J. Biol. Chem., Vol. 260, Issue 23, 12607-12614, Oct, 1985
Self-association mode of a flavoenzyme D-amino acid oxidase from hog kidney. I. Analysis of apparent weight-average molecular weight data for the apoenzyme in terms of models
H Tojo, K Horiike, K Shiga, Y Nishina, H Watari and T Yamano
The self-association of D-amino acid oxidase apoenzyme in 0.1 M sodium
pyrophosphate, pH 8.3, at 25 degrees C was studied by low-angle laser light
scattering. The concentration (c) dependence of the apparent weight-average
molecular weight (Mwapp) was determined over a wide concentration range of
0.04 to 6.1 mg/ml. The extrapolated Mwapp value, to zero enzyme
concentration, corresponded to the Mr value of the monomer. The
self-association mode of the apoenzyme was systematically explored with
nonlinear least-squares analysis of the Mwapp versus c data. The simplest
model that fitted the data well was a model of isodesmic indefinite
self-association of the monomer with the isodesmic association constant of
0.467 +/- 0.034 liter/g. The monomer-dimer model proposed previously, but
only in a low enzyme concentration range of less than 0.9 mg/ml at 5-20
degrees C (Henn, S. W., and Ackers, G. K. (1969) Biochemistry 8,
3829-3838), did not fit the Mwapp versus c data either in the limited low
concentration range or in the whole concentration range examined at 25
degrees C. To test the validity of the chosen model, the observed
sedimentation boundary profiles were compared with the idealized boundary
profiles calculated for the better- fit models. The profile calculated with
the model of the isodesmic indefinite self-association mechanism was
qualitatively consistent with the observed ones. The utility of the
nonlinear least-squares procedure for analyzing self-associating systems
was demonstrated.
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