Mitochondrial respiratory chain of Tetrahymena pyriformis. The thermodynamic and spectral properties.

The mitochondria isolated from the ciliate protozoon Tetrahymena pyriformis carry an oxidative phosphorylation with P/O ratio of 2 for succinate oxidation and P/O ratio of 3 for the oxidation of the NAD-linked substrates. The respiration is more than 90% inhibited with 1 mM cyanide while antimycin A and rotenone inhibit at concentrations of 1000-fold higher than those effective in mammalian mitochondria. Using a combination of spectral studies and potentiometric titrations, the components of the respiratory chain were identified and characterized with respect to the values of their half-reduction potentials. In the cytochrome bc1 region of the chain a cytochrome c was present with an Em7.2 of 0.225 V and two components with absorption maxima at 560 nm and the half-reduction potential values of -0.065 and -0.15 V at pH 7.2. The cytochrome with the more positive half-reduction potential was identified as the analogue of the cytochrome(s) b present in mitochondria of higher organisms, while the cytochrome with the more negative half-reduction potential was tentatively identified as cytochrome o. In addition ubiquinone was present at a concentration of approx. 4 nmol per mg mitochondrial protein. In the spectral region where cytochromes a absorb at least three cytochromes were found. A cytochrome with an absorption maximum at 593 nm and a midpoint potential of -0.085 V at pH 7.2 was identified as cytochrome a1. The absorption change at 615-640 nm, attributed usually to cytochrome a2, was resolved into two components with Em7,2 values of 0,245 and 0.345 V. It is concluded that the terminal oxidase in Tetrahymena pyriformis mitochondria is cytochrome a2 which in its two component structure resembles cytochrome aa3.     

Mitochondrial respiratory chain of Tetrahymena pyriformis The properties of submitochondrial particles and the soluble b and c type pigments

Submitochondrial particles isolated from Tetrahymena pyriformis contain essentially the same redox carriers as those present in parental mitochondria: at pH 7.2 and 22 °C there are two b-type pigments with half-reduction potentials of −0.04 and −0.17 V, a c-type cytochrome with a half reduction potential of 0.215 V, and a two-component cytochrome a₂ with E_m7.2 of 0.245 and 0.345 V.

EPR spectra of the aerobic submitochondrial particles in the absence of substrate show the presence of low spin ferric hemes with g values at 3.4 and 3.0, a high spin ferric heme with g = 6, and a g = 2.0 signal characteristic of oxidized copper. In the reduced submitochondrial particles signals of various iron-sulfur centers are observed.

Cytochrome c₅₅₃ is lost from mitochondria during preparation of the submitochondrial particles. The partially purified cytochrome c₅₅₃ is a negatively charged protein at neutral pH with an E_m7.2 of 0.25 V which binds to the cytochrome c-depleted Tetrahymena mitochondria in the amount of 0.5 nmol/mg protein with a K_D of 0.8 · 10⁻⁶ M. Reduced cytochrome c₅₅₃ serves as an efficient substrate in the reaction with its own oxidase. The EPR spectrum of the partially purified cytochrome c₅₅₃ shows the presence of a low spin ferric heme with the dominant resonance signal at g = 3.28.

A pigment with an absorption maximum at 560 nm can be solubilized from the Tetrahymena cells with butanol. This pigments has a molecular weight of approx. 18 000, and E_m7.2 of −0.17 V and exhibits a high spin ferric heme signal at g = 6.     

Characterization of preribosomal ribonucleoprotein particles from Tetrahymena pyriformis.

