The effect of temperature on the acetylcholinesterase activity of muscle microsomes

Membrane vesicles which constitute the sarcotubular system were separated and the fraction enriched in T-tubules purified by a calcium loading procedure. The preparations of unfractioned microsomes and T-tubules have been analyzed for their relative content of enzyme markers and acetylcholinesterase. The amount of this enzyme in the T-tubule fraction was higher than in mixed microsomes but less than two-fold the value of vesicles derived from sarcoplasmic reticulum. Arrhenius plots of membrane-bound and soluble acetylcholinesterase from either mixed microsomes or fractions enriched in T-tubules show an anomalous behaviour as two break points were obtained. The first discontinuity was found at about 17 degrees C for membrane-bound, and 12-14 degrees C for soluble acetylcholinesterase. The second one being at about 25 degrees C for both particulate and detergent-solubilized enzyme. The changes in activity with temperature suggest that lipid-protein, detergent-protein and protein-protein interactions might be involved in the stabilization of the enzyme both in the natural membrane and in the soluble state.     

Proteolytic stimulation and solubilization of membrane-bound acetylcholinesterase from muscle sarcotubular system

Incubation of membranes derived from sarcotubular system of rabbit skeletal muscle with increasing concentrations of Triton X-100 produced both stimulation of the AChE activity and solubilization of this enzyme. Mild proteolytic treatment of microsomal membranes produced a several fold activation of the still membrane-bound acetylcholinesterase (AChE) activity. Attempts were made to solubilize AChE from microsomal membranes by proteolytic treatment. About 30–40% of the total enzyme activity could be solubilized by means of trypsin or papain. Short trypsin treatment of the microsomal membranes produced first an activation of the membrane-bound enzyme followed by solubilization. Incubation of muscle microsomes for a short time with papain yielded a significant portion of soluble enzyme. Membrane-bound enzyme activation was measured after a prolonged incubation period. These results are compared with those of solubilization obtained by treatment of membranes with progressive concentrations of Triton X-100. The occurrence of molecular forms in protease-solubilized AChE was investigated by means of centrifugation analysis and slab gel electrophoresis. Centrifugation on sucrose gradients revealed two main components of 4.4S and 10–11S in either trypsin or papain-solubilized AChE. These components behaved as hydrophilic species whereas the Triton solubilized AChE showed an amphipatic character. Application of slab gel electrophoresis showed the occurrence of forms with molecular weights of 350,000; 175,000; 165,000; 85,000 and 76,000. The stimulation of membrane-bound AChE by detergents or proteases would indicate that most of the enzyme molecules or their active sites are sequestered into the lipid bilayer through lipid-protein or protein-protein interactions and these are broken by proteolytic digestion of the muscle microsomes.     

Active and inactive ecto-5'-nucleotidase variants in liver of control and dystrophic Lama2dy mice

The ecto-5'-nucleotidase (eNT) activity and the eNT protein content in liver of normal and merosin-deficient dystrophic Lama2dy mice were studied. After the solubilization procedure, the eNT activity in the final extract was 9.2+/-2.5U/mg (nmol of phosphate released from AMP per min and per mg protein) in normal liver, and it rose to 16.1+/-3.9U/mg (P=0.005) in dystrophic liver. The increase of activity was less pronounced in Lama2dy liver (1.7-fold) than the one reported in muscle (four-fold), which probably reflects the lower content of merosin in liver. Similarly to muscle, liver contained active and inactive eNT, as demonstrated by the higher level of immunoreactive protein in normal than in dystrophic liver in Western blots performed with samples containing the same units of eNT activity. PNGase F digestion decreased the size of liver and muscle eNT from 72 and 69kDa, to 63 and 60kDa. Oligoglycan cleavage did not alter eNT activity or the sedimentation coefficient, revealing that oligosaccharides are not required for catalysis or for maintaining the dimeric structure. The eNT protein content in samples of normal liver decreased by 55 or 80% after the trypsinolysis of native or deglycosylated enzyme, but the activity did not change. Such a high proportion of inactive eNT is unlikely to come from aged enzyme, which suggests the involvement of inactive enzyme in non-catalytic actions.     

