Rapid purification and radioimmunoassay of cytosolic malic enzyme

Leigh S. Wise, Charles S. Rubin

Research output: Contribution to journalArticle

12 Citations (Scopus)

Abstract

A very rapid and highly effective procedure has been devised for the isolation of homogeneous malic enzyme from rat liver cytosol. A combination of precipitation with 10 to 20% polyethylene glycol, ion-exchange chromatography on DEAE-cellulose, and affinity chromatography on Procion Red HE-3B Agarose was used to prepare 3 to 4 mg of homogeneous malic enzyme from the livers of two rats in 18 h. In addition to introducing the advantages of simplicity, speed, and high yield (31%) the new method eliminates potentially denaturing steps (heat treatment, ethanol fractionation) and prolonged dialysis procedures used in other purification schemes. Malic enzyme purified by this new method was used to immunize rabbits. The resulting antibodies bound purified rat liver and mouse liver malic enzymes with very similar affinities and also avidly complexed cytosolic malic enzyme from two murine cell lines, 3T3-L1 preadipocytes and 3T3-C2 fibroblasts. When purified malic enzyme was incubated with lactoperoxidase, glucose oxidase and Na 125I 1.8 atoms of 125I were incorporated per molecule of enzyme with full retention of catalytic activity, subunit size, and immunoreactivity. The antiserum, the purified enzyme, and enzymatically iodinated 125I-malic enzyme were used to construct a sensitive, competitive binding radioimmunoassay for the measurement of malic enzyme mass in the range of 1 to 100 ng.

Original languageEnglish (US)
Pages (from-to)256-263
Number of pages8
JournalAnalytical Biochemistry
Volume140
Issue number1
DOIs
StatePublished - 1984

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Radioimmunoassay
Purification
Enzymes
Liver
Rats
Lactoperoxidase
Affinity chromatography
DEAE-Cellulose Chromatography
DEAE-Cellulose
Glucose Oxidase
Competitive Binding
Dialysis
Ion Exchange Chromatography
Fibroblasts
Fractionation
Chromatography
Affinity Chromatography
Cytosol
Sepharose
Immune Sera

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

Cite this

Rapid purification and radioimmunoassay of cytosolic malic enzyme. / Wise, Leigh S.; Rubin, Charles S.

In: Analytical Biochemistry, Vol. 140, No. 1, 1984, p. 256-263.

Research output: Contribution to journalArticle

Wise, Leigh S. ; Rubin, Charles S. / Rapid purification and radioimmunoassay of cytosolic malic enzyme. In: Analytical Biochemistry. 1984 ; Vol. 140, No. 1. pp. 256-263.
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