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.     

Effects of Phospholipases on the Kinetic Properties of Rat Striatal Membrane-Bound Tyrosine Hydroxylase

Abstract: Rat striatal tyrosine hydroxylase can be isolated in both a soluble and a synaptic membrane-bound form. The membrane-bound enzyme, which exhibits lower K _ms for both tyrosine (7 μ M ) and reduced pterin cofactor (110 μ M ) relative to the soluble enzyme (47 μ M and 940 μ M , respectively), can be released from the membrane fraction with mild detergent, and concomitantly its kinetic properties revert to those of the soluble enzyme. Treatment of membrane-bound tyrosine hydroxylase with C. perfringens phospholipase C increased the K _m of the enzyme for tyrosine to 27 μ M and the V _max by 60% without changing the K _m for cofactor. In contrast, treatment of membrane-bound tyrosine hydroxylase with V. russelli phospholipase A₂ increased the K _m for tyrosine to 48 μ M increased the V _max and increased the K _m for cofactor to 560 μ M . The enzyme remained bound to the membrane fraction following both phospholipase treatments. Addition of phospholipids to treated enzyme could partially reverse the effects of phospholipase A₂ treatment, but not the effects of phospholipase C treatment. The kinetic properties of phospholipase-treated, detergent-solubilized tyrosine hydroxylase were identical to those of the control solubilized enzyme. Tyrosine hydroxylase appears to interact with synaptic membrane components to produce at least two separately determined consequences for the kinetic properties of the enzyme.     

Receptor for sex steroid-binding protein of endometrium membranes: solubilization, partial characterization, and role of estradiol in steroid-binding protein-soluble receptor interaction.

The sex steroid-binding protein (SBP) receptor was solubilized from the membranes of human premenopausal endometrium with the zwitterionic detergent CHAPS. The binding activity of the soluble receptor was studied, allowing it to interact with [125I]SBP and precipitating the complex with polyethylene glycol 8,000. The interaction of SBP with the soluble receptor was specific, saturable, and at high affinity. Indeed, the specific binding was definitely improved on the solubilized form of the receptor. The effect exerted by sex steroids on the interaction of SBP with receptor was also examined on both the soluble and membrane-bound forms. At physiologic doses (10(-8) M) estradiol inhibits the binding at a significant extent on the soluble receptor, but not on membrane-bound form. The dose of estradiol required to significantly inhibit the SBP-specific binding was dependent on the form of receptor. In membrane-bound receptor the inhibiting dose of estradiol was higher than its physiologic concentration. Thus, it is likely that, while soluble receptor cannot recognize the complex steroid-SBP, membrane-bound receptor can interact both with "unliganded" SBP and with the estradiol-SBP complex (but not with androgen-SBP complexes) in an estrogen-dependent tissue like human endometrium.     

Interaction of amphipols with sarcoplasmic reticulum Ca2+-ATPase

Amphipols are short-chain amphipathic polymers designed to keep membrane proteins soluble in aqueous solutions. We have evaluated the effects of the interaction of amphipols with sarcoplasmic reticulum Ca(2+)-ATPase either in a membrane-bound or a soluble form. If the addition of amphipols to detergent-solubilized ATPase was followed by removal of detergent, soluble complexes formed, but these complexes retained poor ATPase activity, were not very stable upon long incubation periods, and at high concentrations they experienced aggregation. Nevertheless, adding excess detergent to diluted detergent-free ATPase-amphipol complexes incubated for short periods immediately restored full activity to these complexes, showing that amphipols had protected solubilized ATPase from the rapid and irreversible inactivation that otherwise follows detergent removal. Amphipols also protected solubilized ATPase from the rapid and irreversible inactivation observed in detergent solutions if the ATPase Ca(2+) binding sites remain vacant. Moreover, in the presence of Ca(2+), amphipol/detergent mixtures stabilized concentrated ATPase against inactivation and aggregation, whether in the presence or absence of lipids, for much longer periods of time (days) than detergent alone. Our observations suggest that mixtures of amphipols and detergents are promising media for handling solubilized Ca(2+)-ATPase under conditions that would otherwise lead to its irreversible denaturation and/or aggregation.     