Expression of cholinesterases in human kidney and its variation in renal cell carcinoma types

Despite the aberrant expression of cholinesterases in tumours, the question of their possible contribution to tumorigenesis remains unsolved. The identification in kidney of a cholinergic system has paved the way to functional studies, but details on renal cholinesterases are still lacking. To fill the gap and to determine whether cholinesterases are abnormally expressed in renal tumours, paired pieces of normal kidney and renal cell carcinomas (RCCs) were compared for cholinesterase activity and mRNA levels. In studies with papillary RCC (pRCC), conventional RCC, chromophobe RCC, and renal oncocytoma, acetylcholinesterase activity increased in pRCC (3.92 ± 3.01 mU·mg(-1), P = 0.031) and conventional RCC (2.64 ± 1.49 mU·mg(-1), P = 0.047) with respect to their controls (1.52 ± 0.92 and 1.57 ± 0.44 mU·mg(-1)). Butyrylcholinesterase activity increased in pRCC (5.12 ± 2.61 versus 2.73 ± 1.15 mU·mg(-1), P = 0.031). Glycosylphosphatidylinositol-linked acetylcholinesterase dimers and hydrophilic butyrylcholinesterase tetramers predominated in control and cancerous kidney. Acetylcholinesterase mRNAs with exons E1c and E1e, 3'-alternative T, H and R acetylcholinesterase mRNAs and butyrylcholinesterase mRNA were identified in kidney. The levels of acetylcholinesterase and butyrylcholinesterase mRNAs were nearly 1000-fold lower in human kidney than in colon. Whereas kidney and renal tumours showed comparable levels of acetylcholinesterase mRNA, the content of butyrylcholinesterase mRNA was increased 10-fold in pRCC. The presence of acetylcholinesterase and butyrylcholinesterase mRNAs in kidney supports their synthesis in the organ itself, and the prevalence of glycosylphosphatidylinositol-anchored acetylcholinesterase explains the splicing to acetylcholinesterase-H mRNA. The consequences of butyrylcholinesterase upregulation for pRCC growth are discussed.     

Circadian and ultradian activity rhythms in manatee (Trichechus manatus manatus) in captivity

In this study, we show temporal organization of activity patterns in larger temporal series recording. The objective of this study was to determine the temporal pattern of the rest-activity rhythm in manatee (Trichechus manatus manatus) in captivity. Activity recordings were programmed from August 2010 to September 2011 with actimetry devices, and behavior recordings were conducted in dry and rainy seasons. We showed that the marine manatee presents a complex temporal organization, in which the rest-activity rhythm comprises several frequencies with a predominant circadian component and multiple ultradian components. Our results indicate that the animals were more active during the day with respect to the night. The temporal organization of this cycle entails multiple frequencies that include ultradian rhythms, which may be expressions generated by physiological needs, such as food availability and thermoregulatory requirements. These patterns should be taken into consideration for future studies of biological rhythms in manatee.     

Sleep and rest activity measured by wrist actigraphy in an HIV-infected patient with Steven–Johnson syndrome

The Steven–Johnson syndrome (SJS) is characterized by a sudden onset of mucous membrane erosion (predominantly oral mucosa, lips, and conjunctivae) with widespread blistering of the skin involving up to 10% of the body surface area. It is almost always a drug-related reaction, although it can be caused by infections and immunizations. A 33-year-old man with recent diagnosis of HIV infection developed antiretroviral treatment (ART)-associated SJS. Physical activity and sleep parameters were recorded by wrist actigraphy in four different consecutive scenarios: baseline assessment, first ART regimen, hospitalization, and second ART regimen. Significant differences were observed in physical activity patterns between the four phases. No differences in sleep parameters were found. To our knowledge, this is the first study recording physical activity changes and sleep during a SJS reaction.     

