Molecular form of human lymphocyte membrane-bound acetylcholinesterase

The membrane-bound acetylcholinesterase (AchE) from human peripheral blood lymphocyte gives only one symmetrical peak on sucrose density gradient centrifugation in the presence of Triton X-100 detergent, with the calculated sedimentation coefficient of 6.5 S. However, this dimeric form of AchE was converted to a monomeric 3.8 S form when treated with 2-mercaptoethanol and iodoacetic acid. The results are consistent with studies which have shown by sodium dodecyl sulfate gel electrophoresis that the enzyme is built up of two identical monomers inter-linked by disulfide bond(s). Under reducing conditions, revealed a single species of 70,000 molecular weight, whereas under non-reducing conditions, another species of 140,000 molecular weight of the AchE was found. Polyacrylamide gel electrophoresis indicated a single band with AchE activity in the presence of Triton X-100. In contrast, in the absence of the same detergent multiple band pattern could be observed. These results suggest that membrane-bound AchE enzyme is present in homogenous dimeric form on human lymphocyte membrane.     

Multiple molecular forms of purified human erythrocyte acetylcholinesterase.

1. Human erythrocyte acetylcholinesterase was solubilized by Triton X-100 and purified by affinity chromatography to a specific activity of 3800 IU/mg of protein. The yield of the purified enzyme was 25--45%. 2. Gel filtration on Sepharose 4-B in the presence of Triton X-100 revealed one peak of enzyme activity with a Stokes' radius of 8.7 nm. Density gradient centrifugation in 0.1% Triton X-100 showed one peak of enzyme activity with an S4 value of 6.3S. 3. Isoelectric focusing in Triton X-100 resolved the enzyme into five molecular forms with isoelectric points of 4.55, 4.68, 4.81, 4.98 and 5.18. Upon incubation with neuraminidase the enzyme activity in the first four forms was decreased with a concommitant increase in activity in the form with the higher isoelectric point. 4. After removal of excess Triton X-100 on Bio-Gel HTP, polyacrylamide gel electrophoresis showed seven bands of protein and corresponding bands of enzyme activity. Density gradient centrifugation of the detergent-depleted enzyme at high ionic strength revealed five multiple molecular forms with S4 values of 6.3 S, 10.2 S, 12.2 S, 14.2 S and 16.3 S. At low ionic strength, higher aggregates were observed in addition to the other forms. Dodecylsulfate-polyacrylamide gel electrophoresis gave one subunit only with an apparent molecular weight of 80 000. 5. These results suggest that human erythrocyte acetylcholinesterase, solubilized by Triton X-100, exists in various forms differing in net charge but of apparently similar molecular dimensions. After removal of the detergent, forms with different molecular sizes are observed.     

Purification and characterization of amphiphilic trehalase from rabbit small intestine

Rabbit intestinal trehalase (alpha,alpha-trehalose glucohydrolase, EC 3.2.1.28) was solubilized with Triton X-100 and purified in the presence of EDTA. The purified enzyme was homogeneous on polyacrylamide gel electrophoresis in the presence of Triton X-100 or SDS. It showed amphiphilic properties on gel filtration. polyacrylamide gel electrophoresis, charge-shift electrophoresis and phenyl-Sepharose chromatography. Its molecular weight was estimated to be about 330 000 by gel filtration under nondenaturing conditions and in the presence of Triton X-100, the value being in satisfactory agreement with the sum of the weight of one Triton X-100 micelle and twice the molecular weight (105 000) of purified hydrophilic trehalase which had been deprived of the anchor segment. The two purified trehalases gave almost the same molecular weights (about 75 000) on SDS-polyacrylamide gel electrophoresis. These results suggest that intestinal trehalase consists of two subunits with a molecular weight of 75 000 and that its anchor segment is small (less than 5000). Triton X-100 extracts freshly prepared from intestinal microvilli essentially showed one form of trehalase, which behaved on phenyl-Sepharose and Con A-Sepharose chromatography in the same manner as purified amphiphilic trehalase.     

Purification of the insulin receptor from human placental membranes

Insulin receptors were purified from human placental microsomal membranes by solubilisation with Triton X-100 followed by Sepharose 6B chromatography, phosphate gradient elution from hydroxyapatite and affinity chromatography on concanavalin A-Sepharose. 2000-fold purification was achieved with 63% overall recovery. The purified receptor gave a single band on 3.75% polyacrylamide (0.1% Triton X-100) gel electrophoresis. On sodium dodecyl sulphate-polyacrylamide gel electrophoresis there was a major band at 75,000 and a minor band at 80,000 daltons. The purified receptor rechromatographed on Sepharose 6B with an apparent molecular weight of 300,000.     

