Ceramide mediates nanovesicle shedding and cell death in response to phosphatidylinositol ether lipid analogs and perifosine.

Anticancer phospholipids that inhibit Akt such as the alkylphospholipid perifosine (Per) and phosphatidylinositol ether lipid analogs (PIAs) promote cellular detachment and apoptosis and have a similar cytotoxicity profile against cancer cell lines in the NCI60 panel. While investigating the mechanism of Akt inhibition, we found that short-term incubation with these compounds induced rapid shedding of cellular nanovesicles containing EGFR, IGFR and p-Akt that occurred in vitro and in vivo, while prolonged incubation led to cell detachment and death that depended on sphingomyelinase-mediated generation of ceramide. Pretreatment with sphingomyelinase inhibitors blocked ceramide generation, decreases in phospho-Akt, nanovesicle release and cell detachment in response to alkylphospholipids and PIAs in non-small cell lung cancer cell lines. Similarly, exogenous ceramide also decreased active Akt and induced nanovesicle release. Knockdown of neutral sphingomyelinase decreased, whereas overexpression of neutral or acid sphingomyelinase increased cell detachment and death in response to the compounds. When transferred in vitro, PIA or Per-induced nanovesicles increased ceramide levels and death in recipient cells. These results indicate ceramide generation underlies the Akt inhibition and cytotoxicity of this group of agents, and suggests nanovesicle shedding and uptake might potentially propagate their cytotoxicity in vivo.

Ceramide-mediated apoptosis in lung epithelial cells is regulated by glutathione.

Reactive oxygen species (ROS) are mediators of lung injury, and glutathione (GSH) is the major nonprotein antioxidant that protects the cell from oxidative stress. We have recently shown that H(2)O(2) induces ceramide-mediated apoptosis in human lung epithelial cells. We hypothesized that ROS-mediated depletion of GSH plays a regulatory role in ceramide generation, and thus in the induction of apoptosis. Our present studies demonstrate that GSH at physiologic concentrations (1 to 10 mM) inhibits ceramide production in a time- and dose-dependent manner in A549 human alveolar epithelial cells. On the other hand, buthionine-sulfoximine-mediated depletion of intracellular GSH induces elevation of ceramide levels and apoptosis. In addition, GSH blocks H(2)O(2)-mediated induction of intracellular ceramide generation and apoptosis. These effects were not mimicked by oxidized GSH (GSSG) or other thiol antioxidants, such as dithiothreitol and 2-mercaptoethanol. Moreover, increase of intracellular H(2)O(2), mediated by inhibition of catalase by aminotriazole, also induces ceramide generation and apoptosis. These effects were blocked by N-acetylcysteine. Our results suggest that GSH depletion may be the link between oxidative stress and ceramide-mediated apoptosis in the lung.

Regulation of Staphylococcus aureus pathogenesis via target of RNAIII-activating Protein (TRAP).

Staphylococcus aureus can cause disease through the production of toxins. Toxin production is autoinduced by the protein RNAIII-activating protein (RAP) and by the autoinducing peptide (AIP), and is inhibited by RNAIII-inhibiting peptide (RIP) and by inhibitory AIPs. RAP has been shown to be a useful vaccine target site, and RIP and inhibitory AIPs as therapeutic molecules to prevent and suppress S. aureus infections. Development of therapeutic strategies based on these molecules has been hindered by a lack of knowledge of the molecular mechanisms by which they activate or inhibit virulence. Here, we show that RAP specifically induces the phosphorylation of a novel 21-kDa protein, whereas RIP inhibits its phosphorylation. This protein was termed target of RAP (TRAP). The synthesis of the virulence regulatory molecule, RNAIII, is not activated by RAP in the trap mutant strain, suggesting that RAP activates RNAIII synthesis via TRAP. Phosphoamino acid analysis shows that TRAP is histidine-phosphorylated, suggesting that TRAP may be a sensor of RAP. AIPs up-regulate the synthesis of RNAIII also in trap mutant strains, suggesting that TRAP and AIPs activate RNAIII synthesis via distinct signal transduction pathways. Furthermore, TRAP phosphorylation is down-regulated in the presence of AIP, suggesting that a network of signal transduction pathways regulate S. aureus pathogenesis.

