鸭血清胆碱酯酶的纯化及性质研究

首次采用新技术双水相萃取方法作为鸭血清胆碱酯酶(EC.3.1.1.8 CHE) 纯化的第一步,后经 DEAE-Sephadex A50,sephadex G200 柱层析,获得电泳纯鸭血清胆碱酯酶,提纯倍数1018倍,酶活力回收43.4%,比活274.9U/mg。鸭血清胆碱酯酶性质研究表明:此酶是糖蛋白和酸性蛋白水解酶,等电点 4.2 左右,最适 pH7.5 左右;对底物碘化硫代丁酰胆碱的 K_m=9.8×10^-5mol/L;SDS-PAGE 电泳和聚丙烯酰胺梯度电泳表明,鸭血清胆碱酯酶以相同亚基组成的不同聚合体形式存在,亚基分子量 78000,具有完整的酶活性.不同聚合体带电状态相同.     

Effect of humic acids on the inhibition of pea choline esterase and choline acyltransferase with malathion

Humic acids in a concentration of up to 100 μg ml^-1 affect the activity of choline esterase of pea only weakly during sinapin (choline ester of sinapic acid) hydrolysis. The inhibition is stronger at higher concentrations. A similar course of inhibition with humic acids was also observed during the synthesis of sinapin catalysed with choline acyltransferase present in the same enzyme extract. The organophosphate malathion is a strong inhibitor of both the hydrolysis and the synthesis of sinapin. Thus, for example, at a 3.5 × 10^-5 M concentration it causes 85% inhibition of pea choline esterase. However, in combination with 100 μg of humic acids this inhibition decreases to about 8%. It was observed that humic acids prevent the inhibition of both the hydrolysis and the synthesis of sinapin with malathion even at low concentrations. The interaction of the organophosphate malathion with humic acids seems specific. Humic acids do not affect the inhibition of choline esterase with physostigmine or the inhibition of the hydrolysis and the synthesis of sinapin with chlorocholine chloride, alar and hexamine either. This is also true of the activation of the processes with the K⁺ ions. Humic acids represent competitive inhibitors of choline esterase. Malathion shows an acompetitive — noncompetitive character of the inhibition. The interaction of humic acids with malathion in the roots of the plants may also be assumed in the soil when malathion is applied as an insecticide.     

THE DISTRIBUTION OF ACETYL-CoA IN SPECIFIC AREAS OF THE CNS OF THE RAT AS MEASURED BY A MODIFICATION OF A RADIO-ENZYMATIC ASSAY FOR ACETYLCHOLINE AND CHOLINE1

A rapid and sensitive enzymatic assay for measuring picomole quantities of acetyl-CoA, acetylcholine (ACh), and choline from the same tissue extract has been developed. After ACh and choline were extracted into 15% 1 N formic acid/85% acetone, the pellet was further extracted with 5% trichloroacetic acid (TCA) to remove the remaining acetyl-CoA. The two extraction solvents were pooled and lipids, organic solvents, and TCA were removed first by a heptane-chloroform wash followed by an ether extraction. In the acetyl-CoA assay, endogenous ACh and choline were removed by extractions with sodium tetraphenylboron in butenenitrile prior to the enzymatic reactions. The acetyl-CoA remaining in the aqueous phase was then converted enzymatically to labelled ACh in the presence of [Me-¹⁴C]choline using choline acetyltransferase. The unreacted labelled precursor was converted to choline phosphate by the enzyme choline kinase. The [¹⁴C]ACh formed from acetyl-CoA was extracted into sodium tetraphenylboron in butenenitrile and a portion of the organic phase containing the [¹⁴C]ACh was counted in a scintillation counter. Acetylcholine and choline were assayed from the same tissue extracts by a modification of the procedure by SHEA & APRISON (1973). Acetyl-CoA levels in rat whole brain when killed by the near-freezing procedure were found to be 5.50 ± 0.2 nmol/g. The content of acetyl-CoA was the same whether the rats were killed by the near-freezing method or by total freezing in liquid nitrogen. The levels of acetyl-CoA did not change with time after death when the tissue was maintained at a temperature of ?10°C. In the same tissue extracts from rat whole brain killed by the near-freezing method, the content of ACh was 20.6 ± 0.7 nmol/g and choline 58.2 ± 1.2 nmol/g. Although reproducible, the level reported for choline is high when assayed under this condition. The content of choline however after total freezing was found to be 25.2 ± 2.0 nmol/g. The sensitivity (d. p. m. of sample twice blank) is 10 pmol for the acetyl-CoA assay and 25 pmol for the ACh and choline assays. The regional distribution of these three compounds in the brain of rats as well as the content of acetyl-CoA in heart, liver and kidney are presented.     

