Is the peptidase activity of highly purified human plasma cholinesterase due to a specific cholinesterase isoenzyme or a contaminating dipeptidylaminopeptidase?

The peptidase site of human plasma cholinesterase (butyrylcholinesterase) is distinct from its esteratic site. We found that the number of peptidase sites on an enzyme highly purified from pooled plasma is less than 0.1, as compared with 4 esteratic sites, per tetramer. However, the subunits which carry the peptidase sites are electrophoretically indistinguishable from esteratic subunits. The atypical-silent enzyme (Ea1Es1) had a much higher absolute peptidase activity when substance P was used as the substrate, and we found that the number of peptidase and esteratic sites of this enzyme was roughly the same. This suggests that the mutated esteratic site of the silent possesses a peptidase activity. The esteratic site of the usual allozyme (Eu1Eu1) has no peptidase activity towards substance P. However, a small proportion of peptidase subunits are present in all preparations of enzymes purified from the plasmas of homozygote individuals. The peptidase activity of butyrylcholinesterase might therefore correspond to a specific isoenzyme produced by an epigenetic mechanism or produced by a gene distinct from genes E1 and E2 encoding for cholinesterase subunits. However, the possibility that highly purified cholinesterase contains traces of a dipeptidylaminopeptidase cannot be completely ruled out.     

Possibilitiés d'étude des variantes de la cholinestérase plasmatique humaine par électrophorèse d'affinitéPossibilities of study of the human plasma cholinesterase variants by affinity electrophoresis

Affinity electrophoresis has been applied to the study of the multiple molecular forms of three human plasma cholinesterase phenotypes (usual enzyme U, atypical enzyme A and intermediate UA). Electrophoreses were carried out in polyacrylamide gels containing a water-soluble macromolecular derivative of m-amino-(substituted)-phenyltrimethylammonium immobilized within the gel network.Apparent dissociation constants (K_{D app}) were estimated from the mobilities of the enzymes versus ligand concentration.The ratio of K_{D app} values of the molecular forms of phenotypes A and U which is approximately 2 is consistent with the hypothesis that the anionic site is altered in atypical enzyme.     

The molecular organization of the beta-globin complex of the deer mouse, Peromyscus maniculatus

Recombinant DNA clones have been isolated that contain 80 kb of the beta-globin complex from the deer mouse, Peromyscus maniculatus. Comparisons of this complex with that from the laboratory mouse, Mus domesticus (with an order 5'-Hbby, Hbb-bhO, Hbb-bhl, Hbb-bh2, Hbb-bh3, Hbb-bl, Hbb-b2 3') highlight organizational trends in the beta-globin complex since the two species diverged. Unlike other mammals studied thus far, the deer mouse possesses three adult genes. Partial sequence analysis indicates that each of the three adult genes is intact and hence may be functional. Hybridization of one of the two Mus pseudogenes, Hbb-bh3, to genomic blots from Peromyscus reveals that it has a homologous counterpart in Peromyscus. Homologous genes to the two gamma-like Mus genes, Hbb-bhO and Hbb-bhl, are also found in Peromyscus. The strong hybridization between the Hbb-bhl genes and significant nucleotide similarity between the Hbb-bhO genes suggest that both pairs are important for the ontogeny of these mice although no known product has been identified for the Hbb-bhO genes. The presence of Hbb-bhO and Hbb-bhl in Peromyscus suggests that the duplication that created this related gene set occurred before the two lineages diverged. A single gene for Hbb-y has been isolated from Peromyscus. The adult region in Peromyscus has undergone significant divergence from the same region in Mus, having three rather than two adult genes, the acquisition of at least 15 kb of extra DNA relative to Mus, and possibly the loss of the Hbb-bh2 pseudogene. The nonadult region of the complex, in contrast, contains the same set of genes apparently distributed over the same amount of DNA as in the Mus beta- globin complex. This observation suggests that the embryonic region of the complex is more evolutionarily stable than the adult region.      

Structural and functional studies of the peripheral olfactory nervous system of male and female bumble-bees (Bombus hypnorum and Bombus terrestris)

Volatile chemicals are known to be essential factors that triggeror release different kinds of bumblebees (Bombus sp.) behaviour,in intraspecific and interspecific relationships. Chemical analysesof the involved semio-chemicals are now in progress, and inparallel we have studied the nervous bases of identified odourrecognition, for both males and females (queens and workers)of B. hypnorum and B. terrestris. The insects' antennae areprovided with the sensory equipment for the detection of volatilecompounds; the distribution of placodea-type olfactory sensillawas studied by scanning electron microscopy. Responses of antennalsensory cells to floral and pheromonal components were recorded(EAG). Queens present the most numerous sensillar populationand the highest amplitudes in EAG responses. These results mightbe linked to the polymorphism of the body size and thereforeto the antennal size of the three kinds of insects, the queensbeing the biggest in this insect population. Odour sensitivitydepends on the molecular weight and the chemical function ofthe tested molecules and appears to be higher for some floralcompounds such as vanillin and pheromonal derivatives such asfarnesol.     

Coupling of Na+ with the spermidine transporter protein in NIH BALB/c 3T3 cells

This study demonstrates that polyamine spermidine (Spd) transporter protein is directly coupled with the Na+ in a ternary complex form, Na(+)-Spd-carrier. The Spd is transported with Na+ in a 1:1 stoichiometry relationship. Interestingly, addition of 2-deoxyglucose in the assay medium did not influence significantly the Spd uptake demonstrating the ATP independency of Spd transport.     

Conformational plasticity of butyrylcholinesterase as revealed by high pressure experiments

The ligand binding and kinetic behaviour of butyrylcholinesterase (EC 3.1.1.8, acylcholine acylhydrolase) from human plasma was studied at 35 degrees C under high hydrostatic pressure. The binding of phenyltrimethylammonium was studied by affinity electrophoresis at various pressures ranging from 10(-3) to 2 kbar. The kinetics of enzyme carbamylation with N-methyl(7-dimethylcarbamoxy)quinolinium iodide was studied in single-turnover conditions up to 1.2 kbar using a high-pressure stopped-flow fluorimeter. Experiments were carried out in different media: 1 mM Tris-HCl (pH 8) with water, water containing 0.1 M lithium chloride and deuterium oxide as solvents. The volume changes (delta V and delta V++) associated with each process were determined from the pressure-dependence of the binding and kinetic constants. Kinetic data show that the binding of substrate to the enzyme leads to a pressure-sensitive enzyme conformational state which cannot accomplish the catalytic act. The pressure-induced inhibitory effect is highly cooperative; it depends on both the nature (charged or neutral) and the concentration of the substrate. Also, large solvent effects indicate that enzyme sensitivity to pressure depends on the solvent structure. This findings suggests that the substrate-dependent pressure effect is modulated by the solvation state of the enzyme.