Effect of divalent metal ions on soluble and membrane-bound alkaline phosphatase activity in suckling rat intestine.

EDTA treatment of intestinal brush border membranes (BBM) and epithelial cell supernatant completely inhibited alkaline phosphatase (AP) activity in suckling rat intestine. AP activity was fully regained upon dialysis of the preparations against Zn2+ and to a lesser extent against Co2+, Ca2+ and Mn2+ ions. Other metal ions (Cd2+ and Mg2+) tested were essentially ineffective in restoring the enzyme activity. Considerable differences were observed in kinetic characteristics of the membrane-bound and soluble AP activities in response to various metal ions. There were apparent differences in Km, Vmax, energy of activation (Ea) and thermal stability of the soluble and membrane-bound AP activities, after metal ion substitutions. Nearly 35% AP activity was solubilized on sodium dodecyl sulphate treatment of brush borders (membrane protein: detergent ratio 1:3; w/w). Dialysis of detergent solubilized BBM against different metal ions reconstituted AP activity in the particulate fraction: the order of effectiveness was Zn greater than Ca greater than Mn greater than Co. The kinetic properties of the reconstituted AP were essentially similar to the non-integrated enzyme activity in response to various divalent metal ions examined. But there were apparent differences in Km, Vmax, Ea and thermal stability of the reconstituted AP activity compared to native brush border enzyme. The results suggest the unique requirement of Zn ions for stability and catalytic activity of the soluble and membrane-bound AP activity in suckling rat intestine.     

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.     

Properties of the ATP phosphohydrolase of the facultative thermophile Bacillus coagulans

Abstract The thermostability of the ATP phosphohydrolase of the facultative thermophile Bacillus coagulans has been investigated. Fractionation of disintegrated cell suspensions by differential centrifugation revealed a similar distribution of enzyme activity irrespective of growth temperature. Most of the activity was located in the membrane fraction. Thermostability of solubilized (BF₁) preparation from cells grown at 37°C or 55°C was similar, but membrane-bound BF₀BF₁ from 37°C-grown cells was inactivated at lower temperatures than that from 55°C-grown cells.
Inhibition of the membrane-bound (BF₀BF₁)ATPase by 4-chloro-7-nitro-benzofuran (NbfCl) and quercetin, which both act on the BF₁ portion of the enzyme, was different from that seen with the soluble (BF₁) enzyme. The results show that some modification of BF₁ must occur when the enzyme is membrane-bound.     

Comparison of the membrane-bound hydrogenases from Alcaligenes eutrophus H16 and Alcaligenes eutrophus type strain

Whereas the membrane-bound hydrogenase from Alcaligenes eutrophus H16 is an integral membrane protein and can only be solubilized by detergent treatment, the membrane-bound hydrogenase of Alcaligenes eutrophus type strain was found to be present in a soluble form after cell disruption. For the enzyme of A. eutrophus H16 a new, highly effective purification procedure was developed including phase separation with Triton X-114 and triazine dye chromatography on Procion Blue H-ERD-Sepharose. The purification led to an homogeneous hydrogenase preparation with a specific activity of 269 U/mg protein (methylene blue reduction) and a yield of 45%. During purification and storage the enzyme was optimally stabilized by the presence of 0.2 mM MnCl2. The hydrogenase of A. eutrophus type strain was purified from the soluble extract by a similar procedure, however, with less specific activity and activity yield. Comparison of the two purified enzymes revealed no significant differences: They have the same molecular weight, both consist of two different subunits (Mr = 62,000, 31,000) and both have an isoelectric point near pH 7.0. They have the same electron acceptor specificity reacting with similar high rates and similar Km values. The acceptors reduced include viologen dyes, flavins, quinones, cytochrome c, methylene blue, 2,6-dichlorophenolindophenol, phenazine methosulfate and ferricyanide. Ubiquinones and NAD were not reduced. The two hydrogenases were shown to be immunologically identical and both have identical electrophoretic mobility. For the membrane-bound hydrogenase of A. eutrophus H16 it was demonstrated that this type of hydrogenase in its solubilized, purified state is able to catalyze also the reverse reaction, the H2 evolution from reduced methyl viologen.     

