Lars Ernster

[30] DT-diaphorase: Purification, properties, and function

Publisher Summary DT-diaphorase is a highly active diaphorase in the soluble fraction of rat liver homogenates, which catalyzes the oxidation of nicotine adenine dinucleotide, reduced nicotine adenine dinucleotide hydrogenase (NADH), and nicotinamide adenine dinucleotide phosphate (NADPH) at equal rates. This chapter discusses the purification, properties, and function of DT-diaphorase. Most of the present knowledge about DT-diaphorase has been obtained in studies of the cytosolic enzyme, which was first purified from both rat and beef liver 44 by employing conventional methods. The introduction of affinity chromatography for the purification of DTdiaphorase reduced the number of purification steps considerably. In the method presented in the chapter for the purification of DT-diaphorase, the key isolation step is the biospecific adsorption of the enzyme to immobilized dicoumarol, which is a potent competitive inhibitor of the enzyme with respect to NAD(P)H. A gel with both a high affinity and a high binding capacity for DT-diaphorase is obtained by coupling dicoumarol through an azo linkage to divinyl sulfonate (DVS)-activated Sepharose 6B. In the assay, DT-diaphorase activity is measured routinely with NADH or NADPH as the electron donor and 2,6-dichlorophenol-indophenol (DCPIP) or 2-methyl-l,4-naphthoquinone (menadione) as the electron acceptor. The standard assay system contains 50 m M Tris-HC1, pH 7.5, 0.08% Triton X-100, 0.5 m M NADH or NADPH, and 40 μM DCPIP or 10 μM menadione.

Distribution and redox state of ubiquinones in rat and human tissues

The distribution and redox state of ubiquinone in rat and human tissues have been investigated. A rapid extraction procedure and direct injection onto HPLC were employed. It was found in model experiments that in postmortem tissue neither oxidation nor reduction of ubiquinone occurs. In rat the highest concentrations of ubiquinone-9 were found in the heart, kidney, and liver (130-200 micrograms/g). In brain, spleen, and intestine one-third and in other tissues 10-20% of the total ubiquinone contained 10 isoprene units. In human tissues ubiquinone-10 was also present at highest concentrations in heart, kidney, and liver (60-110 micrograms/g), and in all tissues 2-5% of the total ubiquinone contained 9 isoprene units. High levels of reduction, 70-100%, could be observed in human tissues, with the exception of brain and lung. The extent of reduction displayed a similar pattern in rat, but was generally lower.

[72c] Separation and some enzymatic properties of the inner and outer membranes of rat liver mitochondria

Publisher Summary This chapter describes the separation and enzymatic properties of the inner and outer membranes of rat liver mitochondria. A brief exposure of isolated rat liver mitochondria to sonic oscillation, followed by centrifugation on a sucrose gradient, resulted in the separation of a particulate light subfraction from the bulk of the mitochondria, which exhibited a high rotenone-insensitive NADH-cytochrome c reductase activity, but was devoid of rotenone-sensitive NADH-cytochrome c reductase and other respiratory chain-linked enzyme activities. A similar subfraction was obtained when the mitochondria were subjected to swelling and contraction—rather than sonication—prior to density gradient centrifugation. As both the sonication and the swelling-contraction procedure yielded only partial separation of the rotenone-insensitive NADH-cytochrome c reductase, a combined procedure is described. This results in morphologically well defined outer and inner membrane fractions with a quantitative recovery that makes it suitable for the study of intramitochondrial distribution of enzymes and other chemical constituents. Electron microscopic examination of the heavy and light submitochondrial fractions reveals the following: Osmium-fixed specimens of the heavy subfraction consist of relatively large vesicles bordered by a single membrane. Negatively stained specimens of the same subfraction show mitochondrial images in the stage of bursting, with protrusions of unfolding cristae. The osmium-fixed light subfraction consists of relatively small vesicles (average diameter 0.2 μ ) bordered by a single membrane and devoid of inner structures.

