Acid hydrolases and their Release in Food Vacuole-Less Mutants of Tetrahymena thermophila*

SYNOPSIS. Mutants (NP1 and PSJ5) of Tetrahymena thermophila strains B and D 1968 exist that are unable to construct a functional oral apparatus and form food vacuoles at 37 C but which do so normally at 30 C. Food vacuole-less cells starved in dilute salt solution released similar amounts of acid phosphatase, β-N-acetyl-glucosaminidase and ±-glucosidase activity into the medium as wildtype cells during an 8-h period. Actively growing, food vacuole-less cells had ?50% less total protein, acid phosphatase, β-N-acetyl-glucosamin-idase, and ±-glucosidase per cell than wildtype cells after 72-h growth. During this time food vacuole-less cells released significant amounts of the 3 acid hydrolases into the growth medium. For each hydrolase, the total activity released from growing, food vacuole-less cells was less, on a per cell basis, than the amount released from food vacuole formers. The proportion of the total activity secreted by the mutant and the wildtype cells was the same for acid phosphatase and β-N-acetyl-glucosaminidase and somewhat lower for ±-glucosidase. It is concluded that the release of a significant amount of acid hydrolase activity from Tetrahymena is independent of food vacuole formation and may be analogous to the secretory activity of other nonphagocytic eukaryotic cells.     

Purification and characterization of lysosomal alpha-glucosidase secreted by eukaryote Tetrahymena

A large amount of lysosomal acid hydrolases was released into the medium by Tetrahymena pyriformis strain W during growth. An extracellular lysosomal acid alpha-glucosidase has been purified 500-fold with a 41% yield to homogeneity, as judged by polyacrylamide gel electrophoresis. It was found to be a glycoprotein and to consist of a single 110,000-dalton polypeptide chain. The carbohydrate content of the alpha-glucosidase was equivalent to 2.8% of the total protein content, and the oligosaccharide moiety was composed of mannose and N-acetylglucosamine in a molar ratio of 6.7:2. The optimal pHs for hydrolysis of maltose and p-nitrophenyl-alpha-glucopyranoside, maltose, isomaltose, and glycogen were 1.1 mM, 2.5 mM, 33.0 mM, and 18.5 mg/ml, respectively. This purified enzyme appears to have alpha-1,6-glucosidase as well as alpha-1,4-glucosidase activity. Turanose has a noncompetitive inhibitory effect on the hydrolysis of maltose. The antibody raised against Tetrahymena acid alpha-glucosidase inhibited the hydrolysis of all substrates tested. These properties of Tetrahymena acid alpha-glucosidase were found to be similar to those of the human liver lysosomal alpha-glucosidase.     

Mutants of Tetrahymena thermophila with temperature sensitive food vacuole formation : II. Physiological and morphological studies

We studied 13 independently isolated, nitrosoguanidine-induced mutants of Tetrahymena thermophila having heat-sensitive capacity for food vacuole formation. Mutants belonging to the vacA complementation group have defects in the development of a new oral apparatus (OA) and not in phagocytosis per se; OAs formed at 30 °C are functional for at least three cell generations after transfer to 39 °C, while OAs formed at 39 °C are non-functional with regard to phagocytosis. Morphologically, the mutant OAs appear normal under light microscopy. One mutant forms a functional OA and normal contractile vacuole pores (CVP) at 22 °C, but after transfer to 30 °C these organelles disintegrate and vacuole-less cells swell without dividing. Finally, one mutant may be defective in phagocytosis; all daughters formed during the first three cell generations after transfer from 22 °C to 30 °C make food vacuoles, but at a rate which decreases with time. After 24 h at 30 °C food vacuole formation is practically stopped. With one exception, all mutants grow with wild-type generation times at temperatures restrictive for food vacuole formation in growth medium supplemented with high concentrations of iron, copper and folinic acid. The results indicate the feasibility of using a mutant approach to help dissect the developmental mechanisms responsible for the construction of the OA, and to determine the route of entry for compounds required for cell multiplication.     

The relationship between energy-dependent phagocytosis and the rate of oxygen consumption in Tetrahymena.

