Quantitative determination of cytochrome P-450 in rat liver homogenate

Matsubara, Takashi; Koike, Masahiro; Touchi, Akira; Tochino, Yoshihiro; Sugeno, Koichi

doi:10.1016/0003-2697(76)90114-7

Cited by 346 publications

(117 citation statements)

References 9 publications

Supporting

Mentioning

113

Contrasting

Order By: Relevance

“…The purity of the mitochondrial preparation was checked routinely by assaying the marker enzyme activities and by immunoblot analysis, as described before (13). CYP content of mitoplasts was measured by the sodium dithionite-reduced CO-binding difference spectrum (15) in a buffer system containing 100 mM KH 2 PO 4 , pH 7.4, 20% glycerol (v/v), 0.5% sodium cholate (w/v), and 0.06% Triton N-101 (v/v).…”

Section: Treatment Of Animals and Tissuementioning

confidence: 99%

Role of Protein Kinase C-mediated Protein Phosphorylation in Mitochondrial Translocation of Mouse CYP1A1, Which Contains a Non-canonical Targeting Signal

Dasari

Anandatheerthavarada

Robin

et al. 2006

Journal of Biological Chemistry

View full text Add to dashboard Cite

A large number of mitochondrial proteins lack canonical mitochondrial-targeting signals. The bimodal transport of cytochromes P450 (CYPs) to endoplasmic reticulum and mitochondria (MT), reported previously by us, likely represents one mode of non-canonical protein targeting to MT. Herein, we have studied the mechanism of mouse MT-CYP1A1 targeting to gain insight into the regulatory features and evolutionary conservation of bimodal targeting mechanism. Mouse MT-CYP1A1 consists of two NH 2 -terminal-truncated molecular species, ؉91A1 and ؉331A1. Mutations Pro-2 3 Leu and Tyr-5 3 Leu, which increase the signal recognition particle ( Proteins targeted to mitochondria contain either NH 2 -terminal or internal signals, which include amphipathic ␣-helices, ␤-sheet, and random structures with spaced positively charged residues (1-3). A majority of the NH 2 -terminally located mitochondrial-targeting signals are cleaved by the mitochondrial matrix metalloprotease following their import, although in a number of cases the signals remain uncleaved (1-4). Recent proteomic studies indicate that yeast mitochondria contain as many as 800 proteins while the rodent heart and liver mitochondria may contain well over 1500 proteins (5-8). It is estimated that Ͼ50% of mitochondria-associated proteins in both yeast and mammalian cells lack canonical MT 4 -targeting signals, and the precise mode of targeting of these proteins as well as mechanism of their translocation across the MT membranes remain unclear. The bimodal targeting of CYPs to ER and MT, Alzheimer amyloid precursor protein to plasma membrane and MT, and translocation of cytosolic Glutathione S-transferases to MT (9 -14) may represent one mode of targeting of noncanonical signal containing proteins to the MT compartment.Recent studies from our laboratory showed that different xenobiotic-inducible CYPs such as rat CYP1A1, CYP2E1, and CYP2B1 and others that are widely recognized as microsomal proteins (MC-CYPs) are also targeted to varying degrees to mitochondria (9 -11). These studies led to the concept of a new family of chimeric non-canonical-targeting signals, which function both as ER-targeting and MT-targeting signals, under different physiological conditions. We proposed that bimodal targeting of these CYP proteins is facilitated by the cryptic MTtargeting signal domain (residues 29 -40), located between the transmembrane helical domain (residues 1-30) and the Prorich domain (residues 39 -44) in various members of the CYP1, CYP2, and CYP3 families. We have also shown that the cryptic MT-targeting signals of different CYPs are activated by two distinct mechanisms: (a) endoproteolytic cleavage of the NH 2 -

show abstract

Section: Treatment Of Animals and Tissuementioning

confidence: 99%

Role of Protein Kinase C-mediated Protein Phosphorylation in Mitochondrial Translocation of Mouse CYP1A1, Which Contains a Non-canonical Targeting Signal

Dasari

Anandatheerthavarada

Robin

et al. 2006

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…In our hands, digitonin from Wako Chemicals yielded satisfactory results. The P450 content of mitoplasts was measured by the sodium dithionite-reduced carbon monoxide binding difference spectra (20) in a buffer system containing 100 mM KH 2 PO 4 , pH 7.4, 20% glycerol (v/v), 0.5% sodium cholate (w/v), and 0.4% Triton N-101 (v/v) for measuring the P450 content.…”

Section: Treatment Of Animals and Subcellular Fractionation Of Tissues-mentioning

confidence: 99%

Accumulation of Mitochondrial P450MT2, NH2-terminal Truncated Cytochrome P4501A1 in Rat Brain during Chronic Treatment with β-Naphthoflavone

