Jean Derancourt

Solid phase sequential analysis: Specific linking of acidic peptides by their carboxyl ends to insoluble resins

The success of methods such as the Edman degradation for solid-phase sequential analysis of peptides depends on having efficient methods for attaching peptides to insoluble resins before degradation. Previously described [2,3] attachment procedures using carbonyldiimidazole to couple the C-terminal carboxyl to an amino resin, suffer from the serious problem that side-chain carboxyls also become activated, leading to side reactions. In order to make the solid-phase Edman degradation generally applicable, it is essential to have a procedure for selective activation of the C-terminal carboxyl groups. We wish to report here that a.iV,N’-disubstituted carbodiimide can effect both protection of side-chain carboxyl groups and activation of the C-terminal under suitable experimental conditions (scheme A).

A purified complex from Xenopus oocytes contains a p47 protein, an in vivo substrate of MPF, and a p30 protein respectively homologous to elongation factors EF-1γ and EF-1β

A high molecular mass complex isolated from Xenopus laevis oocytes contains three main proteins, respectively p30, p36 and p47. The p47 protein has been reported to be an in vivo substrate of the cell division control protein kinase p34cdc2. From polypeptide sequencing, we now show that the p30 and the p47 correspond to elongation factor EF‐1β and EF‐1γ. Furthermore, the p30 and p36 proteins were phosphorylated in vitro by casein kinase II.

A chloroplastic RNA-binding protein is a new member of the PPR family

P67, a new protein binding to a specific RNA probe, was purified from radish seedlings [Echeverria, M. and Lahmy, S. (1995) Nucleic Acids Res. 23, 4963–4970]. Amino acid sequence information obtained from P67 microsequencing allowed the isolation of genes encoding P67 in radish and Arabidopsis thaliana. Immunolocalisation experiments in transfected protoplasts demonstrated that this protein is addressed to the chloroplast. The RNA‐binding activity of recombinant P67 was found to be similar to that of the native protein. A significant similarity with the maize protein CRP1 [Fisk, D.G., Walker, M.B. and Barkan, A. (1999) EMBO J. 18, 2621–2630] suggests that P67 belongs to the PPR family and could be involved in chloroplast RNA processing.

Maitotoxin stimulat es the formation of inositol phosphates in rat aortic myocytes

Maitotoxin is the most potent of the known marine toxins. The effect of maitotoxin on muscle contraction or hormone release was consistent with its action on the voltage‐sensitive channel. Indeed, calcium antagonists such as nifedipine or diltiazem were able to reverse the maitotoxin effects. Using smooth muscle cells, we have analysed the effects of maitotoxin on the inositol phosphate metabolism. Maitotoxin stimulates the inositol phosphate formation (5 ± 1.8‐fold in the presence of 10 mM LiCI). Moreover, this effect is not reversed, even partially by calcium antagonists, by α1 antagonists and is not mimicked by Ca2+ ionophores such as A23187 or calcium agonists such as Bay‐K 8644. The action of maitotoxin is further discussed in this paper.

Determination of cardiac and plasma drug levels during long‐term amiodarone therapy

Abstract. A study of plasma and cardiac concentrations of amiodarone during the course of long‐term oral therapy was made possible by the improvement of analytical high performance liquid chromatography of plasma and tissue extracts.

Ontogenic development of liver progesterone metabolism in female sheep. Contribution of cytochrome P4502B and P4503A subfamilies

Age-related changes in progesterone hepatic metabolism were measured in Lacaune ewes in the foetal, neonatal (1 and 4 weeks), growing (7 months), pregnant (11 months) and adult (6 years) stages. 6 beta-Hydroxylation and 20 alpha-reduction were found to be the most efficient metabolic process in ovine microsomes. These activities were detected in 3-month-old foetuses and they increased rapidly during the first month of life, in a similar manner to the developmental expression of the cytochrome P4503A subfamily. 16 alpha- and 21-hydroxylation of progesterone were characterized by low, constant turn over in sheep liver microsomes during development. The hepatic ovine P4502B isozyme was purified to electrophoretic homogeneity by means of successive DEAE cellulose, hydroxylapatite and CM cellulose chromatographic separations. This hemoprotein had an apparent molecular weight of 51 kDa and was characterized by spectral data, NH2-terminal amino-acid sequence, immunological and catalytic properties. The relative contribution of this form and of the previously purified ovine P4503A subfamily was investigated in liver progesterone metabolism by immunoinhibition studies using polyclonal antibodies raised in rabbits and from the existence of induction and of significant correlations between microsomal activity and specific P450 content. In sheep liver microsomes, it would appear that cytochrome P4502B is involved in progesterone 21-hydroxylation whereas P4503A participates in the 6 beta- and 16 alpha-hydroxylation and possibly in the reductive conversion of progesterone in its 20 alpha-hydroxy derivative.

