Biochemical characterization of the tetrodotoxin binding protein from Electrophorus electricus

Biochemical properties of a detergent-solubilized tetrodotoxin binding component from Electrophorus electricus have been examined and compared with those found for the membrane-bound protein. The toxin binding component was solubilized with high efficiency by a variety of nonionic detergents and with lower efficiency by sodium cholate and deoxycholate. Detergent-solubilized preparations bound tetrodotoxin and saxitoxin tightly and specifically, and this binding was observed to be rapidly and irreversibly blocked by carboxylate-modifying reagents. Inactivation by carbodiimide and glycine ester or by a trimethyloxonium salt could be prevented by tetrodotoxin occupancy of the binding site. Tetrodotoxin binding activity in both solubilized preparations and in membranes was found to be highly resistant to proteases. In contrast, the activity was extremely sensitive to the action of phospholipase A2. The biochemical properties of the tetrodotoxin binding component solubilized in mixed lipid-detergent micelles are similar to those found in native membranes, with respect to the characteristics of equilibrium toxin binding and to the sensitivity of toxin binding activity to chemical modification and degradative enzymes. There were some differences with respect to the kinetics of tetrodotoxin binding. In addition, the tetrodotoxin binding component from eel is shown to behave as a glycoprotein, being selectively absorbed to resins coupled to concanavalin A, wheat germ agglutinin, Lens culinaris lectin, and ricin with the appropriate glycoside.     

The binding of tritiated tetrodotoxin to squid giant axons.

The binding of tetrodotoxin to squid gian axons was determined as a function of toxin concentration, using a tritiated toxin preparation of known radiochemical purity and specific activity. From the amount of saturable binding observed, the number of toxin binding sites thought to be sodium channels was found to be 553 plus or minus 119/mum-2 of axon surface.     

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.     

Photoaffinity labeling of the tetrodotoxin binding component ofElectrophorus electricus electroplax

Radioactive azide derivatives of tetrodotoxin (TTX) were synthesized using 2-nitro-4-azidephenyl-[3H]beta alanine for the purpose of photolabeling of the Na channel. Three azide derivatives, N1, N2 and N3, were separated by ion exchange chromatography on Bio-Rex 70 resin and reversed phase high performance liquid chromatography. N3 was more stable and obtained at a higher yield than the other two derivatives. Bioactivity of N3 was one-twentieth of that of TTX. N3 showed reversible binding to membranes of Electrophorus electricus electroplax in the dark with Kd = 30 nM and B max = 5.2 pmol/mg protein. By photoirradiation, irreversible binding of N3 to the membranes was observed. A N3 binding component was solubilized by lubrol PX and partially purified from the electroplax membranes by Sephadex G25 and Sepharose 6B column chromatography. The component, purified 500 fold from the starting membranes, showed molecular weight of 10,000.     

Solubilization and partial characterization of the sex hormone-binding globulin receptor from human prostate

The sex hormone-binding globulin (SHBG) receptor was solubilized from the membranes of human prostate glands with the zwitterionic detergent CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonic acid). The binding activity of the soluble receptor was measured by allowing it to bind to 125I-SHBG and precipitating the complex with polyethylene glycol-8000. The binding activity was stable for at least 4 months at -20 degrees C and had a half-life of 23 days at 4 degrees C. Like the membrane-bound receptor, Scatchard analysis revealed two sets of binding sites for the soluble one. At equilibrium (24 h), the high affinity site had an association constant (KA) of 6.8 x 10(8) M-1 and a binding capacity of 1.4 pmol/mg protein, whereas the low affinity site had a KA of 4.7 x 10(6) M-1 and a binding capacity of 43 pmol/mg protein. At 37 degrees C, the association rate constant (k1) was 8.37 x 10(5) M-1 min-1 and the dissociation rate constant (k2) was 3.43 x 10(-4) min-1. The soluble receptor was retarded on Sepharose CL-6B and had an apparent Mr = 167,000.     

Solubilization and partial characterization of phospholipase A from rat heart sarcoplasmic reticulum

Phospholipase A has been solubilized from the sarcoplasmic reticulum of rat heart by treatment with Tris buffer, potassium chloride, taurodeoxycholate or octyl glucoside. On HPLC gel permeation, two phospholipases were identified at the void volume of a TSK 3000 column and at an apparent molecular mass of 60 kDa. The two activity peaks exhibited a predominance of phospholipase A1 activity (83-91%) and a lesser phospholipase C activity (4-9%) using sonicated 1-palmitoyl-2[1-14C]oleoylphosphatidylcholine liposomes as substrate. The voiding phospholipase A peak, which represented the bulk of the recovered activity, exhibited a requirement for calcium ions in the 0.3-3 microM range. The heat stability and response to mercuric ions was studied and some similarities were noted between the solubilized sarcoplasmic reticulum phospholipases A and the cytosolic phospholipases A of rat heart. It is speculated that the cytosolic phospholipase A which we reported earlier may represent in part phospholipase A released from sarcoplasmic reticulum during isolation of the subcellular membrane fractions.     

