Jean-Pierre Tenu

Binding of nucleotides AMP and ATP to yeast phosphofructokinase:evidence for distinct catalytic and regulatory subunits.

Abstract The binding of ATP to yeast phosphofructokinase, as monitored by flow dialysis, is heterogeneous and is adequately described by assuming two independent classes of three binding sites each per enzyme molecule. Under similar conditions, the binding of 5′AMP is homogeneous and a binding stoichiometry of three 5′AMP molecules per enzyme molecule is evaluated. Displacement experiments show that only the ATP molecules bound to the first class of three tight binding sites are displaced by an excess of 5′AMP. Thus, these tight ATP binding sites can be identified as “regulatory sites” in agreement with kinetic data. Furthermore, molecular weight determinations and electrophoresis results are consistent with an heterologous α 3 β 3 structure of the enzyme oligomer. Therefore the present binding data suggest that yeast phosphofructokinase is constituted by three “catalytic” and three “regulatory” subunits.

Cell binding, uptake and cytosolic partition of HIV anti-gag phosphodiester oligonucleotides 3'-linked to cholesterol derivatives in macrophages.

The purpose of this study is to evaluate the cell interactions of a new class of compounds composed of phosphodiester oligonucleotides linked to the cholesterol group at position 3, 7, or 22 of the steroid structure. The resulting conjugates were assessed for their capacity to bind, penetrate and partition in the cytoplasmic compartment of murine macrophages. The results showed that lipophilic conjugates bind to cells much faster (t(1/2) < or = 10 min) than do underivatized oligomers. Oligomers tethered to the cholesterol at positions 3 and 7 (PO-GEM-3-Chol and PO-GEM-7-Chol) interacted more efficiently with cell membranes and were better internalized than oligomers attached to the cholesterol moiety at position 22 (PO-GEM-22-Chol). The cytosolic fraction of internalized oligomers was studied by a digitonin-based membrane permeabilization method. The recovered fraction of oligomers that can freely diffuse from the cytosol was comparable for GEM-91, a phosphorothioate congener, and for PO-GEM-7-Chol (50-60% of the internalized oligomers), while that of PO-GEM-3-Chol was less (30% of the internalized oligomers) indicating a higher membrane affinity of the latter derivative as compared to the other investigated compounds. Membrane binding and cell internalization correlated well with the hydrophobicity of the conjugates as characterized by their partition coefficients in a water-octanol system. Due to their capacity of rapid binding and cytosolic partition in cells, cholesterol-derivatized oligonucleotides at position 3 or 7 of the steroid molecule appeared as good candidates for systemic delivery of anti-HIV antisense compounds.

Relationship between Phosphatidic Acid Level and Regulation of Protein Transit in Colonic Epithelial Cell Line HT29-cl19A

Colonic epithelial HT29-cl19A cells are polarized and secrete proteins among which α1-antitrypsin represents about 95%. Secretion occurs via a constitutive pathway, so that the rates of secretion directly reflect the rates of protein transit. In this paper we have demonstrated that: 1) in resting cells phospholipase D (PLD) is implicated in the control of apical protein transit; 2) phorbol esters stimulate apical protein transit (stimulation factor 2.2), which is correlated with a PLD-catalyzed production of phosphatidic acid (PA) (2.45-fold increase); 3) the stimulation of cholinergic receptors by carbachol results in an increase (stimulation factor 1.45) of apical protein transit which is independent of protein kinase C and PLD activities, but related to PA formation (1.7-fold increase) via phospholipase(s) C and diacylglycerol kinase activation; 4) an elevation of the cAMP level enhances apical protein transit by a PA-independent mechanism; 5) a trans-Golgi network or post-trans-Golgi network step of the transit is the target for the regulatory events. In conclusion, we have shown that PA can be produced by two independent signaling pathways; whatever the pathway followed, a close relationship between the amount of PA and the level of secretion was observed.

Sequence Dependency of the Internalization and Distribution of Phosphorothioate Oligonucleotides in Vascular Smooth Muscle Cells

Antisense studies imply the utilization of oligonucleotides (ODN) for sequence-specific down-regulation of genes. This usually consists in assessing antisense sequences versus control sequences (mismatched, inverted, scrambled, randomized or any sequence unrelated to the relevant target). Even though the investigated biological effect (knockdown of an unwanted protein) is observed only with the antisense sequence and weakly, if at all, with any of the control sequences, this is a necessary but not a sufficient condition to demonstrate an antisense effect. Indeed, biochemical parameters such as stability, uptake and subcellular compartmentalization of ODN in a given cellular system are most often sequence-dependent processes. In this work, a series of phosphorothioate ODN of different lengths and sequences were evaluated as to their binding, internalization and subcellular distribution properties in vascular smooth muscle cells. In addition to membrane binding and nuclear accumulation, the partition of ODN in the cytosol of cells was measured by a method based upon controlled permeabilization of the plasma membrane, permitting the recovery of the cytosolic content with minimal damage to the membranes of the endocytic vesicles and lysosomes. We found that the tested ODN showed striking differences in their uptake and distribution in smooth muscle cells. Our results gave rise to the problem of validating the observed biological effects when different sequences of ODN were compared. Cellular studies such as the one presented in this work could help in choosing the proper control sequences among ODN exhibiting similar cell interactions as compared to the antisense sequences. Moreover, this method could be useful for the selection of antisense sequences that can be efficiently internalized and preferentially distributed in the appropriate compartments in cells for in vitro antisense studies.

High Specific Radioactivity Labeling of Oligonucleotides with 3H-Succinimidyl Propionate

An easy and rapid method for tritium labeling of deprotected oligonucleotides is proposed. The method consists in performing the reaction of commercial 3H-succinimidyl propionate with a terminal amino group of the oligonucleotide in an organic medium. High specific radioactivity labeling can be achieved with minimal radiolysis during long term storage. The synthesis of the nonradioactive congener having an identical structure to the labeled compound is also described.

Mouvements Moléculaires Consécutifs à la Fixation de Métaux dans la β-Galactosidase

La β-galactosidase de E. coli est activee par les ions Na++ et Mg++. Le mecanisme de l’activation de l’enzyme par Mg++ a ete etudie en detail. D’autres metaux Mn++ et Co++ ont egalement un effet activateur important. L’activation par les metaux, de meme que la reaction inverse correspondent a des phenomenes lents. L’analyse cinetique montre que l’activation est un processus en deux etapes, une premiere fixation rapide du metal sur l’enzyme libre, suivie d’une etape plus lente qui s’accompagne de l’augmentation d’activite impliquant probablement un changement dans la conformation de l’enzyme.