Effect of cetyltrimethylammonium on the human blood cholinesterases activity

The influence of cationic detergent cetyltrimethylammonium on the human blood cholinesterases activity (erythrocyte acetylcholinesterase and plasma butyrylcholinesterase) in reactions of hydrolysis of alpha-thionaphthylacetat and acetylthiocholine is studied. It is shown, that cetyltrimethylammonium is reversible effector for both cholinesterases. This compound competitively inhibited enzymatic hydrolysis of acetylthiocholine by both cholinesterases, and in the reactions of enzymatic hydrolysis alpha-thionaphthylacetat display as the synergistic activator--in experiments with butyrylcholinesterase, and as the reversible inhibitor--in experiments with acetylcholinesterase. Kinetic constants in reaction of acetylcholinesterase inhibition by cetyltrimethylammonium defined by means of different substrates--alpha-thionaphthylacetat and acetylthiocholin. They are close among themselves and amount (2.5 +/- 0.3) x 10(-5) and (2.8 +/- 0.3) x 10(-5) M, accordingly. Butyrylcholinesterase was more sensitive to influence of cetyltrimethylammonium. The kinetic constants defined for this enzyme by the effect of inhibition of acetylthiocholin hydrolysis or activation of alpha-thionaphthylatcetat hydrolysis, are also close among themselves and amount (3.9 +/- 0.4) x 10(-6) and (4.4 +/- 0.4) x 10(-6) M, accordingly.     

Isoenzymes of phosphoglucomutase from human red blood cells: isolation and kinetic properties

A procedure has been developed for the purification of phosphoglucomutase from human red cell (phenotype PGM1 a1 or a3) lysates. It yields homogeneous isoenzyme preparations of the products ("primary" and "secondary") of the two PGM1 and PGM2 loci with distinctive pI (from 6.07 to 5.29). There are substantial differences between PGM1 and PGM2 isoenzymes, having single polypeptide chains of 58,500 and 69,000 Mr respectively and showing different thermostability. The kinetic properties of all the isoenzymes for the phosphoglucomutase reaction are essentially the same (apart from the specific activity of 1089-1263 units/mg for PGM1 forms vs 37-42 units/mg for PGM2 forms), but there are striking differences in substrate specificity. In fact the products of PGM1 locus are "true" phosphoglucomutases, being specific to mutate glucose monophosphates, whereas the PGM2 forms also display phosphoribomutase and glucose 1,6-bisphosphate synthetic activities. Some kinetic properties of these "side activities" are also reported.     

The expression of cholinesterases in human renal tumours varies according to their histological types

The change in the expression of acetylcholinesterase (AChE) and butyrylcholinesterase (BuChE) activities in neoplastic colon and lung prompted us to study the possible effect of cancer on the expression of cholinesterases (ChEs) in kidney. Samples of papillary renal cell carcinoma (pRCC), conventional RCC (cRCC), chromophobe RCC (chRCC) and renal oncocytoma (RON), beside adjacent non-cancerous tissues, were analyzed. In pRCC both AChE and BuChE activities were statistically increased; in cRCC and chRCC only AChE activity increased and in RON neither AChE nor BuChE activities were affected. Abundant amphiphilic AChE dimers (G(2)(A)) and fewer monomers (G(1)(A)) were identified in healthy kidney as well as in all tumour classes. Incubation with PIPLC revealed glycosylphosphatidylinositol in AChE forms. BuChE is distributed between principal G(4)(H), fewer G(1)(H), and much fewer G(4)(A) and G(1)(A) species. RT-PCR showed similar amounts of AChE-H, AChE-T and BuChE mRNAs in healthy kidney. Their levels increased in pRCC but not in the other tumour types. The data support the idea that, as in lung tumours, in renal carcinomas expression of ChE mRNAs, biosynthesis of molecular components and level of enzyme activity change according to the specific kind of cell from which tumours arise.     

Isoenzymes of blood serum alkaline phosphatase under experimental cholemia

The activity and isoenzymic spectrum of alkaline phosphatase of the blood serum and liver under cholemia caused by deoxycholic acid were compared in healthy animals and in animals with the affected liver. It is shown, that under conditions of the bile acids higher content in the organism due to deoxycholic acid, the total activity increases considerably and there appears an isoenzyme absent in the blood serum of healthy animals. Changes in the activity and isoenzymic spectrum of alkaline phosphatase under experimental cholemia developing against a background of the healthy and affected liver are characterized by certain peculiarities.     

