Amphiphilic and hydrophilic molecular forms of acetylcholinesterase in membranes derived from sarcoplasmic reticulum of skeletal muscle

Native molecular forms of acetylcholinesterase (AChE) present in a microsomal fraction enriched in SR of rabbit skeletal muscle were characterized by sedimentation analysis in sucrose gradients and by digestion with phospholipases and proteinases. The hydrophobic properties of AChE forms were studied by phase-partition of Triton X-114 and Triton X-100-solubilized enzyme and by comparing their migration in sucrose gradient containing either Triton X-100 or Brij 96. We found that in the microsomal preparation two hydrophilic 13.5 S and 10.5 S forms and an amphiphilic 4.5 S form exist. The 13.5 S is an asymmetric molecule which by incubation with collagenase and trypsin is converted into a 'lytic' 10.5 S form. The hydrophobic 4.5 S form is the predominant one in extracts prepared with Triton X-100. Proteolytic digestion of the membranes with trypsin brought into solution a significant portion of the total activity. Incubation of the membranes with phospholipase C failed to solubilize the enzyme. The sedimentation coefficient of the amphiphilic 4.5 S form remained unchanged after partial reduction, thus confirming its monomeric structure. Conversion of the monomeric amphiphilic form into a monomeric hydrophilic molecule was performed by incubating the 4.5 S AChE with trypsin. This conversion was not produced by phospholipase treatment.     

INCREASE IN THE APPARENT AChE ACTIVITY IN THE MOUSE DIAPHRAGM INDUCED BY PROTEOLYTIC TREATMENT

Abstract— Isolated endplate regions from the mouse diaphragm were treated with different agents before or after homogenization in order to solubilize junctional AChE and study the effect of solubilization on its apparent activity. Total AChE activity (solubilized + nonsolubilized) of samples treated with collagenase or papain before homogenization was nearly twice as high as in control samples. If collagenase was added after homogenization no increase in apparent activity was observed although in both cases about 70–80% of AChE activity was solubilized. The access of ACh to the membrane-bound enzyme is probably not a limiting factor in the AChE assay as is the case in the electric organ homogenates. Both 1 m -NaCl and Triton X-100 were quite ineffective as solubilizers when applied before homogenization and had an insignificant effect on the apparent AChE activity.
The increase in apparent AChE activity cannot be explained either by a de novo synthesis or by the change in kinetic properties of different species of AChE, or by the release of AChE possibly sequestrated in the membrane vesicles. The possibility is discussed that a part of junctional AChE is inactivated at the beginning of homogenization while it can be preserved by previous solubilization, or that proteolytic treatment may activate some 'silent' AChE sites in motor endplates.
However, the mere fact that the difference does exist suggests that all AChE activity present in intact motor endplates may not be measurable after homogenization.     

Differential effects of ethanol on membrane-bound and soluble acetylcholinesterase from sarcoplasmic reticulum membranes

The action of ethanol on the activity of membrane-bound and soluble acetylcholinesterase (AChE) in sarcoplasmic reticulum of skeletal muscle has been studied. Treatment of membranes with 2.5–12.5% v/v ethanol produced a slight stimulation of the AChE activity and inhibition at higher concentration. The enzyme remained associated with the membranes after these treatments. The enzyme solubilized with Triton X-100 was inhibited by ethanol in a time-independent manner. Isolated 16 S (A₁₂), 10.5 S (G₄) and 4.5 S (G₁) forms of AChE were inhibited by ethanol to a similar extent. Samples were reversibly inhibited by ethanol, up to 12.5% v/v, and irreversibly at higher concentrations. Kinetic studies performed with isolated forms in the presence of 5–12.5% v/v ethanol showed that the solvent behaved as a competitive inhibitor of the asymmetric form but as a mixed inhibitor of the tetrameric and monomeric forms. The results show that the solvent interacts with active and/or regulatory sites of AChE from muscle microsomes.     

