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.     

Purification and characterization of an extracellular ATPase from oviductal secretions.

Oviductal secretions include an ATPase (EC 3.6.1.3) that is transferred from the outer surface of the secretory cells to the surface of the ovulated oocyte. The enzyme has been purified and is a highly labile, very high molecular weight lipoprotein complex (greater than 4-10(6)). It consists of 47% protein and 53% lipid. Lipid composition is limited to phosphatidylcholine, phosphatidylethanolamine and sphingomyelin. The basic protein subunit has a molecular weight of 170 000. The enzyme exhibits many of the characteristics of ectoenzyme ATPase. The enzyme is Mg2+ or Ca2+ dependent; the Mg2+-ATPase has pH optima at 6.0 and 7.8 and the Ca2+-ATPase at 9.0. Substrate specificity is limited to ATP with lesser activity towards GTP, CTP, UPT and ADP. Km for ATP is 0.88 mM and the enzyme is inhibited at substrate concentrations greater than 3 mM ATP.     

Intracellular phospholipase activities of Tetrahymena pyriformis

Phospholipase activity was studied in the protozoan Tetrahymena pyriformis NT-1 by using exogenous phosphatidylethanolamine and phosphatidylcholine. Several phospholipase activities were found in Tetrahymena homogenates. They were distinguished with respect to pH optimum, activity dependence on Ca2+, substrate specificity and positional specificity. Ca2+-Dependent phospholipase activity had an optimal pH around 9 and gave rise to free fatty acid and lysophospholipid. This enzyme hydrolyzes phosphatidylethanolamine but not phosphatidylcholine. The alkaline phospholipase with A1 activity was located mainly in the surface membrane (pellicle fraction). The enzyme activity had a pH optimum ranging from 8 to 9, and required 2 mM CaCl2 for the maximal activity. All detergents tested inhibited the enzyme activity. Ca2+-Independent phospholipase activity had an optimal pH from 4 to 5 and gave rise to free fatty acid, lysophospholipid, diacylglycerol, and monoacylglycerol. We concluded that there are at least three phospholipase in Tetrahymena homogenates, i.e., alkaline phospholipase A and acidic phospholipases A and C.     

Ca2+-dependent modulator proteins from Tetrahymena pyriformis, sea anemone, and scallop and guanylate cyclase activation

Previously, the guanylate cyclase activity of Tetrahymena pyriformis was shown to be activated by an endogenous modulator (calmodulin)-like protein (Na-gao, S., Suzuki, Y., Watanabe, Y., and Nozawa, Y. (1979) Biochem. Biophys. Res. Commun. 90, 261-268). This protein has now been identified as the modulator protein. The identification was based on the capability of this protein to activate the brain modulator-deficient phosphodiesterase and the mobility of this protein upon polyacrylamide gel electrophoresis. The activation of guanylate cyclase was specifically attributable to the Tetrahymena modulator protein since other modulator proteins examined (bovine brain, sea anemone, and scallop) were ineffective. Under the conditions where the activation of Tetrahymena guanylate cyclase occurred, guanylate cyclase activities from other sources, that include rat brain, rat lung, and human platelet, were not affected. In the phosphodiesterase activation, the potencies of scallop and Tetrahymena modulator proteins, which are represented by reciprocals of the quantities of proteins required for half-maximal activation of enzyme, were 66% and 55%, respectively, of that of the brain protein. The same decreasing order was seen for the affinity of these proteins for Ca2+ in enzyme activation. The results suggest a directional change of the modulator protein during the molecular evolution toward an increase in the capability in Ca2+-dependent enzyme activation.     

Identification and purification of a soluble adenosine triphosphatase from Tetrahymena pyriformis .