Ribonucleoprotein particles present in extracts of nuclei prepared from Tetrahymena pyriformis labelled for 1, 2.5, 5 and 10 min with [3H]uridine during exponential growth were analysed by sedimentation through linear 10--30% sucrose gradients. After 1 min of labelling, the early ribosomal RNA precursor (36-S) is found to be associated with slowly sedimenting particles which form a broad peak centred at approximately 50 S. Other kinds of particles sedimenting at 80 S, 66 S, 60 S and 44 S are observed when labelling is carried out for longer periods (2.5, 5 and 10 min). The 80-S particle contains 29-S and 18-S RNA species together with traces of 36-S RNA; the 60-S and 44-S particles contain 26-S and 17-S RNAs respectively. Similar results were obtained when [Me-3H]methionine was used for labelling in place of [3H]uridine. Methylation of the RNA present in slowly sedimenting nuclear components (30-70-S) is rapid, reaching a plateau at 5 min while that of the faster sedimenting (70--90-S) components is still increasing after 10 min. Only three types of ribonucleoprotein particles (80-S, 66-S, and 44-S) were observed when the cells were labelled after prolonged starvation. A scheme of ribosome biogenesis based on these results is presented.     

Localization of an alkyl-acetyl-glycerol-CDP-choline: cholinephosphotransferase activity in submitochondrial fractions of Tetrahymena pyriformis

Tetrahymena pyriformis contains platelet-activating factor (PAF) as a minor lipid, which is biosynthesized de novo. A dithiothreitol-insensitive CDP-choline:cholinephosphotransferase (AAG-CPT), which utilizes alkyl-acetyl-glycerol as a substrate, had been detected in both the mitochondrial and microsomal fractions of the protozoan. In the present report, localization of this enzyme in submitochondrial fractions was studied. Cell fractionation was evaluated with enzyme and morphological markers. In this respect, succinate dehydrogenase, NADPH:cytochrome c reductase, glucose-6-phosphatase, alkaline phosphatase, monoaminoxidase, and cytochrome c oxidase activities were investigated. In the presence of antimycin A, mitochondrial activity of NADPH-cytochrome c reductase, was increased, while the microsomal one was reduced. Cardiolipin was distributed in the inner mitochondrial membrane. Alkaline phosphatase was found exclusively in the cytosol of the protozoan. The main portion of the dithiothreitol-insensitive AAG-CPT was localized in the inner mitochondrial membrane. Our data indicate that mitochondria are able to produce PAF, which might be associated with their function.     

Identification and purification of a soluble adenosine triphosphatase from Tetrahymena pyriformis .

An unusual ATPase isolated from the postribosomal supernatant fraction of $\text{Tetrahymena pyriformis}$ has been purified to homogeneity. The purification procedure consisted of protamine sulfate and heat treatment; column chromatography successively on phosphocellulose, DEAE-cellulose and Sephadex G-150; and isoelectric focusing. The pure enzyme has a molecular weight of 89,000 and requires either Ca²⁺ or Ba²⁺ for maximum activation. Nucleoside triphosphates are hydrolized at decreasing rates in the order: ATP > GTP > ITP > CTP > UTP. The K_m for ATP is 2.5 mM. Because of its properties the enzyme is tentatively classified as a soluble Ca²⁺-activated ATPase.     

UMP pyrophosphorylase of Tetrahymena pyriformis. Partial purification and properties.

UMP pyrophosphorylase (EC 2.4.2.9, UMP:pyrophosphate phosphoribosyltransferase) was purified approximately 85-fold from exponentially growing cells of Tetrahymena pyriformis GL-7. It was found to have a molecular weight of 36,000, and was active over a broad pH range, with an optimum at 7.5. The enzyme exhibited a temperature optimum at 40 °C, above which irreversible inactivation began to occur. The apparent K_m values for uracil and phosphoribosyl pyrophosphate (PRPP) were 0.4 and 6.9 m, respectively. The pyrophosphorylase exhibited a pyrimidine base specificity for uracil, although 5-fluorouracil was utilized by the enzyme. Neither cytosine, orotic acid, nor 6-azauracil competed with uracil for the enzyme or inhibited the production of UMP from uracil and PRPP. Although most triphosphates had little effect on pyrophosphorylase activity, UTP and dUTP, each at a concentration of 1 mm, depressed UMP formation by 86 and 59%, respectively. Thus, UMP pyrophosphorylase may be sensitive to feedback inhibition by the product of the pathway it initiates. UMP pyrophosphorylase specific activity in extracts of Tetrahymena grown in a medium containing uracil as the sole pyrimidine source was threefold higher than that in extracts of cells grown on uridine or UMP.     

