Chemical modification of mitochondria. II. Effect of labeling on oxidative phosphorylation

The labeling of rat liver mitochondria (RLM) by the uncoupler 2,4-dinitro-5-(bromoacetoxyethoxy)phenol (DNBP) was studied and related to the effect of this molecule on oxidative phosphorylation. Alkylation of the cysteine residues was measured both with respect to incubation time of RLM with DNBP and with increasing DNBP concentration. At 3.3 × 10^?5m DNBP, the amount of S-carboxymethyl-cysteine formed was found to level off after about 3 min. The rate of ATP synthesis in RLM is reduced by increasing concentrations of DNBP and falls to zero, with either hydroxybutyrate or succinate as substrate, at 2 × 10^?4m DNBP. To characterize the effect of labeling on oxidative phosphorylation, the $\text{P}\text{O}$ ratio were measured after incubating RLM with DNBP for various times between 10 and 300 sec. The $\text{P}\text{O}$ ratio increases and tends to level off as the incubation time increases. No increase in $\text{P}\text{O}$ ratio was noted when RLM were similarly incubated with the nonlabeling uncoupler 2,4-dinitro-5-(acetoxyethoxy) phenol. Further, the effect of labeling on oxidative phosphorylation was determined with RLM which had been treated with DNBP and then washed free of the excess unreacted uncoupler. DNBP produces specific labeling in RLM which, when related to the effects of this uncoupler on oxidative phosphorylation, suggests that the labeled proteins may be involved in the primary energy transduction process.     

Author index

The effect of the accumulation of β-galactosides on the uptake of P_i into cells and cell nucleotides was examined in ML strains of Escherichia coli. Nonmetabolizable sulfur analogs of lactose, which are accumulated only in the presence of the product of y gene of the Lac operon, inhibited the uptake of P₁ into whole cells and into cell nucleotides. This inhibition was most pronounced in starved cells, those with a low rate of ATP production. When the cell membrane was disrupted by sonication or detergents, the inhibition was lost. No significant inhibition was seen in y^? strains or in inducible y⁺ strains which were not induced. Hence. inhibition of the uptake of phosphate into nucleotides is dependent on the presence of the product of the y gene and a β-galactoside.A technique using ³²P_i and ³³P₁ was developed for simultaneously measuring the turnover and level of nucleotides. β-Galactosides inhibited ATP synthesis in aerobic cells, but stimulated ATP synthesis in anaerobic cells, indicating that an intermediate of oxidative phosphorylation was the source of energy for β-galactoside accumulation.     

In vitro studies of p32 uptake in mouse liver mitochondria

Isolated mouse liver mitochondria were incubated in two types of P³²-labelled sucrose-phosphate buffers. The first contained no added ATP or oxidizable substrate. The second contained added ATP. Samples were taken at specified times, up to 60 minutes, and analyses were made of the mitochondrial TCA-soluble inorganic P³² and the total mitochondrial residue P³¹ and P³². The results of the analyses showed that when the phosphorus inhibition index (the ratio of the amount of incubation inorganic phosphorus to the square of the amount of tyrosine in the mitochondria) was high, inorganic P³² uptake was low and vice versa. In accordance with established data, increased P³² uptake was obtained when ATP was added. ATP was found to stabilize the turnover of TCA-insoluble residue phosphorus as well as to maintain the TCA-soluble orthophosphate pool. These results support findings regarding the inhibitory and controlling effects of incubation medium phosphate in the regulation of inorganic phosphorus uptake.     

The phosphorylation of intramitochondrial AMP: A suggestion for the compartmentation of endogenous Pi pool

