Chemical alterations in cell envelopes of polymyxin-resistant Pseudomonas aeruginosa isolates.

Cell envelopes from Pseudomonas aeruginosa strains resistant to polymyxin were compared with cell envelopes from polymyxin-sensitive strains as to their content of total protein, carbohydrate, and 2-keto-3-deoxyoctonate and as to their protein composition as determined by slab polyacrylamide gel electrophoresis. The cell envelopes of the polymyxin-resistant strains had reduced amounts of lipopolysaccharide, as indicated a reduction in both carbohydrate and 2-keto-3-deoxyoctonate concentrations, and a greatly altered protein composition as shown by polyacrylamide gel electrophoresis. There was a quantitative increase in total cell envelop protein in these strains. However, those protein bands identified as being major outer membrane proteins upon polyacrylamide gel electrophoresis of separated outer and cytoplasmic membranes were reduced greatly in concentration in the polymyxin-resistant cell envelopes. Thus, it appears that polymyxin resistance in these strains is associated with the alteration of the outer membrane through a loss of lipopolysaccharide and outer membrane proteins.     

人红细胞膜带3蛋白的提纯与鉴定

提出了一种分离纯化人红细胞膜带3蛋白的不含血型糖蛋白制剂的改良方法:先后用0.89％NaCl、20mM pH8.0磷酸钠和0.05％TritonX-100处理膜除去膜骨骼蛋白类和血型糖蛋白,再用自行设计的凝胶制备电泳装置进一步纯化。冰冻干燥的制剂是均质的,得率为18.5±2.85％,它的分子量、氨基酸组成和紫外吸收光谱与文献报道基本相同。     

Outer membrane of Pseudomonas aeruginosa: heat- 2-mercaptoethanol-modifiable proteins.

A number of polyacrylamide gel systems and solubilization procedures were studied to define the number and nature of "major" polypeptide bands in the outer membrane of Pseudomonas aeruginosa. It was shown that five of the eight major outer membrane proteins were "heat modifiable" in that their mobility on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was determined by the solubilization temperature. Four of these heat-modifiable proteins had characteristics similar to protein II of the Escherichia coli outer membrane. Addition of lipopolysaccharide subsequent to solubilization caused reversal of the heat modification. The other heat-modifiable protein, the porin protein F, was unusually stable to sodium dodecyl sulfate. Long periods of boiling in sodium dodecyl sulfate were required to cause conversion to the heat-modified form. This was demonstrated both with outer membrane-associated and purified lipopolysaccharide-depleted protein F. Furthermore, lipopolysaccharide treatment had no effect on the mobility of heat-modified protein F. Thus it is concluded that protein F represents a new class of heat-modifiable protein. It was further demonstrated that the electrophoretic mobility of protein F was modified by 2-mercaptoethanol and that the 2-mercaptoethanol and heat modification of mobility were independent of one another. The optimal conditions for the examination of the outer membrane proteins of P. aeruginosa by one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis are discussed.     

Isolation, characterization and protein and polar lipid compositions of Paracoccus denitrificans outer membrane

Sucrose density gradient centrifugation of Paracoccus denitrificans strains ATCC 13543 and ATCC 17741 cell envelopes plus poly-β-hydroxybutyrate, isolated from organisms broken using a French pressure cell, revealed three bands of densities: I, 1.16 g/ml; II, 1.19 g/ml; III, 1.24 g/ml. On the basis of chemical and enzymatic assays and sodium dodecyl sulfate-polyacrylamide gel electrophoresis the bands were identified as: I, cytoplasmic membrane; II, poly-β-hydroxybutyrate; III, outer membrane plus poly-β-hydroxybutyrate. Poly-β-hydroxybutyrate was removed by increased low-speed centrifugation before deposition of cell envelopes. Density gradient centrifugation of cell envelopes gave a simple pattern of two bands, cytoplasmic and outer membranes. In both strains outer membranes showed a broad protein band at M_r 70 000–83 000 upon SDS-polyacrylamide gel electrophoresis of samples solubilized at 25°C, which was not present in samples solubilized at 100°C, where a single major band was present of M_r 32 000 in strain ATCC 13543 and 35 000 in strain ATCC 17741. The major outer membrane protein stained positively for lipid in both strains, as did an M_r 70 000 protein, which was the second major protein in strain ATCC 17741. The second major outer membrane protein of stain ATCC 13543 had an M_r of 20 000 in unheated samples but 23 000 in heated samples. This protein was not present in strain ATCC 17741. Quantitative data on the polar lipid compositions of cell envelope fractions are presented.     