Tiron as a spin-trap for superoxide radicals produced by the respiratory chain of submitochondrial particles

Tiron can be used as a spin-trap for O2 radicals generated by the respiratory chain of submitochondrial particles (SMP). Using this sensitive method, it was shown that the O2 (radical) production by the succinate-oxidizing SMP can be reduced by antimycin or 4-nonyl-2-hydroxyquinoline-N-oxide, the effects of both antibiotics being abolished and prevented by cyanide. It is suggested tht the O2 radicals are produced due to autooxidation of ubisemiquinone which is formed as an intermediate upon one-electron oxidation of CoQH2 by cytochrome c1. The effects of antimycin, 2-nonyl-4-hydroxyquinoline-N-oxide and cyanide on the O2 (radical) generation correlate with the effects of these inhibitors on a steady-state concentration of ubisemiquinone predicted by the Mitchell's Q-cycle hypothesis.     

Dynamic control on the rate of the reduction of the b type cytochromes in submitochondrial particles.

1. In the presence of antimycin and KCN the reduction of cytochrome b in phosphorylating submitochondrial particles followed a biphasic first-order kinetics. The transition from the first, rapid phase to the second, slow phase occurred while the reduction of chtochromes c + c1 and a through or around the antimycin block was still linear with time. Thus, the phase transition was due to a fall-off in the rate of cytochrome b reduction. 2. The biphasic reduction of cytochrome b was observed over a wide temperature range (0--30 degrees C), with succinate of NADH as electron donors and with phosphorylating particles or coupled rat-heart mitochondria. With rat-heart mitochondria the same biphasic reduction was observed in the presence of either carbonyl cyanide p-trifluoromethoxyphenylhydrazone or oligomycin. 3. In both the rapid and the slow phases, the rate of reduction of cytochrome b-561 was equal to that of b-565. Thus both cytochromes b-561 and b-565 were affected by the mechanism which determined the reduction-rate. Furthermore, each of these cytochromes could be reduced individually with rate constants typical of the slow phase. 4. The proportion of rapidly reduced to slowly reduced cytochrome b was independent of the degree of its reducibility and could be controlled by teh experimental conditions. When antimycin was used as the only inhibitor, 96% of the b-type cytochromes were reduced in the rapid phase. If the c and a-type cytochromes were first reduced by ascorbate and tetramethyl-p-phenylenediamine in the presence of KCN and antimycin, all the b-type cytochromes were fully reduced at the slow-rate. 5. With succinate, the rate of the rapid phase depended on the activation level of the succinic-dehydrogenase. The rate constant of the second phase was unaffected by the succinic dehydrogenase activity, if the preparation was more than 20% active. Furthermore, the rate constant of the slow reduction was the same with succinate, NADH, or even with durohydroquinone (which reacted directly with cytochromes b). 6. It is suggested that cytochrome b can exist in two forms: kinetically active or sluggish. The active form is rapidly reduced by the endogenous quinone (QH2) or durohydroquinone. The rate of the reduction of the active form by succinate or NADH is probably determined by the rate of the reduction of Q by the dehydrogenases. The second form of cytochrome b is characterized by its sluggish reduction by QH2 or durohydroquinone. 7. It is proposed that the transformation from the active to the sluggish form is induced by the reduction of a controlling group, named Y, located on the oxygen side of the antimycin inhibition site. When Y is oxidized, cytochrome b is in its active form, and when Y is reduced, cytochrome b is in its sluggish form. The nature of this kinetic control and a comparison with the mechanism controlling the reducibility of cytochrome b are discussed.     

