Enzymatic determination of choline acetyltransferase by coenzyme A cycling and its application to analysis of single mammalian neurons

Abstract: An enzymatic assay for choline acetyltrans-ferase was developed by measuring acetyl-coenzyme A (acetyl-CoA) formed from CoASH and acetylcholine (ACh). This method is extremely sensitive and may be applied to the analysis of microgram to nanogram crude samples. The method is, however, not useful when choline acetyltransferase is present in very low concentrations. The basis of this method is to amplify a small amount of synthesized acetyl-CoA in the assay mixture by using an enzymatic amplification reaction, CoA cycling. This amplification mechanism made it possible to perform microassays (13 nl-2.2 μl of assay volume) of freeze-dried sections prepared from cerebral cortex, striatum, and hippocampus of mice and single cell bodies isolated from freeze-dried sections of rabbit spinal cords. These samples were weighed and added directly to the reaction mixture. The activities of the above cerebral regions, assayed with 1,500–2,000-fold amplification, corresponded well to the results previously reported by other workers. The average activity of single anterior horn cells, determined with 64,000–420,000-fold amplification, was 40-fold higher than that of rabbit cerebral cortex, and the specific activities on a dry weight basis were widely distributed among individual neurons. No activity was detected in the noncholinergic dorsal root ganglion cells or in cerebellar cortex.     

In Vivo Effects of β-Bungarotoxin on the Acetylcholine System in Different Brain Areas of the Rat

The in vivo effects of beta-bungarotoxin (beta-BT) on the acetylcholine (ACh) system were studied in the whole cerebrum and in different brain regions. The effect of beta-BT on cerebral ACh and choline (Ch) contents was time-dependent. The results show that a single intracerebroventricular injection of 1 microgram toxin increased both the ACh and Ch contents in the cortex, hippocampus, and cerebellum, while in the striatum the ACh level was decreased. Ten nanograms of toxin injected into the lateral ventricle twice, on the first and third days, led to a reduced ACh level 2 days after the last treatment. In animals treated with the same dose three times, on the first, third, and fifth days, and sacrificed 2 days after the last injection, the choline acetyltransferase and acetylcholinesterase activities were reduced and the number of muscarinic acetylcholine receptors was decreased. A biphasic effect of the toxin was therefore demonstrated. It is suggested that in the first phase of the toxin effect the increased levels of ACh and Ch may be due to the inhibition of neuronal transmission, while in the second phase, when the elements of the ACh system are reduced, the neuronal degenerating effect of beta-BT plays a significant role.     

Molecular characterization of two newDrosophila melanogaster homologs of human TAFII30

Two new homologs of human (h) TAF_II30, dTAF_II16 and dTAF_II24 were revealed inDrosophila melanogaster. The proteins are encoded by neighboring genes and bind with the TATA-binding protein and other dTAF_II proteins involved in TFIID. Only dTAF_II24 interacts with GCN5 histone acetyltransferase (HAT), which is the first demonstration of the TAF_II-GCN5-HAT complex inD. melanogaster. The two proteins have both common and individual location sites on polytene chromosomes. Possibly, the functions of dTAF_II16 and dTAF_II24 are similar but not identical.     

Effects of Extracellular Choline Concentration and K+ Depolarization on Choline Kinase and Choline Acetyltransferase Activities in Superior Cervical Sympathetic Ganglia Excised from Rats

The activities of choline kinase (CK) and choline acetyltransferase (ChAT) were examined in vitro in superior cervical sympathetic ganglia (SCG) excised from rats following aerobic incubation for 1 h in a medium containing various choline concentrations, with and without application of a high KCl level (70 mM). Ganglionic CK activity was strongly inhibited (by approximately 75%) at low extracellular choline concentrations (1-5 microM) but rose as the choline concentration was raised to 10-50 microM in the incubation medium, then fell and rose again with further increases in choline concentration. A similar but moderate accelerative effect on ganglionic CK activity was also observed after addition of acetylcholine (ACh; 1 mM) without eserine. Whereas specific CK activity did not change significantly in axotomized SCG, in which the ratio of glial cells to neurons is greatly increased for a week after the operation., it was remarkably increased after denervation, in which the preganglionic cholinergic nerve terminals had degenerated. When either a high KCl level or hemicholinium-3 (HC-3; 50 microM) was added to the medium in the presence or absence of choline, ganglionic CK activity was markedly inhibited. On the other hand, ChAT activity in the SCG remained at a significantly high level during incubation with low choline concentrations (1-10 microM), but the enhanced enzyme activity became inhibited as the extracellular choline concentration was raised to 50-100 microM in the medium. Addition of HC-3 to the medium did not alter ganglionic ChAT activity at low choline concentrations. However, application of quinacrine (10 microM) considerably reduced ganglionic CK activity and also suppressed ChAT activity induced by high KCl levels.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thyroid Hormone Actions on a Cholinergic Neuroblastoma Cell Line (S-20Y)

