Protein and Carbohydrate Composition of the Cell Envelope of Halobacterium salinarium

The isolated cell envelope of Halobacterium salinarium strain 1 contained 15 to 20 proteins that were resolved by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. All but one of these proteins had molecular weights of 130,000 or less and together accounted for 50 to 60% of the total envelope protein. The remaining 40 to 50% of the envelope protein was accounted for by a single protein with an apparent molecular weight of approximately 194,000 that stained for carbohydrate with periodate-Schiff reagent. The proteolytic enzymes trypsin and Pronase were used to show that the carbohydrate is covalently bound to the protein. Separation of amino sugar- and hexose-containing tryptic peptides by gel filtration indicated that all of the nonlipid carbohydrate of the cell envelope is covalently bound to protein. The results of partial purification by phenol extraction indicated that both the amino sugar and hexose are bound to the 194,000-molecular-weight protein. Exposure of isolated cell envelopes to low salt concentration resulted in solubilization of a majority of the envelope proteins. A relatively small number of proteins, including the high-molecular-weight, carbohydrate-containing protein, remained bound to the sedimentable cell membrane fraction.     

Lymphocyte-mediated cytolysis of allogeneic tumor cells in vitro. III. Enzyme sensitivity of target cell antigens.

Target tumor cells pretreated with high concentrations of papain or Pronase were resistant to lysis by cytotoxic T lymphocytes (CTL), whereas treatment with trypsin or neuraminidase had no protective effect. Parallel determinations of the H-2 content of target cells following enzyme treatment showed that approximately 80% of surface H-2 was removed by papain or Pronase, 40% by trypsin, and virtually none by neuraminidase treatment. Both susceptibility to lysis by CTL and content of surface H-2 after papain treatment were fully restored by 6 hr at 37 °C in nutrient medium. These findings suggest that lymphocyte-mediated cytolysis (LMC) determinants (target cell antigens bound by CTL) are sensitive to degradation by papain and Pronase but are resistant to the enzymatic action of trypsin and neuraminidase. That a similar pattern of enzyme sensitivity is shown by serologically defined H-2 antigens indicates that both functional classes, LMC and H-2, may have a structural association.     

Differential inhibitory effects of antibiotics on the biosynthesis of envelope proteins of Escherichia coli

Inhibitory effects of six antibiotics (kasugamycin, tetracycline, chloramphenicol, sparsomycin, puromycin and rifampicin) on the biosynthesis of envelope proteins of Escherichia coli were examined and compared with those on the biosynthesis of cytoplasmic proteins. Kasugamycin, puromycin and rifampicin were much more inhibitory to the over-all biosynthesis of cytoplasmic proteins than to that of envelope proteins. On the contrary, tetracycline and sparsomycin showed much stronger inhibitory effects on the biosynthesis of envelope proteins than on that of cytoplasmic proteins. Chloramphenicol showed little difference in its inhibitory effect on the biosynthesis of envelope proteins and cytoplasmic proteins.The envelope proteins were labeled with [³H]arginine in the presence of the antibiotics and separated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The inhibitory effects of the antibiotics on the biosynthesis of individual envelope proteins were then examined. Inhibition patterns were found to be widely different from one envelope protein to the other. For example, the biosynthesis of one major envelope protein of molecular weight 38,000 was more resistant to kasugamycin, chloramphenicol and sparsomycin than that of the other envelope proteins. On the other hand, the biosynthesis of another major envelope protein (lipoprotein) of about 7500 molecular weight was much more resistant to puromycin and rifampicin than that of the other envelope proteins. In the case of tetracycline, little differential inhibitory effect on the biosynthesis of individual envelope proteins was observed.Stability of messenger RNAs for individual envelope proteins was also determined from the inhibitory effect of rifampicin on their biosynthesis. It was found that the average of half lives of mRNAs for major envelope proteins examined (5.5 minutes) is twice as long as the average of those of mRNAs for cytoplasmic proteins (2 minutes), except for the lipoprotein of about 7500 molecular weight which has extremely stable mRNA with a half life of 11.5 minutes. From these results the envelope proteins of E. coli appear to be biosynthesized in a somewhat different manner from that of the cytoplasmic proteins. Furthermore, at least some envelope proteins may have their own specific biosynthetic systems.     