[92a] Microsomal lipid peroxidation

Publisher Summary This chapter discusses the microsomal lipid peroxidation that may be demonstrated by measuring (1) O 2 consumption, (2) NADPH disappearance, and (3) malonaldehyde (MA) formation. Results of an experiment involving the measurement of all three parameters are illustrated. In the experimental procedure, rat liver microsomes are prepared s by sedimenting the 10,000 g supernatant of a 0.25M sucrose homogenate of rat liver at 105,000 g for 60 minutes. O 2 consumption is measured with a Clark O 2 electrode. NADPH disappearance is monitored fluorometrically at 450 mμ with an excitation wavelength of 365mμ. An Eppendorf photometer with fluorometer attachment is a suitable instrument for this purpose. MA formed is measured colorimetrically with the thiobarbituric acid (TBA) reaction. O 2 consumption may alternatively be measured manometrically in the Warburg apparatus. Measurement of NADPH disappearance spectrophotometrically at 340 mμ is complicated by the turbidity of the microsomal suspension which, in addition, may change during the incubation. The maximal rate of NADPH-linked lipid peroxidation at 30° is approximately 160 millimicromoles of O 2 consumed per minute per milligram of microsomal protein. The NADPH disappearance accompanying the microsomal lipid peroxidation ranges between one-third and one-fifth mole of NADPH per mole of O 2 consumed. The maximal extent of lipid peroxidation is approximately one micromole of O 2 consumed per milligram of microsomal protein.

SUBFRACTIONATION AND COMPOSITION OF MICROSOMAL MEMBRANES: A REVIEW

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[5] Use of artificial electron acceptors for abbreviated phosphorylating electron transport: Flavin-cytochrome c

Publisher Summary This chapter discusses the use of artificial electron acceptors for abbreviated phosphorylating electron transport. The second of the three energy-coupling sites of the respiratory chain, coupling site II, is located on the path of electrons from cytochrome b to cytochrome c. The two systems that are described are suited for the study of the phosphorylation originating from coupling site II: the succinate-ferricyanide system, which specifically involves the phosphorylation at coupling site II; and the TMPD shunt, which involves a selective bypass over this site. Ferricyanide is known as the most suitable artificial electron acceptor for phosphorylating electron transport in the cytochrome c region of the respiratory chain. It can be used in connection with both pyridine nucleotide-linked substrates and succinate. In the former case, the electron transport pathway is sensitive to amytal and rotenone and involves coupling sites I and II of the respiratory chain. In the latter case, the system is insensitive to amytal and rotenone, and involves only coupling site II. The succinate-ferricyanide system is the only known specific system for study of coupling site II. It is shown that the use of ferricyanide as an artificial electron acceptor for abbreviated phosphorylating electron transport is limited to intact mitochondria. The chapter also discusses the bypass of coupling site II.

A study of the interaction of a series of substituted barbituric acids with the hepatic microsomal monooxygenase.

1. 1. A series of barbituric acid derivatives have been investigated in an attempt to correlate certain physicochemical parameters, such as lipid solubility and pKa, to their interaction as a substrate with the liver microsomal monooxygenase system. 2. 2. All of the barbiturates gave rise to a type I spectral change when added to suspensions of rat liver microsomes, indicating the formation of a cytochrome P-450-substrate complex. The maximal size of the type I spectral change, however, varied with the different barbiturates and was roughly correlated with their lipid solubility and pKa value. Furthermore, although the allyl-substituted barbiturates produced a type I spectral change at low concentrations, this turned into a modified type II spectral change upon increase in drug concentration. Maximal spectral changes were not additive when different barbiturates in combination were added to the microsomes. 3. 3. Binding affinity for cytochrome P-450, as judged from the spectrally determined dissociation constant (Ks), varied among the different barbiturates studied, and only a rough correlation (correlation coefficient, r = 0.52) was observed between lipid solubility and binding affinity. There was also no good correlation between the rates at which the barbiturates were metabolized by the microsomal monooxygenase system and their lipid solubility (r = 0.42). 4. 4. In contrast, the ability of the barbiturates to act on another membrane-bound enzyme system, the mitochondrial NADH oxidase, revealed a strong correlation (r = 0.92) between lipid solubility and inhibitory efficiency. 5. 5. It is suggested that although lipid solubility is required for a substance to reach the microsomal cytochrome P-450, other properties of the molecule are of importance for determining the affinity with which it will interact with this cytochrome. In the mitochondria, however, lipid solubility alone appears to determine the efficiency with which the various barbiturates exert their inhibitory action on the respiratory chain.

