Characterization of aminopeptidase N from Torpedo marmorata kidney

Summary— A major antigen of the brush border membrane of Torpedo marmorata kidney was identified and purified by immunoprecipitation. The sequence of its 18 N terminal amino acids was determined and found to be very similar to that of mammalian aminopeptidase N (EC 3.4.11.2). Indeed aminopeptidase N activity was efficiently immunoprecipitated by monoclonal antibody 180K1. The purified antigen gives a broad band at 180 kDa after SDS-gel electrophoresis, which, after treatment by endoglycosidase F, is converted to a thinner band at 140 kDa. This antigen is therefore heavily glycosylated. Depending on solubilization conditions, both the antigen and peptidase activity were recovered either as a broad peak with a sedimentation coefficient of 18S (2% CHAPS) or as a single peak of 7.8S (1% CHAPS plus 0.2 % C₁₂E₉), showing that Torpedo aminopeptidase N behaves as an oligomer stabilized by hydrophobic interactions, easily converted into a 160 kDa monomer. The antigen is highly concentrated in the apical membrane of proximal tubule epithelial cells (600 gold particles/μm² of brush border membrane) whereas no labeling could be detected in other cell types or in other membranes of the same cells (basolatéral membranes, vacuoles or vesicles). Monoclonal antibodies prepared here will be useful tools for further functional and structural studies of Torpedo kidney aminopeptidase N.     

Stabilization of fibroblast growth factors by a non-cytotoxic zwitterionic detergent, 3-[(3-cholamidopropyl) dimethylammonio]-1-propane sulfonate (chaps)

Summary The potential usefulness of a zwitterionic detergent, 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate (CHAPS), in the stabilization of acidic and basic fibroblast growth factors (FGFs) was examined. Among several detergents, CHAPS was found to be not only non-cytotoxic but also most useful in handing the diluted preparations of FGFs. The advantages are as follows: 1) at lower concentrations than 0.01% CHAPS did not affect growth factor activity of calf serum (CS) and the growth rate of BLAB/c 3T3 cells. The primary culture of rat prostate epithelium and colony formation of NRK-49F cells were hardly influenced by CHAPS lower than 0.003%; 2) the loss of FGFs that usually occurs due to their adherence to the surface of storage containers was effectively prevented by inclusion of 0.1% CHAPS; 3) the recovery of FGFs after storage or dialysis was significantly enhanced by inclusion of 0.1% CHAPS; 4) CHAPS at lower concentrations than 0.1% does not interfere with amino acid analysis, except that Thr may be misled only when the ratio of protein/CHAPS is low; 5) amino acid sequence analysis was hardly disturbed by CHAPS up to 0.5%. These results indicate that CHAPS is useful as a stabilizing agent for various kinds of polypeptides capable of showing biological activity at a low concentration. Editor's statement Polypeptide growth factors and hormones, especially heparin-binding growth factors (FGF), are notoriously troublesome to handle in purified form at extremely low concentrations. This report demonstrates an interesting solution to this problem.     

Sexual dimorphism of apoptosis in lactotrophs induced by bromocryptine

Previously we found a unique cell surface antigen [Kat1-antigen (Kat1-Ag)] expressed on rat osteoclasts. In the present study, we focused on the expression of this antigen in preosteoclasts, mononuclear precursors of osteoclasts. Immunohistochemical and immunoelectron microscopic observations of the Kat1-Ag expressed in vivo showed the antigen to be present on mononuclear cells having the morphological features of preosteoclasts. The relationship between Kat1-Ag expression and calcitonin receptor (CTR) expression was examined in detail by a double-detection technique for CTR and Kat1-Ag by use of autoradiography and immunocytochemistry, respectively. In a culture system for forming mononuclear preosteoclast-like cells, almost 100% of the mononuclear cells expressing Kat1-Ag also expressed CTR, demonstrating that Kat1-Ag is a reliable immunological marker for identifying preosteoclasts. Interestingly, a significant number of the CTR-positive mononuclear cells did not express the Kat1-Ag. Detection of these cells expressing CTR but not Kat1-Ag strongly suggests the presence of subpopulations in preosteoclasts. We also obtained evidence suggesting that expression of the Kat1-Ag is initiated during the postmitotic stage of the osteoclast progenitors.     

