Presence Of Octylglucoside Research Articles

Crystallization and crystal properties of squid rhodopsin.

Rhodopsin, a photoreceptor membrane protein in the retina, is a prototypical member of the G-protein-coupled receptor family. In this study, rhodopsin from the retina of the squid Todarodes pacificus was treated with V8 protease to remove the C-terminal extension. Truncated rhodopsin was selectively extracted from the microvillar membranes using alkyl glucoside in the presence of zinc ions and was then crystallized by the sitting-drop vapour-diffusion method. Of the various crystals obtained, hexagonal crystals grown in the presence of octylglucoside and ammonium sulfate diffracted to 2.8 A resolution. The diffraction data suggested that the crystal belongs to space group P6(2), with unit-cell parameters a = b = 122.1, c = 158.6 A. Preliminary crystallographic analysis, together with linear dichroism results, suggested that the rhodopsin dimers are packed in such a manner that their transmembrane helices are aligned nearly parallel to the c axis.

Determination of CD59 protein in normal human serum by enzyme immunoassay, using octyl-glucoside detergent to release glycosyl-phosphatidylinositol-CD59 from lipid complex

In this study we have optimised the enzyme immunoassay (ELISA) to quantify CD59 antigen in human serum or plasma. The glycosyl-phosphatidylinositol (GPI)-linked form of CD59 is known to complex with serum high-density lipoprotein. For ELISA optimisation, therefore, we investigated the effect of detergents, added to the sample diluent, on the determined values of CD59. Values obtained in the presence of octyl-glucoside (OG) for 20 adults aged 18–35 years and 17 children 1–5 years old were, respectively, 33–119ng/ml (mean ± S.D.: 66±22ng/ml) and 37–143ng/ml (76±33ng/ml). These results were higher than those measured without OG and were in contrast with published results showing absence, or eight to nine times lower levels, of the protein in serum. A known range for serum concentrations of CD59 in healthy individuals will establish an important reference point for clinical work and for the investigation of diseases involving the complement membrane attack complex (MAC) and its regulation.

Purification and characterization of recombinant microsomal prostaglandin E synthase-1

Recombinant human microsomal prostaglandin E 2 synthase-1 (mPGES-1) was expressed in a baculovirus-Sf9 cell system. The mPGES-1 was solubilized from Sf9 cell membranes with diheptanoylphosphatidylcholine and purified in the presence of octylglucoside using hydroxyapatite column chromatography. The K m values of the substrates PGH 2 and GSH were 14 μM and 0.75 mM, respectively, with the purified enzyme. The specific activity ( 4 μmol/min/mg ) was increased 3–5-fold by non-ionic and zwitterionic detergents. Kinetic analysis showed that dodecylmaltoside increases V max but does not affect the K m values of either substrate. Several other thiol-containing compounds were tested as glutathione replacements, none of which yielded detectable enzyme activity. During enzyme catalysis, glutathione was not oxidized and therefore can be considered an enzyme cofactor. No glutathione transferase or peroxidase activity could be determined with a range of potential substrates. The results show that purified mPGES-1 has a specific activity similar to Cox-2, consistent with its postulated role in Cox-2 mediated PGE 2 formation.

Purification of the Small Mechanosensitive Channel of Escherichia coli (MscS): the Subunit Structure, Conduction, and Gating Characteristicsin Liposomes

The small mechanosensitive channel, MscS, is a part of the turgor-driven solute efflux system that protects bacteria from lysis in the event of osmotic downshift. It has been identified in Escherichia coli as a product of the orphan yggB gene, now called mscS (Levina et al., 1999, EMBO J. 18:1730). Here I show that that the isolated 31-kDa MscS protein is sufficient to form a functional mechanosensitive channel gated directly by tension in the lipid bilayer. MscS-6His complexes purified in the presence of octylglucoside and lipids migrate in a high-resolution gel-filtration column as particles of ∼200 kDa. Consistent with that, the protein cross-linking patterns predict a hexamer. The channel reconstituted in soybean asolectin liposomes was activated by pressures of 20–60mm Hg and displayed the same asymmetric I-V curve and slight anionic preference as in situ. At the same time, the single-channel conductance is proportional to the buffer conductivity in a wide range of salt concentrations. The rate of channel activation in response to increasing pressure gradient across the patch was slower than the rate of closure in response to decreasing steps of pressure gradient. Therefore, the open probability curves were recorded with descending series of pressures. Determination of the curvature of patches by video imaging permitted measurements of the channel activity as a function of membrane tension (γ). Po(γ) curves had the midpoint at 5.5±0.1 dyne/cm and gave estimates for the energy of opening ΔG=11.4±0.5 kT, and the transition-related area change ΔA=8.4±0.4nm2 when fitted with a two-state Boltzmann model. The correspondence between channel properties in the native and reconstituted systems is discussed.

