[8] Purification of cytochrome b5

Strittmatter, Philipp; Fleming, Patrick; Connors, Michael J.; Corcoran, Doris

doi:10.1016/s0076-6879(78)52010-7

Cited by 133 publications

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“…Cytochrome b5 was isolated from rat liver and pig kidney microsomes by octylamine-Sepharose chromatography [7] and was further purified to apparent homogeneity as described by Strittmatter et al [8].…”

Section: Enzyme Purificationmentioning

confidence: 99%

Characterization of pig kidney microsomal cytochrome P-450 catalysing 25-hydroxylation of vitamin D3 and C27 steroids

Bergman

Postlind

1990

Biochemical Journal

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The cytochrome P-450 enzyme which catalyses 25-hydroxylation of vitamin D3 (cytochrome P-45025) from pig kidney microsomes [Postlind & Wikvall (1988) Biochem. J. 253,[549][550][551][552] has been further purified. The specific content of cytochrome P-450 was 15.0 nmol -mg of protein-', and the protein showed a single spot with an apparent isoelectric point of 7.4 and an M, of 50500 upon two-dimensional isoelectric-focusing/SDS/PAGE. The 25-hydroxylase activity towards vitamin D3 was 124pmol min-' nmol of cytochrome P-450-1 and towards la-hydroxyvitamin D3 it was 1375 pmol min-' nmol-1. The preparation also catalysed the 25-hydroxylation of 5/J-cholestane-3a,7a-diol at a rate of 1000 pmol min-' nmol ofcytochrome P-450-1 and w -1 hydroxylation of lauric acid at a rate of 200 pmol min-I nmol of cytochrome P-450-1. A monoclonal antibody raised against the 25-hydroxylating cytochrome P-450, designated mAb 25E5, was prepared. After coupling to Sepharose, the antibody was able to bind to cytochrome P-45025 from kidney as well as from pig liver microsomes, and to immunoprecipitate the activity for 25-hydroxylation of vitamin D3 and 5/3-cholestane-3a,7a-diol when assayed in a reconstituted system. The hydroxylase activity towards lauric acid was not inhibited by the antibody. By SDS/PAGE and immunoblotting with mAb 25E5, cytochrome P-45025 was detected in both pig kidney and pig liver microsomes. These results indicate a similar or the same species of cytochrome P-450 in pig kidney and liver microsomes catalysing 25-hydroxylation of vitamin D3 and C27 steroids. The N-terminal amino acid sequence of the purified cytochrome P-45025 from pig kidney microsomes differed from those of hitherto isolated mammalian cytochromes P-450. INTRODUCTION25-Hydroxylation is involved in the bioactivation of vitamin D and in bile acid metabolism [1,2]. The 25-hydroxylation of vitamin D3is the first step in the bioactivation of vitamin D3 into its hormonal form, and microsomal 25-hydroxylation is involved in an alternative pathway for bile acid synthesis [2]. So far, no extrahepatic enzyme systems able to catalyse 25-hydroxylation of bile acid intermediates have been described.In a previous study [3], the isolation of a cytochrome P-450 (cytochrome P-45025) from pig kidney microsomes which is active in the 25-hydroxylation of vitamin D3 was described. In the present study the enzyme is further characterized with a monoclonal antibody and by determination of the N-terminal amino acid sequence. The properties of the enzyme are compared with those of liver microsomal 25-hydroxylase. EXPERIMENTAL MaterialsTSK DEAE-5PW and ampholines were from LKB, and Mono S HR 10/10 and Protein A-Sepharose were from Pharmacia. Freund's complete adjuvant was from Difco. Standards for IgG subclass determination were kindly donated by Pharmacia. Preparations and sources for steroid analogues have, been described previously [4]. Enzyme purificationCytochrome P-450 catalysing 25-hydroxylation of vitamin D3 and C27 steroids from pig kidney microsomes was purified to...