Ceramide: a potential mediator of apoptosis in human retinal pigment epithelial cells.

PURPOSE: To investigate the signal transduction mechanisms involved in the cell death of human retinal pigment epithelial (RPE) cells after their exposure to either hydrogen peroxide (H(2)O(2)) or tri-butyl hydroxperoxide (tBH). METHODS: Cultured human RPE (hRPE) cells were treated with the chemical oxidants tBH and H(2)O(2) as well as with the synthetic ceramide analogs C(2), C(6), and dihydroceramide for different time periods. Apoptosis was determined by TUNEL staining and annexin-V labeling of phosphatidylserine exposure. Ceramide levels were quantified by the diacylglycerol kinase assay using thin-layer chromatography. RESULTS: H(2)O(2) and tBH caused a high level of apoptosis in the hRPE cells. At the same time, both of these oxidants induced an early and late increase in the intracellular production of ceramide, a lipid second messenger. Moreover, addition of C(2) and C(6) synthetic ceramides caused a high level of apoptosis in these hRPE cells. In contrast, treatment with the immediate precursor of ceramide, dihydroceramide, resulted in no apoptotic response. CONCLUSIONS: The results demonstrate that H(2)O(2) and tBH induce apoptosis in hRPE cells and suggest that the underlying signaling mechanism involves ceramide generation.

Ceramide path in human lung cell death.

Lung epithelium plays a significant role in modulating the inflammatory response to lung injury. Airway epithelial cells are targeted by hydrogen peroxide (H(2)O(2)) and oxygen radicals, which are agents commonly produced during inflammatory processes. The mechanisms and molecular sites affected by H(2)O(2) are largely unknown but may involve the induction of sphingomyelin (SM) hydrolysis to generate ceramide, which serves as a second messenger in initiating an apoptotic response. Here we show that exposure of human airway epithelial (HAE) cells to 50 to 100 microM H(2)O(2) induces within 5 to 10 min a greater than 2-fold activation of neutral sphingomyelinase activity with concomitant SM hydrolysis, ceramide generation, and apoptosis. On the other hand, activation of protein kinase C (PKC) by 12-O-tetradecanoylphorbol-13-acetate inhibits both H(2)O(2)-induced ceramide production and apoptosis. The apoptotic response could be restored by the addition of 25 microM cell-permeant C6-ceramide. These findings indicate that ceramide, the product of SM hydrolysis, plays an important role in H(2)O(2)-induced apoptosis in HAE cells, and that PKC counteracts ceramide-mediated apoptosis in these cells. We suggest that the mediation of epithelial cell apoptosis by ceramide and its inhibition by PKC constitute a central mechanism by which inflammatory processes are modulated in the epithelium of the lung.

Monoclonal anti-endothelial cell antibodies from a patient with Takayasu arteritis activate endothelial cells from large vessels.

OBJECTIVE: To create monoclonal anti-endothelial cell antibodies (mAECA) from a patient with Takayasu arteritis to evaluate their ability to activate human umbilical vein endothelial cells (HUVEC), and to characterize the mechanism of EC activation. METHODS: A panel of mAECA was generated from peripheral blood lymphocytes of a patient with Takayasu arteritis, using Epstein-Barr virus transformation. Activity against macrovascular EC (HUVEC) and microvascular EC (human bone marrow EC immortalized by SV40) antigens was detected by enzyme-linked immunosorbent assay. Inhibition studies were used to select the monoclonal antibodies (mAECA) which share the same EC epitope binding specificity as the total IgG-AECA from the Takayasu arteritis patient. The binding of the mAECA to human aortic EC was studied by immunohistochemistry. The secretion levels of interleukin-6 (IL-6) and von Willebrand factor (vWF) were determined, to serve as markers for EC activation. The activated EC were examined for the adherence of a monocytic cell line (U937), as well as for expression of vascular cell adhesion molecule 1, intercellular adhesion molecule 1, and E-selectin. In addition, nuclear extracts of the mAECA-treated EC were analyzed for the induction of translocation of nuclear factor kappaB (NF-kappaB), using a specific NF-kappaB oligoprobe in an electrophoretic mobility shift assay. RESULTS: Six mAECA were selected, the mixture of which produced 100% inhibition of binding of the original IgG (from the patient with Takayasu arteritis) to HUVEC. All mAECA possessed high activity against macrovascular EC, but none had significant antimicrovascular EC activity. The mAECA, but not normal human IgG, had anti-human aortic EC activity. Four of the 6 mAECA activated EC, manifested by increased IL-6 and vWF secretion. The 4 mAECA induced EC expression of adhesion molecules and increased adhesion of U937 monocytic cells to EC. In addition, these mAECA stimulated the nuclear translocation of the NF-kappaB transcription factor. CONCLUSION: Our findings suggest that AECA may directly stimulate EC in Takayasu arteritis through elevation of adhesion molecule expression associated with NF-kappaB activation and adhesion of monocytes, and may therefore play a pathogenic role in the development of the vasculopathy in Takayasu arteritis.

