Some Stereospecificity Studies with Tritiated Pyridine Nucleotides<sup>*</sup>

Krakow, Gladys; Ludowieg, Julio; Mather, Jane H.; Normore, W. M.; Tosi, Luisa; Udaka, Shigezo; Vennesland, Birgit

doi:10.1021/bi00905a017

Cited by 70 publications

(13 citation statements)

References 22 publications

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“…NAD+-43H (spec. activity 0.7 mc/mmole) was prepared by the method of Krakow et al (38). 1 The detrltiation of NADH was followed by measuring the amount of tritium in water isolated from the reaction mixture as described by Lee et al (37).…”

Section: Stereospedfieity Of Nadh Oxidation In Mltochondriamentioning

confidence: 99%

An Electron-Transport System Associated With the Outer Membrane of Liver Mitochondria

Sottocasa

Kuylenstierna

Ernster

et al. 1967

The Journal of Cell Biology

2,257

584

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Preparations of rat-liver mitochondria catalyze the oxidation of exogenous NADH by added cytochrome c or ferricyanide by a reaction that is insensitive to the respiratory chain inhibitors, antimycin A, amytal, and rotenone, and is not coupled to phosphorylation. Experiments with tritiated NADH are described which demonstrate that this "external" pathway of NADH oxidation resembles stereochemically the NADH-cytochrome c reductase system of liver microsomes, and differs from the respiratory chain-linked NADH dehydrogenase. Enzyme distributation data are presented which substantiate the conclusion that microsomal contamination cannot account for the rotenone-insensitive NADH-cytochrome c reductase activity observed with the mitochondria. A procedure is developed, based on swelling and shrinking of the mitochondria followed by sonication and density gradient centrifugation, which permits the separation of two particulate subfractions, one containing the bulk of the respiratory chain components, and the other the bulk of the rotenone-insensitive NADH-cytochrome c reductase system. Morphological evidence supports the conclusion that the former subfraction consists of mitochondria devoid of outer membrane, and that the latter represents derivatives of the outer membrane. The data indicate that the electron-transport system associated with the mitochondrial outer membrane involves catalytic components similar to, or identical with, the microsomal NADHcytochrome b5 reductase and cytochrome bs.

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Section: Stereospedfieity Of Nadh Oxidation In Mltochondriamentioning

confidence: 99%

An Electron-Transport System Associated With the Outer Membrane of Liver Mitochondria

Sottocasa

Kuylenstierna

Ernster

et al. 1967

The Journal of Cell Biology

2,257

584

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“…The eluate was filtered and evaporated under reduced pressure at 50-60 "C. The residue was dissolved in 10 ml of water and the pyridine nucleotide was precipitated by addition of 100 ml of a mixture of ethanol and acetone (1 : 1, v/v) containing 0.05 ml concentrated HC1. The precipitate was collected by centrifugation, washed with acetone and then ether, and dried in vucuo at room temperature [12]. Recovery of the tritiated pyridine nucleotide was 120 pmol, which represents 30% yield.…”

Section: Preparation Of [4-pr0-r-~ Hinadph and [4-pros3 H I N A Dphmentioning

confidence: 99%

Relationship between Steroids and Pyridine Nucleotides in the Oxido-Reduction Catalyzed by the 17 beta-Hydroxysteroid Dehydrogenase Purified from the Porcine Testicular Microsomal Fraction

