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P C Tai

Publications and source records attributed to P C Tai.

At least 55 records · Page 3Linked to original sources
PubMed1989

Temperature-dependent insertion of prolipoprotein into Escherichia coli membrane vesicles and requirements for ATP, soluble factors, and functional SecY protein for the overall translocation process.

The requirements for the translocation of prolipoprotein into membrane vesicles were examined in an in vitro system. As measured by the eventual modification and processing of the prolipoprotein to form mature lipoprotein, the overall translocation process was found to require ATP hydrolysis, the presence of some heat-labile soluble cytoplasmic translocation factors, and the function of a cytoplasmic membrane protein, SecY/PrlA. However, the initial step of complete insertion of prolipoprotein into the membrane vesicles occurred without apparent requirements of a nucleotide, cytoplasmic translocation factors, or a functional SecY/PrlA membrane protein. Immunopurified prolipoprotein spontaneously inserted into membrane vesicles at elevated temperatures and required ATP and cytoplasmic translocation factors to form mature lipoprotein. The prolipoprotein inserted most efficiently into liposomes made of negatively charged phospholipids, indicating the importance of phospholipids in protein translocation. These results suggest that ATP hydrolysis and the actions of both cytoplasmic translocation factors and a functional SecY/PrlA membrane protein occur at a step(s) after the insertion of the precursors into membrane vesicles. The initial step of spontaneous insertion of prolipoprotein into membranes is in good agreement with membrane trigger hypothesis proposed by W. Wickner (Annu. Rev. Biochem. 48:23-45, 1979) and the helical hairpin hypothesis proposed by D. M. Engleman and T. A. Steitz (Cell 23:411-422, 1981).

Adenosine Triphosphate↗
PubMed1988

SecA protein is required for secretory protein translocation into E. coli membrane vesicles.

The soluble and membrane components of an E. coli in vitro protein translocation system prepared from a secA amber mutant, secA13[Am], contain reduced levels of SecA and are markedly defective in both the cotranslational and posttranslational translocation of OmpA and alkaline phosphatase into membrane vesicles. Moreover, the removal of SecA from soluble components prepared from a wild-type strain by passage through an anti-SecA antibody column similarly abolishes protein translocation. Translocation activity is completely restored by addition of submicrogram amounts of purified SecA protein, implying that the observed defects are solely related to loss of SecA function. Interestingly, the translocation defect can be overcome by reconstitution of SecA into SecA-depleted membranes, suggesting that SecA is an essential, membrane-associated translocation factor.

Alkaline Phosphatase↗
PubMed1988

SecA suppresses the temperature-sensitive SecY24 defect in protein translocation in Escherichia coli membrane vesicles.

Genetic analysis of protein secretion in Escherichia coli has identified secY/prlA and secA as components of the secretory apparatus. We have examined the roles of the secY(prlA) gene product (an integral membrane protein) and the soluble secA gene product in translocation of OmpA and alkaline phosphatase precursors in an in vitro system. The protein translocation defect of the secY24 mutation was recently demonstrated in vitro as was its suppression by an S300 extract. We show here that the extract was essentially inactive in SecY24 suppression when SecA protein was removed from it by immunoaffinity chromatography. Furthermore, purified SecA protein suppressed the SecY24 defect. Preincubation of the inactivated SecY24 membrane vesicles either with S300 containing SecA or with purified SecA protein reconstituted the membranes and restored the translocation activity when assayed in the absence of additional soluble proteins. These results suggest that the SecY24 translocation defect is suppressed by SecA interacting, directly or indirectly, with SecY24 on the cytoplasmic membrane.

Bacterial Proteins↗
PubMed1988

Requirement of heat-labile cytoplasmic protein factors for posttranslational translocation of OmpA protein precursors into Escherichia coli membrane vesicles.

