共查询到20条相似文献,搜索用时 15 毫秒
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J. Randall Slemmon 《Journal of neurochemistry》1989,52(6):1898-1904
The amino terminal sequence of the 13-kilodalton (kD) polypeptide present in purified Drosophila acetyl-CoA: choline-O-acetyltransferase (EC 2.3.1.6) was determined, and its position in the sequence of the intact enzyme was located. Enzyme polypeptides for sequencing were obtained from native enzyme protein by denaturation, followed by fractionation on reverse-phase HPLC. The 13-, 54-, and 67-kD polypeptides recovered from the separation were subjected to amino terminal sequencing. Only the 13-kD fragment yielded a sequence. The 67- and 54-kD polypeptides appeared completely blocked to gas-phase Edman sequencing. The location of the amino terminal sequence from the 13-kD polypeptide in the cDNA-deduced enzyme sequence indicated that this fragment represents the carboxyl portion of the 67-kD enzyme, with the 54-kD polypeptide providing the amino terminal portion. The proteolysis that gave rise to the 13-kD polypeptide occurred at the carboxyl side of a monobasic lysine residue. An earlier comparison of the enzyme from Drosophila and pigs indicated that the cleaved lysine may be a conserved residue in the porcine enzyme. The cleaved enzyme region characterized in this study does not coincide with the regions of high homology found in the two enzymes, but hydrophilicity profiles generated for this area showed similarities. 相似文献
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J. Muñoz-Maines J. Randall Slemmon Mitradas M. Panicker Noel Neighbor Paul M. Salvaterra 《Journal of neurochemistry》1988,50(1):167-175
A fusion protein containing a Drosophila choline acetyltransferase (ChAT) cDNA insert was purified from a lambda gtll lysate of Escherichia coli. The cDNA insert, which contained a 728-amino acid coding region for ChAT, was used for immunizing rabbits. Three different antisera were produced that could recognize native Drosophila ChAT with low titer. In addition, all three antisera stained enzyme polypeptides using the Western blot technique at high titers. The antisera recognized ChAT polypeptides with molecular masses of 67 and 54 kilodaltons in Western blots of partially purified enzyme; these polypeptides had previously been identified using monoclonal anti-ChAT antibodies and are the major components of completely purified enzyme. It was surprising that when these antisera were used to stain Western blots of Drosophila head homogenates, the major immunoreactive band had a molecular mass of 75 kilodaltons. The relationship of this 75-kilodalton polypeptide to ChAT activity was investigated by fractionating fresh fly head homogenates using rapid HPLC gel filtration chromatography. Analysis of column fractions for enzyme activity and immunoreactive polypeptides indicated that the 75- and 67-kilodalton polypeptides can be resolved and are both enzymatically active. In addition, a correlation was observed between the relative immunostaining intensities of both the 75- and 67-kilodalton bands and ChAT activity when supernatants from fresh fly head homogenates were autolyzed at 37 degrees C. Our results indicate that ChAT is present in fresh Drosophila heads primarily as an active enzyme with a molecular mass of 75 kilodaltons.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
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Synaptosomal "Membrane-Bound" Choline Acetyltransferase Is Most Sensitive to Inhibition by Choline Mustard 总被引:3,自引:3,他引:0
R. Jane Rylett 《Journal of neurochemistry》1989,52(3):869-875
The objectives of the present study were to validate the presence of cytoplasmic and membrane-associated pools of choline acetyltransferase (ChAT) in rat brain synaptosomes, and to evaluate inhibition of these different forms of the enzyme by the nitrogen mustard analogue of choline, choline mustard aziridinium ion (ChM Az). The relative distribution of ChAT and lactate dehydrogenase (LDH) was followed in subfractions of synaptosomes to establish whether ChAT activity associated with salt-washed presynaptic membranes represents membrane-bound protein rather than cytosolic enzyme trapped within undisrupted synaptosomes or revesiculated membrane fragments. The percentage of total synaptosomal ChAT activity (14%) recovered in the final membrane pellet always exceeded that of LDH (6%), lending support to the hypothesis that much of the ChAT associated with the membranes was a membrane bound form of the enzyme. Incubation of purified synaptosomes with ChM Az led to irreversible inhibition of ChAT activity; this loss of enzyme activity could not be accounted for by lysis of nerve terminals during incubation in the presence of the mustard analogue. Subfractionation of the ChM Az-treated nerve terminals revealed that the membrane-bound form of ChAT was inhibited to the greatest extent, followed by the ionically membrane-associated enzyme, with the activity of the water-solubilized enzyme not differing significantly from control. Preparation of the synaptosomal ChAT subfractions from untreated nerve terminals prior to incubation with varying concentrations of ChM Az or naphthylvinylpyridine revealed that under these conditions water-solubilized, ionically membrane-associated, and detergent-solubilized membrane-bound pools of ChAT were not differentially inhibited by either compound.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
