No abstract

Inositol 1,4,5-trisphosphate (InsP3) 3-kinase catalyses the ATP-dependent phosphorylation of InsP3 to inositol 1,3,4,5-tetrakisphosphate (InsP4). A method is presented for the rapid purification of InsP3 3-kinase from bovine brain by calmodulin (CaM)-Sepharose affinity chromatography. Maximal activation of the purified InsP3 3-kinase by Ca2+/CaM was 6-7-fold as compared with the activity measured in the presence of EGTA (1 mM) and 10 microM-InsP3. At 10 microM-InsP3 and 0.1 mM free Ca2+, half-maximal activation required about 2 nM-CaM. The mechanism of activation by CaM appeared to be an increase in the maximal velocity of the enzyme without a substantial change in the Km for InsP3. Further purification was achieved by phosphocellulose chromatography eluted with ATP. Specific activity of the purified enzyme at 37 degrees C and 10 microM-InsP3 was 10-20 mumol/min per mg. The apparent Mr of the enzyme, determined by f.p.l.c.-gel filtration, was estimated as about 44,000. The purified InsP3 3-kinase was subjected to SDS/10%-polyacrylamide-gel electrophoresis. InsP3 3-kinase activity was associated with three silver-stained bands, which migrated with apparent Mr values of approx. 52,000, 38,000 and 35,000.

Primary cultures of dog thyroid cells have been used to study the effects of thyrotropin on the synthesis of proteins. The cells were cultured for 4 days in serum-free and thyrotropin-free conditions. Thyrotropin was then added for varying periods of time (6-96 h). In the absence of thyrotropin, the cells have an elongated flattened aspect. Exposure to thyrotropin for 6-24 h produces retraction and rounding up of cells whereas cells incubated with thyrotropin for longer periods of time have an epithelial cuboidal shape. After varying periods of culture the cells were labelled with [35S]methionine for 6 h and then analyzed by one-and two-dimensional gel electrophoresis, followed by autoradiography. The results were as follows.1. After exposure to thyrotropin for 32 h and 48 h, the synthesis of about 18 proteins was increased while that of about 14 others was decreased. After 6 h the labelling of three and five of these proteins was already increased or decreased, respectively.2. Some of the proteins whose synthesis is modified in the presence of thyrotropin were identified. Actin synthesis was markedly decreased with a maximum 24-48 h after the addition of thyrotropin. A modification in the ratio between CI and p tubulins was also observed together with very large changes in a group of proteins having both the relative molecular mass (30000 -40000) and the isoelectric points of tropomyosins.3. Forskolin and cholera toxin caused the same qualitative and quantitative changes as thyrotropin; this suggests that the regulation by thyrotropin of the synthesis of several thyroid cell proteins is mediated by CAMP.In conclusion, the data obtained in this work might help to explain the molecular mechanisms by which thyrotropin (and CAMP) triggers the changes in cell shape which occur during thyroid cell culture. They also indicate that one of the main effects of thyrotropin takes place at the level of several proteins which belong to the cytoskeleton and which are involved in the definition of the cytostructure of the thyroid cells.The activating effect of thyroid-stimulating hormone (thyrotropin) in vivo, on both the synthesis and secretion of thyroid hormones, is associated with marked alterations in thyroid cell morphology. Acute thyrotropin administration produces an extension of pseudopod-like process of the apical cell surface into the follicular lumen (see [I] for a review). Under chronic thyrotropin deprivation the cells are flat, whereas hyperstimulation produces hyperplasia of the thyroid gland and induces 'rounded up' cuboid cells [l]. The establishment of functional primary cultures of thyroid cells 12 -41 and of normal established cell lines [5] have permitted the study of the mechanism(s) responsible for the acute changes in cell morphology. Ultramicroscopic and histoimmunological studies have led to the conclusion that acute thyrotropin administration produces a dramatic reorganization of the microfilament system [6, 71. In contrast, little attention has been devoted to the chronic (or trophic) effects of t...

Rat brain microtubules were prepared at the adult stage and from immature (i.e., 4-day-old) animals. At an early stage of development, the composition of microtubule-associated proteins is qualitatively different from that found at the adult stage [(1982) Eur. J. Biochem. 129, 465-4711. The influence of calmodulin on the time course of assembly of second cycle microtubules was compared at both stages of brain development (i.e., microtubules originating from 4-day-old and adult animals). In the presence of Ca2+ the inhibition of microtubule assembly was more pronounced at a young stage of brain development than at the adult stage. Cross-linking studies with '251-labeled calmodulin further established that the two major microtubule-associated proteins, MAP2 and TAU were able to bind to calmodulin at both stages of brain development but with different intensities. The labeling with '251-labeled calmodulin was Ca2+-dependent, specific, displaced by unlabeled calmodulin and trifluoperazine.CalmoduIin Microtubule