Purification and Characterization of a Membrane-Bound Enkephalin-Forming Carboxypeptidase, "Enkephalin Convertase"

Enkephalin convertase, the enkephalin-synthesizing carboxypeptidase B-like enzyme, has been purified to apparent homogeneity from bovine pituitary and adrenal chromaffin granule membranes. The membrane-bound enkephalin convertase can be solubilized in high yield with 0.5% Triton X-100 in the presence of 1 M NaCl. Extensive purification is achieved by affinity chromatography with p-aminobenzoyl-L-arginine linked to Sepharose 6B. Enzyme purified from both pituitary and adrenal chromaffin granule membranes shows a single band by sodium dodecyl sulfate polyacrylamide gel electrophoresis with an apparent molecular weight of 52,500, whereas enkephalin convertase purified from soluble extracts of these tissues has an apparent molecular weight of 50,000. The regional distribution of the membrane-bound enzyme in the rat brain differs from that of the soluble enzyme. While the soluble enzyme shows 10-fold variations, resembling somewhat the enkephalin peptides, membrane-bound enkephalin convertase is more homogeneously distributed throughout the brain. In rat pituitary glands, membrane-bound enzyme activity is similar in the anterior and posterior lobes, whereas the soluble enzyme is enriched in the anterior lobe. Membrane-bound and soluble forms of enkephalin convertase isolated from either bovine pituitary glands or adrenal chromaffin granules show identical substrate and inhibitor specificities. As with the soluble enzyme, membrane-bound enkephalin convertase hydrolyzes [Met]- and [Leu]enkephalin-Arg6 and -Lys6 to enkephalin, with no further degradation of the pentapeptide.     

Purification and characterization of an arginine-specific carboxypeptidase from Mycoplasma salivarium.

The carboxypeptidase which had been shown to be present exclusively in nonfermentative mycoplasmas was found to be associated with cell membranes of Mycoplasma salivarium. The enzyme was released from the membranes with Triton X-100 and purified by ion-exchange chromatography on DEAE-Sephacel, affinity chromatography on arginine-Sepharose 4B, and chromatofocusing. The purified enzyme had a molecular mass of 218 kilodaltons, as estimated by gel filtration through Sepharose CL-6B, and yielded one band of activity in analytical disc-polyacrylamide gel electrophoresis performed in the presence of 0.5% (wt/vol) Triton X-100. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme treated in the presence or absence of 2-mercaptoethanol revealed one band with a molecular mass of 87 kilodaltons. The enzyme catalyzed selectively the cleavage of the C-terminal arginine residue of peptides such as N-benzoylglycyl-L-arginine, tuftsin, and bradykinin and was inhibited considerably by o-phenanthroline and EDTA but only slightly by NiCl2. The inhibition of the enzyme by EDTA was fully reversed by the addition of ZnCl2, whereas the addition of CoCl2 activated the enzyme.     

Membrane-bound D-gluconate dehydrogenase from Pseudomonas aeruginosa. Purification and structure of cytochrome-binding form.

A membrane-bound D-gluconate dehydrogenase [EC 1.1.99.3] was solubilized from membranes of Pseudomonas aeruginosa and purified to a homogeneous state with the aid of detergents. The solubilized enzyme was a monomer in the presence of at least 0.1% Triton X-100, having a molecular weight of 138,000 on polyacrylamide gel electrophoresis or 124,000--131,000 on sucrose density gradient centrifugation. In the absence of Triton X-100, the enzyme became dimeric, having a molecular weight of 240,000--260,000 on sucrose density gradient centrifugation. Removal of Triton X-100 caused a decrease in enzyme activity. Enzyme activity was stimulated by addition of phospholipid, particularly cardiolipin, in the presence of Triton X-100. The enzyme had a cytochrome c1, c-554(551), which might be a diheme cytochrome, and it also contained a covalently bound flavin but not ubiquinone. In the presence of sodium dodecyl sulfate, the enzyme was dissociated into three components with molecular weights of 66,000, 50,000, and 22,000. The components of 66,000 and 50,000 daltons corresponded to a flavoprotein and cytochrome c1, respectively, but that of 22,000 dalton remained unclear as to its function.     