THE SUBCELLULAR LOCALIZATION OF Ach SYNTHESIS IN RAT STRIATAL SYNAPTOSOMES INVESTIGATED WITH THE USE OF TRITON X-100

—The regulation of [¹⁴C]ACh synthesis was studied in rat striatal synaptosomes incubated in presence of various concentrations of Triton X-100, using [2-¹⁴C]pyruvate or [6-¹⁴C]glucose as precursors. The progressive rupture of the cytoplasmic and mitochondrial compartments induced by the non-ionic detergent was followed by studying the release, into the incubating medium, of lactate dehydrogenase and choline acetyltransferase (ChAc) and of fumarate hydratase, respectively. [³H]Choline uptake (1 μm ) was measured to determine the activity of the high affinity choline permease. ¹⁴CO₂ formation from [2-¹⁴C]pyruvate was used as an index of the Krebs cycle activity. The rate of [¹⁴C]ACh synthesis from [2-¹⁴C] pyruvate was dependent on the Triton X-100 concentration; the ester formation decreased between 0·001% (v/v) and 0·010%, but increased again beyond this concentration of detergent. This last phenomenon was interpreted as the result of an extracellular synthesis of ACh involving pyruvate dehydrogenase and ChAc. At 0·002% Triton X-100 the ¹⁴CO₂ formation was not affected, indicating a normal mitochondrial activity. The decrease of [¹⁴C]ACh synthesis observed up to this detergent concentration could be correlated to the decline of the highaffinity choline permease activity. In these experimental conditions, the ester synthesis could not be restored by the addition of large amounts of choline in the incubating medium suggesting that the molecules of choline must cross the high-affinity choline permease system in order to be acetylated. This could indicate a close association between the permease and choline acetyltransferase.     

CHOLINE ESTERASE AND THE THEORY OF CHEMICAL MEDIATION OF NERVE IMPULSES

The maximum choline esterase activity of the superior cervical ganglion of the cat was measured and found to be, on the average, equivalent to the splitting of 0.10γ of acetyl choline chloride per second per milligram of fresh tissue at a pH of 7.4 and 38°. The least possible time required for destruction of the ester liberated by one nerve impulse was calculated to be 0.015σ. The dissociation constant of the reaction between the enzyme and acetyl choline chloride was determined, and a value of 0.001 was obtained. From the value of the dissociation constant, the time for hydrolysis at the minimum rate was calculated to be about 8 seconds. It was shown that a localization of enzyme and substrate within the ganglion cell would have to exist in order that enzymatic destruction of acetyl choline liberated by nerve impulses occur within the span of the refractory period.     

STUDIES ON ENZYMATIC HISTOCHEMISTRY : XXVI. THE HISTOLOGICAL DISTRIBUTION OF CHOLINE ESTERASE IN THE GASTRIC MUCOSA NORMALLY AND AFTER ADMINISTRATION OF CERTAIN DRUGS

Extraction experiments demonstrated that choline esterase could be removed from microtome sections of tissue with as great facility by 0.9 per cent NaCl as by 30 per cent glycerol. The quantitative distribution of choline esterase through the wall of the pig stomach was studied, and it was found that the epithelial cell region possessed the greatest activity and muscle tissue the least. Pylorus was more active than fundus or cardia. The enzyme activities found were independent of the physiological state of the normal stomach at the time the animal was killed. Neither intramuscular injection of acetyl choline, eserine, nor atropine shortly before killing had significant influence upon the activity in any region of the stomach. The implications of these results were discussed.     