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Studies on the Alkylation of Choline Acetyltransferase by Choline Mustard Aziridinium Ion 总被引:2,自引:2,他引:0
Although a potent irreversible inhibitor of high-affinity choline transport in rat brain synaptosomes, choline mustard aziridinium ion (ChM Az) appeared to be a relatively weak inhibitor of choline acetyltransferase (ChAT) in rat brain homogenates, and evidence for irreversible binding of this compound to the enzyme had not been established. Accordingly, the irreversible inactivation of partially purified rat brain ChAT by ChM Az was studied. This compound is a rather weak inhibitor of the enzyme, with 50% inhibition of ChAT activity achieved following 30 min incubation at 37 degrees C with 0.6 mM ChM Az. This result indicates that although ChM Az has affinity for many nucleophiles there was little diluting effect of the inhibitor in the crude brain homogenate which could be attributed to such reactions (50% inhibition caused by 1.8 mM ChM Az following 10 min incubation). Although the initial binding of ChM Az to ChAT may be of a competitive nature, irreversible bond formation resulted. The time-dependent alkylation reaction conformed to pseudo-first-order kinetics with an observed forward rate constant (kobs) of 0.173 min-1; the half-time (t 1/2) for irreversible binding was about 4 min. The irreversible inactivation of ChAT by ChM Az would appear to be slower than the alkylation of high-affinity choline carriers in synaptosomes by this compound, and the relatively weak inhibitory action of ChM Az against either partially purified ChAT or ChAT activity in crude rat brain homogenates is in striking contrast to previous evidence that ChAT in intact synaptosomes was inhibited irreversibly by lower concentrations of the inhibitor. 相似文献
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Homocholine and Short-Chain N-Alkyl Choline Analogues as Substrates for Torpedo Choline Acetyltransferase 总被引:1,自引:0,他引:1
Abstract: The kinetic parameters, Km and Vmax , for the acetylation of choline and several close analogues were determined by using (a) purified choline acetyltransferase and (b) a hypotonically lysed synaptosomal extract prepared from the electric organ of Torpedo marmorata. Whereas the Km for choline was similar in both cases (0.51 and 0.42 m m ), the crude enzyme showed a three- to fivefold greater affinity for its analogues than the purified enzyme, the activity decreasing rapidly with increased N -alkyl substitution. Homocholine was a poor substrate, but was clearly acetylated by both preparations. The effect of salt on analogue acetylation by the crude enzyme was studied by increasing NaCl concentration from zero to 150 m m . There was an increase in both Km and Vmax for all substrates; choline, N,N,N -dimethylmonoethylaminoethanol, -monomethyldiethylaminoethanol and -dimethylmonobutylaminoethanol showed the greatest changes, whilst N,N,N -triethylaminoethanol and -dimethylmonopropylaminoethanol and homocholine were much less affected. However, in all cases, the kinetic parameter Vmax / Km remained unchanged. The maximal velocities of the different substrates varied more under conditions of high than of low salt. Sodium chloride up to 300 m m had no effect on the amount of enzyme which was bound to membranes in the synaptosomal extract. It is concluded that choline acetyltransferase has a high degree of substrate specificity, which is slightly altered by purification. The effects of salt cannot be explained as a consequence of nonspecific ionic association with membranes. 相似文献
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Expression, Purification, and Characterization of Recombinant Drosophila Choline Acetyltransferase 总被引:1,自引:1,他引:0
Donghai Wu Norbert Schormann Wei Lian Johann Deisenhofer Louis B. Hersh 《Journal of neurochemistry》1993,61(4):1416-1422
Abstract: A cDNA for Drosophila choline acetyltransferase (EC 2.3.1.6; ChAT) was fused with a polyhistidine sequence and expressed in Escherichia coli. The recombinant enzyme was purified to a specific activity of 500 μmol/min/mg of protein using metal affinity chromatography and ion exchange chromatography. Kinetic properties of the recombinant enzyme did not differ significantly from those previously determined. Circular dichroism (CD) spectra revealed that the secondary structure of the enzyme is largely μ-helical. Intrinsic fluorescence spectra of the enzyme indicate that its tryptophan residues are buried. Neither CD nor fluorescence spectra changed significantly in the presence of substrates. The cysteine content of the recombinant Drosophila ChAT was determined to be 16 in the absence and 22 in the presence of 6 M guanidine hydrochloride. Finally, crystallization of recombinant Drosophila ChAT was achieved. 相似文献
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Choline Acetyltransferase: Celebrating Its Fiftieth Year 总被引:7,自引:3,他引:4