Molecular forms of acetylcholinesterase present in the white and grey matter of pig brain

Extraction of the white matter of pig brain with EDTA, lysolecithin or Triton X-100 gave poor yields of soluble acetylcholinesterase although these agents had proved effective at solubilizing the enzyme in the grey matter. This finding, together with the observation that the strong detergent sodium deoxycholate, was needed to solubilize the enzyme, shows that it is more difficult to remove acetylcholinesterase from the white matter of brain than from the grey. This could mean that the enzyme in the white matter is more firmly bound to the membrane than the enzyme in the grey matter.The difference in binding of the enzyme from the two regions of the brain is also reflected in the affinity chromatography experiments which showed a lower recovery for the acetylcholinesterase of white matter compared with the enzyme from grey matter.Starch-block electrophoresis of acetylcholinesterase showed a single negatively charged peak of activity for both the naturally soluble and the deoxycholate solubilized preparations. The presence of only one form on electrophoresis suggests that the molecular species of acetylcholinesterase do not arise from differences in charge.Sucrose density gradient centrifugation of the two preparations from white matter gave a single peak of activity with a sedimentation constant of about 10 S. This corresponds closely to the major species of molecular weight 260,000 detected by gradient gel electrophoresis. Other forms detected in both enzyme preparations by gradient gel electrophoresis were species with molecular weights of 660,000, 180,000, 130,000 and 115,000. The significance of these species in terms of the formation of oligomers is discussed.A comparison was made with the corresponding preparations of acetylcholinesterase from the grey matter and the results showed that acetylcholinesterase from the white and grey matter of pig brain were very similar. The exception to this was the species with a molecular weight of 68,000 which was present in the grey but not the white matter of pig brain.     

Purification of phosphatidylethanolamine N-methyltransferase from rat liver

Phosphatidylethanolamine (PE) N-methyltransferase catalyzes the synthesis of phosphatidylcholine by the stepwise transfer of methyl groups from S-adenosylmethionine to the amino head group of PE. PE N-methyltransferase was solubilized from a microsomal membrane fraction of rat liver using the nonionic detergent Triton X-100 and purified to apparent homogeneity. Specific activities of PE N-methyltransferase with PE, phosphatidyl-N-monomethylethanolamine (PMME), and phosphatidyl-N,N-dimethylethanolamine (PDME) as substrates were 0.63, 8.59, and 3.75 mumol/min/mg protein, respectively. The purified enzyme was composed of a single subunit with a molecular mass of 18.3 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Methylation activities dependent on the presence of PE, PMME, and PDME and the 18.3-kDa protein co-eluted when purified PE N-methyltransferase was subjected to gel filtration on Sephacryl S-300 in the presence of 0.1% Triton X-100. All three methylation activities eluted with a Stokes radius 2.1 A greater than that determined for pure Triton micelles (molecular mass difference of 27.4 kDa). Two-dimensional analysis of PE N-methyltransferase employing nonequilibrium pH gradient gel electrophoresis and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicates that the enzyme is composed of a single isoform. Analysis of enzyme activity using PE, PMME, and PDME at various Triton X-100 concentrations indicated the enzyme follows the "surface dilution" model proposed for other enzymes that act at the surface of mixed micelle substrates. Initial velocity data for all three lipid substrates (at fixed concentrations of Triton X-100) were highly cooperative in nature. Hill numbers for PMME and PDME ranged from 3 at 0.5 mM Triton to 6 at 2.0 mM Triton. All three methylation activities had a pH optimum of 10. These results provide evidence that a single membrane-bound enzyme catalyzes all three methylation steps for the conversion of PE to phosphatidylcholine.     

Amphiphile dependency of the monomeric and dimeric forms of acetylcholinesterase from human erythrocyte membrane

Human erythrocyte membrane-bound acetylcholinesterase was converted to a monomeric species by treatment of ghosts with 2-mercaptoethanol and iodoacetic acid. After solubilization with Triton X-100, the reduced and alkylated enzyme was partially purified by affinity chromatography and separated from residual dimeric enzyme by sucrose density gradient centrifugation in a zonal rotor. Monomeric and dimeric acetylcholinesterase showed full enzymatic activity in presence of Triton X-100 whereas in the absence of detergent, activity was decreased to approx. 20% and 15%, respectively. Preformed egg phosphatidylcholine vesicles fully sustained activity of the monomeric species whereas the dimer was only 80% active. The results suggest that a dimeric structure is not required for manifestation of amphiphile dependency of membrane-bound acetylcholinesterase from human erythrocytes. Furthermore, monomeric enzyme appears to be more easily inserted into phospholipid bilayers than the dimeric species.     

