Georges Sand

The dual genetic control of ornithine transcarbamylase synthesis in Escherichia coli K12

Mutations at 2 distant genes influence ornithine transcarbamylase synthesis in Escherichia coli K12. The wild type alleles of either of them restores to an OTC-less strain the ability to synthesize this enzyme under feedback control by arginine.

In vitro effects of thyroid hormones on red blood cell Ca++-dependent ATPase activity

Thyroid hormones (TH) have been shown to exert a direct stimulatory effect on the Ca++-dependent ATPase from human and other mammalian erythrdcytes. In this in vitro system, T4 has been shown to be more effective than T3. In the present study, TH effects on Ca++-dependent ATPase were investigated, using rabbit and human erythrocyte membranes, after preincubation with 10−10M T4, in the presence or in the absence of exogenous calmodulin (CaM) (5.10−12M to 5.10−9 M). Ca++-dependent ATPase activity was measured as inorganic phosphate (Pi) release from 1 mM ATP. The results showed that basal Ca++-dependent ATPase activity in rabbits was moderately increased by T4 (1.44 ± 0.05 vs 1.32 ± 0.04 μmol Pi/mg protein/90 min, mean ± SE; p<0.05). The time course of Pi release did not show any stimulatory effect of T4 during the first hour of incubation. The effect of T4 became apparent, however, 1 h after the addition of ATP (ΔT4: 15%). With human membranes, T4 induced a relative stimulation of the Ca++-dependent ATPase of 8–10% (p<0.05) in experimental conditions where the enzyme was not maximally stimulated by CaM (A CaM over basal activity: 5–40%). In conditions of high CaM stimulation (Δ CaM: 50–320%), T4 had no effect. These results confirm that Ca++-dependent ATPase activity is increased by T4. The effect of T4 is small, and appears as a late event during incubation with ATP. Stimulation by T4 is expressed in states of low enzyme activation by CaM. In conclusion our results do not favor a direct stimulation of Ca++-dependent ATPase by T4, but suggest an indirect effect on the red blood cell membrane.

Comparison of human thyroxine-binding globulin purification by affinity chromatography procedures

Three procedures for the isolation of thyroxine-binding globulin from human serum, using affinity chromatography on triiodothyronine (T3) linked to Sepharose (A), thyroxine (T4) linked to Sepharose (B) or T3 linked to epoxy-Sepharose (C) as the first purification step, were compared. With the use of additional purification steps, the three procedures yielded pure thyroxine-binding globulin without desialylation. With procedure A, the initial binding of T4-binding globulin to T3-Sepharose was very low, yielding a poor final recovery (17%). Procedure B gave the highest yield (35%) after a three-step purification, with a low T4 content (0.15-0.30 mol/mol). Procedure C also gave a high yield (28%) after only two purification steps, with a T4 content greater than 0.7 mol/mol. The microheterogeneity of T4-binding globulin obtained with these three procedures was demonstrated by isoelectric focusing: five major bands were observed between pH 4.1 and 4.6, and intermediate faint bands (often doublets) in the same pH range. However, with procedures A and C, the most acidic bands (pH 4.10-4.20) were always absent. Thyroxine-binding globulin was preincubated with radioactively labelled T3 or T4 and the hormone-protein complex was analyzed by isoelectric focusing. The binding of T3--compared to that of T4--was reduced in the most acidic protein subspecies. These results suggest differences in the thyroid hormone binding properties of the various subspecies of human T4-binding globulin.

Thyroid Hormone Stimulates Red Blood Cell Ca++-Dependent ATPase Activity Through a “Non-Hormonal” Effect

Thyroid hormones (TH) elicit a wide variety of metabolic and physiological responses in several tissues of higher organisms. The majority of these effects require the interaction of TH with protein receptors located at the cell nucleus. However, exranuclear actions of TH have elicited growing interest during the last decade. Red blood cells have been shown to possess plasma membrane-binding sites for both T3 and T4. Using this as a cellular model, a direct stimulation of erythrocyte membrane Ca++-dependent ATPase (Ca++-ATPase) by physiological concentrations of TH has recently been demonstrated (1,2). Among TH analogues, T4 exerts the highest stimulatory effect on basal enzyme activity which is not dependent upon prior conversion to T3 (3). This effect of T4 requires the mediation of calmodulin (CaM), an ubiquitous cytosolic protein which plays a major role in the regulation of Ca++-ATPase (4).