The membrane-induced structure of melittin is correlated with the fluidity of the lipids

The effect of the bee toxin melittin on DMPC dynamics in fast-tumbling bicelles has been investigated. The (13)C R(1) and (13)C-(1)H NOE relaxation parameters for DMPC were used to monitor the effect of melittin and cholesterol on lipid dynamics. It was found that melittin has the largest effect on the DMPC mobility in DMPC/DHPC bicelles, while less effect was observed in cholesterol-doped bicelles, or in bicelles made with CHAPS, indicating that the rigidity of the membrane affects the melittin-membrane interaction. CD spectra were analysed in terms of cooperativity of the alpha-helix to random coil transition in melittin, and these results also indicated similar differences between the bicelles. The study shows that bicelles can be used to investigate lipid dynamics by spin relaxation, and in particular of peptide-induced changes in membrane fluidity.     

Kinetics and Physical Parameters of Rat Brain Opioid Receptors Solubilized by Digitonin and CHAPS

Rat brain opioid receptors were solubilized with digitonin and a zwitterionic detergent, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS). The yield of solubilization was 70-75% with digitonin and 30-35% with CHAPS. Kinetic and equilibrium studies performed from digitonin extracts resulted in KD values comparable with those of the membrane fractions. Two [3H]naloxone binding sites were obtained in the extracts similarly to membrane fractions. The rank order potency of drugs used in the competition experiments did not change during solubilization. The distributions of mu, delta, and kappa opioid receptor binding sites were similar in membrane and digitonin-solubilized fractions (48-50% mu, 35-37% kappa, and 13-17% delta subtypes). The hydrodynamic properties of digitonin- and CHAPS-solubilized preparations were studied by sucrose density gradient centrifugation and Sepharose-6B chromatography. In all cases, two receptor populations were identified with the following parameters: sedimentation coefficients for the digitonin extracts were 9.2S and 13.2S and for CHAPS extract 8S and 15.6S; the Stokes radii were 45 A and 65A for the digitonin extract and 31A and 76A for the CHAPS-solubilized preparation.     

Effect of a zwitterionic detergent on the state of aggregation and catalytic activity of cytochrome P-450LM2 and NADPH-cytochrome P-450 reductase

The zwitterionic detergent 3-(3-cholamidopropyl)-dimethylammonio-1-propanesulfonate (CHAPS) supports reconstituted cyclohexane hydroxylase activity of cytochrome P-450LM2 and NADPH-cytochrome reductase purified from phenobarbital-induced rabbit liver. Maximum activity (approximately 50% of that with phospholipid) was observed at 2 mM CHAPS. Inhibition took place at higher CHAPS, until at 20 mM CHAPS, no cyclohexane hydroxylase activity was observed. There was little denaturation of the two enzymes under these conditions. At 2 mM CHAPS, P-450LM2 was pentameric (Mr = 250,000) and reductase was dimeric (Mr = 139,500) by sedimentation equilibrium. P-450 was monomeric in 20 mM CHAPS. In addition, a stable complex between the two enzymes was not detected under conditions of maximum activity, even in the presence of saturating substrate. This confirms our previous conclusion that a stable complex between cytochrome P-450LM2 and NADPH-cytochrome P-450 reductase is not a prerequisite for reconstituted xenobiotic hydroxylation (Dean, W. L., and Gray, R. D. (1982) J. Biol. Chem. 257, 14679-14685). Difference spectra of ferric P-450LM2 revealed that below 5 mM CHAPS, the high spin form of the cytochrome was slightly stabilized, while higher CHAPS levels stabilized the low spin form. Monomeric P-450LM2 formed with 20 mM CHAPS catalyzed the hydroxylation of toluene by cumene hydroperoxide. Thus, the reason that monomeric cytochrome P-450LM2 was inactive in NADPH-supported hydroxylation may either be because the bound detergent blocked productive interaction of the cytochrome with reductase or the monomer may be intrinsically incapable of interaction with reductase.     

