Lubrol WX Research Articles

The solubilization of membrane-bound (Na + + K +)-ATPase from rat skeletal muscle

A (Na + + K +)-ATPase (EC 3.6.1.3.) of skeletal muscle was solubilized by treatment with Lubrol WX. Maximum extraction of this enzyme was obtained at a Lubrol WX:protein ratio of 2.5:1. The supernatant fraction of the Lubrol WX extract obtained by centrifugation at 140,000 g for 45 min after NaI treatment had a higher specific activity and greater ouabain sensitivity than that of the NaI-extracted frozen muscle homogenate, which had not been extracted with Lubrol WX.

A simple procedure for the preparation of highly purified (sodium + potassium) adenosinetriphosphatase from the rectal salt gland of Squalus acanthias and the electric organ of Electrophorus electrieus

A simple purification procedure for the Na,K-ATPase from membranes of the rectal gland of Squalus acanthias or crude microsomal fractions from the electric organ of Electrophorus electricus is presented here. The purification procedure consists of solubilization of the Na,K-ATPase with the nonionic detergent. Lubrol WX, chromatography of the diluted Lubrol extract on aminoethyl cellulose, and ammonium sulfate fractionation (1) of the concentrated eluate from the aminoethyl cellulose column. The yields of final purified enzyme are comparable to the earlier purification (1–4) involving the expensive and cumbersome zonal centrifugation stop. The purity of the final enzyme, as attested to by specific activity and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, is as great or greater than that previously reported for the enzyme purified by the procedure involving zonal centrifugation. The simplicity of the present procedure, coupled with the ready commercial availability of electric eels which are quite hardy on shipment, makes purification of the Na,K-ATPase widely available to workers in the field.

Studies of (Na + + K +)-sensitive ATPase activity in pig lymphocytes. Effects of concanavalin A

(Na + + K +)-ATPase activity is demonstrated in plasma membranes from pig mesenteric lymph nodes. After dodecyl sulfate treatment plasma membranes have an 18-fold higher (Na + + K +)-ATPase activity, while their ouabain-insensitive Mg 2+-ATPase is markedly lowered. A solubilized (Na + + K +)-ATPase fraction, obtained by Lubrol WX treatment of the membranes, has very high specific activity (21μmol P i/h per mg protein). Concanavalin A has no effect on these partially purified (Na + + K +)-ATPase, while it inhibits (40%) this activity in less purified fractions which still contain Mg 2+-ATPase activity.

Glucose and cation transport in rat jejunum, ileum and colon in vivo: effects of anionic and nonionic surfactants, and of desoxycholate.

Osmotically balanced solutions of glucose (0.5--300 mM) and sodium chloride, containing surfactants, were instilled into the small or large intestine of anaesthetized rats. Net absorption or secretion of glucose, sodium and potassium was studied. The surfactants tested were dodecylsulphate (3.4--17 mM), dioctylsulphosuccinate (1.8--11 m/M), Lubrol WX (0.1--5%), Triton X 100 (0.25%) and desoxycholate (2.5 mM). Qualitatively, the results were similar to those obtained previously with cationic compounds, suggesting a common mode of action for all surfactants studied. 17 mM dodecylsulphate seemed to abolish completely physiological glucose transport in the jejunum and ileum. At a lower concentration, and witth the other surfactants, normal glucose transport was affected to an intermediate extent.

Solubilization of estrogen synthetase from human term placental microsomes using detergents

