Stabilities of uncomplemented and complemented M15 β-galactosidase (Escherichia coli) and the relationship to α-complementation

被引:5
作者
Gallagher, CN [1 ]
Huber, RE [1 ]
机构
[1] Univ Calgary, Fac Sci, Div Biochem, Calgary, AB T2N 1N4, Canada
来源
BIOCHEMISTRY AND CELL BIOLOGY-BIOCHIMIE ET BIOLOGIE CELLULAIRE | 1999年 / 77卷 / 02期
关键词
beta-galactosidase; alpha-complementation; stability;
D O I
10.1139/bcb-77-2-109
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
M15 beta-galactosidase (Escherichia coli) is a mutant form of beta-galactosidase having residues 11-41 deleted. It is an inactive dimer but can be complemented to the active tetrameric form by the addition of a peptide containing the deleted residues. The activities of uncomplemented and complemented M15 beta-galactosidases decreased starting at 42 degrees C-uncomplemented over a narrow temperature range, complemented over a broad range. This is because uncomplemented protein is a simple dimer while complemented is a mix of interacting oligomers at high temperatures. The effects of added components on stability and alpha-complementation are best explained by binding effects on equilibria between native forms and forms susceptible to inactivation. Mg2+ stabilized complemented protein but destabilized uncomplemented protein (10x less Mg2+ was needed for complemented protein), alpha-Complementation increased somewhat at low Mg2+ but decreased at high Mg2+. These effects can be explained by differential Mg2+ binding to the native and susceptible forms. The enhancement of both stability and alpha-complementation by Na+ can be explained by preferential binding of Na+ to the native forms of both the uncomplemented and complemented proteins. Low 2-mercaptoethanol concentrations stabilized uncomplemented M15 beta-galactosidase, but high concentrations destabilized it. All concentrations destabilized complemented M15 beta-galactosidase. alpha-Complementation was enhanced by 2-mercaptoethanol. Thus, there is a correlation between stability of the uncomplemented protein and alpha-complementation at low 2-mercaptoethanol owing to interactions with native forms. The lack of correlation at higher 2-mercaptoethanol probably results from precipitation by 2-mercaptoethanol. In contrast to irreversible thermal inactivation, differences in reversible stability in urea were small. This suggests that quaternary structure and Mg2+ and Na+ sites are lost at low urea concentrations and are unimportant at the urea concentrations that result in reversible denaturation.
引用
收藏
页码:109 / 118
页数:10
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