Inhibition of energy conservation reactions in chromatophores of Rhodospirillum rubrum by antibiotics

The antibiotics efrapeptin and leucinostatin inhibited photosynthetic and oxidative phosphorylation and related reactions such as the dark and light ATP-P_i exchange reactions and the Mg-ATPase in Rhodospirillum rubrum chromatophores. Higher concentrations of leucinostatin were required for inhibition of the phenazine methosulfate-catalyzed photophosphorylation and light ATP-P_i exchange reaction than for the endogenous or succinate-induced photophosphorylation and dark ATP-P_i exchange reaction. Efrapeptin and leucinostatin inhibited the ATP-driven transhydrogenase while only the latter inhibited the light-driven transhydrogenase, proton gradient formation, and NAD⁺ reduction by succinate in chromatophores. Efrapeptin, but not leucinostatin, inhibited the soluble Ca-ATPase activity of the coupling factor obtained from chromatophores. The inhibition was competitive with ATP. It is concluded that efrapeptin is an effective energy transfer inhibitor whose site of action may be localized in the soluble coupling factor, while the effects of leucinostatin are more complex and cannot be explained as a simple uncoupling.     

Resolution and reconstitution of Rhodospirillum rubrum pyridine dinucleotide transhydrogenase

The Rhodospirillum rubrum pyridine dinucleotide transhydrogenase system is comprised of a membrane-bound component and an easily dissociable soluble factor. Active transhydrogenase complex was solubilized by extraction of chromatophores with lysolecithin. The membrane component was also extracted from membranes depleted of soluble factor. The solubilized membrane component reconstituted transhydrogenase activity upon addition of soluble factor. Various other ionic and non-ionic detergents, including Triton X-100, Lubrol WX, deoxycholate, and digitonin, were ineffectual for solubilization and/or inhibited the enzyme at higher concentrations. The solubilized membrane component was significantly less thermal stable than the membrane-bound component. None of the pyridine dinucleotide substrate affected the thermostability of the solubilized membrane-bound component, whereas NADP⁺ and NADPH afforded protection to membrane-bound component. NADPH stimulated trypsin inactivation of membrane-bound component to a greater extent than NADP⁺, but inactivation of solubilized membrane component was stimulated to the same extent by both pyridine dinucleotides. The solubilized membrane component appears to have a slightly higher affinity for soluble factor than does the membrane-bound component.Abbreviations AcPyAD⁺ oxidized 3-acetylpyridine adenine dinucleotide - BChl bacteriochlorophyll - C_T-particles chromatophores depleted of soluble transhydrogenase factor and devoid of transhydrogenase activity This work was supported by Grant GM 22070 from the National Institutes of Health, United States Public Health Service. Paper I of this series is R. R. Fisher et al. (1975)     

Insulin degradation V. Unmasking of glutathione-insulin transhydrogenase in rat liver microsomal membrane

Glutathione-insulin transhydrogenase (glutathione:protein disulfide oxidoreductase, EC 1.8.4.2) inactivates insulin by cleaving its disulfide bonds. The distribution of GSH-insulin transhydrogenase in subcellular fractions of rat liver homogenates has been studied. From the distribution of insulin-degrading activity and marker enzymes (glucose-6-phosphatase and succinate-INT reductase) (INT, 2-p-iodophenyl-3-p-nitrophenyl-5-phenyl tetrazolium chloride) after cell fractionation by differential centrifugation, the immunological analysis of the isolated subcellular fractions with antibody to purified rat liver GSH-insulin transhydrogenase, and chromatographic analysis (on a column of Sephadex G-75 in 50% acetic acid) of the products formed from ¹²⁵I-labelled insulin after incubation with the isolated subcellular fractions, it is concluded that GSH-insulin transhydrogenase is located primarily in the microsomal fraction of rat liver homogenate. An enzyme(s) that further degrades insulin by proteolysis is located mainly in the soluble fraction; a significant amount of the protease(s) activity is also present in the mitochondrial fraction. The possibility has been discussed that the protease(s) acts upon the intermediate product of insulin degradation, A and B chains of insulin, rather than upon the intact insulin molecule itself.The GSH-insulin transhydrogenase in intact microsomes occurs in a latent state; it is readily released from the microsomal membrane and its activity is greatly increased by treatments which affect the lipoprotein membrane structure of microsomal vesicles. There include homogenization with a Polytron homogenizer, sonication, freezing and thawing, alkaline pH, the nonionic detergent Triton X-100, and phospholipases A and C.     