Detection of fast light-activated H+ release and M intermediate formation from proteorhodopsin.

Proteorhodopsin (pR) is a light-activated proton pump homologous to bacteriorhodopsin and recently discovered in oceanic gamma-proteobacteria. One perplexing difference between these two proteins is the absence in pR of homologues of bR residues Glu-194 and Glu-204. These two residues, along with Arg-82, have been implicated in light-activated fast H+ release to the extracellular medium in bR. It is therefore uncertain that pR carries out its physiological activity using a mechanism that is completely homologous to that of bR. A pR purification procedure is described that utilizes Phenylsepharose and hydroxylapatite columns and yields 85% (w/w) purity. Through SDS-PAGE of the pure protein, the molecular weight of E.-coli-produced pR was determined to be 36,000, approximately 9,000 more than the 27,000 predicted by the DNA sequence. Post-translational modification of one or more of the cysteine residues accounts for 5 kDa of the weight difference as measured on a cys-less pR mutant. At pH 9.5 and in the presence of octylglucoside and diheptanoylphosphotidylcholine, flash photolysis results in fast H+ release and a 400-nm absorbing (M-like) photoproduct. Both of these occur with a similar rise time (4-10 micros) as reported for monomeric bR in detergent. The presence of fast H+ release in pR indicates that either different groups are responsible for fast H+ release in pR and bR (i.e. that the H+ release group is not highly conserved); or, that the H+ release group is conserved and is therefore likely Arg-94 itself in pR (and Arg-82 in bR, correspondingly).

Octyl glucoside induced formation of the molten globule-like state of glutamate dehydrogenase.

The interaction between n-octyl-beta-D-glucopyranoside (octyl glucoside) and bovine liver glutamate dehydrogenase (GDH) was studied using techniques including equilibrium dialysis, UV-spectrophotometry, circular dichroism (CD), fluorescence energy transfer and extrinsic spectrofluorometry in 50 mM sodium phosphate buffer solution (pH 7.6). The equilibrium dialysis experiment showed a higher binding of octyl glucoside to GDH that induces up to 80% enzyme inhibition in 20 mM octyl glucoside solution. The CD study indicated that GDH retains its secondary structure in the presence of octyl glucoside, but loses a degree of its tertiary structure by acquiring a more extended tertiary structure. Measurement of the binding of a hydrophobic fluorescent probe, 1-anilino-naphthalene-8-sulfonate (ANS), to GDH revealed that the binding of ANS to GDH is increased in the presence of octyl glucoside, a finding that may be interpreted in terms of the increment of surface hydrophobic patch(es) of GDH because of its binding to octyl glucoside. Fluorescence energy transfer studies also showed more binding of the reduced coenzyme (NADH) to GDH and the Lineweaver-Burk plots (with respect to NADH) indicate the existence of substrate inhibition in the presence of octyl glucoside. These observations are aimed at explaining the formation of the molten globule-like structure of GDH, which is induced by a non-ionic detergent such as octyl glucoside.