An unusual ATPase isolated from the postribosomal supernatant fraction of $\text{Tetrahymena pyriformis}$ has been purified to homogeneity. The purification procedure consisted of protamine sulfate and heat treatment; column chromatography successively on phosphocellulose, DEAE-cellulose and Sephadex G-150; and isoelectric focusing. The pure enzyme has a molecular weight of 89,000 and requires either Ca²⁺ or Ba²⁺ for maximum activation. Nucleoside triphosphates are hydrolized at decreasing rates in the order: ATP > GTP > ITP > CTP > UTP. The K_m for ATP is 2.5 mM. Because of its properties the enzyme is tentatively classified as a soluble Ca²⁺-activated ATPase.     

Anandamide metabolism by Tetrahymena pyriformis in vitro. Characterization and identification of a 66 kDa fatty acid amidohydrolase

Fatty acid amidohydrolase, a membrane-bound enzyme found in a variety of mammalian cells, is responsible for the catabolism of neuromodulatory fatty acid amides, including anandamide. In an earlier study we reported that Tetrahymena pyriformis was able to secrete a FAAH-like activity in starvation medium (Karava V., Fasia L., Siafaka-Kapadai A., FEBS Lett. 508 (2001) 327-331). In this study the endocannabinoid anandamide, was found to be metabolized by T. pyriformis homogenate by the action of a FAAH-like enzyme, in a time- and concentration-dependent manner. The main metabolic products of [3H]anandamide hydrolysis were [3H]arachidonic acid and ethanolamine. Amidohydrolase activity was maximal at pH 9-10, it was inhibited by phenylmethylsulfonyl fluoride and arachidonyltrifluoromethyl ketone and was Ca2+ and Mg(2+)-independent. Kinetic experiments demonstrated that the enzyme had an apparent K(m) of 2.5 microM and V(max) of 20.6 nmol/min mg. Subcellular fractionation of T. pyriformis homogenate showed that the activity was present in every subcellular fraction with highest specific activity in the microsomal as well as in non-microsomal membrane fraction. Immunoblot analysis of selected subcellular fractions, using an anti-FAAH polyclonal antibody, revealed the presence of an immunoreactive protein with a molecular mass approximately 66 kDa similar to the molecular mass of the mammalian enzyme. In conclusion, this study demonstrates that a FAAH similar to the mammalian enzyme is present in a unicellular eukaryote, indicating the importance of FAAH activity throughout evolution. It also supports the notion that Tetrahymena species may be a suitable model for metabolic studies on endocannabinoids, as well as for the study of drugs targeted towards FAAH.     

Purification and characterization of DNA-dependent ATP phosphohydrolases from KB cells

DNA-dependent ATPases have been purified from logarithmically growing KB cells by chromatography on single-stranded DNA cellulose and phosphocellulose. Phosphocellulose resolved the DNA-dependent ATPases into three activities designated ATPase I, II and III, respectively. From gel filtration and sedimentation analysis ATPases II and III were found to be very similar, both with calculated molecular weights of 78,000. Due to the extreme lability these enzymes were not purified further. The molecular weight of ATPase I determined by gel filtration and sedimentation analysis was calculated to be 140,000. ATPase I was further purified by gradient elution on ATP-agarose, revealing two peaks of activity (IA and IB), and by sucrose gradient sedimentation. Analysis of the fractions from the sucrose gradient by sodium dodecylsulphate gel electrophoresis revealed only one broad polypeptide band co-sedimenting with both ATPase IA and ATPase IB. This band was composed of four closely spaced polypeptides with apparent molecular weights of 66,000, 68,000, 70,000 and 71,000. Comparison of the native molecule weight (140,000) with these results suggests that ATPase I is a dimer. ATPase IA and IB were indistinguishable in their structural and enzymatic properties and presumably represent the same enzyme. The purified enzyme has an apparent Km of 0.5 mM for ATP producing ADP + Pi. A maximum activity of 2,100 molecules of ATP hydrolyzed per enzyme molecular per minute was found. Hydrolysis of ATP requires the presence of divalent cations (Mg2+ greater than Ca2+ greater than Mn2+ greater than Co2+). A broad pH optimum (pH 6--8) was observed. The enzyme uses ATP or dATP preferentially as a substrate, while other deoxyribonucleoside or ribonucleoside triphosphates were inactive. ATPase I prefers denatured DNA as cofactor. The activity with native DNA is 40% of that with denatured DNA.     