Purification and partial characterization of arylsulphatase C from human placental microsomes

Arylsulphatase C (EC 3.1.6.1) has been purified 300-fold from human placental microsomes using a four step procedure involving solubilization with Triton X-100, chromatography on hydroxyapatite, column chromatofocussing and ion-exchange chromatography on DEAE-Sepharose. The purified enzyme is electrophoretically homogeneous and has a molecular weight of 440 000 as determined by polyacrylamide gradient gel electrophoresis. On analysis of the preparation by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate a polypeptide of molecular weight 74 000 was observed, suggesting that the enzyme as purified may be a hexamer. The behaviour of the enzyme during chromatofocussing indicates the enzyme has a pI of 6.56. Steroid sulphatase, as measured by activity towards dehydroepiandrosterone sulphate, co-purifies with arylsulphatase C suggesting that both activities are due to a single enzyme.     

Purification and characterization of an enkephalin aminopeptidase from rat brain membranes

A membrane-bound aminopeptidase was purified from rat brain, and its activity was assayed by high-pressure liquid chromatography with Met-enkephalin as the substrate. The enzyme was extracted with 1% Triton X-100 and purified by chromatography, successively on DEAE-Sepharose CL-6B, Bio-Gel HTP, and Sephadex G-200 columns. The overall purification was about 1200-fold, with 25% yield. The purified enzyme showed one band on disc gel electrophoresis and two bands on sodium dodecyl sulfate electrophoresis with molecular weights of 62 000 and 66 000. The aminopeptidase has a pH optimum of 7.0, a Km of 0.28 mM, and a Vmax of 45 mumol (mg of protein)-1 min-1 for Met-enkephalin. It releases tyrosine from Met-enkephalin, but it does not split the byproduct. It does not hydrolyze gamma- or beta-endorphin, or dynorphin, but it does hydrolyze neutral and basic aminoacyl beta-naphthylamides. The enzyme is inhibited by the aminopeptidase inhibitors amastatin, bestatin, and bestatin-Gly. Its properties, such as its subcellular localization, substrate specificity, pH optimum, and molecular weight, distinguish it from leucine aminopeptidase, aminopeptidase A, aminopeptidase B, aminopeptidase M, and the soluble aminopeptidase for enkephalin degradation.     

Purification of the acetylcholinesterase from human erythrocytes by affinity chromatography (author's transl)]

The acetylcholinesterase from human erythrocytes was released from the plasma membrane with 0.2% Triton X-100 at low ionic strength and purified by two affinity chromatography steps on Sepharose-bound m-[6-(6-amino-caproylamino)caproylamino]phenyltrimethyl-ammonium. The synthesis of the inhibitor is described. The purified, detergent-free acetylcholinesterase was obtained with a specific activity of 4270 U/mg (158000-fold purification) and a 28% yield. The enzyme is a glycoprotein and aggregates in the absence of Triton X-100 into higher molecular complexes. The molecular weight was estimated by sodium dodecylsulfate electrophoresis to be 80000 +/- 3000 in the presence of 2-mercapto-ethanol and 154000 +/- 6000 in its absence.     

Purification and characterization of membrane-bound phosphatidylinositol kinase from rat brain

A membrane-bound phosphatidylinositol (PI) kinase was purified from rat brain. The enzyme was solubilized with Triton X-100 from salt-washed membrane and purified 11,183-fold, with a final specific activity of 150 nmol/min/mg of protein. Purification steps included several chromatography using Q-Sepharose Fast Flow, cellulose phosphate, Toyopearl HW 55 and Affi-Gel Blue. The purified PI kinase had an estimated molecular weight of 80,000 by gel filtration and 76,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified kinase phosphorylated only PI and did not phosphorylate phosphatidylinositol 4-phosphate or diacylglycerol. Km values for PI and ATP were found to be 115 and 150 microM, respectively. The enzyme required Mg2+ (5-20 mM) or Mn2+ (1-2 mM) for activity, was stimulated by 0.1-1.0% (w/v) Triton X-100, and completely inhibited by 0.05% sodium dodecyl sulfate. The enzyme activity showed a broad pH optimum at around 7.4. The enzyme utilized ATP and not GTP as phosphate donor. Nucleoside triphosphates other than ATP and diphosphates significantly inhibited the kinase activity. However, inhibitory effects of adenosine, cAMP, and quercetin were weak.     