Purification and reconstitution into proteoliposomes of the F1F0 ATP synthase from the obligately anaerobic gram-positive bacterium Clostridium thermoautotrophicum

The proton-translocating F1F0 ATP synthase from Clostridium thermoautotrophicum was solubilized from cholate-washed membranes with Zwittergent 3-14 at 58 degrees C and purified in the presence of octylglucoside by sucrose gradient centrifugation and ion-exchange chromatography on a DEAE-5PW column. The purified enzyme hydrolyzed ATP at a rate of 12.6 micromol min(-1) mg(-1) at 58 degrees C and pH 8.5. It was composed of six different polypeptides with molecular masses of 60, 50, 32, 19, 17, and 8 kDa. These were identified as alpha, beta, gamma, delta, epsilon, and c subunits, respectively, as their N-terminal amino acid sequences matched the deduced N-terminal amino acid sequences of the corresponding genes of the atp operon sequenced from Clostridium thermoaceticum (GenBank accession no. U64318), demonstrating the close similarity of the F1F0 complexes from C. thermoaceticum and C. thermoautotrophicum. Four of these subunits, alpha, beta, gamma, and epsilon, constituted the F1-ATPase purified from the latter bacterium. The delta subunit could not be found in the purified F1 although it was present in the F1F0 complex, indicating that the F0 moiety consisted of the delta and the c subunits and lacked the a and b subunits found in many aerobic bacteria. The c subunit was characterized as N,N'-dicyclohexylcarbodiimide reactive. The F1F0 complex of C. thermoautotrophicum consisting of subunits alpha, beta, gamma, delta, epsilon, and c was reconstituted with phospholipids into proteoliposomes which had ATP-Pi exchange, carbonylcyanide p-trifluoromethoxy-phenylhydrazone-stimulated ATPase, and ATP-dependent proton-pumping activities. Immunoblot analyses of the subunits of ATP synthases from C. thermoautotrophicum, C. thermoaceticum, and Escherichia coli revealed antigenic similarities among the F1 subunits from both clostridia and the beta subunit of F1 from E. coli.

Octyl glucoside inhibits [14C]DHP mineralization whereas peroxidase activity is stimulated inPhanerochaete Chrysosporium

Octyl glucoside stimulated peroxidase formation inPhanerochaete chrysosporium ME-446 cultivated in cellulose-based media. Addition of 0.1% of the nonionic surfactant resulted in a ninefold (143 U/L) and sixfold (119 U/L) increase in LiP formation under conditions of N limitation and N excess, respectively. Octyl glucoside also stimulated MnP formation, but to a lesser extent than observed with LiP. The cellobiose-oxidizing enzymes (cellobiose dehydrogenase and cellobiose:quinone oxidoreductase) were stimulated by octyl glucoside when used at a concentration of up to 0.05%, but higher concentrations gave values similar to those for the controls. Little proteolytic activity was detected in the presence of the surfactant. In general, activities of the enzymes studied were of the same order as those seen using Tween-80. In contrast with Tween-80, octyl glucoside markedly inhibited [14C]DHP mineralization. Attempts to account for the observed inhibition of synthetic lignin degradation by P.chrysosporium in the presence of octyl glucoside are discussed.

Thermal Stability of Individual Domains in Platelet Glycoprotein IIbIIIa

Thermal denaturation of platelet glycoprotein IIbIIIa (integrin alpha IIb beta 3) was investigated by spectrofluorimetry and differential scanning calorimetry (DSC). Two forms of the protein were compared: active IIbIIIa, i.e., that fraction that binds to RGD-Sepharose, and inactive IIbIIIa, the non-binding fraction. At pH 8.5 in the presence of octyl glucoside and Ca2+ both forms exhibited a broad complex endotherm consisting of a well expressed low-temperature heat-absorption peak in the range of 40-65 degrees C followed by a broad peak stretching over 65-110 degrees C. Each endotherm could be deconvoluted into at least eight transitions reflecting the melting of at least this many independently folded domains. The first two transitions in the region of the low-temperature peak had similar positions in both forms while at least some of the other transitions occurred at higher temperature in the active protein suggesting that some of the domains are more stable in the latter. When both fractions of IIbIIIa were heated in the fluorometer a sigmoidal transition was observed in the region of the first endothermic peak where the two thermolabile domains melt. This transition was destabilized by 15 degrees C in the presence of EDTA, suggesting that these domains are formed by the 243-468 region of the IIb subunit which contains four Ca(2+)-binding motifs. It was further stabilized by 3 degrees C upon addition of the GRGDSPK peptide in the presence of Ca2+ while in EDTA the peptide had no effect. This is consistent with the involvement of Ca(2+)-binding region in the formation of the ligand-binding site. A 66-kDa chymotryptic fragment, containing the 17-kDa NH2-terminal portion of the IIIa subunit disulfide-linked to its 50-kDa COOH-terminal portion including the cysteine-rich core, exhibited a fluorescence-detected Ca(2+)-independent transition in the region where the higher temperature DSC-detected transitions occur suggesting that some of the latter may be connected with the melting of the corresponding portions of IIbIIIa.

