Changes in cyclic AMP-dependent protein dinase activity in Tetrahymena pyriformis during the growth cycle.

An adenosine 3':5'-monophosphate-dependent protein kinase II (ATP:protein phosphotransferase, EC 2.7.1.37) was partially purified from the cytosol fraction of an exponentially growing culture of Tetrahymena pyriformis. Protein kinase II represented approximately 90% of the cytosolic protein kinase activity. The enzyme had a high degree of substrate specificity for calf thymus and Tetrahymena histones as compared to casein, protamine and phosvitin. The enzyme incorporated the terminal phosphate of ATP into serine and threonine residues of all the histone fractions. The apparent Km of the enzyme for adenosine 3':5'-monophosphate (cyclic AMP) was 1-10-minus 8 M. Protein kinase II was also activated by other cyclic nucleotides with apparent Km values in the range 2.k-10-minus 6 M. Ther specific activity of the cyclic AMP-dependent protein kinase of Tetrahymena decreases markedly from initial high values during the transition from the lag to early log phase of growth. This is followed by a shrp increase in the activity of the enzyme as the log phase of growth progresses. The specific activity of the enzyme increases rapidly during the heat-induced synchronization of Tetrahymena cells. The capacity for rapid phosphorylation of multiple classed of organelle-specific phosphoproteins and the level of cyclic AMP were maximal in Tetrahymena during the earliest phase of growth. These results demonstrate that the cell cycle of Tetrahymena may be coordinated by marked variations in the level of cyclic AMP which in turn regulate the cyclic AMP-dependent protein kinase.     

Regulation of neutral protease activity through the life cycle of Tetrahymena pyriformis

Substantial fluctuations in the intracellular specific activity of neutral proteases, as assayed at pH 8 with azocasein as substrate, occur during the life cycle of the protozoan Tetrahymena pyriformis. Specific activity increases during growth in 2% proteose peptone, despite slow secretion into the medium. The most rapid increase occurs during late stationary phase and appears to be a response to one or more low molecular weight (less than 10 000), heatstable, trypsin-insensitive, polar molecules secreted into the medium. In contrast, intracellular specific activity drops by a factor of 2–5 within the first 2–3 h after transfer to non-nutritive medium. The decrease in activity under these conditions results from an enhanced rate of secretion and the cessation of net synthesis. Its kinetics are unaffected by cycloheximide and concanavalin A.     

Sterol manipulation that modulates the alteration in membrane fluidity of Tetrahymena pyriformis during temperature acclimation

Our previous results [Umeki and Nozawa (1983) Biochem. Biophys. Res. Commun. 113, 96-101] suggested that ergosterol-replaced Tetrahymena cells (ergosterol-cells) accomplish an adaptive modification of fatty-acid composition by a preferential increase in palmitoyl-CoA desaturase activity, which is principally due to the increased content of the terminal component (cyanide-sensitive factor) of the desaturase system. The present study was designed to obtain information as to how the membrane fluidity of ergosterol-cells is changed during cold temperature acclimation. The order parameter (S) of liposomes prepared from ergosterol-cell lipids was reduced more rapidly after a temperature shift-down than that of control liposomes prepared from native cells containing tetrahymanol. These results indicate that, unlike native cells containing tetrahymanol, ergosterol-cells strive to accomplish cold temperature acclimation by undergoing a great modification of membrane fluidity because of the altered microsomal desaturase activity.     

Regulation of neutral protease activity through the life cycles of Tetrahymena pyriformis.

Substantial fluctuations in the intracellular specific activity of neutral proteases, as assayed at pH 8 with azocasein as substrate, occur during the life cycle of the protozoan Tetrahymena pyriformis. Specific activity increases during growth in 2% proteose peptone, despite slow secretion into the medium. The most rapid increase occurs during late stationary phase and appears to be a response to one or more low molecular weight (less than 10 000), heat-stable, trypsin-insensitive, polar molecules secreted into the medium. In contrast, intracellular specific activity drops by a factor of 2--5 within the first 2--3 h after transfer to non-nutritive medium. The decrease in activity under these conditions results from an enhanced rate of secretion and the cessation of net synthesis. Its kinetics are unaffected by cycloheximide and concanavalin A.     

Changes in monoamines in rat hypothalamus during cold acclimation

Action of norepinephrine (NE), serotonin (5-HT) and dopamine (DA) in the hypothalamus have been reported to play key roles in several homeostatic functions, including thermoregulation. The purpose of this study was to clarify differences in concentrations of NE, 5-HT and DA in several hypothalamic regions after cold exposure of different durations. Rats were exposed to a cold environment (5 °C) for 3 hours (3H), 1 day (1D), 7 days (7D), 14 days (14D), or 28 days (28D). After cold exposure, each hypothalamic region was immediately extracted and homogenized. NE, 5-HT and DA in the extract were measured by high-performance liquid chromatography. We observed marked differences in the concentration of NE in each hypothalamic region after cold exposures. NE in the preoptic area was high only in the 3H group, while it was elevated in the 7D, 14D and 28D groups in the ventromedial hypothalamus. On the other hand, NE in the posterior hypothalamus was low in the 3H, 1D, 7D and 14D groups. Cold exposure did not affect concentrations of 5-HT and DA in these hypothalamic regions. Our results suggest the involvement of NE in each hypothalamic region in maintenance of body temperature, and that the neuronally active site in the hypothalamus seems to change during cold acclimation.     