Immune Response against Hamster Erythrocytes in the Low-Responder Mouse Strains

Golden hamsters were used as hosts in this work, and mice of various strains as donors of antigens. 1) There were no strain differences in immunogenicity of erythrocytes from C57BL/6, AKR, SL and CF1 mice. 2) Primary intravenous immunization with mouse erythrocytes (MRBC) induced the production of hemolysin plaque-forming cells (PFC) in a large number, but elicited only in a negligible titer production of 2-mercaptoethanol-resistant antibody. 3) 2-Mercaptoethanol-resistant antibody was produced more efficiently in hamsters pre-sensitized with mouse lymph node (MLN) cells rather than those pre-immunized with MRBC after a booster with MRBC. 4) Numbers of PFC in pre-sensitized hamsters were three-times that of the non-sensitized hamsters after a booster with MRBC, when pre-sensitization was performed intradermally with a small number of MLN cells. 5) Average diameter of the hemolysin plaques in pre-sensitized hamsters was one and a half times larger than that in non-sensitized hamsters. Conclusions agree well with the results in our previous papers that the reversed combination of hosts and antigen donors employed support the concept that certain processes required for delayed hypersensitivity contributed to antibody production under a condition suitable for antibody response.     

Immunity in experimental syphilis. IV. Serological reactivity of antigens extracted from gamm-irradiated Treponema pallidum and Treponema reiteri

Miller, James N. (University of California School of Medicine, Los Angeles), J. H. De Bruijn, and J. H. Bekker. Immunity in experimental syphilis. IV. Serological reactivity of antigens extracted from gamma-irradiated Treponema pallidum and Treponema reiteri. J. Bacteriol. 91:583-587. 1966.-Ultrasonic lysate preparations extracted from virulent Treponema pallidum, Nichols strain, suspensions exposed to 652,800 R of gamma-irradiation exhibited a loss in the serological reactivity of their heat-labile antigens; the heat-stable components of both the lysate and residue antigens were unaffected. The activity of heat-stable, cardiolipin T. pallidum complement-fixing antigen obtained from similarly irradiated organisms was also unaltered. gamma-Irradiation of the cultivable Treponema reiteri with dosages as high as 6,500,000 R failed to alter serologically either the heat-labile or heat-stable component of its lipopolysaccharide-protein (Reiter protein) antigen. The reactivity of the lipopolysaccharide portion of the Reiter protein complex with an antiserum to T. pallidum Nichols indicates previously unsuspected antigenic differences between the rabbit-adapted Nichols strain of the organism and so-called "wild" human strains of T. pallidum in which this antigen is generally absent.     

LPS-induced autoantibody response. I. Ontogenic development of PFC response to bromelain-treated syngeneic erythrocytes

Normal adult mice have been shown to contain a large number of cells secreting antibodies against bromelain-treated syngeneic erythrocytes (Br.MRBC) and the numbers remarkably increase by the stimulation with LPS. In this report development of the anti-Br.MRBC response during ontogeny was examined and it was shown that on the injection of LPS suckling mice responded little to generate splenic plaque-forming cells (PFC) against Br.MRBC in vivo and in vitro. The responsiveness of suckling mice to produce anti-Br.MRBC was shown to be less developed than the anti-TNP response or the mitotic response to LPS. The low responsiveness of suckling mice was analyzed in terms of suppressor activity in the spleen cell population, proliferative capacities of the precursors of anti-Br.MRBC PFC, and their frequencies in the spleen. In the coculture experiment of suckling and adult spleen cells or culture of anti-brain-associated Thy 1-treated, macrophage-depleted spleen cell population, no evidence was obtained to show that suckling spleen cells contained suppressor cells. Kinetic profiles studied in vitro showed that anti-Br.MRBC PFC in the suckling spleen did not increase during the culture as those in the adult spleen. Studies on the precursor frequencies revealed that spleen cells of 15-day-old mice contained precursors of anti-Br.MRBC PFC amounting to 20.5% of the adult precursors whereas the PFC response in vitro by the former was only 4% of the latter. From these experimental data, it was concluded that the low responsiveness of suckling mice was partly due to the low frequency of the precursors in the spleen and, in addition, to the defective nature of the precursors in proliferating to differentiate into PFC.     

