Evidence for substrate‐induced conformational changes in mitochondrial transhydrogenase

Blazyk, John F.; Fisher, Ronald R.

doi:10.1016/0014-5793(75)80494-7

Cited by 17 publications

(6 citation statements)

References 15 publications

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“…All transhydrogenase assays were performed at 23 °C without the use of regenerating systems for substrates. Reverse non-energy-linked transhydrogenase was assayed using the 3-acetylpyridine analogue of N AD+ (AcPyAD+), as described by Blazyk and Fisher (1975). Forward non-energy-linked transhydrogenase was assayed by following the reduction of thio-NADP+ by NADH at 395 nm, assuming a millimolar extinction coefficient of 11.3 (Fisher and Kaplan, 1973).…”

Section: Methodsmentioning

confidence: 99%

Chemical modification of mitochondrial transhydrogenase: evidence for two classes of sulfhydryl groups

Earle¹,

O'Neal²,

Fisher³

1978

Biochemistry

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Chemical-modification studies on submitochondrial particle pyridine dinucleotide transhydrogenase (EC 1.6.1.1) demonstrate the presence of one class of sulfhydryl group in the nicotinamide adenine dinucleotide phosphate (NADP) site and another peripheral to the active site. Reaction of the peripheral sulfhydryl group with N-ethylmaleimide, or both classes with 5,5'-dithiobis(2-nitrobenzoic acid), completely inactivated transhydrogenase. NADP+ or NADPH nearly completely protected against 5,5'-dithiobis(2-nitrobenzoic acid) inactivation and modification of both classes of sulfhydryl groups, while NADP+ only partially protected against and NADPH substantially stimulated N-ethylmaleimide inactivation. Methyl methanethiolsulfonate treatment resulted in methanethiolation at both classes of sulfhydryl groups, and either NADP+ or NADPH protected only the NADP site group. S-Methanethio and S-cyano transhydrogenases were active derivatives with pH optima shifted about 1 unit lower than that of the native enzyme. These experiments indicate that neither class of sulfhydryl group is essential for transhydrogenation. Lack of involvement of either sulfhydryl group in energy coupling to transhydrogenation is suggested by the observations that S-methanethio transhydrogenase is functional in (a) energy-linked transhydrogenation promoted by phenazine methosulfate mediated ascorbate oxidation and (b) the generation of a membrane potential during the reduction of NAD+ by reduced nicotinamide adenine dinucleotide phosphate (NADPH).

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Section: Methodsmentioning

confidence: 99%

Chemical modification of mitochondrial transhydrogenase: evidence for two classes of sulfhydryl groups

Earle¹,

O'Neal²,

Fisher³

1978

Biochemistry

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“…Rat liver pyridine dinucleotide transhydrogenase can be partially inactivated by means of exposure to high temperature or by proteolysis. We reported previously that the degree of inactivation is influenced by the presence of substrates of the enzyme (Blazyk and Fisher, 1975). In thermal experiments, NADPH stabilized while NADP+ labilized the enzyme to heat inactivation.…”

Section: Resultsmentioning

confidence: 92%

“…The degree of tryptic inactivation is linearly dependent on trypsin concentration and is completely inhibited in the presence of excess trypsin inhibitor. Moreover, the inactivation of the enzyme with a fixed trypsin concentration is linear with respect to time (Blazyk and Fisher, 1975).…”

Section: Resultsmentioning

confidence: 99%

“…Rat liver mitochondria were prepared according to the method of Schnaitman and Greenawalt (1968). Submitochondrial particles were prepared as described previously (Blazyk and Fisher, 1975) and stored at -60 °C. Protein was analyzed by the biuret procedure (Jacobs et al, 1956) using recrystallized bovine serum albumin as a standard.…”

Section: Methodsmentioning

confidence: 99%

“…pling to the energy-conservation system (Rydstróm et al, 1970(Rydstróm et al, , 1971bGreen and Ji, 1972;Skulachev, 1974;Blazyk and Fisher, 1975). We have recently provided evidence, through studies of differential thermal and proteolytic inactivation, that conformational changes in transhydrogenase are induced by the binding of at least two of the four substrates (Blazyk and Fisher, 1975). NADPH stabilizes the enzyme to thermal inactivation and markedly increases the degree of tryptic inactivation.…”

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confidence: 99%

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Effects of substrate and inhibitor binding on thermal and proteolytic inactivation of rat liver transhydrogenase

Blazyk¹,

Lam²

1976

Biochemistry

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The thermostability and proteolytic inactivation of rat liver submitochondrial particle transhydrogenase was studied in the presence of pyridine dinucleotide substrates and a variety of divalent metal and nucleotide inhibitors. Relative to the unliganded enzyme, the NADPH-enzyme complex was more thermostable and showed a twofold greater rate of tryptic inactivation, while the NADP+-enzyme complex was more thermolabile and only slightly more susceptible to tryptic inactivation. Neither NAD+ nor NADH significantly affected thermostability or proteolysis. Similar effects of these ligands were observed for the non-energy-linked and energy-linked transhydrogenase reactions, indicating that both activities are catalyzed by the same enzyme. In thermal experiments, acetyl-CoA, 2'-AMP, and NMNH stabilized, palmitoyl-CoAlabilized, and dephospho-CoA, CoA, NMN+, and 5'-AMP had little effect on enzyme stability. Tryptic inactivation was inhibited by 2'-AMP and NMN+ but was not influenced by the other nucleotide inhibitors. Divalent metal ion inhibitors (Mg2+, Ca2+, Mn2+, Ba2+, and Sr2+) stabilized transhydrogenase against thermal inactivation and promoted tryptic inactivation.

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Transhydrogenase

Böger¹

1979

Photosynthesis II

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Evidence for substrate‐induced conformational changes in mitochondrial transhydrogenase

Cited by 17 publications

References 15 publications

Chemical modification of mitochondrial transhydrogenase: evidence for two classes of sulfhydryl groups

Chemical modification of mitochondrial transhydrogenase: evidence for two classes of sulfhydryl groups

Effects of substrate and inhibitor binding on thermal and proteolytic inactivation of rat liver transhydrogenase

Transhydrogenase

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