Properties of the tetrodotoxin binding component in plasma membranes isolated from Electrophorus electricus.

The biochemical properties of the electrically excitable sodium channels in the electroplaque of Electrophorus electricus were investigated using tritiated tetrodotoxin (TTX) as a specific membrane probe. Membrane fragments from the electroplaque were isolated essentially by differential centrifugation and characterized with respect to the plasma membrane markers acetylcholine receptors, acetylcholinesterase, (Na+ + K+)ATPase, and [3H]TTX binding. Equilibrium binding studies showed that [3H]TTX bound to a single population of noninteracting receptor sites with an apparent dissociation constant of 6 +/- 1 X 10(-9) M. The toxin-membrane complex dissociated with a first-order rate constant of 0.012 sec-1. Studies on the pH dependence of complex formation demonstrated the requirement for an ionizable, functional group with a pK of 5.3 and this group has been shown to be a carboxyl. Treatment of the membranes with trimethyloxonium tetrafluoroborate, a carboxyl group modifying reagent, resulted in an irreversible loss in the binding of [3H]TTX, which could be prevented by low concentrations of TTX or saxitoxin. This decrease was due to a reduction in the total number of binding sites and not to a decrease in toxin binding affinities. The relative binding affinities of various monovalent alkali metal and polyatomic cations for the TTX-receptor site showed that this site displayed cation discrimination properties which were similar to those reported previously for the electrically excitable sodium channel in intact nerve fibers. A possible role for this site in the ion selectivity of the sodium channel is proposed.     

Solubilization and characterization of an angiotensin II binding protein from liver

Binding sites with high affinity for angiotensin II were solubilized from hepatic membranes by treatment with digitonin. Binding of radioiodinated angiotensin II was assayed by gel filtration and independently by a technique exploiting the failure of activated charcoal to adsorb the bound ligand. The binding protein was partially purified using ammonium sulfate fractionation followed by gel filtration, and in the presence of protease inhibitors, the isolated binding protein preparation did not catalyze degradation of the angiotensin II. Binding to the membranes as well as to the solubilized preparation was specific and saturable. The membranes exhibited a single set of high-affinity binding sites with a Kd of 0.5 nM. The solubilized preparation, also showed the presence of a single class of high-affinity binding sites (Kd = 10.5 nM). Displacement studies using angiotensin I as well as various fragments, agonists and antagonists of angiotensin II disclosed a structure-activity profile similar to that found with intact membranes. Dissociation of angiotensin II from the soluble macromolecular complex was slow but was enhanced at non-physiological pH values or in the presence of 4.5 M urea, or 1% sodium dodecyl sulfate. Covalent binding of the radioiodinated angiotensin II to a single, specific macromolecular component was achieved by treatment with disuccinimidyl suberate. The apparent molecular weight of this reduced, denatured radioactive protein was estimated at about 68 000 by polyacrylamide gel electrophoresis.     

Solubilization and partial characterization of a phosphoprotein phosphatase from human myelin.

The phosphoprotein phosphatase (phosphoprotein phosphohydrolase, EC 3.1.3.16) solubilized from human central nervous system myelin has been shown to possess a comparatively high degree of specificity towards myelin basic protein, a constituent of the membrane and most likely its natural substrate, rather than the mixed histones. The enzyme has a pH optimum of 7.5. Hydrolysis of both the substrates is stimulated by dithiothreitol and is almost completely dependent upon the presence of divalent metal ions. The maximum rate of dephosphorylation of basic protein is attained in the presence of 125 micrometer Mn2+ whereas a much higher concentration of Mg2+ (50--100 mM) is required for the optimal dephosphorylation of histones. The dephosphorylation of basic protein was also stimulated by Triton X-100 (0.15%, v/v) and was shown to result from a 3-fold increase in the V of the reaction catalyzed by the phosphatase. The apparent Km values for basic protein and histones were unaffected by the presence of Triton X-100 and were found to be approx. 1 and approx. 160 micrometer, respectively. Under optimal conditions of assay, the phosphatase cleaved approx. 32 and approx. 0.7 nmol of orthophosphate.min-1.mg-1 of protein from basic protein and histones, respectively.     

