ISSN:
0887-3585
Keywords:
effective pore's radius
;
α-ketoglutarate dehydrogenase complex
;
branched chain α-keto acid dehydrogenase complex
;
electron microscopy
;
multienzyme complex
;
two-dimensional
;
electrophoresis
;
multienzyme complex
;
aggregation of Pyruvate dehydrogenase complex
;
Chemistry
;
Biochemistry and Biotechnology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Medicine
Notes:
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.
Additional Material:
7 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/prot.340050306
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