Higher Detergent Concentrations Research Articles

Effect of cetyl trimethyl ammonium bromide on the heat‐induced denaturation and protein‐protein interactions of arachin at pH 3.6

The effect of a cationic detergent, cetyl trimethyl ammonium bromide, on the heat‐induced denaturation and protein‐protein interactions of arachin at pH 3.6 was studied by gel melting temperature, turbidity, electrophoresis, u.v. difference and fluorescence spectral measurements. Cetyl trimethyl ammonium bromide decreased the heat‐induced protein‐protein interactions as shown by the melting temperature, turbidity and electrophoretic measurements. Turbidity and electrophoretic measurements also showed that the detergent induces dissociation of the protein before and after heating it at higher detergent concentration (above 1 times 10‐3 M). U.v. difference and fluorescence spectral measurements suggested that the detergent induces unfolding of arachin at room temperature and this increases markedly after heating the protein in the detergent. The promotive effect of cetyl trimethyl ammonium bromide on the heat‐induced denaturation and its preventive effect on the protein‐protein interactions of arachin under the experimental conditions may arise out of the binding of the detergent to protein by primarily hydrophobic interactions.

Effects of detergent on substrate binding and spin state of purified liver microsomal cytochrome P-450LM2 from phenobarbital-treated rabbits.

Spectral changes accompanying the binding of the nonionic detergent n-octyl beta-D-glucopyranoside (n-octyl glucoside) to cytochrome P-450LM2 purified from liver microsomes of phenobarbital-treated rabbits have been compared to changes in catalytic activity obtained in a reconstituted system consisting of various levels of detergent, P-450LM2, and NADPH-cytochrome P-450 reductase. In the absence of substrate and reductase, addition of n-octyl glucoside to 2-3 mM resulted in a difference spectrum (detergent-bound minus detergent-free cytochrome) characterized by a small maximum at 390 nm and a minimum at 410 nm, suggestive of a slight stabilization of the high-spin (S = 5/2) state of the cytochrome. As the detergent concentration was increased to 4-8 mM (corresponding to maximal activity and pentameric or hexameric P-450), a new peak appeared at 427 nm while the minimum remained at 410 nm. Between 10 and 30 mM n-octyl glucoside (conditions which produced catalytically inactive and monomeric P-450) the minimum in the difference spectrum shifted to 390 nm and the maximum to 425 nm, characteristic of a shift in spin equilibrium toward low-spin (S = 1/2) cytochrome. At low and high detergent concentrations, substrate [d-benzphetamine with n-octyl glucoside or cyclohexane with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS)] was bound to P-450LM2 with formation of high-spin P-450, although the increase in high-spin cytochrome was less at high detergent levels than at low. The affinity of P-450 for substrate decreased by 2-3-fold at high detergent.(ABSTRACT TRUNCATED AT 250 WORDS)

On the fractional melting of milk fat and the properties of the fractions

AbstractStandard methods of fractionating milk fat from the melt produce solid fractions that exhibit melting at temperatures considerably lower than that used for the fractionation. The use of high concentrations of aqueous detergent, in the presence of ammonia as an aid to centrifugation, has resulted in this solid material being sub‐fractionated into a semi‐solid, plastic material and a crystalline solid. The technique may be applied directly to milk fat to produce three fractions at a given fractionation temperature. Fatty acid compositions and separations according to triglyceride number are recorded for the parent fats and their fractions. Melting fingerprints, obtained by differential scanning calorimetry, are shown for the various samples, and the heats of melting are discussed.

Evidence for a tubulin-containing lipid-protein structural complex in ciliary membranes.

