Tankyrase Activity in Organs and Tissues of Mice.

Tankyrase, one of the NAD+ ADP-ribosyltransferases, is a target for drugs developed for their anticancer and other pharmacological activities. We designed an assay for estimation of the inhibition or activation of the enzyme in preclinical studies. In mice, the highest specific activity of tankyrase was observed in thymus, spleen, pancreas, and bone marrow. In murine liver, tankyrase is active in ontogenesis and during reparative regeneration; however, the basal activity is hardly detectable in normal liver and most of other organs of adult animals. We suggest that tankyrase is a part of the tissue growth and repair machinery, while its age-dependent inhibition, when an organism stops growing, turns on phenoptosis.

Interaction of afobazole with sigma1-receptors.

In vitro radioligand assay revealed interaction of afobazole with sigma(1)-receptors (Ki=5.9x10(-6) M). Translocation of sigma(1)-receptors from the endoplasmic reticulum to the outer membrane was demonstrated by confocal microscopy. Experiments were performed on the model of HT-22 immortalized hippocampal cells after incubation with afobazole in a concentration of 10(-8) M.

Isolation and physicochemical properties of tankyrase of human embryonic kidney cells of line 293.

We have isolated and purified endogenous cytosolic tankyrase from human embryonic kidney cells of line 293. Our data confirm a model of De Rycker and Price who consider that tankyrase is a master scaffolding protein capable of regulating assembly of large protein complexes. We have also studied kinetic characteristics of tankyrase in the complex, pH dependence of the enzyme activity, and its physicochemical properties.

Polypeptide components of telomere nucleoprotein complex.

Chromosome telomeres of humans and many model organisms contain a structure called a t-loop, which is maintained by TERF, TINF2, Pot1, and other proteins. Increase in TERF1 concentration prevents telomere elongation by telomerase. Decrease in TERF2 concentration (preventing t-loop formation) is accompanied by blockade of proliferation and appearance of other signs of cellular senescence in experiments. Natural regulation of TERF1 involves tankyrase, ATM protein kinase, and fluctuations of the protein level across a cell cycle. The telomere nucleoprotein complex also interacts with various polypeptide macromolecules (e.g., Sir2, PinX1, Rap1, Ku, Rad50/Mre11/Nbs1) responsible for heterochromatin formation, modulation of telomerase activity, DNA repair, and signaling to other cell compartments about telomere state. Study of structure and functioning of telomere nucleoprotein complex may contribute to elucidation of poorly understood mechanisms of aging and processes of tumor transformation of cells.

Soluble tankyrase located in cytosol of human embryonic kidney cell line 293.

We studied the subcellular localization of tankyrase in primary and immortalized human cell cultures. In embryonic kidney cell line 293 the enzyme was excluded from the nuclei and distributed in fractions of soluble cytosolic proteins and low-density microsomes. Newly revealed cytosolic tankyrase in its poly(ADP-ribosyl)ated form was passed through a Sepharose 2B column and eluted as an apparently monomeric protein. The cytosolic localization of the enzyme correlated with its relatively high activity in the 293 cell line in comparison to eight other studied cell types.

Cloning and characterization of TNKL, a member of tankyrase gene family.

By serological screening of a breast tumor cDNA library we have identified a novel human gene, tnkl, encoding an ankyrin-related protein with a high degree of similarity to tankyrase, the poly(ADP-ribose)polymerase associated with human telomeres (Smith et al, Science 282: 1484). The tnkl gene maps to chromosome 10, while the tnks gene encoding tankyrase is located on chromosome 8. The predicted 1166-aa protein product of the tnkl gene is 78% identical to human tankyrase and 62% to a putative D. melanogaster protein. Since the proteins have essentially identical domain structures, the corresponding genes form a distinct gene family. The possible link between TNKL and cancer justifies its further functional analysis.

[Crystallization of three multiple forms of transketolase from baker's yeast]. / Kristallizatsiia trekh mnozhestvennykh form transketolazy iz pekarskikhdrozhzhei.