Isolation and protein composition of the outer membrane ofErwinia amylovora

The outer membrane (OM) ofErwinia amylovora was separated from the cytoplasmic membrane either by isopycnic sucrose density gradient centrifugation or by treating the envelope preparation with sodium lauroylsarcosine. Outer membranes, prepared by using either method, were similar in the content of the major proteins as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The wild-type strains ofE. amylovora (E9, E8, EA178, EA198, EA225, EA273) had—in common in the OM—two major protein bands of apparent molecular weights of 15,800 (protein z) and 38,000 (protein y). The OM protein profiles of the virulent wild-type (E9) and the avirulent mutant (E8) were identical but differed from otherE. amylovora strains by the presence of an additional major protein band of approximately 40,000 (protein x). In strain E9, protein x appeared to be associated with the peptidoglycan, whereas proteins y and z were apparently not peptidoglycan-bound.     

Isolation and partial characterization of Borrelia burgdorferi inner and outer membranes by using isopycnic centrifugation.

In order to characterize the protein composition of the outer membrane of Borrelia burgdorferi, we have isolated inner and outer membranes by using discontinuous sucrose density step gradients. Outer and inner membrane fractions isolated by this method contained less than 1 and 2%, respectively, of the total lactate dehydrogenase activity (soluble marker) in cell lysate. More importantly, the purified outer membranes contained less than 4% contamination by the C subunit of F1/F0 ATPase (inner membrane marker). Very little flagellin protein was present in the outer membrane sample. This indicated that the outer membranes were relatively free of contamination by cytoplasmic, inner membrane or flagellar components. The outer membrane fractions (rho = 1.19 g/cm3) contained 0.15 mg (dry weight) of protein per mg. Inner membrane samples (rho = 1.12 g/cm3) contained 0.60 mg (dry weight) of protein per mg. Freeze-fracture electron microscopy revealed that the outer membrane vesicles contained about 1,700 intramembranous particles per micron 2 while inner membrane densities for inner and outer membranes. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and nonequilibrium pH gel electrophoresis-SDS-PAGE analyses of inner and outer membrane samples revealed several proteins unique to the inner membrane and 20 proteins that localized specifically to the outer membrane. This analysis clearly shows that the inner and outer membranes isolated by this technique are unique structures.     

Pseudomonas aeruginosa outer membrane permeability: isolation of a porin protein F-deficient mutant.

A mutant of Pseudomonas aeruginosa severely deficient in outer membrane protein F levels was isolated by screening heavily mutagenized strains for membrane protein alterations on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. To provide a basis for phenotypic comparison, three independent spontaneous revertants with normal protein F levels were isolated. Neither the protein F-deficient mutant nor its revertants had gross surface alterations as judged by their sensitivities to 31 phages with diverse receptors and their low degrees of leakage of periplasmic beta-lactamase into the supernatant. Outer membrane permeability was measured in whole cells by examining the rates of hydrolysis of a chromogenic beta-lactam, nitrocefin, by periplasmic RP1-encoded beta-lactamase. It was found that the outer membrane permeabilities of wild-type and protein F revertant strains were similar, but low when compared with those of Escherichia coli and an antibiotic-supersusceptible mutant Z61 of P. aeruginosa. The loss of protein F caused a further significant decrease in outer membrane permeability. The results suggest that protein F is a pore-forming protein in vivo and that only a small proportion, as few as 1 in 400, of the protein F molecules form active functional channels in vivo.     

Separation of the cytoplasmic and outer membrane of Pseudomonas aeruginosa PAQ.

A method is described for the isolation of the cytoplasmic and outer membranes of $\text{Pseudomonas aeruginosa}$ PAO.1. The cytoplasmic membrane exhibits nicotinamide adenine dinucleotide oxidoreductase, lactate dehydrogenase DD-carboxypeptidase and succinate dehydrogenase activities. The outer membrane is rich in 2-keto-3-deoxyoctonate and exhibits phospholipase A and DD-carboxypeptidase activity. At least 25 protein species have been detected in the cytoplasmic membrane by polyacrylamide gel electrophoresis. Using the same technique, the outer membrane contains only five protein species of molecular weights, 56,000, 53,000, 38,000, 21,000 and 16,000.     

Separation of inner and outer membranes of Rickettsia prowazeki and characterization of their polypeptide compositions.