The proteins and lipids of the scallop gill ciliary membrane may be reassociated through several cycles of detergent solubilization, detergent removal, and freeze-thaw, without significant change in overall protein composition. Membrane proteins and lipids reassociate to form vesicles of uniform, discrete density classes under a variety of reassociation conditions involving detergent removal and concentration. Freed of the solubilizing detergent during equilibrium centrifugation, a protein-lipid complex equilibrates to a position on a sucrose density gradient characteristic of the original membrane density. When axonemal tubulin is solubilized by dialysis, mixed with 2:1 lecithin/cholesterol dissolved in Nonidet P-40, freed of detergent, and reconstituted by freeze-thaw, vesicles of a density essentially equal to pure lipid result. If the lipid fraction is derived through chloroform-methanol extraction of natural ciliary membranes, a moderate increase in density occurs upon reconstitution, but the protein is adsorbed and most is removed by a simple low ionic strength wash, in contrast to vesicles reconstituted from membrane proteins where even high salt extraction causes no loss of protein. The proteins of the ciliary membrane dissolve with constant composition, regardless of the type, concentration, or efficiency of detergent. Analytical ultracentrifugation demonstrates that monodisperse mixed micelles form at high detergent concentrations, but that membranes are dispersed to large sedimentable aggregates by Nonidet P-40 even at several times the critical micelle concentration, which suggests reasons for the efficacy of certain detergent for the production of ATP-reactivatable cell models. In extracts freed of detergent, structured polydisperse particles, but not membrane vesicles, are seen in negative staining; vesicles form upon concentration of the extract. Membrane tubulin is not in a form that will freely undergo electrophoresis, even in the presence of detergent above the critical micelle concentration. All chromatographic attempts to separate membrane tubulin from other membrane proteins have failed; lipid and protein are excluded together by gel filtration in the presence of high concentrations of detergent. These observations support the idea that a relatively stable lipid-protein complex exists in the ciliary membrane and that in this complex membrane tubulin is tightly associated with lipids and with a number of other proteins.

Effect of sodium dodecyl sulfate on the heat denaturation and aggregation of arachin at pH 3.6

The effect of an anionic detergent, sodium dodecyl sulfate, on the heat‐induced denaturation and protein‐protein interactions of arachin at pH 3.6 was studied by melting temperature, turbidity, electrophoresis and fluorescence spectral measurements. Sodium dodecyl sulfate induced aggregation in arachin at room temperature, in the concentration range 1 times 10‐5 to 1 times 10‐2 M, as shown by turbidity, electrophoresis and fluorescence spectral measurements. However, higher detergent concentrations (> 1 times 10‐2 M SDS) decreased the aggregation. Electrophoretic experiments at pH 3.6 showed that sodium dodecyl sulfate at a concentration of 5 times 10‐2 M changes the protein charge from positive to negative. Fluroescence spectral measurements suggested that the detergent at 5 times 10‐2 M level unfolds arachin. Heating and cooling arachin at pH 3.6 in the presence of sodium dodecyl sulfate increased the protein‐protein interactions as compared to those at room temperature, in the detergent concentration range 1 times 10‐5 to 1 times 10‐2 M; at higher detergent concentrations there was a sharp decrease. The observed effects of sodium dodecyl sulfate on the structure and conformation and heat‐induced protein‐protein interactions of arachin at the experimental pH may arise out of the detergent binding to the protein involving both ionic and hydrophobic interactions.

Studies on the nature and activation of O2(-)-forming NADPH oxidase of leukocytes. Identification of a phosphorylated component of the active enzyme.

Highly active superoxide (O2-)-forming NADPH oxidase was extracted from plasmamembranes of phorbol-12-myristate-13-acetate-activated pig neutrophils and was partially purified by gel filtration chromatography. Oxidase activity copurified with cytochrome b-245 in an aggregate containing phospholipids and was almost completely separated from FAD and NAD(P)H-cytochrome c reductase. A polypeptide with molecular weight of 31,500 strictly paralleled the purification of NADPH oxidase, suggesting that it is a major component of the enzyme. The enzyme complex was then dissociated by high detergent and salt concentration and cytochrome b-245 was isolated by a further gel filtration chromatography, with a 147 fold purification with respect to the initial preparation. The cytochrome b-245 showed a 31,500 molecular weight by SDS electrophoresis, indicating that it is actually the component previously identified in the partially purified enzyme. The 31,500 protein was phosphorylated in enzyme preparations from activated but not from resting neutrophils, suggesting that phosphorylation of cytochrome b-245 is involved in the activation mechanism of the O2(-) -forming enzyme responsible for the respiratory burst in phagocytes.