Identification of the pore forming element of Semliki Forest virus spikes

Pore formation at mildly acidic pH by SFV spike proteins was investigated using isolated and modified virions. Modification of the virions was performed by limited proteolysis in presence of octylglucoside and resulted in the formation of E1 particles and spikeless particles, respectively. Pore formation was detected by measuring the influx of propidium iodide into the viral particles. The results obtained clearly showed that the presence of E1 alone is sufficient to promote pore formation at mildly acidic pH. Thus E1 represents the pore forming element of the viral spike proteins.

Isoelectric focusing in immobilized pH gradients of the glucose transporter from human red cell membranes.

Isoelectric focusing of the human red cell glucose transporter (a transmembrane protein) was performed in immobilized pH gradients. Isoelectric focusing of integral membrane proteins presents problems that are related to the amphiphilic nature of these proteins. Solubilizing additives must be used to counteract hydrophobic effects. In our case, urea and the nonionic detergent, Triton X-100, were used. Focusing was done at 15 degrees C. The isoelectric point (pI) of the glucose transporter (freshly purified by anion-exchange chromatography in the presence of octyl glucoside) was determined at 8.4 +/- 0.05 (n = 9), in good agreement with our earlier determinations by two-dimensional electrophoresis with isoelectric focusing in the presence of carrier ampholytes in the first dimension. The width of the focused zone was approximately 0.1 pH unit, more narrow than after focusing with carrier ampholytes. In an immobilized pH gradient from pH 7 to 10, the transporter region at pH 8.4 comprised one major and one or two minor zones. The pH interval 4-10 was also used and showed a single transporter zone. The glucose transporter tends to self-associate in detergent solution. Octyl glucoside-purified glucose transporter formed oligomers during incubation at 37 degrees C. Upon focusing, these oligomers appeared in a wide pH interval far below pH 8.4.

Subunit interactions of vesicular stomatitis virus envelope glycoprotein influenced by detergent micelles and lipid bilayers.

The envelope glycoprotein (G protein) of vesicular stomatitis virus is a transmembrane protein that exists as a trimer of identical subunits in the virus envelope. We have examined the effect of modifying the environment surrounding the membrane-spanning sequence on the association of G protein subunits using resonance energy transfer. G protein subunits were labeled with either fluorescein isothiocyanate or rhodamine isothiocyanate. When the labeled G proteins were mixed in the presence of the detergent octyl glucoside, mixed trimers containing both fluorescent labels were formed as a result of subunit exchange, as shown by resonance energy transfer between the two labels. In contrast when fluorescein- and rhodamine-labeled G proteins were mixed in the presence of Triton X-100, no resonance energy transfer was observed, indicating that subunit exchange did not occur in Triton X-100 micelles. However, if labeled G proteins were first mixed in the presence of octyl glucoside, energy transfer persisted after dilution with buffer containing Triton X-100. This result indicates that the G protein subunits remained associated in Triton X-100 micelles and that the failure to undergo subunit exchange was due to lack of dissociation of G protein subunits. Chemical cross-linking experiments confirmed that G protein was trimeric in the presence of Triton X-100. The efficiency of resonance energy transfer between labeled G protein was higher when G proteins were incorporated into dimyristoylphosphatidylcholine liposomes compared to detergent micelles. This result indicates that the labels exist in a more favorable environment for energy transfer in membranes than in detergent micelles.(ABSTRACT TRUNCATED AT 250 WORDS)

48] Acyl/alkyldihydroxyacetone phosphate reductase from guinea pig liver peroxisomes