Horse kidney neutral alpha-D-glucosidase: purification of the detergent-solubilized enzyme; comparison with the proteinase-solubilized forms

Neutral alpha-D-glucosidase (alpha-D-glucoside glucohydrolase, EC 3.2.1.20) from horse kidney brush-border membranes was solubilized using Emulphogene BC 720 and purified by an affinity chromatography technique. The enzyme preparation (390-fold purified), which was free of other known microvillus hydrolases, exhibited one precipitate line in crossed immunoelectrophoresis and migrated as a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis. Several criteria (charge-shift crossed immunoelectrophoresis and hydrophobic chromatography) revealed the purified detergent form of the enzyme to be an amphipathic molecule. The papain treatment of either brush-border membrane vesicles or the purified detergent form of neutral alpha-D-glucosidase released an enzymatic form devoid of these amphipathic properties. Conversely, after trypsin treatment of the "d' form of the enzyme, two enzymatic forms were obtained: the first and major form retained these amphipathic properties; the second form exhibiting the same properties as the papain-released form. Furthermore, only a very small amount of neutral alpha-D-glucosidase can be released after trypsin solubilization of brush-border membrane vesicles, and the released enzyme did not exhibit amphipathic properties. These results were interpreted as meaning that the trypsin attack site on the detergent form of the enzyme had either poor affinity for, or obstructed access to, the proteinase when the enzyme was integrated in native membrane or in Triton X-100 micelles, whereas the proteolytic site of the papain was always accessible.     

THE DISSOCIATION OF RAT BRAIN MEMBRANES BEARING ACETYLCHOLINESTERASE BY THE NON-IONIC DETERGENT TRITON X-100 AND AN EXAMINATION OF THE PRODUCT

Abstract— The action of Triton X-100 on a membrane preparation from rat brain was studied with reference to the solubilization of acetylcholinesterase and the product was characterized by exclusion chromatography. The AChE and membrane protein were readily solubilized to form particles corresponding to a mol. wt. of about 5 × 10⁵. The solubility of these particles depended on the continued presence of the detergent. It was concluded that these soluble particles formed an intermediate stage in organization between membrane-bound AChE and the soluble protein enzyme, and perhaps represented preexisting lipoprotein subunits of the membranes.     

Characterization and partial purification of squalene-2,3-oxide cyclase from Saccharomyces cerevisiae.

The membrane nature of squalene oxide cyclase from Saccharomyces cerevisiae was investigated by comparing properties of the enzyme recovered from both microsomes and the soluble fraction of the yeast homogenate. The "apparent soluble" form and microsomal form of the enzyme were both stimulated by the presence of mammalian soluble cytoplasm and corresponded to one another in response to detergents Triton X-100 and Triton X-114. The observed strong dependence of the enzyme activity on the presence of detergents and the behavior of the enzyme after Triton X-114 phase separation were peculiar to a lipophilic membrane-bound enzyme. A study of the conditions required to extract the enzyme from microsomes confirmed the lipophilic character of the enzyme. Microsomes, exposed to ipotonic conditions to remove peripheral membrane proteins, retained most of the enzyme activity within the integral protein fraction. Quantitative dissociation of the enzyme from membranes occurred only if microsomes were treated with detergents (Triton X-100 or octylglucoside) at concentrations which alter membrane integrity. The squalene oxide cyclase was purified 140 times from yeast microsomes by (a) removal of peripheral proteins, (b) extraction of the enzyme from the integral protein fraction with octylglucoside, and (c) separation of the solubilized proteins by DEAE Bio-Gel A chromatography. Removal of the peripheral proteins seemed to be a key step necessary for obtaining high yields.     

Characterization of human somatotropin binding to detergent-solubilized lactogenic receptors from rat liver.