Wechselwirkungen der Aldehyddehydrogenase aus Acinetobacter calcoaceticus mit Membranlipiden I. Einfluß von Detergentien, Proteinasen und Phospholipasen auf die Solubilisierung und Aktivität des Enzyms

AbstractThe NADP+‐dependent aldehyde dehydrogenase of Acinetobacter calcoaceticus was inducible by alkanes. It was located in the membranes surrounding the intracellular alkane inclusions. The enzyme could not be solubilized by changing of the pH or the ionic strength but by detergents at concentrations around the critical micelle concentration. High concentrations of detergents inactivated the enzyme; reactivation was possible by addition of natural membrane phospholipids. The enzyme in intact membranes gave lower Km values than the enzyme in the micelle form. The size of the particles of cell envelopes (including the inclusion membranes) and of micelles was controlled by measurements of the wavelength dependence of turbidity. Proteinases were unable to solubilize the enzyme; in the presence of detergent they inactivated it irreversibly. Phospholipase A2 and D inactivated the enzyme reversibly.

Complex glycoproteins associated with the detergent-resistant membrane matrix of the rhabdomeral microvilli of crayfish photoreceptors

The lectin-binding properties of crayfish photoreceptor membrane were examined using isolated rhabdoms and frozen sections of whole retinas after permeabilization. Fluorescently-labelled concanavalin A (Con A) and wheat germ agglutinin (WGA) bind specifically to the rhabdoms, the accessory pigment cells, and the basement membrane of the retina in permeabilized frozen sections. Con A also binds to the clear zone in sectioned material. Peanut agglutinin (PA) binds strongly to domains associated with the periphery of rhabdoms. Intact isolated rhabdoms also bind Con A and WGA with high affinity. The major lectin-binding proteins of the photoreceptor membrane were characterized on nitrocellulose blots after SDS electrophoresis of purified photoreceptor membrane. Two major glycoproteins were found to bind a number of lectins. A 130 000 molecular weight peptide (GPII) specifically bound Con A, WGA, Dolichos biflorus agglutinin, (DBA) Ulex europaeus agglutinin (UEA) and PA, whereas the major 142 000 d protein (GPI) of the photoreceptor membrane bound only Con A and WGA implying a rather simple hexose chain consisting mainly of mannose and N-acetylglucosamine. Photoreceptor membrane vesicles, extracted with a high concentration of non-ionic detergent and centrifuged, show a specific enrichment of both proteins as well as actin and a 40 000 d component. Significant amounts of both glycoproteins are removed from the membrane residue by 0·6 m KI, even under conditions known to stabilize actin-based cytoskeletons. Both the stoichiometric amounts of these peptides in relation to the actin complement and their physicochemical properties are consonant with the hypothesis that one or both may be involved in linking a submembranous, actin-based cytoskeleton to the lipid bilayer. Rhodopsin, the major component of photoreceptor membrane, appears to be a glycoprotein by periodic acid-Schiff (PAS) staining methods but fails to bind significant amounts of lectins on Western blots. These findings are discussed in the light of post-translational processing and intracellular pathways for photoreceptor membrane components in the compound eye.

Optimal conditions for detection of reverse transcriptase activity in human placentas

Optimal conditions for detecting reverse transcriptase activity in human placental extracts are described. They vary with the state of the placenta at birth and are influenced by relative amounts of detergent, monovalent cation, and protein in the reaction mixture. Demonstrating activity of the placental enzyme requires detergent, but the enzyme is sensitive to high detergent concentrations. This sensitivity can be be altered by lowering the monovalent cation concentration from 0.154 to 0.034 M and by adding protein to the reaction mixture. The detection of reverse transcriptase in Rauscher murine leukemia virus and baboon endogenous type C virus, but not in the Mason-Pfizer monkey type D virus, shows similar requirements.