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Choline acetyltransferase (ChAT) catalyzes synthesis of acetylcholine (ACh) in cholinergic neurons. ACh synthesis is regulated by availability of precursors choline and acetyl coenzyme A or by activity of ChAT; ChAT regulates ACh synthesis under some conditions. Posttranslational phosphorylation is a common mechanism for regulating the function of proteins. Analysis of the primary sequence of 69-kD human ChAT indicates that it has putative phosphorylation consensus sequences for multiple protein kinases. ChAT is phosphorylated on serine-440 and threonine-456 by protein kinase C and CaM kinase II, respectively. These phosphorylation events regulate activity of the enzyme, as well as its binding to plasma membrane and interaction with other cellular proteins. It is relevant to investigate differences in constitutive and inducible patterns of phosphorylation of ChAT under physiological conditions and in response to challenges that cholinergic neurons may be exposed to, and to determine how changes in phosphorylation relate to changes in neurochemical transmission. 相似文献
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Victoria N. Luine William Rostene† Joseph Rhodes Bruce S. McEwen 《Journal of neurochemistry》1984,42(4):1131-1134
Addition of vasoactive intestinal peptide (VIP) to brain homogenates increased the activity of choline acetyltransferase (ChAT) but not that of acetylcholinesterase or glucose-6-phosphate dehydrogenase. Activity of ChAT was increased in the anterior hypothalamus and in the dorsal and ventral hippocampus, but not in the parietal cortex or posterior hypothalamus. Increased activity occurred rapidly after VIP addition to homogenates and was maximal at 10(-7)M concentration. Kinetic analysis indicates that the Vmax of the enzyme is increased and the Km for choline, but not acetyl-coenzyme A, is decreased in the presence of VIP. Results support a possible VIP-cholinergic interaction in the CNS. 相似文献
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Functional Analysis of Conserved Histidines in Choline Acetyltransferase by Site-Directed Mutagenesis 总被引:2,自引:1,他引:1
Abstract: The choline acetyltransferase (ChAT) reaction involves the transfer of the acetyl group of acetyl-CoA to choline, in which an active site histidine is believed to act as a general acid/base catalyst. A comparison of the deduced amino acid sequences of the enzyme from Drosophila , pig, rat, and Caernohabditis elegans revealed three conserved histidines: Drosophila His268 , His393 , and His426 . Each of these histidines was replaced by a leucine and a glutamine, and the kinetic properties of each of the recombinant mutant enzymes were determined. The mutations yielded active His268 Leu-ChAT, HisZ68 Gln-ChAT, and His393 Gln-ChAT and inactive His393 Leu-ChAT, His426 Leu- ChAT, and His426 Gln-ChAT. The kinetic constants Km(CoA) , Km(acetyloholine) . and Vmax were essentially the same for all of the active mutants. When the integrity of the CoASAc binding site was investigated in the inactive mutants, the data suggested that the binding site in His393 Leu-ChAT is disrupted but conserved in His426 Leu-ChAT and His426 Gln- ChAT. These results suggest that His426 is an essential catalytic residue and could serve as an acid/base catalyst. 相似文献
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Properties and Partial Purification of Choline Acetyltransferase from the Nematode Caenorhabditis elegans 总被引:4,自引:4,他引:0
We have stabilized and studied choline acetyltransferase from the nematode Caenorhabditis elegans. The enzyme is soluble, and two discrete forms were resolved by gel filtration. The larger of these two forms (MW approximately 154,000) was somewhat unstable and in the presence of 0.5 M NaI was converted to a form indistinguishable from the "native" small form (MW approximately 71,000). We have purified the small form of the enzyme greater than 3,300-fold by a combination of gel filtration, ion-exchange chromatography, and nucleotide affinity chromatography. The purified preparation has a measured specific activity of 3.74 mumol/min/mg protein, and is free of acetylcholinesterase and acetyl-CoA hydrolase activities. The Vmax of the purified enzyme is stimulated by NaCl, with half-maximal stimulation at 80 mM NaCl. The Km for each substrate is also affected by salt, but in different manners from each other and the Vmax; the kinetic parameter Vmax/Km thus changes significantly as a function of the salt concentration. 相似文献
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Conservation of Amino Acid Sequences Between Human and Porcine Choline Acetyltransferase 总被引:2,自引:2,他引:0
Louis B. Hersh Karen Takane Karen Gylys Carolyn Moomaw Clive Slaughter 《Journal of neurochemistry》1988,51(6):1843-1845
Abstract: The amino acid sequence of 11 peptides generated from human placental choline acetyltransferase was compared to the corresponding amino acid sequences predicted from the nucleotide sequence of a recently cloned porcine choline acetyltransferase cDNA. These peptides, which were generated by cyanogen bromide cleavage or tryptic digestion, accounted for 23% of the amino acids in the enzyme. Of the 145 amino acids sequenced eight differed between the two species, yielding an identity of 94% over the regions sampled.
