Michel Morange

Firefly luciferase luminescence assays using scintillation counters for quantitation in transfected mammalian cells

The firefly enzyme luciferase catalyzes the luminescent reaction of luciferin with ATP and oxygen. The luciferase gene has recently been cloned and proposed as a reporter gene in procaryotic and eucaryotic cells. We present here a luciferase activity assay which relies on luminescence detection using a standard scintillation counter. This technique is simple, fast, inexpensive, and still very sensitive: as little as 0.02 pg (250,000 molecules) of enzyme is readily detected. The technique is optimized for the luciferase assay in mammalian cell lysates. Thus, the luciferase gene may become a very useful tool for gene regulation studies.

Altered expression of heat shock proteins in embryonal carcinoma and mouse early embryonic cells.

In a previous paper, we have shown that in the absence of stress, mouse embryonal carcinoma cells, like mouse early embryo multipotent cells, synthesize high levels of 89- and 70-kilodalton heat shock proteins (HSP)(O. Bensaude and M. Morange, EMBO J. 2:173-177, 1983). We report here the pattern of proteins synthesized after a short period of hyperthermia in various mouse embryonal carcinoma cell lines and early mouse embryo cells. Among the various cell lines tested, two of them, PCC4-Aza R1 and PCC7-S-1009, showed an unusual response in that stimulation of HSP synthesis was not observed in these cells after hyperthermia. However, inducibility of 68- and 105-kilodalton HSP can be restored in PCC7-S-1009 cells after in vitro differentiation triggered by retinoic acid. Similarly, in the early mouse embryo, hyperthermia does not induce the synthesis of nonconstitutive HSP at the eight-cell stage, but induction of the 68-kilodalton HSP does occur at the blastocyst stage. Such a transition in the expression of HSP has already been described for Drosophila melanogaster and sea urchin embryos and recently for mouse embryos. It may be a general property of early embryonic cells.

Mouse 89 kD heat shock protein: Two polypeptides with distinct developmental regulation☆

Unstressed early mouse embryos have been previously shown [1] to synthesize at very high rates 70 and 89 kD proteins belonging to the heat shock protein (HSP) family. But it was not clear whether expression of heat shock-inducible or non-inducible (cognate) genes accounted for this spontaneous synthesis. In this report we show that the 89 kD mouse HSP can be separated into two proteins by high resolution PAGE. These two components show distinct but related peptide pattern after limited proteolysis. They are synthesized from distinct mRNAs. One of these proteins--HSP89f--is synthesized at a high rate by unstressed cells and its synthesis is rather insensitive to stress, whereas synthesis of the other protein--HSP89s--is strongly stimulated by heat shock in fibroblasts. Both HSP89f and HSP89s are major proteins synthesized in unstressed mouse preimplantation embryos and embryonal carcinoma (EC) cells. After in vitro differentiation of the EC cells the spontaneous synthesis of HSP89s decreases. Thus spontaneous expression of a mammalian inducible HSP is developmentally regulated.

Unusual levels of heat shock element-binding activity in embryonal carcinoma cells.

In contrast to differentiated somatic cells, mouse embryonal carcinoma (EC) cell lines spontaneously express high levels of major members of the heat shock protein (HSP) family. In addition, some EC cell lines (noninducible) are not able to induce HSP gene transcription and HSP synthesis after a stress. However, after in vitro differentiation, constitutive HSP expression decreases and the differentiated derivatives become able to induce HSP gene transcription after a stress. These cells were tested by gel shift assays for the presence of an activity able to bind the heat shock element (HSE) before and after a stress. Control fibroblasts grown at 37 degrees C did not contain significant levels of HSE-binding activity, but heat shock dramatically increased the level of HSE-binding activity. In contrast to control fibroblasts, all EC cells contained significant levels of HSE-binding activity at 37 degrees C. In the inducible EC cell line F9, as in fibroblasts, heat shock strongly increased the level of HSE-binding activity. In the noninducible EC cells, however, HSE-binding activity markedly decreased upon heat shock. During in vitro differentiation of the noninducible cell line PCC7-S-1009, the constitutive HSE-binding activity found at 37 degrees C disappeared and heat induction of the HSE-binding activity appeared. Therefore, a good correlation exists between the high spontaneous expression of some members of the HSP family and the constitutive level of HSE-binding activity in EC cells at 37 degrees C. Heat induction of HSP gene transcription correlates with a strong increase in HSE-binding activity, whereas a deficiency in heat induction of HSP gene transcription is associated with a loss of HSE-binding activity upon heat shock.

Phosphorylation of the 90 kDa heat shock protein in heat shocked HeLa cell lysates

The 90 kDa heat shock protein (hsp 90) is a major phosphorylated protein under normal growth conditions. However, it does not incorporate detectable levels of phosphate by incubation of control HeLa cell lysates with [γ‐32P]ATP in vitro. In this paper we show that strong phosphorylation of hsp 90 occurs in lysates prepared from heat shocked HeLa cells. Possible involvement of the eukaryotic initiation factor 2 kinase of the heme‐controlled repressor of translation is discussed.

Purification of muscle glycogen particles by glycerol-gradient centrifugation.

In the recent years, several reports of the association of muscle glycogen with specific proteins have appeared [l-3] . This assembly has been a useful tool for the purification of glycogen metabolizing enzymes [4] . The observation that the catalytic and regulatory properties of the enzymes are different when they are attached in the particle or free in solution suggests furthermore that this assembly can be a physiologically important one. The regulation of phosphorylase kinase [S] and phosphatase [6] by calcium ions in solution and in the glycogen organelle is quite different, whereas at the level of phosphorylase [7] itself, of debranching enzyme [8] and of phosphatase a distinctive maximal catalytic activity of the enzymes has been reported. The nature of the stimulus which triggers this different behavior is still uncompletely understood although these properties have been allotted to protein-protein interactions, suggesting the importance of the supramolecular structure of the glycogen particle. In the present communication, we describe an improved purification procedure which allows to obtain in less than 6 h particles completely devoid of contaminating sarcoplasmic reticulum membranes and suitable for structural studies.

Early Effects of Heat Shock on Enzymes: Heat Denaturation of Reporter Proteins and Activation of a Protein Kinase which Phosphorylates the C-terminal Domain of RNA Polymerase II

It is a common statement that heat-shock impairs cellular functions because some essential proteins are heat-denaturated. This statement relies mostly on indirect arguments developped by several workers (Hightower, 1980). The fate of such denatured proteins is matter for discussion: are they degraded or renatured? A priming non-lethal heat-shock stimulates transiently the synthesis of the heat-shock proteins (HSP) and increases transiently the cell resistance against a second challenging stress (Subjeck and Sciandra, 1982). Is this thermotolerant state due to a protective effect against thermal denaturation or to a better repair of the damaged proteins? What is the behaviour of the heat-shock proteins?

Application of a metrizamide density‐gradient to the purification of glycogen particles

Different enzymes, related to the glycogen metabolism have been reported to be associated in the rabbit skeletal muscle within a ‘glycogen particle’ [l-5]. In this purified particle one can detect the presence of glycogen phosphorylase [ 1,351, phosphorylase kinase [ 1,5] , phosphorylase phosphatase [l] , amylase [l] , myokinase [l] , adenylic deaminase [ 11, the enzymes of the glycolytic pathway [l] , g

Protein denaturation during heat shock and related stress. Escherichia coli beta-galactosidase and Photinus pyralis luciferase inactivation in mouse cells.

xgen synthetase [3,5] and amylo I-6-glucosidase