1. The mitochondrial level of AMP gradually diminishes during incubation of mitochondria with glutamate but does not with succinate. This decline of AMP, associated with stoichiometric increase in ADP and/or ATP, is accelerated by the addition of electron acceptors or 2,4-dinitrophenol, while arsenite, arsenate and rotenone are inhibitory. These results are in agreement with the view that AMP is phosphorylated to ADP in the inner space of rat liver mitochondria via succinyl-CoA synthetase (succinate: CoA ligase (GDP), EC 6.2.1.4) and GTP:AMP phosphotransferase dependent on the oxidation of 2-oxoglutarate, which is promoted by the transfer of electron from NADH to the respiratory chain.2. Studies of the periodical changes of chemical quantities of adenine nucleotides as well as of their labelling with ³²P_i reveals the following characteristics concerning mitochondrial phosphorylation. (i) In contrast to the mass action ratio of ATP to ADP, the ratio of ADP to AMP is not affected by the intramitochondrial concentration of P_i. (ii) ³²P_i, externally added, is incorporated into ADP much more slowly than into γ-phosphate of ATP. (iii) Conversely, ATP loses its radioactivity from γ-phosphate position more rapidly than [³²P]ADP when ³²P-labelled mitochondria are incubated with non-radioactive P_i.3. In order to elucidate the above characteristic properties of phosphorylation, a hypothetical scheme is proposed which postulates the two separate compartments in the intramitochondrial pool of P_i; one readily communicates with external P_i and is utilized for the phosphorylation of ADP in oxidative phosphorylation, while the other less readily communicates with external P_i and serves as the precursor of ADP via succinyl-CoA synthetase and GTP:AMP phosphotransferase.     

The incorporation of 32Pi into intramitochondrial ADP fraction dependent on the substrate-level phosphorylation

1. A significant amount of ³²P_i is incorporated into ADP fraction if mitochondrial phosphorylation is allowed to proceed solely dependent on the endogenous adenine nucleotides even in the absence of uncouplers or inhibitors of oxidative phosphorylation. This formation of [³²P]ADP is accompanied by a significant labelling of the GTP fraction as well as by a decrease in mitochondrial AMP.2. A good correlation, highly significant on a statistical basis, is obtained between the incorporation of ³²P_i into ADP on the one hand and the oxidation of [1-¹⁴C]glutamate to ¹⁴CO₂ on the other, under a wide variety of conditions of respiration, suggesting that the substrate-level phosphorylation linked to the oxidation of 2-oxoglutarate leads to the phosphorylation of AMP in rat liver mitochondria.3. Since intramitochondrial GTP is not directly labelled by the [³²P]ATP added, it is concluded that neither nucleoside diphosphokinase (ATP:nucleoside diphosphate phosphotransferase, EC 2.7.4.6) nor adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3) is functioning in such an EDTA-containing medium as employed in the present study because of lack of the enzymes inside the inner membrane. This not only indicates that ATP never serves as a phosphate donor for the observed phosphorylation of AMP, but also, along with several other lines of evidence, lends strong support to the view that [³²P]GTP generated as a result of the substrate-level phosphorylation is a direct precursor of [³²P]ADP through the mediation of GTP:AMP phosphotransferase, which has been verified to be located inside the inner membrane by the significant labelling of GTP by [³²P]ADP.     

Evidence for detection of AT 32 P bound at the coupling sites of mitochondrial oxidative phosphorylation

A protein-bound ³²P-labeled substance previously detected in rat-liver mitochondria under conditions chosen to reveal possible energy-rich intermediates of oxidative phosphorylation has been identified as ³²P-γ-labeled ATP. The acid-precipitable protein-bound ATP (E·ATP) appears to equilibrate with medium ATP at the time of acid denaturation. After acid denaturation, the ³²P label of E·ATP is only slowly removed by exposure to perchloric acid containing ATP or PP_i. E·ATP is discharged in aurovertin-inhibited mitochondria during a short exposure to an uncoupler of oxidative phosphorylation in the absence of any change in the endogenous ATP pool. Under optimal energy conditions about one E·ATP is observed per two cytochrome oxidase. The results are consistent with the binding of ATP at the coupling sites of oxidative phosphorylation.     

Zur Wirkung von Sauerstoff auf die 32P-Markierung von Polyphosphaten und organischen Phosphaten bei Ankistrodesmus braunii im Licht