Isolation and Partial Properties of a Porin-Like Protein from Vibrio parahaemolyticus Cell Envelope

The cell envelope of Vibrio parahaemolyticus pilot strain K-11 contains a major protein with an apparent molecular weight of 35,000 which was not solubilized with 2% sodium dodecyl sulfate (SDS) at 50 C for 30 min and was resistant to trypsin. The protein was extracted from the SDS-insoluble envelope with SDS containing 0.4 m NaCl and purified by acetone precipitation and gel filtration. The purified protein was completely dissociated into a monomer with a molecular weight of 35,000 in SDS at 60 C. The amino acid composition of the protein was nearly the same as that of porins from Escherichia coli and Salmonella typhimurium. Thus the protein seems to be porin-like.     

Biochemical studies of the excitable membrane of Paramecium tetraurelia. V. Effects of proteases on the ciliary membrane

The swimming behavior of Paramecium is regulated by an excitable membrane that covers the body and cilia of the protozoan. In order to obtain information on the topology and function of ciliary membrane proteins, Paramecia were treated with trypsin, chymotrypsin or pronase and the effects of these proteases were analyzed using electron microscopy, gel electrophoresis of ciliary fractions and behavioral tests. At the concentrations used, trypsin and chymotrypsin had little or no effect on the cells while pronase removed the cell surface coat, visible as fuzzy material covering the cell membrane. The same pronase treatment caused the specific removal of a high molecular weight protein (250 000), as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis. This protein, the 'immobilization antigen', constitutes the major protein of the ciliary membrane. Although the immobilization antigen was removed (or markedly decreased), no marked and reproducible difference was observed in the swimming behavior of the treated cells. We also determined the effects of proteases on isolated ciliary fractions to explore the sidedness of ciliary membrane proteins. A set of proteins relatively resistant to protease digestion was identified; they may be intrinsic membrane proteins.     

Protein Composition of the Cell Wall and Cytoplasmic Membrane of Escherichia coli

Envelope preparations obtained by passing Escherichia coli cells through a French pressure cell were separated by sucrose density gradient centrifugation into two distinct particulate fractions. The fraction with the higher density was enriched in fragments derived from the cell wall, as indicated by the high content of lipopolysaccharide, the low content of cytochromes, and the similar morphology of the fragments and intact cell walls. The less-dense fraction was enriched in vesicles derived from the cytoplasmic membrane, as indicated by the enrichment of cytochromes, the enzymes lactic and succinic dehydrogenase and nitrate reductase, and the morphological similarity of the vesicles to intact cytoplasmic membrane. Both fractions were rich in phospholipid. The protein composition was compared by mixing the cytoplasmic membrane-enriched fraction from a (3)H-labeled culture with the cell wall-enriched fraction from a (14)C-labeled culture and examining the resulting mixture by gel electrophoresis. Thirty-four bands of radioactive protein were resolved; of these, 27 were increased two- to fourfold in the cytoplasmic membrane-enriched fraction, whereas 6 were similarly increased in the cell wall-enriched fraction. One of the proteins which is clearly localized in the cell wall is the protein with a molecular weight of 44,000, which is the major component of the envelope. This protein accounted for 70% of the total protein of the cell wall, and its occurrence in the envelope from spheroplasts suggests that it is a structural protein of the outer membranous component of the cell wall.     