THE SYNTHESIS OF PHOSPHOLIPIDS IN THE NUCLEUS AND NUCLEAR MEMBRANE OF SYNCHRONIZED HeLa CELLS

HeLa cells were synchronized by a double thymidine block and pulse labeled at different stages of the cell cycle with ³H-choline. The specific activity of phospholipids extracted from the cell, the nucleus and the nuclear membrane showed a progressive increase from S to G₁; the incorporation of choline into phospholipids of asynchronous cells showed a specific activity intermediate between the values of S and G₁ cells. Similar results were obtained when ³²phosphorus was used as a precursor instead of choline. Thin layer chromatographic analysis of phospholipids extracted from cells in S and from cells in G₁ failed to show any difference in the distribution of radioactivity among the various phospholipid classes. Choline uptake by HeLa cells in different phases of the cell cycle did not show significant variations. However, during the synchronization process, shortly after the addition of excess thymidine, an increased uptake of choline by cells and an increased incorporation of choline into phospholipids were found. The results indicate that some of the changes occurring in phospholipids synthesis may not be cell cycle dependent, but may be the effect of the synchronizing process.     

The effect of phenylacetic acid on cholinesterase activity and on some isoenzymes of esterases and cholinesterases of peain vitro

Phenylacetic acid at 1.5 × 10^-3 M inhibits the activity of some esterase isoenzymes from pea leaves separated by means of polyacrylamide gel electrophoresis. Some of the inhibited esterases show cholinesterase activity. Inhibition of the total activity has been demonstrated with a partially purified protein fraction from pea leaves containing choline esterase. The inhibition constant established after Dixon was 7.9 × 10^-3 M and the type of inhibition was competitive.     

High-Affinity Choline Transporter

The cholinergic neurons have long been a model for biochemical studies of neurotransmission. The components responsible for cholinergic neurotransmission, such as choline acetyltransferase, vesicular acetylcholine transporter, nicotinic and muscarinic acetylcholine receptors, and acetylcholine esterase, have long been defined as functional units and then identified as molecular entities. Another essential component in the cholinergic synapses is the one responsible for choline uptake from the synaptic cleft, which is thought to be the rate-limiting step in acetylcholine synthesis. A choline uptake system with a high affinity for choline has long been assumed to be present in cholinergic neurons. Very recently, the molecular entity for the high-affinity choline transporter was identified and is designated CHT1. CHT1 mediates Na⁺- and Cl^–-dependent choline uptake with high sensitivity to hemicholinium-3. CHT1 has been characterized both at the molecular and functional levels and was confirmed to be specifically expressed in cholinergic neurons.     

MULTIPLE FORMS OF CHOLINE ACETYLTRANSFERASE AND THE HIGH AFFINITY UPTAKE OF CHOLINE IN BRAIN OF DEVELOPING AND ADULT RATS

The multiple molecular forms of choline acetyltransferase (ChAT) were analysed during the postnatal development of rat brain. Changes in the sodium-dependent, high affinity uptake of [³H]choline (HAUC) and in the efficiency of conversion of labelled choline into ACh in vitro were also examined. Both mature and 7-day old brain contained three molecular forms of ChAT, with isoelectric points of pH 7.3, 7.9 and 8.3, but the immature brain appeared to contain smaller concentrations of the most basic form of the enzyme (pI = 8.3). Of the total choline uptake measured in slices of frontal cortex, adult samples exhibited a greater proportion of HAUC than 7-day samples and appeared to acetylate more efficiently the [³H]choline accumulated by high affinity uptake. This evidence suggests a basic molecular form of ChAT, appearing in rat brain during postnatal development, might be responsible for the efficient coupling of the high affinity uptake and subsequent acetylation of choline in cholinergic nerve terminals.     

