Effect of detergents on the reliability of a chemical assay for P-700

A chemical assay for P-700 was developed using 0.36 mM potassium ferricyanide as oxidant and 1.6 mM sodium ascorbate as reductant. The major difference from other chemical assays for P-700 is procedural. The method is designed to take advantage of the availability of microprocessor-linked spectrophotometers to obtain greater accuracy by minimizing the spectral changes due to irreversibly oxidized antenna chlorophyll molecules. The value measured for the P-700 concentration in a sample of chloroplasts was not changed by the presence of EDTA, Mg²⁺ or sucrose in the assayed solution. Similarly, half of the detergents tested (Triton X-100, Nonidet P-40, digitonin, Deriphat 160, Miranol S2M-SF and Miranol M2M) did not alter the value when added to the chloroplasts. The remainder of the detergents examined caused a significant decrease or increase in the value for P-700 content. Sodium dodecyl sulfate, of particular interest due to its widespread use, caused a doubling in the amount of apparent P-700. This effect may be due to this detergent and some others enabling an additional long wavelength form of chlorophyll, possibly an intermediary electron acceptor in Photosystem I, to be chemically oxidized and reduced under the assay conditions.     

Effect of detergents on the enzyme activities of the rat liver plasma membrane

The anionic detergents sodium dodecyl sulfate (SDS) and Alipal CO-433 and the non-ionic detergent Trition X-100 at concentrations of 0.02–0.10% cause a more rapid solubilization of phospholipid than proteins in isolated rat liver plasma membranes. All three detergents cause an increase in membrane turbidity at low detergent concentration (0.01–0.04%) but then decrease the turbidity at higher detergent concentration (0.04–0.10%). Each detergent gives a characteristic turbidity-detergent concentration profile which is pH dependent.The activities of the membrane-bound enzymes Mg²⁺ ATPase, 5′-nucleotidase and acid and aklaline phosphatase were influenced by each detergent to a different extent. Each enzyme gave a characteristic activity-detergent concentration profile. Mg²⁺ ATPase was inhibited by all detergents. 5′-Nucleotidase was stimulated by Triton and Alipal but inhibited by SDS. Alkaline phosphatase was stimulated by Alipal and SDS and not influenced by Triton. Acid phosphatase was stimulated by Triton and inhibited by Alipal and SDS. 56% of the total membrane-bound alkaline phosphatase and 23% of the total membrane-bound 5′-nucleotidase was solubilized in an active form by 0.06% and 0.05% SDS respectively.     

Highly purified sarcoplasmic reticulum vesicles are devoid of Ca2+-independent (‘basal’) ATPase activity

On solubilization with Triton X-100 of sarcoplasmic reticulum vesicles isolated by differential centrifugation, the Ca²⁺-ATPase is selectively extracted while approximately half of the initial Mg²⁺-, or ‘basal’, ATPase remains in the Triton X-100 insoluble residue. The insoluble fraction, which does not contain the 100 000 dalton polypeptide of the Ca²⁺-ATPase, contains high levels of cytochrome c oxidase. Furthermore, its Mg²⁺-ATPase activity is inhibited by specific inhibitors of mitochondrial ATPase, indicating that the ‘basal’ ATPase separated from the Ca²⁺-ATPase by detergent extraction originates from mitochondrial contaminants.To minimize mitochondrial contamination, sarcoplasmic reticulum vesicles were fractionated by sedimentation in discontinuous sucrose density gradients into four fractions: heavy, intermediate and light, comprising among them 90–95% of the initial sarcoplasmic reticulum protein, and a very light fraction, which contains high levels of Mg²⁺-ATPase. Only the heavy, intermediate and light fractions originate from sarcoplasmic reticulum; the very light fraction is of surface membrane origin. Each fraction of sarcoplasmic reticulum origin was incubated with calcium phosphate in the presence of ATP and the loaded fractions were separated from the unloaded fractions by sedimentation in discontinuous sucrose density gradients. It was found that vesicles from the intermediate fraction had, after loading, minimal amounts of mitochondrial and surface membrane contamination, and displayed little or no Ca²⁺-independent basal ATPase activity. This shows conclusively that the basal ATPase is not an intrinsic enzymatic activity of the sarcoplasmic reticulum membrane, but probably originates from variable amounts of mitochondrial and surface membrane contamination in sarcoplasmic reticulum preparations isolated by conventional procedures.     