Peroxynitrite induces covalent dimerization of epidermal growth factor receptors in A431 epidermoid carcinoma cells.

Irreversible tyrosine modifications by inflammatory oxidants such as peroxynitrite (ONOO-) can affect signal transduction pathways involving tyrosine phosphorylation. The epidermal growth factor receptor (EGFR), a member of the c-ErbB receptor tyrosine kinase family, is involved in regulation of epithelial cell growth and differentiation, and possible modulation of EGFR-dependent signaling by ONOO- was studied. Exposure of epidermoid carcinoma A431 cells to 0.1-1.0 mM ONOO- resulted in tyrosine nitration on EGFR and other proteins but did not significantly affect EGFR tyrosine autophosphorylation. A high molecular mass tyrosine-phosphorylated protein (approximately 340 kDa) was detected in A431 cell lysates after exposure to ONOO-, most likely representing a covalently dimerized form of EGFR, based on immunoprecipitation and/or immunoblotting with alpha-EGFR antibodies and co-migration with ligand-induced EGFR dimers cross-linked with 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide. Covalent EGFR dimerization by ONOO- probably involved intermolecular dityrosine cross-linking and was enhanced after receptor activation with epidermal growth factor. Furthermore, irreversibly cross-linked EGFR was more extensively tyrosine-phosphorylated compared with the monomeric form, indicating that ONOO- preferentially cross-links activated EGFR. Exposure of A431 cells to ONOO- markedly reduced the kinetics of tyrosine phosphorylation of a downstream EGFR substrate, phospholipase C-gamma1, which may be related to covalent alterations in EGFR. Alteration of EGFR signaling by covalent EGFR dimerization by inflammatory oxidants such as ONOO- may affect conditions of increased EGFR activation such as epithelial repair or tumorigenesis.

EGF-Receptor phosphorylation and signaling are targeted by H2O2 redox stress.

Inflammation of the respiratory tract is associated with the production of reactive oxygen species, such as hydrogen peroxide (H2O2) and superoxide (O2-), which contribute extensively to lung injury in diseases of the respiratory tract. The mechanisms and target molecules of these oxidants are mainly unknown but may involve modifications of growth-factor receptors. We have shown that H2O2 induces epidermal growth factor (EGF)-receptor tyrosine phosphorylation in intact cells as well as in membranes of A549 lung epithelial cells. On the whole, total phosphorylation of the EGF receptor induced by H2O2 was lower than that induced by the ligand EGF. Phosphorylation was confined to tyrosine residues and was inhibited by addition of genistein, indicating that it was due to the activation of protein tyrosine kinase (PTK). Phosphoamino acid analysis revealed that although the ligand, EGF, enhanced the phosphorylation of serine, threonine, and tyrosine residues, H2O2 preferentially enhanced tyrosine phosphorylation of the EGF receptor. Serine and threonine phosphorylation did not occur, and the turnover rate of the EGF receptor was slower after H2O2 exposure. Selective H2O2-mediated phosphorylation of tyrosine residues on the EGF receptor was sufficient to activate phosphorylation of an SH2-group-bearing substrate, phospholipase C-gamma (PLC-gamma), but did not increase mitogen-activated protein (MAP) kinase activity. Moreover, H2O2 exposure decreased protein kinase C (PKC)-alpha activity by causing translocation of PKC-alpha from the membrane to the cytoplasm. These studies provide novel insights into the capacity of a reactive oxidant, such as H2O2, to modulate EGF-receptor function and its downstream signaling. The H2O2-induced increase in tyrosine phosphorylation of the EGF receptor, and the receptor's slower rate of turnover and altered downstream phosphorylation signals may represent a mechanism by which EGF-receptor signaling can be modulated during inflammatory processes, thereby affecting cell proliferation and thus having implications in wound repair or tumor formation.