Molecular properties of acetylcholinesterase in mouse spleen

The presence of acetylcholinesterase (AChE) mRNA and activity in the tissues and cells involved in immune responses prompted us to investigate the level and pattern of AChE components in spleen. AChE activity was higher in mouse spleen (0.46 +/- 0.13 micromol of acetylthiocholine split per hour and per mg protein) than in muscle or heart, but lower than in brain. The spleen was essentially free of butyrylcholinesterase (BuChE) activity. About 40% of spleen AChE was extracted with a saline buffer, and a further 40% with 1% Triton X-100. Sedimentation analyses, the splitting of subunits in AChE dimers, phosphatidylinositol-specific phospholipase C (PIPLC) exposure, and phenyl-agarose chromatography showed that hydrophilic (G1H, 43%) and amphiphilic AChE monomers (G1A, 36%), as well as amphiphilic dimers (G2A, 21%), occurred in spleen. All these molecules bound to fasciculin-2-Sepharose, although the extent of binding was higher for G1H (77%) than for G1A (63%) or G2A (48%) forms. Differences in the extent to which wheat germ lectin (WGA) adsorbed with AChE of mouse spleen and of erythrocyte allowed us to discard the blood origin of spleen AChE activity. A 62 kDa protein was labeled in spleen samples using antibodies against human AChE. The protein was attributed to AChE monomers since its size was the same, regardless of whether disulfide bonds were reduced or not. Since cholinergic stimulation modulates proliferation/maturation of lymphoid cells, AChE may be important for regulating the level of acetylcholine (ACh) in the neighborhood of cholinergic receptors (AChR) in spleen and other lymphoid tissues.     

Muscular dystrophy by merosin deficiency decreases acetylcholinesterase activity in thymus of Lama2dy mice

Half of congenital muscular dystrophy cases arise from laminin alpha2 (merosin) deficiency, and merosin-deficient mice (Lama2dy) exhibit a dystrophic phenotype. The abnormal development of thymus in Lama2dy mice, the occurrence of acetylcholinesterase (AChE) in the gland and the impaired distribution of AChE molecules in skeletal muscle of the mouse mutant prompted us to compare the levels of AChE mRNAs and enzyme species in thymus of control and Lama2dy mice. AChE activity in normal thymus (mean +/- SD 1.42 +/- 0.28 micromol acetylthiocholine/h/mg protein, U/mg) was decreased by approximately 50% in dystrophic thymus (0.77 +/- 0.23 U/mg) (p = 0.007), whereas butyrylcholinesterase activity was little affected. RT-PCR assays revealed variable levels of R, H and T AChE mRNAs in thymus, bone marrow and spinal cord. Control thymus contained amphiphilic AChE dimers (G2A, 64%) and monomers (G1A, 19%), as well as hydrophilic tetramers (G4H, 9%) and monomers (G1H, 8%). The dimers consisted of glycosylphosphatidylinositol-anchored H subunits. Western blot assays with anti-AChE antibodies suggested the occurrence of inactive AChE in mouse thymus. Despite the decrease in AChE activity in Lama2dy thymus, no differences between thymuses from control and dystrophic mice were observed in the distribution of AChE forms, phosphatidylinositol-specific phospholipase C sensitivity, binding to lectins and size of AChE subunits.     

Variations in the locomotor activity of the Mexican wolf (Canis lupus baileyi) respect to moon periodicity: a study in an outdoor enclosure

It is assumed that the response of the regulatory system of mammal activity depends on the changes in light intensity throughout the 24-h cycle. The aim of this study was to determine whether the moon luminosity cycle exerts an effect on the locomotor activity of the Mexican wolf (C. lupus baileyi). Data collection was carried out with the actimetry, of 11 individuals were analyzed using ANOVA to determine the effect of the lunar cycle. Significant differences were encountered between moon phases (p = 0.001), with a decrement of activity during new and full moon. However, effects were dependent also on the age of the individuals and the daylight period. On the other hand, it is a possible regulation of the activity pattern by the effect of lunar periodicity. This periodicity needs a more detailed examination to determine its adaptive function.     

The level of aryl acylamidase activity displayed by human butyrylcholinesterase depends on its molecular distribution

Butyrylcholinesterase (BuChE) and acetylcholinesterase (AChE) display both esterase and aryl acylamidase (AAA) activities. Their AAA activity can be measured using o-nitroacetanilide (ONA). In human samples depleted of acetylcholinesterase, we noticed that the ratio of amidase to esterase activities varied depending on the source, despite both activities being due to BuChE. Searching for an explanation, we compared the activities of BuChE molecular forms in samples of human colon, kidney and serum, and observed that BuChE monomers (G(1)) hydrolyzed o-nitroacetanilide much faster than tetramers (G(4)). This fact suggested that association might cause differences in the AAA site between single and polymerized subunits. This and other post-translational modifications in BuChE subunits probably determine their level of AAA activity. The higher amidase activity of monomers could justify the presence of single BuChE subunits in cells as a way to preserve the AAA activity of BuChE, which could be lost by oligomerization.