Comparison of physico-chemical properties of two forms of dopamine-beta-hydroxylase from chromaffin granules

The purification procedures of both soluble and membrane-bound forms of dopamine-beta-hydroxylase have been developed. The membrane-bound form was solubilized by a detergent. Both preparations have been obtained in electrophoretically homogeneous form. The yield of soluble and membrane-bound enzyme forms by a described procedure was 22 mg and 15 mg, correspondingly, from 100 g of chromaffin granules paste. A comparative analysis of the main physico-chemical properties of the two enzyme forms has shown their identity. The effects of pH, ionic strength, oxidants and reducers on the EPR spectra of the two forms of dopamine-beta-hydroxylase have been investigated. A comparison of the EPR spectra of the two forms of the enzyme suggests that the copper environment of soluble and membrane-bound dopamine-beta-hydroxylases is practically identical.     

Effect of phospholipid, detergent and protein-protein interaction on stability and phosphoenzyme isomerization of soluble sarcoplasmic reticulum Ca-ATPase

The purpose of the present study was to elucidate the separate roles of lipid, detergent and protein-protein interaction for stability and catalytic properties of sarcoplasmic reticulum Ca-ATPase solubilized in the non-ionic detergent octa(ethylene glycol) monododecyl ether (C12E8). The use of large-zone high-performance liquid chromatography permitted us to define the self-association state of Ca-ATPase peptide at various detergent, phospholipid and protein concentrations, and also during enzymatic turnover with ATP. Conditions were established for monomerization of Ca-ATPase in the presence of a high concentration of phospholipid relative to detergent. The lipid-saturated monomeric preparation was relatively resistant to inactivation in the absence of Ca2+, whereas delipidated enzyme in monomeric or in oligomeric form was prone to inactivation. Kinetics of phosphoenzyme turnover were examined in the presence and absence of Mg2+. Dephosphorylation rates were sensitive to Mg2+, irrespective of whether the peptide was present in soluble monomeric form or was membrane-bound. C12E8-solubilized monomer without added phospholipid was, however, characterized by a fast initial phase of dephosphorylation in the absence of Mg2+. This was not observed with monomer saturated with phospholipid or with monomer solubilized in myristoylglycerophosphocholine or deoxycholate. The mechanism underlying this difference was shown to be a C12E8-induced acceleration of conversion of ADP-sensitive phosphoenzyme (E1P) to ADP-insensitive phosphoenzyme (E2P). The phosphoenzyme isomerization rate was also found to be enhanced by low-affinity binding of ATP. This was demonstrated both in membrane-bound and in soluble monomeric Ca-ATPase. Our results indicate that a single peptide chain constitutes the target for modulation of phosphoenzyme turnover by Mg2+ and ATP, and that detergent effects, distinct from those arising from disruption of protein-protein contacts, are the major determinants of kinetic differences between C12E8-solubilized and membrane-bound enzyme preparations.     

Purification and characterization of a novel glycan-phosphatidylinositol-specific phospholipase C from Trypanosoma brucei

A novel membrane-bound glycan-phosphatidylinositol-specific phospholipase C, which catalyzes the conversion of membrane form variant surface glycoproteins to soluble variant surface glycoproteins, with the release of sn-1,2-dimyristylglycerol, has been isolated from Trypanosoma brucei. The activity was solubilized from trypanosome membrane fractions in non-ionic detergent and purified by anion exchange chromatography on DEAE-cellulose followed by chromatography on phosphatidylinositol-Sepharose. The enzyme constitutes about 0.1% of the total cellular protein and has an apparent molecular weight of 39,800. The enzyme shows a head group specificity for molecules containing carbohydrate covalently linked to glycan-phosphatidylinositol, but can also act on the monoacyl derivative of membrane form variant surface glycoprotein. It shows no specific ion requirements but is stimulated by thiol-reducing agents and inhibited by ions that thiols chelate.     

