Phosphatidylserine decarboxylase from Clostridium butyricum

Phosphatidylserine decarboxylase activity has been characterized in membrane preparations from Clostridium butyricum ATCC 19398. A particulate fraction was shown to catalyze the formation of phosphatidylethanolamine and plasmenylethanolamine when vesicles containing phosphatidylserine and plasmenylserine were used as substrate. No plasmenylethanolamine was formed when phosphatidylserine alone was used as substrate. The activity with phosphatidylserine was activated by divalent cations and was optimal under anaerobic conditions. Ionic detergents inhibited phosphatidylethanolamine formation strongly and nonionic detergents inhibited partially. In the presence of Triton X-100, phosphate from [32P]phosphatidylserine appeared in three unidentified lipid products, in addition to phosphatidylethanolamine. The formation of these products was time- and Triton X-100 concentration-dependent. Hydroxylamine inhibited phosphatidylserine decarboxylase, but did not prevent the reactions stimulated by Triton X-100.     

Partial purification and properties of mammalian phosphatidylglycerophosphatase

The phosphatidylglycerophosphatase (EC 3.1.3.27) activity of rat liver mitochondria was investigated by assaying the conversion of 14C-labelled phosphatidylglycerophosphate to phosphatidylglycerol. The activity was associated with a mitochondrial membrane fraction and could not be released into solution employing techniques applicable to a peripheral membrane protein. The enzyme was partially purified by sonication, pH 5.0 precipitation, and gel filtration. Various ionic and nonionic detergents as well as numerous divalent cations inhibited the phosphatase. The enzyme displayed a high affinity for phosphatidylglycerophosphate.     

Membrane attachment of Slr0006 in Synechocystis sp. PCC 6803 is determined by divalent ions

Slr0006 is one of the Synechocystis sp. PCC 6803 proteins strongly induced under carbon limiting conditions. Slr0006 has no predicted transmembrane helices or signal peptide sequence, yet it was exclusively recovered in the membrane fraction of Synechocystis, when the cells were broken in isolation buffers which contain divalent cations and are generally used for photosynthesis studies. Even subsequent washing of the membranes with high salt or various detergents did not release Slr0006, indicating strong binding of the Slr0006 protein to the membranes. Further, DNAse or RNAse treatment did not disturb the tight binding of Slr0006 protein to the membranes. Nevertheless, when the cells were broken in the absence of divalent cations, Slr0006 remained completely soluble. Binding of the Slr0006 to the membrane could not be properly reconstituted if the cations were added after breaking the cells in the absence of divalent ions. This unusual phenomenon has to be considered in identification and localization of other yet uncharacterized cyanobacterial proteins.     

CTP-dependent lipid kinases of yeast

Membrane fractions from yeast Saccharomyces cerevisiae catalyzed a transfer of gamma-phosphate from [gamma-32P]CTP into membranous lipids. Phosphorylated compounds were identified as phosphatidic acid and dolichyl phosphate (DolP). The membrane fraction also catalyzed phosphorylation of the exogenous dolichol. The activity of the phosphorylating enzymes could be modified by the yeast growing conditions; i.e., the enzyme from yeast grown aerobically favored the synthesis of phosphatidate over dolichyl phosphate in the ratio of 3:1, whereas the membrane fraction from anaerobically grown yeast synthesized PA and DolP in the ratio of 0.5:1. The activity of the phosphorylating enzymes could also be modified by divalent cations and the concentration of detergents. Phosphorylation of lipids does not occur in the presence of [gamma-32P]ATP and is not influenced by the presence of UTP or GTP. This result points to the specific role of CTP as a gamma-phosphate donor for the synthesis of phosphatidate and dolichyl phosphates in the yeast system.     

Oxyethylene chain-cation complexation: nonionic polyoxyethylene detergents attain a positive charge and demonstrate electrostatic head group interactions.