The Two Km's for ATP of Corn-Root H+-ATPase and the Use of Glucose-6-Phosphate and Hexokinase as an ATP-Regenerating System

Plasma membrane vesicles derived from corn (Zea mays L.) roots retain a membrane-bound H+-ATPase that is able to form a H+ gradient across the vesicle membranes. The activity of this ATPase is enhanced 2- to 3-fold when Triton X-100 or lysophosphatidylcholine is added to the medium at a protein:detergent ratio of 2:1 (w/w). In the absence of detergent, the ATPase exhibits only one Km for ATP (0.1-0.2 mM), which is the same as for the pumping of H+. After the addition of either Triton X-100 or lysophosphatidylcholine, two Km's for ATP are detected, one in the range of 1 to 3 [mu]M and a second in the range of 0.1 to 0.2 mM. The Vmax of the second Km for ATP increases as the temperature of the assay medium is raised from 15[deg]C to 38[deg]C. The Arrhenius plot reveals a single break at 30[deg]C, both in the absence and in the presence of detergents. In the presence of Triton X-100 the H+-ATPase catalyzes the cleavage of glucose-6-phosphate when both hexokinase and ADP are included in the assay medium. There is no measurable cleavage when the apparent affinity for ATP of the H+-ATPase is not enhanced by Triton X-100 or when 1 mM glucose is included in the assay medium. These data indicate that when the high-affinity Km for ATP is unmasked with the use of detergent, the ATPase can use glucose-6-phosphate and hexokinase as an ATP-regenerating system.     

MAINTENANCE OF IONS,PROTEINS AND WATER IN LENS FIBER CELLS BEFORE AND AFTER TREATMENT WITH NON-IONIC DETERGENTS

If the plasma membrane and its associated transport proteins are solely responsible for maintenance of the asymmetric solute distribution then disruption of the plasma membrane would quickly lead to the symmetric distribution of all unattached inorganic ions between the cell and the extracellular environment. To test this hypothesis fresh pig lenses were incubated in Hanks ’ balanced salt solution in either absence or presence of non-ionic detergents (0.2 % Triton X-100 or 0.2 % Brij 58). Both detergents caused permeabilization of every lens fiber cell as shown by electron microscopy. The flux kinetics of K⁺, Mg^{2 +}, Na⁺, Ca^{2 +}, water and protein out of and into the permeabilized lens fiber cells was measured. Triton X-100 caused a faster flux rate of all solutes than did Brij 58. The Triton X-100 induced flux of solutes and water was associated with a decrease in lens ATP. Incubation of untreated lenses in solutions of different osmotic pressures at 0 °C demonstrated that the major fraction of lens water was osmotically unresponsive. Thus the asymmetric distribution of solutes in lens fiber cells is dependent on an intact plasma membrane and on a co-operative ATP-dependent association between K⁺, Mg^{2 +}, water and cytomatrix proteins.     

A comparative study of the effect of various detergents on the structure and function of sarcoplasmic reticulum vesicles

Summary The effects produced by the detergents Triton X-100, sodium dodecylsulphate and sodium cholate on sarcoplasmic reticulum vesicles have been comparatively studied. In all cases, maximal effects are found 5 min after detergent addition. Triton X-100 and SDS are approximately ten times more effective than cholate in protein and phospholipid solubilization. Both Triton X-100 and SDS maintain Ca⁺⁺ accumulation in SR vesicles at detergent concentrations below 10^–3 M; higher concentrations cause a strong inhibition. On the other hand, cholate produces a gradual inhibition of Ca⁺⁺ accumulation in the concentration range between 10^–4 M and 2.5 × 10^–2 M. Triton X-100 and SDS produce a gradual solubilization of the specific Ca⁺⁺-ATPase activity up to a 10^–3 M detergent concentration, above which a strong inactivation occurs, while the enzyme solubilization increases with the presence of cholate in the whole concentration range under study. The different behaviour of sodium cholate, when compared to SDS or Triton X-100, is discussed in relation to the surfactant molecular structures. The possibility of membrane lysis and reassembly in the presence of some detergents is also considered.Abbreviations SR sarcoplasmic reticulum - SDS sodium dodecylsulphate - DTT dithiothreitol - EGTA ethyleneglycoltetraacetate - PEP phosphoenolpyruvate     