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Section: Enzyme Purificationmentioning

confidence: 99%

Characterization of pig kidney microsomal cytochrome P-450 catalysing 25-hydroxylation of vitamin D3 and C27 steroids

Bergman

Postlind

1990

Biochemical Journal

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“…The catalytic domain can be separated from the holenzyme by limited proteolytic digestion ofthe microsomal fraction (9, 21). Alternately, the intact enzyme can be obtained by detergent solubilization (16,20,24 Unfortunately, much less is known about the corresponding electron transport and P-450 monoxygenase systems from plant tissue. Galle et al (5) have recently purified an NADH-ferricyanide reductase from potato tuber that is apparently involved in the desaturation of oleate to form linoleic acid in higher plants.…”

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confidence: 99%

“…This system includes NADH-Cyt b5 reductase, a flavoprotein with a single FAD prosthetic group, and the heme protein Cyt b5. Both the NADPH and NADH flavoprotein reductases as well as Cyt b5 have been purified to homogeneity from a variety of mammalian microsomal fractions and have received intensive study during the past decade (20,23,24). Both of these proteins of the NADH linked chain are amphipathic (15) in nature, consisting oftwo definite domains, a water soluble catalytic portion and a hydrophobic fragment that binds the protein to the microsomal membrane.…”

mentioning

confidence: 99%

“…The catalytic domain can be separated from the holenzyme by limited proteolytic digestion ofthe microsomal fraction (9,21). Alternately, the intact enzyme can be obtained by detergent solubilization (16,20,24). Biophysical and biochemical characterizations of the mammalian proteins involved in these mixed function oxidative biotransformations have utilized numerous spectroscopic and structural probes, the primary amino acid sequence of the NADPH Cyt P-450 reductase, NADH-Cyt b5 reductase, and numerous Cyt b5 ' Supported by the University of Illinois and by a grant from the United States Department of Agriculture to M. A. S.…”

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confidence: 99%

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Purification and Characterization of Microsomal Cytochrome b₅ and NADH Cytochrome b₅ Reductase from Pisum sativum

Jollie

Sligar²,

Schuler³

1987

Plant Physiol.

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In this communication we document the reproducible protocols for the purification of milligram quantities of cytochrome b5 and NADH-cytochrome b5 reductase from the microsomal fraction of Pisum sativum. The cytochrome b5 component of this NADH linked electron transport chain was found to have a molecular mass of 16,400 daltons and the reductase a molecular mass of 34,500 daltons. These components could be reconstituted into a functional NADH oxidase activity active in the reduction of exogenous cytochrome c or ferricyanide. In the latter assay the purified reductase exhibited a turnover number of 22,000 per minute. The aminoterminal amino acid sequence of the cytochrome b5 component was determined by sequential Edmund degredation, thus providing crucial information for the efficient cloning of this central protein of plant microsomal electron transfer.The microsomal electron transport systems of mammalian tissue play important roles in a variety of oxidative reactions including fatty acid desaturation and the support of mixed function oxidative biotransformations catalyzed by the Cyt P-450 monooxygenases. Reductive reactions of the latter class involve NADPH dehydrogenation by a flavoprotein, NADPH-Cyt P-450 reductase, containing both FAD and FMN prosthetic groups, followed by sequential one electron tansfers to the cytochrome P450 heme center. In addition, some P450 biotransformations can be mediated by electron transfer from a short redox transfer chain which links to the dephosphorylated form of pyridine nucleotide, NADH. This system includes NADH-Cyt b5 reductase, a flavoprotein with a single FAD prosthetic group, and the heme protein Cyt b5. Both the NADPH and NADH flavoprotein reductases as well as Cyt b5 have been purified to homogeneity from a variety of mammalian microsomal fractions and have received intensive study during the past decade (20,23,24). Both of these proteins of the NADH linked chain are amphipathic (15) in nature, consisting oftwo definite domains, a water soluble catalytic portion and a hydrophobic fragment that binds the protein to the microsomal membrane. The catalytic domain can be separated from the holenzyme by limited proteolytic digestion ofthe microsomal fraction (9, 21). Alternately, the intact enzyme can be obtained by detergent solubilization (16,20,24 Unfortunately, much less is known about the corresponding electron transport and P-450 monoxygenase systems from plant tissue. Galle et al. (5) have recently purified an NADH-ferricyanide reductase from potato tuber that is apparently involved in the desaturation of oleate to form linoleic acid in higher plants. Two mitochondrial forms of a reductase have also been isolated by Klein and Burke (8), the first of which contained two peptide fragments and was specific for NADH whereas the second was apparently comprised of five polypeptides, displayed a broad pH maxima, and utilized both NADH and NADPH. Efforts at the isolation of a Cyt P-450 reductase from the microsomal fraction of higher plants began with the partial pur...