We report literature data indicating that the polyoxyethylene chain of polyoxyethylene detergents attracts cations via dipole-ion interactions thereby attaining a positive charge character. This implies that nonionic polyoxyethylene detergents like Triton X-100 and C12E8 may interact electrostatically with phospholipid head groups. We describe how a positive charge character of Triton X-100 and C12E8 can explain their hitherto mysterious stomatocytogenic shape altering effect in human erythrocytes.     

Studies on the quaternary structure of class I major histocompatibility complex antigens. Effect of different agents on the interaction between subunits

The effect of temperature, urea, guanidine HCl, ionic and nonionic detergents, organic solvents, chaotropic salts, pH, and divalent cations has been investigated on purified human histocompatibility antigens solubilized by papain (HLApap) or solubilized by sodium cholate (HLAchol). HLApap and HLAchol are fairly stable proteins to agents acting predominantly on hydrogen bonds (temperature, urea) or hydrophobic forces (ionic and nonionic detergents). However, agents which affect ionic interactions (pH, salts, divalent cations) dissociate the molecules into subunits. A single binding site for beta 2-microglobulin with an affinity constant of 1.0 X 10(7) M-1 was found for the alpha chain of HLAchol. The dissociated subunits can be separated by affinity chromatography on Sepharose-rabbit IgG anti-human beta 2-microglobulin and reassociate in vitro when incubated under the appropriate conditions. The results point toward an important role of ionic interactions between subunits in the stabilization of the quaternary structure of HLA.     

The influence of detergents on the availability of pertussis toxin substrates

Pertussis toxin-dependent ADP-ribosylation of rat heart and human mononuclear leukocyte membranes was found to be markedly enhanced in the presence of detergents. The order of potency for this effect of detergents was Triton X-100 approximately Lubrol PX greater than digitonin much greater than cholate greater than 3-[(3-cholamidopropyl)dimethylammonia]propanesulfonic acid. Exposure of membranes to increasing concentrations of detergents increased the proportion of pertussis toxin substrate demonstrable in the supernatant fraction whereas the substrate remaining in the pellet fraction demonstrated a complicated relationship with the concentration of detergent. In complementary experiments, it was found that immunochemical detection of G proteins in the pellet fraction from suspensions previously incubated with a maximal concentration of detergent revealed a reduced presence of G proteins with a concomitant increase in the concentration of G proteins in the supernatant fraction; this situation was not observed at submaximal concentrations of detergent during the preincubation of myocardial membranes. The results suggest that the detergent-mediated enhancement of pertussis toxin's action to ADP-ribosylate susceptible G proteins is a complicated process that includes concentration-dependent creation of conditions favorable to the actions of the toxin as well as solubilization of the substrates for the toxin.     

Solubilization of thyroid peroxidase by nonionic detergents.

We have examined the ability of nonionic detergents to solubilize thyroid peroxidase from a porcine thyroid particulate fraction, as measured by the release of peroxidase activity into the supernatant fraction after centrifugation at 105,000 X g for 1 hour and the retardation of the supernatant peroxidase of Sepharose 6B. The parameters of peroxidase solubilization by Triton X-100 have been investigated in detail. Under optimum conditions, 60 to 95% of the thryoid peroxidase and about 50% of the total protein is released into the 105,000 X g, 1-hour supernatant. Under the optimum conditions established with Triton X-100, a series of Brij detergents of different chemical structure were equally effective in releasing peroxidase and protein. The protein patterns of the supernatants obtained with these detergents were similar on sodium dodecyl sulfate-polyacrylamide electrophoresis gels, suggesting that the detergents studied release similar membrane proteins. The Triton X-100 and Brij 58 supernatants were chromatographed separately on Sepharose 6B equilibrated with 0.1% Triton X-100 or Brij 58, respectively. In both cases, 75 to 80% of the peroxidase activity was retarded, thereby indicating that the nonionic detergents effect solubilization of the peroxidase rather than dispersal of nonsedimentable membrane fragments. These studies report the first successful solubilization of thyroid peroxidase by nonionic detergents. Together with previous evidence from our laboratory, these experiments indicate that thyroid peroxidase is an integral membrane protein.     