Sensitivity of the (Na+ + K+)-ATPase to state-dependent inhibitors: Effects of digitonin and triton X-100

Treatment of a purified (Na⁺ + K⁺)-ATPase preparation from dog kidney with digitonin reduced enzymatic activity, with the (Na⁺ + K⁺)-ATPase reaction inhibited more than the K⁺-phosphatase reaction that is also catalyzed by this enzyme. Under the usual assay conditions oligomycin inhibits the (Na⁺ + K⁺)-ATPase reaction but not the K⁺-phosphatase reaction; however, treatment with digitonin made the K⁺-phosphatase reaction almost as sensitive to oligomycin as the (Na⁺ + K⁺)-ATPase reaction. The non-ionic detergents, Triton X-100, Lubrol WX and Tween 20, also conferred sensitivity to oligomycin on the K⁺-phosphatase reaction (in the absence of oligomycin all these detergents, unlike digitonin, inhibited the K⁺-phosphatase reaction more than the (Na⁺ + K⁺)-ATPase reaction). Both digitonin and Triton markedly increased the K_0.5 for K⁺ as activator of the K⁺-phosphatase reaction, with little effect on the K_0.5 for K⁺ as activator of the (Na⁺ + K⁺)-ATPase reaction. In contrast, increasing the K_0.5 for K⁺ in the K⁺-phosphatase reaction by treatment of the enzyme with acetic anhydride did not confer sensitivity to oligomycin. Both digitonin and Triton also increased the inhibition of the K⁺-phosphatase reaction by ATP and decreased the inhibition by inorganic phosphate and vanadate. These observations are interpreted as digitonin and Triton favoring the E₁ conformational state of the enzyme (manifested by sensitivity to oligomycin and a greater affinity for ATP at the low-affinity substrate sites), as opposed to the E₂ state (manifested by insensitivity to oligomycin, greater sensitivity to phosphate and vanadate, and a lower K_0.5 for K⁺ in the K⁺-phosphatase reaction). In addition, digitonin blocked activation of the phosphatase reaction by Na⁺ plus CTP. This effect is consistent with digitonin dissociating the catalytic subunits of the enzyme, the interaction of which may be essential for activation by Na⁺ plus nucleotide.     

Reconstitution of the sarcoplasmic reticulum Ca(2+)-ATPase: mechanisms of membrane protein insertion into liposomes during reconstitution procedures involving the use of detergents.