Abstract The microsomal fraction of term placenta was incubated with a range of concentrations of several bile acids and non-ionic detergents (deoxycholate, cholate, taurocholate, Triton X-100, Tween 80, Lubrol PX, Lubrol WX and digitonin) and then centrifuged at 120,000 g for 1 h. Aromatase activity was progressively depleted from the pellet with increasing detergent concentration for each detergent except Tween 80 (up to 5% concentration). Since a cytochrome P-450 monoxygenase system is assumed to be involved in androgen aromatization, two detergents, deoxycholate and Triton X-100, were also examined for their ability to remove NADPH cytochrome c reductase activity and cytochrome P-450 from the microsomes. Although the cytochrome was removed from microsomes at detergent concentrations similar to those for aromatase, approximately 2- to 3-fold higher detergent concentrations were required to deplete the microsomes of reductase activity. Each detergent used inhibited aromatase activity in the microsomal homogenate in a concentration-dependent manner that roughly paralleled the concentration-dependent depletion of aromatase activity from the pellet. Aromatase activity was measured in the supernatant (120,000 g-h) from the cholate- and deoxycholate-treated microsomes by diluting the supernatant to lower detergent concentrations. Significant amounts of aromatase activity could not be measured in the 120,000 g-h supernatant from non-ionic detergent-treated microsomes even after dilution to lower detergent concentrations. Dilution of the cholate- and deoxycholate-soluble prepaations to lower detergent concentrations resulted in an aromatase-active precipitate with a 3-fold higher specific activity than the original microsomes. Deoxycholate- and cholate-solubilized aromatase activity is included in a Sepharose 6B column, eluting at an approximate molecular weight of 100,000 to 120,000 daltons.

Mise en évidence, études cinétiques et solubilisation de l'adénylate cyclase particulaire de Nocardia restricta

An adenylate cyclase (EC r.6.1.1.) was found in cell-free extracts of several Nocardia species. The enzyme from Nocardia restricta has been specially studied. It is a membrane enzyme which exhibits a strong specific activity, one hundred times greater than that of mammals. It has an optimal pH of 8.5 in Tris buffer and an absolute requirement for divalent ions, Mg++ or Mn++ (Mn++ ions are the most efficient). The kinetic properties of this adenylate cyclase are similar to those that could be expected of an allosteric enzyme having, as a substrate, the ATP-Mg++ complex and, as an activator, free Mn++ ions. Ca++ ions are activators: they set up the maximum velocity without modification of the KM. GTP is a competitive inhibitor (KI = 5.10(-5) M). Fluoride ions have no detectable effect on activity. Non-ionic detergents, Lubrol WX and Triton X 100, are inhibitors of the enzyme which has been partially solubilized by repeated freezing and thawing, following by brief ultra-sonic treatment. Catalytic sites are not modified after the solubilization, but cooperative effects between moles of substrate ATP-Mn++ are diminished: the KM becomes smaller and the sigmoidal shape of the curve v = f (ATP-Mn++) is attenuated.

Ouabain binding to phospholipid-dependent adenosine triphosphatase.

The role of phospholipid in the binding of ouabain to the (Na+ + K+)-dependent adenosine triphosphatase was studied. Enzyme preparations obtained from rabbit kidney were treated with Lubrol WX to remove the phospholipid component essential for ATPase activity. Reconstituted enzyme samples were prepared by the addition of phosphatidylserine and sedimentation of an enzymically active lipid-protein complex. The binding of ouabain to both kinds of preparations was measured under equilibrium conditions with the use of 3H-labelled ouabain and initial ouabain concentrations in the range 0.01-1 micrometer. The main findings were: (i) (Mg2+ + Pi) promoted binding of significant quantities of ouabain only to the reconstituted enzyme; (ii) the absence of added Na+, (Mg2+ + ATP) similarly promoted binding only to the reconstituted samples; (iii) the addition of Na+ in the presence of (Mg2+ + ATP) increased the amount of ouabain bound to the reconstituted enzyme when the ouabain concentration was below about 0.1 micrometer, but it had no effect when the ouabain concentration was about 1 micrometer; (iv) (Mg2+ + ATP) induced ouabain binding to the depleted enzyme only when Na+ was also added; (v) the amount of ouabain bound to both depleted and reconstituted enzymes was the same in the presence of (Mg2+ + ATP + Na+); (vi) the reconstituted enzyme appeared to have a greater affinity for Na+ than did the depleted enzyme.

A simple and rapid method for the reversible removal of lipids from a membrane-bound enzyme.