Rickettsia prowazeki were disrupted in a French pressure cell and fractionated into soluble (cytoplasm) and envelope fractions. The envelope contained 25% of the cell protein, with the cytoplasm containing 75%. Upon density gradient centrifugation, the envelope fraction separated into a heavy band (1.23 g/cm3) and a lighter band (1.19 g/cm3). The heavy band had a high content of 2-keto-3-deoxyoctulosonic acid, a marker for bacterial lipopolysaccharide, but had no succinic dehydrogenase, a marker for cytoplasmic membrane activity, and therefore represented outer membrane. The lighter band exhibited a high succinate dehydrogenase activity, and thus contained inner (cytoplasmic) membrane. Outer membrane purified by this method was less than 5% contaiminated by cytoplasmic membrane; however, inner membrane from the gradient was as much as 30% contaminated by outer membrane. The protein composition of each cellular fraction was characterized by sodium dodecyl sulfate--polyacrylamide gel electrophoresis. The outer membrane contained four major proteins, which were also major proteins of the whole cell. The cytoplasmic membrane and soluble cytoplasm exhibited a more complex pattern on gels.     

Influence of sodium dodecyl sulphate quality on the electrophoretic mobility of the outer membrane proteins of mucoid and non-mucoid Pseudomonas aeruginosa

The electrophoretic mobilities of proteins F and H1 from the outer membrane of Pseudomonas aeruginosa (mucoid and non-mucoid) in polyacrylamide gel electrophoresis were affected by the quality of sodium dodecyl sulphate (SDS) used. In particular, the sodium tetradecyl sulphate impurity present in crude SDS influenced the mobilities of F and H1. These observations explain conflicting reports on changes in outer membrane proteins with strain and growth conditions. Synthesis of H1 was induced by growth in magnesium depleted medium but repressed when calcium or manganese were added to magnesium depleted medium.     

Protein alterations in isolated outer membranes of polymyxin-resistantPseudomonas aeruginosa isolates

Isolated outer membranes fromPseudomonas aeruginosa strains resistant to polymyxin were compared with isolated outer membranes from polymyxin-sensitive strains as to their protein composition as determined by slab polyacrylamide gel electrophoresis. Both the porin protein and the H1 protein were decreased greatly in concentration in the outer membranes from the polymyxin-resistant strains. This confirms previous findings reduced concentrations of these proteins in cell envelopes of these strains. No evidence was found for these decreases being the result of an artifactual loss of these proteins from the outer membrane due to conditions used for growth or for preparation of cell envelopes. Nevertheless, the role of these outer membrane proteins in mediating polymyxin resistance is uncertain.     

Heterogeneity of human erythrocyte band 3 analyzed by two-dimensional gel electrophoresis

Human erythrocyte membrane and purified band 3 were separated initially by isoelectric focusing and then examined in a second dimension by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Band 3 was segregated into three major bands whether the protein was contained within the membranes or was present in the isolated state. The isoelectric points of these major bands were 5.25, 5.35 and 5.70. Of chymotryptic fragments of band 3, the 60-kDa fragment was also separated into three major bands whose pI values were 4.75, 5.10 and 5.30. The multiplicity of band 3 appears to be due to different charges carried by the peptide(s) and is not ascribed to oxidation of band 3 during its preparation. Isoelectric points of the purified 60-kDa fragment were different from the pI values of the fragment coexisting with the complementary 35-kDa fragment, in which case the pI values were exactly the same as those of intact band 3. This suggests that these fragments interact tightly in situ even after being cleaved by chymotrypsin, and the tight interaction must still be present during electrophoresis in the first dimension.     

Identification of pilin pools in the membranes of Pseudomonas aeruginosa.

The proteins of purified inner and outer membranes obtained from Pseudomonas aeruginosa strains PAK and PAK/2Pfs were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and treated with antiserum raised against pure pili. Bound antipilus antibodies were visualized by reaction with 125I-labeled protein A from Staphylococcus aureus. The results showed that there are pools of pilin in both the inner and outer membranes of P. aeruginosa and that the pool size in the multipiliated strain is comparable with that of the wild-type strain.     