Topology of glutathione-insulin transhydrogenase in rat liver microsomes

Glutathione-insulin transhydrogenase (EC 1.8.4.2) catalyzes the inactivation of insulin through scission of the disulfide bonds to form insulin A and B chains. In the liver, the transhydrogenase occurs primarily in the microsomal fraction where most of the enzyme is present in a latent (‘inactive’) state. We have isolated rat hepatic microsomes with latent transhydrogenase activity being an integral part of the vesicles. We have used these vesicles to study the topological location of glutathione-insulin transhydrogenase by investigating the effects of detergents (Triton X-100 and sodium deoxycholate), phospholipase A₂ and proteinases (trypsin and thermolysin) on the latent enzyme activity. Treatment of intact vesicles with variable concentrations of detergents and phospholipase A₂ resulted in the unmasking of latent transhydrogenase activity. The extent of unmasking of transhydrogenase activity is dependent upon the concentration of detergent or phospholipase used and is accompanied by a parallel release of the enzyme into the soluble fraction. Activation of the transhydrogenase by phospholipase A₂ is partially inhibited by bovine serum albumin and the extent of inhibition is inversely proportional to the phospholipase concentration. In intact vesicles, latent transhydrogenase activity is resistant to proteolytic inactivation by both trypsin and thermolysin, while in semipermeable and permeable vesicles these proteases inactivate 60 and 25% of the total transhydrogenase activity, respectively. Together these results indicate that in microsomes transhydrogenase is probably weakly bound to membrane phospholipid components and that most of the enzyme is present on the cisternal surface (i.e., the luminal surface of endoplasmic reticulum) of microsomes. Each detergent and phospholipase apparently unmasks glutathione-insulin transhydrogenase activity through disruption of the phospholipid-enzyme interaction followed by translocation of the enzyme to the soluble (cytoplasmic) fraction and not through increases in substrate availability.     

Specificity of the transhydrogenase factor for chromatophores of Rhodopseudomonas spheroides and Rhodospirillum rubrum

Extensive washing of chromatophores of Rhodospirillum rubrum and Rhodopseudomonas spheroides with dilute buffer results in a complete loss of the energylinked transhydrogenase activities of Rsp. rubrum but only a partial loss of the light-driven reaction in chromatophores of Rps. spheroides. It was not possible to reactivate the Rps. spheroides transhydrogenation with the Rsp. rubrum transhydrogenase factor nor with a protein fraction of Rps. spheroides isolated by procedures identical to that used for the isolation of the Rsp. rubrum transhydrogenase factor. The Rsp. rubrum factor is highly specific and cannot be replaced by a number of sulfhydryl compounds tested for reconstitution of Rsp. rubrum transhydrogenation. A published procedure for the isolation of a “transhydrogenase factor” from Rps. spheroides chromatophores yields a preparation having energy-dependent transhydrogenation when supplemented with dithiothreitol in the absence of added chromatophores.     

The proton-translocating nicotinamide–dinucleotide (phosphate) transhydrogenase of rat liver mitochondria

1. The NAD(P) transhydrogenase activity of the soluble fraction of sonicated rat liver mitochondrial preparations was greater than the NAD-linked isocitrate dehydrogenase activity, and the NAD-linked and NADP-linked isocitrate dehydrogenase activities were not additive. The NAD-linked isocitrate dehydrogenase activity was destroyed by an endogenous autolytic system or by added nucleotide pyrophosphatase, and was restored by a catalytic amount of NADP. 2. We concluded that the isocitrate dehydrogenase of rat liver mitochondria was exclusively NADP-specific, and that the oxoglutarate/isocitrate couple could therefore be used unequivocally as redox reactant for NADP in experiments designed to operate only the NAD(P) transhydrogenase (or loop 0) segment of the respiratory chain in intact mitochondria. 3. During oxidation of isocitrate by acetoacetate in intact, anaerobic, mitochondria via the rhein-sensitive, but rotenone- and arsenite-insensitive, NAD(P) transhydrogenase, measurements of the rates of carbonyl cyanide p-trifluoromethoxyphenylhydrazone-sensitive and carbonyl cyanide p-trifluoromethoxyphenylhydrazone-insensitive pH change in the presence of various oxoglutarate/isocitrate concentration ratios gave an -->H(+)/2e(-) quotient of 1.94+/-0.12 for outward proton translocation by the NAD(P) transhydrogenase. 4. Measurements with a K(+)-sensitive electrode confirmed that the electrogenicity of the NAD(P) transhydrogenase reaction corresponded to the translocation of one positive charge per acid equivalent. 5. Sluggish reversal of the NAD(P) transhydrogenase reaction resulted in a significant inward proton translocation. 6. The possibility that isocitrate might normally be oxidized via loop 0 at a redox potential of -450mV, or even more negative, is discussed, and implies that a P/O quotient of 4 for isocitrate oxidation might be expected.     