Thyroid hormone (T3) has a multiplicity of effects on the developing nervous system. We have investigated T3 action using a cholinergic neuroblastoma cell line (S-20Y) as a model. S-20Y contains a nuclear receptor for T3 with binding properties similar to those of other T3 target tissues. In addition, these cells can carry out 5'-deiodination, which is necessary to produce active thyroid hormone in vivo. The enzyme involved in this process appears to be a type I deiodinase, based on its reaction kinetics and its susceptibility to inhibition by propylthiouracil. S-20Y cells maintained in T3-depleted medium showed decreased choline acetyltransferase (ChAT) activity. ChAT activity was restored to the control level in a dose-dependent manner by T3 repletion. Neither cell density nor viability was influenced by the hypothyroid state. The presence of a T3 receptor and the enzyme activity for T3 production, together with an effect of T3 on ChAT activity, demonstrate that S-20Y cells are a target for T3 action and suggest that these cells represent an excellent model system for studies of T3 effects on nervous tissues.     

pH dependence of the multiline, manganese EPR signal for the 'S2' state in PS II particles. Absence of proton release during the S1----S2 electron transfer step of the oxygen evolving system

The pH dependence of oxygen evolution rates, 2,6-dichlorophenolindophenol (DCIP) reduction rates and the intensity of the multiline manganese EPR signal associated with the S2K ok state has been studied using oxygen-evolving spinach (PS) II particles. The oxygen evolution and DCIP reduction rates are found to be very sensitive to pH, with the maximal rates occurring at pH 6.5-7.0. Both the rate and yield of the S2 multiline manganese EPR signal intensity, produced by single flash excitation at room temperature or by continuous illumination at 200 K, are found to be independent of pH, indicating that no proton is released from this manganese site during the S1----S2 electron transfer. These results agree with those from other laboratories showing no proton release on this transition, but using techniques monitoring other species.     

Anatomical mapping of choline acetyltransferase (ChAT)-like and glutamate decarboxylase (GAD)-like immunoreactivity in outer hair cell efferents in adult rats

Summary The distribution of choline acetyltransferase (ChAT)-like and glutamate decarboxylase (GAD)-like immunoreactivity in the cochleae of 15 adult Wistar white rats was investigated using the peroxidase-antiperoxidase (PAP) technique. A monoclonal antibody to ChAT and a polyclonal antiserum to GAD were used. Immunoreaction was investigated quantitatively, in the electron microscope, on tangential sections of the tunnel of Corti and the rows of outer hair cells. ChAT-like and GAD-like immunoreactivity was found in all efferent nerve fibres in the tunnel of Corti and in all efferent synapses on the outer hair cells. A coexistence of ChAT and GAD in the efferent system to the outer hair cells of the rat is therefore assumed.     

Induction of choline acetyltransferase in the neuroblastoma x glioma cell line NG108-15

The mechanism of the induction of choline acetyltransferase activity in the hybrid cell line NG108-15 was studied. Induction by cyclic AMP analogs, forskolin, and prostaglandin E₁ + theophylline was found to be rapid with an increase in choline acetyltransferase specific activity detectable within 8 hrs and maximal after 24 hrs. Immunoblot analysis was used to demonstrate that the increase in choline acetyltransferase specific activity induced by prostaglandin E₁ + theophylline was due to an increase in enzyme protein. Cycloheximide effectively blocked the induction of choline acetyltransferase by prostaglandin E₁ + theophylline. These results demonstrate that the induction of choline acetyltransferase activity involves the synthesis of new enzyme protein. Attempts to measure choline acetyltransferase turnover by blocking its synthesis with cycloheximide indicated that this enzyme is a relatively stable protein with a half-life of greater than 24 hrs.     

Isolation of fetal mouse motor neurons on discontinuous percoll density gradients

Summary The spinal cords of fetal NIH∶CR mice, gestational age Day 12 to 14, were dissected free of meninges and dorsal root ganglia, chemically dissociated, and layered onto discontinuous Percoll gradients at densities 1.040, 1.050, and 1.060 g/ml. After centrifugation (800 Xg for 15 min at 4° C), three morphologically, biochemically, and immunohistologically distinct cell populations were collected from the gradient interfaces. The first interface, located at a density of 1.040 g/ml, was choline acetyltransferase enriched (0.86±0.08) compared to the second and third fractions (0.42±0.01 and 0 pmol acetylcholine synthesized/μg protein, respectively). When simultaneously cultured with fetal mouse cardiac muscle on a gelatin-polylysine-laminin substrate in serum-free medium, these cells developed the characteristics of motor neurons. Dr. Strong is supported in part by a Research Fellowship from the Medical Research Council of Canada.     

A Corynebacterium glutamicum gene encoding a two-domain protein similar to biotin carboxylases and biotin-carboxyl-carrier proteins

Following the analysis of transposon Tn5432-induced mutants of Corynebacterium glutamicum ATCC 13032, a gene encoding a protein with a biotin-binding motif was cloned. The DNA sequence of this gene revealed an open reading frame encoding 591 amino acids with a calculated mol. mass of 63.4 kDa. The protein is composed of two domains, an N-terminal biotin carboxylase and a C-terminal biotin-carboxyl-carrier protein, that are highly similar to corresponding subunits from prokaryotic and eukaryotic biotin enzymes. Over 70% identity was found to a protein from Mycobacterium leprae proposed to be part of an acyl-CoA carboxylase. Since it was not possible to inactivate the C. glutamicum gene, the gene most likely encodes a subunit of the essential acetyl-CoA carboxylase, which catalyzes the committed step in fatty acid synthesis. Received: 2 December 1995 / Accepted: 20 May 1996