Postsynaptic acetylcholine receptor efficacy is similarly increased by detergents and acetylcholinesterase inhibitors at an Aplysia synapse

At Aplysia H- and D-type cholinergic neuro-neuronal synapses, application of high concentrations of detergents (Triton X-100 and sodium deoxycholate) depressed synaptic transmission and the postsynaptic response to ionophoretic application of acetylcholine (ACh) or carbachol. However, when very low concentrations of detergents (of the order of 10 −9 M for sodium deoxycholate) were used, the nerve-evoked response as well as the ACh and carbachol ionophoretic responses were facilitated (by at least 200%), but only in H-type cells. This facilitation was similar to that previously observed in the same receptor type when acetylcholinesterase (AChE) was inhibited by various organophosphate or carbamate acetylcholinesterase inhibitor (AChEIs) 3. Indeed, the effects of AChEI and detergents were not cumulative. We propose that on H-type synapses detergents may perturb a hypothetical molecular interaction between AChE and the acetylcholine receptor (AChR) by which AChE modulates the ability of the AChR to be activated by ACh or carbachol.

Proteoglycans synthesized by rat ovarian granulosa cells in culture. Isolation, fractionation, and characterization of proteoglycans associated with the cell layer.

Rat ovarian granulosa cells were isolated from immature female rats after stimulation with pregnant mare's serum gonadotropin and maintained in culture. Proteoglycans were labeled with [35S]sulfate, [3H]glucosamine, or [3H]serine as precursors. Proteoglycans associated with the cell layer were extracted with 4 M guanidine HCl buffer containing 2% Triton X-100. Labeled proteoglycans were isolated using sequential DEAE-Sephacel and Sepharose CL-4B chromatography under dissociative conditions in the presence of detergent. The cell layer contained three different hydrodynamic size species of dermatan sulfate proteoglycans, DS-Ic, DS-IIc, and DS-IIIc, and two different hydrodynamic size species of heparan sulfate proteoglycans, HS-Ic and HS-IIc, when analyzed on Sepharose CL-4B, DS-Ic, DS-IIc, and HS-Ic were indistinguishable from corresponding species in the medium fraction in terms of (a) hydrodynamic size analyzed on Sepharose CL-2B or Sepharose CL-4B, (b) buoyant density distribution in dissociative CsCl gradients, and (c) glycosaminoglycan and oligosaccharide compositions. The majority of these three proteoglycan species could be removed by brief trypsin treatment indicating their cell surface localization. Heparin released a part of the trypsin-removable proteoglycan population, which mostly consisted of DS-Ic with smaller amounts of DS-IIc and HS-Ic. Cell layer extracted with 4 M guanidine HCl without detergent contained a large hydrodynamic size structure excluded from Sepharose CL-2B, which contained DS-IIc and HS-Ic proteoglycans with large amounts of exogenous proteins. Proteoglycans were dissociated from this supramolecular structure in the presence of high concentrations of detergents (2% sodium dodecyl sulfate or Triton X-100), indicating the hydrophobic nature of this structure, probably a part of the plasma membrane, and suggesting that DS-IIc and HS-Ic are intercalated into membrane. Both DS-IIIc and HS-IIc were intracellular species, which were not released into the medium by the living cells or removed by either trypsin or heparin. DS-IIIc species was a single glycosaminoglycan chain having the same hydrodynamic size and sulfation pattern as those of glycosaminoglycan chains on DS-II. HS-IIc species was also a single glycosaminoglycan chain with an average molecular size only 1/3-1/4 that of chains on HS-I but heparan sulfate chains from both species showed a similar average sulfation pattern when analyzed by nitrous acid fragmentation. Both DS-IIIc and HS-IIc had small amounts of covalently attached peptides.

