Danièle Carré

Purification and properties of Escherichia coli CTP(ATP)-tRNA nucleotidyltransferase

Abstract 1. CTP(ATP)-tRNA nucleotidyltransferase has been purified 1000-fold from Escherichia coli. It appears homogeneous on analytical centrifugation and about 60% pure on disc electrophoresis. 2. The enzyme requires Mg2+ or Mn2+. 3. The molecular weight of the enzyme is 37000 and the sedimentation coefficient 2.9 S. 4. The Michaelis constants are 3.3 · 10−4 M and 1.7 · 10−5 M for ATP and CTP, respectively. 5. ATP is a non-competitive inhibitor of CMP incorporation into tRNA and CTP a non-competitive inhibitor of AMP incorporation. Ki for ATP is 3.7 · 10−4 M and for CTP 2.0 · 10−4 M.

Enzymes involved in DNA replication in the axolotl: I. Analysis of the forms and activities of DNA polymerase and DNA ligase during development☆

Abstract DNA polymerases and DNA ligases have been studied during development of the amphibian, axolotl. Three forms of DNA polymerase, I, II, and III, with sedimentation coefficients in sucrose of 9, 6, and 3.1 S, respectively, have been found in the axolotl egg. The activity of these three DNA polymerases is unchanged during early embryonic development. The activity of DNA polymerase III then increases significantly, beginning at the tailbud stage, while the activity of DNA polymerase II increases at the larval stage. DNA polymerase I does not show significant variations during this time. On the basis of their catalytic properties, it appears that DNA polymerases I and II are α-type DNA polymerases whereas DNA polymerase III is a β-type enzyme. Two different DNA ligases are found in the axolotl, one showing a sedimentation coefficient in sucrose of 8.2 S (heavy form) and the other, 6 S (light form). The 6 S enzyme is the major DNA ligase activity found in the egg before and after fertilization. Its activity then decreases during embryonic development. It can be observed again, as the only DNA ligase activity, in some adult tissues. The 8.2 S enzyme appears during the first division cycle of the fertilized egg, is present at all stages of embryonic development, and is absent from the adult tissues tested. Properties of the two DNA ligases at different stages of embryonic development have also been compared.

Etude du cnidome et de la cnidogenese chez Apolemia uvaria (lesueur, 1811) (Siphonophore physonecte)

Abstract The development of different types of cnidocystes in Apolemia uvaria (Siphonophore physonecte) has been observed in vivo, using a phase contrast microscope. The main steps of the morphological differentiation of birhopaloides, stenoteles, mastigophores and isorhizes are described. In all cases, the Golgi apparatus secretes a capsule and a long external tube at the anterior apex of this capsule. When the tube has reached a certain length, it invaginates in the capsule and becomes the definitive nematocystic filament. In all types of cnidocysts, the external tube, before its invagination, has the same shape as the invaginate tube. The constitution of cnidome of Apolemia uvaria , not well known up till now, is defined.

Study of the Escherichia coli tRNA nucleotidyltransferase: Effect of inorganic ions and thiol blocking reagents on enzyme activity

Abstract The effect of different divalent and monovalent cations on reactions catalyzed by the Escherichia coli tRNA nucleotidyltransferase has been examined. The highest rates of incorporation of AMP into tRNA—pX—C—C or tRNA—pX and of CMP into tRNA—pX are observed in the presence of Mg2+. Mn2+ is less efficient than Mg2+ for AMP incorporation; it is inefficient for CMP incorporation; in its presence UMP is incorporated instead of CMP. Co2+ shows only low efficiency in all cases examined. In the presence of optimal Mg2+ concentration, Mn2+ decreases the rate of incorporation of CMP and, to a lower extent, of AMP, but increases the rate of UMP incorporation. K+ at 0.1 M concentration stimulates CMP incorporation in both tRNA—pX and tRNA—pX—C whereas it has an inhibitory effect on AMP and UMP incorporation in all cases. The role of thiol groups on enzyme activity has been examined using —SH blocking reagents. 5,5′-Dithiobis(2-nitrobenzoic) acid or N-ethylmaleimide inhibit the incorporation of AMP into tRNA—pX—C—C but are without effect on the incorporation of CMP or UMP into tRNA—pX.

Study of the Escherichia coli tRNA nucleotidyltransferase. Interactions of the enzyme with tRNA.

Abstract A complex between Escherichia coli tRNA nucleotidyltransferase and tRNA has been isolated by centrifugation in a glycerol gradient (sedimentation constant 5.3 S). The complex is dissociated in the presence of ethylenediamine tetraacetate or high salt concentrations. The apparent K m of the enzyme for E. coli tRNA—pX—C—C in the reaction of incorporation of AMP is 1.5 · 10 −6 M; tRNA with an intact 3′ terminus is a competitive inhibitor of the tRNA substrate in this reaction, with a K i value of 0.9 · 10 −6 M. tRNA protects tRNA nucleotidyltransferase against thermal inactivation. The activation energy of the reaction of inactivation of tRNA-complexed tRNA nucleotidyltransferase is twice that of the free enzyme. The dissociation constant of the tRNA—tRNA nucleotidyltransferase complex deduced from the protection is 0.3 · 10 −6 M for tRNA—pX—C—C—A. Intercalation of ethidium bromide into tRNA molecules reduces the rate of the reaction catalyzed by tRNA nucleotidyltransferase but not the extent of the addition of nucleotides; the addition of this dye to the tRNA phosphate groups decreases both. Besides the synthesis of the 3′ terminal —pC—C—A sequence of tRNA molecules and of certain viral RNAs, the tRNA nucleotidyltransferase exhibits a low activity of incorporation of CMP into ribosomal RNA partially degraded by phosphodiesterase.

Study of the Escherichia coli tRNA nucleotidyltransferase. Specificity of the enzyme for nucleoside triphosphates

Summary Besides the main reactions leading to the repair of tRNA molecules deprived of part or all of their 3′ terminal -pCpCpA sequence, purified E. coli tRNA nucleotidyltransferase catalyzes in vitro , under certain conditions the synthesis of sequences not found in natural tRNAs. In the absence of CTP, AMP is incorporated directly into tRNA-pX or tRNA-pXpC leading to tRNA-pXpA or tRNA-pXpCpA respectively. In the absence of ATP one extra CMP is added to tRNA-pXpCpC to form tRNA-pXpCpCpC. UMP can be incorporated instead of CMP and the sequence -pXpU and -pXpCpU formed. The incorporation of UMP cannot be followed by the incorporation of either a second UMP or an AMP. In all cases, the rate of misincorporation is lower than the rate of the synthesis of the normal sequence. The apparent K M of the enzyme for UTP is 3.0 10 −4 M. CTP inhibits competitively the incorporation of UMP into tRNA-pX with a K i value (1.6 10 −5 M) close to its apparent K M .