The Ca(2+)-ATPase from skeletal muscle sarcoplasmic reticulum was reconstituted into sealed phospholipid vesicles using the method recently developed for bacteriorhodopsin (Rigaud, J.L., Paternostre, M.T. and Bluzat, A. (1988) Biochemistry 27, 2677-2688). Liposomes prepared by reverse-phase evaporation were treated with various amounts of Triton X-100, octyl glucoside, sodium cholate or dodecyl octa(oxyethylene) glycol ether (C12E8) and protein incorporation was studied at each step of the liposome solubilization process by each of these detergents. After detergent removal by SM-2 Bio-Beads the resulting vesicles were analyzed with respect to protein incorporation by freeze-fracture electron microscopy, sucrose density gradients and Ca2+ pumping measurements. The nature of the detergent used for reconstitution proved to be important for determining the mechanism of protein insertion. With octyl glucoside, direct incorporation of Ca(2+)-ATPase into preformed liposomes destabilized by saturating levels of this detergent was observed and gave proteoliposomes homogeneous in regard to protein distribution. With the other detergents, optimal Ca(2+)-ATPase pumping activities were obtained when starting from Ca(2+)-ATPase/detergent/phospholipid micellar solutions. However, the homogeneity of the resulting recombinants was shown to be dependent upon the detergent used and in the presence of Triton X-100 or C12E8 different populations were clearly evidenced. It was further demonstrated that the rate of detergent removal drastically influenced the composition of resulting proteoliposomes: upon slow detergent removal from samples solubilized with Triton X-100 or C12E8, Ca(2+)-ATPase was found seggregated and/or aggregated in very few liposomes while upon rapid detergent removal compositionally homogeneous proteoliposomes were obtained with high Ca2+ pumping activities. The reconstitution process was further analyzed by centrifugation experiments and the results demonstrated that the different mechanisms of reconstitution were driven predominantly by the tendency for self-aggregation of the Ca(2+)-ATPase. A model for Ca(2+)-ATPase reconstitution was proposed which accounted for all our results. In summary, the advantage of the systematic studies reported in this paper was to allow a rapid and easy determination of the experimental conditions for optimal detergent-mediated reconstitution of Ca(2+)-ATPase. Proteoliposomes prepared by the present simple method exhibited the highest Ca2+ pumping activities reported to date in Ca(2+)-ATPase reconstitution experiments performed in the absence of Ca2+ precipitating agents.     

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

The properties of Ca²⁺-ATPase purified and reconstituted from bovine pulmonary artery smooth muscle microsomes {enriched with endoplasmic reticulum (ER)} were studied using the detergents 1,2-diheptanoyl-sn-phosphatidylcholine (DHPC), poly(oxy-ethylene)8-lauryl ether (C₁₂E₈) and Triton X-100 as the solubilizing agents. Solubilization with DHPC consistently gave higher yields of purified Ca²⁺-ATPase with a greater specific activity than solubilization with C₁₂E₈ or Triton X-100. DHPC was determined to be superior to C₁₂E₈; while that the C₁₂E₈ was determined to be better than Triton X-100 in active enzyme yields and specific activity. DHPC solubilized and purified Ca²⁺-ATPase retained the E1Ca−E1*Ca conformational transition as that observed for native microsomes; whereas the C₁₂E₈ and Triton X-100 solubilized preparations did not fully retain this transition. The coupling of Ca²⁺ transported to ATP hydrolyzed in the DHPC purified enzyme reconstituted in liposomes was similar to that of the native micosomes, whereas that the coupling was much lower for the C₁₂E₈ and Triton X-100 purified enzyme reconstituted in liposomes. The specific activity of Ca²⁺-ATPase reconstituted into dioleoyl-phosphatidylcholine (DOPC) vesicles with DHPC was 2.5-fold and 3-fold greater than that achieved with C₁₂E₈ and Triton X-100, respectively. Addition of the protonophore, FCCP caused a marked increase in Ca²⁺ uptake in the reconstituted proteoliposomes compared with the untreated liposomes. Circular dichroism analysis of the three detergents solubilized and purified enzyme preparations showed that the increased negative ellipticity at 223 nm is well correlated with decreased specific activity. It, therefore, appears that the DHPC purified Ca²⁺-ATPase retained more organized and native secondary conformation compared to C₁₂E₈ and Triton X-100 solubilized and purified preparations. The size distribution of the reconstituted liposomes measured by quasi-elastic light scattering indicated that DHPC preparation has nearly similar size to that of the native microsomal vesicles whereas C₁₂E₈ and Triton X-100 preparations have to some extent smaller size. These studies suggest that the Ca²⁺-ATPase solubilized, purified and reconstituted with DHPC is superior to that obtained with C₁₂E₈ and Triton X-100 in many ways, which is suitable for detailed studies on the mechanism of ion transport and the role of protein–lipid interactions in the function of the membrane-bound enzyme.     