A microsomal ATP-activated pyridine nucleotide transhydrogenase

An ATP-activated transhydrogenase which catalyzes the reduction of TPN⁺ by DPNH has been demonstrated in the microsomal fraction from the endosperm of immature Echinocystis macrocarpa seeds. The activity is specifically dependent on the presence of ATP (K_m of approximately 0.1 mm) of several nucleotides tested. The reaction is stimulated by MgCl₂ addition up to concentrations of 6 mm. When 10^?2m EDTA is added to the assay mixture in the absence of added MgCl₂, a transhydrogenation reaction is observed which no longer shows any dependence on added ATP. A TPN⁺-dependent ATPase activity can be demonstrated in these preparations, but no fixed stoichiometry between ATP cleavage and TPNH formation could be established. A lag in attaining the maximal rate of transhydrogenation is seen unless the enzyme is preincubated for 10 min with ATP before initiating the reaction. It can further be shown that preincubation of the enzyme with ATP followed by removal of the ATP on a Dowex 1 column produces an enzyme capable of catalyzing the transhydrogenation without the further addition of ATP. 2,4-Dinitrophenol and thyroxin are effective inhibitors of the transhydrogenase and 2,4-dinitrophenol was shown to inhibit the activating effect of ATP during the preincubation period. It is concluded that the role of ATP is in the modification of the enzyme rather than direct participation in the transhydrogenation. The transhydrogenase is inhibited by ADP and AMP. This results in a response of the enzyme to adenylate energy charge in a manner characteristic for regulatory enzymes which participate in ATP-utilizing metabolic sequences.     

Inactivation of Rhodospirillum rubrum coupling factor by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole. Modification of a tyrosine protected by phosphate

Chemical modification of Rhodospirillum rubrum chromatophores by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) results in inactivation of photophosphorylation, Mg²⁺-ATPase, oxidative phosphorylation and ATP-driven transhydrogenase, with apparent first-order kinetics. Other energy-linked reactions such as light-driven transhydrogenase and light-dependent proton uptake were insensitive to NBD-Cl. The Ca²⁺-ATPase activity of the soluble coupling factor from chromatophores (R. rubrum F₁) was inactivated by NBD-Cl with kinetics resembling those described for Mg²⁺-ATPase and photophosphorylation activities of chromatophores. Both NBD-chromatophores and NBD-R. rubrum F₁ fully recovered their activities when subjected to thiolysis by dithioerythritol. Phosphoryl transfer reactions of chromatophores and Ca²⁺-ATPase activity of R. rubrum F₁ were fully protected by 5 mM P_i against modification by NBD-Cl. ADP or ATP afforded partial protection. Analysis of the protection of Ca²⁺-ATPase activity by P_i indicated that NBD-Cl and P_i are mutually exclusive ligands. Spectroscopic studies revealed that tyrosine and sulfhydryl residues in R. rubrum F₁ underwent modification by NBD-Cl. However, the inactivation was only related to the modification of tyrosine groups.     