Regulation of naturally occurring autoantibody-producing cells: detection of a suppressive substance which inhibits the secretion of antibodies directed against enzyme-treated isologous erythrocytes

Normal mice possess spleen cells capable of forming hemolytic plaques against bromelain-treated autologous red cells (Br MRBC). There is present in the serum of these same mice a substance which can inhibit the formation of these plaques. This substance is inhibitory to the secretion of these antibodies following incubation of spleen cells in 20% serum at 4 degrees C for 5 min. This substance is not inhibitory to the formation of anti-sheep erythrocyte plaques from mice either immunized or nonimmunized with sheep erythrocytes. Characterization of the substance indicates that it is neither soluble antigen nor specific antibody. However, inclusion of nanogram amounts of soluble antigen from bromelain-treated red cells in the assay mixture effectively neutralized the suppression. In addition, passage of serum through a mouse anti-Br MRBC antibody immunoadsorbent effectively removed the suppressive activity of the serum while suppression could be recovered in the acid eluate from such a column. This suggests that the mechanism of suppression brought about by incubation in serum is due to the action of a molecule possessing anti-idiotypic activity directed against the cell surface receptors of anti-Br MRBC B cells. Attempts to isolate the molecule based on the postulate that it is immunoglobulin in nature have been unsuccessful.     

Posttranslational modification at the N terminus of the human adenovirus type 12 E1A 235R tumor antigen.

The adenovirus E1A transforming region, which encodes immortalization, partial cell transformation, and gene activation functions, expresses two early mRNAs, 13S and 12S. Multiple-T antigen species with different electrophoretic mobilities are formed from each mRNA, presumably by unknown posttranslational modifications. The adenovirus type 12 (Ad12) 13S and 12S mRNAs encode E1A T antigens of 266 and 235 amino acid residues (266R and 235R), respectively. To study possible posttranslational processing at the N and C termini and to distinguish between the Ad12 266R and 235R T antigens, we prepared antibodies targeted to synthetic peptides encoded at the common C (peptide 204) and N (peptide 202) termini of the 266R and 235R T antigens and at the unique internal domain of the 266R T antigen (peptide 206). The specificity of each anti-peptide antibody was confirmed by immunoprecipitation of the 266R and 235R T antigens produced in Escherichia coli. Immunoprecipitation analysis of the E1A T antigens synthesized in Ad12-infected KB cells revealed the following. Antibody to the common C terminus recognized three T antigens with apparent Mrs of 43,000, 42,000, and 39,000 (43K, 42K, and 39K). All three forms were phosphorylated and were present in both the nucleus and the cytoplasm. The 43K and 42K T antigens were rapidly synthesized during a 10-min pulse with [35S]methionine in Ad12-infected cells. The 43K T antigen had a half-life of 20 min, the 42K T antigen had a longer half-life of about 40 min, and the 39K T antigen became the predominant E1A T antigen. Antibodies to the unique region immunoprecipitated the 43K T antigen but not the 42K and 39K T antigens. Antibody to the N terminus immunoprecipitated the 43K and 42K T antigens but not the 39K T antigen, suggesting that the 39K T antigen possessed a modified N terminus. Partial N-terminal amino acid sequence analysis showed that the 43K and 42K T antigens contain methionine at residues 1 and 5, as predicted from the DNA sequence, whereas no methionine was released from the 39K T antigen during the first six cycles of Edman degradation. We propose that the short-lived 43K T antigen is the primary product of the 13S mRNA, the 266R T antigen; the somewhat more stable 42K T antigen is the primary product of the 12S mRNA, the 235R T antigen.(ABSTRACT TRUNCATED AT 400 WORDS)     

Bcl-xL expression interferes with the effects of L-glutamine supplementation on hybridoma cultures

While feeding protocols and ectopic expression of anti-apoptotic genes have been used to improve the viability of hybridoma cell lines, the effect of the expression levels of survival genes on the behavior of hybridomas following nutrient supplementation is unknown. In this study, we compared the behavior of the Sp2/0-Ag14 hybridoma (Bcl-xL(low)) and the P3x63-Ag8.653 myeloma (Bcl-xL(high)) following culture supplementation with the amino acid L-glutamine (L-Gln). Our data revealed that L-Gln addition substantially increased Sp2/0-Ag14 cell viability and total cell density, concomitant with a decrease in the rate of cell death. This effect was not seen when other amino acids or D-glucose (D-Glc) replaced L-Gln. The improvement in the culture behavior of Sp2/0-Ag14 cells was attributed to a reduction in the rate of accumulation of apoptotic cells. On the other hand, L-Gln supplementation had only a limited effect on the growth of the P3x63-Ag8.653 cells. Interestingly, Sp2/0-Ag14 cells over-expressing Bcl-xL showed a culture behavior upon L-Gln complementation that was similar to the P3x63-Ag8.653 myeloma. These results suggest that the anti-apoptotic gene expression profile of hybridoma cells can markedly impact on the beneficial effects afforded by nutrient supplementation.     