Effects of detergents on ribosomal precursor subunits of Bacillus megaterium.

Cell extracts prepared by osmotic lysis of protoplasts were analyzed by sucrose gradient sedimentation. In the absence of detergents, ribosomal precursor particles were found in a gradient fraction which sedimented faster than mature 50S subunits and in two other fractions coincident with mature 50S and 30S ribosomal subunits. Phospholipid, an indicator of membrane, was shown to be associated with only the fastest-sedimenting ribosomal precursor particle fraction. After the extracts were treated with detergents, all phospholipid was found at the top of the gradients. Brij 58, Triton X-100, and Nonidet P-40 did not cause a change in the sedimentation values of precursors; however, the detergents deoxycholate or LOC (Amway Corp.) disrupted the fastest-sedimenting precursor and converted the ribosomal precursor subunits which sedimented at the 50S and 30S positions to five different classes of more slowly sedimenting particles. Earlier reports on the in vivo assembly of ribosomal subunits have shown that several stages of ribosomal precursor subunits exist, and, in the presence of the detergents deoxycholate and LOC, which had been used to prepare cell extracts, the precursors sedimented more slowly. Our data are consistent with the hypothesis that those detergents selectively modify the structure of ribosomal precursors and lend further support to the hypothesis that the in vivo ribosomal precursor subunits have 50S and 30S sedimentation values. In addition, these data support the idea that the ribosomal precursor particles found in the fast-sedimenting fraction may constitute a unique precursor fraction.     

alpha-D-Mannosidase of Saccharomyces cerevisiae. Characterization and modulation of activity.

A unique and interesting alpha-D-mannosidase (alpha-D-mannoside mannohydrolase EC 3.2.1.24) activity has been isolated from Saccharomyces cerevisiae. The enzyme was localized in a crude particulate fraction of the cell extract and was not solubilized by treatment with detergents or high ionic strength NaCl. The enzyme had a pH optimum of 6.3, Km 50 micron with p-nitrophenyl-alpha-D-mannopyranoside, and was competitively inhibited by D-mannose (Ki 20 mM). The enzyme is not affected by ethylenediaminetetraacetic acid, a number of different cations, or sulfhydryl reagents. It was inhibited by p-chloromercuriphenyl sulfonic acid and this inhibition is prevented by the addition of substrate. The cellular concentration of alpha-D-mannosidase is inversely proportional to growth rate, suggesting that the enzyme is under catabolite repression. The level of enzyme was found to increase approx. 8-fold during sporulation. This is apparently due to de novo synthesis, since inhibition of protein synthesis by cycloheximide prevents the increase in enzyme activity.     

Solubilization and characteristics of the thyroid NADPH-dependent H2O2 generating system

Solubilization of the thyroid particulate-associated NADPH-dependent H2O2 generating system has been tested with different detergents; (3-(3-cholamidopropyl)-dimethylammonio)1-propane sulfonate (CHAPS) was found to be the best of the six detergents tested. The ratio of H2O2 generation to NADPH oxidation was similar for CHAPS extract and native particulate material. CHAPS was also the only detergent able to preserve the Ca++-sensitivity of the NADPH oxidase. Solubilization of this enzyme allowed the determination of some of its characteristics: specificity for divalent cations, apparent Km for NADPH, optimum pH and sensitivity to SH- reagents.     

