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

Basaran, N
Arinc, E
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.


Simultaneous purification and characterization of cytochrome b5 reductase and cytochrome b5 from sheep liver
Arinc, E; Cakir, D (Elsevier BV, 1999-02-01)
Cytochrome b5 was purified from detergent solubilized sheep liver microsomes by using three successive DEAE-cellulose, and Sephadex G-100 column chromatographies. It was purified 54-fold and the yield was 23.5% with respect to microsomes. The apparent Mr of cytochrome b5 was estimated to be 16,200 +/- 500 by SDS-PAGE. Absolute absorption spectrum of the purified cytochrome b5 showed maximal absorption at 412 nm and dithionite-reduced cytochrome b5 gave peaks at 557, 526.5 and 423 nm. The ability of the puri...
Purification and characterization of two forms of endo-beta-1,4-mannanase from a thermotolerant fungus, Aspergillus fumigatus IMI 385708 (formerly Thermomyces lanuginosus IMI 158749)
Puchart, V; Vrsanska, M; Svoboda, P; Pohl, J; Ögel, Zümrüt Begüm; Biely, P (Elsevier BV, 2004-11-01)
Two extracellular endo-β-1,4-mannanases, MAN I (major form) and MAN II (minor form), were purified to electrophoretic homogeneity from a locust bean gum-spent culture fluid of Aspergillus fumigatus IMI 385708 (formerly Thermomyces lanuginosus IMI 158749). Molecular weights of MAN I and MAN II estimated by SDS-PAGE were 60 and 63 kDa, respectively. IEF afforded several glycoprotein bands with pI values in the range of 4.9–5.2 for MAN I and 4.75–4.9 for MAN II, each exhibiting enzyme activity. MAN I as well a...
Characterization and modulation by drugs of sheep liver microsomal flavin monooxygenase activity
Demirdöǧen, Birsen Can; Adalı, Orhan (Wiley, 2005-07-01)
The flavin monooxygenases (FMO) catalyse the NADPH and oxygen-dependent oxidation of a wide range of nucleophilic nitrogen-, sulfur-, phosphorus-, and selenium heteroatom-containing chemicals, drugs, and agricultural agents. In the present study, sheep liver microsomal FMO activity was determined by measuring the S-oxidation rate of methimazole and the average specific activity obtained from different microsomal preparations was found to be 3.8 +/- 1.5 nmol methimazole oxidized min(-1) mg(-1) microsomal pro...
Characterization of proteome alterations in Phanerochaete chrysosporium in response to lead exposure
Yildirim, Volkan; ÖZCAN, Servet; Becher, Doerte; Buettner, Knut; Hecker, Michael; Özcengiz, Gülay (Springer Science and Business Media LLC, 2011-03-09)
Background: Total soluble proteome alterations of white rot fungus Phanerochaete chrysosporium in response to different doses (25, 50 and 100 mu M) of Pb (II) were characterized by 2DE in combination with MALDI-TOF-MS.
ARINC, E; Güray, Nülüfer Tülün; SAPLAKOGLU, U; Adalı, Orhan (Elsevier BV, 1992-01-01)
1. Two forms of soluble NADH cytochrome b5 reductase were purified from human erythrocytes. Two distinct fractions both having the NADH cytochrome b5 reductase activity eluted from the second DEAE-cellulose column were further purified by ultrafiltration and 5'-ADP-agarose affinity chromatography.
Citation Formats
N. Basaran and E. Arinc, “Purification and characterization of two forms of microsomal cytochrome b5 from sheep lung,” INTERNATIONAL JOURNAL OF BIOCHEMISTRY & CELL BIOLOGY, pp. 719–734, 1998, Accessed: 00, 2020. [Online]. Available: https://hdl.handle.net/11511/66171.