Summary Short time incorporation of ³²P was carried out with synchronised algae (young cells) depleted of phosphate. For the separation and determination of the acid-insoluble phosphate fractions of the cells an improved fractionation procedure was applied. In order to exclude competition by carbon dioxide all experiments were done in the absence of CO₂.Compared with nitrogen, CO₂-free air produces an increase in the labelling of phosphorylated compounds in the light. In strong white light, at high pH, air effects a remarkable increase of ³²P in the acid-insoluble phosphate (P _u), mainly in inorganic polyphosphates (P _ul), whereas the total phosphate uptake remains almost unchanged. The increase in labelling of acid-insoluble phosphate is, therefore, accompanied by a substantial decrease in the labelling of acid-soluble compounds (P _l). In weak white light or in far-red light, at low pH even in strong white or red light, an increase of phosphate uptake and an increased labelling of the acid-stable organic acid-soluble fraction (P _os) is observed instead. The effect of oxygen increases somewhat with increasing light intensity up to light saturation, and it increases markedly with increasing oxygen concentration.An essential contribution by oxidative phosphorylation to this oxygen effect can be ruled out on account of its much higher sensitivity to oxygen. Pseudocyclic photophosphorylation is also not regarded as the main force because of its higher oxygen affinity. Occurrence of photorespiration has not been clearly established so far in related algae (Chlorella), and its use for phosphorylation is unknown. A better, although not complete explanation is given by comparing the oxygen effect with the well-known inhibition of photosynthesis by oxygen (Warburg effect), which leads to an increase in glycolate formation and a simultaneous decrease in the pool sizes of carbon reduction cycle intermediates, even in the absence of CO₂. Since the photophosphorylation process, as well as the photosynthetic electron flow, seem unaffected by high oxygen concentrations whereas the formation of organic phosphate compounds is partially inhibited, excess ATP may be available for polyphosphate synthesis. This explanation would be consistent with the assumption that polyphosphate-ADP kinase mediates an equilibrium between ATP and polyphosphates, mainly at higher pH. At low pH and in other cases the excess ATP might be available for an increased phosphate uptake and for phosphorylation of endogenous carbohydrates.

---

---

Herrn Prof. Dr. W. Simonis zum 60. Geburtstag gewidmet.     

The incorporation of 32P into spectrin aggregates following incubation of erythrocytes in 32P-labelled inorganic phosphate

³²P was incorporated into spectrin by incubation of fresh erythrocytes with ³²P_i and glucose. The dimer and tetramer aggregates revealed only covalently-bound incorporation of phosphorus, while a higher aggregate of spectrin revealed both covalent and non-covalent incorporation. The specific activity of the covalently-bound phosphorus in all oligomers was identical, suggesting that the state of association is independent of phosphorylation. The non-covalent incorporation was shown to be due to the association of ATP with this higher aggregate. The nucleotide appears not to be bound directly to spectrin but rather to component 5 (erythrocyte actin) which is also found to be associated with this highly aggregated spectrin structure.     

Respiration Rate and Adenosine Triphosphate Formation in Tissues4

When leaves of Nicotiana tabacum L. var. Xanthi were inoculatedwith TMV, local lesion formation gave enhanced ATP concentrationaccompanied by increased respiration. Phosphorus metabolismin healthy and infected leaves was investigated either by floatingleaf tissue on ³²P₁-phosphate buffer or by allowing intact leavesto take up ³²P₁-phosphate buffer through the petioles. Bothmethods gave increased ³²P₁ incorporation into inorganic andorganic acid-soluble phosphorus fractions of the infected leaf.Comparison of the specific activity of ³²P₁-phosphate buffersupplied with the specific activity of the -phosphate groupof ATP demonstrated that 50 per cent of the ATP in the healthyleaf compared with over 78 per cent of the ATP in the infectedleaf was metabolically active in respiration. As the ATP concentrationin the infected leaf is much greater than that in the healthyleaf, this means that the amount of ATP being utilized and resynthesizedas a result of respiratory metabolism in the infected leaf ismore than twice that in the healthy leaf. Pulse-labelling experimentsdemonstrated that the rate of ATP turnover was very similarin healthy and infected leaves; therefore the increased respirationin the infected leaf results from the larger ATP pool with aturnover rate of ATP similar to that in the healthy leaf. Withtobacco callus tissue cultures infected by TMV, where infectiondoes not result in local-lesion formation, phosphorus metabolismwas unaltered in the infected tissue. It is concluded that necroticlocal-lesion formation results from increased availability ofATP at sites of ATP utilization, and this is aggravated by over-productionof ATP in the infected leaf.     

The incorporation of P into spectrin aggregates following incubation of erythrocytes in P-labelled inorganic phosphate

³²P was incorporated into spectrin by incubation of fresh erythrocytes with ³²P_i and glucose. The dimer and tetramer aggregates revealed only covalently-bound incorporation of phosphorus, while a higher aggregate of spectrin revealed both covalent and non-covalent incorporation. The specific activity of the covalently-bound phosphorus in all oligomers was identical, suggesting that the state of association is independent of phosphorylation. The non-covalent incorporation was shown to be due to the association of ATP with this higher aggregate. The nucleotide appears not to be bound directly to spectrin but rather to component 5 (erythrocyte actin) which is also found to be associated with this highly aggregated spectrin structure.     