Structural changes in (Na+ + K+)-ATPase accompanying detergent inactivation

Structural changes in the purified (Na+ + K+)-ATPase accompanying detergent inactivation were investigated by monitoring changes in light scattering, intrinsic protein fluorescence, and tryptophan to beta-parinaric acid fluorescence resonance energy transfer. Two phases of inactivation were observed using the non-ionic detergents, digitonin, Lubrol WX and Triton X-100. The rapid phase involves detergent monomer insertion but little change in protein structure or little displacement of closely associated lipids as judged by intrinsic protein fluorescence and fluorescence resonance energy transfer. Lubrol WX and Triton X-100 also caused membrane fragmentation during the rapid phase. The slower phase of inactivation results in a completely inactive enzyme in a particle of 400 000 daltons with 20 mol/mol of associated phospholipid. Fluorescence changes during the course of the slow phase indicate some dissociation of protein-associated lipids and an accompanying protein conformational change. It is concluded that non-parallel inhibition of (Na+ + K+)-ATPase and p-nitrophenylphosphate activity by digitonin (which occurs during the rapid phase of inactivation) is unlikey to require a change in the oligomeric state of the enzyme. It is also concluded that at least 20 mol/mol of tightly associated lipid are necessary for either (Na+ + K+)-ATPase or p-nitrophenylphosphatase activity and that the rate-limiting step in the slow inactivation phase involves dissociation of an essential lipid.     

Nonionic detergents increase the stoichiometry of ligand binding to the rat hepatic galactosyl receptor

When digitonin is used to expose intracellular galactosyl (Gal) receptors in isolated rat hepatocytes, only about half of the binding activity for 125I-asialoorosomucoid (ASOR) is found as compared to cells solubilized with Triton X-100. The increased ligand binding in the presence of detergent is not due to a decrease in Kd but could be due either to an increase in the number of ASORs bound per receptor or to exposure of additional receptors. Several experiments support the former explanation. No additional activity is exposed even when 80% of the total cell protein is solubilized with 0.4% digitonin. It is, therefore, unlikely that receptors are in intracellular compartments not permeabilized by digitonin and inaccessible to 125I-ASOR. Digitonin-treated cells are not solubilized by Triton X-100 if they are first treated with glutaraldehyde under conditions that retain specific binding activity. 125I-ASOR binding to these permeabilized/fixed cells increases about 2-fold in the presence of Triton X-100 and a variety of other detergents (e.g., Triton X-114, Nonidet P-40, Brij-58, and octyl glucoside) but not with the Tween series, saponin, or other detergents. When these fixed cells are washed to remove detergent, 125I-ASOR binding decreases almost to the initial level. Affinity-purified Gal receptor linked to Sepharose 4B binds approximately twice as much 125I-ASOR in the presence of Triton X-100 as in its absence. The results suggest that the increase in Gal receptor activity in the presence of nonionic detergents is due to an increase in the valency of the receptor rather than to exposure of additional receptors.     

Adenosine triphosphatase activity in the neural lobe of the bovine pituitary gland

1. Homogenates of neural lobes of bovine pituitary glands were fractionated by differential and density-gradient ultracentrifugation and the distribution of adenosine triphosphatase (ATPase) activity was studied. It was shown that all the activity was membrane-bound. 2. On the basis of ionic requirements the ATPase activity was grouped into three categories: (a) Mg²⁺-dependent, (b) Ca²⁺-dependent and (c) Mg²⁺+Na⁺+K⁺-dependent (ouabain-sensitive) ATPases. The activity in the absence of bivalent cations was negligible. The ratio between the activities of the three ATPases varied between the different subcellular fractions. 3. Preincubation of the subcellular fractions with deoxycholate increased the activity of the Mg²⁺+Na⁺+K⁺-dependent enzyme, whereas the Mg²⁺- and Ca²⁺-activated ATPases were either unaffected or slightly inhibited. Triton X-100 solubilized the Mg²⁺- and Ca²⁺-ATPases; however, the activity of the Mg²⁺+Na⁺+K⁺-ATPase was abolished by the concentration of Triton X-100 used. 4. All the subfractions displayed unspecific nucleotide triphosphatase activity towards GTP, ITP and UTP. These substrates inhibited the hydrolysis of ATP by all three ATPases. ADP also inhibited the ATPases. 5. Polyacrylamide-gel electrophoresis of extracts containing the Mg²⁺- and Ca²⁺-dependent ATPase activity solubilized by Triton X-100 revealed the presence of two enzymes; one activated by either Mg²⁺ or Ca²⁺ and the other activated only by Ca²⁺. 6. In sucrose density gradients the distribution of vasopressin was different from that of all three types of ATPases. It is therefore suggested that the neurosecretory granules do not possess ATPase activity.     

