present in kidney. The possibility that these peptides were artifacts of homogenization was ruled out as follows: 125I-labeled tagged purified native aldolase was added to the buffer prior to liver homogenization. The homogenates were than subjected to NaDodSO4/PAGE followed by autoradiography, and the labeled enzyme was shown to remain intact. This method is suggested for general use in the search for degradation products of other cellular proteins.

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