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“…All of the Cyt forms described in this study were spectrally identical to the native form 51 and were quantified from the Soret absorption peaks using absorbance coefficients of 185 mM À1 cm À1 for the dithiothreitol-reduced form. 52 Complete absorption spectra of the solution appropriately diluted in 10 mM Tris-acetate pH 8 buffer were monitored by scanning from 350 to 450 nm in a 1-cm light path cuvette.…”

Section: Cloning Screening and Bacterial Culturingmentioning

confidence: 99%

Export of a hyperexpressed mammalian globular cytochrome b₅ precursor in Escherichia coli is dramatically affected by the nature of the amino acid flanking the secretory signal sequence cleavage bond

2010

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Export of a hyperexpressed mammalian globular cytochrome b 5 precursor in Escherichia coli is dramatically affected by the nature of the amino acid flanking the secretory signal sequence cleavage bond Naheed N. Kaderbhai,* Khalil Ahmed, and Mustak A. Kaderbhai Abstract: A chimeric mammalian globular cytochrome b 5 fused to Escherichia coli alkaline phosphatase signal sequence (SS) was used as a model probe to investigate the influence of substituting each one of the standard 20 amino acids at its N-terminus on the Sec-dependent export of the precursor to the periplasmic space of E. coli. Substituting the native Met 11 of the passenger protein flanking the SS with any one of the remaining 19 amino acids introduced significant changes in the export of cytochrome b 5 without jamming the Sec-dependent translocon. Acidic and hydrophilic residues proved to be the most efficient promoters of export. Small, nonbulky and basic residues yielded intermediate levels of the hemoprotein export. Replacement with a Cys 11 residue generated significant quantities of both monomeric and disulfide-linked dimeric forms. However, bulky, aromatic and hydrophobic residues caused a significant decline in the rates of secretion. In expectation with their absences in the natural periplasmically secreted proteins, Pro and Ile-tagged cytochrome b 5 precursors failed to generate any detectable secreted recombinant products. Although Ala, amongst the native E. coli periplasmic proteins, is the preferred X 11 residue with an occurrence of 50% frequency, it proved half as effective in promoting export when inserted proximally to the SS of cytochrome b 5 . The mechanisms involved for these export variations are discussed. The findings will prove beneficial for high-level generation of recombinant proteins by secretory means for pharmaceutical and related biotechnological applications.

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[8] Purification of cytochrome b5

Cited by 133 publications

References 11 publications

Characterization of pig kidney microsomal cytochrome P-450 catalysing 25-hydroxylation of vitamin D3 and C27 steroids

Characterization of pig kidney microsomal cytochrome P-450 catalysing 25-hydroxylation of vitamin D3 and C27 steroids

Purification and Characterization of Microsomal Cytochrome b₅ and NADH Cytochrome b₅ Reductase from Pisum sativum

Export of a hyperexpressed mammalian globular cytochrome b₅ precursor in Escherichia coli is dramatically affected by the nature of the amino acid flanking the secretory signal sequence cleavage bond

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[8] Purification of cytochrome b5

Cited by 133 publications

References 11 publications

Characterization of pig kidney microsomal cytochrome P-450 catalysing 25-hydroxylation of vitamin D3 and C27 steroids

Characterization of pig kidney microsomal cytochrome P-450 catalysing 25-hydroxylation of vitamin D3 and C27 steroids

Purification and Characterization of Microsomal Cytochrome b5 and NADH Cytochrome b5 Reductase from Pisum sativum

Export of a hyperexpressed mammalian globular cytochrome b5 precursor in Escherichia coli is dramatically affected by the nature of the amino acid flanking the secretory signal sequence cleavage bond

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Purification and Characterization of Microsomal Cytochrome b₅ and NADH Cytochrome b₅ Reductase from Pisum sativum

Export of a hyperexpressed mammalian globular cytochrome b₅ precursor in Escherichia coli is dramatically affected by the nature of the amino acid flanking the secretory signal sequence cleavage bond