Interaction between the chemotactic cAMP receptor and a detergent-insoluble membrane residue of Dictyostelium discoideum. Modulation by guanine nucleotides

Cells from Dictyostelium discoideum carry chemotactic cAMP receptors on their surface. Kinetic studies have revealed the existence of two slowly dissociating, high affinity receptor forms (SS and S) and one or more fast dissociating, low affinity forms (F) (Van Haastert, P.J.M., and De Wit, R.J.W. (1984) J. Biol. Chem. 259, 13321-13328). We have studied the interaction of these different cAMP-receptor types with a detergent-insoluble membrane residue. Isolated D. discoideum membranes were extracted with the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS), which was previously shown to be the only detergent in the presence of which cAMP receptor binding is completely preserved (Janssens, P. M. W., and Van Driel, R. (1986) Biochim. Biophys. Acta 885, 91-101). The protein composition of the CHAPS-insoluble membrane residue appeared to be similar to that of the Triton X-100-insoluble membrane skeleton. Cyclic AMP binding studies revealed a specific association of the slowly dissociating cAMP receptors (SS and S forms) with this CHAPS-insoluble residue. All fast dissociating (F type) receptors were solubilized by CHAPS. GTP induced a transition of 75% of the SS and S receptors to faster dissociating forms. This transition was accompanied by the release of an equal number of receptors from the residue. These effects of GTP required that the cAMP receptor was occupied, and were completely reversible. After removal of the guanine nucleotide SS and S type receptors reappeared, bound to the residue, with a t1/2 of 5-10 min at 0 degrees C. We conclude that a detergent-insoluble membrane residue is involved in signal transduction via the chemotactic cAMP receptor. Both receptor occupation and a guanine nucleotide binding protein control receptor-residue interaction.     

A soluble interleukin 2 receptor produced by a normal alloreactive human T cell clone binds interleukin 2 with low affinity

Several alloreactive human T cell clones derived from a rejected kidney graft were found to produce in their culture supernatants soluble interleukin 2 receptors (IL-2R) upon specific antigenic challenge (irradiated B cell line from the graft's donor). Among them, the 2B11, a high producer clone, was used to purify a soluble IL-2R preparation which was analyzed, in comparison with the high and low affinity cell-surface IL-2R expressed by 2B11 cells, for its parameters of interaction with a set of anti-IL-2R monoclonal antibodies (mAb) and IL-2. This soluble receptor purified by affinity chromatography (anti-IL-2R mAb column) and sodium dodecyl sulfate gel electrophoresis is composed of a single chain of 35,000 to 45,000 Da. Immunoradiometric assays (IRMA) at equilibrium were set up, using pairs of mAb directed against two separate epitopes on the Tac antigen of the human IL-2R, to measure the respective dissociation constant of these mAb for the soluble IL-2R. They were found to be identical to those found on the cell-surface IL-2R. A 1:1 stoichiometry between the two epitopes were found both on the membrane and soluble species. Competition experiments between membrane and soluble IL-2R for binding the mAb allowed the quantitative analysis of the concentration of soluble IL-2R without the need of amino acid analysis on purified material and set up a quantitative IRMA for the human soluble IL-2R (detection limit 5 pM). The affinity of the soluble IL-2R for IL-2 was determined by various techniques including an IRMA using an anti-IL-2R mAb and radiolabeled IL-2. The results obtained led us to conclude that the soluble IL-2R binds IL-2 with a dissociation constant (KD = 30 nM) identical to that found for the binding of IL-2 to low affinity cell-surface IL-2R (Tac antigen). Whereas 2.5% of cell-surface IL-2R expressed 2 days after antigenic stimulation of 2B11 cells were of high affinity for IL-2 (KD = 25 pM), no (less than 0.07%) high affinity binding sites could be detected on the purified soluble IL-2R. This soluble IL-2R therefore likely corresponds to a truncated, extracellular part of the membrane Tac antigen. The amounts of soluble Tac antigen produced by the 2B11 alloreactive human T cell clone did not exceed 1 nM and, as expected from the binding studies, did not affect IL-2-induced T cell proliferation. The physiologic and pathologic implications of our results are discussed.     

