Noboru Sasaki

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).

Immunochemical evidence for participation of NADPH-cytochrome c reductase in microsomal fatty acyl-CoA desaturation of Tetrahymena cells lacking in cytochrome P-450

Rabbit antibody was prepared against NADPH-cytochrome c reductase of Tetrahymena microsomes. When examined by the Ouchterlony double diffusion test, anti-NADPH-cytochrome c reductase immunoglobulin formed a single precipitation line with Tetrahymena reductase but not rat liver one. The antibody inhibited the NADPH-cytochrome c reductase activity of Tetrahymena microsomes, but it did not affect either NADH-ferricyanide or NADH-cytochrome c reductase activity of Tetrahymena microsomes. The NADPH-dependent desaturation of stearoyl-CoA in Tetrahymena microsomes was inhibited by anti-reductase immunoglobuline, while the NADH-dependent desaturation was affected by neither anti-reductase nor control immunoglobuline. It was suggested that the temperature associated-alteration of NADPH-cytochrome c reductase activities would be important for regulation of microsomal NADPH-dependent desaturase activities in Tetrahymena which contains no cytochrome P-450.

Changes in microsomal hemoprotein content and glucose 6-phosphatase activity during low-temperature acclimation of Tetrahymena pyriformis

To examine the temperture‐associated alterations in microsomal enzymes in an eukaryote, Tetrahymena cells were transferred from 39.5–15°C. The content of cytochrome b‐560ms, a cytochrome similar to but not identical with liver microsomal cytochrome b 5, was slightly increased within 2 h after temperature shift‐down, then gradually decreased, and reached the cytochrome b‐560ms level of the 15°C‐isothermally grown cells within 10 h following the temperature‐shift. Cytochrome P‐450 was not detected in Tetrahymena microsomes. The presence of a co‐binding hemoprotein with a main peak at 420 nm was, however, observed. The amount of this hemoprotein was unchanged for 2 h after cold‐shift. Later, it increased gradually and reached the maximum level at 10 h. Glucose 6‐phosphatase activity increased slightly at 10 h, when cell division had already been regained. We conclude that cytochrome b‐560ms, which is linked to the desaturase, may be essential for short‐term adaptation (2 h after shift), while the amount of co‐binding hemoprotein and the activity of glucose 6‐phosphatase would be associated with long‐term adaptation (10 h after temperature shift) in Tetrahymena cells.

Studies on Tetrahymena microsomal electron transport systems: Solubilization of microsomal electron transport enzymes involved in fatty acid desaturation

Tetrahymena microsomes were solubilized with five different detergents and the effect on electron transport enzymes involved in fatty acid desaturation was studied. Cytochrome b560ms and NADPH-cytochrome c reductase were solubilized with a low concentration detergent (0.25%), in the order of sodium deoxycholate greater than Renex 690 greater than Triton X-100 greater than octylglucoside greater than sodium cholate, whereas all of these detergents at the high concentration (1%) could solubilize preferentially both enzymes (70-100%). Increasing the concentration of various detergents from 0.5 to 1.0% did not produce an incremental change in NADH-ferricyanide reductase solubilization. NADH-cytochrome c reductase system, which would be catalyzed by the cooperation action of NADH-ferricyanide and cytochrome b560ms, was relatively inactivated by all detergents. Compared to the other four detergents, octylglucoside has a much higher recovery of stearoyl-CoA desaturase activities in the supernatant. Our study suggests that octylglucoside may be more useful for the isolation in active form of cyanide-sensitive factor (CSF) from Tetrahymena microsomes.

Effects of taurolipids on lysosomal enzymes in Tetrahymena

Abstract Effects of novel taurolipid A and B localized in Tetrahymena lysosomes on the activities of lysosomal enzymes purified from Tetrahymena were investigated. Both taurolipids activated acid phosphatase, while they did not affect α-glucosidase and β-hexosaminidase. The acid phosphatase activity was activated approximately 3-fold by both taurolipids A and B, with the half-maximum activations for taurolipid A and B being at approximately 1.03·10 −4 and 0.72·10 −4 M, respectively. When the purified acid phosphatase was incubated at 37°C in citrate-phosphate buffer (pH 5.0) its activity was rapidly inactivated, but the inactivation was prevented to a remarkable extent by the addition of taurolipids to the incubation medium. These results thus suggest that the taurolipids may be involved in activating and stabilizing acid phosphatase in Tetrahymena lysosomes.

