Expression of human acylphosphatase in Escherichia coli affects intracellular calcium levels.
In vitro experiments demonstrated the ability of acylphosphatase to hydrolyze the phosphorylated intermediate that is formed during the activity of Na+, K(+)- and Ca(2+)-ATPases of mammalian cells membranes. In order to investigate the effect of this enzyme on intracellular cation levels, a synthetic gene for human muscle acylphosphatase has been expressed in E. coli strains BL21 and JM101. Intracellular total steady-state calcium concentration, as measured by isotopic exchange, was significantly higher in transformed cells as compared to controls and the rising was dependent on the level of acylphosphatase expression. Accordingly also free intracellular calcium concentration, as measured by Fura-2 fluorescence, increased in transformed cells. On the other hand, phosphate levels were not affected by the expression of acylphosphatase, while sodium and rubidium levels increase in transformed cells. Intracellular pH resulted to be slightly affected by the expression of acylphosphatase, cytoplasm of transformed JM101 bacteria being more alkaline (pH 7.45) as compared to control cells (pH 7.40). On the basis of these results, it can be suggested that acylphosphatase acts in vivo by regulating the cation transport in E. coli.
Liguri
G
G
Department of Biochemical Sciences, University of Florence, Italy.
Cecchi
C
C
Pieri
A
A
Raugei
G
G
Vecchi
M
M
Modesti
A
A
Nassi
P
P
Ramponi
G
G
D017766:Acid Anhydride Hydrolases / Q000096:biosynthesis* / Q000235:genetics; D001693:Biological Transport, Active / Q000235:genetics; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D016257:Fura-2; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D009132:Muscles / Q000201:enzymology; D010766:Phosphorylation / Q000187:drug effects; D011188:Potassium / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D000097024:Acylphosphatase
D017766:Acid Anhydride Hydrolases; D002118:Calcium; D000252:Calcium-Transporting ATPases; D016257:Fura-2; D011188:Potassium; D012964:Sodium; D000254:Sodium-Potassium-Exchanging ATPase; D000097024:Acylphosphatase