A simple, rapid and reproducible method for the reversible removal of lipids from a membrane-bound enzyme is described. Essentially, a membrane preparation containing (Na+ + K+)-dependent adenosine triphosphatase was extracted with the non-ionic detergent Lubrol WX in the presence of glycerol, and partial separation of protein from lipid was achieved with the use of only two centrifugations. About 74% of the endogenous phospholipid and 79% of the cholesterol were removed, concomitant with a virtually complete loss of ouabain-sensitive adenosine triphosphatase activity, but with retention of 60-100% of the K+-dependent phosphatase activity. The addition of pure phosphatidylserine re-activated the enzyme to more than 80% of the initial activity, and up to 30% of the protein was recovered. Excess of phosphatidylserine could be washed off the enzyme to give a stable 'reconstituted' preparation. The effects of variation in the experimental conditions were examined, and the results are discussed with respect to the possibility of adapting the method to the study of other lipid-dependent enzymes bound to membranes.

The role of tightly bound phospholipid in the activity of erythrocyte acetylcholinesterase

Acetylcholinesterase was extracted from bovine erythrocyte ghosts in a hypotonic (25 m0sm) phosphate buffer (pH 7.4) and solubilized with Lubrol WX (2 mg/ml). Solubilization resulted in a decrease of the partial specific volume of the acetylcholinesterase from 0.895 ml/g in the particulate fraction to 0.793 ml/g in the Lubrol WX solubilized fraction. Both the particulate fraction and the solubilized fractions gave Arrhenius plots with a discontinuity in the activation energy at 20°C. Phospholipase A2 or C did not abolish the discontinuity. Treatment with 1.77 M NaCl resulted in a linear Arrhenius plot of intermediate activation energy. The high salt treatment removed phospholipid not previously extracted by chloroform/methanol. The extracted phospholipid corresponded to cardiolipin on one and two dimensional silica gel chromatography. Addition of cardiolipin to a high salt-treated preparation of acetylcholinesterase restored the discontinuity in the Arrhenius plot. It is concluded that the physical state of cardiolipin, tightly bound to acetylcholinesterase by ionic interactions, modulates the catalytic activity of acetylcholinesterase.

Differential effects of detergents and dimethylsulfoxide on membrane prostaglandin E 1 and F 2α receptors

Pretreatment of membranes for 1 hr at 4° with up to 0.1% Triton X-100 (TX-100) and sodium desoxycholate (SDC), resulted in a greater loss of [ 3H] prostaglandin (PG)F 2α binding compared to E 1 binding. Lubrol WX (LWX) tended to cause a greater loss of [ 3H]PGF 2α than E 1 binding. However, the differential loss was not as marked as with TX-100 or SDC. Triton X-305 was relatively ineffective, but loss of [ 3H]PGE 1 binding was greater than for PGF 2α. Increasing concentrations of dimethylsulfoxide (DMSO) progressively inhibited PGF 2α binding without affecting PGE 1 binding. The detergent, but not DMSO, induced losses of membrane PG binding were due to solubilization of the receptors. Greater amounts of membrane protein and phospholipids were solubilized at detergent (TX-100 and SDC) concentrations that solubilized 100% of PGE 1 receptors compared to 100% solubilization of F 2α receptors. Neither the duration of preincubation nor the amount of membrane protein chosen were responsible for differential PGE 1 and F 2α receptor losses. These differential membrane PG receptor losses raise the possibility of differences in PGE 1 and F 2α receptors association with the membrane structure.

Steroid glucuronyltransferases of rat liver. Properties of oestrone and testosterone glucuronyltransferases and the effect of ovariectomy, castration and administration of steroids on the enzymes.

