Acetylation of Choline and Homocholine by Membrane-Bound Choline-O-Acetyltransferase in Mouse Forebrain Nerve Endings

Abstract: The choline analog homocholine is not acetylated in vitro by choline- O -acetyltransferase (ChAT, EC 2.3.1.6), which is solubilized by 100 mM-sodium phosphate buffer washes of a crude vesicular fraction of mouse forebrain. However, both homocholine and choline are acetylated by a form of ChAT which is nonionically associated with a subcellular fraction of mouse forebrain containing membrane-associated organelles and occluded acetylcho-line (P₄). Acetylation of homocholine by membrane-associated ChAT is saturable. 4-(1-Naphthylvinyl)pyridine (NVP) inhibits the acetylation of both choline (60%) and homocholine (40%) by membrane-associated ChAT but reduces the acetylation of choline alone by soluble ChAT (76%). Choline and homocholine serve as competitive alternative substrates for the same membrane-associated ChAT, whereas homocholine acts only as a competitive inhibitor of choline acetylation by soluble ChAT. Acetylhomocholine competitively inhibits the acetylation of choline by both soluble and membrane-associated ChAT more dramatically than does the natural end product, acetylcholine.     

Immunoassay and Activity of Calcium-Activated Neutral Proteinase (mCANP): Distribution in Soluble and Membrane-Associated Fractions in Human and Mouse Brain

The millimolar form of calcium-activated neutral proteinase (mCANP) is generally regarded as a cytosolic enzyme in nonneuronal systems, although its subcellular localization in brain is less well established. To resolve conflicting reports on the localization of mCANP based on activity measurements, we developed an immunoassay for CANP and compared the content and activity of the molecule in soluble and membrane fractions of mouse and human brain. Western blot immunoassays, using two different antibodies specific for mCANP, demonstrated that mCANP content is 4.5 ng/g in human or mouse brain, about 0.0005% of the total protein. More than 95% of the total immunoreactive mCANP remained in the soluble fraction after 15,000 g centrifugation of the whole homogenate. mCANP activity was determined with [14C]azocasein as substrate after removing endogenous CANP inhibitor(s) by ion-exchange chromatography on DEAE-cellulose. Caseinolytic activity was detected only in fractions derived from the supernatant extract. The distribution of mCANP content and enzyme activity were unchanged when tissues were extracted with different concentrations of Triton X-100. These findings establish the usefulness and validity of the CANP immunoassay and demonstrate that mCANP in mouse and human brain is localized predominantly within the cytosol.     

Postnatal Development of Cholinergic Enzymes and Receptors in Mouse Brain

The developmental profiles for the cholinergic enzymes acetylcholinesterase and choline acetyltransferase, and the muscarinic and nicotinic receptors were determined in whole mouse brain. The enzyme activities (per milligram of protein) increased steadily from birth, reaching adult levels at 20 days of age. These increases were primarily due to increases in Vmax. Muscarinic receptor numbers, measured by [3H]quinuclidinyl benzilate binding, also increased from birth to 25 days of age. Brain nicotinic receptors were measured with the ligands L-[3H]nicotine and alpha-[125I]-bungarotoxin. Neonatal mouse brain had approximately twice the number of alpha-bungarotoxin binding sites found in adult mouse brain. Binding site numbers rose slightly until 10 days of age, after which they decreased to adult values, which were reached at 25 days of age. The nicotine binding site was found in neonatal brain at concentrations comparable to those at the alpha-bungarotoxin site followed by a steady decline in nicotine binding until adult values were reached. Thus, brain nicotinic and muscarinic systems develop in totally different fashions; the quantity of muscarinic receptors increases with age, while the quantity of nicotinic receptors decreases. It is conceivable that nicotinic receptors play an important role in directing the development of the cholinergic system.     

Calcium Effects on the Solubilization of Membrane-Bound Histidine Decarboxylase in the Rat Brain

In a previous work we have shown that histidine decarboxylase (HD) activity is found in a soluble and a membrane-bound form. A major part (82%) of the membrane-bound HD activity in the crude mitochondrial fraction (P2) was present in the synaptic plasma membrane-containing subfraction. Physiological concentrations of Ca2+ had no direct effect on HD activity but caused a solubilization of approximately 50% of membrane-bound HD in the P2 fraction. Mg2+ had similar but lower effects (20% solubilization) than Ca2+. Incubation with depolarizing concentrations of K+ in the presence of 1 mM CaCl2 caused a significant (30%) solubilization of HD.     