A PAF-acetylhydrolase activity in Tetrahymena pyriformis cells

Our study provides evidence for the existence of an acylhydrolase activity in Tetrahymena pyriformis cells, capable of hydrolyzing the sn-2 ester bond of the PAF molecule. This activity is mainly distributed in the microsomal fraction (76.5% of total) and has properties similar to the mammalian PAF-acetylhydrolase since it is Ca(2+)-independent, acid-labile, is inhibited by DFP and PMSF but it is not affected by egg yolk phosphatidylcholine. This microsomal acylhydrolase has apparent Km and Vmax values of 1.56 microM and 373 pmols.mg.min respectively. This is the first report of the existence of a PAF-acetylhydrolase activity in a non-mammalian cell.     

Correlation between fluidity and fatty acid composition of phospholipid species in Tetrahymena pyriformis during temperature acclimation.

The correlation between the fluidity of phospholipids and their fatty acid composition was studied by spin label technique and gas-liquid chromatography for three major phospholipid species in Tetrahymena pyriformis during temperature acclimation. The fluidity of 2-aminoethylphosphonolipid increased within the first 10 h of the cold-acclimation when the content of gamma-linolenic acid in 2-aminoethylphosphonolipid was highest, and it then decreased up to 24 h. On the other hand, the fluidities of phosphatidylethanolamine and phosphatidylcholine showed a gradual decrease up to 24 h after the temperature shift, although gamma-linolenic acid contents were highest at 10 h after the temperature shift. Thus the fluidity changes of these two phospholipids were interpreted as resulting from the altered content of other fatty acids in addition to gamma-linolenic acid, since the gamma-linolenic acid content was smaller than that of 2-aminoethylphosphonolipid. The results suggest that the content of gamma-linolenic acid in 2-aminoethylphosphonolipid plays a role in regulating the thermal adaptation process.     

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.     

Changes in thermal phase transition of various membranes during temperature acclimation in Tetrahymena

Changes in the thermal phase transition temperature of membrane lipids were studied by X-ray wide-angle diffraction during adaptation of Tetrahymena pyriformis to a lower growth temperature. After a shift in growth temperature from 39 to 15 degrees C, the phase transition temperature was lowered gradually in microsomal and pellicular phospholipids, whereas that in mitochondrial phospholipids was unchanged for 10 h after the temperature shift. Only a small decrease in the transition temperature of mitochondrial phospholipids was observed, even after 24 h following the shift. Transition temperatures of microsomal, pellicular and mitochondrial phospholipids reached the growth temperature (15 degrees C) about 6, 10 and 24 h after the temperature shift. The temperature dependence of the solid phase in membrane phospholipids was estimated from the 4.2 A peak of the X-ray diffraction pattern. In the case of the phospholipids extracted from cells grown at 39 degrees C, the solid phase was increased upon lowering temperature in a similar manner in all three membrane fractions: mitochondria, pellicles and microsomes. However, in the case of the phospholipids from cells exposed to a lower growth temperature (15 degrees C) for 10 h, the increase in the solid phase was significantly smaller in mitochondrial phospholipids than in two other membrane fractions. The difference in the thermal behaviour of mitochondrial lipid from pellicular and microsomal lipids is discussed in terms of phase transition and phase separation.     

Biochemical and Ultrastructural Changes in Tetrahymena pyriformis During Starvation*

SYNOPSIS. Certain of the ultrastructural and biochemical changes occurring during the first 25 hr of starvation in Tetrahymena pyriformis were studied. Ultrastructurally, numerous profiles of degenerating mitochondria were seen in the early stages of starvation. The presence of oxidizable substrate such as glucose and acetate did not prevent this degeneration. Numerous large nucleoli were formed, many of which seemed to be passing into the cytoplasm as forming autophagic vacuoles. There was a transient increase in Oil Red O-positive bodies, presumably lipid (triglycerides). The extent and duration of this increase were pronounced in the presence of acetate. The lipid droplets appeared to arise within the cisternae of the endoplasmic reticulum. Lipid reserves were apparently utilized prior to carbohydrates, as the disappearance of lipid droplets preceded glycogen utilization, both in the presence of acetate and in the absence of exogenous substrate. A considerable loss of cellular protein also occurred. In cells from inorganic medium supplemented with glucose, glycogen occupied much of the cell, leaving only islands of cell organelles. Acid phosphatase was localized, ultrastructurally, mainly in autophagic vacuoles which contained mitochondria and other cell organelles, and in association with small, double-membraned structures which seemed to be sequestering small areas of cytoplasm. Such sequestered areas also appeared within larger autophagic vacuoles. Residual bodies containing concentric whorls of myelin-like membranes surrounding a more solid core accumulated during starvation. Acid phosphatase activity decreased in amount but not in specific activity. The specific activity of cathespin doubled or tripled, but there was little change in total enzyme.     

Changes in glutathione content during cold acclimation in Cornus sericea and Citrus sinensis

Glutathione content was evaluated in relation to freezing tolerance in red osier dogwood stems and Valencia orange leaves. Exposure of dogwood and citrus to cold-acclimating conditions in controlled environments led to increases in reduced glutathione (GSH) content which were correlated with freezing tolerance. GSH did not accumulate in field-grown dogwood stems during cold acclimation in fall, but did increase in content prior to deacclimation in late winter. Further studies showed that accumulation of GSH in dogwood at low temperatures is dependent on adequate levels of sulfate in the soil. In citrus, modulation of GSH content by infiltration of leaf tissue with various compounds including GSH did not alter freezing tolerance. Root treatment with N,N-diallyl-2,2-dichloroacetamide (R-25788) increased leaf GSH content, but not hardiness. Evidence presented indicates that glutathione accumulates in plant tissues exposed to low temperatures, but that GSH accumulation is not associated with freezing tolerance.