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Title
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Expression and characterization of a recombinant maize CK-2 alpha subunit.
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Authors
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B.Boldyreff,
F.Meggio,
G.Dobrowolska,
L.A.Pinna,
O.G.Issinger.
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Ref.
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Biochim Biophys Acta, 1993,
1173,
32-38.
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PubMed id
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Abstract
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CKIIB, one of the CK-2 like enzymes which have been isolated from maize, has
been shown to be a monomeric enzyme that cross-reacts with anti CK-2 alpha
specific antibodies suggesting a possible relationship between the two proteins
(Dobrowolska et al. (1992) Eur. J. Biochem. 204, 299-303). In order to support
the immunological data also by biochemical and biophysical experiments the
availability of a recombinant CK-2 alpha from maize was a prerequisite. A maize
cDNA clone of maize CK-2 alpha was expressed in the bacterial strain BL21 (DE3).
The recombinant protein was purified to homogeneity; its molecular mass on
one-dimensional SDS PAGE was estimated to be 36.5 kDa. The calculated molecular
mass according to the amino acid composition is 39,228 Da (332 amino acids). The
recombinant maize CK-2 alpha (rmCK-2 alpha) exhibited mostly the same properties
as the recombinant human CK-2 alpha (rhCK-2 alpha). In several respects it
behaved differently from CKIIB, thus supporting the notion that either CKIIB is
encoded by another gene or it undergoes extensive posttranscriptional and/or
posttranslational alterations. Three observations in particular disprove any
close relatedness between CKIIB and rmCK-2 alpha, namely: (a) the
phosphorylation of calmodulin by CKIIB is dramatically stimulated by polylysine,
whereas polylysine inhibits rather than stimulating the phosphorylation of
calmodulin by rmCK-2 alpha (and by rhCK-2 alpha). (b) Addition of rhCK-2 beta
has no significant influence on the stimulation of the calmodulin
phosphorylation by CKIIB whereas in the case of rmCK-2 alpha and rhCK-2 alpha
addition of rhCK-2 beta is required for optimal stimulation by polylysine. (c)
CKIIB does not self-assemble with rhCK-2 beta to form a high molecular mass
complex as it is demonstrated for rmCK-2 alpha.
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