1. Microsomal preparations from rat liver, kidney and intestine were tested for UDP-glucuronyltransferase activity by using oestrone, oestradiol-17 beta, oestriol, testosterone, cortisol, cortisone, corticosterone, aldosterone, tetrahydrocortisol and tetrahydrocortisone as substrates. The microsomal preparation from the liver glucuronidated oestrone, oestradiol-17 beta and testosterone. 2. The specific activity of the enzyme was significantly higher in livers from female rats than in those from male rats. 3. Testosterone was actively glucuronidated by both sexes. Cortisol, cortisone, corticosterone, aldosterone, tetrahydrocortisol and tetrahydrocortisone were not glucuronidated by any of the three tissues. 4. The non-ionic detergent Lubrol WX activates liver microsomal UDP-glucuronyltransferase 2-3-fold with oestrone and testosterone as substrates. 5. Oestrone glucuronyltransferase was inhibited by oestradiol-17 beta, predominantly competitively and by testosterone non-competitively. Bilirubin was a non-competitive inhibitor of oestrone glucuronidation. p-Nitrophenol had no effect. 6. Oestrone glucuronyltransferase could not be stimulated by either acute or prolonged treatment of animals with phenobarbital, whereas a single dose of 3-methylcholanthrene led to a moderate stimulation. 7. Ovariectomy leads to a 56% decrease in oestrone glucuronyltransferase activity; administration of oestradiol-17 beta induces the enzyme to normal activity after 12 days, and after 15 days the activity is twice the control value. Actinomycin D and cycloheximide block the oestradiol-17 beta-induced increase in enzyme activity. 8. Castration has no effect on the activity of testosterone glucuronyltransferase, nor does administration of testosterone influence enzyme activity. The results provide strong evidence for the existence of multiple steroid glucuronyltransferases in the liver of the rat.

Properties of detergent-solubilized adenylate cyclase and gonadotropin receptors of testis and ovary

The relationship between solubilized hormone-binding sites and adenylate cyclase was examined in detergent extracts of particulate testis and ovarian fractions. Both basal and fluoride-stimulated activities of the particulate enzyme were markedly increased in the presence of detergents, and about 60% of the enzyme activity was recovered as the soluble form in the 300,000 g supernatant. Enhancement of adenylate cyclase activity was more marked with Lubrol PX and WX than with Triton X-100, and the highest recovery and activation of adenylate cyclase were obtained with 0.5% Lubrol PX. The particulate and solubilized testicular enzymes were more active in the presence of Mn 2+, and the detergent-extracted soluble ovarian cyclase showed a small and inconstant response to gonadotropin. Fractionation of Lubrol-solubilized testis and ovarian preparations on Sepharose 6B showed two peaks of free gonadotropin receptors. The binding activity eluted with K av of 0.32 corresponded to the receptor sites previously characterized in detergent-solubilized gonadal particles, and was coincident with the elution profile of adenylate cyclase activity. An additional peak of binding activity with K av of 0.56 was not accompanied by detectable adenylate cyclase activity. These observations suggest that the peak of larger molecular size could represent a gonadotropin receptor-cyclase complex, while the cyclase-free second peak could represent dissociated receptors or binding sites which were not coupled to adenylate cyclase.

Purification and properties of microsomal UDP-glucuronosyltransferase from rat liver.

p-Nitrophenol conjugating activity associated with liver microsomal UDP-glucuronosyltransferase (EC 2.4.1.17) was purified 150- to 200-fold from cell-free homogenates. The purification scheme included solubilization with the nonionic detergent Lubrol WX, anion exchange chromatography at pH 6.0 and 7.5, and affinity chromatography with UDP-hexanolamine Sepharose 4B. The enzyme purified as a phospholipid-protein complex and was shown to consist of a single polypeptide chain of molecular weight 59,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino acid analysis indicated approximately 531 mol of amino acids/59,000 g of enzyme and a molar ratio of nonpolar to polar residues of 1.08. During fractionation, the enzyme displayed instability with such steps as gel filtration, dialysis, or ultrafiltration of dilute samples; however, upon adsorption to ion exchange resins or storage in concentrated form, the enzyme was reasonably stable. The active lipoprotein complex showed both size and charge heterogeneity as judged by gel filtration and electrofocusing. Three forms of the enzyme resolved by isoelectric focusing had isoelectric points which averaged pH 6.68, 6.56, and 6.31. Polypeptide compositions of these electrophoretically distinct phospholipid protein complexes were indistinguishable on the basis of sodium dodecyl sulfate-polyacryl-amide gel electrophoresis, suggesting that the charge heterogeneity may be the result of differences in the phospholipid content of the lipoprotein complex.

