A new fiber-forming protein from Tetrahymena pyriformis

A new fiber-forming protein was isolated from the acetone powder of Tetrahymena pyriformis by co-precipitating with skeletal muscle myosin while trials were made to find actin or actin-like protein in Tetrahymena. It has a molecular weight of 38000 D and forms a tetramer (140000 D, 9 S) in physiological conditions. Its isoelectric point (pH 6.7), amino acid composition and antigenic determinant(s) differ significantly from those of non-muscle actin and skeletal muscle actin. It does not undergo G-F conversion while actin does, and does not activate Mg²⁺-ATPase of skeletal muscle myosin. The protein localizes in the oral apparatus and division furrow as revealed by fluorescent antibody method. The protein can be assembled into 14-nm filaments in a reassembly buffer. The in vitro filaments appear to correspond to some filaments included in the oral apparatus and the contractile ring. The fiber-forming protein from Tetrahymena may play important roles in cell motility including cell division.     

Isolation of nucleoli from Tetrahymena pyriformis.

A procedure is described to isolate nucleoli from Tetrahymena pyriformis which contain extrachromosomal ribosomal DNA. Macronuclei isolated by the Nonidet procedure were sonicated at a reduced magnesium concentration, and the sonicate was fractionated by isopycnic centrifugation in a metrizamide density gradient. The heaviest band, designated Band IIb, contains exclusively ribosomal DNA, thus constituting the nucleolar fraction. The purity of the nucleolar fraction on a DNA basis, which is defined as the percentage of ribosomal DNA and determined by equilibrium centrifugation in a CsCl density gradient, was around 70%. Electron microscopic examination revealed that the isolated nucleoli retained fairly well the ultrastructure of the in situ nucleoli. Some of the biochemical properties of the isolated nucleoli are also presented.     

A CO-Binding Hemoprotein Derived from Tetrahymena pyriformis

A CO-binding hemoprotein was purified from Tetrahymena pyriformis and some of its properties were studied.

The hemoprotein possessed protoheme, its molecular weight was about 11,000, and its isoelectric point was at pH 8.2. The oxidized form of the hemoprotein showed the Soret band at 406 nm and had no distinct peaks in the region of α- and β-bands, while the reduced form showed the peaks at 426, 527 and 560 nm. The hemoprotein reacted with CO resulting in shift of the Soret band from 426 to 420 nm. The CO-compound showed a broad band from 537 to 573 nm. The hemoprotein was not autoxidizable or oxygenated either. It did not show either of the cytochrome oxidase, peroxidase and NADH oxidase activities.

It should be carefully determined whether or not cytochrome o is functioning as the terminal oxidase in T. pyriformis, as the CO-binding hemoprotein which does not react with molecular oxygen exists in the organism.     

Dolichyl phosphate phosphatase from Tetrahymena pyriformis.

A soluble dolichyl phosphate phosphatase from Tetrahymena pyriformis was purified about 68-fold. The enzyme appeared to be specific for dolichyl phosphate and existed in two interrelated forms, one of mol.wt. about 500000 and the other of mol.wt. about 63000. The enzyme was strongly inhibited by 5 mM-Mn2+ and was strongly stimulated by Mg2+. Tetrahymena in the exponential growth phase contained more of this enzymic activity than cells in stationary or lag phase. The dolichyl phosphate phosphatase may be loosely bound to mitochondrial membranes. Two roles proposed for this enzyme are (1) that of releasing dolichol from its phosphorylated biosynthetic form for its use in the cell as unesterified dolichol or dolichyl ester and/or (2) that of regulation of synthesis of glycoproteins or some other glycosylated compound.     

Characterization of polyriboadenylate polymerase from Tetrahymena pyriformis.