The mitochondrial adenosine triphosphatase of Acanthamoeba castellanii. Partial characterization and changes in activity during exponential growth

1. The mitochondrial adenosine triphosphatase (ATPase) of Acanthamoeba castellanii is Mg2+-requiring (optimum cation: ATP ratio of 1.5) and has two pH optima of activity (at pH 6.6 and 8.1). 2. ATPase activity of submitochondrial particles is effectively inhibited by twelve different inhibitors of energy conservation suggesting similarities in inhibitor-binding sites to other previously characterized complexes. 3. Gel filtration by passage through Sephadex G-50 increases ATPase activity of submitochondrial particles between 1.5 and 3.5 fold indicating the presence of a low molecular weight inhibitor protein. 4. After removal of the inhibitor protein, sensitivity to inhibitors of energy conservation decreases by between 1.5 and 14 fold. Crude F1-inhibitor preparations from A. castellanii, Schizosaccharomyces pombe, Tetrahymena pyriformis and bovine heart also inhibit ATPase activity. 5. Large variations in ATPase activity, F1-inhibitor protein activity, and amounts of immunologically-determined ATPase protein were observed during exponential growth, and the correlation between changes in these measurements is discussed. 6. The results are also discussed highlighting the similarities between the mitochondrial ATPase of A. castellanii and other mitochondrial ATPases.     

Functional characteristics of calcium-sensitive adenylyl cyclase of ciliate Tetrahymena pyriformis

Calcium-sensitive forms of adenylyl cyclase (AC) were revealed in most vertebrates and invertebrates and also in some unicellular organisms, in particular ciliates. We have shown for the first time that calcium cations influence the AC activity of ciliate Tetrahymena pyriformis. These cations at the concentrations of 0.2-20 microM stimulated the enzyme activity, and maximum of catalytic effect was observed at 2 microM Ca2+. Calcium cations at a concentrations of 100 microM or higher inhibited the AC activity. Calmodulin antagonists W-5 and W-7 at the concentrations of 20-100 microM inhibited the catalytic effect induced by 5 microM Ca2+ and blocked the effect at higher concentrations of Ca2+. Chloropromazine, another calmodulin antagonist, reduced Ca2+-stimulated AC activity only at the concentrations of 200-1000 microM. AC stimulating effects of serotonin, EGF and cAMP increased in the presence of 5 microM Ca2+. AC stimulating effects of EGF, cAMP and insulin decreased in the presence of 100 microM Ca2+, and AC stimulating effect of cAMP decreased also in the presence of calmodulin antagonists (1 mM). At the same time, stimulating effect of D-glucose in the presence of Ca2+ and calmodulin antagonists did not change essentially. The data obtained speak in favor of the presence of calcium-sensitive forms of AC in ciliate T. pyriformis which mediate enzyme stimulation by EGF, cAMP, insulin, and serotonin.     