Lactogenic receptors from rat liver microsomal fraction ('microsomes') were extracted by treatment with 1% (w/v) Triton X-100. Triton X-100 exerts an inhibitory effect on both the binding reaction and the separation of the free hormone from the complex. The association and dissociation of 125I-labelled human somatotropin are time- and temperature-dependent processes. The association rate constant, k1, is 6.7 x 10(6) mol . litre-1 . min-1 at 25 decrees C, and the dissociation rate constant, k-1, is 1.1 x 10(-3) min-1 at 25 degrees C. Scatchard analysis of saturation data reveals the existence of a single class of receptors and that solubilization leads to a slight decrease in affinity and a sharp increase in binding capacity. The dissociation constant, Kd, of the solubilized preparation is 0.22 nM and the binding capacity 2900 fmol/mg of protein. Similar results were obtained from competition experiments. Binding of 125I-labelled human somatotropin to the solubilized receptors is specifically inhibited by hormones with lactogenic activity. Incubation of the solubilized preparation with trypsin resulted in an 80% decrease in binding activity. The solubilized form of the receptor has a slightly increased sensitivity to the inactivation by trypsin, heat and extremes of pH, with respect to the membrane-bound form.     

Acetylcholinesterase is orientated facing the cytoplasmic side in membranes derived from sarcoplasmic reticulum

(1) Microsomal membranes from white rabbit muscle enriched in sarcoplasmic reticulum (SR) were used to investigate the preferential localization of acetylcholinesterase (AChE) in these membranes. (2) Integrity and orientation of the vesicles was assessed by measuring the inulin-inaccessible space of the vesicles and its calcium-loading capacity. (3) Treatment of the membranes with diisopropyl phosphorofluoridate (DFP), an irreversible inhibitor which is free soluble in lipid, produced an almost complete inactivation of AChE. The inhibition was prevented in assays performed with the non-permeant reversible inhibitor BW 284c51 (BW). (4) Similar results were obtained if echothiophate iodide (ECHO), an irreversible and poorly permeant inhibitor, instead of DFP was used. (5) Sedimentation profiles of enzyme solubilized with Triton X-100 from membranes inhibited by DFP after protection with BW showed a minor reduction in the relative proportion of a 4.5 S (G1) form. (6) Treatment of intact or saponin-permeabilized membranes with concanavalin A (ConA) produced enzyme-lectin complexes. In both cases, most of the enzyme was recovered in the sedimented complexes after centrifugation of the Triton-solubilized membranes. (7) Incubation of intact membranes with the antibody AE1 led to the formation of immuno complexes. Sedimentation analyses of the molecular forms of AChE revealed a shift in the sedimentation coefficients, whether the antibody was added before or after solubilization of the enzyme. (8) These results firmly establish an external localization of AChE in SR, most of the protein backbone facing the cytoplasmic side of the membrane.     

Lignoceroyl-CoA ligase activity in rat brain microsomal fraction: topographical localization and effect of detergents and alpha-cyclodextrin

Lignoceroyl-CoA ligase activity has been detected in microsomal fractions prepared from rat brain. The synthesis of lignoceroyl-CoA from [1-14C]lignoceric acid and CoASH by this enzyme had an absolute dependence on ATP and Mg2+; ATP could not be replaced by GTP [I. Singh, M. S. Kang, and L. Phillips (1982) Fed. Proc. 41, 1192]. The product has been characterized as lignoceroyl-CoA by the following criteria: Rf on thin-layer chromatography; incorporation of [1-14C]lignoceric acid and [3H]CoASH into the product; acid hydrolysis and identification of the radiolabel in lignoceric acid; and methanolysis and identification of the radiolabel in methyl lignocerate by thin-layer chromatography. The optimal concentrations for CoASH, ATP, and Mg2+ were about 100 microM, 10 mM, and 5 mM, respectively. Lignoceric acid, solubilized by alpha-cyclodextrin, Triton X-100, and deoxycholate, was utilized by the lignoceroyl-CoA ligase, but lignoceric acid solubilized by Triton WR-1339 was not. Topographical localization of lignoceroyl-CoA ligase in the plane of rat brain microsomal membranes was determined by the use of Triton X-100, trypsin, and mercury-Dextran, and was compared with the marker enzymes, ethanol acyltransferase and thiamine pyrophosphatase, which are known to be localized on the luminal (inner) surface of the microsomal vesicles. Mercury-Dextran (100 microM) and trypsin (trypsin:microsomes, 1:56 w/w) treatment of the microsomes inhibited the lignoceroyl-CoA ligase activity by 70 and 90% without disrupting the microsomal vesicles. Disruption of the vesicles with Triton X-100 increased the activity of both ethanol acyltransferase and thiamine pyrophosphatase by 400% but there was no increase in lignoceroyl-CoA ligase activity. These results suggest that lignoceroyl-CoA ligase is localized on the cytoplasmic surface of the microsomal vesicles.     