Poly(A) polymerase [polyadenylate nucleotidyltransferase, EC 2.7.7.19] was extracted from Tetrahymena pyriformis. The enzyme was demonstrated to be present in three forms by column chromatography on DEAE-cellulose, and they were termed poly(A) polymerase Ia, Ib, and II in order of increasing affinity to the column. The properties of enzymes Ia and Ib were similar except that Ia utilizes poly(A) as a primer rather efficiently. Enzyme II differed from enzymes Ia and Ib not only in elution profile on DEAE-cellulose column chromatography but also in pH and temperature preferences, molecular weight, requirement for divalent cations, sensitivity to salts at high ionic strength, optimal primer concentration, and subcellular localization. The molecular weights of enzymes Ia and Ib measured by gel filtration were both 43,000, and that of enzyme II was 95,000. All three enzymes required Mn2+ for maximal activity; Mg2+ could replace Mn2+ in the reaction of enzyme II, but only partially. In the presence of 0.1 M ammonium sulfate the activities of enzymes Ia and Ib were both completely inhibited, whereas enzyme II still showed 42% of its original activity. These findings suggest that there are two distinct types of poly(A) polymerase in Tetrahymena pyriformis.     

Isolation and characterization of dolichols from Tetrahymena pyriformis

Dolichols of Tetrahymena pyriformis were isolated and characterized by TLC, HPLC and mass spectrometry. Four strains of Tetrahymena were studied and found to have relatively small amounts of dolichol, from 0.26 to 2.60 mg dolichol/kg wet weight. All four strains had approximately the same relative proportions of isoprenologs, dolichol-13 (2%), dolichol-14 (74%), dolichol-15 (23%), and dolichol-16 (less than 1%). Tetrahymena dolichols were found mainly in the mitochondrial subcellular fraction (86%). The pellicle fraction contained 9% and the microsomal fraction, 5% of the remaining dolichol. Free dolichol has also been found in the mitochondrial fraction of four other organisms. We were not able to demonstrate dolichyl esters in these organisms, but their presence is inferred, because reduced yields of dolichol were obtained if the lipid extracts were not saponified prior to HPLC assay.     

Macrophage Activation by Tetrahymena pyriformis II. Active Protein Fractions from Tetrahymena

ABSTRACT. Water-soluble and 0.6 M KCl-soluble protein fractions prepared from Tetrahymena pyriformis , when inoculated into mice, could effectively induce activated macrophages having the ability to kill Toxoplasma gondii in vitro. This effect was not induced by other proteins tested, such as bovine serum albumin, pepsin from porcine stomach mucosa and chicken egg-white lysozyme, nor by muramyl dipeptide (MDP), a potent immunoadjuvant. Five fractions obtained by DEAE-Sephadex chromatography of the water-soluble protein fraction were compared with regard to induction of toxoplasmacidal activity in macrophages. The first peak was most effective for activation of macrophages. Five fractions obtained by chromatography of the 0.6 M KCl-soluble protein fraction were also examined and it was found that the first peak had the activity. No marked difference in activity was observed between the active fractions of water-soluble and 0.6 M KCl-soluble protein fractions. For practical use, we focused on the water-soluble active fraction. The minimum effective dose of the active fraction was 100 μg and the fraction could activate macrophages directly in vitro. Four fractions obtained by gel filtration of the active fraction on Sephadex G-200 were compared and the first peak had the activity. The first peak contained a single protein, revealed by SDS-polyacrylamide gel electrophoresis; its apparent molecular weight was 64,000.     

Isocitrate dehydrogenase of Tetrahymena pyriformis

Summary We have studied the isocitrate dehydrogenase ofTetrahymena pyriformis. This enzyme is able to utilize both NAD and NADP, but kinetic studies suggest that the enzymatic activity with NAD is not of physiological significance.Some of the factors that might regulate the NADP-dependent isocitrate dehydrogenase were also studied. This enzyme has an absolute requirement for divalent cations; Mg²⁺ and Mn²⁺ will serve as cofactors but the latter is more effective than the former.It is known that this enzyme is subject to a concerted inhibition by oxaloacetate and glyoxylate. Either glyoxylate or oxaloacetate alone also are capable of inhibiting the enzyme although higher concentrations are required. We have found concerted inhibition also for the NAD-dependent isocitrate dehydrogenase from rat liver and yeast. The activity of theTetrahymena pyriformis enzyme is inhibited by NADPH. This inhibition is competitive with NADP. The Ki and Km values are, respectively, 23µ m and 18µ m.