Structural and functional properties of a Ca2+-ATPase from human platelets

An antibody prepared against highly purified rabbit muscle Ca2+-ATPase from sarcoplasmic reticulum has been observed to cross-react with proteins in human platelet membrane vesicles. The antibody specifically precipitated Ca2+-ATPase activity from solubilized human platelet membranes and recognized two platelet polypeptides denatured in sodium dodecyl sulfate with Mr = 107,000 and 101,000. Ca2+-ATPase activity from Brij 78-solubilized platelet membranes was purified up to 10-fold. The purified preparation consisted mainly of two polypeptides with Mr approximately 100,000, and 40,000. The lower molecular weight protein appeared unrelated to Ca2+-ATPase activity. The Ca2+-ATPase in human platelet membrane vesicles exhibited "negative cooperativity" with respect to the kinetics of ATP hydrolysis. The apparent Km for Ca2+ activation of ATPase activity was 0.1 microM. Ca2+-dependent phosphorylation of platelet vesicles by [gamma-32P]ATP at 0 degrees C yielded a maximum of 0.2-0.4 nmol of PO4/mg of protein that was labile at pH 7.0 and 20 degrees C. This result suggests that only about 2-4% of the total protein in platelet membrane vesicles is the Ca2+-ATPase, which agrees with an estimate based on the specific activity of the Ca2+-ATPase in platelet membranes (20-50 nmol of ATP hydrolyzed/min/mg of protein at 30 degrees C). Calmodulin resulted in only a 1.6-fold stimulation of Ca2+-ATPase activity even after extensive washing of membranes with a calcium chelator or chlorpromazine. It is concluded that human platelets contain a Ca2+-ATPase immunochemically related to the Ca2+ pump from rabbit sarcoplasmic reticulum and that the enzymatic characteristics and molecular weight of the platelet ATPase are quite similar to those of the muscle ATPase.     

The plasma membrane Ca2+-ATPase protein from red blood cells is not modified in preeclampsia

Plasma membrane Ca2+-ATPase activity diminishes by about 50% in red blood cells during preeclampsia. We investigated whether the number of Ca2+-ATPase molecules is modified in red cell membranes from preeclamptic pregnant women by measuring the specific phosphorylated intermediate of this enzyme. Also, we isolated the Ca2+-ATPase protein from both normotensive and preeclamptic pregnant women and estimated its molecular weight, and its cross-reactions with specific polyclonal and monoclonal (5F10) antibodies against it. We measured the Ca2+-ATPase activity in a purified state and the effect of known modulators of this ATPase. It was found that the phosphorylated intermediate associated with PMCA is similar for red cell ghosts from normotensive and preeclamptic women, suggesting a similar number of ATPase molecules in these membranes. The molecular weight of the Ca2+-ATPase is around 140 kDa for both normotensive and preeclamptic membranes, and its cross-reactions with specific antibodies is similar, suggesting that the protein structure remains intact in preeclampsia. Calmodulin, ethanol, or both calmodulin plus ethanol, stimulated the Ca2+-ATPase activity to the same extent for both normotensive and preeclamptic preparations. Our results showed that the reduced Ca2+-ATPase activity of the red cell membranes from preeclamptic women is not associated with a defective enzyme, but rather with a high level of lipid peroxidation.     

Presence of two DNA polymerases in Tetrahymena pyriformis.

Two DNA polymerases were detected in Tetrahymena pyriformis, strain GL. One (enzyme I) was sensitive to N-ethylmaleimide, while the other (enzyme II) was insensitive. The molecular weight of the enzymes, as determined by glycerol gradient centrifugation analysis, were approximately 130,000 and 70,000, respectively. Optimal concentration of MgCl2 was 10mM for enzyme I and 18mM for enzyme II. KCl inhibited enzyme I but stimulated enzyme II. Poly (dA-dT) served effectively as a template for enzyme I, while poly(dA).(dT)12-18 was an effective template for enzyme II. Enzyme I activity increased with cell growth and sharply declined after the cells reached the stationary phase. On the other hand, enzyme II activity appeared only at the end of log phase. In cells synchronized by starvation-refeeding technique enzyme I was markedly stimulated in correspondence to the rate of DNA synthesis, whereas the level of enzyme II activity changed to lesser extent. By ethidium bromide treatment, only enzyme I activity was induced.     

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.     

