Lysosomal enzymes produced by immobilized Tetrahymena thermophila

Summary The ciliated protozoon Tetrahymena thermophila was immobilized for production of secreted lysosomal enzymes in two ways. Cells entrapped in solid Ca-alginate spheres survived but were unable to grow and multiply. However, when encapsulated in hollow Ca-alginate spheres Tetrahymena multiplied well, reaching 0.9 × 10⁷ cells/ml. These immobilized cells secreted large amounts of lysosomal enzymes when the medium was changed daily. This system was transferred to a reactor scale using a conical bubble column reactor for semicontinuous cultivation of the encapsulated cells. Under these conditions -glucosidase, -glucosidase, -hexosaminidase and acid phosphatase were produced for at least 4 weeks. The hollow spheres were stable for 3 months and contained living and secreting Tetrahymena cells during this time. Immobilized T. thermophila cells can thus serve as a good source for production of commercially interesting enzymes. Offprint requests to: A. Tiedtke     

Constitutive Secretion of Acid Hydrolases in Tetrahymena thermophila

THE role of lysosomal enzymes in intracellular digestion is now well established [11]. Most often we think of lysosomal hydrolases in catabolism of endogenous or foreign material taken up by endocytosis. There is however, a number of reports dealing with the release of acid hydrolases into the extracellular fluid in a variety of eukaryote cells. These cells range from Saccharomyces cerevisiae [15], Dictyostelium discoideum [10], Leishmania donovani [20], Acanthamoeba castellani [22], Entamoeba histolytica [12, 31], and species of Tetrahymena [1–3, 6] to mammalian cells in culture [49]. Concerning the latter, fibroblasts and hepatocytes in culture release acid hydrolases to the extracellular medium, but only if the synthesis of a specific recognition marker is impaired in the cells. This marker (man-nose-6-phosphate) is used for receptor mediated segregation of lysosomal enzymes into the lysosomal compartments. If the receptor or the marker are lacking, the hydrolases fail to enter the lysosomal compartment, and are secreted in immature form together with molecules belonging to the constitutive secretory pathway of the cells [8, 49]. Such a release of acid hydrolases seems to occur spontaneously from mammalian osteoclasts [4]. Macrophages, on the other hand, need a specific stimulation for their release process [40]. In lower eukaryotes the release may     

An assessment of proteolytic enzymes in Tetrahymena thermophila.

Cellular extracts of Tetrahymena thermophila were found to contain substantial levels of proteolytic activity. Protein digestion occurred over broad ranges of pH, ionic strength, and temperature and was stimulated by treatment with thiol reductants, EDTA and sodium dodecyl sulfate. Incubation at temperatures > or = 60 degrees C or with high concentrations of chaotropic reagents such as 10 M urea or 6 M guanidine-HCl caused an apparent irreversible loss of activity. Activity was also strongly diminished by increasing concentrations of divalent cations. Several peptide aldehydes, p-hydroxymercuribenzoate, and alkylating reagents such as iodoacetate, N-tosyl-L-lysine chloromethyl ketone, N-tosyl-L-phenylalanine chloromethyl ketone, N-methylmaleimide, and trans-epoxysuccinyl-L-leucylamido-(4-guanidino)-butane were potent inhibitors of proteolytic activity. Aprotinin diminished activity by approximately 40% while benzamidine, 3,4-dichlorosocoumarin, and trypsin inhibitors from soy bean, lima bean, and chicken egg caused relatively modest inhibition of proteolytic activity. Phenylmethanesulfonyl fluoride had no apparent effect. Electrophoretic separation of proteins on SDS-polyacrylamide gels copolymerized with gelatin substrate revealed that at least eight active proteolytic enzymes were present in cell extracts ranging in apparent molecular weight from 45,000 to 110,000. Five of these apparent proteases were detected in 70% ammonium sulfate precipitates. Gelatinase activity was not detectable when extracts were pretreated with iodoacetate or E-64, indicating that all of the enzymes observed in activity gels were sensitive to thiol alkylation. Cellular extracts of T. thermophila appeared to contain multiple forms of proteolytic enzymes which were stimulated by thiol reductants and inhibited by thiol modifying reagents. Accordingly, the proteolytic enzymes present in cell extracts appear to be predominantly cysteine proteinases.     