On the presence of a nicotinamide nucleotide transhydrogenase in mitochondria from potato tuber

Mitochondria isolated from potato (Solanum tuberosum L.) tuber were investigated for the presence of a nicotinamide nucleotide transhydrogenase activity. Submitochondrial particles derived from these mitochondria by sonication catalyzed a reduction of NAD⁺ or 3-acetylpyridine-NAD⁺ by NADPH, which showed a maximum of about 50 to 150 nanomoles/minute·milligram protein at pH 5 to 6. The K_m values for 3-acetylpyridine-NAD⁺ and NADPH were about 24 and 55 micromolar, respectively. Intact mitochondria showed a negligible activity in the absence of detergents. However, in the presence of detergents the specific activity approached about 30% of that seen with submitochondrial particles. The potato mitochondria transhydrogenase activity was sensitive to trypsin and phenylarsine oxide, both agents that are known to inhibit the mammalian transhydrogenase. Antibodies raised against rat liver transhydrogenase crossreacted with two peptides in potato tuber mitochondrial membranes with a molecular mass of 100 to 115 kilodaltons. The observed transhydrogenase activities may be due to an unspecific activity of dehydrogenases and/or to a genuine transhydrogenase. The activity contributions by NADH dehydrogenases and transhydrogenase to the total transhydrogenase activity were investigated by determining their relative sensitivities to trypsin. It is concluded that, at high or neutral pH, the observed transhydrogenase activity in potato tuber submitochondrial particles is due to the presence of a genuine and specific high molecular weight transhydrogenase. At low pH an unspecific reaction of an NADH dehydrogenase with NADPH contributes to the total trans-hydrogenase activity.     

Resolution and reconstitution of Rhodospirillum rubrum pyridine dinucleotide transhydrogenase. Proteolytic and thermal inactivation of the membrane component.

Pyridine dinucleotide transhydrogenase of the Rhodospirillum rubrum chromatophore membrane was readily resolved by a washing procedure into two inactive components, a soluble transhydrogenase factor protein and an insoluble membrane-bound factor. Transhydrogenation was reconstituted on reassociation of these components. The capacity of the membrane factor to reconstitute enzymatic activity was lost after proteolysis of soluble transhydrogenase factor-depleted membranes with trypsin. NADP+ or NADPH, but neither NAD+ nor NADH, stimulated by several fold the rate of trypsin-dependent inactivation of the membrane factor. Substantial protection of the membrane factor from proteolytic inactivation was observed in the presence of Mg2+ ions, an inhibitor of transhydrogenation, or when the soluble transhydrogenase factor was bound to the membrane. Coincident with the loss of enzymatic reconstitutive capacity of the membrane factor was a loss in the ability of the membranes to bind the soluble transhydrogenase factor in a stable complex. The membrane component was inactivated by preincubating soluble transhydrogenase factor-depleted membranes at temperatures above 45 degrees. NADP+, NADPH, or Mg2+ ions, but neither NAD+ nor NADH, protected against inactivation. These studies indicate that (a) the binding of NADP+ or NADPH to the membrane factor promotes a conformational alteration in the protein such that its themostability and susceptibility to proteolysis are increased, and (b) the inhibitory Mg2+ ion-binding site resides in the membrane component.     

Membrane-associated, energy-linked reactions in Bdellovibrio bacteriovorus.

Disrupted cells of Bdellovibrio bacteriovorus exhibited adenosine triphosphatase activity, 60 to 80% of which was in the soluble fraction. Dicyclohexylcarbodiimide did not inhibit the adenosine triphosphatase activity in membrane particles. The particles did not show energy-linked transhydrogenase activity. The activity of non-energy-linked transhydrogenase as well as the rate of oxygen consumption were higher in membrane particles of the host-independent strain than in the host-dependent strains. The uptake of amino acid uptake was inhibited by cyanide and by carbonyl cyanide p-trifluoromethoxyphenyl hydrazone. Valinomycin, in the presence of K+, did not inhibit the uptake, and only partial inhibition was exerted by arsenate and dicyclohexylarbodiimide. Sulfhydryl reagents inhibited amino acid uptake.     

Colicins and bacterial membranes: structures and functions. II. Studies on reconstituted homologous and hybrid membranes prepared from cytoplasmic membranes of untreated and colicin K-treated bacteria.