Publisher Summary Acylglycerone-phosphate reductase—a membrane-bound enzyme present in animal tissues—catalyzes the reduction of long-chain acyldihydroxyacetone phosphate (acyI-DHAP) or long-chain alkyl-DHAP by NADPH to the corresponding sn -glycerol 3-phosphate derivatives. In liver and other organs, the major fraction of the enzyme is present in peroxisomes. The enzyme has been solubilized and partially purified from Ehrlich ascites cell microsomes and purified to homogeneity from guinea pig liver peroxisomes. This chapter describes a method for the preparation of the homogeneous enzyme from guinea pig liver peroxisomes. The enzyme activity is assayed by measuring the radioactive lipid product formed after incubating alkyl-DHAP with B-[4- 3 H] NADPH. The purification of alkyldihydroxyacetone phosphate is discussed in the chapter. On sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, the purified enzyme shows a single protein band of relative mobility corresponding to 60,000 molecular weight. On gel filtration, in the presence of octylglucoside, the molecular weight of the enzyme is estimated at 75,000. The enzyme has a broad pH optimum between pH 6.5 and 7.5.

Active and monomeric human red cell glucose transporter after high performance molecular-sieve chromatography in the presence of octyl glucoside and phosphatidylserine or phosphatidylcholine

The human red cell glucose transporter (Glut 1) was purified by ion-exchange chromatography in the presence of octyl glucoside. The state of association of the protein was studied, and the transport activity was determined after exchange of copurified membrane lipids for phosphatidylserine (PS) or phosphatidylcholine (PC). The purpose was to analyze the Glut 1 preparation for homogeneity and activity prior to attempts at crystallization. Analyses by high performance molecular-sieve chromatography showed that the Glut 1 was monomeric immediately after the ion-exchange purification: the M r of the Glut 1 polypeptide was estimated to be 49 000 ± 6000 by TSKgel G3000SW chromatography monitored by low-angle laser light-scattering photometry, differential refractometry and UV photometry. This required determination of the absorption coefficient of the Glut 1, which was measured to be 1.13 ± 0.03ml mg −1 cm −1 at 280 nm, referring to the polypeptide concentration. The M r value is consistent with the cDNA-deduced M r 54117 of the very similar HepG2 glucose transporter polypeptide. At 2°C, pH 7 and an ionic strength of 0.06 M, the Glut 1 associated gradually during three days to form oligomers. These formed much more rapidly at room temperature or at high ionic strength. Freshly prepared Glut 1 retained high activity after separation from membrane lipids on a TSKgel G3000SW column in the presence of 40 mM octyl glucoside and 1 mM PS or PC. In contrast, most of the activity was lost when the membrane lipids were separated from the protein in the absence of eluent lipids. The presence of a phospholipid was thus essential for retention of high activity of the Glut 1 in octyl glucoside and PC was nearly as effective as PS.

Water-soluble proteins do not bind octyl glucoside as judged by molecular sieve chromatographic techniques

It is well known that the non-ionic detergent octyl glucoside (1-O- n-octyl-β- d-glucopyranoside) solubilizes biological membrane components. It forms complexes with membrane-spanning proteins by hydrophobic interactions and it forms mixed micelles with membrane lipids. In contrast, non-ionic detergents usually do not bing to water-soluble proteins. According to a recent report, substantial and cooperative binding of octyl glucoside to several water-soluble proteins does occur near the critical micelle concentration. However, data have been obtained that contradict this report. No decrease was found in the elution volumes of five water-soluble proteins on molecular sieve chromatography on two Superose columns in tandem when 35 m M octyl glucoside was included in the eluent. No binding of the detergent to these proteins was observed at 20 or 22.5 m M octyl glucoside on molecular sieve chromatography on a TSK SW guard column as determined by differential refractometry and UV spectrophotometry of the proteins in the absence or presence of octyl glucoside. The experiments were done with the same buffer system and with six of the proteins used in the reported study. It is concluded that, as expected, there is no binding of octyl glucoside to water-soluble proteins above the detection limit (0.1 g detergent/g protein) of the refractometric method. The binding of, on average, 1.3 ± 0.2 g of detergent per gram of was observed at 20 m M octyl glucoside in the reported study is not consistent with the present results.