ANALYSIS OF THE PREFERENTIAL RELEASE OF NEWLY SYNTHESIZED ACETYLCHOLINE BY CORTICAL SLICES FROM RAT BRAIN WITH THE AID OF TWO DIFFERENT LABELLED PRECURSORS

—The release of newly synthesized acetylcholine (ACh) by cortical slices from rat brain in the presence of 25 mm -KCl was studied. The slices were incubated for 5 min in a medium containing both [2-¹⁴C]pyruvate and choline labelled with 3 deuterium atoms (choline-d₃) in order to label at the same time the acetyl moiety and the choline moiety of ACh. The non-labelled ACh and the ACh-d₃ were measured by pyrolysis-gas chromatography/mass spectrometry, and the [^I4C]ACh by liquid scintillation counting. It was found that the newly formed [⁴C]ACh as well as the newly formed ACh-d₃ had a more than 2.5 times greater probability of being released than the preformed non-labelled ACh. These findings strongly suggest that it is not simply the ACh synthesized immediately inside the nerve ending membrane from incoming undiluted labelled choline, which is preferentially released, but that all newly formed ACh has a greater probability of being released than preformed ACh. No preferential release of newly formed ACh was observed when the incubation medium contained 5.6 mm -pyruvate instead of 10 mm -glucose + 0.6 mm -pyruvate. The cause of this difference remains unexplained.     

HIGH AFFINITY CHOLINE TRANSPORT IN GUINEA PIG BRAIN AND THE EFFECT OF NOREPINEPHRINE

—The influence of 1-norepinephrine on the accumulation of [¹⁴C]choline by nuclei-free homogenates and synaptosomes of guinea-pig brain was studied. Kinetic analysis of choline accumulation by guinea-pig brain resulted in both high and low affinity Michaelis constants. Norepinephrine stimulated the high affinity choline transport process but not the low and the magnitude of its stimulation in 3 different brain regions was correlated with the choline acetyltransferase activity of those regions. Depletion of norepinephrine from the brainstem by pretreatment with the catecholamine depleter alpha-methyl-para-tyrosine significantly decreased the maximal velocity of choline transport. Both the alpha adrenergic receptor blocker phentolamine and the beta adrenergic receptor blocker propranalol inhibited norepinephrine induced stimulation of choline transport. Cocaine stimulated choline transport at low concentrations and pretreatment of animals with reserpine significantly antagonized cocaine's stimulation of choline transport. The results suggest that endogenous norepinephrine may modify the high affinity choline transport process in guinea-pig brain.     

蜜蜂脑乙酰胆碱免疫反应阳性神经元的分布

通过免疫组织化学方法-PAP法,使用乙酰胆碱(ACh)抗体,研究了中华蜂(Apis sinensis)和意大利蜂(Apis mellifera L)脑中具有乙酰胆碱免疫阳性反应的神经元胞体的形态、分布及神经元类型.并和已知的在其他昆虫脑中用ACh的合成酶ChAT和其水解酶AChE的抗体免疫组化法所获得的结果进行了比较.     

STUDIES IN THE MODE OF ACTION OF INSECTICIDES

Acetylcholine, choline chloride, acetyl-β-methylcholine, benzoylcholine, carbamyl choline, adrenaline and d -tubocurarine are non-toxic when injected into the locust. Prostigmine is also non-toxic, and eserine considerably less toxic to the locust than to man.
The toxic effect of tetraethyl pyrophosphate (TEPP) cannot be antagonized by injection of atropine or enhanced by d -tubocurarine.
The injection of acetylcholine chloride following injection of TEPP does not affect subsequent mortality.
These findings are discussed, and it is suggested that the physiology of the nervous system of the insect is unlike that of the mammal, neither cholinesters nor adrenaline being concerned in it. Phosphorus insecticides are thought to inhibit a general esterase not specifically connected with cholinesters.     