Cytoplasmic membranes of Escherichia coli K12 C600 treated and not treated with colicin K were dissociated into unsolubilized and solubilized fractions. Neither fraction catalyzed ATP-linked transhydrogenase activity. Mixtures of unsolubilized fractions of the untreated bacteria with solubilized fractions of either the treated or untreated bacteria yielded reconstituted membranes with restored ATP-linked transhydrogenase activity. The level of the activity was similar to that of the undissociated membranes of untreated bacteria. The membranes which were reconstituted from unsolubilized fractions of the treated bacteria and the solubilized fraction of the treated or the untreated bacteria showed impairment of activity. The impairment is not due to an inability to bind ATPase of the soluble fraction or to an incorrect binding of the ATPase. The impaired, reconstituted membranes showed striking decreases in the relative amounts of three proteins with apparent molecular weights of 122,000, 73,000, and 62,000. The affected proteins were found to be components of the unsolubilized membrane fraction. It is, thus, concluded that the impaired activity is due to the defective nature of the unsolubilized membrane fraction of colicin-treated cells.     

The udhA Gene of Escherichia coli Encodes a Soluble Pyridine Nucleotide Transhydrogenase

The udhA gene of Escherichia coli was cloned and expressed in E. coli and found to encode an enzyme with soluble pyridine nucleotide transhydrogenase activity. The N-terminal end of the enzyme contains the fingerprint motif of a dinucleotide binding domain, not present in published E. coli genome sequences due to a sequencing error. E. coli is hereby the first organism reported to possess both a soluble and a membrane-bound pyridine nucleotide transhydrogenase.     

Pyridine nucleotide transhydrogenase activity of soluble cardiac NADH dehydrogenase and particulate NADH-ubiquinone reductase

Pyridine nucleotide transhydrogenase activities of a highly purified soluble NADH dehydrogenase and particulate NADH-ubiquinone reductase (Complex I) differ in their pH optima (5.0 and 6.0, respectively) and in their sensitivity to inhibition by Mg²⁺ and ATP. The oxidation of NADPH with ferricyanide as acceptor is very similar in both preparations with a pH optimum around 5.0. It is concluded that Complex I possesses two types of transhydrogenase activity, whereas only one has been found in the soluble dehydrogenase.     

Cofactor Regeneration by a Soluble Pyridine Nucleotide Transhydrogenase for Biological Production of Hydromorphone

We have applied the soluble pyridine nucleotide transhydrogenase of Pseudomonas fluorescens to a cell-free system for the regeneration of the nicotinamide cofactors NAD and NADP in the biological production of the important semisynthetic opiate drug hydromorphone. The original recombinant whole-cell system suffered from cofactor depletion resulting from the action of an NADP⁺-dependent morphine dehydrogenase and an NADH-dependent morphinone reductase. By applying a soluble pyridine nucleotide transhydrogenase, which can transfer reducing equivalents between NAD and NADP, we demonstrate with a cell-free system that efficient cofactor cycling in the presence of catalytic amounts of cofactors occurs, resulting in high yields of hydromorphone. The ratio of morphine dehydrogenase, morphinone reductase, and soluble pyridine nucleotide transhydrogenase is critical for diminishing the production of the unwanted by-product dihydromorphine and for optimum hydromorphone yields. Application of the soluble pyridine nucleotide transhydrogenase to the whole-cell system resulted in an improved biocatalyst with an extended lifetime. These results demonstrate the usefulness of the soluble pyridine nucleotide transhydrogenase and its wider application as a tool in metabolic engineering and biocatalysis.     

Resolution and reconstitution of Rhodospirillum rubrum pyridine dinucleotide transhydrogenase: localization of substrate binding sites.

Butanedione in the presence of borate buffer reversibly inhibits Rhodospirillum rubrum chromatophore transhydrogenase complex and the separated membrane-bound and soluble factor components of the complex. NADP⁺ completely protected against inactivation of the membrane-bound component, whereas NAD⁺ was without effect. Soluble factor was maximally protected only partially by either NAD⁺ or NADP⁺, but a mixture of the substrates afforded complete protection. NADP⁺-dependent association of soluble factor with factor-depleted membranes was markedly decreased after incubation of membranes with butanedione in the absence, but not in the presence, of NADP⁺. Soluble factor was bound to agarose-NAD and was eluted by NAD⁺, but not by NADP⁺. These results demonstrate the presence of at least three nicotinamide adenine dinucleotide binding sites on R. rubrum transhydrogenase complex, including separate NADP and NAD binding sites on soluble factor and a NADP binding site on the membrane-bound component.     