Three forms of baker's yeast transketolase have been revealed. These forms differed in thermal stability and elution profiles during chromatography on a phosphocellulose column and migrated with identical rates during electrophoresis in the presence of sodium dodecyl sulfate. The same forms in yeast, pig and rat liver and in different organs and tissues of the rabbit were found to be similar in their thermal stability and chromatographic properties. The relative amounts of the forms appeared to depend on the physiological state of the organism. Crystals of the three pure forms were grown using ammonium sulfate as the precipitating agent. These crystals differed morphologically and by stability upon storage. The possibility of interconversion of the transketolase forms is discussed.

Morphological differences in crystals of multiple forms of yeast transketolase.

Monocrystals of three individual multiple forms of yeast transketolase (A, B and C) differing in their thermostability have been obtained. Ammonium sulfate was used as a precipitating agent. Crystals of the mentioned forms were found to possess different morphology and stability during storage. Single crystals growing from the enzyme form C within 4-7 days were subsequently destroyed. Simultaneously, in the preparation, microcrystals started to grow in a great number. They were found to correspond morphologically to crystals obtained from transketolase A. A possibility of interconversions of the enzyme forms in sequence C----A----B is discussed.

Inhibition of transketolase by N-acetylimidazole.

Transketolase from baker's yeast is rapidly inactivated in the presence of N-acetylimidazole. According to kinetic data, acetylation of one amino acid residue of the protein per active site is sufficient for TK* inactivation. The holoenzyme is inhibited more slowly than is apotransketolase. The presence of a tyrosine residue in the enzyme's active site, essential for activity, is suggested.

Conversion of Woodward's Reagent K in an aqueous medium: mathematical analysis applied to enzyme modification.

A change in the optical density of Woodward's Reagent K solution at 340 nm has been shown. It is observed after the reagent has been dissolved in a weakly acidic medium. The optical density correlates with the reagent's ability to inhibit transketolase. A method for assay of the inhibitor concentration changes in the medium during enzyme modification is suggested.

[Functional carboxylic group in the active center of transketolase]. / Funktsional'naia karboksil'naia gruppa v aktivnom tsentre transketolazy.

Baker's yeast transketolase is rapidly inactivated in the presence of carboxylic group modifiers, i.e., 1-ethyl-3(3'-dimethylaminopropyl)-carbodiimide or Woodward's reagent K. This inactivation is due to modification of the carboxylic group in the enzyme active center. The essential groups localized in the two active centers of transketolase differ in the rate of modification; accordingly, the inactivation kinetics appears as biphasic. A complete loss of the enzyme activity occurs as a result of modification of one carboxylic group per enzyme active center. The pKa value of modifiable groups is equal to about 6.5. This modification decreases by two orders of magnitude the affinity of the substrate for the active center. The carboxylic groups are not directly involved in the interaction with the substrates; their modification does not significantly affect the coenzyme binding. It is supposed that these groups are responsible for the deprotonation of the second carbon in the thiamine pyrophosphate thiazolium ring.

An investigation of the carboxyl group function in the active center of transketolase.

Transketolase from baker's yeast is rapidly inactivated in the presence of 1-ethyl-3 (3'-dimethylaminopropyl)-carbodiimide. pKa of the modified carboxyl groups is approximately 6.5. An investigation of the initial steps of enzymatic catalysis monitored by a changes in the circular dichroism spectra and in an oxidation reaction with ferricyanide made it possible to conclude that the modification interferes with the donor substrate attachment to the enzyme. Evidence obtained was suggesting that the carboxyl group of the active center facilitates dissociation of a proton from the carbon atom in the second position of the thiamine pyrophosphate thiazolium ring.

The carboxyl group in the active center of transketolase.

Transketolase from baker's yeast is rapidly inactivated in the presence of 1-ethyl-3 (3'-dimethylaminopropyl)-carbodiimide or Woodward's reagent K. In both cases the kinetics of inactivation is biphasic, which agrees with the presence of two active centers in the enzyme molecule differing in their sensitivity to the inhibitors. There is some evidence that inactivation of transketolase is due to modification of carboxyl groups of enzyme. Complete inactivation is achieved by modification of one carboxyl per active site of the enzyme. The experimental results suggest that the carboxyl group is essential for the enzymatic activity of transketolase.