SODIUM-DEPENDENT HIGH AFFINITY CHOLINE UPTAKE: A REGULATORY STEP IN THE SYNTHESIS OF ACETYLCHOLINE

The sodium-dependent high affinity choline uptake into synaptosomes from rat brain has been studied after in vivo treatments which would alter the activity of cholinergic neurons. We utilized a number of treatments to reduce the activity of cholinergc neurons in the brain. Administration of pentobarbital (65 mg/kg), chloral hydrate (40 mg/kg) and γbutyrelactone (750 mg/kg) caused a 50-80% reduction in sodium-dependent high affinity choline uptake in several brain regions (30 min). This depression was not found 24 h after injection. Interruption of the cholinergic septal-hippocampal or habenuleinterpeduncular tracts by lesions (10 min-1 h) also caused a similar, large reduction in sodium-dependent high affinity choline uptake in the hippocampus and the interpeduncular nucleus respectively. We reversed the inactivity after pentobarbital administration by direct electrical stimulation of the cholinergic septal-hippocampal tract. Stimulation (40 Hz) for 10-15 min completely reversed the depression in sodium-dependent high affinity choline uptake. Stimulation at lower frequencies or for shorter times caused a partial reversal. Administration of pentylenetetrazol (75 mg/kg), a convulsant, was utilized to increase the activity of central cholinergic neurons. After drug administration, we found a large (60%) increase in sodium-de-pendent high affinity choline uptake. This increase was not found in the hippocampus when cholinergic afferents were interrupted by septal lesion prior to drug administration. We also examined the uptake after administration of cholinergic drugs. Oxotremorine (0.75 mg/kg), a muscarinic agonist which reduces acetylcholine release and turnover, caused a reduction in uptake. On the other hand, administration of scopolamine (5 mg/kg), a cholinergic antagonist which increases acetylcholine turnover, caused an increase in sodium-dependent high affinity choline uptake. Addition of any drug utilized, drectly to uptake samples, did not alter uptake. We examined the conversion of [³H]choline to [³H]acetylcholine in hippocampal synaptosomes after septal lesion, pentylenetetrazol administration and in untreated controls. In all cases, 60-70% of the total sodium-dependent tritium content was present as [³H]acetylcholine. Evidence was presented that homoexchange is not or is less involved in choline uptake than in GABA uptake. A kinetic analysis of sodium-dependent high affinity choline uptake was performed after all treatments. We found changes in V_max, after all treatments, which were consistently in the same direction as the alterations in activity. The proposal is made that the sodium-dependent high affinity choline uptake is coupled to cholinergic activity in such a way as to regulate the entry of choline for the maintenance of acetylcholine synthesis. The findings also lead us to propose that sodium-dependent high affinity choline uptake in vitro be utilized as a rapid, relative measure of the activity of cholinergic nerve terminals in vivo.     

LACK OF PHOSPHOLIPID TRANSPORT MECHANISMS IN CELL MEMBRANES OF THE CNS1

The possible transport role of phospholipid-protein complexes, present in the cell supernatant of rat brain was investigated using labelled choline as precursor of phosphatidyl choline. Results obtained after the intracranial injection of choline gave no indication of a sequence of events compatible with a transport of phospholipid molecules from the possible site of synthesis (microsomes) to the supernatant and subsequently to myelin. Chase experiments using rat brain slices incubated in vitro with radioactive choline agreed well with the above mentioned results. Contrariwise, when Na³⁵₂SO₄ was used as precursor, the results clearly indicated that synthesis of sulphatides takes place in microsomes, followed by transfer of the radioactive lipid to sulphatide-containing lipoproteins in the supernatant and finally to myelin. Results presented in this paper seem to give further support to the idea that other subcellular fractions, besides microsomes, can autonomously synthesize part of their own provision of phospholipids. Possible reasons which might explain the marked differences between the mechanisms of addition of phospholipids and sulphatides to myelin are discussed in relation to results obtained by other investigators.     

