Rat cerebral-cortex adenosine deaminase activity and its subcellular distribution.
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ABSTRACT:
SUBMITTER: Pull I
PROVIDER: S-EPMC1168461 | biostudies-other | 1974 Oct
REPOSITORIES: biostudies-other
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Project description:1. Rat cerebral-cortex slices were incubated with (32)P(i), acetylcholine and eserine for periods of 10min and 2h. The specific radioactivity of phosphatidylinositol was elevated during these treatments by 36 and 106% respectively. 2. The specific radioactivities of the phosphatidylinositol in different cell structures were determined after subcellular fractionation. They were highest in the nuclear, microsomal and synaptic-vesicle fractions and lowest in myelin, both in the controls and in the acetylcholine-treated slices. 3. The stimulated labelling of phosphatidylinositol was relatively evenly distributed: no subcellular fraction showed a stimulation markedly higher than that in the homogenate. 4. Studies of the distributions and activities of marker enzymes indicated that the subcellular fractionation achieved was similar to that with fresh tissue. 5. The results are discussed in relation to the previous report that the stimulation is observed throughout the neuronal cell-bodies and in relation to the hypothesis that the labelled phosphatidylinositol produced by stimulation is a component of an acetylcholine-receptor proteolipid localized in the synaptic junction.
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Project description:1. Guanylate cyclase of every fraction studied showed an absolute requirement for Mn2+ ions for optimal activity; with Mg2+ or Ca2+ reaction was barely detectable. Triton X-100 stimulated the particulate enzyme much more than the supernatant enzyme and solubilized the particulate-enzyme activity. 2. Substantial amounts of guanylate cyclase were recovered with the washed particulate fractions of cardiac muscle (63-98%), skeletal muscle (77-93%), cerebral cortex (62-88%) and liver (60-75%) of various species. The supernatants of these tissues contained 7-38% of total activities. In frog heart, the bulk of guanylate cyclase was present in the supernatant fluid. 3. Plasma-membrane fractions contained 26, 21, 22 and 40% respectively of the total homogenate guanylate cyclase activities present in skeletal muscle (rabbit), cardiac muscle (guinea pig), liver (rat) and cerebral cortex (rat). In each case, the specific activity of this enzyme in plasma membranes showed a five- to ten-fold enrichment when compared with homogenate specific activity. 4. These results suggest that guanylate cyclase, like adenylate cyclase, and ouabain-sensitive Na+ + K+-dependent ATPase (adenosine triphosphatase), is associated with the surface membranes of cardiac muscle, skeletal muscle, liver and cerebral cortex; however, considerable activities are also present in the supernatant fractions of these tissues which contain very little adenylate cyclase or ouabain-sensitive Na+ + K+-dependent ATPase activities.
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Project description:Background:Loss of UBE3A causes Angelman syndrome, whereas excess UBE3A activity appears to increase the risk for autism. Despite this powerful association with neurodevelopmental disorders, there is still much to be learned about UBE3A, including its cellular and subcellular organization in the human brain. The issue is important, since UBE3A's localization is integral to its function. Methods:We used light and electron microscopic immunohistochemistry to study the cellular and subcellular distribution of UBE3A in the adult human cerebral cortex. Experiments were performed on multiple tissue sources, but our results focused on optimally preserved material, using surgically resected human temporal cortex of high ultrastructural quality from nine individuals. Results:We demonstrate that UBE3A is expressed in both glutamatergic and GABAergic neurons, and to a lesser extent in glial cells. We find that UBE3A in neurons has a non-uniform subcellular distribution. In somata, UBE3A preferentially concentrates in euchromatin-rich domains within the nucleus. Electron microscopy reveals that labeling concentrates in the head and neck of dendritic spines and is excluded from the PSD. Strongest labeling within the neuropil was found in axon terminals. Conclusions:By highlighting the subcellular compartments in which UBE3A is likely to function in the human neocortex, our data provide insight into the diverse functional capacities of this E3 ligase. These anatomical data may help to elucidate the role of UBE3A in Angelman syndrome and autism spectrum disorder.
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Project description:1. Slices of rat cerebral cortex, incubated anaerobically at 37 degrees , lost K(+) from an initial concentration of 102m-equiv./kg. to a concentration of 57m-equiv./kg. after 10min. On subsequent aerobic incubation they regained K(+) rapidly at a rate that varied with the K(+) concentration of the medium. 2. Lower aliphatic alcohols, present at equal thermodynamic activity, produced approximately equal degrees of inhibition of K(+) uptake during the aerobic incubation. This inhibition was reduced by an increase in K(+) content of the medium. Ethanol did not affect the rate of K(+) loss during anaerobic incubation. 3. Li(+), in concentrations of 1-10mm, also inhibited K(+) uptake by brain-cortex slices, the degree of inhibition varying with the Li(+) concentration. Ouabain also inhibited K(+) uptake. 4. The same series of alcohols, at equal thermodynamic activity, produced comparable degrees of inhibition of Na(+),K(+),Mg(2+)-stimulated adenosine-triphosphatase activity in brain microsomes. 5. It is suggested that inhibition of cation transport is an important, but not a primary, mechanism in the production of central nervous depression by alcohols and other substances.