H2O2 acts on cellular membranes to generate ceramide signaling and initiate apoptosis in tracheobronchial epithelial cells.

Hydrogen peroxide (H2O2) is an inflammatory oxidant which contributes to the pathogenesis of chronic diseases such as lung injury of the respiratory tract, atherosclerosis and cancer. The mechanisms and target sites of this reactive oxidant are mainly unknown. So far there are opposing reports as to whether reactive oxidants inhibit or promote apoptosis. We activated the death pathway in primary tracheobronchial epithelial (TBE) cells with H2O2 (20-200 microM) and observed the morphological changes, DNA laddering patterns, and DNA fragmentation associated with apoptosis. Elevation of ceramide with exogenous ceramide analogs was sufficient for apoptosis induction with the same characteristics and in the same time frame. H2O2 induced rapid sphingomyelin hydrolysis to ceramide, the elevation of which paralleled the induction of apoptosis. Furthermore, H2O2 acted directly on TBE cells membrane preparations devoid of nuclei, stimulating sphingomyelin hydrolysis through a neutral Mg2+ dependent sphingomyelinase (SMase). These data suggest that the formation of ceramide from sphingomyelin in the plasma membrane is a key event in H2O2-induced apoptosis in tracheobronchial epithelial cells.

Autoinducer of virulence as a target for vaccine and therapy against Staphylococcus aureus.

Staphylococcus aureus causes pathologies ranging from minor skin infections to life-threatening diseases. Pathogenic effects are largely due to production of bacterial toxin, which is regulated by an RNA molecule, RNAIII. The S. aureus protein called RAP (RNAIII activating protein) activates RNAIII, and a peptide called RIP (RNAIII inhibiting peptide), produced by a nonpathogenic bacteria, inhibits RNAIII. Mice vaccinated with RAP or treated with purified or synthetic RIP were protected from S. aureus pathology. Thus, these two molecules may provide useful approaches for the prevention and treatment of diseases caused by S. aureus.

EGF receptor phosphorylation is affected by ionizing radiation.

Eukaryotic cells respond to ionizing radiation with cell cycle arrest, activation of DNA repair mechanisms, and lethality. However, little is known about the molecular mechanisms that constitute these responses. Here we report that ionizing radiation enhances epidermal growth factor (EGF) receptor tyrosine phosphorylation in intact cells as well as in isolated membranes of A431 cells. Phosphoamino acid analysis revealed that ionizing radiation preferentially enhances tyrosine phosphorylation, while EGF enhances the phosphorylation of all three phosphoamino acids (serine, threonine and tyrosine) of the EGF receptor. In addition, radiation reduces the turnover rate of the EGF receptor, while EGF increases the rate of the receptor turnover and down-regulation. Moreover, the confined radiation-induced phosphorylation of tyrosine residues is inhibited by genistein, indicating that this phosphorylation of EGF receptor is due to protein tyrosine kinase activation. These studies provide novel insights into the capacity of radiation to modulate EGF receptor phosphorylation and function. The radiation-induced elevation in the EGF receptor tyrosine phosphorylation and the receptor's slower rate of turnover are discussed in terms of their possible role in cell growth and apoptosis modulation.

The rise and fall of ceramide and 1,2-diacylglycerol (DAG): modulation by transforming growth factor-beta 1 (TGF beta 1) and by epidermal growth factor (EGF).