RELATIVE IMPORTANCE OF CHOLINE TRANSPORT TO SPONTANEOUS AND POTASSIUM DEPOLARIZED RELEASE OF ACh

—The importance of extracellular choline transport to spontaneous and K⁺ depolarized release of ACh was studied using mouse brain cortex minces. The results suggest that extracellular choline transport is not essential to spontaneously released ACh but is essential to K⁺ depolarized ACh release. Similar cumulative amounts of choline and ACh were found in the incubation media following incubation of minces in either Krebs or 35 mm -K⁺ Krebs suggesting the same production of free choline during both conditions. Double reciprocal plots of choline accumulation by non-depolarized cortex minces yield high and low affinity components. Conversely, similar analysis of choline accumulation by depolarized minces yields a single Michaelis constant (68 μm ) similar to the low affinity (50 μm ) Michaelis constant determined for choline accumulation by non-depolarized minces. Kinetic analysis of ACh release as a function of extracellular choline concentration during K⁺ depolarization also yields a Michaelis constant of 68 μm These data suggest a link between choline transport and ACh release during K⁺ depolarization.     

THE EFFECTS OF BOTULINUM TOXIN ON ACETYLCHOLINE METABOLISM IN MOUSE BRAIN SLICES AND SYNAPTOSOMES

The effects of Type A botulinum toxin on acetylcholine metabolism were studied using mouse brain slice and synaptosome preparations. Brain slices that had been incubated with the toxin for 2h exhibited a decreased release of acetylcholine into high K⁺ media. Botulinum toxin did not affect acetylcholine efflux from slices in normal K⁺ media. When labeled choline was present during the release incubation, a‘newly-synthesized’pool of acetylcholine was formed in the tissue. In toxin-treated slices exposed to high K⁺, both the production and the release of this‘newly-synthesized’acetylcholine were depressed. A possible explanation for these actions of botulinum toxin would be via an inhibition of the high affinity uptake of choline. This hypothesis was tested by measuring the high affinity uptake of [³H]choline into synaptosomes prepared from brain slices. Previous exposure of slices to botulinum toxin caused a significant reduction in the accumulation of label by the synaptosomes. These data are discussed in terms of our current understanding of the mechanism of action of botulinum toxin and the toxin's interaction with the mechanisms regulating acetylcholine turnover.     

EFFECT OF OXOTREMORINE ON THE APPARENT REGIONAL TURNOVER OF ACETYLCHOLINE IN MOUSE BRAIN

The effect of oxotremorine (1 mg kg^-1 i.p.) on the steady state concentration of acetylcholine (ACh) and choline (Ch) and the transformation of radioactive choline ([³H]Ch) was studied in different brain regions of the mouse following death by microwave irradiation of the head. Oxotremorine significantly increased the concentration of endogenous ACh in the cortex and hippocampus and of endogenous Ch in the cortex. Pretreatment with atropine (5 mg kg^-1 i.p.) prevented the increase in ACh. The biosynthesis of radioactive ACh ([³H]ACh) was decreased in all brain regions. Atropine (5 mg kg^-1) pretreatment counteracted this effect of oxotremorine (1 mg kg^-1), while methylatropine (5 mg kg^-1) had no effect except in the striatum. A calculation of the apparent turnover rate of ACh showed that oxotremorine (1 mg kg^-1) decreased the turnover in the cortex, hippocampus, midbrain. and striatum.     

THE EFFECT OF THIAMINE DEFICIENCY ON THE ACETYLCOENZYMEA AND ACETYLCHOLINE LEVELS IN THE RAT BRAIN

Abstract— It is shown that transketolase activities in red blood cells and whole brain of normal and thiamine-deficient rats correlate well with heart frequencies.
The effect of thiamine depletion on the levels of acetylcoenzyme A (acetyl-CoA) and acetylcholine (ACh), and on the activities of pyruvate dehydrogenase, choline acetyl-transferase and acetylcholine esterase was studied in whole brains of thiamine-deficient, thiamine-supplemented ad libitum and pair-fed rats. The concentrations of acetyl-CoA and ACh decreased in thiamine-deficient brains by 42 and 35 per cent, respectively.
Total pyruvate dehydrogenase activity did not change during vitamin B₁ deficiency. The 'resolved' enzyme, reconstituted with thiamine diphosphate, had an association constant of 5.4 × 10⁻⁶ m . Choline acetyltransferase and acetylcholine esterase activities remained unchanged in thiamine deficiency.
Possible mechanisms which could explain the reduced Ach levels in vitamin B₁ deficiency are discussed.