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Spinach nitrate reductase: purification, molecular weight, and subunit composition.

H Nakagawa1, Y Yonemura, H Yamamoto

  • 1Department of Agricultural Chemistry, Faculty of Horticulture, Chiba University, Matsudo, Chiba 271, Japan.

Plant Physiology
|January 1, 1985
PubMed
Summary

Spinach leaves contain nitrate reductase, an enzyme crucial for plant growth. This study successfully purified the enzyme, revealing it is composed of two identical subunits, each weighing approximately 110,000 to 120,000 Daltons.

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Area of Science:

  • Plant biochemistry
  • Enzyme purification
  • Molecular biology

Background:

  • Nitrate reductase is a key enzyme in plant nitrogen assimilation.
  • Understanding its structure is vital for comprehending plant physiology and metabolism.

Purpose of the Study:

  • To purify nitrate reductase from spinach leaves.
  • To characterize the molecular properties of the purified enzyme, including its subunit composition and molecular weight.

Main Methods:

  • Enzyme purification using multiple chromatography techniques (butyl Toyopearl, hydroxyapatite-brushite, blue Sepharose CL-6B).
  • Polyacrylamide gel electrophoresis (PAGE) under native and denaturing conditions.
  • Sucrose density gradient centrifugation and gel filtration for molecular weight estimation.

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Main Results:

  • Nitrate reductase was purified approximately 3,000-fold with high specific activity.
  • Native PAGE confirmed enzyme homogeneity.
  • Molecular weight estimation indicated a native enzyme of ~270,000 Da.
  • SDS-PAGE revealed subunits of ~114,000 Da, suggesting a dimeric structure.

Conclusions:

  • Spinach nitrate reductase is a homodimer.
  • Each subunit has a molecular weight between 110,000 and 120,000 Daltons.
  • This structural information provides insights into the enzyme's function in nitrogen assimilation.