Fructose-bisphosphatase, Zinc-Free, from Rabbit Liver

Margaret M. Demaine; Carol A. Caperelli; Stephen J. Benkovic

doi:10.1016/S0076-6879(82)90149-5

Fructose-bisphosphatase, Zinc-Free, from Rabbit Liver

Margaret M. Demaine
, Carol A. Caperelli
, Stephen J. Benkovic

Chemistry

Research output: Contribution to journal › Article › peer-review

1 Scopus citations

Abstract

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.

Original language	English (US)
Pages (from-to)	327-329
Number of pages	3
Journal	Methods in enzymology
Volume	90
Issue number	C
DOIs	https://doi.org/10.1016/S0076-6879(82)90149-5
State	Published - Jan 1 1982

All Science Journal Classification (ASJC) codes

Biochemistry
Molecular Biology

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10.1016/S0076-6879(82)90149-5

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@article{73eecbb827204739bd09d95875a254c2,

title = "Fructose-bisphosphatase, Zinc-Free, from Rabbit Liver",

abstract = "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.",

author = "Demaine, \{Margaret M.\} and Caperelli, \{Carol A.\} and Benkovic, \{Stephen J.\}",

note = "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.",

year = "1982",

month = jan,

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doi = "10.1016/S0076-6879(82)90149-5",

language = "English (US)",

volume = "90",

pages = "327--329",

journal = "Methods in enzymology",

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publisher = "Academic Press Inc.",

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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 -

Fructose-bisphosphatase, Zinc-Free, from Rabbit Liver

Abstract

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