Synthesis of novel phosphorothioates and phosphorodithioates and their differential inhibition of cholinesterases

The anticholinesterase activities of newly synthesized phosphorothioates and phosphorodithioates were investigated. The compounds were evaluated for their acetylcholinesterase (AChE) and butyrylcholinesterase (BChE) inhibition potency through IC₅₀ determination. The selectivities of the synthesized compounds toward both enzymes were determined and compared in terms of their molecular structures.     

Tissue cholinesterases. A comparative study of their kinetic properties

The substrate saturation and temperature-dependent kinetic properties of soluble and membrane-bound forms of acetylcholinestarase (AChE) from brain and butyrylcholinesterase (BChE) from heart and liver were examined. In simultaneous studies these parameters were also measured for AChE in erythrocyte membranes and for BChE in the serum from rat and humans. For both soluble and membrane-bound forms of the enzyme from the three tissues, two components were discernible. In the brain, Km of component I (high affinity) and component II (low affinity) was somewhat higher in membrane-bound form than that of the soluble form components, while the Vmax values were significantly higher by about five fold. In the heart, Km of component II was lower in membrane-bound form than in the soluble form, while Vmax for both the components was about four to six fold higher in the membrane-bound form. In the liver, Vmax was marginally higher for the two components of the membrane-bound enzyme; the Km only of component I was higher by a factor of 2. In the rat erythrocyte membranes three components of AChE were present showing increasing values of Km and Vmax. In contrast, in the human erythrocyte membranes only two components could be detected; the one corresponding to component II of rat erythrocyte membranes was absent. In the rat serum two components of BChE were present while the human serum was found to possess three components. Component I of the human serum was missing in the rat serum. Temperature kinetics studies revealed that the Arrhenius plots were biphasic for most of the systems except for human serum. Membrane binding of the enzyme resulted in decreased energy of activation with shift in phase transition temperature (Tt) to near physiological temperature.     

Isoenzymes of sphingomyelinase and the genetic defect in Niemann-Pick disease, type C

Isoenzymes of sphingomyelinase have been resolved by isoelectric focusing. The two major species (I and II) in human liver have distinct isoelectric points, pH optima and Km values. Liver from Niemann-Pick disease Type C contained isoenzyme I (pI 4.6) while isoenzyme II (pI 5.2) was absent. The absence of isoenzyme II likely constitutes the genetic defect in this disease.     

Molecular biological search for human genes encoding cholinesterases

Cholinesterases (ChEs) are highly polymorphic proteins, capable of rapidly hydrolyzing the neurotransmitter acetylcholine and involved in terminating neurotransmission in neuromuscular junctions and cholinergic synapses. In an attempt to delineate the structure and detailed properties of the human protein(s) and the gene(s) coding for the acetylcholine hydrolyzing enzymes, a human cDNA coding for ChE was isolated by use of oligodeoxynucleotide screening of cDNA libraries. For this purpose, a method for increasing the effectiveness of oligonucleotide screening by introducing deoxyinosine in sites of codon ambiguity and using tetramethyl-ammonium salt washes to remove false-positive hybrids was employed. The resulting isolated 2.4-kilobase (kb) cholinesterase cDNA sequences encode for the entire mature secretory protein, preceded by an N-terminal signal peptide. The human ChE primary sequence shows almost no homology to other serine hydrolases, with the exception of a hexapeptide at the active site. In contrast, it displays extensive homology with acetylcholinesterase form Torpedo californica and Drosophila melanogaster as well as with bovine thyroglobulin. These extensive homologies probably suggest the need of the entire coding sequence for the physiological function(s) fulfilled by the enzyme and further suggest a common, unique, ancestral gene for these cDNAs. In turn, the cDNA was used as a probe to isolate genomic DNA sequences for the 5'-region of the human ChE gene. The genomic DNA fragment encoding part of the 5'-region of ChEcDNA was detected by DNA blot hybridization, enriched 70-fold by gel electrophoresis and electroelution, cloned in lambda phage and isolated. Sequencing of the cloned DNA revealed that it did indeed include part of the 5'-region of ChEcDNA, starting at an adjacent 5'-position to the nucleotides coding for the initiator methionine, and ending with an EcoRI restriction site inherent to the ChEcDNA sequence. The isolated fragment of the human cholinesterase gene is currently employed to complete the structural characterization of this and related genes.