Inactivation and Disassembly of the Pyruvate Dehydrogenase Multienzyme Complex from Bovine Kidney by Limited Proteolysis with an Enzyme from Rat Liver

Abstract

Mammalian pyruvate dehydrogenase multienzyme complex is inactivated when treated with a leupeptin-sensitive enzyme (termed 'inactivase') obtained from rat liver lysosomes. However, the inactivation of the overall reaction does not affect any of the component activities of the enzyme complex.By several methods it is demonstrated that treatment with the inactivase provokes the disassembly of the complex into its constituent enzyme components which, though being enzymatically active when assayed separately, are unable to catalyze the coordinated reaction sequence of pyruvate oxidation. The dissociation occurs as a consequence of limited proteolysis of the lipoate acetyltransferase core of the multienzyme complex. Isolated nicked acetyltransferase retains its complete enzymatic activity and behaves as a high-molecular-weight aggregate. The lipoamide dehydrogcnasc and pyruvate dehydrogenase components, however, are not cleaved by the inactivase.The pyruvate dehydrogenase complex catalyzes the oxidative decarboxylation of pyruvate according to Eqn (1):Pyruvate + NAD+ + CoASH -+

Pyruvate Dehydrogenase Complex from Baker's Yeast. 2. Molecular Structure, Dissociation, and Implications for the Origin of Mitochondria

“…A comparison of the complexes from yeast and bovine kidney (Fig. 2, lanes 2 and 3) shows that in the kindey complex, El2 is somewhat smaller ( M , 41000 [14]) and E1/3 a little larger (MI 35500 [14]) than in yeast. The yeast complex is very different from the complexes of gram-negative bacteria which contain one large E l chain instead of two E l subunits [I -31.…”

Limited proteolysis of 2‐oxoglutarate dehydrogenase multienzyme complex from bovine kidney

“…The experimental conditions for protease treatment of OGDC were identical as described for PDC in [3,7].…”

“…In contrast, during treatment with trypsin or bromelain, the El band was cleaved faster than E2. Just as with bovine kidney PDC [3,4,7] OGDC was disassembled into its enzyme components during treatment with papain or elastase. This is demonstrated in fig.3 showing the results of sucrose density gradient centrifugation of papain-treated OGDC.…”

“…The isolation procedure was modified by the addition of a sucrose density gradient centrifugation step as in [7]. The complex had spec.…”

“…were assayed as in [7]. 2-Oxoglutarate dehydrogenase (El) was assayed either spectrophotometrically as in [9] or by measuring the decarboxylation of [l-14C]-oxoglutarate (NEN) under the conditions given in [lo].…”