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 E_m7.2 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 E_m7.2 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 degree 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 Em7.2 of 0.245 and 0.345 V. EPR spectra of the aerobic submitochondrial particles in the absence of substrate show the presence of low spine 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 Em7.2 of 0.25 V which binds to the cytochrome c-depleted Tetrahymena mitochondria in the amount of 0.5 nmol/mg protein with KD 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 alpha 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 Em7.2 of--0.17 V and exhibits a high spin ferric heme signal at g=6.     

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.     

Mitochondrial deoxyribonucleic acid from Tetrahymena pyriformis and its kinetic complexity

1. Mitochondrial DNA from Tetrahymena pyriformis strain T has a buoyant density (rho) of 1.685 compared with rho1.688 for whole cell DNA. Mitochondrial preparations from T. pyriformis strain W show an enrichment of a light satellite (rho1.686), although this is not obtained free from nuclear DNA (rho1.692). 2. T. pyriformis mitochondrial DNA renatures rapidly and the kinetics of this process indicate a complexity of approx. 3x10(7) daltons. 3. The base-pairing in the renaturation product is of a precise nature, since the ;melting' temperature (80.5 degrees C) is indistinguishable from that of the native DNA (80.5 degrees C). 4. Centrifugation of mitochondrial DNA in an alkaline caesium chloride density gradient gives two bands, implying the separation of the complementary strands.     

The transsulfuration pathway in Tetrahymena pyriformis.

Four enzymes necessary for the metabolism of methionine by the trans-sulfuration pathway, methionine adenosyltransferase (EC 2.5.1.6), adenosylhomocysteinase (EC 3.3.1.1), cystathionine beta-synthase (EC 4.2.1.22) and cystathionine gamma-lyase (EC 4.4.1.1) were identified in Tetrahymean pyriformis. The ability of these cells to transfer 35S from E135S]methionine to form [35S] cysteine was also observed and taken as direct evidence for the functional existence of this pathway in Tetrahymena. An intermediate in the pathway and an active methyl donor, S-adenosylmethionine, was qualitatively identified in Tetrahymena and its concentration was found to be greater in late stationary phase cells than in early stationary phase cells.     

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.     

The Distribution of Tetrahymena pyriformis1

SYNOPSIS. This paper is a brief account of both amicronucleate and sexually active strains of Tetrahymena pyriformis and their distribution with some comments on their possible evolution.     

Monotertiarybutylhydroquinone effects on Tetrahymena pyriformis.

The molecular toxicity of monotertiarybutylhydroquionone (TBHQ) was studied using $\text{Tetrahymena}$$\text{pyriformis}$ as a model cell system. TBHQ at 26 ppm in the media inhibited cell growth by 50%. TBHQ inhibited the oxidation of ¹⁴C-acetate to ¹⁴CO₂. In addition, increasing concentrations of TBHQ decreased the incorporation of ¹⁴C-acetate into lipids and protein, ¹⁴C-amino acids into protein, ³H-uridine into RNA and ³H-thymidine into DNA. The incorporation of ¹⁴C-acetate into glycogen increased with concentrations up to 20 ppm TBHQ in the media while glycogen synthesis decreased with 40 ppm TBHQ.     

Purification and properties of a membrane-bound L-asparaginase of Tetrahymena pyriformis

L-Asparaginase activity reaches maximal values at the stationary phase of growth of Tetrahymena pyriformis and fluctuates upon the growth conditions and the composition of the medium. Most of the L-asparaginase activity (80%) is associated with the endoplasmic reticulum, and the remaining with the pellicles. Detergents either alone or in combination with NaCl up to 0.5 M concentration failed to solubilize L-asparaginase. Solubilization can be accomplished by means of either the chaotropic agents KSCN and NaClO₄, or 0.1 M sodium phosphate buffer pH 8.0, following pretreatment of the particulates with 2% w/v Triton X₁₀₀. L-Asparaginase has been purified to near homogeneity by hydrophobic and gel filtration chromatography. The native enzyme has a relative molecular weight of 230000. It is a multiple subunit enzyme, with subunit size of 39000. Its isoelectric point is at pH 6.8. It acts optimally at pH 8.6 with a Km of 2.2 mM. It does not hydrolyse L-glutamine and its reaction is inhibited competitively by D-aspartic acid and D-asparagine as well as by Ir asparagine analogues with substituents at the 0 position.     

Isolation of nucleoli from Tetrahymena pyriformis.

A procedure is described to isolate nucleoli from Tetrahymena pyriformis which contain extrachromosomal ribosomal DNA. Macronuclei isolated by the Nonidet procedure were sonicated at a reduced magnesium concentration, and the sonicate was fractionated by isopycnic centrifugation in a metrizamide density gradient. The heaviest band, designated Band IIb, contains exclusively ribosomal DNA, thus constituting the nucleolar fraction. The purity of the nucleolar fraction on a DNA basis, which is defined as the percentage of ribosomal DNA and determined by equilibrium centrifugation in a CsCl density gradient, was around 70%. Electron microscopic examination revealed that the isolated nucleoli retained fairly well the ultrastructure of the in situ nucleoli. Some of the biochemical properties of the isolated nucleoli are also presented.     

The purine and pyrimidine metabolism of Tetrahymena pyriformis

The metabolism of purines and pyrimidines by the ciliated protozoan Tetrahymena was investigated with the use of enzymatic assays and radioactive tracers. A survey of enzymes involved in purine metabolism revealed that the activities of inosine and guanosine phosphorylase (purine nucleoside: orthophosphate ribosyltransferase, E.C. 2.4.2.1) were high, but adenosine phosphorylase activity could not be demonstrated. The apparent K_m for guanosine in the system catalyzing its phosphorolysis was 4.1 ± 0.6 × 10^?3 M. Pyrophosphorylase activities for IMP and GMP (GMP: pyrophosphate phosphoribosyltransferase, E.C. 2.4.2.8), AMP (AMP: pyrophosphate phosphoribosyltransferase, E.C. 2.4.2.7), and 6-mercaptopurine ribonucleotide were also found in this organism; but a number of purine and pyrimidine analogs did not function as substrates for these enzymes. The metabolism of labeled guanine and hypoxanthine by intact cells was consistent with the presence of the phosphorylases and pyrophosphorylases of purine metabolism found by enzymatic studies. Assays for adenosine kinase (ATP: adenosine 5'-phosphotransferase, E.C. 2.7.1.20) inosine kinase, guanosine kinase, xanthine oxidase (xanthine: O₂ oxidoreductase, E.C. 1.2.3.2), and GMP reductase (reduced-NADP: GMP oxidoreductase [deaminating], E.C. 1.6.6.8) were all negative. In pyrimidine metabolism, cytidine-deoxycytidine deaminase (cytidine aminohydrolase, E.C. 3.5.4.5), thymidine phosphorylase (thymidine: orthophosphate ribosyltransferase, E.C. 2.4.2.4), and uridine-deoxyuridine phosphorylase (uridine: orthophosphate ribosyltransferase, E.C. 2.4.2.3) were active; but cytidine kinase, uridine kinase (ATP: uridine 5'-phosphotransferase, E.C. 2.7.1.48), and CMP pyrophosphorylase could not be demonstrated.