Sequence Homologies between p36, the substrate of pp60src tyrosine kinase and a 67 kDa protein isolated from bovine aorta

A 67 kDa actin-binding protein was isolated from bovine aorta. Partial amino acid sequence determination of two large thermolysin peptides were used to compare 67 kDa bovine aorta protein and p36 the substrate of pp60src tyrosine kinase. Sequence analysis shows that 67 kDa bovine aorta protein shares common domains with p36 and possesses the consensus aminoacid sequences of mammalian Ca2+-dependent membrane-binding protein and p36/gelsolin.

The effects of maitotoxin on phosphoinositides and calcium metabolism in a primary culture of aortic smooth muscle cells

Maitotoxin, a potent marine toxin isolated from toxic tropical dinoflagellates and poisonous fishes induces contraction of different smooth muscle preparations. Actions of maitotoxin on phosphoinositides and calcium metabolism were studied using a primary culture of aortic smooth muscle cells. Maitotoxin induced a very large increase of cytosolic calcium concentration as evaluated by fura-2 acetoxymethyl ester fluorescence. This increase was concomitant with stimulation of inositol-phosphate accumulation and loss of viability of aortic smooth muscle cells. These responses to maitotoxin were abolished in Ca2+-free medium, and were mimicked by saponin. Calcium ionophores or K+ depolarisation did not induce inositol-phosphate formation. These results suggest that maitotoxin acts by altering smooth muscle cells permeability allowing a sustained calcium influx which is able to activate inositol-phosphate formation and which is lethal for the cells.

Base catalyzed aminolysis of carbodithioic esters and its interest in solid phase sequential analysis of peptides

The finding that a peptide terminal N-thioacyl derivative can be split off by the action of TFA yielding the 2-substituted thiazol-5(4H)-one which identifies the N-terminal amino acid and the shortened peptide constitutes the base of a new route for the sequential analysis of polypeptide chains [l-3]. The mild reaction conditions required for the release of the N-terminal thiazolinones together with the chemical and physical properties favourable for their identification constitute the most promising aspect in view of a complete automatization of the analytical procedure. The thioacylation reaction is brought about by carbodithioic esters (sometimes by thionic esters) because there are no other generally satisfactory reagents for thioacylating amino groups. Nevertheless these compounds are not particularly good thioacylating reagents and this constituted the most important drawback for a satisfactory procedure of sequential analysis. In order to overcome this difficulty the use of active esters of dithiobenzoic acid has been proposed [2] and the results obtained led one to recognise that some esters containing a good leaving group could be usefully choosen after a systematic exploration. An alternative possibility of general application to increase the rate of the coupling reaction between amino groups and thioacylating reagents is proposed in this work. We report here that aminolysis of carbodithioic esters is susceptible to base catalysis and we describe the experimental conditions by which

N‐aminoethyl polyacrylamide as support for solid‐phase sequencing of proteins

The automatic sequencing of proteins starting from N-terminus by solid-phase technique should be performed following the same principles as for peptides [l] . In this case the solid-phase sequencing could entirely cover the sequencing field from smaller to larger peptides and proteins and became an alternative rather than a complementary route for automatic sequence analysis if comparated to the homogenous phase technique. However, the serious difficultiwwhich arise when large peptides are to be linked to a solid support for their subsequent sequential analysis constitute the principal limit for routine work. Nevertheless the chemical and physical problems associated with the insolubilization step seems to be eliminated or minimised as shown by the successful sequencing of proteins linked to aminated porous glass [2]. Despite some inconveniencies, essentially a low degree of substitution [3] and the potential lability of the -%-O-C linkage, the aminated porous glass possesses the general properties of an ideal support for large polypeptide sequencing. In other words the best results should be obtained using an inert and rigid support possessing a high concentration of suitable functional groups on its surface. On the basis of these considerations we synthetised N-aminoethyl polyacrylamide starting from a commercial polyacrylic resin: