DISTRIBUTION OF ACETYLCHOLINE, CHOLINE, CHOLINE ACETYLTRANSFERASE AND ACETYLCHOLINESTERASE IN REGIONS AND SINGLE IDENTIFIED AXONS OF THE LOBSTER NERVOUS SYSTEM

Abstract— Acetylcholine, its precursor (choline), and the enzymes of its biosynthesis and degradation (choline acetyltransferase and acetylcholinesterase, respectively) have been studied and quantified in extracts of several regions of the nervous system of the lobster and in single, isolated axons of identified efferent excitatory, efferent inhibitory and afferent sensory neurons. The choline acetyltransferase is a soluble enzyme similar to that from other species. The predominant acetylcholine-hydrolysing enzyme is largely membrane-bound and has been characterized as a specific acetylcholinesterase. A single peak of acetylcholinesterase activity can be detected upon velocity sedimentation analysis of Triton X-100-treated extracts of all regions of the nervous system. Choline acetyltransferase distribution parallels that of sensory neural elements, and its specific activity shows nearly a 500-fold difference from the richest to the poorest neural source. Acetylcholinesterase levels span only a 23-fold range, and activity is found in all neural regions, including those free of known sensory components. A radiochemical microassay for choline and acetylcholine in the range of 20–2000 pmol is described in detail. All 3 types of axons contain comparable levels of choline ( ca. 2 pmol/μg protein), but acetylcholine is asymmetrically distributed. Efferent axons contain no detectable acetylcholine, while sensory axons from abdominal muscle receptor organs have an average of 1·9 pmol/μg protein. Choline acetyltransferase is similarly distributed; sensory axons show at least 500-fold greater activity than efferent axons. Acetylcholinesterase is nearly uniformly distributed among the three types of fibres. These results are discussed in terms of a general view of transmitter accumulation in single neurons.     

Choline kinase in Cuscuta reflexa

1. Choline kinase is a mitochondrial enzyme in Cuscuta reflexa. It can be solubilized from the particles by treatment with 350mm-sodium chloride, or by freezing and thawing. 2. Choline kinase of C. reflexa was purified by starting from the crude mitochondrial fraction. A 33-52% recovery of the enzyme, on the basis of the activity in the original homogenate, in 1200-2250-fold enrichment, was effected. 3. The purified preparation of choline kinase had a sigmoid saturation curve with respect to choline, with a Hill number of 2.3, and was inhibited by ADP (competitive in nature and allosteric in binding, with a Hill number of 2.7) and by phosphorylcholine (non-competitive and non-allosteric). The kinetic characteristics of the enzyme were consistent with the K type allosteric model of Monod et al. (1965). 4. The enzyme was desensitized, with respect to choline regulation, by prolonged storage in the cold, was activated significantly on warming before assay and was inactivated by high concentrations of sodium chloride. 5. The significance of allostery in choline kinase in relation to the intracellular regulation of phospholipid synthesis is discussed.     

INHIBITION OF BACTERIAL AND MAMMALIAN CHOLINE ACETYLTRANSFERASES BY STYRYLPYRIDINE ANALOGUES

—Choline acetyltransferase was extracted from Lactobacillus plantarum by relatively gentle procedures involving penicillin treatment, osmotic shock and passage through a French pressure cell. After partial purification, the extract was compared with choline acetyltransferase of calf caudate nucleus for kinetic properties and response to a class of inhibitors which consists of analogues of styrylpyridine. Both enzymes obeyed a sequential mechanism with Michaelis constants for the bacterial enzyme, K_m= 8 μm vs. acetyl-CoA and 0·44 mm vs. choline; and for the caudate nucleus enzyme, K_m= 15 μm vs. acetyl-CoA and 0·8 mm vs. choline. Both were stabilized by dithiothreitol and EDTA. The extracts differed in that the bacterial enzyme was more labile and apparently was susceptible to conformational changes, which modified its response to the styrylpyridinetype inhibitors. The use of intact cells of Lactobacillus plantarum as an in vivo system for studying the inhibition of choline acetyltransferase by styrylpyridines was possible only for non-quaternary analogues, which exist as an equilibrium mixture of charged and uncharged species.     

Allosteric activation of the hydrolysis of specific substrates by chymotrypsin.

A variety of azobenzene compounds having bis-quaternary nitrogens have been shown to accelerate the hydrolysis by chymotrypsin of certain specific substrates by an allosteric mechanism. One of the most potent, 2,2'-bis[alpha-(benzyldimethylammonium)methyl]azobenzene dibromide (2,2'-QBzl) accelerated the hydrolysis of glutaryl-L-phenylalanine p-nitroanilide 40-fold at saturating concentration. Acceleration was by increasing kcat without altering Km. The hydrolysis of acetyl-L-tyrosine p-nitroanilide and acetyl-L-tyrosine anilide was also accelerated by Q-Bzl (25-fold and 1.8-fold respectively) while the hydrolysis of hemoglobin, azocoll and a number of esters was not affected. The inactivation of chymotrypsin by diphenylcarbamyl chloride and diphenylcarbamyl fluoride was accelerated by 2,2'-Q-Bzl. Reac;ivation in the presence of NH2OH was also accelerated, but in the absence of added nucleophile (i.e. of NH20H) no increase in rate was detectable. An allosteric effector was covalently attached to chymotrypsinogen A by reaction with 2,2'-bis[alpha-(o-bromomethylbenzyldimethylammonium)methyl]azobenezene dibromide. The product, when converted to active enzyme, was about 4 times more active than chymotrypsin as a result of an increase in kcat of hydrolysis; Km was unaffected. The mechanism of the allosteric acceleration process is not known but, because for all of the substrates affected acylation of the enzyme is rate-limitimg, it is tentatively suggested that the effectors facilitate proton transfer to the leaving group by an inductive effect on the 'charge relay system'. Spectral studies indicate that the allosteric site is a portion of the enzyme with a polarity near that of water, possibly on the outside surface of the enzyme molecule.     

PARTIAL PURIFICATION AND PROPERTIES OF CHOLINE KINASE (EC 2.7.1.32) FROM RABBIT BRAIN: MEASUREMENT OF ACETYLCHOLINE

Choline kinase (EC 2.7.1.32; ATP: choline phosphotransferase) was purified 200-fold from an extract of acetone powder of rabbit brain by a combination of acid precipitation, ammonium sulphate precipitation, DEAE cellulose chromatography, and ultrafiltration. Maximal activity of 243 nmol of phosphorylcholine synthesized. min^?1 mg^?l of protein occurred at pH 9.5–10.0 in the presence of 10 mm MgS0₄, 10 mm choline and 0.005% (w/v) bovine serum albumin. 2-Aminoethanol, 2-methylaminoethanol, and 2-dimethylaminoethanol were also phosphorlyated by the enzyme preparation. The enzyme quantitatively converted low concentrations of choline (2.5–50 μm ) to phosphorylcholine [³²P] in the presence of ATP [y³²P], and may, therefore, be used to measure small amounts of choline acetylcholine. There were two K_m values for choline at pH 9.5; 32 μm and 0.31 mm . At pH 7.4, the higher K_m was not observed and enzyme activity was maximal with 0.1 mm choline. The K_m for ATP was 1.1 mm . Enzyme activity was inhibited by ATP (20 mm ), AMP, ADP, cytidine diphosphocholine (1 or 10 mm ), and activated by choline esters (1.0 mm ), NaCl or KCl(200 mm ).     

Phosphofructokinase. II. Role of ligands in pH-dependent structural changes of the rabbit muscle enzyme.

The effect of ligands, including substrates and allosteric effectors, on the pH-dependent inactivation and reactivation of rabbit muscle phosphofructokinase has been examined in terms of the mechanism proposed previously (Bock, P.E. and Fireden, C. (1976) J. Biol. Chem. 251, 5630-5636). It is concluded thatt many ligands exert their effect by binding preferentially to either protonated or unprotonated forms of the enzyme and thus shifting an apparent pK for the inactivation or reactivation process. ATP and fructose 6-phosphate influence the apparent pK to different extents and in different directions, with ATP binding preferentially to the protonated forms and fructose 6-phosphate to the unprotonated forms. Enzyme inactivated by ATP can be reactivated by the addition of fructose 6-phosphate. The experiments indicate that inactivation and reactivation in the presence of these ligands can occur by kinetically different pathways as has been found for these processes in the absence of ligands. The results are discussed in relation to what might be expected for ligand binding properties of the enzyme as a function of pH, temperature, and enzyme concentration. The effect of ATP and MgATP is complex, perhaps representing more than one site of binding. Citrate appears to bind preferentially to protonated forms of the enzyme while fructose 1,6-bisphosphate and AMP bind preferentially to the unprotonated forms. ADP, K+, and NH4+ appear to have little or no preference in binding to different enzyme forms.     

Reactivation and aging of diphenyl phosphoryl acetylcholinesterase.

Acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) is readily in hibited by 10(-5) M diphenylphosphorochloridate even though the inhibitor hydrolyzes in a few seconds. The fluoridate is a much weaker inhibitor. The inhibited enzyme, diphenyl phosphoryl enzyme spontaneously recovers only about 50% of its activity with a half time of about 17 min at pH 7.0 and 6 min at pH 8.0. The fact that only 50% of the original activity returns is due to aging. The rates of reactivation and aging can be very greatly increased by a few percent of an organic solvent. Depending on the solvent even 1% may increase the rates by a factor of 5 or 6. The highest increase in rate was 70-fold. Quaternary NH+4 also increases the rates. Organic solvents and NH+4 also accelerate the reactivation of the much more stable diethyl phosphoryl enzyme derivative.     

Evidence for the existence of a single enzyme catalyzing the phosphorylation of choline and ethanolamine in primate lung.

Choline kinase (ATP:choline phosphotransferase, EC 2.7.1.32) has been isolated and purified 1000-fold from adult African Green monkey lung with a yield of 10%. The purified enzyme also phosphorylated ethanolamine (ratio of ethanolamine kinase to choline kinase = 0.30). This ratio remained constant throughout the purification procedure. The Km for choline (3.0 - 10(-5) M) was lower than that of ethanolamine (1.2 - 10(-3) M.) Choline was also found to inhibit ethanolamine kinase activity by 50% at a concentration of 0.005 mM, while ethanolamine inhibited choline only at very high concentrations (100--150 mM). When the enzyme was subjected to inactivation by heat, hemicholinium-3, trypsin digestion, and p-hydroxymercuribenzoate, both ethanolamine kinase and choline kinase activities were destroyed at the same rate. Freezing and thawing in the absence of glycerol also destroyed both activities at the same rate. Based on these findings, we conclude that in adult African Green monkey lung tissue, there is only one enzyme for the phosphorylation of ethanolamine and choline, and that choline phosphorylation predominates.     

Enzymatic determination of phospholipase D activity with choline oxidase

A new enzymatic method was developed for the assay of phospholipase D [phosphatidylcholine phosphatidohydrolase EC 3.1.4.4] from cabbage leaves using choline oxidase from Arthrobacter globiformis cells. The method was based on the estimation of choline by the following series of enzymatic reactions after ending the phospholipase D reaction: Choline + 202 + h2o Choline oxidase Betaine + 2H2O2 2H2O2 + Phenol + 4-Aminoantipyrine Peroxidase Quinoneimine dye + 4H2O The amount of choline was proportional to the amount of resulting quinoneimine dye with an absorbance maximum at 500 nm. The phospholipase D reaction (choline liberation) was carried out at pH 5.5 in the presence of Ca2+ ions and ended by adding EDTA in conc. Tris-HCl buffer, pH 8, to give a final pH of around 8. The initial rate of the phospholipase D reaction was proportional to the enzyme concentration over the absorbance change range of 0 to 0.25 (equivalent to 0-21 micron of choline) under the optimal reaction conditions.     

胆碱脱氢酶的底物保护作用

胆碱脱氢酶（ＣＤＨ）是线粒体电子传递酶系的一个重要组成,它位于线粒体内膜。膜固有的ＣＤＨ与用去垢剂从线粒体上增溶下来的酶在性质上有一定差异,本文研究了温度、ＳＤＳ对增溶ＣＤＨ的失活作用,发现底物胆碱的存在有明显的保护作用,说明底物诱导ＣＤＨ产主构象变化．     

Autotaxin structure–activity relationships revealed through lysophosphatidylcholine analogs

Autotaxin (ATX) catalyzes the hydrolysis of lysophosphatidylcholine (LPC) to form the bioactive lipid lysophosphatidic acid (LPA). LPA stimulates cell proliferation, cell survival, and cell migration and is involved in obesity, rheumatoid arthritis, neuropathic pain, atherosclerosis and various cancers, suggesting that ATX inhibitors have broad therapeutic potential. Product feedback inhibition of ATX by LPA has stimulated structure–activity studies focused on LPA analogs. However, LPA displays mixed mode inhibition, indicating that it can bind to both the enzyme and the enzyme–substrate complex. This suggests that LPA may not interact solely with the catalytic site. In this report we have prepared LPC analogs to help map out substrate structure–activity relationships. The structural variances include length and unsaturation of the fatty tail, choline and polar linker presence, acyl versus ether linkage of the hydrocarbon chain, and methylene and nitrogen replacement of the choline oxygen. All LPC analogs were assayed in competition with the synthetic substrate, FS-3, to show the preference ATX has for each alteration. Choline presence and methylene replacement of the choline oxygen were detrimental to ATX recognition. These findings provide insights into the structure of the enzyme in the vicinity of the catalytic site as well as suggesting that ATX produces rate enhancement, at least in part, by substrate destabilization.     

Nucleotide binding to glycogen phosphorylase b in the crystal.

The binding of the allosteric activator, AMP, and the inhibitor, ATP, to glycogen phosphorylase b has been studied in the crystal at 3 Å resolution. The nucleotides bind to two sites on the enzyme which are identified as site N, the allosteric effector site which is close to the subunit-subunit interface, and site I, a nucleoside inhibitor site which blocks the entrance to the active site crevasse. AMP when bound at the allosteric effector site makes several defined interactions with the enzyme in agreement with the results of solution studies. The contacts involve the N-10 position of the base, the 2′ hydroxyl of the ribose and the phosphate. IMP, analysed at 4 Å resolution, appears to bind in an identical conformation to AMP. At 3 Å resolution no well defined conformational changes are observed on binding AMP, although there are indications of a disturbance of the crystal lattice. It is concluded that the forces which stabilise the crystal lattice prevent the allosteric response of the enzyme in the crystal.     

Multiple forms of choline acetyltransferase in several species demonstrated by isoelectric focusing

1. The behaviour of choline acetyltransferase from pigeon, guinea-pig, rat and cat brain on isoelectric focusing was studied. 2. Choline acetyltransferase from pigeon and guinea-pig brain showed single peaks with isoelectric points at pH6.6 and 6.8 respectively. Only one molecular form of the enzyme was therefore detected in these species. 3. Three peaks of choline acetyltransferase activities with isoelectric points 7.3-7.6, 7.7-7.9 and 8.3 were obtained with enzyme preparations from rat brain. 4. The separate identities of each of the three forms were confirmed by refocusing. 5. Choline acetyltransferase activity from a high-speed supernatant of rat brain homogenate was distributed similarly to a partially purified enzyme preparation from rat brain in the isoelectric gradient. 6. The enzyme activities from cat brain were separated into two distinct peaks with isoelectric points 7.0 and 8.4, and a possible third peak with isoelectric point 7.6. 7. The two main peaks showed considerable differences in stability on storage, and their identities were confirmed by refocusing. 8. The distribution of the enzyme activities was unaltered by isoelectric focusing in the presence of 3m-urea. 9. The apparent K(m) for choline of choline acetyltransferase from rat, cat and guinea-pig brain was 0.8mm, whereas for the pigeon enzyme it was 0.4mm.     

Intermediates on the reassociation pathway of phosphofructokinase I from Escherichia coli

The folding and association pathway of the allosteric phosphofructokinase from Escherichia coli has been investigated after complete denaturation of the protein in guanidine hydrochloride by spectroscopical methods, fluorescence and circular dichroism. Three successive processes can be observed during the renaturation of this protein. First, a fast reaction, detected by fluorescence, results in the formation of a (partially) structured monomer. Second, two monomers associate into a dimeric species. This step involves the shielding of the unique tryptophan residue, Trp 311, from the aqueous solvent, and it corresponds to the formation of the interface containing the effector binding site. The presence of ATP during renaturation increases the rate of formation of this dimeric species. The other ligands of the enzyme have no effect on this reaction as well as on the whole reactivation. Finally, the enzymatic activity is regained during the third slowest step. This last reaction is due to the association of two dimers into the native tetrameric structure. The presence of fructose 6-phosphate does not increase the rate of reactivation, even though this ligand strongly stabilizes the native enzyme against denaturation by bridging the interface corresponding to the active site. The self-assembly of phosphofructokinase from E. coli from its unfolded and separated chains follows a specific order in the formation of the interactions between subunits and involves a dimeric intermediate with a defined geometry.     

