Pierre Jouan

Stimulation by estradiol-17β of thymidine kinase activity in the rat uterus

Abstract The action of estradiol-17β(E 2 ) on thymidine kinase (TK) activity was studied in uteri from immature female rats. It was demonstrated that a single injection of E 2 highly stimulated the enzyme activity which reached its maximum level 24 h after hormone administration. Physiological amounts of E 2 were efficient and changes in TK activity were observed exclusively in uterus and liver. A single injection of Tamoxifen produced the same effect as E 2 but repeated administration resulted in the complete inhibition of enzyme activity. Using antibiotics it was demonstrated that E 2 induced the synthesis of new enzyme molecules rather than an increase in enzyme activity. This statement was corroborated by the fact that after hormone administration the increase in TK activity was preceded by an increase in RNA-polymerase activity and followed by that in DNA-polymerase α activity. Moreover, the separation of TK isoenzymes on DEAE-Sephadex and the use of d -CTP as inhibitor of the adult isozyme suggested that E 2 induced the “fetal” form of the enzyme. In addition, it was demonstrated that TK activity in uteri from ovariectomized adult female rats was enhanced by E 2 administration, and that the increase was due to the stimulation of the fetal isoenzyme. It was suggested that TK could be used as a marker of the action of estrogens and antiestrogens in target organs.

A Rapid Two-Step Purification of Rat Liver Fetal Thymidine Kinase

The fetal isoenzyme of thymidine kinase was purified to apparent homogeneity from cytosols of rat fetuses liver. A two-step purification including anion exchange chromatography and affinity chromatography was developed. The purified enzyme appears as oligomeric with a relative molecular weight of 71 kDa. In denaturing media its molecular weight was 24 kDa, and its pHi 8.3.

A SPECIFIC TESTOSTERONE RECEPTOR IN THE CYTOSOL OF RAT ANTERIOR HYPOPHYSIS

A study was designed to measure the specificity of the testosterone binding in the cytosol of the anterior pituitary of immature male rats (40 day old rats). The binding at equilibrium was studied after cytosol incubation at 0°C with a constant dose of [3H]-testosterone at various times. The binding was measured after gel filtration on Sephadex G 25 medium. Equilibrium was reached at 5 h and remained constant during 24 h. No significant testosterone metabolism was detected. The binding specificity was determined using different concentrations of [3H]-testosterone. Cytosols were incubated for 5 h at 0°C with concentrations of [3H]-testosterone ranging from 10-9 M to 10-6 M. The protein-testosterone complex was isolated by gel filtration on Sephadex G 25 medium. A Scatchard plot of the binding data produced a curved line showing that more than one binding component was involved. This curve was resolved into two linear components by the Rosenthal method. The dissociation constant (KD) for the specific binding was 2.3 × 10-9 M and the estimated amount of specific receptor was 8 × 10-11 M. The specific binding capacity was estimated to be 2 × 10-14 moles per mg of protein.