Boopathi

Anandatheerthavarada²,

Bhagwat³

et al. 2000

Journal of Biological Chemistry

View full text Add to dashboard Cite

The biochemical and molecular characteristics of cytochrome P4501A1 targeted to rat brain mitochondria was studied to determine the generality of the targeting mechanism previously described for mitochondrial cytochrome P450MT2 (P450MT2) from rat liver. In rat brain and C6 glioma cells chronically exposed to ␤-naphoflavone (BNF), P450MT2 content reached 50 and 95% of the total cellular pool, respectively. P450MT2 from 10 days of BNF-treated rat brain was purified to over 85% purity using hydrophobic chromatography followed by adrenodoxin affinity binding. Purified brain P450MT2 consisted of two distinct molecular species with NH 2 termini identical to liver mitochondrial forms. These results confirm the specificity of endoprotease-processing sites. The purified P450MT2 showed a preference for adrenodoxin ؉ adrenodoxin reductase electron donor system and exhibited high erythromycin N-demethylation activity. Brain mitoplasts from 10-day BNF-treated rats and also purified P450MT2 exhibited high N-demethylation activities for a number of neuroactive drugs, including trycyclic anti-depressants, anti-convulsants, and opiates. At 10 days of BNF treatment, the mitochondrial metabolism of these neuroactive drugs represented about 85% of the total tissue activity. These results provide new insights on the role of P450MT2 in modulating the pharmacological potencies of different neuroactive drugs in chronically exposed individuals.Cytochrome P450 (P450) 1 enzymes play a critical role in the metabolism of an array of endogenous as well as exogenous substrates (1-3). The xenobiotic inducible forms with roles in the metabolism of carcinogens, pollutants, and drugs were thought to be exclusively associated with the endoplasmic reticulum (hereafter referred to as microsomes) of liver, brain, and other tissues. In contrast to this general belief, recent reports from our laboratory showed that the BNF-inducible P4501A1 and phenobarbital-inducible P4502B1 are also targeted to mitochondria under both in vitro and in vivo cell transfection conditions. These results are consistent with previous studies from our, as well as, other laboratories showing the presence of P450 proteins cross-reacting with antibodies to the major microsomal forms, in liver and brain mitochondria from inducer treated and untreated rats, and also insects (4 -10). Direct sequencing of hepatic mitochondrial P450 proteins purified from BNF-treated rats suggested the occurrence of two forms of P450MT2, both of which were NH 2 -terminal cleaved versions of P4501A1 (11). The molecular form cleaved past the 4th amino acid residue (ϩ5/1A1) represents a minor component while that cleaved past residue 32 (ϩ33/1A1) represents the major component in mitochondria from BNF-treated rat liver. This major form will be routinely referred to as P450MT2 throughout this paper.The mitochondrial targeted P450MT2 exhibited many molecular and biochemical properties distinct from the parent microsomal P4501A1 (12): 1) P450MT2 interacted with Adx with an affinity of 0.6 M K d , 2) funct...

show abstract

“…Cytochrome P-450 was determined according to Omura and Sato [19] as modified by Matsubara et al [20] using a Pye Unicam SP 1750 spectrophotometer.…”

Section: Methodsmentioning

confidence: 99%

Cytochrome P‐450‐dependent H₂O₂ production demonstrated in vivo

1986

View full text Add to dashboard Cite

By administration of allylisopropylacetamide, an inhibitor of cytochrome P‐450, we demonstrated that cytochrome P‐450 is involved in the production of H2O2 during aminopyrine metabolism and phenobarbital induction in both the unanaesthetized guinea pig and rat. In the guinea pig we also found evidence for the existence of a basal cytochrome P‐450‐dependent H2O2 production, i.e. in the absence of exogenous substrate. Catalase participates in the decomposition of H2O2 produced in the endoplasmic reticulum where cytochrome P‐450 is localized.

show abstract

Quantitative determination of cytochrome P-450 in rat liver homogenate

Cited by 346 publications

References 9 publications

Role of Protein Kinase C-mediated Protein Phosphorylation in Mitochondrial Translocation of Mouse CYP1A1, Which Contains a Non-canonical Targeting Signal

Role of Protein Kinase C-mediated Protein Phosphorylation in Mitochondrial Translocation of Mouse CYP1A1, Which Contains a Non-canonical Targeting Signal

Accumulation of Mitochondrial P450MT2, NH2-terminal Truncated Cytochrome P4501A1 in Rat Brain during Chronic Treatment with β-Naphthoflavone

Cytochrome P‐450‐dependent H₂O₂ production demonstrated in vivo

Contact Info

Product

Resources

About

Quantitative determination of cytochrome P-450 in rat liver homogenate

Cited by 346 publications

References 9 publications

Role of Protein Kinase C-mediated Protein Phosphorylation in Mitochondrial Translocation of Mouse CYP1A1, Which Contains a Non-canonical Targeting Signal

Role of Protein Kinase C-mediated Protein Phosphorylation in Mitochondrial Translocation of Mouse CYP1A1, Which Contains a Non-canonical Targeting Signal

Accumulation of Mitochondrial P450MT2, NH2-terminal Truncated Cytochrome P4501A1 in Rat Brain during Chronic Treatment with β-Naphthoflavone

Cytochrome P‐450‐dependent H2O2 production demonstrated in vivo

Contact Info

Product

Resources

About

Cytochrome P‐450‐dependent H₂O₂ production demonstrated in vivo