Action of amobarbital on microsomal and mitochondrial respiratory state in perfused rat liver with and without phenobarbital induction

Brauser, B.; Sies, Helmut; Bücher, Th.

doi:10.1016/0014-5793(69)80009-8

Cited by 16 publications

(10 citation statements)

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“…Furthermore, when electron flow to the mixed function oxidase is blocked by antimycin in a liver of a fasted rat ( [15]; see below), a decrease of reduced P-450-CO is observed (Fig.9, lower part), similarly amounting to about 8 O l 0 of the total P-450-CO. After addition of aminopyrine as external substrate the degree of reduction increased to 0.1 [14]. As shown in Table 3, more extensive reduction occurred when hexobarbital served as external substrate.…”

Section: Formation Of the Reduced Cytochrome P-450-co Corrlplexmentioning

confidence: 73%

“…Recently, it was demonstrated that in the presence of low amounts of carbon monoxide (3.5 vol.-O/,) the reduced P-450-CO complex can be continuously monitored in perfused liver by dual wavelength reading of AA450--463 (or also AA450-461) without inhibition of respiration [2,3]. While information about the kinetic as well as equilibrium constants of the P-450-CO reaction is not available for the liver enzyme, making an exact calculation of the indicator reaction impossible, we shall tentatively calculate realizing that several problems exist for the exact numerical assignment of this term, especially of the numerator.…”

Section: Formation Of the Reduced Cytochrome P-450-co Corrlplexmentioning

confidence: 99%

“…This figure was calculated as follows. P-450 reducible by anoxia was measured as the absorbance difference between the anoxia increment at the wavelength pair 450-461 nm in the presence and in the absence of CO, representing P-450 plus cytochrome oxidase and cytochrome oxidase alone, respectively [3]. The increase of dA,,-,,, …”

Section: Formation Of the Reduced Cytochrome P-450-co Corrlplexmentioning

confidence: 99%

“…The perfusion fluid (100 ml) was equilibrated with an oxygen-carbon dioxide (95 :5, v/v) mixture in a temperature regulated (33" on liver surface) rotating cylinder oxygenator and passed through the liver via vena portae and vena cava. Where indicated, carbon monoxide was added to the gas mixture to give 0,-C0,-CO (91.5: 5.0 : 3.5)in order to monitor the reduced cytochrome P-45040 complex [2,3]. For anoxia, oxygen was replaced by argon.…”

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confidence: 99%

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Interaction of Mixed Function Oxidase with its Substrates and Associated Redox Transitions of Cytochrome P‐450 and Pyridine Nucleotides in Perfused Rat Liver

Sies¹,

Brauser²

1970

European Journal of Biochemistry

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1. The interactions of a 'Type I' compound, hexobarbital, and of a 'Type 11' compound, metyrapone, with the mixed function oxidase were demonstrated by spectrophotometric techniques in hemoglobin-free perfused livers from phenobarbital-treated rats. The binding signal for hexobarbital occurred when cytochrome P-450 was mainly oxidized (normoxic perfusion) or reduced (anoxic perfusion), The binding signal disappeared as hexobarbital was metabolized.2. Functional aspects were demonstrated by simultaneous readings of (a) the reduced cytochrome P-450-CO complex, providing an indication of the degree of reduction of cytochrome P-450; (b) surface fluorescence (366 --f >420 nm); and (c) oxygen uptake.The steady state level of reduced P-450-CO was increased when substrate was metabolized. Surface fluorescence intensity decreased following hexobarbital addition. This decrease was explained partly by a decrease of the tissue level of NADPH, and partly also by interference of the P-450-hexobarbital complex a t the excitation wavelength. Extra oxygen uptake was 2.2-2.4 moles per mole of hexobarbital. Half-maximal concentration of hexobarbital was 59 and 75 pM for extra oxygen uptake and binding to the oxidase, respectively, in agreement with data for enzymatic activity and binding from isolated liver microsomes.The parameters (a, b, c) returned to their original level as hexobarbital was metabolized, similar to findings with the steroid 21 -hydroxylase system of isolated adrenocortical microsomes.3. The overall degree of reduction of the NADPH system, NADPH/NADPH+NADP+, decreased from 0.8 to 0.7 in livers from phenobarbital-treated rats when hexobarbital was added. I n contrast, the free cytosolic NADH system did not undergo redox changes as indicated by a constant ratio of lactatelpyruvate in the perfusate.4. Mitochondria1 NADH dehydrogenase was shielded against the barbiturate by the endoplasmic reticulum as a consequence of phenobarbital induction.A wealth of information about the hepatic mixed function oxidase system has accumulated during recent years from studies with isolated microsomal fractions [1, 1 a]. Direct insight into this system within the intact organ is possible by spectrophotometric and fluorometric techniques in the hemoglobin-free perfused rat liver [2,3]. I n the present study, simultaneous measurement of the effect of external substrate on four parameters is described, furthering the understanding of this system when , integrated in the dynamics of the cell: (a) spectral interaction of external substrate with cytochrome P-450, the terminal oxidase ; secondly, the steady state level of the reduced P-450-CO complex, both Enzyme. Mixed function oxidase (EC 1.99.1.1.). 35.measured by spectrophotometry of perfused liver in the transmitted light; (b) effect of external substrate on reduced pyridine nucleotide surface fluorescence; and (c) oxygen uptake, serving as a measure of the rate of mixed function oxidation.The interrelations between mixed function oxidase and other cellular redox systems such as the ...

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Section: Formation Of the Reduced Cytochrome P-450-co Corrlplexmentioning

confidence: 73%

Section: Formation Of the Reduced Cytochrome P-450-co Corrlplexmentioning

confidence: 99%

Section: Formation Of the Reduced Cytochrome P-450-co Corrlplexmentioning

confidence: 99%

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confidence: 99%

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Interaction of Mixed Function Oxidase with its Substrates and Associated Redox Transitions of Cytochrome P‐450 and Pyridine Nucleotides in Perfused Rat Liver

Sies¹,

Brauser²

1970

European Journal of Biochemistry

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“…Studies with the perfused rat liver by Thurman and Scholz (4j 5^) as well as Sies, et al (6,7) indicate that approximately 60 percent of cellular respiration by this organ is sensitive to inhibition by Antimycin A -a chemical well recognized as a powerful inhibitor of the mitochondrial respiratory chain. Of the remaining 40 percent of cellular respiration approximately half is sensitive to the inhibitor sodium cyanide.…”

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confidence: 99%