Changes in structure of isolated chloroplasts ofCodium fragile andCaulerpa filiformis in response to osmotic shock and detergent treatment

The ultrastructure of chloroplasts from two genera of coenocytic green algae, Codium and Caulerpa, were examined after suspension in hypotonic solution and in detergent at various concentrations. The capacity of the suspensions to carry out CO2-dependent and ferricyanide-dependent O2 evolution was measured under the same conditions of osmotic strength and detergent concentration. The chloroplasts in the preparations were in the form of cytoplasts and gave rates of O2 evolution comparable with those expected from undamaged chloroplasts. Suspension in hypotonic solution depressed the rate of CO2-dependent O2 evolution in both species, but this was partially restored in the Codium chloroplasts when these were re-suspended in iso-osmotic solutions. Major structural changes were observed only after suspension in buffer when the Codium chloroplasts lost their outer envelope, most of their stroma, and the thylakoids became swollen. Caulerpa chloroplasts were more variable in their response and, even when suspended in buffer only, the proportion of the plastids which had lost all of their stroma and thylakoid swelling was never as common as in Codium chloroplasts. However, once suspended in hyper-osmotic medium below 700 mosmolar, Caulerpa chloroplasts could not regain their capacity for CO2-dependent O2 evolution. Detergent treatment removed the cytoplast membrane but not the cytoplasmic material adhering to the chloroplast envelope. High concentrations of detergent were needed to cause loss of the chloroplast envelope, loss of stromal contents and unstacking of the thylakoids. Caulerpa chloroplasts were less sensitive to detergent than those of Codium. There was no indication that specific structures such as the thylakoid organizing body were resistant to detergent action. The results show that exposure to hypotonic solutions and to detergent results in less damage to these chloroplasts than it would to those of higher plants. It is proposed that the basis of this unusual resistance is not due to the properties of the chloroplast membranes but to the presence of material which coats the organelles during isolation. This material is likely to be identical with the sulphated xylo-mannogalactan isolated from the vacuole contents of these algae and which has the visco-elastic properties essential to allow the organelles to resist disruption by osmotic forces and disintegration by detergents.

Kinetic properties of C 12E 8-solubilized (Na ++K +)-ATPase

(1) The properties of the rectal gland (Na + + K +)-ATPase (ATP phosphohydrolase, EC 3.6.1.8) solubilized in octaethyleneglycol dodecylmonoether (C 12E 8) have been investigated. (2) The Kinetic properties of the solubilized enzyme resemble those of the membrane-bound enzyme to a large extent. The main difference is that K m for ATP for the (Na + + K +)-ATPase is about 30 μM for the solubilized enzyme and about 100 μM for the membrane-bound enzyme. (3) The Na +-form (E 1) and the K +-form (E 2) can also be distinguished in the solubilized enzyme, as seen from tryptic digestion, the intrinsic fluorescence and cosin fluorescence responses to Na + and K +. (4) The number of vanadate-binding sites is unchanged upon solubilization, and it is shown that vanadate binding is much more resistant to detergent inactivation than the enzymatic activities. The number of phosphorylation sites on the 95–100% pure supernantant enzyme is about 3.8 nmol/mg, and is equal to the number of vanadate sites. (5) Inactivation of the enzyme by high concentrations of detergent can be shown to be related to the C 12E 8/protein ratio, with a weight ratio of about 4 being a threshold for the onset of inactivation at low ionic strength. At high ionic strength, more C 12E 8 is required both for solubilization and inactivation. (6) It is observed that the commercially available detergent polyoxyethylene 10-lauryl ether is much less deleterious than C 12E 8, and its advantages in the assay of detergent-solubilized (Na + + K +)-ATPase are discussed. (7) The results show that (Na + + K +)-ATPase can be solubilized in C 12E 8 in an active form, and that most of the kinetic and conformational properties of the membrane-bound enzyme are conserved upon solubilization. C 12E 8-solubilized (Na + + K +)-ATPase is therefore a good model system for a solubilized membrane protein.