The plasma membrane of yeast protoplasts exposed to hypotonicity becomes porous but does not disintegrate in the presence of protons or polyvalent cations

Protoplasts of Saccharomyces cerevisiae swelled, lysed and disintegrated when exposed to hypotonic solutions at neutral pH. At pH 4.5 or lower the hypotonically treated protoplasts did not disintegrate and they retained their intracellular proteins, nucleic acids and nucleotides. However, they became leaky for K+ and Ca2+, indicating that pores had been created in the surface membrane, relaxing the osmotic stress. Upon readjustment of pH to neutral, the hypotonically treated protoplasts released the intracellular content and disintegrated. Also, at low pH, protoplasts did not swell in isotonic ammonium acetate and were refractory to the permeabilizing effect of nystatin and to lysis with low concentrations of detergents. Protoplasts were similarly protected against lysis and disintegration by hypotonic treatment or by detergents, even at neutral pH, if the incubation media contained polyvalent cations, especially Zn2+, La3+, spermine, and Ca2+ chelated with EDTA. The protoplasts exposed to hypotonic stress at low pH did not respire and could not regenerate into viable cells. Effects of H+ and polyvalent cations on intramembrane forces acting between molecules of membrane phospholipids are considered along with possible changes in interactions between membrane proteins.     

Differential effects of detergents, fatty acids, cations and heating on ostrich skeletal muscle 20S proteasome

The 20S proteasome, the catalytic core of the 26S proteasome, has previously been isolated, purified and partially characterised from ostrich skeletal muscle (Thomas, A.R., Oosthuizen, V., Naude, R.J., Muramoto, K. 2002. Biol. Chem. 383, 1267-1270). Due to the apparent latency of the 20S proteasome purified from various sources, this study focuses on further characterising the ostrich enzyme in terms of the effects of selected detergents, fatty acids and cations, as well as heating at 60 degrees C, on four of its activities. Results showed that ostrich skeletal muscle 20S proteasome was affected in a non-concentration-dependent manner by the selected detergents and fatty acids. Monounsaturated fatty acids, unlike unsaturated fatty acids, showed no major effects on the activities of the ostrich enzyme. The enzyme did not show sensitivity towards monovalent cations and the only divalent cations that showed a relevant effect were Ca2+ and Mg2+. Heating at 60 degrees C for 1-2 min had a substantial activating effect only on the peptidylglutamylpeptide-hydrolase (PGPH) and caseinolytic activities. In conclusion, many of the effects by the abovementioned reagents and conditions were noticeably different to those shown on different sources of the enzyme, further demonstrating the unique kinetic characteristics of the ostrich skeletal muscle 20S proteasome.     

A hemagglutinin specific for sialic acids in a rat brain synaptic vesicle-enriched fraction

A hemagglutinating activity was detected in a synaptic vesicle-enriched fraction prepared from adult rat brain, using trypsinized glutaraldehyde-fixed rabbit erythrocytes. The specific activity of the fraction, in two series of experiments, was 7.5 and 16-fold higher than in the other subcellular fractions. The activity was absent from the synaptosome cytosol. In a study using twenty-five different carbohydrates and glycoproteins, best inhibitors were N-acetylneuraminic acid and N-glycolylneuraminic acid, together with bovine submaxillary mucin and a glycopeptide fraction prepared from rabbit erythrocyte membranes. The activity was thermolabile and very sensitive to proteolytic enzymes (but insensitive to neuraminidase) indicating that a protein (agglutinin) is responsible for the activity. Experiments using detergents and high ionic strength showed that the agglutinin is tightly bound to membranes, inactivated by the so-called non denaturing detergents, and stable in diluted sodium dodecyl sulphate. Hypotheses are discussed on the possible function of the agglutinin.     