Immunoelectron microscopic detection of band 3 protein during erythroid cell differentiation by a monoclonal antibody

We created a monoclonal antibody, designated EB1 (IgM, kappa), that reacts with erythroblasts by fusion of P3-X63-Ag8.653 with splenocytes of rats immunized with erythroblastic islands isolated from mice spleens. Western blotting revealed that EB1 reacted with the band 3 protein of the erythrocytic membrane. It stained erythrocytes and erythroblasts, forming clusters in the bone marrow, splenic red pulp, and fetal liver, but did not stain other tissues in the cryostat sections. The EB1 antigen was detected during dimethyl sulfoxide-induced differentiation of murine erythroleukemia cells. Immunoelectron microscopy revealed that the EB1 antigen was expressed from the basophilic erythroblasts during normal erythroid differentiation. Preferential segregation of the EB1 antigen on the cell membrane of the nucleating erythroblasts was not observed. These results suggest that EB1 is specific for erythrocyte band 3 protein and may be useful for studying erythroid cell differentiation.     

Molecular structure of the toxin domain of heat-stable enterotoxin produced by a pathogenic strain of Escherichia coli. A putative binding site for a binding protein on rat intestinal epithelial cell membranes

Heat-stable enterotoxins are a family of toxin peptides that are produced by enterotoxigenic Escherichia coli and consist of 18 and 19 amino acid residues (Aimoto, S., Takao, T., Shimonishi, Y., Hara, S., Takeda, T., Takeda, Y., and Miwatani, T. (1982) Eur. J. Biochem. 129, 257-263). A synthetic fully toxic analog of the enterotoxin, Mpr5-STp(5-17), where Mpr is beta-mercaptopropionic acid and which consists of 13 amino acid residues from Cys5 to Cys17 in a heat-stable enterotoxin but is deaminated at its N terminus (Kubota, H., Hidaka, Y., Ozaki, H., Ito, H., Hirayama, T., Takeda, Y., and Shimonishi, Y. (1989) Biochem. Biophys. Res. Commun. 161, 229-235), has been crystalized from water, and its crystal structure has been solved by a direct method and refined by least square procedures to give an R factor of 0.089. The crystal belongs to the orthorhombic space group P2(1)2(1)2(1) with unit cell constants a = 21.010 (2) A, b = 27.621 (4) A, and c = 12.781 (1) A. The asymmetric unit of the crystals contains one peptide molecule with 13 water molecules. A right-hand spiral peptide backbone extends throughout the molecule. Three beta-turns are located along this spiral and fixed tightly by three intramolecular disulfide linkages. The actual structure predicts the biniding region on the enterotoxin to the receptor protein on the membrane of rat intestinal epithelial cells.     

Guanylyl cyclase is a heat-stable enterotoxin receptor.

Plasma membrane forms of guanylyl cyclase have been shown to function as natriuretic peptide receptors. We describe a new clone (GC-C) encoding a guanylyl cyclase receptor for heat-stable enterotoxin. GC-C encodes a protein containing an extracellular amino acid sequence divergent from that of previously cloned guanylyl cyclases; however, the protein retains the intracellular protein kinase-like and cyclase catalytic domains. Expression of GC-C in COS-7 cells results in high guanylyl cyclase activity. In addition, heat-stable enterotoxin from E. coli, but not natriuretic peptides, causes marked elevations of cyclic GMP and is specifically bound by cells transfected with GC-C. The enterotoxin fails to elevate cyclic GMP in nontransfected cells or in cells transfected with the natriuretic peptide/guanylyl cyclase receptors. These results show that a heat-stable enterotoxin receptor responsible for acute diarrhea is a plasma membrane form of guanylyl cyclase.     

pH6 antigen of Yersinia pestis interacts with plasma lipoproteins and cell membranes