Labeling of succinate-cytochrome c reductase with 125I. Accessibility of the peptides to the aqueous phases on the cytosolic and matrix sides of the mitochondrial membrane

Lactoperoxidase-catalyzed radioiodination was used to study the arrangement of the component peptides of succinate-cytochrome c reductase with respect to the aqueous phases on each side of the mitochondrial inner membrane. Mitochondria depleted of their outer membrane and inside-out vesicles purified from submitochondrial particles by the lectin-affinity procedure (D'Souza, M. P., and Lindsay, J. G. (1981) Biochim. Biophys. Acta 640, 463-472) were iodinated using immobilized preparations of lactoperoxidase. The labeled membranes were solubilized in detergent and the succinate-cytochrome c reductase was purified by immunoprecipitation with specific IgG. Analysis of the radioiodine distribution after sodium dodecyl sulfate-polyacrylamide gel electrophoresis and comparison with peptide stain patterns show that bands 2 (64 kilodaltons), 6 (30 kilodaltons), 9 (15 kilodaltons), and 11 (less than 10 kilodaltons) are labeled from the cytoplasmic surface of the membrane. Bands 1 (72 kilodaltons), 4 (48 kilodaltons), and 8 (20 kilodaltons) appear to be labeled on the matrix side of the membrane, while bands 3 (52 kilodaltons), 5 (35 kilodaltons), 7 (25 kilodaltons), and 10 (11 kilodaltons) are labeled from both sides of the membrane. Tentative identification of the labeled bands suggests that band 1 is the large subunit of succinate dehydrogenase. Bands 3 and 4 represent proteins which have been referred to as core proteins I and II. Bands 5 and 6 are the proteins associated with cytochromes b and c1, respectively; band 7 is the Rieske iron-sulfur protein.     

Isolation and partial characterization of inner and outer membrane fractions of Nitrobacter hamburgensis

Abstract Highly purified preparations of inner, i.e. cytoplasmic and intracytoplasmic, membranes and outer membranes were isolated from Nitrobacter hamburgensis strain X14 by sucrose density-gradient centrifugation of cell-free extracts. The two membrane fractions differed markedly in morphology, density, and protein composition as determined by polyacrylamide gel electrophoresis. The inner membrane fraction was enriched in NADH oxidase and nitrite oxidase activity. It contained four major protein bands of apparent M _rs of 28 000, 32 000, 70 000, and 116000. The outer membrane fraction was characterized by the presence of 2-keto-3-deoxyoctonate and contained two major proteins of apparent M _rs of 13 000 and 50 000. There was no evidence for differences between cytoplasmic and intracytoplasmic membranes.     

Differences in the Membrane Skeleton Proteins of RBC of AMoL Patients

We report here the different components of erythrocyte membrane skeleton proteins between acute monocytic leukemic (AMoL) patients and normal people. The bands in the 2 region of ghost membrane from AMoL patients exhibited significant differences on SDS gel electrophoresis. Band 2.2 was found to be missing and a “new” band with molecular weight (MW) 161 000 appeared. Also band 4.9 was missing, and the amounts of spectrin, actin, and band 4.8 of AMoL patients were decreased markedly. No such alterations can be seen in normal individuals, even in acute myeloid leukemic (AML) and chronic myeloid leukemic (CML) patients     

Differences in the membrane skeleton proteins of RBC of AMoL patients

We report here the different components of erythrocyte membrane skeleton proteins between acute monocytic leukemic (AMoL) patients and normal people. The bands in the 2 region of ghost membrane from AMoL patients exhibited significant differences on SDS gel electrophoresis. Band 2.2 was found to be missing and a "new" band with molecular weight (MW) 161,000 appeared. Also band 4.9 was missing, and the amounts of spectrin, actin, and band 4.8 of AMoL patients were decreased markedly. No such alterations can be seen in normal individuals, even in acute myeloid leukemic (AML) and chronic myeloid leukemic (CML) patients.     

Topography of protein kinase C substrates analyzed by membrane splitting

We have used the methods of planar cell and membrane monolayer formation and monolayer splitting to study structural details of the transmembrane signaling process mediated by protein kinase C. We analyzed human red cell membrane proteins phosphorylated by phorbol ester activation of protein kinase C. Planar single membrane preparations, extraction procedures, and gel electrophoresis coupled with silver staining and autoradiography confirmed that two bands in the 100 kDa region, and bands 4.1, and 4.9, were peripheral and phosphorylated by treatment with 12-O-tetradecanoylphorbol 13-acetate (TPA). TPA also stimulated minor incorporation of [32 P]Pi into most integral membrane proteins, including band 3, glycophorin A, the band 4.5 region (glucose transporter) and band 7. Planar cell and membrane-splitting methods revealed that neither integral nor peripheral phosphorylated polypeptides were cleaved by freeze fracture, that all phosphorylated peripheral proteins partitioned intact with the cytoplasmic side of the membrane, and that the percentages of [32P]Pi-labeled peripheral proteins were the same in split membrane cytoplasmic leaflets as in intact membranes. As a unique approach to examining protein topographies membrane splitting provides strong evidence that the major phosphorylated products of the polyphosphatidylinositide pathway are topographically associated with the cytoplasmic leaflet of the human erythrocyte plasma membrane. We further conclude that TPA-induced phosphorylation of red cell peripheral proteins does not significantly alter their transbilayer partitioning patterns after membrane splitting.     