The induction of high rates of food vacuole formation in Tetrahymena pyriformis increased the rate of respiration in exponentially growing cells by 17% and in starving cells by 47.5%. The increased rate of oxygen uptake was caused by phagocytosis itself, as shown by comparing the rates of respiration of a Tetrahymena mutant exposed to particles at the permissive or restrictive temperatures for food vacuole formation. During cell division, heat-synchronized cells in rich, particle-supplemented medium showed a significant decrease in the rate of respiration. Furthermore, dimethyl sulphoxide, in concentrations sufficient to block food vacuole formation, suppressed the rate of respiration to a level similar to that of starved cells. Cytochalasin B, fowever, did not reduce the rate of oxygen uptake despite the inability of the cells to complete the formation of food vacuoles during treatment; a possible explanation for this finding is discussed. There was a strong correlation between formation of food vacuoles and a high metabolic rate in Tetrahymena.     

Secretion heterogeneity of lysosomal enzymes in Tetrahymena pyriformis

A number of lysosomal enzymes are secreted from Tetrahymena pyriformis during growth and during starvation. The secretion is energy-dependent and kinetically different among hydrolases. On the basis of the secretion kinetics under starvation conditions, Tetrahymena hydrolases can be separated into three classes. The first group containing acid phosphatase, beta-glucosidase and alpha-galactosidase, are secreted slowly. Within this group about 4% of the initial cellular activity is released per hour. The second group of enzymes, including alpha-glucosidase, alpha-mannosidase and beta-galactosidase, exhibit moderate secretion (11-15% of the initial cellular activity per hour). The third group, N-acetyl-beta-hexosaminidase, has the highest rate of secretion (22% of the initial cellular activity per hour). N-Acetyl-beta-hexosaminidase shows a continuous increase in overall activity during starvation, which is completely blocked by adding cycloheximide; its secretion is also suppressed. Such involvement of enzyme biosynthesis was not seen in the first and second groups. Furthermore, treatment with weak bases caused inhibited secretion of differing degree among acid phosphatase (group I), alpha-glucosidase (group II) and N-acetyl-beta-hexosaminidase (group III).     

Dual capacity for nutrient uptake in Tetrahymena. II. Role of the two systems in vitamin uptake.

Previously, we found that a mutant strain of Tetrahymena pyriformis without food vacuoles failed to grow unless the nutrient media were richly supplemented with vitamins and trace metals. Here we show that calcium folinate alone can replace the extra vitamin supplementation. The mutant requires approximately 90-fold higher concentration of folinate than the wild-type cells to give similar growth responses in a chemically defined medium. We infer that the food vacuole is an important route of uptake for this vitamin in the wild-type cells. We found no difference between mutant and wild-type cells in their requirements for nicotinic acid, pantothenic acid, riboflavin-monophosphate, and pyridoxal. We infer that an extravacuolar route contributes importantly to uptake of these 4 compounds.     

Dual Capacity for Nutrient Uptake in Tetrahymena. II. Role of the Two Systems in Vitamin Uptake

SYNOPSIS Previously, we found that a mutant strain of Tetrahymena pyriformis without food vacuoles failed to grow unless the nutrient media were richly supplemented with vitamins and trace metals. Here we show that calcium folinate alone can replace the extra vitamin supplementation. The mutant requires ∼ 90-fold higher concentrations of folinate than the wild-type cells to give similar growth responses in a chemically defined medium. We infer that the food vacuole is an important route of uptake for this vitamin in the wild-type cells. We found no difference between mutant and wild-type cells in their requirements for nicotinic acid, pantothenic acid, riboflavin-monophosphate, and pyridoxal. We infer that an extravacuolar route contributes importantly to uptake of these 4 compounds.     