Solubilization and conversion of hepatic adenylate cyclase to a form requiring MnATP as substrate

A general feature of membrane-bound adenylate cyclase systems is the “lability” of the basal enzyme to dispersion by detergents. A stable form of the detergentsolubilized enzyme is obtained only if the membrane-bound enzyme is first pretreated with fluoride or Gpp(NH)p. However, we have found with the basal hepatic enzyme that the lability is evident primarily when MgATP is used as substrate; substitution of MnATP for MgATP reveals that substantial basal activity survives detergent treatment. This effect is independent of the detergent; it is seen with either Lubrol PX or with deoxycholate. In addition to the altered substrate requirement, the membrane-bound and solubilized forms of the basal enzyme exhibit other differences. In contrast to the membrane-bound form, the solubilized enzyme shows (1) weak stimulation by Gpp(NH)p; (2) little inhibition by adenosine, (3) strong inhibition by P_i or PP_i, and (4) and apparent loss of the Me²⁺-reactive regulatory site. Such dissimilarities between membranebound and solubilized cyclase are not seen if the membranes are pretreated with Gpp(NH)p prior to exposure to detergents. The characteristics of the solubilized basal hepatic enzyme are similar to those of the naturally occurring soluble adenylate cyclase found in mature rat testes. It would appear that separation of adenylate cyclase from components that confer regulation by divalent cation and guanine nucleotides produces a form of the enzyme that will turnover only MnATP; this may represent the free catalytic moiety. Such preparations could be useful in reconstructing some of the regulatory functions of adenylate cyclase seen in its membrane-bound form.     

Characterization of Membrane-Bound and Soluble Catechol-O-Methyltransferase from Human Frontal Cortex

Abstract: Catechol- O -methyltransferase (COMT; E.C. 2.1.1.6) from human frontal cortex occurs in both a soluble and membrane-bound form. Attempts to solubilize the membrane-bound transferase by repeated washing or by extraction into solutions of high ionic strength were unsuccessful. The finding that Triton X-100 was capable of solubilizing membrane-bound COMT suggested that the membrane-bound transferase is an integral membrane protein. The membrane-bound and soluble enzymes did not differ in their requirements for magnesium ions or in their pH-activity profiles; both enzymes showed an optimum near pH 8.0 when assayed in phosphate buffer. In addition, the two enzymes did not differ in the degree of inhibition caused by CaCl₂, both enzymes displaying 65% inhibition at 2.5 m M CaCl₂. The competitive inhibitors tropolone and nordihydroguaiaretic acid displayed K _i values for the membrane-bound transferase five- to 10-fold lower than those observed for the soluble transferase. Solubilization of membrane-bound COMT in Triton X-100 resulted in an increase in the apparent K _m value of the membrane-bound transferase for dopamine. The increase in K _m appeared to be due to apparent competitive inhibition by Triton X-100 and reached a limiting value of approximately 80 μM. These results confirm that membrane-bound COMT is an integral membrane protein that may be structurally distinct from soluble COMT.     