The involvement of possible cytoplasmic factors in ATP-dependent postttranslational translocation of proteins into Escherichia coli membrane vesicles was examined. The precursor of OmpA protein was partially purified by DEAE-cellulose chromatography, and its translocation was found to require material from the soluble cytoplasmic fraction. The fractionated active cytoplasmic translocation factor (CTF) was protease sensitive, micrococcal nuclease insensitive, N-ethylmaleimide resistant, and heat labile. The heat sensitivity of the CTF allowed its specific and preferential inactivation in the crude-precursor synthesis mixture, which provided a simple and rapid assay procedure for the factor during purification. Two active fractions were detected upon further fractionation: the major one was about 8S in sucrose gradient centrifugation and 120 kilodaltons by Sephadex filtration, whereas the other was about 4S and 60 kilodaltons in sucrose gradient centrifugation and by Sephadex filtration, respectively. The active fractions could also be fractionated by DEAE-Sepharose chromatography. These CTFs are apparently different from the previously reported 12S export factor (M. Muller and G. Blobel, Proc. Natl. Acad. Sci. USA 81:7737-7741, 1984).

Bacterial Outer Membrane Proteins↗
PubMed1987

Synergism between eosinophil cationic protein and oxygen metabolites in killing of schistosomula of Schistosoma mansoni.

To study the cytotoxic reactions responsible for mediating eosinophil damage to schistosomula of Schistosoma mansoni, we have used cytoplasts (eosinophil or neutrophil vesicles devoid of granules and nuclei, with an intact oxidase in their plasma membrane) in combination with purified eosinophil cationic protein (ECP) or major basic protein (MBP) in a cytotoxicity test toward schistosomula. Suboptimal concentrations of ECP (10(-6) M) or MBP (10(-6) M) resulting in less than 10% killing were used in combination with cytoplasts. Cytoplasts alone in the presence of immune serum tested over a wide range of cytoplast:schistosomula ratios generated superoxide and hydrogen peroxide, but were unable to damage schistosomula. However, when a suboptimal ECP concentration (10(-6) M) was combined with neutroplasts or eosinoplasts, 43.9% +/- 8.5 (n = 7) and 24.7% +/- 9.8 (n = 3), respectively, of the schistosomula were killed. Oxygen metabolites were responsible for the synergism, because cytoplasts from a patient with chronic granulomatous disease were unable to act in synergy with ECP. In contrast to ECP, no synergism was found between cytoplasts and MBP (10(-6) to 2 X 10(-5)M). These results show that oxygen metabolites are important for the killing of schistosomula by lowering the concentration of ECP needed to inflict damage.

Animals↗
PubMed1987

Deposits of eosinophil granule proteins in cardiac tissues of patients with eosinophilic endomyocardial disease.

Eosinophilic endomyocardial disease is a complication of the hypereosinophilic syndrome and of several other disorders associated with high blood eosinophil counts. Eosinophil granule proteins may be involved in the development of these lesions. This multi-centre study investigated whether these proteins could be demonstrated within the cardiac tissues of eighteen patients with eosinophilic endomyocardial disease. Serial sections of tissue taken at necropsy or at cardiac biopsy were stained for eosinophil major basic protein by indirect immunofluorescence and for eosinophil cationic protein, eosinophil protein-X, and activated eosinophils by means of alkaline-phosphatase-linked monoclonal antibodies. Activated eosinophils and secreted eosinophil granule proteins were most evident within the necrotic and later stage thrombotic lesions and were found mainly within the areas of acute tissue damage in the endocardium and in the walls of small blood vessels. These findings suggest that eosinophil granule proteins are involved in cardiac injury, producing muscle damage and vascular injury which lead to the development of endomyocardial fibrosis.

Acute Disease↗
PubMed1987

Protein translocation into Escherichia coli membrane vesicles is inhibited by functional synthetic signal peptides.

