Purification and characterization of two forms of microsomal cytochrome b5 from sheep lung


Basaran N., Arinc E.

INTERNATIONAL JOURNAL OF BIOCHEMISTRY & CELL BIOLOGY, cilt.30, ss.719-734, 1998 (SCI İndekslerine Giren Dergi) identifier identifier identifier

  • Cilt numarası: 30 Konu: 6
  • Basım Tarihi: 1998
  • Doi Numarası: 10.1016/s1357-2725(98)00017-x
  • Dergi Adı: INTERNATIONAL JOURNAL OF BIOCHEMISTRY & CELL BIOLOGY
  • Sayfa Sayıları: ss.719-734

Özet

Two forms of cytochrome b5 were purified from detergent solubilized sheep lung microsomes by three successive DEAE-cellulose, Sephadex G-100 and Sephadex G-200 column chromatographies. The specific contents of cytochromes b5-I and b5-II were determined to be 45.4 and 43.8 nmol b5/mg protein, which represented up to 567 and 547-fold purification compared with that of the lung microsomes. The most striking difference between b5-I and b5-II was observed in their elution pattern from the third DEAE-cellulose column. Cytochromes gave one protein band with a Mr of 16400 +/- 500 on SDS-PAGE. Both forms of reduced b5 showed a major peak at 423 nm while reduced b5-I had two minor peaks at 527 and 556 and reduced b5-II gave two well-defined peaks at 526 and 555 nm. The ability of the purified b5-I and b5-II fractions to transfer the electrons from NADH-cytochrome b5 reductase to cytochrome c was investigated. Apparent K-m, 0.055 mu M, of b5-II was found to be 38% lower than that of b5-I. In addition, cytochrome b5-I was found to be more sensitive to heat treatment than b5-II when cytochromes were subjected to 62 degrees C for varying periods of time and the coupling of b5 reduction to cytochrome c reduction was determined. These results may indicate that two forms may exist in lung endoplasmic reticulum. (C) 1998 Elsevier Science Ltd. All rights reserved.