Demonstration of a high affinity Ca2+ ATPase in rat liver plasma membranes

Rat liver plasma membranes contained a high affinity Ca²⁺-ATPase which had an apparent half saturation constant of 0.2 μM for calcium. The Ca²⁺-ATPase was not stimulated by adding magnesium and/or calmodulin. Conversely, the addition of these substances diminished the calcium-stimulation of the ATPase. Orthovanadate (7 nM-2 mM), mitochondrial ATPase blockers (NaN₃, KCN, dicyclohexylcarbodiimide), Na⁺, K⁺ and ouabain had no effect on the ATPase activity. The ATPase was separated from nonspecific divalent cation stimulatable ATPase (Mg²⁺-ATPase) by solubilization with Triton X-100 followed by a Sephadex G-200 column chromatography and showed an apparent molecular weight of 200,000.     

The effects of cations on the structure and photochemistry of the photosystem II core complex

We have studied the effects of cations and detergents on the structure (molecular weight) and photochemistry of Triton X-100 Photosystem II subchloroplast particles (TSF-IIa). The effect of Mg²⁺ ions on activity depended on the Triton X-100 content of the preparation. If the residual Triton X-100 was not removed prior to assay, MgCl₂ increased the rate of electron transport, acting at a site on the reducing side of Photosystem II. Lowering the pH also increased the rate of electron transport. If the Triton X-100 was removed from the particles, both MgCl₂ and NaCl caused a decrease in the rate of electron transport. Addition of Triton X-100 caused a reversible decrease in the number of active Photosystem II reaction centers. Both cations and Triton X-100 had a profound effect on the molecular weight of the Photosystem II particles as determined by gel filtration. At 20 °C, addition of 0.05% Triton X-100 decreased the molecular weight from a high value (≥800,000) to 250,000. At 4 °C, addition of 1 mm MgCl₂ or 100 mm NaCl increased the molecular weight of the complex. In the absence of these salts 67% of the protein eluted with a molecular weight of 460,000 (the rest was >800,000-in the void volume). In the presence of these salts all of the material had a molecular weight of ≥800,000. A similar effect was observed when the pH was lowered from 8 to 6. Further work is needed to determine whether there is a correlation between the changes in molecular weight and activity.     

Interaction of Triton X-100 with particulate cardiac guanylate cyclase: comparison between Mg2+- and Mn2+-supported enzyme activities in particulate. detergent-solubilized and detergent-insoluble fractions

Guanylate cyclase activity was determined in a 1000g particulate fraction derived from rabbit heart homogenates using Mg²⁺ or Mn²⁺ as sole cation in the presence and absence of Triton X-100. With Mg²⁺, very little guanylate cyclase activity could be detected in the original particulate fraction assayed with or without Triton, or in the particulate fraction treated with varying concentrations of Triton (detergent-treated mixture) prior to enzyme assay. However, the detergent-solubilized supernatants as well as the detergent-insoluble residues (pellets) derived from detergent-treated mixtures possessed appreciable Mg²⁺-supported enzyme activity. With Mn²⁺, significant enzyme activity was detectable in the original particulate fraction assayed without Triton. Much higher activity was seen in particulate fraction assayed with Triton and in detergent-treated mixtures; the supernatants but not the pellets derived from detergent-treated mixtures possessed even greater activity. The sum of enzyme activity in pellet and supernatant fractions greatly exceeded that of the mixture. When the pellets and supernatants derived from detergenttreated mixtures were recombined, measured enzyme activities were similar to those of the original mixture. With Mg²⁺ or Mn²⁺, the specific activity of guanylate cyclase in pellet and supernatant fractions varied considerably depending on the concentration of Triton used for treatment of the particulate fraction; treatment with low concentrations of Triton (0.2–0.7 μmol/mg protein) gave supernatants showing high activity whereas treatment with relatively greater concentrations of the detergent (>0.7 μmol/mg protein) gave pellets showing high activity. The relative distribution of guanylate cyclase in pellet and supernatant fractions expressed as a function of Triton concentration during treatment (of the particulate fraction) showed that 50 to 80% of the recovered enzyme activity remained in supernatants at low detergent concentrations whereas 50 to 80% of the recovered activity resided in the pellets at higher detergent concentrations. Inclusion of excess Triton in the enzyme assay medium did not alter the specific activity profiles and the relative distribution patterns of the cyclase in pellet versus supernatant fractions. The results demonstrate the inherent potential of cardiac particulate guanylate cyclase to utilize Mg²⁺ in catalyzing the synthesis of cyclic GMP. However, it appears that some factor(s) endogenous to the cardiac particulate fraction severely impairs the expression of Mg²⁺-dependent activity; Mn²⁺-dependent activity is also affected by such factor(s) but apparently less severely. Further, the results suggest that previously reported activities of cardiac particulate guanylate cyclase, despite being assayed with Mn²⁺ and in the presence of Triton X-100, represent underestimation of what otherwise appears to be a highly active enzyme system capable of utilizing physiologically relevant divalent cation such as Mg²⁺.     