Of the eight amino acids that differed six could represent single base changes in the DNA sequence. These findings demonstrate strong sequence similarity between porcine and human choline acetyltransferase and indicate that they are closely related evolutionarily. 相似文献
Of the eight amino acids that differed six could represent single base changes in the DNA sequence. These findings demonstrate strong sequence similarity between porcine and human choline acetyltransferase and indicate that they are closely related evolutionarily. 相似文献
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To understand the developmental regulation of acetylcholine (ACh) synthesis in the Xenopus retina, the properties of choline acetyltransferase (CAT) and cholinesterase (ChE), as well as histochemical localization of ChE in the retina, were studied during development. CAT activity first became detectable in the developing eyecup at stages 35/36. This was followed by a rapid, 50-fold rise in specific activity between stages 35/36 and 44. Since this rapid rise coincided with an almost identical increase in total ACh synthesis in whole retinae found in previous studies, it is suggested that this increase was sufficient to account for the rapid increase in total ACh synthesis. Moreover, it also correlated with increased rates of synaptogenesis in both the inner and the outer plexiform layers. Total ChE was resolved into specific and nonspecific ChE by the use of tetraisopropylpyrophosphoramide. Total ChE activities first became detectable at stages 35/36. Specific ChE [acetylcholinesterase (AChE)] increased from 50% at stage 39 to 95% of total ChE activities at stage 66. Again, the most rapid increase in both total ChE and AChE activities occurred between stages 35/36 and 44. Histochemical studies showed that AChE was localized predominantly in the two plexiform layers, with the inner plexiform layer more heavily stained at all stages. Moreover, a stratified staining pattern, clearly discerned in the inner plexiform layer, also correlated with synaptogenesis during this early period of retinal development. 相似文献
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Choline Acetyltransferase Activity in the Rat Trigeminal System 总被引:1,自引:0,他引:1
Choline acetyltransferase activity was investigated in the superior cervical ganglia and in six microdissected regions of the medulla oblongata of the rat ipsilateral and contralateral to electrolytic lesions of the trigeminal sensory ganglia (Gasserian). Electrolytic lesions of the Gasserian ganglia failed to modify levels of enzymatic activity in all structures studied. This result would be an argument against the existence of a major cholinergic population of sensory neurones in the trigeminal system. 相似文献
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Immunoaffinity Purification of Human Choline Acetyltransferase: Comparison of the Brain and Placental Enzymes 总被引:10,自引:4,他引:6
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. 相似文献
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Three hybridomas producing monoclonal antibodies to bovine brain choline acetyltransferase (ChAT) have been established by fusion of the spleen cells from a mouse immunized with purified enzyme with myeloma NS-1 cells. All three clones produced IgGl antibodies that reacted with enzyme protein denatured with sodium dodecyl sulfate. By using one of the monoclonal antibodies, a rapid and efficient immunoaffinity purification procedure of bovine ChAT has been established. Immunoblot analysis and immunoaffinity purification indicated that bovine ChAT is a single 68-kilodalton protein. The monoclonal antibodies will offer us a good tool to isolate the cDNA clones encoding ChAT. 相似文献
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Takahiko Kato 《Journal of neurochemistry》1989,52(2):636-640
Activities of choline acetyltransferase (ChAT) were microassayed in individual cell bodies of motor neurons, isolated from freeze-dried sections after autopsy of lumbar spinal cords from four patients with sporadic amyotrophic lateral sclerosis (ALS) and four control patients with nonneurological diseases. Numerous large neurons were found in the anterior horn at the early degeneration stage of ALS, but the cell bodies atrophied and decreased in number at the late advanced stage. The small, atrophied neurons were very fragile and were easily destroyed during the isolation procedure with a microknife. The average activity, expressed on a dry weight basis, of 58 ALS neurons was lower than that of 67 control neurons. The large, well-preserved neurons at the early nonadvanced stage had markedly lower ChAT activities than control neurons. The specific activity gradually increased with the progress of atrophy but did not return to the control level. 相似文献