Trapping choline oxidase in a nonfunctional conformation by freezing at low pH

Choline oxidase is a flavin-dependent enzyme that catalyzes the oxidation of choline to glycine-betaine, with oxygen as electron acceptor. Storage at pH 6 and -20 degrees C resulted in a change in the conformation of choline oxidase, which was associated with complete loss of catalytic activity when the enzyme was assayed at pH 6. Incubation of the inactive enzyme at pH values > or = 6.5 and 25 degrees C resulted in a fast and partial reactivation of the enzyme, which occurred with slow onset of steady state during enzymatic turnover. The rate of approaching steady state was independent of the concentrations of choline and enzyme, but increased to a limiting value with increasing pH, defining a pKa value of approximately 7.3 for an unprotonated group required for enzyme activation. Prolonged incubation of the inactive enzyme at pH 6 and temperatures > or = 20 degrees C, at which no hysteretic behavior was observed, resulted in the slow and full recovery of activity over 3 h, associated with a conformational change that reverted the enzyme to the native form. Activation of the enzyme at pH 6 was enthalpy-driven with deltaH(double dagger) and TdeltaS(double dagger) values of approximately 112 kJ mol(-1) and approximately 20 kJ mol(-1) determined at 25 degrees C. These data suggest that freezing the enzyme at low pH induces a localized and reversible conformational change that is associated with the complete and reversible loss of catalytic activity.     

Amino acid effector binding to rabbit muscle pyruvate kinase

l-Phenylalanine, an allosteric inhibitor of rabbit muscle pyruvate kinase, is shown to bind to the tetrameric enzyme in a ratio of 4 moles effector per mole of tetramer. This binding is slightly cooperative in the absence of divalent cation activators, but the cooperativity is strongly increased when measured in the presence of 2.5 mm Mg²⁺ or Mn²⁺. The effector affinity is somewhat decreased under these conditions. l-Alanine was known to antagonize all measured phenylalanine effects and is shown here to also bind to 4 sites on the protein. The binding is noncooperative and little affected by the presence of the divalent activating cations. Competition experiments with phenylalanine and alanine suggest competition for the same site. Substrate kinetic measurements at P-enolpyruvate and Mg²⁺ concentrations under 100 μm show considerable inhibition of the enzyme at phenylalanine concentrations around 100 μm, near the serum levels of the free amino acid. The approach to the phenylalanine-inhibited velocity occurs with half-times less than 1 sec.     

Renaturation of the allosteric phosphofructokinase from Escherichia coli

The allosteric phosphofructokinase from Escherichia coli has been renatured after complete unfolding in concentrated guanidine hydrochloride. The enzyme regains both its catalytic and regulatory abilities quantitatively. The kinetics of reactivation are biphasic and are consistent with a two-step mechanism in which a monomolecular reaction precedes a bimolecular one. The presence of ATP during reactivation increases the rate at which phosphofructokinase is renatured; the second order rate constant of the bimolecular step increases from about 10(4) M-1 S-1 in the absence of ATP to about 2 X 10(5) M-1 S-1 in the presence of 1 mM ATP. The other ligands of the enzyme have no effect on reactivation. It is tentatively proposed that a folded monomer is the intermediate species which already possesses a functional ATP-binding site and that the rate-limiting association step is the formation of dimeric species. This interpretation is compatible with the known three-dimensional structure of another bacterial phosphofructokinase, that from Bacillus stearothermophilus.     

Inactivation of yeast phosphoglycerate kinase by Cr-ATP complexes and its implications on the conformation of the enzyme active site.

