CHOLINE acetyltransferase (acetyl-CoA: choline 0-acetyltransferase, I.U.B. 2.3.1.6) is an enzyme of major interest in neurochemistry. Until recently, studies with this enzyme were limited by the availability of the bioassays, described by CHANG and GADDUM (1933) and the much less sensitive colorimetric method of HESTRIN (1949). SCHUBERTH (1963, 1966) developed a radiochemical assay for the enzyme, but the method required an acetyl-CoA generating system since it did not distinguish acetyl-CoA from ACh. Selective precipitation methods were developed by MCCAMAN and HUNT (1965) and ALPERT, KISLIUK and SHUSTER (1966), in which ACh is precipitated as the reineckate, and by FONNUM (1966), in which the tetraphenylborate salt is formed. These precipitation methods are specific and their sensitivity is limited only by the available specific activity of the substrate, but the tedium of washing and centrifuging the precipitated product remains. A gas chromatographic method designed by STAVINOHA and RYAN (1965) is somewhat cumbersome for several concurrent assays or for use in enzyme purification studies.We have designed and evaluated an assay method for choline acetyltransferase which is rapid and precise. It was originally developed for use with the enzyme from Lactobacillus arabinosis (STEPHENSON and ROWATT, 1947; GIRVIN and STEPHENSON, 1954; ALPERT et al., 1966), and has been applied with equal facility to the enzyme from mouse brain. The method takes advantage of the fact that the enzyme transfers an acetyl moiety from an anionic substrate, acetyl-CoA, to a cationic substrate, choline, resulting in a cationic product, ACh. The two classes of ions can be separated readily by use of an anion exchange resin. If the acetyl moiety of the substrate is labelled with radioactive carbon, the incubation mixture can be applied to a small column of the resin, and the radioactive product, which passes through the column, can be determined directly by scintillation counting. MATERIALS A N D METHODSA stock culture of LactobacilIus planfarum No. 8014 (synonym: Lactobacillus arabinosus 17-5 was kindly provided by A. Alpert, and was maintained on a synthetic medium. A 140 gal batch of these bacteria was cultured for us by the New England Enzyme Center. Dowex 1 X 8 chloride (200-400 mesh) was purchased from J. T. Baker, Phillipsburg, N.J., and washed 4 times with distilled water before use. Male C57 B1/6J mice were obtained from Jackson Laboratories, Bar Harbor, Maine. [l-*4C1Acetyl-CoA (0.5-1 .O mc/m-mole) was purchased from New England Nuclear,
Activity of the enzyme choline acetyltransferase (CAT), which mediates the synthesis of the neurotransmitter, acetylcholine, was increased up to 20-fold in spinal cord (SC) cells grown in culture with muscle cells for 2 wk. This increase was directly related to the duration of co-culture as well as to the cell density of both the SC and muscle involved and was not affected by the presence of the acetylcholine receptor blocking agent, ct-bungarotoxin. Glutamic acid decarboxylase (GAD) activity was often markedly decreased in SC-muscle cultures while the activities of acetylcholinesterase and several other enzymes were little changed.Increased CAT activity was also observed when SC cultures were maintained in medium which had been conditioned by muscle cells or by undifferentiated cells from embryonic muscle. Muscle-conditioned medium (CM) did not affect the activities of SC cell GAD or acetylcholinesterase. Dilution or concentration of the CM directly affected its ability to increase SC CAT activity, as did the duration and timing of exposure of the SC cells to the CM. The medium could be conditioned by muscle cells in the presence or the absence of serum, and remained effective after dialysis or heating to 58~ Membrane filtration data were consistent with the conclusion that the active material(s) in CM had a molecular weight in excess of 50,000 daltons. We conclude that large molecular weight material that is released by muscle cells is capable of producing a specific increase in CAT activity of SC cells.Neurons and their target cells appear to interact in a complex reciprocal manner. These interactions have been most readily established in the neuromuscular system, where denervation results in numerous changes in muscle cell physiology (1, 6), in the visual system, where spatial and temporal relationships influence retinotectal innervation patterns (8, 23), and in the sympathetic nervous system, where the postganglionic axonal pathways appear to subserve bidirectionally significant regulatory processes (2,7,20). While these types of interactions may be mediated by neurotransmitters such as acetylcholine, or by larger molecules, such as nerve growth factor, the constraints imposed in vivo by the close physical association between cells whose interactions are demonstrable, militate against characterization of the trophic molecules involved. In contrast, the study of interacting cell systems in vitro obviates this difficulty and, as a result, a variety of techniques-circumfu-16
Abstract— The sequence complexities of rat brain RNAs were measured by RNA‐driven hybridization reactions with nonrepetitive rat DNA. The total sequence complexity of rat brain HnRNA was estimated to be 6.61 x 108 nucleotides while rat brain poly(A)‐mRNA sequence complexity was 1.32 x 108 nucleotides. Up to 33.7% of the total transcribable nonrepetitive DNA was expressed in the nuclear RNA. The nuclear RNAs reacted with complex kinetics over at least 4.5 decades of equivalent Rot (product of RNA concentration and time), with an apparent division into three major RNA abundance classes. The abundances of average nuclear RNA species in these classes ranged from 2.9 x 109 copies per brain (18 copies per cell) to 2.4 x 105 copies per brain (1.5 x 10−3 copies per cell). Poly(A)‐mRNA diversity was sufficient to code for 8.8 x 104 polypeptides of 50,000 daltons. There were also three distinguishable abundance classes of poly(A)‐mRNA with frequencies which ranged from 8.9 x 108 copies per brain (5.5 copies per cell) to 3.2 x 105 copies per brain (2 x 10−3 copies per cell). Evidence for compartmentalization of expressed RNA sequences supports the concept that the extensive morphological and physiological specialization evident in brain parallels extensive transcriptional specialization at the cellular level. Brain and liver RNA diversities were measured under double‐blind experimental conditions in three experiments with rats raised in experientially enriched (EC) or impoverished (IC) environments. Liver RNA diversity of EC animals was not different from that of IC animals. Brain total RNA of EC animals, at equivalent R0ts of 184,000‐212,000, hybridized to 10.6% of rat unique DNA (mean of 11 separate groups of rats). The average hybridization of brain RNA from 11 groups of IC animals in the same range of equivalent Rot was 8.2% of the unique DNA. The difference was statistically significant at P < 0.02. Of 10 groups of 3 littermate pairs (paired across EC and IC groups) brain RNA diversity was greater in EC animals in 8 cases. A least squares fit of the kinetics of hybridization to a pseudo first order reaction showed that, at saturation, the RNA from brains of EC animals was complementary to 16.4% of the unique DNA while that from IC animals was complementary to 9.1%. This difference was found in the least abundant class of rat brain RNA. These changes in sequence diversity reflected either an increase in the number of diverse RNA species present or an increase in the number of copies of certain RNA species in the rats raised in an enriched environment. A change in brain RNA populations of this magnitude may reflect a significant difference in brain function between EC and IC animals.
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