Glutathione in Tetrahymena thermophila

In Tetrahymena , glutathione is synthesized from the same precursors as it is in higher animals and is present in similar intracellular concentrations. The intracellular thiol-disulfide ratio is also identical to that of mammalian tissues, due to the activity of glutathione reductase. The intracellular GSH-level was found to be dependent on the sulfur-containing amino acids in the chemically defined medium.     

Glutathione in Tetrahymena thermophila

In Tetrahymena, glutathione is synthesized from the same precursors as it is in higher animals and is present in similar intracellular concentrations. The intracellular thiol-disulfide ratio is also identical to that of mammalian tissues, due to the activity of glutathione reductase. The intracellular GSH-level was found to be dependent on the sulfur-containing amino acids in the chemically defined medium.     

Structural and functional insights into CST tethering in Tetrahymena thermophila telomerase

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Small RNAs of Tetrahymena thermophila

Highly purified nuclear and cytoplasmic RNAs were obtained from Tetrahymena thermophila BVII containing only a minimal amount of cross-contamination. In the nuclear RNA fraction we have detected at least 6 distinct snRNAs. Some of the RNA species showed microheterogeneity. SnRNAs of Tetrahymena thermophila are very similar to rat snRNAs, as far as length is concerned. Our cytoplasmic small RNA fraction contained two RNAs, 7S and T7, reported recently (18) as nuclear, particularly nucleolar RNAs. Moreover, we could detect only one cytoplasmic small RNA species Tc1, Tc2 was not observed.Neither the nuclear nor the cytoplasmic small RNA species are degradation products of ribosomal RNA as was shown by Northern blotting and following hybridization with pGY17 containing the entire transcribed region of the ribosomal DNA of Tetrahymena thermophila.     

嗜热四膜虫——具有发展潜力的功能基因组学研究模型

在真核生物的分子生物学和遗传学研究方面,纤毛类原生动物嗜热四膜虫(Tetrahymenathermophila)已经被证明是一种有价值的生物学模型。通过对它的研究,人们发现并且掌握了核酶的分子机制、RNA的自我拼接、端粒的结构和功能、DNA序列重组等机理。这种原生动物的基因组功能分别由两个细胞核执行,即二倍体的小核与生殖过程有关,而多倍体的大核决定细胞的基因转录,并为转化基因的表达提供了强有力的手段。     

The Tetrahymena thermophila phagosome proteome

In vertebrates, phagocytosis occurs mainly in specialized cells of the immune system and serves as a primary defense against invading pathogens, but it also plays a role in clearing apoptotic cells and in tissue remodeling during development. In contrast, unicellular eukaryotes, such as the ciliate Tetrahymena thermophila, employ phagocytosis to ingest and degrade other microorganisms to meet their nutritional needs. To learn more about the protein components of the multistep process of phagocytosis, we carried out an analysis of the Tetrahymena phagosome proteome. Tetrahymena cells were fed polystyrene beads, which allowed for the efficient purification of phagosomes. The protein composition of purified phagosomes was then analyzed by multidimensional separation coupled with tandem mass spectrometry. A total of 453 peptides were identified that resulted in the identification of 73 putative phagosome proteins. Twenty-eight of the proteins have been implicated in phagocytosis in other organisms, indicating that key aspects of phagocytosis were conserved during evolution. Other identified proteins have not previously been associated with phagocytosis, including some of unknown function. Live-cell confocal fluorescence imaging of Tetrahymena strains expressing green fluorescent protein-tagged versions of four of the identified phagosome proteins provided evidence that at least three of the proteins (including two with unknown functions) are associated with phagosomes, indicating that the bulk of the proteins identified in the analyses are indeed phagosome associated.     

Unidirectional co-stimulation by a non-mating strain of Tetrahymena thermophila

We report that a fatty acid auxotroph of Tetrahymena thermophila (RH179E1) fails to mate, yet retains the ability to co-stimulate normal cells unidirectionally. Thus, co-stimulation can be analyzed experimentally in the absence of pair formation. We show that the co-stimulation of normal cells of one mating type is sufficient to shorten the waiting period for pair formation of those cells with initiated cells. This is the first evidence that co-stimulation causes a hyperinduction of mating reactivity in T. thermophila, generating in turn a positive feedback mechanism for (presumably) gamone production. Co-stimulation by the variant strain is at a maximum after 3-4 h of exposure when the variant and wild-type cells are at a ratio of 1:1. When mixed with wild-type cells, RH179E1 induces the formation of progeny (at low frequency) which inherit exclusively genetic material of the wild-type cells.     