Transforming growth factor beta 1 (TGF beta 1) increases the phosphorylation of the epidermal growth factor (EGF) receptor and inhibits the growth of A431 cells, but the mechanism of TGF beta 1 signaling is unknown. Recent studies from this and other laboratories suggest a novel sphingomyelin signal transduction pathway (1-4). Ceramide, which is generated by sphingomyelinase action, can be deacylated to sphingoid bases, which are potential inhibitors of protein kinase C (PKC). Ceramide appears to have bioeffector properties. Cell-permeable ceramide analogs stimulate monocytic differentiation of human leukemia (HL60) cells (1), as well as the phosphorylation of the EGF receptor at Thr669 in A431 human epidermoid carcinoma cells (2). Further studies (3,4) demonstrate the existence of a ceramide-activated protein kinase (CAPK) that may mediate some of these aspects. The present studies aim to investigate the mechanism of TGF beta 1 signaling and to explore whether TGF beta 1's pathway involves activation of PKC by 1,2-Diacylglycerol (DAG) and/or stimulation of a CAPK by ceramide. Ceramide and DAG levels of A431 cells are determined by thin layer chromatography (TLC) after treatment with either TGF beta 1 or with EGF. 100 pM TGF beta 1 treatment for 1 hr increases the cellular contents of DAG 2-fold. 20 nM EGF treatment for 15 min decreases it 0.5-fold. Ceramide levels are reduced 2-fold by TGF beta 1 and almost unaffected by EGF. To evaluate the involvement of other components of signal transduction, the effects of TGF beta 1 and EGF on PKC activity are studied. 20 nM EGF decreases membrane PKC activity to 0.5-fold of controls, whereas 100 pM TGF beta 1 treatment of A431 cells increases this activity 4-fold. Modulation of PKC activity is paralled by translocation of the enzyme between the cytosol and the membrane as determined by Western immunoblot analysis. These studies suggest that TGF beta 1 and EGF may have regulatory effects on both sphingolipid and phospholipid metabolisms which could transmodulate both the CAPK and the PKC mediated signal tranduction pathways.

The effect of ionizing radiation on signal transduction: antibodies to EGF receptor sensitize A431 cells to radiation.

What determines the degree of cell-resistance or sensitivity to ionizing radiation is not yet known. As a corollary to the ability of ceramide to induce apoptosis, some questions arise as to whether malignant cells escape apoptosis because of their inability to mount a ceramide response to inducers of apoptosis. To shed more light on the molecular mechanisms of tumor cell response to radiation, we tested whether exposure to ionizing radiation (of 200-1000 cGy) is associated with changes in ceramide levels in A431 tumor epithelial cells and whether the ability of ceramide to induce apoptosis is inhibited by protein kinase C (PKC) activation. Our studies demonstrate an immediate decrease in cellular levels of ceramide in response to radiation, while sphingosine levels increase. Under the same conditions the cellular 1,2-diacylglycerol (DAG) levels decrease as well, being accompanied by the translocation of PKC alpha from the membrane to the cytoplasm. Elevation of membrane PKC levels by 12-O-tetradecanoylphorbol 13-acetate (TPA) treatment had no effect on cell survival after irradiation, while treatment with EGF during and after irradiation augmented cell survival. Moreover, monoclonal antibodies to the EGF receptor (EGFR) sensitize cells to radiation by facilitating radiation-induced apoptosis. It is thus plausible that in human Squamous carcinoma cells, radiation activates predominantly the EGFR to induce resistance, while both sphingomyelin and PKC signal transduction pathways are deactivated and demonstrate no significant role in the modulation of the sensitivity or the resistance of A431 cells to ionizing radiation.

Transforming growth factor beta modulates phosphorylation of the epidermal growth factor receptor and proliferation of A431 cells.

Transforming growth factor beta (TGF-beta) increased the phosphorylation of the epidermal growth factor (EGF) receptor and inhibited the growth of A431 cells. Incubation with TGF-beta induced maximal EGF receptor phosphorylation to levels 1.5-fold higher than controls. Phosphorylation increased more prominently (4-5-fold) on tyrosine residues as determined by phosphoamino acid analysis and antiphosphotyrosine antibody immunoblotting. The kinase activity of EGF receptor was also elevated 2.5-fold when cells were cultured in the presence of TGF-beta. The antiproliferative effect of TGF-beta on A431 cells was accompanied by prolongation of G0-G1 phase and by morphological changes. TGF-beta augmented the growth inhibition of A431 cells which could be induced by EGF. In parallel, the specific EGF-induced increase in total phosphorylation of the EGF receptor was also augmented in the presence of TGF-beta. In cells cultured with TGF-beta, the phosphorylation of EGF receptor tyrosines induced by 20-min exposure to EGF was further increased 2-3-fold, suggesting additive effects upon receptor phosphorylation. EGF receptor activation by TGF-beta is characterized by kinetics quite distinct from that induced by EGF and therefore appears to take place through an independent mechanism. The TGF-beta-induced elevation in the phosphorylation of the EGF receptor may have a role in the augmented growth inhibition of A431 cells observed in the presence of EGF and TGF-beta.