The effects of proteases on proteins and glycoproteins of Dictyostelium discoideum plasma membranes

Dictyostelium discoideum cells were incubated with proteases, the plasma membranes subsequently isolated and changes in proteins and glycoproteins examined with dodecylsulfate gel electrophoresis. Low papain concentrations gave rise to a protein band which apparently derived from actin. Since actin was the only protein attacked, the results suggest some part of the actin is exposed on the outer surface of the cell. Higher papain concentrations released a substantial portion of actin from the plasma membrane and partially digested some of the glycoproteins. Since the new actin-derived band was not further digested, the glycoproteins may be required to stabilize the actin polymer rather than anchor those actin molecules which are directly associated with the plasma membrane. Pronase treatment released the two myosin heavy chains from the plasma membrane, in particular the higher molecular weight chain. Actin was not affected. Some glycoproteins were digested. Trypsin attacked many of the plasma membrane proteins, and the myosin heavy chains were completely removed. Actin was only moderately affected. However, the glycoproteins were entirely resistant to trypsin. Apparently the myosin heavy chains are attacked either due to their partial exposure on the cell surface or the exposure of proteins which anchor them in the membrane. These anchoring proteins cannot be glycoproteins or actin. Proteins and glycoproteins were largely digested when isolated plasma membranes were incubated with papain and pronase. The effects of trypsin on whole cells and isolated plasma membranes were similar.     

Proteins of the human erythrocyte membrane as modified by pronase

Pronase degrades proteins on the outer surface of the human erythrocyte membrane which run in polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulfate at a molecular weight of approximately 125,000. Carbohydrate and sialic acid are removed, but fragments of molecular weight 50,000 to 100,000 remain attached to the membrane. The most prominent fragment, one of molecular weight about 73,000, can be labeled with a membrane-impermeable reagent (sulfanilic acid diazonium salt), so it is still accessible from the outside of the cell. Pronase rapidly inactivates membrane-bound acetylcholinesterase, but it has relatively little effect on the facilitated diffusion of glucose; both are inhibited by the diazonium salt. Extensive digestion leads to potassium loss and osmotic lysis. Ghosts prepared in 15 mosm-Tris (pH 7.6) are extensively degraded by pronase: essentially all the protein shifts to low molecular weight. Pronase is even more potent in 3% sodium dodecyl sulfate. Ghosts prepared from intact cells which have been treated with the enzyme hydrolyze when dissolved in the detergent unless steps are taken to inhibit proteolysis.     

Characterization of the membrane matrix derived from the microsomal fraction of rat hepatocytes

A highly purified membrane preparation derived from the microsomal fraction of rat hepatocytes has been chemically characterized and fractionated by means of gel filtration. The preparation has been freed of ribosomes and intravesicular protein and has a composition on a w/w basis of 52.1% protein, 45.0% phospholipid, 2.9% carbohydrate and no RNA. $\text{97 ± 2\%}$ of the total membrane phosphorus is accounted for as phospholipid phosphorus.Determination of the molecular weight distribution of the constituent polypeptides by sodium dodecyl sulfate-polyacrylamide gel electrophoresis gave values ranging from 171 000 to 16 000 for the major classes of proteins. Although several membrane glycoproteins have been identified, the most prominent species has an apparent molecular weight of 171 000, 40% of the total microsomal protein is present' in the 49 000–60 000 molecular weight region. Examination of the intrinsic polypeptide composition of membranes obtained from smooth and degranulated rough endoplasmic reticulum revealed no detectable qualitative differences.Sodium dodecyl sulfate-solubilized microsomal membrane proteins were separated by gel filtration into much simplified molecular weight classes, some of which showed predominantly a single electrophoretic component. Amino acid analysis of individual fractions showed a noticeable trend toward a decreasing ratio of acidic to basic residues with decreasing molecular weight.Membrane phosphorus was distributed between two chromatographic fractions: one containing the membrane phospholipid (97% of the total) as well as essentially all the cholesterol, the other, at the inclusion volume of the gel filtration system, containing small molecular weight species (3% of the total phosphorus). The absence of a ribonuclease-resistant RNA component eluting near the void volume clearly distinguishes the microsomal membrane from the nuclear envelope.     

