TY - JOUR
T1 - Fructose-bisphosphatase, Zinc-Free, from Rabbit Liver
AU - Demaine, Margaret M.
AU - Caperelli, Carol A.
AU - Benkovic, Stephen J.
N1 - Funding Information:
Financial support for this research was provided by Grant GM 13306 from the U.S. Public Health Service. Margaret de Maine is an awardee of an Eloise Gerry Fellowship from Sigma Delta Epsilon.
PY - 1982/1/1
Y1 - 1982/1/1
N2 - This chapter describes an assay method and the purification procedure for fructose-bisphosphatase (FBPase) from rabbit liver. The enzyme is assayed spectrophotometrically by following the rate of NADPH accumulation at 340 nm in the presence of excess glucose-6-phosphate dehydrogenase and glucose-6-phosphate isomerase. Neutral FBPase is purified from frozen livers of young, 24-hr-fasted rabbits. For Zn analysis, atomic absorption spectroscopy is employed to measure the concentration of the stock Zn2+ solution and the levels of Zn2+ (and Mn2+) in buffer solutions, water, and FBPase samples. Removal of Zn2+ from FBPase involves various steps; the removal of the tightly bound Zn2+ does not irreversibly alter the FBPase structure. However, experimental studies of other properties of zinc-free FBPase are technically difficult, because the exposure of the enzyme to buffer or other reagents immediately results in binding of the adventitious Zn2+ by FBPase.
AB - This chapter describes an assay method and the purification procedure for fructose-bisphosphatase (FBPase) from rabbit liver. The enzyme is assayed spectrophotometrically by following the rate of NADPH accumulation at 340 nm in the presence of excess glucose-6-phosphate dehydrogenase and glucose-6-phosphate isomerase. Neutral FBPase is purified from frozen livers of young, 24-hr-fasted rabbits. For Zn analysis, atomic absorption spectroscopy is employed to measure the concentration of the stock Zn2+ solution and the levels of Zn2+ (and Mn2+) in buffer solutions, water, and FBPase samples. Removal of Zn2+ from FBPase involves various steps; the removal of the tightly bound Zn2+ does not irreversibly alter the FBPase structure. However, experimental studies of other properties of zinc-free FBPase are technically difficult, because the exposure of the enzyme to buffer or other reagents immediately results in binding of the adventitious Zn2+ by FBPase.
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U2 - 10.1016/S0076-6879(82)90149-5
DO - 10.1016/S0076-6879(82)90149-5
M3 - Article
C2 - 6296604
AN - SCOPUS:0020400817
SN - 0076-6879
VL - 90
SP - 327
EP - 329
JO - Methods in enzymology
JF - Methods in enzymology
IS - C
ER -