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C Gache

Publications and source records attributed to C Gache.

29 records · Page 2Linked to original sources
PubMed1983

Monoclonal antibodies to the sea urchin egg vitelline layer inhibit fertilization by blocking sperm adhesion.

Thirty-one mouse hybridomas were produced against the vitelline layer (VL) of the egg of the sea urchin S. purpuratus. Ascites fluids of eight of the 31 bound to the VL surface in the high ionic strength conditions of sea water. Binding was specific to the VL, since immunofluorescence showed that the antibodies elevated from the egg surface with the fertilization envelope after activation with ionophore A23187. Antibody binding was strictly species-specific, the eggs of L. pictus showing no reaction. An immunoperoxidase surface-binding assay showed a wide range in the amount of each monoclonal antibody binding to the VL surface at saturation. All eight monoclonals inhibit fertilization by inhibiting the binding of sperm to the VL. None of the eight ascites fluids reacted with egg jelly. The inhibition of fertilization correlates positively with amount of antibody binding the egg surface. In contrast to the effects of polyclonal rabbit antisera raised against whole eggs or egg cortices, these eight monoclonal antibodies to the VL do not induce the wrinkling of the egg, the cortical granule reaction, the centering of pronuclei, or any other visual indication of metabolic activation.

Animals↗
PubMed1980

Affinity labeling of the digitalis receptor with p-nitrophenyltriazene-ouabain, a highly specific alkylating agent.

Three derivatives of ouabain have been synthesized which alkylate the digitalis receptor. These derivatives were formed through reductive amination of p-nitrophenyltriazene (NPT) ethylenediamine to the periodate-oxidized rhamnose moiety of ouabain. The non-covalent binding of the ouabain derivatives (NPT-ouabain, designated I, II, and III) was followed (i) by their ability to inhibit the activity of sodium- and potassium-activated ATPase ((Na+,K+)-ATPase) purified from the electric organ of Electrophorus electricus, (ii) by the binding of [3H]NPT-ouabain I to the enzyme, and (iii) by the inhibition of [3H]ouabain binding with unlabeled NPT-ouabain I. Covalent modification of the digitalis site of (Na+,K+)-ATPase occurs after long periods of time. At pH 7.5 (25 degrees C) the best alkylating derivative, NPT-ouabain I, gives maximum covalent labeling after 6 h. Only the large polypeptide chain (Mr = 93,000) of the purified enzyme is specifically labeled with [3H]NPT-ouabain I while the glycoprotein chain (Mr = 47,000) is not significantly labeled. Labeling of a microsomal fraction of the electric organ with [3H]NPT-ouabain I gave the same type of gel pattern as that observed with the purified enzyme. [3H]NPT-ouabain I was also used to label the digitalis receptor in highly purified axonal membranes and in cardiac membranes prepared from embryonic chick heart. Although the (Na+,K+)-ATPase in both types of membranes has a low affinity for ouabain, [3H]NPT-ouabain I proved to be a very efficient affinity label for the digitalis receptor. In the complex mixture of polypeptides found in these membrane preparations, only a single polypeptide chain having a Mr = 93,000 is specifically labeled by [3H]NPT-ouabain I.

Affinity Labels↗
PubMed1978

The role of serum albumin in the uptake of fatty acids by cultured cardiac cells from chick embryo.

Fatty acids enter the cultured cardiac cells from chicken embryo through two mechanisms, one involving a saturable process, the other resembling passive diffusion. Studies of the saturable component, at fixed concentrations of palmitate or of oleate and at increasing concentrations of albumin, show that this protein increases the rate of palmitate uptake with a maximum at palmitate/albumin molar ratios between 7 and 10 while it decreases the rate of oleate uptake under all conditions. Albumin cannot be replaced by other serum proteins; its effect is specific to saturated fatty acids, can be mimicked by the detergent Tween 40 and involves the binding of the fatty acid to the protein, prior to its delivery to the cell. Both with labelled saturated and unsaturated fatty acids the presence of albumin lowers the proportion of unesterified fatty acids and enhances the proportion of esterified fatty acids recovered in the cardiac cell after uptake. A similar effect of albumin was also found with hepatocytes and permanent cell lines. A specific role for serum albumin is presented, which assumes a 'dispersing' effect of this protein towards dimers (or higher aggregates) of saturated fatty acids and the entry of fatty acids into the cell as monomers.

Animals↗
PubMed1976

(Na+, K+)-activated adenosinetriphosphatase of axonal membranes, cooperativity and control. Steady-state analysis.

1. The ATP sites. Homotropic interactions between ATP sites have been studied in a very large range of Na+ and K+ concentrations. The ( Na+, K+)-activated ATPase displays Michaelis-Menten kinetics for ATP under standard concentration conditions of Na+ (100 mM) and K+ (10 mM). The steady-state kinetics behavior changes at very low concentrations of K+ where negative cooperativity is observed. The existence of a high affinity and a low affinity site for ATP was clearly demonstrated from the study of the ATP stimulated hydrolysis of p-nitrophenylphosphate in the presence of Na+ and K+. The ratio of apparent affinities of high and low affinity sites for ATP is 86 at pH 7.5. 2. The Na+ sites. The binding of Na+ to its specific stimulatory sites (internal sites) is characterized by positive cooperativity with a Hill coefficient n(H(Na+))=2.0. Homotropic interactions between Na+ sites are unaffected by variations of the K+ concentration. 3. The K+ sites. (a) Binding of K+ to the (external) stimulatory site of the ATPase has been analyzed by following the (Na+, K+)-ATPase activity as well as the p-nitrophenylphosphatase activity in the presence of Na+ and K+ (with or without ATP). Binding is characterized by a Hill coefficient of 1.0 and a K(0.5(K+))=0.1 to 0.8 mM. The absence of positive or negative cooperativity persists between 5 mM and 100 mM Na+. (b) The analysis of the p-nitrophenylphosphatase or of the 2, 4 dinitrophenylphosphatase activity in the presence of K+ alone indicates the existence of low affinity sites for K+ with positive homotropic interactions. The characteristics of stimulation in that case are, K(0.5)=5 mM, n(H)=1.9. The properties of this family of site(s) are the following: firstly, saturation of the low affinity site(s) by K+ prevents ATP binding to its high affinity internal site. Secondly, saturation of the low affinity sites for K+ prevents binding of Na+ to its internal sites. Thirdly, this family of sites disappears in the presence of ATP, p-nitrophenylphosphate or of both substrates, when Na+ binds to its internal sites. Na+ binding to its specific stimulatory sites provokes the formation of the high affinity type of site for K+. 4. Mg2+ stimulation of the (Na+, K+)-ATPase is characterized by a Hill coefficient n(H(Mg2+))=1.0 and a K(0.5(Mg2+))=1 mM stimulation is essentially a V effect. Heterotropic effects between binding of Mg2+ and substrate to their respective sites are small. Heterotropic interactions between the Ms2+, Na+ and K+ sites are also small. 5. The fluidity of membrane lipids also controls the (Na+, K+)-ATPase activity. Phase transitions or separations in the membrane hardly affect recognition properties of substrates, Na+, K+ and Mg2+ for their respective sites on both sides of the membrane. Only the rate of the catalytic transformation is affected.

Acetylcholinesterase↗