Quantitative immunoelectrophoresis of proteins in human erythrocyte membranes. Analysis of protein bands obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

1. We have defined conditions that permit quantitative immunoelectrophoresis in agarose gels of dodecyl sulfate-solubilized erythrocyte membrane proteins. 2. Using human serum albumin, transferrin, MN-glycoprotein (glycophorin) and crude spectrin as test proteins, we found that accurate analyses are possible if samples and gels are 1% in non-ionic detergent (Berol EMU-043) or Triton X-100) and if no more than 100 nmol free dodecyl sulfate is applied per sample. 3. Dodecyl sulfate treated membranes analyzed by crossed immunoelectrophoresis using rabbit antibodies against membrane material yielded optimal precipitation patterns in gels containing 1% of non-ionic detergent. 4. Crossed immunoelectrophoresis in the presence of 1% of Berol revealed precipitates when 10 protein bands defined and isolated by preparative dodecyl sulfate-polyacrylamide gel electrophoresis were run against anti-membrane antibodies. Seven of these bands showed more than one precipitation arc, indicating the presence of more than one antigenic component. 5. Crossed-line immunoelectrophoresis showed that dodecyl sulfate-polyacrylamide gel electrophoresis bands 1, 2 and 2.1 shared common antigenic components. The MN-glycoprotein was present in bands 3, 4A, 4B and 5, where antigenic components of the major intrinsic erythrocyte membrane protein, band 3, were also found. 6. After absorption of the anti-membrane antibody with intact erythrocytes, immunoelectrophoresis showed the disappearance of the MN-glycoprotein precipitates. An increase in the area below the precipitate corresponding to the major intrinsic protein (band 3) was also observed, indicating exposure of some antigens of this protein on the outer surface of intact cells. 7. After absorption of the antibody preparation with washed erythrocyte membranes, immunoprecipitates were not seen in any experiments, indicating that all antigenic determinants observed are exposed at one or both surfaces of the membrane. 8. Our analyses indicate that the peptide moieties of serum lipoproteins do not constitute a significant component of erythrocyte membranes.     

Isolation and electrophoretic analysis of nucleoli, phenol-soluble nuclear proteins, and outer cyst walls from Acanthamoeba castellanii during encystation initiation

A technique is described for isolating nuceoli from Acanthamoeba castellanii. Nuclei isolated by a modification of the technique of F. J. Chlapowski and R. N. Band (1971) are sonicated in a surcrose-Tris-MgSO4-KC1-Triton X-100 buffer and centrifuged on a linear sucrose gradient extending from 1.3 M to 1.5 M with a 2.6 M cushion, at 41000 rpm for 90 min. The only apparent contaminants in the nucleolar preparation are outer cyst walls. A procedure is described for the isolation of chemically pure outer cyst walls, and a comparison of the proteins with the nucleolar preparation reveals that outer cyst walls represent negligible contaminants. The ultrastructure of these isolated nucleoli examined with transmission electron microscopy is found to be identical with that of nucleoli from whole cells, fixed in an identical manner. The 50 nucleolar proteins separated by SDS gel electrophoresis have been examined throughout the growth cycle of Acanthamoeba and into the strat of induced encystment, at which time 10 protein bands disappear, 11 bands are observed to decrease, and 8 are seen to increase in concentration. Phenol-soluble proteins are extracted from the nucleolus which correspond to 29 of the 50 nucleolar proteins, with 17 of these proteins corresponding to nucleolar proteins that change at the onset of encystment. Thes nucleolar proteins are also compared with those of rat liver nucleoli by gel electrophoresis, resulting in the observation that extremely few protein homologies exist between the two. Numerous quantitative and qualitative changes in the gel pattern of phenol-soluble nuclear proteins during early and late log phase growth and the onset of stationary phase were also observed.