Non-specific biosynthesis of gammacerane derivatives by a cell-free system from the protozoon Tetrahymena pyriformis. Conformations of squalene, (3S)-squalene epoxide and (3R)-squalene epoxide during the cyclization

1. A cell-free system from the protozoon Tetrahymena pyriformis was incubated with either [12-3H]squalene or (RS)-2,3-epoxy-2,3-dihydro-[12,13-3H]squalene. Squalene was cyclized into tetrahymanol whereas racemic squalene epoxide was transformed into gammacerane-3 alpha,21 alpha-diol and gammacerane-3 beta,21 alpha-diol. After cyclization of (RS)-2,3-epoxy-2,3-dihydro-[3-3H]squalene, both epimeric gammaceranediols were labelled with a tritium atom located at C-3, showing that no isomerization via a 3-oxo compound occurred. 2. The proton NMR spectra of the cyclization products of synthetic (2E, 22E)-(1,1,1,24,24,24-2H6)squalene and (RS)-(22E)-2,3-epoxy-2,3-dihydro-(1,1,1,24,24,24-2H6)squalene show that squalene and the (3S)enantiomer of its epoxide are cyclized in an all pre-chair conformation, whereas the (3R) enantiomer of squalene epoxide is cyclized in a pre-boat conformation as concerns the cycle A. 3. The squalene cyclase of T. pyriformis presents the same lack of substrate specificity as the cyclase of Acetobacter pasteurianum: in addition to squalene, its normal substrate, it also cyclizes both enantiomers of its epoxide. This conformational versatility is characteristic of squalene cyclases but no longer exists in the squalene epoxide cyclases from eukaryotes.     

Preparation and partial characterization of cell-free protein-synthesizing extracts from Tetrahymena pyriformis.

1. We have examined methods necessary for preparing post-mitochondrial supernatants from Tetrahymena pyriformis strain HSM that are capable of efficient cell-free protein synthesis. 2. The requirements for optimum synthesis in these extracts are described. 3. Data relating to the kinetics of protein synthesis and the initiation capacity of these supernatants are presented.     

The ultrastructural characteristics of the interaction of Legionella pneumophila with the infusorian protozoon Tetrahymena pyriformis

In this work the morphological features of the interaction of L. pneumophila virulent strain and T. pyriformis have been studied on the submicroscopic level in the time course of the process. The study has shown the process of the destruction of the bacterial population and the penetration of individual intact Legionella cells from the phagosome into the endoplasm of T. pyriformis after 6-9 hours of interaction in the form of the budding of the phagosome and further multiplication of Legionella in the endoplasm. As revealed in this study, T. pyriformis have two types of phagosomes characterized by different variants of the destruction of Legionella. In T. pyriformis lysosomes-like granules, mitochondria and the granular endoplasmatic network take part in the process of interaction. The process of interaction has been found to end by day 7 in the death of all protozoal cells taking part in interaction.     

Enzymatic cyclization of neryl pyrophosphate to -terpineol by cell-free extracts from peppermint

A cell-free system prepared from peppermint ($\text{Mentha piperita}$ L.) shoot tips catalyzed the cyclization of neryl pyrophosphate to α-terpineol. Cyclization could be demonstrated in the absence of added cofactors, but addition of NaF inhibited competing phosphatase/pyrophosphatase activity, resulting in much higher levels of α-terpineol formation. Under certain conditions cyclization was stimulated by Mg⁺⁺. Similar enzyme preparations were obtained from spearmint ($\text{Mentha spicata}$ L.) leaves and carrot ($\text{Daucus carota}$ L.) storage organ. The cyclization of neryl pyrophosphate to α-terpineol appears to be a key reaction in the biosynthesis of cyclohexanoid monoterpenes.     

Studies on the biosynthesis of tetrahymanol in Tetrahymena pyriformis. The mechanism of inhibition by cholesterol

Tetrahymanol biosynthesis by the protozoan Tetrahymena pyriformis was progressively inhibited by the inclusion of cholesterol in the growth medium. Studies with labelled precursors of tetrahymanol have established that there are two major sites of inhibition in whole cells. The inhibition at the first site, between acetate and mevalonate, occurred rapidly after addition of cholesterol. The activity of 3-hydroxy-3-methylglutaryl-CoA reductase (EC 1.1.1.34), a predominantly cytosolic enzyme in this organism, was not inhibited in cholesterol-grown cells nor by addition of cholesterol directly to the assay medium. The second major site of inhibition in whole cells is between mevalonate and squalene and this is accompanied by inhibition of the enzyme that converts farnesyl-pyrophosphate into squalene (squalene synthetase). Squalene cyclase is partially inhibited. The conversion of mevalonate into tetrahymanol in vitro was not inhibited by the addition of cholesterol to the assay medium. Tetrahymanol added to the culture medium is taken up by the cells but does not inhibit endogenous biosynthesis. It is suggested that cholesterol inhibits the later stages of tetrahymanol biosynthesis by causing a change in membrane structure and function which alters the activity of membrane-bound enzymes.     

The effect of cholesterol on ubiquinone and tetrahymanol biosynthesis in Tetrahymena pyriformis.