The distribution of C 12E 8-solubilized oligomers of the (Na + + K +-ATPase

(1) Gel filtration of (Na + + K +)-ATPase (ATP phosphohydrolase, EC 3.6.1.8) solublized in octaethyleneglycol dodecylmonoether (C 12E 8) has been performed under conditions where active (α⨿) 2 dimers ( M r 265 000) are obtained, and under conditions where dissociation into αβ monomers occurs without appreciable loss of activity. (2) It is shown that the αβ monomers aggregate with time to form (αβ) 2 dimers at low detergent concentrations with no change in enzymatic activity. At high detergent concentrations the aggregation is much slower, but the enzymatic activity is lost rapidly. (3) Polyacrylamide gel electrophoresis in the presence of C 12E 8 also suggest that high concentrations the smaller particles, and conditions for gel electrophoresis ar described. (4) The inactivating effect of C 12E 8 at high C 12E 8/protein ratios can be related to a delipidation of the enzyme, with about 0.19 mg Phospholipid required per mg protein for optimal activity.(5) The experiments suggest that the solublized (Na + + K +-ATPase can be disrupted into particles containing only on α-chain and one or two β-chains without irreversible loss of activity, and that the stable form of the enzyme is an (αβ) 2 dimer.

Proton ATPase of rat liver mitochondria. Preparation and visualization of a functional complex using the novel zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate.

The proton ATPase of rat liver mitochondria has been purified by a simple procedure which involves the use of the novel, zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate to solubilize the membrane-bound complex. The purified enzyme has a high, oligomycin-sensitive ATPase activity (11.3 +/- 2.9 mumol/min/mg) in the absence of added phospholipids. It shows, in four different gel electrophoretic systems, the five bands characteristic of the F1 portion of the complex and three additional Coomassie blue-stainable bands which have apparent molecular weights of 28,000, 19,000, and 13,600. A fourth Coomassie blue-stainable component of about 10,000-12,500 daltons comigrates with the delta subunit, whereas a fifth component, detectable only by absorption at 280 nm, is observed between the dye front and the 10,000-dalton species. The enzyme complex has been reconstituted into liposomal vesicles of asolectin. Under these conditions the enzyme catalyzes an ATP-Pi exchange reaction and is capable of translocating protons in an ATP-dependent manner as assayed by quenching of 9-amino-6-chloro-2-methoxyacridine. Both activities are inhibited by the addition of oligomycin, uncoupler, dicyclohexylcarbodiimide, and cadmium. At high detergent concentration, the complex appears in negative stain electron microscopy in a dispersed state. The tripartite structure is clearly visible in monomeric, dimeric, or trimeric forms of the molecule. At the low detergent concentration, the proton ATPase tends to cluster into densely packed arrays. This represents the first report of the properties of a functionally active proton ATPase solubilized and purified in the presence of a zwitterionic detergent.

A new sensitive assay for the calcium-dependent binding of C-reactive protein to phosphorylcholine

A new sensitive assay for the calcium-dependent binding of rabbit C-reactive protein to phosphorylcholine has been developed. The assay involves coating the wells of polyvinylchloride microtiter plates with a bovine serum albumin-phosphorylcholine conjugate followed by the addition of C-reactive protein. The quantity of C-reactive protein is determined by indirect enzyme-linked immunoadsorbant assay using first, affinity purified goat anti-C-reactive protein immunoglobulin followed by a commercial rabbit anti-goat immunoglobulin-alkaline phosphatase conjugate. The binding of rabbit C-reactive protein to the bovine serum albumin-phosphorylcholine conjugate is completely inhibited by free phosphorylcholine, by pneumococcal C-polysaccharide and by calcium chelators but not by high concentrations of neutral, cationic or zwitterionic detergents. The assay is sensitive to 2 ng C-reactive protein.