A synthetic peptide corresponding to the signal sequence of wild type Escherichia coli lambda-receptor protein (LamB) inhibits in vitro translocation of precursors of both alkaline phosphatase and outer membrane protein A into E. coli membrane vesicles (half-maximal inhibition at 1-2 microM). By contrast, the inhibitory effect was nearly absent in a synthetic peptide corresponding to the signal sequence from a mutant strain that harbors a deletion mutation in the LamB signal region and displays an export-defective phenotype for this protein in vivo. Two peptides derived from pseudorevertant strains that arose from the deletion mutant and exported LamB in vivo were found to inhibit in vitro translocation with effectiveness that correlated with their in vivo export ability. Controls indicated that these synthetic signal peptides did not disrupt the E. coli membrane vesicles. These results can be interpreted to indicate that the presequences of exported proteins interact specifically with a receptor either in the E. coli inner membrane or in the cytoplasmic fraction. However, biophysical data for the family of signal peptides studied here reveal that they will spontaneously insert into a lipid membrane at concentrations comparable to those that cause inhibition. Hence, an indirect effect mediated by the lipid bilayer of the membrane must be considered.

Alkaline Phosphatase↗
PubMed1987

Biochemical evidence for the secY24 defect in Escherichia coli protein translocation and its suppression by soluble cytoplasmic factors.

The secY (prlA) gene product is an integral membrane protein that has been identified genetically as one of the central components of the Escherichia coli protein translocation machinery. We have examined the effect of the secY24 (temperature-sensitive) mutation on the protein translocation activity of E. coli inverted membrane vesicles. Vesicles isolated from cells carrying this allele and grown at the nonpermissive temperature (42 degrees C) were less than 1% as active in translocation as vesicles isolated from an isogenic secY+ strain under the same conditions. Vesicles from the mutant strain grown at the permissive temperature (32 degrees C) were partially active, but those vesicles preincubated at 40 degrees C lost 90% of their activity. Moreover, the secY24 translocation defect on in vivo- or in vitro-inactivated vesicles was suppressed, or compensated, by an S300 soluble fraction from wild-type cells or from secY24 cells grown at nonpermissive temperature. The suppressing factor(s) was heat-labile and sensitive to proteinase K. These results provide biochemical evidence for the essential role of SecY in the translocation process and indicate that the translocation defect of SecY24 membranes can be compensated for by supplementing with additional soluble cytoplasmic proteins.

Bacterial Proteins↗
PubMed1987

Killing of Plasmodium falciparum by eosinophil secretory products.

The multiplication of two strains of Plasmodium falciparum in culture, as measured by [3H]hypoxanthine incorporation, was inhibited in a dose-dependent manner by granule proteins secreted by purified eosinophils obtained from patients with the hypereosinophilic syndrome. Morphological examination revealed the presence of abnormal parasites inside erythrocytes, indicating that they were killed in situ, and the later stages of the developmental cycle were found to be most susceptible to these toxic effects. A monoclonal antibody against eosinophil cationic protein partially blocked the inhibitory effect, suggesting that it was caused by more than one of the eosinophil granule proteins. Thus some of the antimalarial effects of molecules such as the tumor necrosis factor, which activates eosinophils, may be mediated through the enhanced production of eosinophil secretion products.

Antibodies, Monoclonal↗
PubMed1987

Nucleotide sequences and expression in Escherichia coli of the in-phase overlapping Pseudomonas aeruginosa plcR genes.

The translation products of chromosomal DNAs of Pseudomonas aeruginosa encoding phospholipase C (heat-labile hemolysin) have been examined in T7 promoter plasmid vectors and expressed in Escherichia coli cells. A plasmid carrying a 4.7-kilobase (kb) DNA fragment was found to encode the 80-kilodalton (kDa) phospholipase C as well as two more proteins with an apparent molecular mass of 26 and 19 kDa. Expression directed by this DNA fragment with various deletions suggested that the coding region for the two smaller proteins was contained in a 1-kb DNA region. Moreover, the size of both proteins was reduced by the same amount by an internal BglII-BglII DNA deletion, suggesting that they were translated from overlapping genes. Similar results were obtained with another independently cloned 6.1-kb Pseudomonas DNA, which in addition coded for a 31-kDa protein of opposite orientation. The nucleotide sequence of the 1-kb region above revealed an open reading frame with a signal sequence typical of secretory proteins and a potential in-phase internal translation initiation site. Pulse-chase and localization studies in E. coli showed that the 26-kDa protein was a precursor of a secreted periplasmic 23-kDa protein (PlcR1) while the 19-kDa protein (PlcR2) was mostly cytoplasmic. These results indicate the expression of Pseudomonas in-phase overlapping genes in E. coli.