Partial characterization and inactivation of membrane-bound phosphofructokinase from Tetrahymena pyriformis

In Tetrahymena pyriformis, 6-phosphofructokinase (ATP:D-fructose-6-phosphate 1-phosphotransferase, EC 2.7.1.11) is membrane-bound. Enzyme activity is solubilized by treatment of membranes with Triton X-100 or by high ionic strength in the presence of a chelator. The solubilized enzyme has an approximate molecular weight of 300 000. Both the membrane-bound enzyme and the solubilized enzyme exhibit maximum activity over a wide pH range. At low pH, the membrane-bound form of the enzyme is irreversibly inactivated, whereas the solubilized enzyme is not. The membrane-bound enzyme is inactivated by incubation with Mg2+, ATP, fluoride and a soluble factor that is heat labile, nondialysis, (NH4)2SO4 precipitable and sensitive to trypsin. The solubilized enzyme is not inactivated under similar conditions.     

Different reactivity to lysophosphatidylcholine, DIDS and trypsin of two brain sialyltransferases specific for O-glycans: a consequence of their topography in the endoplasmic membranes

Some properties of two distinct rat brain sialyltransferases, acting on fetuin and asialofetuin, respectively, were investigated. These two membrane-bound enzymes were both strongly inhibited by charged phospholipids. Neutral phospholipids were without effect except lysophosphatidylcholine (lysoPC) which modulated these two enzymes in a different way. At 5 mM lysoPC, the fetuin sialyltransferase was solubilized and highly activated while the asialofetuin sialyltransferase was inhibited. Preincubation of brain microsomes with 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), known as a specific anion inhibitor and a non-penetrating probe, led to a moderate inhibition of the asialofetuin sialyltransferase just as in the case of the ovomucoid galactosyltransferase (used here as a marker for the luminal side of the Golgi membrane); under similar conditions, the fetuin sialyltransferase was strongly inhibited. In the presence of Triton X-100, which induced a disruption of membranes, all three enzymes were strongly inhibited by DIDS. Trypsin action on intact membranes showed that asialofetuin sialyltransferase, galactosyltransferase and fetuin sialyltransferase were all slightly inhibited. After membrane disruption by Triton X-100, the first two enzymes were completely inactivated by trypsin while the fetuin sialyltransferase was quite insensitive to trypsin treatment. From these data, we suggest that the fetuin sialyltransferase, accessible to DIDS, is an external enzyme, oriented closely towards the cytoplasmic side of the brain microsomal vesicles (endoplasmic and Golgi membranes), whereas the asialofetuin sialyltransferase is an internal enzyme, oriented in a similar manner to the galactosyltransferase. Moreover, the anion site (nucleotide sugar binding site) of the fetuin sialyltransferase must be different from its active site, as this enzyme, when solubilized, is strongly inhibited by DIDS while no degradation is observed in the presence of trypsin.     

Solubilization and partial characterization of acetylcholinesterase from the sarcotubular system of skeletal muscle