Pentachlorophenol inhibition of succinate oxidation by the respiratory chain in submitochondrial particles from the bovine heart

Study on the effect of pentachlorophenol on the succinate oxidase activity of submitochondrial particles and on the reduction level of cytochromes b revealed that the Ki value for PCP is equal to 2-4 microM. The succinate-DCPIP-reductase activity is noncompetitively inhibited with PCP (by 75-85%) (Ki = 3.6 microM). In the case of the succinate-PMS-reductase activity PCP at micromolar concentrations decreases the value of V only by 40% (C50 = 2 microM) with a simultaneous increase of the Km value for PMS. The identity of Ki values for PCP under these conditions suggests that the effect of PCP is due to the inhibitor interaction with the same component of the succinate dehydrogenase complex. The type of action of PCP on the succinate-acceptor-reductase activities indicates that the inhibiting effect of PCP on succinate oxidations is similar to that exerted by traditional inhibitors of succinate dehydrogenase--tenoyltrifluoroacetone and carboxins. Since PCP inhibits succinate dehydrogenase at low concentrations, it seems likely that the biological (pesticidal) effect of PCP is provided for not only by its uncoupling action but also by the inhibition of succinate oxidation in the respiratory chain.     

Amino acid sequence of cytochrome c from Tetrahymena pyriformis Phenoset A.

The cytochrome c of Tetrahymena pyriformis GL (Phenoset A) had an isoelectric point of 6.5 and by sequence the following composition: Asp(7) Asn(2) Thr(4) Ser(8) Glu(6) Gln(2) Pro(7) Gly(13) Ala(13) Val(7) Met(2) Ile(5) Leu(6) Tyr(2) Phe(5) Lys(11) His(3) Trp(1) Arg(3) Cys(2) (total 109 residues). The peptides derived from the protein afforded complete overlap, so a complete sequence could be determined without reference of homologous proteins. Alignment with other mitochondrial cytochromes c required two internal deletions totalling three residues and an N-terminal region two residues longer than, and a C-terminal region one residue shorter than, the previously known limits. The sequence was the most divergent of the known mitochondrial cytochromes c, suggesting a distant relationship of ciliates to other eukaryotes. Details of the sequence data have been deposited as Supplementary Publication no. SUP 50068 (37 pages) at The British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7 BQ, U.K., from whom copies can be obtained on the terms given in Biochem. J. (1976) 153,5.     

Catalytic activity of cytochromes c and c1 in mitochondria and submitochondrial particles.

1. Beef heart mitochondria have a cytochrome c1:c:aa3 ratio of 0.65:1.0:1.0 as isolated; Keilin-Hartree submitochondrial particles ahve a ratio of 0.65:0.4:1.0. More than 50% of the submitochondrial particle membrane is in the 'inverted' configuration, shielding the catalytically active cytochrome c. The 'endogenous' cytochrome c of particles turns over at a maximal rate between 450 and 550 s-1 during the oxidation of succinate or ascorbate plus TMPD; the maximal turnover rate for cytochrome c in mitochondria is 300-400 s-1, at 28 degrees-30 degrees C, pH 7.4. 2. Ascorbate plus N,N,N',N'-tetramethyl-p-phenylene diamine added to antimycin-treated particles induces anomalous absorption increases between 555 and 565 nm during the aerobic steady state, which disappear upon anaerobiosis; succinate addition abolishes this cycle and permits the partial resolution of cytochrome c1 and cytochrome c steady states at 552.5-547 nm and 550-556.5 nm, respectively. 3. Cytochrome c1 is rather more reduced than cytochrome c during the oxidation of succinate and of ascorbate + N,N,N',N'-tetramethyl-p-phenylene diamine in both mitochondria and submitochondrial particles; a near equilibrium condition exists between cytochromes c1 and c in the aerobic steady state, with a rate constant for the c1 leads to c reduction step greater than 10(3) s-1. 4. The greater apparent response of the c/aa3 electron transfer step to salts, the hyperbolic inhibition of succinate oxidation by azide and cyanide, and the kinetic behaviour of the succinate-cytochrome c reductase system, are all explicable in terms of a near-equilibrium condition prevailing at the c1/c step. Endogenous cytochrome c of mitochondria and submitochondrial particles is apparently largely bound to cytochrome aa3 units in situ. Cytochrome c1 can either reduce the cytochrome c-cytochrome aa3 complex directly, or requires only a small extra amount of cytochrome c to carry the full electron transfer flux.     