Phosphorus incorporation in the ehrlich ascites tumor cell

Data from isotopic uptake experiments were used to measure the kinetics of labelling of cellular phosphate, ATP and ADP in the Ehrlich ascites tumor cell. The results show that steady state phosphate exchange flux was 0.333 ± 0.052 (S.E.) μmoles per 10⁷ cells per hour at 37°, and that the specific activity of phosphate was the same as P_γ ATP. Metabolic inhibition reduced the phosphate flux by 30–50%. A model, based on oxidative phosphorylation and the adenylate kinase reaction is used to interpret the labelling sequence of P_β ATP and P_β ADP, and its dependence on P_γ ATP.     

Photosynthesis and phosphorylation of light-harvesting chlorophyll a/b—protein in intact chloroplasts: Effects of uncouplers

Protein phosphorylation in isolated, intact pea chloroplasts was measured during the onset of CO₂-dependent O₂ evolution. Total incorporation of ³²P (from ³²P_i) into the light-harvesting chlorophyll a/b—protein was found to be less sensitive than O₂ evolution to inhibition by the uncouplers FCCP and NH₄C1 It is concluded that changes in the rate of ATP synthesis cannot affect protein phosphorylation without also affecting the rate of CO₂-fixation in this system. The ATP/ADP ratio is therefore unlikely to regulate photosynthetic protein phosphorylation under normal physiology conditions.     

Regulation of rat liver phosphofructokinase by glucagon-induced phosphorylation

In perfused rat liver, the effects of various hormones on the stimulation of phosphorylation and allosteric properties of purified phosphorfructokinase were investigated. Rat livers were perfused with [³²P]phosphate followed with various hormones or cyclicAMP, and ³²P-labeled phosphofructokinase was isolated. ³²P incorporation into the enzyme and enzyme inhibition by ATP or citrate were determined. Only glucagon increased the ³²P incorporation into phosphofructokinase and this increase was approximately threefold. The cyclicAMP level was increased simultaneously approximately four- to fivefold compared to the control perfused liver. Similar results were obtained by perfusing the liver with cyclicAMP (0.1 mm). The phosphorylated phosphofructokinase showed a decrease in the K_i values for ATP (from 0.4 to 0.2 mm) and citrate (from 2 to 0.6 mm). Neither epinephrine nor insulin affected the extent of phosphorylation or the allosteric properties of the enzyme. The half-maximal concentration of glucagon required for phosphorylation of phosphofructokinase and modification of its allosteric properties was approximately 6 × 10^?11m. It is concluded that glucagon increases the inhibition of liver phosphofructokinase by ATP and citrate through phosphorylation of the enzyme involving a β-receptor-mediated cyclicAMP-dependent mechanism.     

Rapid protein kinase assay using phosphocellulose-paper absorption.

Protein kinase (EC 2.7.1.37) catalyzes the phosphorylation of serine and threonine residues of a number of proteins. Histone is widely used as an acceptor substrate in measuring the activity of this enzyme isolated from a variety of sources. We have devised a rapid procedure for resolving phosphohistone from ATP and its metabolites based on the specific absorption of phosphorylated histone onto phosphocellulose paper. Using [γ-³²P]ATP as the phosphoryl donor, aliquots of the protein kinase assay mixture are applied to phosphocellulose-paper disks that are then immersed in water which elutes [γ-³²P]ATP and metabolites. After brief organic solvent extraction and drying, bound radioactivity is measured by liquid scintillation spectrometry.     

Tightly bound nucleotides of the energy-transducing ATPase,and their role in oxidative phosphorylation. I. The Paracoccus denitrificans system

1. The coupling ATPase of Paracoccus denitrificans can be removed from the membrane by washing coupled membrane fragments at low salt concentrations.2. This ATPase resembles coupling ATPases of mitochondria, chloroplasts and other bacteria. It is a negatively charged protein of molecular weight about 300 000. An inhibitor protein is bound tightly to the ATPase in vivo, and can be destroyed by trypsin treatment.3. ATP and ADP are found tightly bound to the coupling ATPase of P. denitrificans, both in its membrane-bound and isolated state. The ATP/ADP ratio on the enzyme is greater than one.4. Under de-energised conditions, the bound nucleotides are not available to the suspending medium. When the membrane is energised however, the bound nucleotides can exchange with added nucleotides and incorporate ³²P_i. ³²P_i is incorporated into the β and γ positions of the bound nucleotides, but β-labelling probably does not occur on the coupling ATPase.5. Uncouplers inhibit the exchange of the free nucleotides or ³²P_i into the bound nucleotides, while venturicidin (an energy transfer inhibitor) and aurovertin stimulate the exchange.6. The response of the bound nucleotides to energisation is consistent with their being involved directly in the mechanism of oxidative phosphorylation.     