The biosynthesis of ubiquinone-8 from radioactive mevalonate by cultures of Tetrahymena pyriformis is demonstrated. Under normal conditions the incorporation of this radioactive precursor into ubiquinone and the triterpenoid alcohol tetrahymanol reflects the amounts of these two compounds in the cell. Growth of T. pyriformis in the presence of cholesterol results in a complete inhibition of incorporation of radioactive mevalonate into tetrahymanol while there is a corresponding increase of radioactive incorporation into ubiquinone. This increased incorporation of mevalonic acid into ubiquinone must reflect a reduced level of mevalonic acid in the cell under these conditions and is not due to increased ubiquinone biosynthesis, indicating tight regulation of the pathway prior to mevalonate formation.     

The effect of excess mevalonic acid on ubiquinone and tetrahymanol biosynthesis in Tetrahymena pyriformis.

When T. pyriformis is grown in the presence of 10(-2)M-mevalonic acid, the uptake exceeds the cell's requirement for this biosynthetic intermediate. The majority of the excess mevalonic acid is diverted into ubiquinone-8 biosynthesis whereas the biosynthesis of tetrahymanol, the major product of the mevalonic acid pathway, is unchanged. In the presence of excess external mevalonic acid, the biosynthesis of mevalonic acid by the cell is inhibited. It is proposed that ubiquinone biosynthesis is normally regulated by mevalonic acid availability, whereas tetrahymanol biosynthesis is regulated primarily at a later point in the pathway.     

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.     

The biosynthesis of a choline nucleotide by a cell-free extract from Streptococcus pneumoniae.

Choline, a component of the wall teichoic acid of Streptococcus pneumoniae, was converted to cytidine diphosphocholine via choline phosphate by enzymes which were identified in cell-free extracts of the pneumococcus. The first enzyme, choline kinase, was investigated in some detail. It appeared to have a pH optimum of 7.3 to 7.4 and was stimulated by Mg2+. Kinetic studies gave an apparent Michaelis constant (Km) for ATP of I mM, and for choline of 0.19 mM, with Vmax values of 3 nmol min-1 (mg protein)-1 and 0.5 nmol min-1 (mg protein)-1 respectively. The second enzyme, CDPcholine pyrophosphorylase was specific for CTP and had a requirement for Mg2+ with an optimum at 7 mM.     

The termination of the central microtubules from the cilia of Tetrahymena pyriformis.

In frayed axonemes of cilia isolated from Tetrahymena pyriformis, observed in negative stain, the central apparatus remains intact, stabilized in part by the sheath projections that encircle the two singlet central microtubules. The projections terminate ca. 1.5 +/- 0.5 micron before the microtubules themselves end. The microtubules are capped together at their tips by a distinct structure, the central pair cap. The cap, ca. 50 nm across and 90 nm long, consists of a stack of two disks and a ball, similar in shape to a finial. The cap is the only part of the axoneme that extends to the distalmost point of the ciliary membrane and, therefore, it may be of significance in length determination or in shaping the ciliary tip.     

The biosynthesis of gramicidin S in a cell-free system

1. A cell-free system prepared from Bacillus brevis cells, harvested in the late phase of growth and consisting of the 11000g supernatant, has been shown to incorporate into gramicidin S the five constituent amino acids added in labelled form. The results are consistent with complete synthesis and not merely a completion of pre-existing intermediate peptides. 2. The incorporation of ¹⁴C-labelled amino acids by the 11000g supernatant into gramicidin S requires an energy source. Omission of phosphoenolpyruvate and pyruvate kinase from the incubation mixture prevents incorporation into gramicidin S. The cell-free system incorporates [¹⁴C]-leucine, -proline and -phenylalanine over a period of 4hr. With [¹⁴C]leucine, incorporation into gramicidin S takes place in the range pH6–9 with maximum incorporation at pH7·0. High concentrations of chloramphenicol or puromycin decreased the incorporation into gramicidin S by only about 20%. 3. The 50000g supernatant exhibited no decrease in ability of incorporating [¹⁴C]valine into gramicidin S as compared with the 11000g supernatant. About 40% of the incorporating ability remained in the 105000g supernatant after 3hr. centrifugation. When recombining the 105000g sediment with the 105000g supernatant, some increase in incorporation over that obtained with the supernatant alone was obtained. The findings tend to support the view that gramicidin S is synthesized in a different manner from that of proteins.     

Purification and characterization of a secreted protease from Tetrahymena pyriformis

A simple major protease, secreted into the medium during growth of Tetrahymena pyriformis strain W, has been purified about 4000-fold by (NH4)2SO4 precipitation, ion-exchange chromatography, gel filtration and affinity chromatography on organomercurial-Sepharose. The purified protease was homogeneous as judged by polyacrylamide gel electrophoresis and was a monomeric protein with a molecular weight of 22 000-23 000. Amino acid analysis showed that the enzyme was rich in acidic amino acids. In addition, the purified Tetrahymena protease consists of multiple forms with isoelectric point between pH 5.3 and 6.3. Optimum activity of the purified enzyme was in the pH range 6.5-8.0 with alpha-N-benzoyl-DL-arginine-p-nitroanilide and with azocasein, while it was in the lower pH range (4.5-5.5) for denatured hemoglobins. The purified enzyme was inhibited by compounds effective against thiol proteases. Leupeptin and chymostatin were potent inhibitors but pepstatin was without effect. This enzyme is similar to cathepsin B and appears to be a major proteolytic enzyme in Tetrahymena.