Purification of the bovine lens isozymes which reduce fructose diphosphate to sorbitol diphosphate

Satish Srivastava, Naseem Ansari, Sidney Lerman

Research output: Contribution to journalArticle

1 Citation (Scopus)

Abstract

Three isozymes of an enzyme which reduce fructose 1, 6-diphosphate (FDP) to sorbitol 1, 6-diphosphate (SDP) in the presence of NADH have been purified from bovine lens. The isozymes were fractionated by acid precipitation of the lens homogenate followed by DE-52 column chromatography. This step separated the FDP reducing activity into three major peaks, peak 1, peak 2, and peak 3. Each of these peaks were further purified by affinity chromatography using Reactive blue-2-agarose, Sephadex G-150 gel filtration, and DE-52 column chromatography. Polyacrylamide dise gel electrophoresis demonstrated the presence of one major isozyme and one minor isozyme in each of the three peaks. The Km values for FDP were 8·0, 5·7, and 4·7 mM for peaks 1, 2, and 3 respectively. The reaction product SDP was characterized by nuclear magnetic resonance spectroscopy. All the isozymes utilized pyruvate as substrate with the Km for peaks 1, 2, and 3 being 0·63, 0·20, and 0·09 mm respectively. These studies therefore indicate that FDP reducing activity and lactate dehydrogenase activity co-purify and may be expressed by the same enzyme protein.

Original languageEnglish (US)
Pages (from-to)669-677
Number of pages9
JournalExperimental Eye Research
Volume43
Issue number4
DOIs
StatePublished - 1986

Fingerprint

Sorbitol
Diphosphates
Fructose
Lenses
Isoenzymes
Cibacron Blue F 3GA
Chromatography
Enzymes
Pyruvic Acid
Affinity Chromatography
L-Lactate Dehydrogenase
NAD
Sepharose
Gel Chromatography
Polyacrylamide Gel Electrophoresis
Magnetic Resonance Spectroscopy
Acids
fructose-1,6-diphosphate
Proteins

Keywords

  • cataract
  • diabetes
  • fructose diphosphate
  • lactate dehydrogenase
  • sorbitol diphosphate
  • sorbitol pathway

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems

Cite this

Purification of the bovine lens isozymes which reduce fructose diphosphate to sorbitol diphosphate. / Srivastava, Satish; Ansari, Naseem; Lerman, Sidney.

In: Experimental Eye Research, Vol. 43, No. 4, 1986, p. 669-677.

Research output: Contribution to journalArticle

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