The effect of concanavalin A on egestion of food vacuoles in Tetrahymena

The ability of concanavalin A (conA) to disrupt food vacuole elimination at the cytoproct of Tetrahymena pyriformis, strain GL-C, was investigated using fluorescence microscopy and thin section electron microscopy. ConA was found to induce "tails" in Tetrahymena. These tails were specifically stained by fluorescent conA. Thin section observations of conA-treated cells revealed that these tails were the result of abnormal egestion of food vacuole contents at the cytoproct. Tail formation appears to result from an inhibition of endocytosis of food vacuole membrane during egestion. Instead, the food vacuole membrane appears to be cast out of the cell, along with the contents of the vacuole. The mechanism of this inhibition may be related to an apparent absence of microtubules or microfilamentous mat in the cytoproct region of conA-treated cells. Although conA is ingested into food vacuoles in large amounts, conA appears to affect endocytosis only from outside the cell; ingested conA does not appear to be effective. ConA may exert its influence by binding to the cytoproct region. The ability of conA to induce tail formation is inhibited by sugars specific to it. Numerous membranous vesicles are found in association with the oral cilia and cytoproct region of conA-treated cells. These vesicles may be the conA-binding material reported to be secreted by Tetrahymena.     

Effects of metabolites present during growth of Tetrahymena pyriformis on the subsequent secretion of lysosomal hydrolases

Tetrahymena were grown in proteose-peptone medium supplemented with glucose, mannose, fructose, galactose, acetate, succinate, or pyruvate and then washed and resuspended in a non-nutrient salt solution and the amounts of 7 acid hydrolases secreted into the medium in a one hour incubation were measured. Cells that had been grown in the presence of glucose secreted about half the amounts of acid phosphatase, β-N-acetylglucosaminidase and acid protease as did control cells grown in unsupplemented medium. Pyruvate was about as effective as glucose and both were slightly more effective than acetate or fructose. Succinate had little effect. Similar experiments showed that α-mannosidase, β-fucosidase, and β-galactosidase are secreted into the salt solution and that secretion is reduced by prior growth of the cells in medium supplemented with glucose or mannose but not galactose. Except for α-mannosidase, these reductions in amounts of hydrolase secreted were not accompanied by appreciable changes in intracellular activity, and therefore demonstrate a persistent effect of growth in the presence of certain metabolites on the subsequent secretion of lysosomal hydrolases. Since the inhibition of subsequent secretion depended on both the individual metabolite and the particular hydrolase examined, it appears that the effect of metabolites is not limited to a general inhibition of secretion but may differentially alter some properties of lysosomal subpopulations. A preliminary characterization of the secreted acid protease of Tetrahymena suggests that there may be two acid proteases released, since up to 25% of the activity was not inhibited by high concentrations of pepstatin, leupeptin, or chymostatin.     

Pleiotropic Effects of a Temperature-Sensitive Mouthless Mutation in Tetrahymena thermophila on the Excretion of Extracellular Enzymes

The mode of enzyme excretion was investigated during balanced growth in wild type Tetrahymena thermophila and in a temperature-sensitive mutant that did not form a mouth and food vacuoles at the restrictive temperature. The mutant was used to determine which of the extracellular enzymes are normally excreted by defecation. During balanced growth at the permissive temperature the excretion of enzymes was constant, and maximal in complex medium. No protease activity against azocasein was detected. Changes in the growth temperature of the wild type cells only affected the release of 3′-nucleotidase. For the mutant, however, the excretion changed markedly with increasing temperature: (a) ribonuclease, deoxyribonuclease, α-glucosidase, and β-glucosidase were detected in 0–10% of normal amounts; (b) 3′- and 5′-nucleotidase, not measured previously in Tetrahymena, were found in 10- to 14-fold of normal amounts; (c) excretion of acid phosphatase was unaffected. We therefore conclude that the extracellular enzymes (a) are released by defecation, that 3′- and 5′-nucleotidase are secreted through the membrane system, and that acid phosphatase is released by lysosomes emptied through pinocytosis. It is proposed that the lysosomes used for the formation of digestive vacuoles are different from those used for the formation of pinocytotic vacuoles and for autophagic vacuoles.     

Control of phosphonic acid and phosphonolipid synthesis in Tetrahymena.

The phosphonolipid content of the protozoan Tetrahymena pyriformis was increased by growing the organism on a medium containing increasing amounts of 2-aminoethylphosphonic acid. With levels of 0, 1, 5 and 10 mM 2-amino-ethylphosphonic acid, the phosphonolipid content was 23, 25, 31 and 37% of the total cellular phospholipids, respectively. This increase was accompanied by a reciprocal decrease in phosphatidylethanolamine. With 32Pi in the growth medium along with the 2-aminoethylphosphonic acid, the incorporation of the radioactivity into new molecules of 2-aminoethylphosphonic acid was almost totally inhibited, indicating a feedback control on phosphonic acid synthesis.     