Attempts were made to solubilize acetylcholinesterase (AChE) from microsomal membranes isolated from rabbit white muscle. The preparative procedure included a step in which the microsomes were incubated in a solution containing high salt concentration (0.6 M KCl). About 15% of the total enzyme activity could be solubilized with dilute buffer. Addition of EDTA (1 mM), EGTA (1 mM) or NaCl (0.5 and 1 M) to the extraction buffer did not improve the solubilization yield. Several non-ionic detergents and biliary salts were then used to bring the enzyme into solution. Triton X-100, C₁₂E₉ (dodecylnonaethylenglycol monoether) and biliary salt, above their critical micellar concentration, proved to be very effective as solubilizing agents. The occurrence of multiple molecular forms in detergent-soluble AChE was investigated by means of molecular sieving, centrifugation analysis, and slab gel electrophoresis. Experiments on gel filtration showed that, during the process, half of the enzyme was transformed into aggregates, the rest of the activity appearing as peaks with Stokes radii ranging from 3.7 to 7.9 nm. Both ionic strength and detergent nature modify the number and relative proportion of these peaks. Centrifugation analysis of Triton-saline-soluble AChE yielded molecular forms of 4.8S, 10–11S, and 13.5S, whereas deoxycholate extracts revealed species of 4.8S, 10S, and 15S, providing that gradients were prepared with 0.5 M NaCl. In the absence of salt, forms of 6.5–7.5S, 10S, and 15S were measured. The lightest species was always the predominant form. Slab gel electrophoresis showed several bands (68,000–445,000). The 4.8S component only yielded bands of 65,000–70,000. The results suggest that the monomeric form of AChE (4.8S), the most abundant species in muscle microsomes, has a Stokes radius of 3.3 nm and a molecular weight in the range of 70,000.     

Studies on the transverse localization of lysophospholipase in bovine liver microsomes using proteolytic enzymes.

1. Sonication of bovine liver microsomes completely solubilized the membrane-bound lysophospholipase II (EC 3.1.1.5). Co-chromatography with purified 125I-labelled lysophospholipase indicated that the enzyme was solubilized from microsomes in a lipid-free state. 2. In the presence of residual microsomal membranes, the solubilized lysophospholipase could only be partly degraded by trypsin (EC 3.4.21.4). Therefore, trypsin could not be used to study the transmembrane disposition of lysophospholipase in intact microsomes. 3. Chymotrypsin (EC 3.4.21.1) destroyed the solubilized lysophospholipase activity, even in the presence of residual microsomal membranes. 4. Lysophospholipase in intact microsomal vesicles was resistant to chymotrypsin digestion. 5. When microsomal vesicles were made leaky with lysophosphatidylcholine, chymotrypsin destroyed more than 95% of the lysophospholipase activity. 6. It is concluded from these experiments that at least the active center of lysophospholipase is located at the luminal side of the bovine liver microsomal membrane.     

Solubilization, purification and reconstitution of Ca(2+)-ATPase from bovine pulmonary artery smooth muscle microsomes by different detergents: preservation of native structure and function of the enzyme by DHPC

The properties of Ca(2+)-ATPase purified and reconstituted from bovine pulmonary artery smooth muscle microsomes {enriched with endoplasmic reticulum (ER)} were studied using the detergents 1,2-diheptanoyl-sn-phosphatidylcholine (DHPC), poly(oxy-ethylene)8-lauryl ether (C(12)E(8)) and Triton X-100 as the solubilizing agents. Solubilization with DHPC consistently gave higher yields of purified Ca(2+)-ATPase with a greater specific activity than solubilization with C(12)E(8) or Triton X-100. DHPC was determined to be superior to C(12)E(8); while that the C(12)E(8) was determined to be better than Triton X-100 in active enzyme yields and specific activity. DHPC solubilized and purified Ca(2+)-ATPase retained the E1Ca-E1*Ca conformational transition as that observed for native microsomes; whereas the C(12)E(8) and Triton X-100 solubilized preparations did not fully retain this transition. The coupling of Ca(2+) transported to ATP hydrolyzed in the DHPC purified enzyme reconstituted in liposomes was similar to that of the native micosomes, whereas that the coupling was much lower for the C(12)E(8) and Triton X-100 purified enzyme reconstituted in liposomes. The specific activity of Ca(2+)-ATPase reconstituted into dioleoyl-phosphatidylcholine (DOPC) vesicles with DHPC was 2.5-fold and 3-fold greater than that achieved with C(12)E(8) and Triton X-100, respectively. Addition of the protonophore, FCCP caused a marked increase in Ca(2+) uptake in the reconstituted proteoliposomes compared with the untreated liposomes. Circular dichroism analysis of the three detergents solubilized and purified enzyme preparations showed that the increased negative ellipticity at 223 nm is well correlated with decreased specific activity. It, therefore, appears that the DHPC purified Ca(2+)-ATPase retained more organized and native secondary conformation compared to C(12)E(8) and Triton X-100 solubilized and purified preparations. The size distribution of the reconstituted liposomes measured by quasi-elastic light scattering indicated that DHPC preparation has nearly similar size to that of the native microsomal vesicles whereas C(12)E(8) and Triton X-100 preparations have to some extent smaller size. These studies suggest that the Ca(2+)-ATPase solubilized, purified and reconstituted with DHPC is superior to that obtained with C(12)E(8) and Triton X-100 in many ways, which is suitable for detailed studies on the mechanism of ion transport and the role of protein-lipid interactions in the function of the membrane-bound enzyme.     