Identity Between Cytoplasmic and Membrane-Bound S-100 Proteins Purified from Bovine and Rat Brain

Cytoplasmic and membrane-bound S-100 proteins were purified to homogeneity from bovine and rat brain. Cytoplasmic and membrane-bound S-100 from single species are identical by immunological, electrophoretic, spectrophotometric, and functional criteria. Cytoplasmic and membrane-bound S-100 from bovine brain consists of nearly equal amounts of S-100a and S-100b, whereas cytoplasmic and membrane-bound S-100 from rat brain consists mostly of S-100b. The functional role of membrane-bound S-100 remains to be elucidated.     

Multiple Forms of Choline-O-Acetyltransferase in Mouse and Rat Brain: Solubilization and Characterization

Three forms of acetyl coenzyme A: choline-O-acetyltransferase (EC 2.3.1.6, ChAT) have been isolated from mouse and rat forebrain synaptosomes with a 100 mM sodium phosphate (NaP) buffer of pH 7.4, a high-salt solution (500 mM NaCl), and a 2% Triton DN-65 solution, respectively. The Triton-solubilized form of ChAT differed from the other two forms in its capacity to acetylate homocholine, its pH profile, and its sensitivity to denaturation. NaCl-solubilized ChAT could be distinguished from the other two forms with respect to pH profile, sensitivity to inhibition by 4-(1-naphthylvinyl) pyridine (in the presence of Triton), and apparent Km value for choline acetylation. The caudate and putamen of rat brain contained the highest amount of ChAT activity, based on tissue wet weight, and the cerebellum contained the least of the brain regions examined; only the cerebellum had more membrane-bound than soluble ChAT. Septal lesion reduced ChAT activity in the NaP- and Triton-solubilized fractions prepared from hippocampus by 68% and 64%, respectively, whereas it reduced the activity of the NaCl-solubilized fraction by only 21%. These results suggest that three different forms of ChAT may exist in both mouse and rat brain.     

Cytosolic Choline Acetyltransferase Binds Specifically to Cholinergic Plasma Membrane of Rat Brain Synaptosomes to Generate Membrane-Bound Enzyme

Uncovering the way membrane-bound choline acetyltransferase (ChAT) interacts with membranes and with which membrane in cholinergic neurons may help in understanding its role in acetylcholine metabolism. Subfractionation of rat hippocampal synaptosomes aiming to separate synaptic vesicles from plasma membranes shows that membrane-bound ChAT is bound to plasma membrane. Either detergents or urea and alkali can solubilize membrane-bound enzyme. Detergent-solubilized enzyme has a higher sedimentation rate than urea-alkali solubilized or cytosolic ChAT. Once dissociated, membrane-bound ChAT reassociates specifically with cholinergic plasma membranes, a process that was abolished by previous treatment of membranes with trypsin. Cytosolic ChAT behaves similarly. Thus, in cholinergic synaptosomes, ChAT exists as cytosolic and peripheral activity. Cytosolic ChAT generates peripheral enzyme most probably by interacting with a protein of plasma membrane of cholinergic nerve terminals. This receptor protein might regulate the amount of membrane-bound ChAT in cholinergic neurons.     

Developmental Regulation of Microtubule-Associated Protein 2 Expression in Regions of Mouse Brain

The relative levels of microtubule-associated protein 2(MAP2) were determined during postnatal development of the mouse in six different discrete brain regions: cerebellum, cortex, hippocampus, olfactory bulb, brainstem, and hypothalamus. Brain homogenates were electrophoresed on sodium dodecyl sulfate-containing gels and analyzed by immunoblotting with MAP2-specific antibodies. The levels of MAP2 in each region were determined using radiolabeled secondary antibodies and densitometric quantification of the autoradiograms over a range that was determined to have a linear response. The results indicated that in all regions and at all ages there was only one high-molecular-weight polypeptide of MAP2, which did not change in electrophoretic mobility after dephosphorylation. In most regions, the levels of MAP2 increased during the first 2 postnatal weeks. However, there were differences in the time course and relative levels of MAP2 between regions. In addition, all regions of the brain expressed the low-molecular-weight form of MAP2 (MAP2c) that was present at birth as a heterogeneous group of polypeptides with an apparent molecular weight of 70K. Most of the heterogeneity of MAP2c, however, was eliminated after dephosphorylation. The levels of MAP2c decreased dramatically after 2 weeks postnatally, except for the olfactory bulb, where the levels of MAP2c remained relatively high even in adults.     