Regio- and stereo-specific nitrile hydrolysis by the nitrile hydratase from Rhodococcus AJ270

Abstract Acid phosphatase activity was measured in individual cells by determining their optical densities through a scanning confocal laser microscope. The naphthol AS-TR (3-hydroxy-2-naphtoic acid 4'-chloro-2'-methylanilide) phosphate-hexazotized para-rosanilin method was used to visualise the acid phosphatase content in the light microscope. Evidence was obtained that the amount of enzyme varied in exponential growth phase cells as the fission age increased. By comparing the acid phosphatase activity with the rate of food vacuole formation, it appeared that the amount of enzyme inside the cells decreased in early clonal life, whereas the rate of food uptake increased. It was assumed that the reduction of acid phosphatase content could lead to a more extended life of vacuoles and to a decreased membrane recycling rate. In turn, the reduced supply of membrane available for food vacuole formation could partly be responsible for the decrease of the food uptake rate observed after the initial increase.     

Formation and fate of extranuclear chromatin bodies in Tetrahymena.

The fate of extranuclear chromatin bodies (ECBs) formed by exclusion of macronuclear material at the time of karyokinesis was followed quantitatively in Tetrahymena pyriformis strain GL-I. In a logarithmic growth phase culture, 51% of the dividing cells produced one (43%) or more (8%) ECBs. Most of these gradually disappear before the next cell division, but approximately 13% are retained and carried into subsequent cell cycles. The random distribution of ECBs into anterior or posterior daughter cells, their staining and morphological characteristics, and their rapid loss in cells in starvation medium, all indicate that ECBs play no more of a role in cellular activity than that of an internally produced food vacuole.     

Secretory hydrolases of Entamoeba histolytica

Cells of Entamoeba histolytica grown over a period of four days contained NADP+-dependent alcohol dehydrogenase exclusively inside the cells. No activity of this enzyme could be found in the growth medium after harvesting the cells. Under the same conditions, acid phosphatase, beta-N-acetylglucosaminidase, esterase, alpha-glucosidase, and different amylases of the parasite were found both inside the cells and in the medium. The activities present in the cell homogenate and in the medium before and after growth of the amoebas were partially separated by gel filtration on Sephadex G150 and G75, respectively. The comparison of the elution diagrams revealed that NADP+-dependent alcohol dehydrogenase, acid phosphatase, esterase, and amylases occurred as multiple forms inside the cells. These activities, as well as beta-N-acetylglucosaminidase and alpha-glucosidase, were released into the extracellular environment to a different degree. The enzymes originating from the parasite were identified and distinguished from those of the ingredients of the growth medium according to their molecular mass and pH optimum. Furthermore, the amoebic origin of the secreted enzymes was shown on the basis of their inhibition by antibodies prepared against the supernatant fraction of the homogenate.     

Formation and Fate of Extranuclear Chromatin Bodies in Tetrahymena

The fate of extranuclear chromatin bodies (ECBs) formed by exclusion of macronuclear material at the time of karyokinesis was followed quantitatively in Tetrahymena pyriformis strain GL-I. In a logarithmic growth phase culture, 51% of the dividing cells produced one (43%) or more (8%) ECBs. Most of these gradually disappear before the next cell division, but ? 13% are retained and carried into subsequent cell cycles. The random distribution of ECBs into anterior or posterior daughter cells, their staining and morphological characteristics, and their rapid loss in cells in starvation medium, all indicate that ECBs play no more of a role in cellular activity than that of an internally produced food vacuole.     

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.     

Partial purification and characterization of a bacteriolytic enzyme secreted by Tetrahymena.