Ceramide stimulates epidermal growth factor receptor phosphorylation in A431 human epidermoid carcinoma cells. Evidence that ceramide may mediate sphingosine action.

Recent studies suggest the existence of a signal transduction pathway involving sphingomyelin and derivatives (Kolesnick, R. N. (1989) J. Biol. Chem. 264, 7617-7623). The present studies compare effects of ceramide, sphingosine, and N,N-dimethylsphingosine on epidermal growth factor (EGF) receptor phosphorylation in A431 human epidermoid carcinoma cells. To increase ceramide solubility, a ceramide containing octanoic acid at the second position (C8-cer) was synthesized. C8-cer induced time- and concentration-dependent EGF receptor phosphorylation. This event was detectable by 2 min and maximal by 10 min. As little as 0.1 microM C8-cer was effective, and 3 microM C8-cer induced maximal phosphorylation to 1.9-fold of control. EGF (20 nM) increased phosphorylation to 2.1-fold of control. Sphingosine stimulated receptor phosphorylation over the same concentration range (0.03-3 microM) and to the same extent (1.8-fold of control) as ceramide. The effects of C8-cer and sphingosine were similar by three separate criteria, phosphoamino acid analysis, anti-phosphotyrosine antibody immunoblotting, and phosphopeptide mapping by high performance liquid chromatography. Phosphorylation occurred specifically on threonine residues. N,N-Dimethylsphingosine, a potential derivative of sphingosine, was less effective. Since sphingosine and ceramide are interconvertible, the level of each compound was measured under conditions sufficient for EGF receptor phosphorylation. C8-cer (0.1-1 microM) induced dose-responsive elevation of cellular ceramide from 132 to 232 pmol.10(6) cells-1. In contrast, cellular sphingosine levels did not rise. This suggests that C8-cer acts without conversion to sphingosine. Exogenous sphingosine (0.1-1 microM) also increased cellular ceramide levels to 227 pmol.10(6) cells-1, but did not increase its own cellular level of 12 pmol.10(6) cells-1. Higher sphingosine concentrations that induced no further increase in EGF receptor phosphorylation produced very large elevations in cellular sphingosine. Hence, at effective concentrations, both compounds elevated cellular ceramide but not sphingosine levels. Additional studies performed with [3H]sphingosine demonstrated that cells contain substantially less N,N-dimethylsphingosine than free sphingosine and, during short term incubation, convert less than 5% of added sphingosine to N,N-dimethylsphingosine. These studies provide evidence that ceramide may have bioeffector properties and suggest sphingosine may act in part by conversion to ceramide.

A simple and efficient enzymatic method for covalent attachment of DNA to cellulose. Application for hybridization-restriction analysis and for in vitro synthesis of DNA probes.

Single-stranded DNAs (ssDNAs) were covalently bound by a simple and efficient enzymatic method to a solid support matrix and used to develop several new procedures for gene analysis. The novel procedure to prepare a ssDNA stably coupled to a solid support employed T4 DNA ligase to link covalently oligo (dT)-cellulose and (dA)-tailed DNA. Beginning with essentially any double stranded DNA the procedure generates a ssDNA linked by its 5' end to a cellulose matrix in a concentration of over 500 ng per mg. DNA from the plasmid pBR322 (4300 bp) and a fragment of the beta-globin gene (1800 bp) were coupled to the solid support and used for several experiments. The ssDNAs on the cellulose efficiently hybridized with as little as 5 pg of complementary double-stranded DNAs. The DNA hybrids formed on the solid support were specifically and efficiently cleaved by restriction endonucleases. These specific restriction cuts were utilized for the diagnosis of correct sequences. In addition, the ssDNA on the solid support served as an efficient template for the synthesis of complementary ssDNAs. The complementary synthesized ssDNAs were uniformly labeled, more than two kilobases in size, and largely full length. About 85% of the ssDNA linked to cellulose was available for the synthesis of complementary DNA, and after strand-separation, the preparation was reusable for the synthesis of additional complementary DNA.