Activation of brain adenosine triphosphatase activities with lubrol WX

1. 1. (Na + + K +-ATPase and Mg 2+-ATPase. in crude and partially purified (microsomal, NaI-treated) preparations derived from mouse brain cortex, were activated by the nonionic detergent, Lubrol WX. 2. 2. The ligand requirements and activatability of the crude and purified preparations were markedly different. 3. 3. In the crude enzyme preparation, both the total and Mg 2+-dependent ATPase activities were activated 2-fold with 0.01% Lubrol in a similar manner. 4. 4. Also, the activatable activities were similarly destroyed by heating at 50°C and could not be regenerated by cooling at 0°C. 5. 5. These observations suggest that the Lubrol activatable activity of the (Na + + K +)-ATPase and Mg 2+-ATPase is one and the same.

Resolution and reconstitution of Rhodospirillum rubrum pyridine dinucleotide transhydrogenase. II. Solubilization of the membrane-bound component.

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 that 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.

Trehalase transformation in silkworm midgut during metamorphosis

The particulate trehalase from silkworm larval midgut was effectively solubilized by repeated freezing and thawing, and by incubation with snake venom and non-ionic detergents (Lubrol PX and WX and Triton X-100). With solubilization the activity was enhanced and the activation behaviour was dependent upon the developmental stage of silkworms, being highest (up to about 3-fold) at the spinning stage.

Molecular mechanism of cardiotoxin action on axonal membranes

Cardiotoxin isolated from Naja mossambica mossambica selectively deactivates the sodium-potassium activated adenosine triphosphatase of axonal membranes. Tetrodotoxin binding and acetylcholinesterase activities are unaffected by cardiotoxin treatment. The details of association of cardiotoxin with the axonal membrane were studied by following the deactivation of the sodium-potassium activated adenosine triphosphatase and by direct binding measurements with a tritiated derivative of the native cardiotoxin. The maximal binding capacity of the membrane is 42-50 nmol of cardiotoxin/mg of membrane protein. The high amount of binding suggests association of the toxin with the lipid phase of the membrane. It has been shown that cardiotoxin first associates rapidly and reversibly to membrane lipids, then, in a second step, it induces a rearrangement of the membrane structure which produces and irreversible deactivation of the sodium-potassium activated adenosine triphosphatase. Solubilization of the membrane-bound ATPase with Lubrol WX gives an active enzyme species that is resistant to cardiotoxin-induced deactivation. Cardiotoxin binding to the membrane is prevented by high concentrations of Ca 2+ and dibucaine. Although cardiotoxins and neurotoxins of cobra venom have large sequence homologies, their mode of action on membranes is very different. The cardiotoxin seems to bind to the lipid phase of the axonal membrane and inhibits the sodium-potassium activated adenosine triphosphatase, whereas the neurotoxin associates with a protein receptor in the post-synaptic membrane and blocks acetylcholine transmission.

Sea urchin sperm guanylate cyclase. Purification and loss of cooperativity.