Structural studies of fc receptors. II. The effect of dissociation and reassociation of phospholipids on the activity of Fc gamma receptors of macrophages

The receptor for the Fc portion of immunoglobulin G (Fc receptor) of guinea pig macrophages was solubilized with a detergent and partially delipidated to the point where the ligand binding activity was essentially lost. Delipidation of the Fc receptor was done by fractionating the macrophage lysate by gel filtration in the presence of detergent. The elution behavior of Fc receptor-detergent complex and phospholipid-detergent mixed micelles varied depending on the kind of detergents used for membrane solubilization and for gel filtration. Separation of phospholipids from Fc receptor was best achieved when octylglucoside-solubilized fraction was chromatographed on Sepharose CL-6B in the presence of deoxycholate; the phospholipid peak emerged at Kav = 0.55 and the Fc receptor at Kav = 0.45. The fraction of Kav = 0.45 showed only a marginal activity when the activity was measured after removal of detergents, but activity was clearly shown when phospholipid fraction was added to this fraction prior to removal of the detergents. Reappearance of the Fc receptor activity was shown to be due to association of phospholipids with the Fc receptor. Three kinds of phospholipids with different polar head groups examined, phosphatidylcholine, phosphatidylserine, and phosphatidylethanolamine, were all able to reconstitute active Fc receptor, although phosphatidylethanolamine was somewhat less effective than the others. Thus, our study demonstrated the amphipathic nature of the Fc receptor, the binding of which is dependent on the interaction with phospholipids.     

The renal degradation of myelin basic protein peptide 43-88 by two enzymes in different subcellular fractions.

Previous studies have demonstrated that the kidney is the major site for clearance and catabolism of a peptide (residues 43-88) of encephalitogenic or basic protein (BP) derived from central-nervous-system myelin. In the present investigation rat renal tissue was shown to be capable of degrading human BP peptide 43-88 over the pH range 4-11.5 with peaks of activity at pH5 and pH9. The enzymic activity at pH5 was localized mainly to the 5900 g pellet (crude mitochondrial fraction) and, on the basis of its sulphydryl features, was inferred to be cathepsin B. The enzyme activity at pH9 was greatly enriched in the 100 000 g pellet (microsomal brush-border fraction), and its sensitivity to inhibitors suggested that it was a metalloproteinase. The activity at alkaline pH in the 100 000 g pellet was stimulated 3-fold by non-ionic detergents and 20-fold by ATP and polyphosphates. Through a series of experiments the ATP stimulation of the alkaline proteinase activity was concluded to be the result of a reversal of inhibition imposed by the presence of another cationic protein, methylated bovine serum albumin. Inhibition by certain bivalent cations, the irregular effects of chelators and the effects of poly-L-lysine supported this conclusion. These studies indicate the availability of renal enzymes of different types and in different cellular compartments that are capable of degrading BP peptide 43-88. In particular, the relative amounts of bivalent cations, anions and charged proteins and peptides are likely to be major influences on the activity of the alkaline proteinase in vivo. The control of this degradation as well as the features of the smaller fragments of the peptide formed may determine biological and immune events subsequent to the release of this potentially autoantigenic material.     

Characterization of phospholipase C-mediated phosphatidylinositol degradation in rat heart ventricle

Phosphoinositide-specific phospholipase C (PI-PLC) activity was investigated in the rat heart ventricle. Incubation of ventricle homogenate or 100,000g supernatant fraction with [3H]myoinositol or [3H]arachidonate-labeled phosphatidylinositol in the presence of Ca2+ resulted in a decrease in phosphatidylinositol with a concomitant increase in water-soluble [3H]inositol phosphate or [3H]diglyceride, respectively. Total overt homogenate PI-PLC activity could be accounted for in the supernatant fraction. Neutral, zwitterionic, cationic, or anionic detergents did not unmask membrane-associated activity. While cytosolic phospholipase C was active against a pure phosphatidylinositol substrate in the presence of Ca2+, no hydrolytic activity was detected when phosphatidylinositol was presented as a component (4-5%) of a mixture of phospholipids. However, addition of deoxycholate to the incubation mixture (pH 6.5, Ca2+ 10(-3) M) containing mixed phospholipids resulted in the exclusive hydrolysis of inositol phospholipids. Ventricular supernatant phospholipase C-mediated phosphatidylinositol degradation has a sharp pH optimum at 5.5 and a specific requirement for Ca2+. Activity is maximal at 1 to 2 X 10(-3) M Ca2+, with inhibition occurring at higher levels. Under optimized conditions phosphatidylinositol is hydrolyzed at a rate of 20-25 nmol/min/mg protein. Multivalent cations inhibit Ca2+-dependent PI-PLC activity while monovalent cations and anions have no effect. There is no apparent selectivity for specific fatty acid moieties on phosphatidylinositol. Soluble PI-PLC is inhibited by sulfhydryl reagents, neomycin, mepacrine, trifluoperazine, and propranolol. Chlorpromazine, dibucaine, and tetracaine exert a biphasic influence, stimulating at lower and inhibiting at higher concentrations.     