Studies on lipid metabolism in Tetrahymena pyriformis: properties of acyltransferase systems.

Membrane preparations from Tetrahymena pyriformis catalyzed the acylations of glycerophosphate, isomeric monoacylglycerophosphate, and 1-acylglycerylphosphoryl-choline. Under the optimal conditions, glycerophosphate acyltransferase and 1-acylgly-cerophosphate acyltransferase used saturated and unsaturated acyl-CoA at comparable rates. The specificities of these acyltransferase systems for various acyl-CoAs as compared with the respective maximal velocities do not directly explain the fatty acid distribution in glycerophospholipids. However, the acylation of 2-acylglycerophosphate was highly selective for palmitate when the incubations were carried out in the presence of palmitoyl-CoA, oleoyl-CoA, 1-acylglycerophosphate, and 2-acylglycerophosphate. The 1-acylglycerylphosphorylcholine acyltransferase system showed relatively higher specificity for unsaturated acyl-CoA, which is consistent with the fatty acid pattern of phospholipids. Significant amounts of diglyceride and triglyceride were formed together with phosphatidic acid from acyl-CoA and glycerophosphate, indicating that the enzymes involved in triglyceride synthesis are closely associated with acyltransferase systems involved in phosphatidate synthesis in microsomes. These acyltransferase activities were found mainly in microsomes, and to a lesser extent, in pellicles, too. No significant difference was observed in the properties of acyltransferase systems in microsomes and pellicles.     

Specificity and other properties of three ribonucleases of Tetrahymena pyriformis

Three ribonucleases, RNase I, RNase II and RNase III, were purified from the 109,000 X g supernate of detergent-treated Tetrahymena pyriformis strain W. RNases I and II act optimally at pH 5.5-6.0 and are inhibited by increasing concentrations of salts of monovalent cations. RNase III acts optimally at pH 7.5 and is activated 1.5-fold by millimolar concentrations of ZnSO4 and 5-fold by 50 mM KCl. RNases II and III are activated approximately 100% in the presence of 3 M and 5 M urea respectively. All enzymes are heat-sensitive and acid-resistant. They are endonucleases forming 2',3'-cyclic products. Their base specificity, as tested against ribosomal RNAs of known sequence, is as follows: RNase I hydrolyzes preferentially YpN and secondarily GpN bonds, RNase II is highly specific for RpN bonds, though the preparation can also hydrolyze the UpU sequence. Finally the principal targets of RNase III are YpR sequences and secondarily YpY sequences. A shorthand visualization of base specificity of nucleases in the form of right isosceles triangles is presented. The triangles are constructed by subdividing each of the two perpendicular sides in as many units as the maximum number of times the most abundant dinucleotide appears in all substrates employed and plotting the frequency of hydrolysis of each dinucleotide sequence by the enzyme under study. The proximity of each dinucleotide sequence to the hypotenuse or to one of the perpendicular sides is indicative of its susceptibility or resistance to the enzyme's action.     

Studies of the catalytic properties of an endonuclease isolated from Tetrahymena pyriformis.

An acid endonuclease hydrolyzing both DNA and RNA was purified from Tetrahymena pyriformis, strain E. The enzyme is distributed in all major subcellular compartments and is excreted into the growth medium towards the middle of the logarithmic phase. It hydrolyzes DNA to penta or hexanucleotides, on the average, bearing the monoesterified phosphate at the 3'-position. Particularly in early phases of the reaction it shows a very pronounced specificity for bases with a keto group at position 4 of the pyrimidine ring, such as guanine and thymine.