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Project description:The enzymatic conversion of isothiazolo[4,3-d]pyrimidine-based adenosine (tz A) and 2-aminoadenosine (tz 2-AA) analogues to the corresponding isothiazolo[4,3-d]pyrimidine-based inosine (tz I) and guanosine (tz G) derivatives is evaluated and compared to the conversion of native adenosine to inosine. Henri-Michaelis-Menten analyses provides the foundation for a high-throughput screening assay, and the efficacy of the assay is showcased by fluorescence-based analysis of tz A conversion to tz I in the presence of known and newly synthesized inhibitors.
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Project description:Pre-incubation of various subcellular fractions from rat brain caused a significant decrease in the phosphorylation of individual polypeptides. The rate and extent of this loss of labelling was not uniform and polypeptides whose phosphorylation was independent of cyclic AMP were primarily affected, whereas substrate availability remained unaltered. It is recommended that pre-incubation effects must be carefully monitored if valid conclusions are to be made about the physiological relevance of changes in protein phosphorylation observed in vitro.
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Project description:1. The subcellular distribution of the phosphoprotein fraction of grey matter from guinea-pig brain has been determined. Phosphoproteins occurred in all fractions of the tissue, the mitochondrial fraction containing 29%, supernatant 26%, microsomal fraction 23% and nuclear fraction 15%. The quantity of phosphoproteins per unit quantity of protein was highest in the microsomal fraction and lowest (by a factor of nearly 50%) in the mitochondrial fraction. 2. Observations are reported on the nature of the nuclear subfraction believed from earlier work to contain a phosphoprotein sensitive to the effects of electrical pulses applied to cerebral slices in vitro. With respect to its content of phosphoprotein, cholesterol, ribonucleic acid and succinate-dehydrogenase activity, the subfraction resembles the nerve-ending fraction of the mitochondrial fraction, from which it appears to be derived by cross-contamination. Electron microscopy tended to confirm this impression. Reasons are adduced for discounting the earlier claim that the sensitive phosphoprotein is contained in a new subcellular structure distinct from nuclei, mitochondria or microsomes.
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Project description:BackgroundHuman adenosine deaminase 2 (ADA2) is an extracellular enzyme that negatively regulates adenosine-mediated cell signaling by converting adenosine to inosine. Altered ADA2 enzyme activity has been associated with some viral infections and rheumatic diseases. The potential utility of ADA2 as a biomarker is, however, limited by the absence of established ranges of ADA2 concentration and enzyme activity in the healthy population. It is known that ADA2 enzyme activity is lower in adults, but when (and why) this decline happens is not known. The purpose of this study was to establish normative ranges of ADA2 enzyme activity and protein concentration in the healthy pediatric population.MethodsWe modified a commercially available ADA2 enzyme activity assay to enable higher throughput analysis of fresh, frozen and hemolyzed blood samples. With this assay and ADA2 protein immunoblotting, we analyzed ADA2 enzyme activity and protein concentration in blood plasma from a cohort of children and adolescents (n = 94) aged 5 months to 18 years. One-way ANOVA with subsequent Tukey multiple comparison test was used to analyze group differences. Reference intervals were generated using the central 95% of the population (2-97.5 percentiles).ResultsADA2 enzyme activity was consistent in fresh, frozen, and hemolyzed sera and plasma as measured by our modified assay. Analysis of plasma samples from the healthy pediatric cohort revealed that ADA2 enzyme activity is significantly lower in older children than in younger children (p < 0.0001). In contrast, there was no significant correlation between ADA2 protein concentration and either age or ADA2 enzyme activity.ConclusionWe observed that ADA2 enzyme activity, but not ADA2 protein concentration, negatively correlates with age in a cohort of children and adolescents. Our findings stress the importance of appropriate age-matched controls for assessing ADA2 enzyme activity in the clinical setting.
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Project description:1. Breakdown of phosphatidylinositol was studied in homogenates and subcellular fractions of rat cerebral cortex by using both membrane-bound and externally added [(32)P]phosphatidylinositol as substrate. 2. In the presence of deoxycholate breakdown followed first-order kinetics at low substrate concentrations ([unk]1mm) and zero-order kinetics at higher concentrations (6-9mm). 3. Maximum breakdown by cerebral-cortex homogenates was approximately 0.5mumol/h per mg of protein and occurred at pH7.0 in the presence of 8mm-phosphatidylinositol, 2mm-CaCl(2) and 2mg of deoxycholate/ml. Activity was abolished by 1mm-ethanedioxybis(ethylamine)tetra-acetate. 4. The products of phosphatidylinositol breakdown were 1,2-diacylglycerol and a mixture of d-myoinositol 1:2-cyclic phosphate (55%) and d-myoinositol 1-phosphate (45%). The two phosphate esters appeared to be produced together and in constant proportions. 5. Some 51% of the activity was particle-bound, with the highest activities in small nerve endings, microsomal material and two synaptic membrane fractions (fractions Mic(20), Mic(100), M(1) 1.0 and M(1) 0.9 respectively), all of which were also rich in acetylcholinesterase and which have been shown to be rich in other surface-membrane enzymes. Much of the particle-bound activity therefore appears to be present in cerebral-cortex plasma membranes. 6. The results are discussed in relation to previously described soluble activities that catalyse the same reaction, and to a possible role of the membrane-bound enzyme in enhanced phosphatidylinositol turnover in externally stimulated cells.
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