Kinetic Studies of Mouse Brain Transketolase

Abstract: The activity of transketolase in mouse brain was 5.7 nmol/min/mg protein measured by an enzyme-coupled spectrophotometric assay. The apparent K_m for ribose-5-phosphate was 330 μ M , for d -xylulose-5-phosphate was 120 μ M , and for thiamine pyrophosphate was 7 μ M . However, thiamine pyrophosphate remained tightly bound to transketolase in homogenates in which it dissociated completely from another thiamine pyrophosphate- dependent enzyme, the pyruvate dehydrogenase complex. These data suggest that loss of transketolase activity is likely to be a later consequence of thiamine deficiency in mammalian brain than is decreased activity of pyruvate dehydrogenase complex.     

Properties and Ontogenic Development of Membrane-Bound Histidine Decarboxylase from Rat Brain

Abstract: Histidine decarboxylase (HD) activity was determined in high-speed fractions (100,000 g for 60 min) obtained from whole rat brain homogenates. Twenty-eight percent of the HD activity was associated with membranes, and the remaining was soluble. Several properties of the soluble and membrane-bound HD were compared. No significant differences in the values of K _m for histidine and pyridoxal 5'-phosphate were observed. The solubilization of membrane-bound HD with Triton X-100 resulted in an increase of 60% over the nonsolubilized activity with no changes in the K _m for substrate and cofactor. The proportion of free pyridoxal 5'-phosphate-independent activity was identical in both fractions. The soluble and membrane-bound forms of the enzyme differ slightly in their pH-activity profiles, although both enzymes showed an optimum pH near 6.5. The HD activities present in soluble and membrane fractions were determined at different postnatal ages. The soluble activity increased until day 90, whereas the membrane-bound activity became stabilized from day 20.     

Prenatal Monosodium Glutamate Causes Long-Lasting Cholinergic and Adrenergic Changes in Various Brain Regions

Prenatal monosodium glutamate (MSG) given through the mother's diet was found previously to cause behavioral changes in the offspring, including learning disabilities. In the present study, neurochemical parameters were measured in the brains of prenatally exposed rats at various ages throughout development up to adulthood. At 15 days of age, choline uptake and choline acetyltransferase (ChAT) activity in the frontal cortex were significantly reduced (by 80 and 25%, respectively) in MSG-exposed animals, whereas the same cholinergic parameters in hippocampus were not changed. During later development, choline uptake gradually increased, until in adulthood it became significantly higher in MSG-exposed animals than in the controls. This enhancement was found in both males and females. Our previous study showed that only the male offspring were learning disabled. Choline uptake and ChAT activity were enhanced in the hippocampus of adult male animals. Norepinephrine (NE) uptake was reduced (by 25%) in the frontal cortex of males only. There was no change in NE uptake in the hypothalamus.     

不同年龄小白鼠全脑细胞质氨酰tRNA合成酶活力的比较

从不同年龄(20天,30天,1年)的小白鼠全脑制得细胞质混合氨酰tRNA合成酶。用异源体系(即用酵母tRNA和小白鼠全脑氨酰tRNA合成酶)测定了氨酰tRNA合成酶分别载运~3H标记的Asp、Gly、Glu、Lys和Ala的活力。结果表明除未检出tRNA~(Glu)的合成酶活力外,对其余四种氨基酸都有明显的活力,特别是年龄20天小白鼠的氨酰tRNA合成酶对~3H-Gly具有高达35％的载运活力。对~3H-Gly、~3H-Lys和~3H-Ala的载运活力有随增龄而下降的趋势,但对~3H-Asp的载运活力则随年龄增长而增高。     

Arginine Metabolism in Mouse Brain Synaptosomes

In a study employing mouse brain synaptosomes and synaptosomal sonicates, the complete metabolic machinery was found to be present for transport of arginine into synaptosomes, its conversion to ornithine, and the formation from the latter of glutamic acid, gamma-aminobutyric acid, and proline. The results show that a delicate balance probably exists between the flows of metabolites. This balance, which probably determines the steady-state levels of these substances in nerve terminals, can be altered by concentrations of the metabolites themselves through feedback inhibition as well as by levels of cofactors.     

Characterization of Mono- and Polyclonal Antibodies Against Highly Purified Choline Acetyltransferase: A Monoclonal Antibody Shows Reactivity in Human Brain

Choline acetyltransferase (ChAT) from porcine brain was purified by immunoaffinity chromatography, and the highly purified enzyme was subsequently used for immunization of mice and rabbits. After fusion of mouse spleen cells, 32 cultures producing monoclonal antibodies directed against ChAT were detected by an enzyme-linked immunosorbent assay (ELISA) with immunoaffinity-purified ChAT. Of these original 32, the most active 11 cultures were cloned and used for ascites production. The 11 clones generated monoclonal antibodies of the immunoglobulin (Ig) M class (three), the IgG1 subclass (seven), and the IgG2b subclass (one). The isoelectric points of the antibodies of the IgG class were different in each case. The monoclonal antibodies exhibited different binding characteristics in the above ELISA and on western blots. Two monoclonal antibodies demonstrated excellent immunohistological results with neurons of rat brain and spinal cord. One of them reacted well immunohistochemically with neurons of human brain and also recognized partially purified human placenta ChAT in the ELISA.     