Exchange-inert beta, gamma-bidentate Cr(H2O)x(NH3)y ATP complexes inactivate yeast phosphoglycerate kinase (PGK) by forming a coordination complex at the enzyme active site. The observed inactivation rates ranged from 0.019 min-1 to 0.118 min-1 for Cr(NH3)4ATP and Cr(H2O)4ATP, respectively. Incorporation of one mol of Cr-ATP to the enzyme was sufficient for complete inactivation of the enzyme. The presence of Mg-ATP protected the enzyme against inactivation by Cr-ATP. The other substrate 3-phosphoglycerate (3-PGA), when present, reduced the observed inactivation rates. The reduction of the k(obs) by 3-PGA was proportional to the number of NH3 ligands present in the coordination sphere of Cr3+ in the Cr-ATP complex, suggesting that in the ternary enzyme-Cr-ATP-3-PGA complex 3-PGA may be coordinated to the metal ion. When the effector sulfate ion was present, the presence of 3-PGA did not cause any further effects on the observed inactivation rates. This suggests that bound substrates are in a different arrangement at the active site when sulfate is present and therefore 3-PGA may not need to displace a ligand from Cr3+. Additionally, PGK exhibited a stereoselectivity for the binding of Cr(H2O)4ATP. delta diastereomer of Cr(H2O)4ATP yielded an order of magnitude smaller Ki value compared to the value observed with the lambda isomer. The recovery of enzyme activity was observed over a period of a few hours upon removal of excess Cr-ATP. The presence of substrates and/or effector ion sulfate did not alter the observed reactivation rate. There was no difference in the reactivation rates of the enzyme which was inactivated with Cr(H2O)4ATP or Cr(NH3)4ATP with and without 3-PGA. Increasing the ligand exchange rates of Cr3+ of Cr-ATP by increasing the pH value of the recovery medium from 5.9 to 6.8 increased the rate of recovery by a factor of 8. The pH dependence of the reactivation indicated that one hydroxyl group is involved in the recovery of the enzyme activity in enzyme CrATP and enzyme.CrATP.3-PGA complexes.     

SEPARATION OF APPARENT MULTIPLE FORMS OF HUMAN BRAIN CHOLINE ACETYLTRANSFERASE BY ISOELECTRIC FOCUSING

Abstract— Choline acetyltransferase (acetyl-CoA: choline O -acetyl transferase; EC 2.3.1.6; ChAc) purified from human brain (basal ganglia) and sciatic nerve were separated into apparent multiple enzyme forms by the method of isoelectric focusing (pH gradient 3-10) on acrylamide gel. A preparative separation of enzyme forms of human brain was accomplished by the column method, by using a sucrose gradient. When each separated form was re-electrofocused, only a portion of the ChAc activity was observed in its original pH region while more than one-half of the recovered activity for each fraction appeared at pH 7.8-8. Gel filtration and kinetic studies of separated forms indicated that the more acidic forms might be aggregates, while more basic forms might be configurational isomers. Human ChAc of sciatic nerve did not exhibit acidic forms on electrofocusing, but otherwise yielded an electrofocusing profile similar to that of human brain. ChAc of rabbit brain and sciatic nerve each exhibited only a single form at pH 7.1 ± 0.2. Although ChAc differs among species, the enzyme of brain and sciatic nerve of the same species cannot be clearly distinguished by electrofocusing.     

Choline transport activity in Staphylococcus aureus induced by osmotic stress and low phosphate concentrations.

Uptake of [14C]choline upon hyperosmotic stress of exponential-phase Staphylococcus aureus cultures in a complex medium occurred after a delay of 2.5 to 3.5 h. This uptake could be prevented by chloramphenicol, suggesting that it occurred via an inducible transport system. Radioactivity from [14C]choline was accumulated as [14C]glycine betaine. However, neither choline nor glycine betaine could act as the major carbon and energy source for the organism, suggesting that choline was not metabolized beyond glycine betaine. Assay of choline transport activity in cells grown under different conditions in defined media revealed that osmotic stress was mainly responsible for the induction, but choline gave a further increase in induction. The system was not induced in anaerobically grown cells. Choline transport activity was repressed by glycine betaine and proline betaine, suggesting that these compounds are corepressors. Choline transport activity was not induced in cells osmotically stressed by 1 M potassium phosphate or 0.5 M sodium phosphate, but was induced in cells grown in low-phosphate medium in the absence of osmotic stress. This suggests that there is a connection between the phosphate and osmotic stress regulons. Choline transport was energy and Na+ dependent and had a Km of 46 microM and a maximum rate of transport (Vmax) of 54 nmol/min/mg (dry weight). The results of competition studies suggested that N-methyl and an alcohol group or aldehyde groups at the ends of the molecule were important in its recognition by the system. Glycine betaine was not a highly effective competitor, suggesting that its transport system and the choline transport system were distinct from each other. Choline transport was highly susceptible to a variety of inhibitors, which may be related to the greater dependence on respiratory metabolism of cells grown in the presence of high NaC1 concentrations.