CORTEX PROTEIN OF SEA URCHIN EGGS I. ITS PURIFICATION AND OTHER PROTEIN COMPONENTS OF THE CORTEX

Isolated cortical hull of the sea urchin egg consisted of a gel layer having 3–4 μ in thickness which could be dispersed with 0.6 m KCl. After removing a protein fraction soluble in 10 m m Tris-HCl buffer (pH 7.0–7.2) containing 1 m m ATP or EDTA and 1 m m GSH, so called KCl-soluble protein of the cortices was obtained. After purifying the "cortex protein", it was homogeneous so far as checked by ultracentrifugation and electrophoresis on a polyacrylamide gel. The cortex protein had a thiol-disulfide exchange activity to Ca-insoluble protein in the ATP-extract of the cortices catalyzed by a transhydrogenase. Neither ovoactin nor actomyosin-like protein was detected in the ATP-extract or the 0.6 m KCl-extract of the cortices respectively. Hyalin was not detected in our KCl-soluble protein fractions of isolated cortices.     

Energy-linked and energy-independent transhydrogenase activities inEscherichia coli vesicles

Active transport vesicles ofEscherichia coli were shown to possess low levels of energy-independent and energy-dependent nicotinamide nucleotide transhydrogenase activities. Breakage of such vesicles in a French pressure cell resulted in a fraction which had an 8–10-fold increased respiration- and ATP-driven transhydrogenase activities.Stimulation of the ATPase activity in vesicles with Triton X-100 was also paralleled by a 2-fold increase in the energy-independent transhydrogenase.Disruption of the vesicles similarly resulted in increases in the energy-independent transhydrogenase, NADH and succinate oxidase activities but a decrease in succinate supported proline uptake.In the light of these findings, the ‘sidedness’ of the vesicle membranes is discussed.     

Insulin degradation by human skeletal muscle

Although previous studies from this and other laboratories have extensively characterized insulin degrading activity in animal tissues, little information has been available on insulin responsive human tissues. The present study describes the insulin degrading activity in skeletal muscle from normal human subjects. Fractionation of a sucrose homogenate of skeletal muscle demonstrated that 97% of the total neutral insulin degrading activity was in the 100 000 × g supernatant with no detectable glutathione-insulin transhydrogenase activity. The 100 000×g pellet contained 85% of the total acid protease activity and all the glutathione-insulin transhydrogenase activity. The soluble insulin degrading activity was purified 1400-fold by ammonium sulfate fractionation, molecular exclusion, ion-exchange and affinity chromatography. Enzymatic activity was determined by measuring an increase in trichloroacetic acid-soluble products of the ¹²⁵I-labeled hormone substrates. The purified enzyme showed marked proteolytic specificity for insulin with a K_m of 1.63·10^?7 M (±0.32) and was competitively inhibited by proinsulin and glucagon with K_i values of 2.1 · 10?⁶ M and 4.0 · 10^?6 M, respectively. This insulin protease exhibited a pH optimum between 7 and 8, a molecular weight of 120 000 and was capable of degrading glucagon. Inhibition studies demonstrated that a sulfhydryl group is essential for activity. Molecular exclusion chromatography of [¹²⁵I]insulin degraded products revealed a time-dependent increase in degradation products with molecular weights intermediate between intact insulin and iodotyrosine. These studies demonstrate that the major enzymatic system responsible for insulin degrading activity is a soluble cysteine protease capable of rapidly metabolizing insulin under physiologic conditions.     

2,3-Dihydroxybenzoate 2,3-oxygenase from the chloroplast fraction of Tecoma stans

2,3-Dihydroxybenzoate-2,3-oxygenase is mainly localized in the soluble and the chloroplast fractions of Tecoma leaves. It is associated with the lamellar structure of the chloroplast fraction. The chloroplast enzyme has properties similar to those of the soluble enzyme, but it has a longer half-life and is more stable to dialysis than the soluble enzyme. It is inhibited by sulfhydryl reagents and the inhibition is reversed by the addition of reduced glutathione. The chloroplast enzyme is insensitive to iron-chelating agents. The enzyme loses activity on dialysis against copper-chelating agents and the activity is completely recovered on the addition of copper; addition of iron does not restore the activity. Polyphenol oxidase is probably present only in the active form in the Tecoma chloroplast but it is not involved in the intradiol cleavage of 2,3-dihydroxybenzoic acid.