MDMPR:小鼠发育代谢表型库

小鼠发育代谢表型库(Mouse Developmental and Metabolic Phenotype Repository,MDMPR)是一个致力于小鼠资源和表型数据实时共享的开放性平台,它依托于科技部重点研发计划“发育编程及其代谢调节”专项项目“建立小鼠发育代谢表型库”。该项目预计在5年内完成500个发育代谢相关小鼠敲除模型的建立,并对其表型数据进行标准化的解析、建立表型数据库。MDMPR作为一个资源及数据集成的库,由多个子系统作为支撑,包括ES细胞数据库、项目管理系统、繁育管理系统、精子库管理系统、表型分析系统,信息化管理深入到项目中每个环节,从基因突变ES细胞制备、基因突变小鼠制备、小鼠繁育,精子冻存到最终的表型分析、数据处理及展示,保证了MDMPR产生数据的真实性及实时性。MDMPR除了不断地推进项目进行,增加自身产生的数据外,也在积极的整合其他的资源及数据,如人特异性基因敲除ES细胞库、蛋白相互作用数据库(STRING)、核心转录调节环路(dbCoRc)和Enhancer-Indel数据库,今后还将进一步整合,帮助发育代谢及其他领域的研究人员能够一站式的获取所需资源和数据、加快研究进程,最终服务于全人类的医疗事业。     

Developmental Expression of the Glycine Transporters GLYT1 and GLYT2 in Mouse Brain

Abstract: Using immunocytochemical localization, the distribution of the glycine transporters GLYT1 and GLYT2 in the developing mouse brain was studied. GLYT1 and GLYT2 immunoreactivity begins during the period of fiber outgrow and synaptogenesis. GLYT2 is first expressed in spinal and spinothalamic white matter and is followed by the expression of synaptophysin. In the postnatal stages, GLYT2 staining in the white matter disappears, and a punctuated pattern in the gray matter emerges. In contrast, in the fetal brain GLYT1 immunoreactivity coincides with gray matter neuropil and processes of radial glia. GLYT1 is distributed over a much wider area of the brain than GLYT2. However, the distribution of these two GLYTs implies that GLYT1 and GLYT2 operate in concert within the area where both are present. At the day 12 embryo stage, GLYT1 antibodies stain the liver, and later they also react with the pancreas and the gastroduodenal junction. No other organs exhibit significant GLYT1 immunoreactivity. We additionally observed the presence of GLYT1 in rat fetal cerebral cortex and hippocampus, which was not detected in fetal mouse brain. Moreover, GLYT1 immunoreactivity was found in the mouse floor plate and the ventral commissure but was not present in the same regions in rats. These findings suggest possible differences in the expression of GLYT1 between these two species.     

Immunoaffinity Purification of Human Choline Acetyltransferase: Comparison of the Brain and Placental Enzymes

A rapid and efficient immunoaffinity purification procedure has been developed for human placental choline acetyltransferase (ChAT). Using this procedure, human placental ChAT was purified to homogeneity with high recovery of enzyme activity (50-60%). Purified ChAT was used to raise a monospecific anti-human ChAT polyclonal antibody in rabbits. A comparison of the physical properties of ChAT was made between the enzymes purified from human brain and human placenta. Only one form of the enzyme exists in either tissue, having identical molecular weights of 68,000 and a single apparent pI of 8.1. A more detailed comparison of the two enzymes using peptide mapping and epitope mapping indicates identity between the brain and placental enzymes.     

Distribution of Activity Converting 4-Aminobutyraldehyde to γ-Aminobutyric Acid in Subcellular Fractions of Mouse Brain

The subcellular distribution of activity of 4-aminobutyraldehyde dehydrogenase (ABAL-DH) was studied in mouse brain. ABAL-DH was localized mainly in the crude mitochondrial fraction; most of the activity in this fraction was found in the subfraction containing synaptosomes, and the remainder was in the mitochondrial fraction. After osmotic disruption of synaptosomes, most of the activity was located in the synaptic cytosol, and the remainder was in the synaptic mitochondria. Sucrose density subfractionation of synaptosomes revealed that gamma-aminobutyric acid, glutamic acid decarboxylase, and ABAL-DH localized in a denser region of gradient fraction than the region containing acetylcholinesterase and choline acetyltransferase.