Enhancement and inhibition of enzymes of heme metabolism by diethyl maleate in the rat kidney

The acylation of sn-glycerol 3-phosphate with palmityl-CoA was compared in mitochondria and microsomes isolated from rat liver. Polymyxin B, an antibiotic known to alter bacterial membrane structure, stimulated the mitochondrial glycerophosphate acyltransferase but inhibited the microsomal enzyme. When mitochondrial and microsomal fractions were incubated at 4–6 °C for up to 4 h, the mitochondrial enzyme remained virtually unchanged while the microsomal enzyme lost about one-half of its activity. Incubations at higher temperatures also revealed that the mitochondrial enzyme was comparatively more stable under the conditions employed. The mitochondrial acyltransferase showed no sensitivity to bromelain, papain, Pronase, and trypsin, all of which strongly inhibited the microsomal enzyme. The differential sensitivity to trypsin was observed in mitochondria and microsomes isolated from other rat organs. However, the liver mitochondrial glycerophosphate acyltransferase was inhibited by trypsin in the presence of either 0.05% deoxycholate or 0.1% Triton X-100. The trypsin sensitivity of the mitochondrial glycerophosphate acyltransferase in the presence of detergent was not due to the presence, in the mitochondrial fraction, of a trypsin inhibitor which became inactivated by Triton X-100 or deoxycholate. The results suggest that the catalytic site of mitochondrial glycerophosphate acyltransferase is not exposed to the cytosolic side and it is located in the inner aspect of the outer membrane.     

A hydrophobic form of the small-intestinal sucrase-isomaltase complex

A large scale preparation of brush border membranes is described. Solubilized by either papain or Triton X-100, the sucrase-isomaltase complex is purified in a three-step procedure, including differential centrifugation, Sephadex G-200 and DEAE-cellulose chromatography. Detergent solubilized and protease solubilized sucrase-isomaltase differ in the tendency to aggregate but not in enzymatic characteristics. The chemical composition and the molecular weight of the two enzyme complexes are almost identical. Limited digestion of the Triton-solubilized sucrase-isomaltase complex by papain produces a protein electrophoretically indistinguishable from papain-solubilized sucrase-isomaltase together with low molecular proteolytic fragments.     

A neutral proteinase of monkey liver microsomes. Solubilization, partial purification, and properties.

Conditions for the solubilization of membrane-bound neutral proteinase associated with monkey liver microsomes were investigated. Among the reagents tested, deoxycholate, cholate, and some nonionic detergents, including Triton X-100, with hydrophilic-lipophilic balance values of around 13, were effective. The solubilization profile indicated that the enzyme is bound to the microsomal membranes by strong hydrophobic interaction. The enzyme was partially purified from monkey liver microsomal fraction, previously washed with 1 M KCl and 0.05% sodium dodecyl sulfate, by Triton X-100 extraction, followed by chromatography on columns of hydroxylapatite and Sepharose CL-6B. The apparent molecular weight of the enzyme was estimated to be about 88,000 from the elution position on Sepharose CL-6B column chromatography in the presence of 0.5% sodium cholate. It was optimally active at pH 8.0 with heat-denatured casein as a substrate. It was strongly inhibited by diisopropyl phosphorofluoridate and phenylmethanesulfonyl fluoride, indicating that the enzyme is a serine proteinase. EDTA, EGTA, and chymostatin also inhibited the enzyme strongly. Among urea-denatured protein substrates tested, calf thymus histone was hydrolyzed most rapidly, followed by casein, hemoglobin, and bovine serum albumin, whereas practically no hydrolysis occurred with denatured ovalbumin, fibrinogen, and gamma-globulin as substrates.     