Itoh¹,

Tamaoki²

1975

Eur J Biochem

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The 17 P-hydroxysteroid dehydrogenase which was purified from porcine testicular microsomal fraction [Inano, H. and Tamaoki, B. (1974) Eur. J. Biochem. 44,[13][14][15][16][17][18][19][20][21][22][23] catalyzed the reduction 'of androstenedione to testosterone with the accompanying oxidation of equimolar NADPH. For the oxido-reduction of the steroids, the 17 P-hydroxysteroid dehydrogenase preferred NADP(H) to NAD(H). Transhydrogenation from NADPH to NAD' or NADH to NADP' through the cyclic oxido-reduction of the steroids by the purified 17 P-hydroxysteroid dehydrogenase preparation was not spectrophotometrically detectable, because of selective preference of the testicular 17 P-hydroxysteroid dehydrogenase against NADP(H). To examine stereospecific transfer of the hydrogen from NADPH to androstenedione by the purified 17 P-hydroxysteroid dehydrogenase, the following tritiated cofactors were synthesized : [4-3H]NADP' was prepared by catalytic replacement from nonradioactive NADP' and 3H,0 in the presence of potassium cyanide. Then, [~-~~o -R -~H ] N A D P H was enzymatically synthesized from the [4-3H]NADP' by glucose 6-phosphate and its dehydrogenase. On the other hand, [~-~~O -S -~H ] N A D P Hwas prepared from the [4-3H]NADP' by isocitrate and isocitrate dehydrogenase. When androstenedione was incubated with the 17 P-hydroxysteroid dehydrogenase in the presence of these stereospecifically 3H-labeled cofactors, only the tritium located at 4-pro-S position of the nicotinamide moiety of NADPH was transferred to testosterone. The location of the tritium in the testosterone molecule produced, 17~-position of the steroid, was assigned by the fact that the tritium of the testosterone remained in its molecule after acetylation, but was completely lost by oxidation.The 17P-hydroxysteroid dehydrogenase of the testicular microsomal membrane was solubilized by sonication and then purified to apparent homogeneity, as checked by polyacrylamide gel electrophoresis and thin-layer gel chromatography [I]. EnzymesEnzjwzes. l7b-Hydroxysteroid dehydrogenase (EC 1 .I .I .64) ; estradiol-17P dehydrogenase (EC 1.1.1.62); glucose-6-phosphate dehydrogenase (EC 1.1.1.49) ; isocitrate dehydrogenase (EC 1.1.1 .-42) ; 3a-hydroxysteroid dehydrogenase (EC 1.1.1.51) ; 20a-hydroxysteroid dehydrogenase (EC 1. I . 1.149), 20P-hydroxysteroid dehydrogenase (EC 1.1.1.53); steroid llg-hydroxylase (EC 1.14.15.4) and alcohol dehydrogenase (EC 1.1.1.1).Trivial Names. Androstenedione, androst-4-ene-3,17-dione; testosterone, 17jj-hydroxyandrost-4-en-3-one; estrone, 3-hydroxyestra-1,3,5(10)-trien-17-one; estradiol-l7g, estra-l,3,5(1O)-triene-3,178-diol; dehydroepiandrosterone, 3b-hydroxyandrost-5-en-17-one; 5P-dihydrotestosterone, 17P-hydroxy-5~-androsten-3-one; 11-deoxycorticosterone, 21-hydroxypregn-4-ene-3,20-dione; corticosterone, 1 18,21 -dihydroxypregn-4-ene-3,20-dione.with the similar function have been reported to exist in placenta [2-41, ovary [5], skin [6], kidney [7], and erythrocytes [S], and the properties of those purified enzymes have been r...

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“…The isotope effect was further shown by mixing tritium-labeled 5~-[24-2H,]cholestane-3a,7a,12a-triol with tritiumlabeled 5j3-[24-1H2]cholestane-3a,7a,12a-triol. 24Hydroxylation of this mixture yielded 5jhholestane-3a,7a,12a,24-tetrol with a ratio of unlabeled to labeled molecules which was about 4 times higher than the ratio in the 5j3-cholestane-3a,7a,l2a-triol incubated ( Table 2). The isotope effect was essentially unchanged when the hydroxylation was inhibited by about 40°/, in an atmosphere of 40' 1, carbon monoxide.…”