Studies on cyclic nucleotide metabolism in Tetrahymena pyriformis: partial characterization of cyclic AMP- and cyclic GMP-dependent phosphodiesterases

Cyclic nucleotide phosphodiesterase [EC 3.1.4.17] was examined in tetrahymena pyriformis strain NT-1. Enzymic activity was associated with the soluble and the particulate fractions, whereas most of the cyclic GMP phosphodiesterase activity was localized in the soluble fraction; the activities were optimal at pH 8.0-9.0. Although very low activities were detected in the absence of divalent cations, they were significantly increased by the addition of either Mg2+ or Mn2+. A kinetic analysis of the properties of the enzymes yielded 2 apparent K(m) values ranging in concentration from 0.5 to 50 micron and from 0.1 to 62 micron for cyclic AMP and GMP, respectively. A Ca2+ -dependent activating factor for cyclic nucleotide phosphodiesterase was extracted from Tetrahymena cells, but this factor did not stimulate guanylate cyclase [EC 4.6.1.2] activity in this organism. On the other hand, tetrahymena also contained a protein activator which stimulated guanylate cyclase in the presence of Ca2+, although this activator did not stimulate the phosphodiesterase. The results suggested that Tetrahymena might contain 2 types of Ca2+ -dependent activators, one specific for phosphodiesterase and the other for guanylate cyclase.     

Purification and some properties of a new Ca2+-binding protein (TCBP-10) present in tetrahymena cilium

A new Ca2+-binding protein, different from calmodulin, has been detected in the cilium and cell body of Tetrahymena. This protein, designated as TCBP-10, has been purified from the cells to homogeneity. TCBP-10 is an acidic protein (pI = 4.5) which shows a Ca2+-dependent mobility shift in alkali-glycerol-polyacrylamide gel electrophoresis. The protein is resistant to heat and trichloroacetic acid. The molecular weight of the protein is 10,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 22,000 by Sephadex G-50 gel filtration, suggesting that the native form of the protein is a dimer. The protein has a molar extinction coefficient of 6,500 at 282 nm. Equilibrium dialysis experiments revealed that the protein binds 1 mol of Ca2+/mol of protein with a dissociation constant of 27 microM. The protein contains a relatively large quantity of acidic amino acids, single residues of cysteine, histidine, and tryptophan, and no methionine. These properties are similar to those of some low molecular weight Ca2+-binding proteins belonging to the calmodulin family. Thus, the cilium of Tetrahymena contains a second Ca2+-binding protein in addition to calmodulin. We consider that TCBP-10 and calmodulin may play important cooperative roles in the Ca2+-regulation of ciliary movement in Tetrahymena.     

ATPase activity and Ca2+ transport by reconstituted tryptic fragments of the Ca2+ pump of the erythrocyte plasma membrane

The purified Ca2+ ATPase of the erythrocyte plasma membrane has been submitted to controlled trypsin proteolysis under conditions that favor either its (putative) E1 or E2 configurations. The former configuration has been forced by treating the enzyme with Ca2+-saturated calmodulin, the latter with vanadate and Mg2+. The E1 conformation leads to the accumulation of a polypeptide of Mr 85 KDa which still binds calmodulin, the E2 conformation to the accumulation of one of Mr 81 KDa which does not. Both fragments arise from the hydrolysis of a transient 90 KDa product which has Ca2+-calmodulin dependent ATPase activity, and which retains the ability to pump Ca2+ in reconstituted liposomes. Highly enriched preparations of the 85 and 81 KDa fragments have been obtained and reconstituted into liposomes. The former has limited ATPase and Ca2+ transport ability and is not stimulated by calmodulin. The latter has much higher ATPase and Ca2+ transport activity. It is proposed that the Ca2+ pumping ATPase of erythrocytes plasma membrane contains a 9 KDa domain which is essential for the interaction of the enzyme with calmodulin and for the full expression of the hydrolytic and transport activity. This putative 9 KDa sequence contains a 4 KDa "inhibitory" domain which limits the activity of the ATPase. In the presence of this 4 KDa sequence, i.e., when the enzyme is degraded to the 85 KDa product, calmodulin can still be bound, but no longer stimulates ATPase and Ca2+ transport.     