Effect of triton X-100 treatment on the lipid composition,biochemical and functional properties of hake (Merluccius hubbsi) myofibrils purified from pre- and post-spawned fish

• 1.1. The effects of Triton X-100 treatment on the lipid contents and functional properties of hake myofibrils from pre- and post-spawned fish were investigated.
• 2.2. Differences in lipids, biochemical and functional properties of hake myofibrils related to the gonadal condition of fish were observed.
• 3.3. Triton X-100 treatment removed 65% of polar lipids in myofibrils from pre-spawned fish and only 10% in myofibrils from post-spawned fish.
• 4.4. Triton X-100 increased the Hill coefficient to 1.5 in an allosteric type of reaction for the myofibrillar Mg²⁺-ATPase from pre-spawned hake.
• 5.5. The detergent effect observed on the contraction response was greater in myofibrils from prespawned fish than in post-spawned fish.

     

Diacylglycerol kinase from suspension cultured plant cells : purification and properties

Diacylglycerol kinase (ATP:1,2-diacylglycerol 3-phosphotransferase, EC 2.7.1.107) from suspension-cultured Catharanthus roseus cells was extracted from a membrane fraction with 0.6% Triton X-100 and 150 millimolar NaCl and was purified about 900-fold by DEAE-cellulose, blue Sepharose, gel permeation, and phenyl-Sepharose chromatography. The enzyme is obviously membrane bound as activity in the cytosol could not be detected. In the presence of detergents such as Triton X-100 (3-[3-cholamidopropyl]dimethylamino)-1-propanesulfonate (Chaps), or deoxycholate, a molecular weight of about 250,000 was determined by gel filtration. In glycerol density gradients, the enzyme sedimented slightly more slowly than bovine serum albumin, indicating a molecular weight of less than 68,000. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis enzyme activity could be assigned to a protein of 51,000 daltons. As found previously for bacterial and animal diacylglycerol kinases, the purified enzyme was completely devoid of activity without the addition of phospholipids or deoxycholate. Cardiolipin was found to be most effective, whereas higher amounts of detergent were inhibitory. The enzyme needs divalent cations for activity, with Mg²⁺ ions being the most effective. Apparent K_m values for ATP and diacylglycerol were determined as 100 and 250 micromolar, respectively.     

