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

Abstract

A method has been developed that enables us to identify intracellular degradation intermediates of fructosebisphosphate 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 1251-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 possibilit that these peptides were artifacts of homogenization was ruled out as follows: 1251-labeled tagged purified native aldolase was added to the buffer prior to liver homogenization. The homogenates were then 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.The various stages that comprise the process of intracellular protein degradation and the proteases involved have remained obscure despite extensive studies in many laboratories (1, 2). So far no high molecular weight intermediates of intracellular degradation of specific native proteins have been identified in eukaryotes. The identification of such intermediates is obviously necessary in order to elucidate the mechanism of intracellular degradation of proteins. Several previous experimental results from our laboratory have indicated that a direct search for degradation intermediates of specific proteins might be feasible for the following reasons:

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