Highly efficient immunoliposomes prepared with a method which is compatible with various lipid compositions

Monoclonal antibody was conjugated to N-glutaryl-phosphatidylethanolamine in the presence of octylglucoside by using N-hydroxysulfosuccinimide as a carboxyl-activation reagent. The conjugated antibody was then incorporated into liposomes by a simple dialysis method. The method is mild and is compatible with various lipid compositions of the liposomes. We have prepared immunoliposomes containing a lung endothelium-specific monoclonal antibody and showed excellent target binding (∼75% injected dose) of the immunoliposomes in mouse. Immunoliposomes can be prepared to contain other acidic lipids such as phosphatidylserine and various amounts of cholesterol. The presence of 20% or more cholesterol in liposomes resulted in high level of target binding. We have used in these experiments a new radioactive lipid-phase marker, 111In-DTPA-SA, which was very stable in vivo . The halflife of clearance in mouse exceeded 3 weeks.

Solubilization, purification and characterization of lysoplasmalogen alkenylhydrolase (lysoplasmalogenase) from rat liver microsomes

Alkenylhydrolase (EC 3.3.2.2; EC 3.3.2.5) has been purified 200-fold to a specific activity of 8.0 μmol/min per mg from rat liver microsomes with 51% of the activity recovered. Purification was accomplished by solubilization of the membrane-associated enzyme with octylglucoside and chromatographic resolution on sequential DEAE cellulose and hydroxylapatite (HPLC) columns in the presence of octylglucoside. The partially purified enzyme, specific for the 2-deacylated plasmalogen, lysoplasmalogen (1-alk-1′-enyl- sn-glycero-3-phosphocholine or ethanolamine), had no hydrolytic activity with intact plasmalogens or 1-acyl- sn-glycero-3-phosphoethanolamine. Kinetic analyses of enzymic activity demonstrated apparent K m values of 5.5 and 42 μM for 1-alk-1′-enyl- sn-glycero-3-phosphocholine and 1-alk-1′-enyl- sn-glycero- 3-phosphoethanolamine, respectively. The V max values were 11.7 and 13.6 μmol/min per mg with the choline and ethanolamine substrates, respectively. The optimal pH range was between 6.6 and 7.1 with both substrates; the energy of activation for the purified enzyme was 15 200 cal. The enzyme required no cofactors and was unaffected by low millimolar concentrations of Ca 2+, Mg 2+ or EDTA. It was inhibited by the sulfhydryl-reacting reagent, p-chloromercuribenzoate. Mono- or diradylglycerophospholipids or sphingomyelin did not affect the enzymic activity at 37° C. Activity of the purified enzyme, destroyed by freezing at −20°C, was preserved if stored at this temperature in the presence of 300–600 μM diradylglycerophosphocholine or 50% glycerol. A continuous spectrophotometric assay, adapted in our laboratory for the assay of liver alkenylhydrolase, faciitated this purification. This is the first reported purification of alkenylhydrolase.

Semliki forest virus particles containing only the E 1 envelope glycoprotein are infectious and can induce cell-cell fusion

Hydrophobic interaction chromatography (phenyl- and octyl-Sepharose) was performed with Semliki Forest virus to investigate the effect of low pH on its hydrophobicity. At neutral pH, the virus could be bound to the column and completely eluted by the detergent NP-40. Low pH treatment of virus prior to application to the column resulted in stronger binding as reflected by the increased amount of detergent necessary to totally elute the virus. If, however, the low pH treatment was done after binding of the virus to the column, only 15% of the input virus could be eluted by the detergent, indicating a drastic increase in hydrophobicity. Thus binding of the virus to a hydrophobic environment potentiates the effect of low pH on viral hydrophobicity. Trypsin digestion of column-bound virus after low pH treatment resulted in complete digestion of E 2 and E 3; however, E 1 was totally resistant. From this result, we conclude that E 1 alone is responsible for the hydrophobic interaction. We have made use of these observations to produce viral particles which were devoid of E 2 and E 3 by trypsin digestion in the presence of octyl glucoside. These E 1 viral particles were infectious and could induce membrane fusion. We conclude that only E 1 is necessary and sufficient to mediate membrane fusion. Acid pH induces a drastic increase in the hydrophobicity of E 1 which probably facilitates its interaction with the lipid bilayers during the fusion event in endosomes.