Stability Properties of Photopolymerized Liposomes

Lipid vesicles (liposomes) can be formed from [1,2–(methacryloyloxy)dodecanoyl]–L–α–phosphatidylcholine, a photopolymerizable analog of phosphatidylcholine. As compared to conventional liposomes, the photopolymerized vesicles display enhanced mechanical stability and enhanced retention of lipophilic compounds. Thus, while detergents solubilize conventional vesicles and release incorporated lipophilic markers such as cholesterol oleate, the photopolymerized vesicles retain lipophilic compounds even at extremely high detergent concentrations. Photopolymerized vesicles may prove to be useful in certain aspects of the formulation and controlled delivery of hydrophilic and lipophilic drugs.

Isoelectric focusing with reduced cathodic drift and migration into the anode chamber

An apparatus has been developed to reduce cathodic drift and migration into the anode chamber in vertical gel rod isoelectric focusing (IEF). In contrast to commercially available apparatuses, this apparatus can easily handle many more gels at one time, and the length, diameter and shape of its gel can be arbitrarily changed. In addition, high concentrations of detergent can be used to dissolve the protein samples, and removal of the gel cylinders from the glass tubes is easy.

Characterization of the membrane bound Mg 2+-ATPase of rat skeletal muscle

A procedure was developed to isolate a membrane fraction of rat skeletal muscle which contains a highly active Mg 2+-ATPase (5–25 μmol P i/mg min). The rate of ATP hydrolysis by the Mg 2+-ATPase was nonlinear but decayed exponentially (first-order rate constant ≥0.2 s −1 at 37°C). The rapid decline in the ATPase activity depended on the presence of ATP or its nonhydrolyzable analog 5′-adenylyl imidodiphosphate (Ado PP[NH] P). Once inactivated, removal of ATP from the medium did not immediately restore the original activity. ATP- or Ado PP[NH] P-dependent inactivation could be blocked by concanavalin A, wheat germ agglutinin or rabbit antiserum against the membrane. Additions of these proteins after ATP addition prevented further inactivation but did not restore the original activity. Low concentrations of ionic and nonionic detergents increased the rate of ATP-dependent inactivation. Higher concentrations of detergents, which solubilize the membrane completely, inactivated the Mg 2+-ATPase. Cross-linking the membrane components with glutaraldehyde prevented ATP-dependent inactivation and decreased the sensitivity of the Mg 2+-ATPase to detergents. It is proposed that the regulation of the Mg 2+-ATPase by ATP requires the mobility of proteins within the membrane. Cross-linking the membrane proteins with lectins, antiserum or glutaraldehyde prevents inactivation; increasing the mobility with detergents accelerates ATP-dependent inactivation.

Solubilization of the catecholamine carrier of chromaffin granule membranes in a form that binds substrates and inhibitors of uptake.

The component of bovine chromaffin granule membrane responsible for the binding of [2-3H]dihydrotetrabenazine has been solubilized by treatment with detergents. The binding activity of the soluble material was measured either by equilibrium dialysis or by precipitation of the protein by poly(ethylene glycol) and filtration of the precipitate. The best yields and activities were obtained with sodium cholate, which solubilized more than 70% of the binding sites; deoxycholate had deleterious effects on the soluble activity, and among nonionic detergents, octyl beta-glucoside was the most efficient. With cholate, [3H]dihydrotetrabenazine binding occurred on soluble sites as judged by centrifugation of the bound material and by filtration on Sepharose 6B columns. Binding could be measured in the presence of cholate, but high detergent concentrations had a reversible inhibitory effect. The [3H]dihydrotetrabenazine binding characteristics of the soluble material were similar to those of the granule membrane, with one class of binding sites (KD = 23 nM, Bmax = 90 pmol/mg of protein). The association and dissociation rate constants were 0.072 X 10(6) M-1 s-1 and 1.1 X 10(-3) s-1, respectively. The pharmacological properties of the binding sites were also similar to those of the membranes. Our results thus support the view that the catecholamine carrier has been solubilized in an active conformation that binds substrates and inhibitors of uptake.