Size determination of multienzyme complexes using two‐dimensional agarose gel electrophoresis

Richard A. Easom, Michael S. Debuysere, Merle S. Olson, Philip Serwer

Research output: Contribution to journalArticle

9 Scopus citations

Abstract

In the studies of the size and structure of multienzyme complexes, a procedure complementary to electron microscopy for determining the molecular dimensions of hydrated multisubunit complexes is needed. For some applications this procedure must be capable of detecting aggregation of complexes and must be applicable to impure preparations. In the present study, a procedure of two‐dimensional agarose gel electrophoresis (2d‐AGE) (Serwer, P. et al. Anal. Biochem. 152: 339–345, 1986) was modified and employed to provide accurate sizemeasurements of several classical multienzyme complexes. To improve band clarity and to achieve required gel pore sizes, a hydroxyethylated agarose was used. The effective pore's radius (PE) as a function of gel concentration was determined for this agarose inthe range of PE value needed for multienzyme complexes (effective radius, R = 10–30 nm). Appropriate conditions wereestablished to measure R value ± 1% of the pyruvate (PDC), α‐ketoglutarate (α‐KGDC), and the branched chain α‐keto acid (BCDC) dehydrogenase multienzyme complexes; the accuracy of R was limited by the accuracy of the determinations of the R value for the sizestandards. The PDC from bovine heart was found to have an R = 22.4 ± 0.2 nm following cross‐linking with glutaraldehyde that was necessary for stabilization of the complex. Dimers and trimers of PDC, present in the preparations used, were separated from monomeric PDCduring 2d‐AGE. All R values for the enzyme complexes studied were agreement with, though more accurate than, R valuesobtained by use of electron microscopy. In contrast to this statement, the internal dihydrolipoyl transacetylase core of PDC (E2) had an R of 18.8 ± 0.2 nm using 2d‐AGE, but 10.5 nm by electron microscopy. This observation confirms the proposal that the core of the PDC has externally projecting fibrous domains invisibleto electron microscopy.

Original languageEnglish (US)
Pages (from-to)224-232
Number of pages9
JournalProteins: Structure, Function, and Bioinformatics
Volume5
Issue number3
DOIs
StatePublished - 1989

Keywords

  • aggregation of Pyruvate dehydrogenase complex
  • branched chain α‐keto acid dehydrogenase complex
  • effective pore's radius
  • electron microscopy
  • electrophoresis
  • multienzyme complex
  • multienzyme complex
  • two‐dimensional
  • α‐ketoglutarate dehydrogenase complex

ASJC Scopus subject areas

  • Structural Biology
  • Biochemistry
  • Molecular Biology

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