[112] Energy-linked reduction of NAD+ by succinate

Publisher Summary This chapter examines the energy-linked reduction of NAD + by succinate. Succinate-linked NAD + reduction involves a reversal of electron transport through coupling site 1 of the respiratory chain, with the expenditure of one high-energy bond per molecule of NAD + reduced by succinate. Amytal, rotenone, and malonate are diagnostic inhibitors of succinate-linked NAD + reduction. Uncouplers of oxidative phosphorylation such as 2, 4- dinitrophenol, dicoumarol, etc., which probably cause a splitting of the C ∼ I compounds, are general inhibitors of succinate-linked NAD + reduction. The energy-transfer inhibitor, oligomycin, characteristically inhibits succinate-linked NAD + reduction when this is driven by ATP, but not when it is driven by energy derived directly from the respiratory chain. Another inhibitor of oxidative phosphorylation, aurovertin, inhibits the ATP-driven succinate-linked NAD + reduction only at levels much higher than those needed for blocking oxidative phosphorylation. The electron- and energy-transfer inhibitor, octylguanidine, abolishes succinate-linked NAD + -reduction under all conditions. Mg ++ inhibits the ATP-driven succinate-linked NAD + reduction in intact mitochondria or submitochoadrial particles prepared with digitonin. Succinate-linked NAD + reduction may be assayed with different reaction systems, depending on whether the assay is performed (1) with mitochondria or submitochondrial particles; (2) with energy derived from electron transport through the respiratory chain or from ATP; and (3) by measuring the NADH formed directly or by transferring hydrogen from the NADH formed to a terminal acceptor and measuring the disappearance of the acceptor or the appearance of the reduced product.

Sodium periodate, sodium chlorite, and organic hydroperoxides as hydroxylating agents in steroid hydroxylation reactions catalyzed by adrenocortical microsomal and mitochondrial cytochrome P450.

Abstract This study has investigated the mechanism of steroid hydroxylation in bovine adrenocortical microsomes and mitochondria by employing NaIO 4 , NaClO 2 , and various organic hydroperoxides as hydroxylating agents and comparing the reaction rates and steroid products formed with those of the NADPH-dependent reaction. In the microsomal hydroxylating system, progesterone, 17α-hydroxyprogesterone, and androstenedione were found to act as substrates. Progesterone was chosen as the model substrate and was converted mainly to the 21-hydroxylated derivative in the presence of microsomal fractions fortified with hydroxylating agent. Using saturating levels of hydroxylating agent, NaIO 4 was found to be the most effective in promoting progesterone hydroxylation followed by cumene hydroperoxide, t-butyl hydroperoxide, NADPH, NaClO 2 , and pregnenolone 17α-hydroperoxide. Evidence for cytochrome P 450 involvement included a marked inhibition of the activity by substrates and modifiers of cytochrome P 450 and by reagents that convert cytochrome P 450 to cytochrome P 420 . Steroid hydroxylation was studied in adrenocortical mitochondria that had been previously depleted of endogenous pyridine nucleotides by aging for 1 h at 30 dgC in a phosphate-supplemented medium. Androstenedione was converted to its respective 6β-, 11β-, 16β-, and 19-hydroxylated derivatives when incubated with aged mitochondrial fractions fortified with hydroxylating agent whereas progesterone was hydroxylated in the 1β-, 6β-, and 15β- positions. These hydroxylations were completely abolished by preheating the mitochondria for 5 min at 95 dgC prior to assay, indicating the enzymic nature of the reactions. Deoxycorticosterone and deoxycortisol were effective substrates for NADPH-dependent enzymic 11β-hydroxylation but were extensively degraded nonenzymically to unidentified products in the presence of NaIO 4 and hydroxylating agents other than NADPH and consequently could not be utilized as substrates in these reactions. Using androstenedione as substrate, NaIO 4 was the most effective hydroxylating agent, followed by cumene hydroperoxide, NaClO 2 , t-butyl hydroperoxide, and NADPH. These hydroxylations were inhibited by substrates and modifiers of cytochrome P 450 and by reagents that convert cytochrome P 450 to cytochrome P 420 . A mechanism for steroid hydroxylation in adrenocortical microsomes and mitochondria is proposed in which the ferryl ion (compound I) of cytochrome P 450 functions as the common “activated oxygen” species.

Preparation and characterization of total, rough and smooth microsomes from the lung of control and methylcholanthrene-treated rats

Optimal conditions for the preparation of relatively pure microsomes and microsomal subfractions from rat lung have been determined. The most importnat of these conditions is homogenization of a 20% (w/v) suspension of lung tissue in 0.44 M sucrose/1% (w/v) bovine serum albumin with four up-and-down strokes at 440 rev./min in a Potter-Elvehjem homogenizer. The 10000 X g supernatant prepared from this homogenate can be centrifuged at 105000 X g to obtain total microsomes or subfractionated into rough and smooth microsomes on a Cs+-containing discontinuous sucrose gradient. The total, rough and smooth microsomes have been characterized in terms of their chemical composition, enzymatic activity, and morphology. These preparations should prove useful in studies of various enzymes in lung (e.g. benzpyrene monooxygenase, epoxide hydrase, enzymes of phospholipid and ascorbic acid synthesis) and in subfractionations designed to reveal heterogeneites in the lateral plane of the lung endoplasmic reticulum.