Identifying functional regions of rRNA by insertion mutagenesis and complete gene replacement in Tetrahymena thermophila.

The free, linear macronuclear ribosomal RNA genes (rDNA) of Tetrahymena are derived from a unique copy of micronuclear rDNA during development. We have injected cloned copies of the micronuclear rDNA that have been altered in vitro into developing macronuclei and obtained transformants that express the paromomycin-resistant phenotype specified by the injected rDNA. In most cases, these transformants contain almost exclusively the injected rDNA which has been accurately processed into macronuclear rDNA. Mutants with a 119 bp insertion at three points in the transcribed spacers and at two points in the 26S rRNA coding region were tested. Cells containing these spacer mutant rDNAs are viable, although one of them grows slowly. This slow-growing line contains the insertion between the 5.8S and 26S rRNA coding regions and accumulates more rRNA processing intermediates than control lines. One of the 26S rRNA mutants failed to generate transformants, but the other did. These transformants grew normally, and produced 26S rRNA containing the inserted sequence. A longer insertion (2.3 kb) at the same four points either abolished transformation or generated transformants that retained at least some wild-type rDNA. This study reveals that some rRNA sequences can be altered without significantly affecting cell growth.     

Ciliary Membrane Proteins of Tetrahymena thermophila

SYNOPSIS SDS polyacrylamide gels of the ciliary membrane proteins of Tetrahymena thermophila revealed 5 major peaks and 11 minor protein peaks ranging in molecular weight from below 20,000 to above 250,000. The peaks resembled those found for ciliary membrane proteins of Paramecium aurelia. .     

Phagocytosis in Tetrahymena thermophila: naloxone-reversible inhibition by opiates

1. Nanomolar concentrations of opiates inhibit phagocytosis in the ciliated protozoan Tetrahymena thermophila. 2. Naloxone and naltrexone counteract the effect of the opiate agonists tested. 3. The dose-response curves are U-shaped, with no detectable effect at low or high concentrations. 4. An increase in extracellular calcium and dopamine counteract the inhibition caused by metenkephalin. 5. The recognition mechanism for opiates in Tetrahymena cannot be classified as belonging to any of the mammalian opiate receptor subtypes and is perhaps a primitive receptor.     

Secretion of Hexosaminidase Isozymes by Tetrahymena*

SYNOPSIS. Tetrahymena pyriformis strain HSM secrete large quantities of lysosomal acid hydrolases into the medium. The finding that 2 isozymes of β-N-acetylhexosaminidase (2-acetamido-2-deoxy-β-D-glucoside acetamidodeoxyglucohydrolase; EC 3.2.1.30) could be resolved by DEAE ion exchange chromatography and of possible differences between the secreted mixture and the intralysosomal hexosaminidase activity suggested that Tetrahymena might prove useful for studies of the control of lysosomal hydrolase isozyme secretion. In the present paper, we report a considerable purification of these isozymes and describe a number of their kinetic properties. Four isozymes were isolated into 2 major forms, A₁ and B₁, and 2 minor forms, A₂ and B₂, which were similar to the respective major forms in all kinetic properties tested. Hexosaminidase B₁ has a molecular weight of ?93,000 daltons and is inhibited by high concentrations of p-nitrophenyl-N-acetyl-β-D-glucosaminide. The inhibition is reversed by ethanol. Hexosaminidase A₁ has a molecular weight of ?170,000 and is not inhibited by high concentrations of substrate. The A forms are relatively less active against p-nitrophenyl-N-acetyl-β-D-galactosaminide than the B forms. Neither hexosaminidases A₁ or B₁ has any endo-β-N-acetylhexosaminidase activity. Comparison of the properties of the 2 major isozymes suggested that measurements of activity obtained under different assay conditions could be used to quantitate the amount of each isozyme in a mixture of the two. Log- and early stationary-phase cells secrete ?20% of isozyme A and 80% of isozyme B into the medium or into a dilute salt solution. With increasing culture age the fraction of isozyme A secreted rises to over 90%. Supplementation of the proteose-peptone growth medium with glucose causes a decrease in total hexosaminidase subsequently secreted but with no change in proportion of each isozyme. Cells suspended in a dilute salt solution containing 0.1 mM L-propranolol secrete slightly more isozyme A than do control cells suspended without L-propranolol. Phenoxy-benzamine (0.2 mM) causes a slight decrease in the proportion of isozyme A released.     