Solubilization and partial characterization of cell membrane Fc receptors

We assayed detergent cell lysates and culture supernatant fluid of mouse cells for the presence of soluble FcR by means of a radioligand bioassay. Strict correlation was found between the amount of FcR present in soluble form and that found on intact cells. The soluble FcR of all cells tested, except mouse macrophages, behaved as lipoprotein by buoyant density centrifugation analysis and enzyme treatment. In the absence of detergent, soluble FcR sedimented as a 20S macromolecule; in detergent the material was 7 to 9S. It was readily bound by insolubilized IgG but not by insolubilized BSA or F(ab')2 fragments of IgG.     

Solubilization and partial purification of a cell surface component of Leishmania braziliensis

A simple procedure that allowed the extraction and partial purification of a component of 65,000 mol.wt. from the surface of Leishmania braziliensis promastigotes is described. Iodinated cell surface membrane fractions were solubilized using Triton X-100 followed by Nonidet P-40. The macromolecular components were then freed of the detergents by passage of the extracts through a column of DE52 cellulose. The component of 65,000 mol.wt. was eluted from the column with 1 m NaCl. This component in whole parasites was immunoprecipitated by sera from patients with cutaneous, mucocutaneous, and diffuse cutaneous leishmaniasis and kala-azar. None of the major surface determinants reacted with sera from normal individuals with antibovine rabbit serum. The relevance and the possible applications on the immunoprophylaxis of the disease are briefly discussed.     

Solubilization and partial characterization of homogalacturonan-methyltransferase from microsomal membranes of suspension-cultured tobacco cells.

The transfer of a methyl group from S-adenosyl-L-methionine onto the carboxyl group of alpha-1,4-linked-galactosyluronic acid residues in the pectic polysaccharide homogalacturonan (HGA) is catalyzed by an enzyme commonly referred to as pectin methyltransferase. A pectin methyltransferase from microsomal membranes of tobacco (Nicotiana tabacum) was previously characterized (F. Goubet, L.N. Council, D. Mohnen [1998] Plant Physiol 116: 337-347) and named HGA methyltransferase (HGA-MT). We report the solubilization of HGA-MT from tobacco membranes. Approximately 22% of the HGA-MT activity in total membranes was solubilized by 0.65% (w/v) 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid containing 1 mM dithioerythritol. The addition of phosphatidylcholine and the methyl acceptors HGA or pectin (30% degree of esterification) to solubilized enzyme increased HGA-MT activity to 35% of total membrane-bound HGA-MT activity. Solubilized HGA-MT has a pH optimum of 7.8, an apparent K(m) for S-adenosyl-L-methionine of 18 microM, and an apparent V(max) of 0. 121 pkat mg(-1) of protein. The apparent K(m) for HGA and for pectin is 0.1 to 0.2 mg mL(-1). Methylated product was solubilized with boiling water and ammonium oxalate, two conditions used to solubilize pectin from the cell wall. The release of 75% to 90% of the radioactivity from the product pellet by mild base treatment showed that the methyl group was incorporated as a methyl ester rather than a methyl ether. The fragmentation of at least 55% to 70% of the radiolabeled product by endopolygalacturonase, and the loss of radioactivity from the product by treatment with pectin methylesterase, demonstrated that the bulk of the methylated product produced by the solubilized enzyme was pectin.     

Solubilization and partial characterization of rat liver squalene epoxidase.

The microsomal enzyme system from rat liver which catalyzes squalene epoxidation requires a supernatant protein and phospholipids (Tai, H., and Bloch, K. (1972) J. Biol. Chem. 247, 3767). It has now been found that these two cytoplasmic components can be replaced by Triton X-100. The same detergent solubilizes the microsomal squalene epoxidase and the resulting supernatant can be separated into two components, A and B, by DEAE-cellulose chromatography. Neither Fraction A nor B alone has significant squalene epoxidase activity but combining the two affords a reconstituted system 5-fold higher in specific epoxidase activity than that of the original microsomes. FAD and Triton X-100 in addition to molecular oxygen and NADPH are required in the reconstituted system. Subjecting Fraction A to a second DEAE-cellulose chromatography does not change its specific activity but lowers NADH-ferricyanide reductase activity and the protoheme content to 1/25 and 1/4, respectively. When Fraction B was chromatographed on Sephadex G-200, the specific epoxidase activity tested in the presence of Fraction A was increased 3-fold. This procedure also raised the specific activity of NADPH-cytochrome c reductase activity in Fraction B 3-fold. The reconstituted epoxidase system is not inhibited by either carbon monoxide, potassium cyanide, or o-phenanthrolien but Tiron at 1 mM was inhibitory (50%). Erythrocuprein has no effect on epoxidation. No evidence has been found for the participation of hemoproteins (P450 or cytochrome b5) in squalene epoxidation. Component B appears to be identical with the flavoprotein NADPH-cytochrome c reductase. Component A may be a flavoprotein with an easily dissociable prosthetic group.