The Lubrol-dispersed guanylate cyclase from sea urchin sperm was purified and isolated essentially free of detergent by GTP affinity chromatography, DEAE-Sephadex chromatography, and gel filtration. After removal of the detergent, the enzyme remained in solution in the presence of 20% glycerol. The specific activity of the purified enzyme was about 12 mumol of guanosine 3':5'-monophosphate (cyclic GMP) formed - min-1 - mg of protein-1 at 30 degrees, an activity about 4600 times that of a soluble guanylate cyclase purified recently from Escherichia coli (Macchia V., Varrone, S., Weissbach, H., Miller, D.L., and Pastan, I. (1975) J. Biol. Chem. 250, 6214-6217). The cyclic GMP phosphodiesterase activity was negligible and adenosine 3':5'-monophosphate (cyclic AMP) phosphodiesterase was not detectable in the purified preparation. Cyclic AMP formation from ATP occurred at a rate of 0.002% of that of guanylate cyclase. In the absence of phosphodiesterase or guanosine triphosphatase inhibitors, 100% of the added GTP was converted to cyclic GMP. The purified enzyme required Mn2+ for maximum activity, the relative rates in the presence of Mg2+ or Ca2+ being less than 0.6% of the rates with Mn2+. The purified enzyme displayed classical Michaelis-Menten kinetics with respect to MnGTP (apparent Km is approximately equal to 170 muM) in contrast to the positively cooperative kinetic behavior displayed by the unpurified, detergent-dispersed, or particulate guanylate cyclase. The molecular weight of the purified enzyme was approximately 182,000 as estimated on Bio-Gel A-0.5m columns equilibrated in the presence or absence of 0.1 M NaCl. The unpurified, detergent-dispersed enzyme also migrated with an apparent molecular weight of 182,000 on columns equilibrated with 0.5% Lubrol WX and 0.1 M NaCl, but it migrated as a large aggregate (molecular weight is greater than 5 X 10(5)) on columns equilibrated in the absence of either the detergent of NaCl. After gel filtration, the unpurified, dispersed enzyme still yielded positive cooperative kinetic patterns as a function of MnGTP. Na dodecyl-SO4 gel electrophoresis of the enzyme after the DEAE-Sephadex or the gel filtration steps resulted in two major protein bands with estimated molecular weights of 118,000 and 75,000. Whether or not these protein bands represent the subunit molecular weights of guanylate cyclase is unknown at present.

An improved assay for steroid glucuronyltransferase in rat liver microsomes

A simple and economical method of assaying rat liver microsomal estrone and testosterone glucuronyltransferase activity has been developed. Liver microsomes were activated by pretreatment with Lubrol WX. The incubation was carried out at 37°C for 30 min and contained 30–600 μ m steroid, 1–2 m m UDP-glucuronic acid, 10 m m MgCl 2, and 80–150 μg of microsomal protein. Enzyme activities showed a maximum at pH 8.8 with Tris-HCl buffer. After incubation the unreacted substrate was quantitatively removed by a single extraction with dichloromethane. The glucuronide was estimated by counting an aliquot of the aqueous phase in a liquid scintillation counter. The variation coefficients with estrone and testosterone as substrates were 6.0 and 4.0%, respectively.

Sedimentation behavior of solubilized gonadotropin receptor from plasma membranes of bovine corpus luteum

The gonadotropin receptors associated with plasma membrane fractions were solubilized by detergents, including Triton X-100, Lubrol WX, Lubrol PX and sodium deoxycholate before and after equilibration with 125I-labelled human chorionic gonadotropin. The binding activity remained in solution even after centrifugation at 300 000 × g for 3 h. The solubilized gonadotropin receptor or gonadotropin receptor complex was characterized by gel filtration and sucrose density gradient centrifugation. Sucrose density gradient centrifugation of solubilized gonadotropin-receptor complex in the presence of Triton X-100 had a sedimentation coefficient of 6.5 S whereas the solubilized uncomplexed receptor had a sedimentation coefficient of 5.1 S. In the absence of the detergent, solubilized hormone receptor complex from plasma membrane fractions I and II sedimented with a apparent sedimentation coefficient of 6.6 S and 7.4 S, respectively. Similary, the free receptor also showed higher sedimentation profile with a apparent sedimentation coefficient of 6.7 S for fraction I and 7.2 S for fraction II. Treatment of plasma membranes with phospholipase A and C inhibited the binding of 125I-labelled human chorionic gonadotropin in a dose dependent manner, whereas phospholipase D was without any effect. Doses of 1.4 mI.U. of phospholipase A or 0.6 mI.U. of phospholipase C were required to produce 50% inhibition of the binding activity. These phospholipases had no effect on the performed 125I-labelled human chorionic gonadotropin-receptor complex nor on the sedimentation profile of solubilized gonadotropin receptor complex.