Solubilization of phencyclidine receptors from rat cerebral cortex in an active ligand binding state

A phencyclidine (PCP) receptor binding site has been solubilized in an active ligand-binding state from rat cerebral cortical membranes with sodium deoxycholate. Optimal receptor solubilization occurs at a detergent/protein ratio of 0.5 (w/w); for 5 mg protein/ml solubilized with 0.25% sodium deoxycholate, about 60% of the protein and 25% of the receptor is solubilized. Specific binding of either [3H]-N-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP) or [3H]MK-801 is measurable by filtration through Sephadex G-50 columns or glass fiber filters; more than 60% of the binding activity is stable after 48 h at 4 degrees C. In the presence of detergent, [3H]TCP binding exhibits a Kd of 250 nM, a Bmax of 0.56 pmol/mg protein, and a pharmacological profile consistent with that of the membrane-bound PCP receptor, although most drugs bind with affinities 2 to 8 fold lower than in membranes. Upon reduction of detergent concentration, binding parameters approximate those for the membrane-bound receptor ([3H]TCP binding: Kd = 48 nM, Bmax = 1.13 pmol/mg protein).     

Isolation and characterization of mannose 6-phosphate/insulin-like growth factor II receptor from bovine serum.

A convenient means was devised for the purification of milligram quantities of a soluble form of the mannose 6-phosphate/insulin-like growth factor II receptor (Man-6-P/IGF II receptor). The receptor was purified to near homogeneity from bovine serum by affinity chromatography on agarose-pentamannosephosphate in the absence of detergent. Approximately 2.5 mg of receptor were obtained from 500 ml of fetal calf serum. The concentration of receptor in serum decreased sharply with development. Fetal calf serum Man-6-P/IGF II receptor was immunologically similar to detergent-solubilized, membrane-bound Man-6-P/IGF II receptor from bovine liver. N-Terminal sequence analysis revealed that the purified serum receptor, but not the solubilized, membrane-associated receptor, contains stoichiometric amounts of bound IGF II. The results of sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and gel chromatography studies suggest that the fetal calf serum receptor (in contrast to the solubilized, membrane-bound bovine testis receptor) does not aggregate. The affinity of the fetal calf serum receptor for bovine testis beta-galactosidase approximated one-half that observed for solubilized, membrane-bound bovine testis receptor.     

Resolution and reconstitution of Rhodospirillum rubrum pyridine dinucleotide transhydrogenase

The Rhodospirillum rubrum pyridine dinucleotide transhydrogenase system is comprised of a membrane-bound component and an easily dissociable soluble factor. Active transhydrogenase complex was solubilized by extraction of chromatophores with lysolecithin. The membrane component was also extracted from membranes depleted of soluble factor. The solubilized membrane component reconstituted transhydrogenase activity upon addition of soluble factor. Various other ionic and non-ionic detergents, including Triton X-100, Lubrol WX, deoxycholate, and digitonin, were ineffectual for solubilization and/or inhibited the enzyme at higher concentrations. The solubilized membrane component was significantly less thermal stable than the membrane-bound component. None of the pyridine dinucleotide substrate affected the thermostability of the solubilized membrane-bound component, whereas NADP⁺ and NADPH afforded protection to membrane-bound component. NADPH stimulated trypsin inactivation of membrane-bound component to a greater extent than NADP⁺, but inactivation of solubilized membrane component was stimulated to the same extent by both pyridine dinucleotides. The solubilized membrane component appears to have a slightly higher affinity for soluble factor than does the membrane-bound component.Abbreviations AcPyAD⁺ oxidized 3-acetylpyridine adenine dinucleotide - BChl bacteriochlorophyll - C_T-particles chromatophores depleted of soluble transhydrogenase factor and devoid of transhydrogenase activity This work was supported by Grant GM 22070 from the National Institutes of Health, United States Public Health Service. Paper I of this series is R. R. Fisher et al. (1975)     

An improved procedure for the purification of ethanolaminephosphotransferase. Reconstitution of the purified enzyme with lipids

Ethanolaminephosphotransferase (CDPethanolamine:1,2-diacylglycerol ethanolaminephosphotransferase, EC 2.7.8.1) has been purified in active form from rat brain microsomes by a two-step chromatographic procedure. Enzyme preparations characterized by high specific activity and stability were obtained supplementing the solubilization and elution buffers, containing 1% Triton X-100, with 0.01% 2,6-di-tert-butyl-4-methylphenol. The specific activity of the purified enzyme was about 1200-times higher than that of the crude solubilized enzyme. The lipid dependence of ethanolaminephosphotransferase was studied both in the presence of Triton X-100 and in detergent-free enzyme preparations. The activity of the detergent-solubilized ethanolaminephosphotransferase was strongly modified by phospholipids. The kinetic behaviour of the enzyme was also dependent on the lipids contained in the aggregates obtained by removal of the detergent from detergent/lipid/protein suspensions. A regulatory role of phospholipids on the activity of the membrane-bound ethanolaminephosphotransferase is discussed.     