Section: 0)mentioning

confidence: 98%

On the Rate-Limiting Step in Microsomal Hydroxylation of Steroids

Björkhem

1972

Eur J Biochem

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The rate-limiting step in the microsomal, NADPH-dependent 24-and 26-hydroxylations of 5@-cholestane-3a,7aI,12a-triol, 60-hydroxylation of testosterone and 16n-hydroxylation of pregnenolone has been studied. Possible isotope effects were determined when the hydrogen in the substrate which is removed in the hydroxylation was substituted with deuterium; when the 4A-or 4B-hydrogen in NADPH was substituted with deuterium ; and when the incubation was carried out in deuterated water. 3. When the reactions were carried out in deuterated water, there was no significant inhibition of 24-hydroxylation of 5@-cholestane-3a,7a, 12a-tri01, whereas 16a-hydroxylation of pregnenolone was inhibited by 150/,, 6t3-hydroxylation of testosterone by 30°/, and 26-hydroxylation of 50-cholestane-3a,7a, 12a-trio1 by 60°/,.As splitting of a C-2H bond in the rate-limiting step is expected to decrease the rate of the overall reaction to less than one-half, it was concluded that the rate-limiting step in the 16a-hydroxylation of pregnenolone and 24-hydroxylation of 5/?-cholestane-3a,7a,l2a-triol is the splitting of the C-H bond in the substrate. Some other step is rate-limiting in the 6@-hydroxylation of testosterone and the 26-hydroxylation of 5,4cholestane-3a,7a, 12n-triol. I n the case of 26-hydroxylation of 5@-cholestane-3~,7a,12a-triol, water might participate in the rate-limiting step by hydration or protonolysis of the enzyme. It is suggested that a common feature of hydroxylation in which splitting of the C-H bond in the substrate is rate-limiting is a relatively low sensitivity towards carbon monoxide.The rate-limiting step in most microsomal hydroxylations has not been defined. Kinetic studies have shown that in microsomal NADPH-dependent hydroxylations of some drugs, the rate of reduction Enzymes. 3a-Hydroxysteroid dehydrogenase, fa-hydroxysteroid: NADP oxidoreductase (EC 1.1.1.50).of cytochrome P-450 -substrate complex [2-41 or the rate of oxygen activation [4] may be rate-limiting in the over-all hydroxylation reaction. I n recent work from this laboratory [5,6], it was suggested that breaking of the C-H bond in the substrate might be a rate-limiting step in the microsomal hydroxylation of some steroids and fatty acids. It was shown that 7a-hydroxylation of [7a-3H]-plus [24-14C]-labeled taurodeoxycholic acid and 9-hydroxylation of [9-2H]decanoic acid were accompanied by a kinetic isotope effect of such magnitude that it was likely that splitting of the C-3H and C 2 H bonds repectively was rate-limiting. On the other hand,

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Some Stereospecificity Studies with Tritiated Pyridine Nucleotides^*

Cited by 70 publications

References 22 publications

An Electron-Transport System Associated With the Outer Membrane of Liver Mitochondria

An Electron-Transport System Associated With the Outer Membrane of Liver Mitochondria

Relationship between Steroids and Pyridine Nucleotides in the Oxido-Reduction Catalyzed by the 17 beta-Hydroxysteroid Dehydrogenase Purified from the Porcine Testicular Microsomal Fraction

On the Rate-Limiting Step in Microsomal Hydroxylation of Steroids

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Some Stereospecificity Studies with Tritiated Pyridine Nucleotides*

Cited by 70 publications

References 22 publications

An Electron-Transport System Associated With the Outer Membrane of Liver Mitochondria

An Electron-Transport System Associated With the Outer Membrane of Liver Mitochondria

Relationship between Steroids and Pyridine Nucleotides in the Oxido-Reduction Catalyzed by the 17 beta-Hydroxysteroid Dehydrogenase Purified from the Porcine Testicular Microsomal Fraction

On the Rate-Limiting Step in Microsomal Hydroxylation of Steroids

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Some Stereospecificity Studies with Tritiated Pyridine Nucleotides^*