Purification of the Primary Dihydroorotate Dehydrogenase (Oxidase) from Rat Liver Mitochondria

Dihydroorotate dehydrogenase was purified to homogeneity from rat liver mitochondria by Triton X-100 solubilization, diethylaminoethyl cellulose chromatography and gel electrophoresis. The overall yield was 30 percent. The enzyme has a subunit molecular weight of 61, 000.     

Isolation of L-dynurenine 3-hydroxylase from the mitochondrial outer membrane of rat liver.

Outer membrane preparations of rat liver mitochondria were isolated, after the mitochondria had been prepared by mild digitonin treatment under isotonic conditions. L-Kynurenine 3-hydroxylase [EC 1.14.13.9] was solubilized on a large scale from outer membrane by mixing with 1% digitonin or 1% Triton X-100, followed by fractionation into a minor fraction I and a major fraction II by DEAE-cellulose column chromatography. The distribution of total L-Dynurenine 3-hydroxylase was roughly 20 and 80% in fraction I and II, respectively. Fraction I consisted of crude enzyme loosely bound to anion exchanger. In the present investigation, fraction I was not used because of its low activity and rapid inactivation. In contrast, fraction II consisted of crude enzyme with high activity, excluded from DEAE-cellulose column chromatography in the presence of 1 M KC1. In addition, fraction II was purified by Sephadex G-200 gel filtration and DEAE-Sephadex A-50 column chromatography with linear gradient elution, adding 1 M KC1 and 1% Triton X-100 to 0.05 M Tris-acetate buffer, pH 8.1. After isoelectric focusing, the purified enzyme preparation was proved to be homogeneous, since the L-kynurenine 3-hydroxylase fraction gave a single band on disc gel electrophoresis. The molecular weight of this enzyme was estimated to be approximately 200,000 or more by SDS-polyacrylamide gel electrophoresis and from the elution pattern on Sephadex G-200 gel filtration. A 16-Fold increase of the enzyme activity was obtained compared with that of the mitochondrial outer membrane. The isoelectric point of the enzyme was determined to be pH 5.4 by Ampholine isoelectric focusing.     

Biochemical characterization of human umbilical vein endothelial cell membrane bound acetylcholinesterase

Acetylcholinesterase is an enzyme whose best-known function is to hydrolyze the neurotransmitter acetylcholine. Acetylcholinesterase is expressed in several noncholinergic tissues. Accordingly, we report for the first time the identification of acetylcholinesterase in human umbilical cord vein endothelial cells. Here we further performed an electrophoretic and biochemical characterization of this enzyme, using protein extracts obtained by solubilization of human endothelial cell membranes with Triton X-100. These extracts were analyzed under polyacrylamide gel electrophoresis in the presence of Triton X-100 and under nondenaturing conditions, followed by specific staining for cholinesterase or acetylcholinesterase activity. The gels revealed one enzymatically active acetylcholinesterase band in the extracts that disappeared when staining was performed in the presence of eserine (an acetylcholinesterase inhibitor). Performing western blotting with the C-terminal anti-acetylcholinesterase IgG, we identified a single protein band of approximately 70 kDa, the molecular mass characteristic of the human monomeric form of acetylcholinesterase. The western blotting with the N-terminal anti-acetylcholinesterase IgG antibody revealed a double band around 66-70 kDa. Using the Ellman's method to measure the cholinesterase activity in human umbilical vein endothelial cells, regarding its substrate specificity, we confirmed the existence of an acetylcholinesterase enzyme. Our studies revealed a predominance of acetylcholinesterase over other cholinesterases in human endothelial cells. In conclusion, we have demonstrated the existence of a membrane-bound acetylcholinesterase in human endothelial cells. In future studies, we will investigate the role of this protein in the endothelial vascular system.     