A simplified method for the preparation of detergent-free lipid rafts

Lipid rafts are small plasma membrane domains that contain high levels of cholesterol and sphingolipids. Traditional methods for the biochemical isolation of lipid rafts involve the extraction of cells with nonionic detergents followed by the separation of a low-density, detergent-resistant membrane fraction on density gradients. Because of concerns regarding the possible introduction of artifacts through the use of detergents, it is important to develop procedures for the isolation of lipid rafts that do not involve detergent extraction. We report here a simplified method for the purification of detergent-free lipid rafts that requires only one short density gradient centrifugation, but yields a membrane fraction that is highly enriched in cholesterol and protein markers of lipid rafts, with no contamination from nonraft plasma membrane or intracellular membranes.     

Glucose and nucleoside transporters of human erythrocytes: effects of detergents on immunoadsorption of a membrane protein to its monoclonal antibody.

Immunoadsorption of membrane proteins solubilized in detergents has been used widely for identification, purification and quantitation of transporters and receptors. In an effort to separate the glucose and nucleoside nucleoside transporters of human erythrocytes (GT and NT, respectively) that copurify in a membrane protein fraction band 4.5, we examined in the present study the effects of seven different detergents on the immunoadsorption of GT to its monoclonal antibody, 65D4 (Craik, et al. (1988) Biochem. Cell Biol. 66, 839-852). The following results were obtained. (1) The maximum extent of the immunoadsorption of GT by 65D4 varied between 52 to 98% in different detergents. For non-ionic detergents, there was an apparent inverse correlation between the maximum immunoreactivity of GT and the aggregation number or micellar size of detergents. (2) The immunoprecipitate of GT by 65D4 was contaminated with nucleoside transporter to an extent that varied from 2 to 35 mol% in different detergents. There is an inverse correlation between the extent of the contamination and the detergent aggregation number. However, this contamination was quantitatively accounted for by a time-dependent, non-specific aggregation of NT with GT in detergents. (3) A high degree of purification of NT in band 4.5 by immunoadsorptive removal of GT with 65D4 was achieved in C12E8 as predicted by the observed low NT-GT aggregation and the relatively high epitope-accessibility of GT in this detergent. Based on these findings, we conclude that certain detergents can reduce the immunoreactivity of membrane proteins significantly by modulating epitope accessibility, and may also produce a false immuno-cross-reactivity by inducing nonspecific protein aggregation.     

Inorganic polyphosphates and sensitivity of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> cells to membrane-damaging agents

The leakage of ATP and potassium ions from the cells of Saccharomyces cerevisiae with different levels of inorganic polyphosphate was studied under the action of two detergents (natural cellobiose lipid 16-[6-O-acetyl-2′-O-(3-hydroxyhexanoyl)-β-cellobiosyloxy)-2,15-dihydroxyhexadecanoic acid and sodium dodecyl sulfate) and silver cations. Cellobiose lipid had practically the same membrane-damaging activity against the cells grown in phosphate-containing medium, under phosphate starvation, and under polyphosphate hypercompensation. The cells grown under the latter conditions were less sensitive to sodium dodecyl sulfate and silver cations. The possible protective action of polyphosphates against the membrane-damaging agents under study is discussed.