Amino Acid Sequence↗
PubMed1987

Effects of antibiotics and other inhibitors on ATP-dependent protein translocation into membrane vesicles.

The effects of several membrane antibiotics and other agents on ATP-dependent protein translocation were examined in membrane vesicles under conditions where no significant proton motive force was present. The membrane perturbants ethanol and procaine abolished ATP-dependent protein translocation. Phenethyl alcohol at low concentrations abolished translocation, whereas at high concentrations it allowed precursors to be translocated but inhibited their processing. Translocation of precursors promoted by phenethyl alcohol was temperature dependent and occurred without an added energy source but was enhanced by ATP. However, such precursors could not be further processed to mature forms upon removal of the alcohol. The membrane-active antibiotics polymyxin B and gramicidin S were strong inhibitors of translocation, whereas gramicidin D, cerulenin, and mycobacillin had no effect even at higher concentrations, indicating some specificity in interference with protein translocation. Duramycin, an antibiotic previously shown to affect protein-lipid interaction, severely impaired protein translocation. These results showed that membrane structures play important roles, either directly or indirectly, in protein translocation. Chelating agents 1,10-phenanthroline and EDTA, but not EGTA [ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid], also abolished protein translocation.

Adenosine Triphosphate↗
PubMed1986

Role of specific IgE antibodies in peroxidase (EPO) release from human eosinophils.

After the demonstration of cytophilic IgE immunoglobulins (Ig) on human blood and lung eosinophils, their role in cell activation was studied by eosinophil peroxidase (EPO) assay. Hypodense human eosinophils from filariasis-infected patients were activated by anti-human Ig or various antigens. A selective release of EPO occurred after incubation with anti-human IgE, but not with anti-human IgG. The activation by antigens showed a strict antibody specificity of cytophilic IgE antibodies. The direct involvement of IgE antibodies in activation by the specific antigen was evidenced by inhibition experiments with aggregated human IgE myeloma protein. Circulating IgE antibodies exhibiting the same specificity and able to induce EPO release were detected in the sera from filariasis patients by a passive sensitization assay. Only the hypodense eosinophils were able to release EPO after IgE-dependent activation both in the direct assay and in the passive sensitization test, confirming the functional heterogeneity of human eosinophils. These results suggest that the interaction between IgE antibodies and human eosinophils can play a role both in protective immunity and pathology by releasing active pharmacologic mediators.

Antibodies, Anti-Idiotypic↗
PubMed1986

Misread protein creates membrane channels: an essential step in the bactericidal action of aminoglycosides.

Among the pleiotropic effects of aminoglycosides, their irreversible uptake and their blockade of initiating ribosomes have appeared to explain their bactericidal action, while the contributions of translational misreading and membrane damage and the mechanism of that damage have remained uncertain. We now present evidence that incorporation of misread proteins into the membrane can account for the membrane damage. The bactericidal action thus appears to result from the following sequence, in which each step is essential: slight initial entry of the antibiotic; interaction with chain-elongating ribosomes, resulting in misreading; incorporation of misread protein into the membrane, creating abnormal channels; increased (and irreversible) entry through these channels, and hence increased misreading and formation of channels; and, finally, blockade of initiating ribosomes. This mechanism can account for several previously unexplained observations: that streptomycin uptake requires protein synthesis during, but not after, the lag before the membrane damage; that streptomycin-resistant cells, which fail to take up streptomycin, can do so after treatment by another aminoglycoside; and that puromycin at moderate concentrations accelerates streptomycin uptake, while high concentrations (which release shorter chains) prevent it. In addition, puromycin, prematurely releasing polypeptides of normal sequence, also evidently creates channels, since it is reported to promote streptomycin uptake even in streptomycin-resistant cells. These findings imply that normal membrane proteins must be selected not only for a hydrophobic anchoring surface, but also for a tight fit in the membrane.