Multiple forms of acetylcholinesterase from pig brain

1. A number of methods of solubilization of pig brain acetylcholinesterase (EC 3.1.1.7) were studied. The multiple enzymic forms of the resultant preparations were examined by polyacrylamide-gel electrophoresis. 2. Butanol extraction, Nagarase treatment and ultrasonication proved unsuitable as preparatory methods, but detergent treatment (Triton X-100, Triton X-100-KCl and lysolecithin) gave good yields. 3. Separation of soluble enzyme in three systems of polyacrylamide-gel electrophoresis were compared and the relative advantages are discussed. 4. By using a 6% (w/v) gel and continuous buffer system two forms of acetylcholinesterase were detected in Triton X-100-solubilized enzyme, but the incorporation of a sample and spacer gel and a discontinuous buffer system resolved this into four components. The forms of the soluble enzyme extracted by different methods differed in mobility. 5. With gradient polyacrylamide-gel electrophoresis between two and six forms were detected, depending on the method used for extraction. The average molecular weights of the five forms most frequently found were 60000, 130000, 198000, 266000 and 350000. 6. Treatment of the Triton X-100-extracted enzyme with 2.5m-urea altered the pattern and evidence of dissociation was observed. 7. The results are discussed in the light of present theories on the molecular structure of acetylcholinesterase.     

Purification and properties of acyl/alkyl dihydroxyacetone-phosphate reductase from guinea pig liver peroxisomes

The peroxisomal acyl/alkyl dihydroxyacetone-phosphate reductase (EC 1.1.1.101) was solubilized and purified 5500-fold from guinea pig liver. The enzyme could be solubilized by detergents only at high ionic strengths in presence of the cosubstrate NADPH. Peroxisomes, isolated from liver by a Nycodenz step density gradient centrifugation, were first treated with 0.2% Triton X-100 to remove the soluble and a large fraction of the membrane-bound proteins. The enzyme was solubilized from the resulting residue by 0.05% Triton X-100, 1 M KCl, 0.3 mM NADPH, and 2 mM dithiothreitol in Tris-HCl buffer (10 mM) at pH 7.5. The enzyme was further purified after precipitating it by dialyzing out the KCl and then resolubilized with 0.8% octyl glucoside in 1 M KCl (plus NADPH and dithiothreitol). The second solubilized enzyme was purified to homogeneity (370-fold from peroxisomes) by gel filtration in a Sepharose CL-6B column followed by affinity chromatography on an NADPH-agarose gel matrix. NADPH-agarose was prepared by reacting periodate-oxidized NADP+ to adipic acid dihydrazide-agarose and then reducing the immobilized NADP+ with NaBH4. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified enzyme showed a single homogeneous band with an apparent molecular weight of 60,000. The molecular weight of the native enzyme was estimated to be 75,000 by size exclusion chromatography. Amino acid analysis of the purified protein showed that hydrophobic amino acid comprised 27% of the molecule. The Km value of the purified enzyme for hexadecyldihydroxyacetone phosphate (DHAP) was 21 microM, and the Vmax value in the presence of 0.07 mM NADPH was 67 mumol/min/mg. The turnover number (Kcat), after correcting for the isotope effect of the cosubstrate NADP3H, was calculated to be 6,000 mol/min/mol of enzyme, assuming the enzyme has a molecular weight of 60,000. The purified enzyme also used palmitoyldihydroxyactone phosphate as a substrate (Km = 15.4 microM, and Vmax = 75 mumol/min/mg). Palmitoyl-DHAP competitively inhibited the reduction of hexadecyl-DHAP, indicating that the same enzyme catalyzes the reduction of both acyl-DHAP and alkyl-DHAP. NADH can substitute for NADPH, but the Km of the enzyme for NADH (1.7 mM) is much higher than that for NADPH (20 microM). The purified enzyme is competitively (against NADPH) inhibited by NADP+ and palmitoyl-CoA. The enzyme is stable on storage at 4 degrees C in the presence of NADPH and dithiothreitol.     

Amphiphilic Detergent-Soluble Acetylcholinesterase from Torpedo marmorata: Characterization and Conversion by Proteolysis to a Hydrophilic Form

The membrane-bound acetylcholinesterase (AChE) from the electric organ of Torpedo marmorata was solubilized by Triton X-100 or by treatment with proteinase K and purified to apparent homogeneity by affinity chromatography. Although the two forms differed only slightly in their subunit molecular weight (66,000 and 65,000 daltons, respectively), considerable differences existed between native and digested detergent-soluble AChE. The native enzyme sedimented at 6.5 S in the presence of Triton X-100 and formed aggregates in the absence of detergent. The digested enzyme sedimented at 7.5 S in the absence and in the presence of detergent. In contrast to the detergent-solubilized AChE, the proteolytically derived form neither bound detergent nor required amphiphilic molecules for the expression of catalytic activity. This led to the conclusion that limited digestion of detergent-soluble AChE results in the removal of a small hydrophobic peptide which in vivo is responsible for anchoring the protein to the lipid bilayer.