Modulating restriction endonuclease activities and specificities using neutral detergents

It is well known that type II restriction enzyme activities and specificities can be modulated by altering solution conditions. The addition of co-solvents such as dimethyl sulfoxide (DMSO), alcohols and polyols can promote star activity, which is the cleavage of non-cognate sequences. While neutral detergents are often used to control protein aggregation, little is known about the effect of neutral detergents on restriction enzyme activities and specificities. We report here that BamHI, BglI, BglII, EcoRI, EcoRV, HindIII, MluI, PvuII, SalI and XhoI restriction endonucleases are remarkably tolerant of high concentrations of neutral detergents Triton X-100, CHAPS and octyl glucoside. In most cases, lambda DNA cleavage rates were comparable to those observed in the absence of detergent. Indeed, the specific activities of SalI and XhoI were appreciably increased in the presence of Triton X-100. For all enzymes active in the presence of detergents, sequence specificity toward lambda DNA was not compromised. Assays of star cleavage of pUC18 by EcoRI, PvuII and BamHI endonucleases in equimolar concentrations of Triton X-100 and sucrose revealed reduced star activity in the detergent relative to the sucrose co-solvent. Interestingly, under star activity-promoting conditions, PvuII endonuclease displayed greater fidelity in Triton X-100 than in conventional buffer. Taken altogether, these results suggest that in some cases, neutral detergents can be used to manipulate restriction endonuclease reaction rates and specificities.     

Role of phospholipid and protein-protein associations in activation and stabilization of soluble Ca2+-ATPase of sarcoplasmic reticulum

The effect of increasing concentrations of the nonionic detergent Triton X-100 on catalytic activity, stability, phospholipid content, and aggregational state of solubilized Ca2+ ion activated adenosinetriphosphatase (Ca2+-ATPase) of sarcoplasmic reticulum has been investigated. Increasing concentrations of Triton X-100 in the range 0.2-0.6% (w/v) inhibited ATP hydrolysis and p-nitrophenyl phosphate hydrolysis in parallel to the extent of 50% and 95%, respectively. Inactivation of p-nitrophenyl phosphate hydrolysis by preincubation in excess ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) at 25 degrees C was monophasic and first order at all concentrations of Triton X-100. The rate constant for inactivation increased sharply in the range 0.1-0.6% Triton X-100. At higher concentrations, the increase was less marked. Protein-protein associations of the solubilized ATPase were assessed by glutaraldehyde cross-linking and by ultracentrifugation in sucrose gradients. Both methods indicated a decrease in these associations in the 0.1-0.5% range. Cross-linking studies established that above 0.5% Triton X-100 the enzyme is greater than 90% monomeric. The amount of phospholipid associated with the ATPase, recovered from sucrose gradients, decreased from about 50 mol of phospholipid/mol of ATPase at 0.1% Triton X-100 to about 3 mol of phospholipid/mol of ATPase at 0.5% and higher concentrations. Monomeric ATPase and aggregated ATPase isolated from equilibrium mixtures of these components had similar phospholipid/protein ratios. The results indicated that with increasing Triton X-100 concentrations, inhibition of catalysis, destabilization, loss of protein-protein associations, and loss of phospholipid occur concurrently.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of (Mg,Ca)-ATPase activity in rat pancreatic plasma membranes.

1. Pancreatic plasma membranes containing a high adenylate cyclase activity and a low contamination by cytochrome c oxidase were isolated from the rat by sucrose density centrifugation. The preparation contained an (Mg,Ca)-ATPase of high activity with the following characteristics. 2. The ATPase activity was shown to have two apparent Km values for Mg-ATP (0.24 +/- 0.09 mM and 1.15 +/- 0.21 mM) and two apparent Km values for Ca-ATP (0.14 +/- 0.09 mM and 0.68 +/- 0.10 mM). Mg-GTP and Ca-GTP were also hydrolysed by the preparation. The phase transition temperature was 19.3 +/- 1.0 degrees C for the Mg-ATPase and 22.6 +/- 1.1 degrees C for the Ca-ATPase activities. 3. Three lines of evidence suggest that Mg-ATP and Ca-ATP were substrates for the same enzyme: Mg-dependent and Ca-dependent activities were not additive; the two activities showed the same pH optimum at 8.0; and the nonionic detergents Triton X-100, Triton X-305, Triton N-101, Lubrol P 12 A, and digitonin, produced a parallel solubilization of the two activities. 4. Enzyme activities were insensitive to potassium, sodium, ouabain, pancreozymin, carbamoyl-choline, secretin, concanavalin A, wheat germ agglutinin, and soybean lectin.