Alkaline Phosphatase↗
PubMed1986

Role of ribosome degradation in the death of starved Escherichia coli cells.

In Escherichia coli cultures limited for phosphate, the number of ribosomal particles was reduced to a small percentage of its earlier peak value by the time the viable cell count began to drop; the 30S subunits decreased more than the 50S subunits. Moreover, the ribosomal activity was reduced even more: these cells no longer synthesized protein, and their extracts could not translate phage RNA unless ribosomes were added. The translation initiation factors also disappeared, suggesting that they become less stable when released from their normal attachment to 30S subunits. In contrast, elongation factors, aminoacyl-tRNA synthetases, and tRNA persisted. During further incubation, until viability was reduced to 10(-5), the ribosomal particles disappeared altogether, while tRNA continued to be preserved. These results suggest that an excessive loss of ribosomes (and of initiation factors) may be a major cause of cell death during prolonged phosphate starvation.

Bacterial Proteins↗
PubMed1986

Roles of H+-ATPase and proton motive force in ATP-dependent protein translocation in vitro.

Membrane vesicles from an Escherichia coli mutant with a deletion of the uncBC operon required ATP to translocate proteins, thus ruling out an essential role of F1F0-H+-ATPase in ATP-dependent protein translocation. Moreover, proteins could be translocated in the absence of proton motive force. At suboptimal ATP concentrations, D-lactate stimulated protein translocation, indicating that proton motive force, although insufficient to support translocation, could facilitate the process.

Adenosine Triphosphate↗
PubMed1986

Effects of nucleotides on ATP-dependent protein translocation into Escherichia coli membrane vesicles.

We have shown previously that Escherichia coli can translocate the same protein either co- or posttranslationally and that ATP hydrolysis is essential for the posttranslational translocation of the precursors of alkaline phosphatase and OmpA protein into inverted E. coli membrane vesicles. ATP-dependent protein translocation has now been further characterized. In the absence of exogenous Mg2+, dATP, formycin A-5'-triphosphate, ATP-alpha-S, and N1-oxide-ATP could replace ATP, but many other nucleotides were not only ineffective but inhibited ATP-dependent translocation. The inhibitors included nonhydrolyzable ATP analogs, ATP-gamma-S, 8-azido-ATP, AMP, ADP, cyclic AMP, PPi, and tripolyphosphate. On the other hand, adenosine, adenosine 5'-tetraphosphate, and N1,N6-etheno-ATP neither supported nor inhibited translocation. Moreover, photoaffinity labeling of azido-adenine nucleotides rendered membranes inactive for subsequent ATP-dependent protein translocation. These results suggest that protein translocation involves at least an ATP-binding site in the membrane and hydrolysis of ATP and that both the adenosine and phosphate moieties of ATP play a role.

Adenosine Triphosphate↗
PubMed1986

The in vivo cardiotoxic effect of eosinophilic cationic protein in an animal preparation.

We studied the in vivo effects of eosinophilic cationic protein (ECP) on DBA/2 mice, and compared the cardiac lesions caused by ECP with those caused by encephalomyocarditis (EMC) virus. ECP caused myocarditis in two of five mice (40%), and EMC virus did so in five of five mice (100%). Cardiac lesions of ECP were mild and limited to the right ventricular wall, which differed from those in the mice with EMC virus inoculation, where both the right and left ventricles were involved. This experiment is the first demonstration of the in vivo cardiotoxicity of ECP.

Animals↗