Modification of microsomal lipid composition and electron transport enzyme activities in isovalerate-supplemented cells of novelTetrahymena ISO

Tetrahymena ISO cells, which have an unusually high level ofiso odd-numbered fatty acids, were grown medium supplemented with various concentrations of isovalerate. There was a marked increase in the total proportion ofiso odd-numbered fatty acids in supplemented whole cells (28.9→70.3%) and microsomes (37.7→84%), with a corresponding decrease in normal fatty acids, although no significant alteration of phospholipid composition was observe during 11 hr isovalerate-supplementation. Microsomal palmitoyl-CoA and stearoyl-CoA desaturase activities in isovalerate-supplemented cells decreased by 45.7% and 30.6% during 11 hr, respectively. NADH-cytochrome c reductase and NADH-ferricyanide reductase activities as well as the content of cytochrome b560ms, which is similar to mammalian microsomal cytochrome b5, were reduced in microsomes from 11 hr-supplemented cells, whereas NADPH-cytochrome c reductase activity was constant. It is suggested that the alteration of the cross-sectional area of lipid molecules in the bilayer, which results from the replacement of normal fatty acids withiso-15∶0 andiso-17∶1, would result in the decline of palmitoyl- and stearoyl-CoA desaturation in the isovalerate-supplemented cells, in order to maintain membrane fluidity at a functional level.

Properties of lysosomal β-hexosaminidase accumulated in Niemann-Pick mouse liver

Abstract Various lysosomal acid hydrolases from tissues of Niemann-Pick mice, a mutant strain of C57BL/KsJ mice spm/spm, were examined and compared to those from control mice. Activities of β-hexosaminidase, β-galactosidase, acid phosphatase, and cathepsin L were elevated in the liver and spleen of the affected mice, whereas no significant changes in β-glucosidase and acid α-glucosidase were observed. α-Mannosidase and neutral α-glucosidase activities were rather decreased in the affected mouse liver. The level of β-hexosaminidase in the Niemann-Pick mice was raised sixfold in the liver and two- to threefold in the spleen and brain, whereas its total activity was decreased in the kidney. Sixty to ninety percent of total activity of lysosomal hydrolases was solubilized with 0.1% Triton X-100 in control mice, but most of the β-hexosaminidase activity of the Niemann-Pick mice remained associated with the membrane fraction of liver lysosomes. The β-hexosaminidase of the Niemann-Pick mice was appreciably stable when heated at 55°C, while hydrolases of the affected mice and all of the enzymes tested in control mice were heat labile. The relative content of two β-hexosaminidase fractions separated by DEAE-cellulose column chromatography was 8% for β-hexosaminidase I and 92% for β-hexosaminidase II in the case of the control mouse liver. The isozyme pattern of hexosaminidases in Niemann-Pick mice was similar to that of control enzymes. However, the β-hexosaminidase II accumulated in Niemann-Pick mouse liver was different from that of the control in optimum pH, Km values and thermostability.

Effects of various inhibitors on desaturase and reductase enzyme activities in Tetrahymena microsomal electron transport system

Abstract 1. 1. The effects of several inhibitors on the Tetrahymena microsomal desaturase and cytochrome reductase activity were studied. 2. 2. Cyanide strongly inhibited Tetrahymena microsomal desaturation supported by either NADH or NADPH. Moreover, NADH- and NADPH-dependent desaturase activity was inhibited by cytochrome c and ferricyanide which are artificial electron acceptors. 2′-AMP, a competitive inhibitor versus NADPH, inhibited NADPH-dependent desaturation, while NADH-desaturase activity was not affected by this nucleotide. 3. 3. NADH-ferricyanide and NADPH-cytochrome c reductase activities as well as microsomal desaturase activity were sensitive to p -chloromercuribenzoate, mercuric chloride and N -ethylmaleimide respectively. This indicated that the sulfhydryl groups of NADH-ferricyanide reductase and NADPH-cytochrome c reductase are essential for the interaction of nucleotides with these flavoproteins in Tetrahymena microsomes.

Isolation of detergent-solubilized NADPH-cytochrome c reductase from Tetrahymena microsomes, with regard to thermoadaptive regulation of fatty acyl-CoA desaturase activity

Abstract 1. 1. A microsomal NADPH-cytochrome c reductase was purified from the protozoan Tetrahymena pyriformis by column chromatography on DEAE-cellulose, 2′,5′-ADP-Sepharose 4B, and Sephacryl S-200 in the presence of detergents. 2. 2. The purified reductase enzyme represented a 1000-fold purification from the microsomal-bound state. 3. 3. SDS-polyacrylamide gel electrophoresis revealed a single band of 70,000 mol. wt for the purified enzyme which contains FAD and FMN. 4. 4. The reductase was shown to reduce detergent-solubilized cytochrome b56ms from Tetrahymena microsomes, which is similar to mammalian cytochrome b5, in the presence of NADPH.

Species-specific antibodies of Tetrahymena acid α-glucosidase

Abstract 1. 1. Tetrahymena acid α-glucosidases A and B were purified from T. pyriformis W and T. thermophila 399, respectively. The acid α-glucosidases A and B were different in immunological properties and thermostability. 2. 2. The acid α-glucosidases A and B reflected specific distribution between T. pyriformis and T. thermophila. 3. 3. Type A and B of taurolipid showed a species-specific distribution pattern between T. pyriformis and T. thermophila.