Secretion of hexosaminidase isozymes by Tetrahymena.

Tetrahymena pyriformis strain HSM secrete large quantities of lysosomal acid hydrolases into the medium. The finding that 2 isozymes of beta-N-acetylhexosaminidase (2-acetamido-2-deoxy-beta-D-glucoside acetamidodeoxyglucohydrolase; EC 3.2.1.30) could be resolved by DEAE ion exchange chromatography and of possible differences between the secreted mixture and the intralysosomal hexosaminidase activity suggested that Tetrahymena might prove useful for studies of the control of lysosomal hydrolase isozyme secretion. In the present paper, we report a considerable purification of these isozymes and describe a number of their kinetic properties. Four isozymes were isolated into 2 major forms, A1 and B1, and 2 minor forms, A2 and B2, which were similar to the respective major forms in all kinetic properties tested. Hexosaminidase B1 has a molecular weight of approximately 93,000 daltons and is inhibited by high concentrations of p-nitrophenyl-N-acetyl-beta-D-glucosaminide. The inhibition is reversed by ethanol. Hexosaminidase B1 has a molecular weight of approximately 93,000 daltons and is inhibited by high concentrations of p-nitrophenyl-N-acetyl-beta-D-glucosaminide. The inhibition is reversed by ethanol. Hexosaminidase A1 has a molecular weight of approximately 170,000 and is not inhibited by high concentrations of substrate. The A forms are relatively less active against p-nitrophenyl-N-acetyl-beta-D-galactosaminide than the B forms. Neither hexosaminidases A1 or B1 has any endo-beta-N-acetylhexosaminidase activity. Comparison of the properties of the 2 major isozymes suggested that measurements of activity obtained under different assay conditions could be used to quantitate the amount of each isozyme in a mixture of the two. Log- and early stationary-phase cells secrete approximately 20% of isozyme A and 80% of isozyme B into the medium or into a dilute salt solution. With increasing culture age the fraction of isozyme A secreted rises to over 90%. Supplementation of the proteose-peptone growth medium with glucose causes a decrease in total hexosaminidase subsequently secreted but with no change in proportion of each isozyme. Cells suspended in a dilute salt solution containing 0.1 mM L-propranolol secrete slightly more isozyme A than do control cells suspended without L-propranolol. Phenoxybenzamine (0.2 mM) causes a slight decrease in the proportion of isozyme A released.     

Cilia-Mediated Oriented Chemokinesis in Tetrahymena thermophila

The role of the cilia in the locomotion (“gliding”) of Tetrahymena thermophila in a semi-solid medium has been studied when cells were migrating in gradients of attractant. Video recordings and computer-aided motion analysis of migrating cells and their ciliary activity show that Tetrahymena thermophila migrate by swimming forward in semi-solid methyl cellulose, using their cilia. Ciliary reversals occur at certain intervals and cause a termination (“stop”) of cellular migration. Cells with reversed cilia resume forward migration when normal ciliary beating resumes. In gradients of attractants, cells migrating towards the attractant suppress ciliary reversals, which leads to longer runs between stops than in control cells. Cells migrating away from the attractant have a higher frequency of ciliary reversals than the control cells resulting in shorter runs. Stimulated cells adapt to a particular ambient concentration of attractant several times during migration in the gradient. Adaptation is followed by de-adaptation, which occurs during the “stop”. In the presence of cycloheximide, a strong inhibitor of chemoattraction, the attractant-induced suppression of ciliary reversal is abolished (cells become desensitized to the attractant). It is concluded that Tetrahymena has a short-term memory during adaptation. This is important for the efficiency of migration towards an attractant.     

Putrescine biosynthesis in Tetrahymena thermophila.

The putrescine-biosynthesis pathway in Tetrahymena thermophila was delineated by studying crude extracts prepared from exponentially growing cultures. A pyridoxal phosphate-stimulated ornithine decarboxylase activity competitively inhibited by putrescine was detected. CO2 was also liberated from L-arginine, but analyses by t.l.c. and enzyme studies suggested that the activity was not due to arginine decarboxylase, nor could enzyme activities converting agmatine into putrescine be detected. We conclude that the decarboxylation of L-ornithine is probably the only major route for putrescine biosynthesis in this organism during exponential growth.