A hydrophobic form of the small-intestinal sucrase-isomaltase complex.

A large scale preparation of brush border membranes is described. Solubilized by either papain or Triton X-100, the sucrase-isomaltase complex is purified in a three-step procedure, including differential centrifugation, Sephadex G-200 and DEAE-cellulose chromatography. Detergent solubilized and protease solubilized sucrase-isomaltase differ in tendency to aggregate but not in enzymatic characteristics. The chemical composition and the molecular weight of the two enzyme complexes are almost identical. Limited digestion of the Triton-solubilized sucrase-isomaltase complex by papain produces a protein electrophoretically indistinguishable from papain-solubilized sucrase-isomaltase together with low molecular proteolytic fragments.     

PROPERTIES OF MEMBRANE-BOUND DOPAMINES-HYDROXYLASE IN CHROMAFFIN GRANULES FROM BOVINE ADRENAL MEDULLA

Abstract— Properties of membrane-bound and soluble dopamine-β-hydroxylase were studied. Both enzyme forms have identical affinities for tyramine as the substrate. Arrhenius plots of the membrane-bound activity displayed a discontinuity at 29°C, the activation energy changing from 20,500 cal/mol below 29°C to 9500 cal/mol above 29°C. The soluble enzyme, like the purified enzyme, did not show discontinuities in Arrhenius plots, the activation energies being 18,500 cal/mol and 16,500 cal/mol respectively. The membrane-bound enzyme showed a discontinuity in the p K^m for tyramine versus reciprocal temperature plot, with a transition at 29°C, whereas the soluble enzyme failed to show such transition.
The membrane-bound dopamine-β-hydroxylase is solubilized by Triton X-100 as well as by lysolecithin. Of lecithin, lysophosphatidyl ethanolamine and phosphatidyl serine, lysolecithin was the only phospholipid to induce solubilization of membrane-bound dopamines β-hydroxylase. The 29°C-transition was not removed by treatment either with lysolecithin or with Triton X-100 used at concentration of up to 2%. This could indicate a solubilization of dopamine-β-hydroxylase with an associated lipid moiety which cannot be dispersed from the enzyme molecule and which still affects the activation energy and Michaelis constant for tyramine.
Results are discussed in terms of the relationship between lipid organization and enzyme activities; the significance of the solubilization by lysolecithin during exocytosis is considered in terms of the exocytosis process and fate of the chromaffin granule.     

Heterogeneity of Rat Brain Acetylcholinesterase: A Study by Gel Filtration and Gradient Centrifugation

Abstract: According to their solubilization properties, two classes of acetyl-cholinesterases (AChE) can be detected in the adult rat brain: a "soluble" species (easily solubilized without detergent), and a membrane-bound species (solubilized only in the presence of detergent). The latter was found to be homogeneous by gel filtration (Stokes radius 8.05 ± 0.35 nm) and sucrose gradient centrifugation (9.75 ± 0.2 S) in the presence of Triton X-100. The "soluble" AChE gives three stable species in the presence of the same detergent with Stokes radii and sedimentation constants of 10.9 ± 0.5 nm and 16 ± 2 S; 6.75 ± 0.30 nm and 10.7 ± 0.4 S; 5.37 ± 0.35 nm and 4.37 ± 0.1 S. Co-chromatography and co-sedimentation or the reduction and alkylation of disulfide bridges show that all the soluble species are different from the membrane-bound AChE. The possibility that soluble and membrane-bound AChE are completely different molecules is discussed.