Studies on the role of RNA synthesis in auxin induction of cell enlargement

Selective inhibitors were used to study the connection between nucleic acid synthesis and indoleacetic acid (IAA) induction of cell enlargement. Actinomycin D (act D) and azaguanine (azaG) almost completely inhibit IAA-induced growth in aged artichoke tuber disks when they are added simultaneously with IAA. In contrast, when they are added 24 hours after the hormone, these inhibitors have little or no effect on the induced growth which continues for 48 hours or more with little or no inhibition. Inhibitors of protein synthesis still stop growth when applied 24 hours after the IAA, thus protein synthesis and presumably supporting metabolism are still essential.

In corn coleoptile sections auxin-induced growth did not show any pronounced tendency to become less sensitive to act D as the IAA treatment progressed. Act D did not completely inhibit the response to IAA unless the sections were pretreated with act D for 6 hours. In contrast to act D, cordycepin produced almost complete inhibition of IAA-induced growth when added with the IAA.

Although IAA has a very large and very rapid stimulatory effect (within 10 min) on incorporation of ³²P-orthophosphate into RNA in disks, it did not cause a detectable change in the base composition of the RNA synthesized. Furthermore, the promotive effect could be accounted for through increased uptake of the ³²P. That much of the RNA synthesis in these tissues is not necessary for auxin action is indicated by the results with fluorouracil (FU). FU strongly inhibits RNA synthesis, probably acting preferentially on ribosomal RNA synthesis, without inhibiting auxin-induced growth in the disks or coleoptile sections. FU also strongly inhibited respiration in auxin-treated disks indicating that the large promotion of respiration by auxin likewise may not be entirely necessary for growth.

At least in the artichoke disks, RNA synthesis is required for auxin induction of cell enlargement and not for cell enlargement itself.

The possible relationships of auxin induction of cell enlargement and RNA synthesis are discussed.

     

Intermediate complex of ATP hydrolysis and synthesis by muscle proteins

Myosin catalyzed exchange between ³²P_i and ATP in reaction medium during its enzymatic hydrolysis of ATP only by a very small amount. Addition of actin increased to a great extent the rate of incorporation of ³²P_i in the presence of Mg. Glycerinated smooth muscle fibers also exhibited the ability to exchange ³²P_i and ATP upon the application of external force (repeated stretching and releasing). A schematic mechanism of the action of actin and external force on acceleration of ³²P_i incorporation is proposed and the importance of the M*-ADP complex for force generation is suggested.     

Does AMP participate in photosynthetic phosphorylation?

Osmotically disrupted chloroplasts catalyze a rapid, light and AMP and ATP dependent ³²P_i incorporation into ATP. Light does not stimulate [¹⁴C] AMP incorporation into ATP in this system. AMP in the presence of P_i inhibits electron flow in a manner analogous to ADP inhibition in the absence of P_i. The inhibition of AMP + P_i is reversed on addition of ADP.     

Protein phosphorylation in a dopamine-sensitive homogenate of rat caudate: Effect of adenosine 3′,5′-cyclic monophosphate and putative neurotransmitters

Adenosine 3′,5′-cyclic monophosphate (cyclic AMP) and its 8-methylthio derivative stimulate the incorporation of ³²P into proteins endogenous to a homogenate of rat caudate nucleus when 4 μM [γ?³²P] ATP is usedas substrate. Higher concentrations of ATP reduced the effect of the cyclic nucleotide until at 400 μM no significant increase in protein phosphorylation was seen.Incubation of the homogenate with 400 μM ATP and 100 μM dopamine resulted in an approx. 2-fold increase in cyclic AMP but did not alter caudate protein phosphorylation suggesting that the catecholamine could not stimulate protein phosphorylation under the experimental conditions used in the present study.