The bacterial pathogen Yersinia pestis expresses a potential adhesin, the pH6 antigen (pH6-Ag), which appears as fimbria-like structures after exposure of the bacteria to low pH. pH6-Ag was previously shown to agglutinate erythrocytes and to bind to certain galactocerebrosides. We demonstrate that purified pH6-Ag selectively binds to apolipoprotein B (apoB)-containing lipoproteins in human plasma, mainly LDL. Binding was not prevented by antibodies to apoB. pH6-Ag interacted also with liposomes and with a lipid emulsion, indicating that the lipid moiety of the lipoprotein was responsible for the interaction. Both apoB-containing lipoproteins and liposomes prevented binding of pH6-Ag to THP-I monocyte-derived macrophages as well as pH6-Ag-mediated agglutination of erythrocytes. Binding of pH6-Ag to macrophages was not dependent on the presence of LDL receptors. Treatment of the cells with Triton X-100 or with methyl-beta-cyclodextrin indicated that the binding of pH6-Ag was partly dependent on lipid rafts. We suggest that interaction of pH6-Ag with apoB-containing lipoproteins could be of importance for the establishment of Y. pestis infections. Binding of lipoproteins to the bacterial surface could prevent recognition of the pathogen by the host defence systems. This might be important for the ability of the pathogen to replicate in the susceptible host.     

Comparative study of the interaction of CHAPS and Triton X-100 with the erythrocyte membrane

The zwitterionic detergent CHAPS, a derivative of the bile salts, is widely used in membrane protein solubilization. It is a “facial” detergent, having a hydrophilic side and a hydrophobic back. The objective of this work is to characterize the interaction of CHAPS with a cell membrane. To this aim, erythrocytes were incubated with a wide range of detergent concentrations in order to determine CHAPS partition behavior, and its effects on membrane lipid order, hemolytic effects, and the solubilization of membrane phospholipids and cholesterol. The results were compared with those obtained with the nonionic detergent Triton X-100. It was found that CHAPS has a low affinity for the erythrocyte membrane (partition coefficient K = 0.06 mM^− 1), and at sub-hemolytic concentrations it causes little effect on membrane lipid order. CHAPS hemolysis and phospholipid solubilization are closely correlated. On the other side, binding of Triton X-100 disorders the membrane at all levels, and has independent mechanisms for hemolysis and solubilization. Differential behavior was observed in the solubilization of phospholipids and cholesterol. Thus, the detergent resistant membranes (DRM) obtained with the two detergents will have different composition. The behaviors of the two detergents are related to the differences in their molecular structures, suggesting that CHAPS does not penetrate the lipid bilayer but binds in a flat position on the erythrocyte surface, both in intact and cholesterol depleted erythrocytes. A relevant result for Triton X-100 is that hemolysis is not directly correlated with the solubilization of membrane lipids, as it is usually assumed.     

Zwitterionic detergent mediated interaction of purified cytochrome P-450LM4 from 5,6-benzoflavone-treated rabbits with MADPH-cytochrome P-450 reductase

Hydroxylation of acetanilide catalyzed by purified cytochrome P-450LM4 and NADPH-cytochrome P-450 reductase was reconstituted with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). The optimum rate of production of 4-hydroxyacetanilide was observed between 3 and 7 mM CHAPS and was about half that with 0.05 mM dilauroylglyceryl-3-phosphocholine (di-12-GPC). At higher detergent concentrations, hydroxylase activity decreased until at 15-20 mM CHAPS the system was inactive. The effect of CHAPS on the state of aggregation of P-450LM4 and on interaction between the cytochrome and P-450 reductase alone and under turnover conditions was investigated by ultracentrifugation. At 4 mM CHAPS, P-450LM4 was hexameric to heptameric (Mr 369,000). Neither reductase nor reductase plus acetanilide and NADPH altered the state of P-450LM4 aggregation, suggesting that a stable 1:1 P-450/reductase complex did not form under turnover conditions. Replacing CHAPS with 0.05 mM di-12-GPC resulted in formation of heterogeneous P-450 oligomers (Mr greater than 480,000). At CHAPS concentrations where substrate hydroxylation did not occur (15 and 22 mM), P-450LM4 was shown by sedimentation equilibrium measurements to be dimeric and monomeric, respectively. P-450 reductase was shown to reduce monomeric P-450LM4 in the presence of NADPH. Thus, the dependence of hydroxylase activity on [CHAPS] may be related to the state of aggregation of the cytochrome. An apparent correlation between P-450 aggregation state and NADPH-supported hydroxylation was also observed with phenobarbital-inducible P-450LM2 in the presence of detergents [Dean, W.L., & Gray, R.D. (1982) J. Biol. Chem. 257, 14679-14685; Wagner, S.L., Dean, W.L., & Gray, R.D. (1984) J. Biol. Chem. 259, 2390-2395].