Solubilization by CHAPS detergent of barbiturate-enhanced benzodiazepine-GABA receptor complex

Abstract: Barbiturates enhance the binding of [³H]flunitrazepam to benzodiazepine receptors solubilized with the detergent 3-[(3-cholamidopropyl)-dimethylammonio]propanesulfonate (CHAPS) from bovine cortex. The enhancement by the barbiturates is seen as a decrease in the dissociation constant, K _d , for specific benzodiazepine binding, with no effect on the number of binding sites. The effect of the barbiturates is facilitated by chloride ions, is concentration-dependent, and has a specificity that correlates well with the anesthetic potency of barbiturates. [³H]Flunitrazepam binding activity is stable with storage at 4°C., but barbiturate enhancement of soluble benzodiazepine binding activity decayed rapidly ( t _1/2= 48 h). [³H]Muscimol binding (GABA receptor) activity was also enhanced by barbiturates. Agarose gel filtration column chromatography of the CHAPS-solubilized receptor proteins showed the same elution profile as receptors solubilized with sodium deoxycholate, and enhancement by barbiturates was observed for both the benzodiazepine and GABA binding activities.     

Differential inhibition of soluble and membrane-bound acetylcholinesterase forms from mouse brain by choline esters with an acyl moiety of an intermediate size

Differential inhibitions of soluble and membrane-bound acetylcholinesterase forms purified from mouse brain were examined by the comparison of kinetic constants such as a K _m value, a K_ss value (substrate inhibition constant), and IC₅₀ values of active site-selective ligands including choline esters. Membrane-bound acetylcholinesterase form (solubilized only in the presence of detergent) showed lower K_m and K_ss values than soluble acetylcholinesterase form (easily solubilized without detergent). Edrophonium expressed a slightly but significantly (p<0.01) higher inhibition of detergent-soluble acetylcholinesterase form than aqueous-soluble acetylcholinesterase form, while physostigmine inhibited both forms with a similar potency. A remarkable difference in inhibition was observed using choline esters; although choline esters with acyl chain of a short size (acetyl-to butyrylcholine) or a long size (heptanoyl- to decanoylcholine) showed a similar inhibitory potency for two forms of acetylcholinesterase, pentanoylcholine and hexanoylcholine inhibited more strongly aqueous-soluble acetylcholinesterase than detergent-soluble acetylcholinesterase. Thus, it is suggested that the two forms of AChE may be distinguished kinetically by pentanoyl- or hexanoylcholine.This work was supported in part by Agency for Defense Development.     

Solubilization of human platelet vasopressin receptors

The human platelet membrane receptor for vasopressin (AVP) has been solubilized with the cholic acid derivative detergent 3-( [3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate. Rapid and simple separation of free tritiated AVP ( [3H]AVP) from the solubilized receptor-hormone complex was done by filtration through polyethylenimine-treated filters. [3H]AVP binds to this soluble receptor with an equilibrium dissociation constant of 11.03 +/- 1.86 nM and a maximal number of binding sites = 288 +/- 66 fmol/mg protein while the corresponding values of the membrane-bound receptor are 1.62 +/- 0.21 nM and 237 +/- 38 fmol/mg of protein, respectively. The Ki value for native AVP derived from competition experiments is 11.02 +/- 2.05 nM for the soluble receptor. Competition experiments with specific vascular and renal antagonists confirm that the solubilized receptor belongs to the V1-vascular subtype.