Tetrahymena pyriformis strain HSM secretes large quantities of acid hydrolases into the culture medium. An enzyme secreted by the ciliate and capable of degrading walls of streptococci was identified and purified to a considerable degree. The pH optimum of this enzyme was 3--4, and it was eluted after cytochrome c from Sephadex G-75 columns. Unlike lysozyme, the enzyme was thermolabile at pH 2.9, but relatively thermostable at pH 8.1. It degraded 14C-labeled cell walls of streptococci releasing reducing groups. Cell walls prepared from different strains of streptococci differed in susceptibility to this enzyme, the most sensitive strain tested being of group A, type T12. It was shown in immunologic studies that this hydrolase released the group-specific carbohydrate from the walls. Secretions of Tetrahymena from early stationary-phase cultures had more bacteriolytic activity than those from cells from late stationary-phase cultures. Further, cells from cultures grown in glucose-supplemented medium secreted less of the enzyme than ciliates of comparable age grown in unsupplemented proteose-peptone. The newly isolated bacteriolytic enzyme, presumably of lysosomal origin, may be helpful in characterizing streptococcal cell walls.     

Differential increase in activity of acid phosphatase induced by phosphate starvation in Tetrahymena.

We have studied the effects of phosphate starvation on the levels and distributions of activities of acid phosphatase and beta-hexosaminidase in cultures of Tetrahymena thermophila. The cells were grown in synthetic nutrient medium and refed every day with fresh medium. After 4 days of growth in the complete medium, the cultures were divided into two portions. One received complete medium and the other phosphate-free, but otherwise complete, medium. Population densities and activities of acid phosphatase and beta-hexosaminidase in cells plus medium and in cell-free samples were determined in aliquots removed every day before medium replacement. In cultures having complete medium the enzyme levels remained fairly constant; in the phosphate-starved cultures both total and extracellular activities of acid phosphatase increased sixfold. beta-Hexosaminidase levels remained essentially unaltered in both cases. These results indicate that phosphate starvation can induce differential increase in acid phosphatase activity in cultures of Tetrahymena. Somewhat less than 50% of the total activities of both enzymes are found in the cell-free extracellular fluid at any time.     

The Tetrahymena homolog of bacterial and mammalian 4-hydroxyphenylpyruvate dioxygenases localizes to membranes of the endoplasmic reticulum

The expression and intracellular localization of the Tetrahymena homolog of 4-hydroxyphenylpyruvate dioxygenase (HPPD) were investigated in wild-type Tetrahymena thermophila strain B1868 VII and the mutant strains IIG8, defective in food vacuole formation, MS-1, blocked in secretion of lysosomal enzymes, and SB 281, defective in mucocyst maturation. Immunoelectron microscopy and confocal laser scanning microscopy demonstrated that Tetrahymena HPPD primarily localized to membranes of the endoplasmic reticulum. In addition, Tetrahymena HPPD was detected in association with membranes of the Golgi apparatus, and transport vesicles in exponentially growing wild-type and mutant strains. In starved cells, Tetrahymena HPPD localized exclusively to membranes of small vesicles. Since no de novo synthesis of Tetrahymena HPPD takes place in cells starved for more than 30 min, these results suggest that there is a flow of Tetrahymena HPPD from the endoplasmic reticulum to small vesicles, possibly via the Golgi apparatus, and that Tetrahymena HPPD contains a signal for vesicle membrane retrieval or retention.     

Endocytosis and the adaptive acid hydrolases in Tetrahymena pyriformis GL

Endocytosis of yeast cells by Tetrahymena pyriformis GL for a period of 2.5 h produced changes in cellular acid hydrolases. Acid phosphatase, acid deoxyribonuclease and acid proteinase activities were markedly increased, whereas there was a decrease in acid ribonuclease activity and little change in -glucosidase activity. These alterations do not appear to be due to any alteration in the rates of secretion of these enzymes into the milieu. Evidence is presented that the cellular enzyme increases found upon endocytosis of yeast reflect changes in lysosomal enzymes, because it was shown that the acid phosphatase activity increase resulted in an increased amount of latent enzyme within the cell. The results also support the idea that there are at least 3 distinct populations of lysosomes, in addition to phagolysosomes, present in Tetrahymena pyriformis GL, with different modes of formation. There appears to be a large excess of lysosomes, uncombined with phagosomes, present in these fed cells since latency averaged 66% in broken-cell preparations which contained very few intact phagolysosomes. The phagolysosomal acid phophatase activity cannot account for more than 34% of that present in the cell. The endocytosis of yeast in the presence of growth medium resulted in a marked drop in the rate of cell division as compared to cells growing in the growth medium alone. The results are discussed.