High-performance agarose gel chromatography in octyl glucoside of integral membrane proteins from human red cells, with special reference to the glucose transporter

Integral membrane proteins and lipids from human red cells were fractionated in the presence of octyl glucoside by high-performance gel chromatography on a 22-ml column of the small-bead cross-linked agarose gel Superose 6, at 5°C, pH 7.6 and 30–50 m M detergent. To avoid aggregation a relatively high flow-rate, 9 ml/h, was chosen. At low ionic strength four main fractions were resolved, which contained anion transporter multimers(I), glycophorin oligomers(II), glucose transporter dimers(III) and phospholipids(IV). In 0.5 M sodium chloride the resolution was lower but the yield of the glucose transporter was markedly higher, and chromatography of partially purified glucose transporter gave a protein recovery of about 90%. The apparent M r values for the octyl glucoside complexes of the main components were: anion transporter, 900 000; glycophorin A, 210 000–360 000, dependent on ionic strength; glucose transporter, 110 000–160 000; lipids, 70 000. Some components aggreated with time: at a flow-rate of 1 ml/h mainly glycophorins and the glucose transporter were eluted, but no anion transporter, and fractionation performed 20 h after solubilization showed extensive aggregation of proteins. Superose-6 chromatography of glucose transporter and membrane lipids that had been isolated on DEAE-cellulose partially resolved the transporter and the phospholipid fractions. In this case, the resolution was better with 50 than with 30 m M detergent. The maximum glucose transport activity was approximately one-tenth of that observed before fractionation and appeared in two main fractions, at the main transporter fraction as well as at the overlap between the transporter and the lipids. The activity level was the same in both fractions, although the protein concentration was much lower in the second one. Addition of 2 m M egg-yolk phospholipids to the eluent did not increase the activity. This strongly indicates that the glucose transporter needs some specific membrane lipids to retain high transport activity. At the concentration of ca. 0.3 mg/ml used, the glucose transporter was probably eluted as a dimer in the absence of phospholipids and as a monomer in their presence.

Purification and characterization of a Ca2+-dependent membrane peptidase involved in the signaling of mating pheromone in Rhodosporidium toruloides.

A mating-type-specific, membrane thiol peptidase (referred to as trigger peptidase) that seems to play a key role in the transmembrane signaling of the lipopeptidyl mating pheromone rhodotorucine A at the cell surface of mating type a cells of Rhodosporidium toruloides (T. Miyakawa, M. Kaji, T. Yasutake, Y.K. Jeong, E. Tsuchiya, and S. Fukui, J. Bacteriol. 162:294-299, 1985) was purified to homogeneity and characterized. The following lines of evidence support the contention that the enzyme we purified was the trigger peptidase: the identical specificity of hydrolysis at the Arg-Asn sequence of rhodotorucine A and the sensitivity of the reaction to sulfhydryl-blocking reagents; the identical specificity for the substrate, with a strict requirement for the presence of the lipid moiety; and the absence of the corresponding activity in the pheromone-producing strain (mating type A) and in a sterile mutant strain, M-39 (type a), that lacks trigger peptidase activity in vivo. The apparent molecular weight of trigger peptidase was estimated to be 68,000 by Sepharose 6B gel filtration in the presence of octylglucoside and 63,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Trigger peptidase alone was inactive but exhibited enzymatic activity with the simultaneous addition of Ca2+, membrane phospholipids, and a nonionic detergent such as octylglucoside. The concentration of Ca2+ required for maximum activation was approximately 1 mM. Only Mn2+ could replace Ca2+ at comparable concentrations. Among the phospholipids tested, only phosphatidylserine and phosphatidylethanolamine supported trigger peptidase activation. Solubilized trigger peptidase was strongly inhibited by antipain and phosphoramidon.