The influence of the EGF on proliferative signal transduction in ciliate Tetrahymena pyriformis

It has been shown that the transduction of the proliferative signal induced by EGF in ciliata Tetrahymena pyriformis cells is not connected with autophosphorylation of the receptor tyrosine kinases. The results obtained indicate that EGF in ciliata cells initiates the mitogenic pathway including the membrane proteins of the tyrosine kinases-like type (without tyrosine phosphorylation sites), adenylate cyclase, tyrosine- and Ca2+ -dependent ERK-like kinases.     

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.     

Purification and properties of a vanadate- and N-ethylmaleimide-sensitive ATPase from chromaffin granule membranes

A vanadate- and N-ethylmaleimide-sensitive ATPase was purified about 500-fold from chromaffin granule membranes. The purified preparation contained a single major polypeptide with an apparent molecular mass of about 115 kDa, which was copurified with the ATPase activity. Immunological studies revealed that this polypeptide has no relation to subunit I (115 kDa) of the H+-ATPase from chromaffin granules. The ATPase activity of the enzyme is inhibited about 50% by 100 microM N-ethylmaleimide or 5 microM vanadate. The enzyme is not sensitive to dicyclohexylcarbodiimide, ouabain, SCH28080, and omeprazole, which distinguishes it from Na+/K+-ATPase and the gastric K+/H+-ATPase. ATP and 2-deoxy ATP are equally effective substrates for the enzyme. However, the enzyme exhibited only 10% activity with GTP as a substrate. UV illumination of the purified enzyme in the presence of [alpha-32P]ATP exclusively labeled the 115 kDa protein. This labeling was increased by Mg2+ and strongly inhibited by Ca2+ ions. Similarly, the ATPase activity was dependent on Mg2+ and inhibited by the presence of Ca2+ ions. The ATPase activity of the enzyme was largely insensitive to monovalent anions and cations, except for F-, which inhibited the vanadate-sensitive ATPase. Incubation of the enzyme in the presence of [14C]N-ethylmaleimide labeled the 115-kDa polypeptide, and this labeling could be prevented by the addition of ATP during the incubation. A reciprocal experiment showed that preincubation with N-ethylmaleimide inhibited the labeling of the 115-kDa polypeptide by [alpha-32P]ATP by UV illumination. This suggests a close proximity between the ATP-binding site and an essential sulfhydryl group. A possible connection between the isolated ATPase and organelle movement is discussed.     

Poly(A) synthesis in T2L phage-infected Escherichia coli. A combination of polynucleotide phosphorylase and ATPase.

In crude extracts of T2L phage-infected Escherichia coli cells an enzyme activity was found that produced poly(A) from ATP as substrate. Purification of the extract led to the isolation of two enzymes, a polynucleotide phosphorylase and an ATPase. The polynucleotide phosphorylase possessed the same properties as the well-known enzyme from uninfected cells and its molecular weight was about 265 000. The ATPase was purified to over 90% purity; its molecular weight was estimated to be about 165 000 with three subunits of 55 000. The characterization of this enzyme showed that it was different from any ATPase known so far. Mg2+ cannot be replaced by Ca2+, as it can from the membrane-bound ATPases. The only product yielded by the enzyme was ADP; it was very specific for ATP, other ribonucleotide triphosphates being practically unaffected. The rate of ATP splitting was found to be very high, the turnover number being 2.51 X 10(4) min-1 at 37 degrees C. Even at 0 degree C the enzyme was still active. The optimal assay conditions for ATPase turned out to be very similar to those of polynucleotide phosphorylase. Thus the combination of the two enzymes very efficiently produced poly(A) from ATP. In this combination the polynucleotide phosphorylase was the rate-limiting enzyme, since its turnover number was about 40 times lower than that of the ATPase. The evaluation of a variety of properties of the poly(A)-synthesizing constituent found in the crude extracts led us to conclude that this activity arises from the combined action of ATPase and polynucleotide phosphorylase, and is not due to a poly(A) polymerase.