Functional molecular weight of the lac carrier protein from Escherichia coli as studied by radiation inactivation analysis.

Cytoplasmic membrane vesicles prepared from Escherichia coli containing multiple copies of the lac y gene were frozen in liquid nitrogen before or after generation of a proton electrochemical gradient (interior negative and alkaline) and irradiated with a high-energy electron beam at -135 degrees C. Subsequently, the lac carrier protein was extracted into octyl beta-D-glucopyranoside, reconstituted into proteoliposomes, and assayed for transport activity. Under all conditions tested, activity decreased as a single exponential function of radiation dosage, allowing straightforward application of target theory for determination of functional molecular mass. When lac carrier activity solubilized from nonenergized vesicles was assayed, the results obtained were consistent with a functional molecular size of 45-50 kDa, a value similar to the size of the protein as determined by other means. Similar values were obtained when the octyl beta-D-glucopyranoside extract was irradiated, and the target size observed for D-lactate dehydrogenase was in good agreement with the molecular size of this enzyme. Strikingly, when the same procedures were carried out with vesicles that were energized with appropriate electron donors prior to freezing and irradiation, a functional molecular size of 85-100 kDa was obtained for the lac carrier with no change in the target size of D-lactate dehydrogenase. In contrast, when the vesicles were energized under conditions in which the proton electrochemical gradient was collapsed, the target mass of the lac carrier returned to 45-50 kDa. The results indicate that the functional mass of the lac carrier protein is no greater than a dimer and suggest that the proton electrochemical gradient may cause an alteration in subunit interactions.

Topology of the lac carrier protein in the membrane of Escherichia coli.

Proteolysis of topologically sealed right-side-out and inside-out membrane vesicles from Escherichia coli with chymotrypsin, trypsin, or papain inactivates lac carrier function in a symmetrical manner. Concomitantly, the electrophoretic mobility of lac carrier protein photoaffinity labeled in situ with p-nitro[2-3H]phenyl-alpha-D-galactopyranoside is altered from a relative Mr of 33,000 to 20,000, and the time course of proteolysis is almost identical in vesicles of opposite polarities. In contrast, solubilization of the vesicles in NaDodSO4 followed by proteolysis causes fragmentation of the Mr 33,000 band into material that electrophoreses at the solvent front. Notably, proteolysis has no effect whatsoever on the ability of the lac carrier protein to bind substrate, as judged by photoaffinity-labeling experiments. Furthermore, the electrophoretic patterns of samples proteolyzed prior to photoaffinity labeling are the same as those observed when the procedures are reversed. These results show that the lac carrier protein spans the membrane and indicate that the binding site resides within a segment that is embedded in the bilayer.

Preparation, characterization, and properties of monoclonal antibodies against the lac carrier protein from Escherichia coli.

Monoclonal antibodies directed against the lac carrier protein purified from the membrane of Escherichia coli were prepared by somatic cell fusion of mouse myeloma cells with splenocytes from an immunized mouse. Several clones produce antibodies that react with the purified protein as demonstrated by solid-phase radioimmunoassay and by immunoblotting experiments; culture supernatants from the clones inhibit active transport of lactose in isolated membrane vesicles. Five stable clones were selected for expansion, formal cloning, and production of ascites fluid, and the antibodies secreted in vivo by each clone also were found to inhibit lactose transport. Antibody from hybridoma 4B1, an IgG2a immunoglobulin, inhibits active transport of lactose in proteoliposomes reconstituted with purified lac carrier and in right-side-out membrane vesicles. In contrast, the antibody has no effect on the generation of the proton electrochemical gradient by membrane vesicles nor does it alter the ability of vesicles containing the lac carrier to bind p-nitrophenyl-alpha-D-galactopyranoside. In order to achieve 50% inhibition of transport activity, a 2- to 3-fold molar excess of antibody to lac carrier is required, regardless of the amount of lac carrier in the membrane. Thus, the concentration of antibody required for a given degree of inhibition is proportional to the amount of lac carrier in the membrane. Finally, antibody-induced inhibition occurs within seconds, an observation suggesting that the epitope is accessible on the surface of the membrane.