Biochemical studies of the excitable membrane of Paramecium tetraurelia. V. effects of proteases on the ciliary membrane

The swimming behavior of Paramecium is regulated by an excitable membrane that covers the body and cilia of the protozoan. In order to obtain information on the topology and function of ciliary membrane proteins, Paramecia were treated with trypsin, chymotrypsin or pronase and the effects of these proteases were analyzed using electron microscopy, gel electrophoresis of ciliary fractions and behavioral tests. At the concentrations used, trypsin and chymotrypsin had little or no effect on the cells while pronase removed the cell surface coat, visible as fuzzy material covering the cell membrane. The same pronase treatment caused the specific removal of a high molecular weight protein (250 000), as judged by sodium dodecyl sulfate polyacrylamide gel electrophoresis. This protein, the ‘immobilization antigen’, constitutes the major protein of the ciliary membrane. Although the immobilization antigen was removed (or markedly decreased), no marked and reproducible difference was observed in the swimming behavior of the treated cells. We also determined the effects of proteases on isolated ciliary fractions to explore the sidedness of ciliary membrane proteins. A set of proteins relatively resistant to protease digestion was identified; they may be intrinsic membrane proteins.     

Reconstitution of the d-glucose transporter of bovine thymocyte plasma membrane: Partial purification of transport activity by chromatography on agarose lentil lectin and agarose ethanethiol

The d-glucose transporter of bovine-thymocyte plasma membrane was partially purified using several procedures in sequence. Dimethylmaleic anhydride extraction removed extrinsic membrane proteins (approximately 50% of the total membrane protein) after which sodium cholate solubilized 40% of the residual protein. Reconstitution of solubilized proteins into phospholipid liposomes indicated a 2.5-fold increase in sugar transport specific activity relative to membrane solubilized without dimethylmaleic anhydride extraction. Detergent removal by gel filtration on G-50 Sephadex resulted in reaggregation of intrinsic membrane proteins. Ultracentrifugation of the reaggregated proteins generated a particulate fraction (pellet 1) which contained about 50% of the total d-glucose transport activity of the preparation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of pellet 1 demonstrated removal of a major band at 68,000 daltons and two minor bands not removed by dimethylmaleic anhydride. The 68,000-dalton protein was not removed by any other method tested. Chromatography of resolubilized pellet 1 on a tandem-bed column of agarose ethanethiol and agarose lentil lectin resulted in a 6-fold increase in transport specific activity of nonabsorbed proteins relative to pellet 1. Approximately 15% of the protein (80–90% of the transport activity) applied to the tandem-bed column was recovered in the nonabsorbed fraction. Sodium dodecyl sulfate-gel electrophoresis of proteins in the nonabsorbed fraction showed apparent enrichment of a diffuse zone at 52,00045,000 daltons. The overall increase in specific activity of the partially purified preparation was about 12-fold relative to unpurified solubilized proteins.     

Chemical characterization of the proteins and glycoproteins of mouse liver plasma membranes solubilized by sequential extraction with aqueous and organic solvents

1. A procedure for the stepwise fractionation of the proteins of mouse liver plasma membranes is described. 2. Of the membrane protein 20-25% was soluble in 50mm-sodium carbonate-bicarbonate buffer (pH9.7). This fraction contained a large number of proteins but only 1 major glycoprotein. It was low in sialic acid, amino sugars and phospholipid. 3. Extraction of the alkali-insoluble residue with aq. 33% pyridine solubilized an additional 30-35% of the membrane protein. The pyridine-soluble membrane components were enriched in sialic acid and glucosamine and it was shown that this procedure resulted in the selective extraction of glycoproteins. 4. Gel filtration in sodium dodecyl sulphate resolved the pyridine-soluble proteins into five fractions of decreasing molecular weight and an inverse relationship between molecular weight and sialic acid content was indicated.     

Surface polymers of the nematode-trapping fungus Arthrobotrys oligospora

The nematophagous fungus Arthrobotrys oligospora captures nematodes using adhesive polymers present on special hyphae (traps) which form a three-dimensional network. To understand further the adhesion mechanisms, A. oligospora surface polymers were visualized by transmission electron microscopy and characterized by chemical methods. Both traps and hyphae were surrounded by a fibrillar layer of extracellular polymers which stained with ruthenium red. The polymer layer was resistant to most of the chemicals and enzymes tested. However, part of the layer was removed by sonication in a Tris-buffer or by extraction in a chaotropic salt solution (LiCl), and the structure of the polymers was modified by treatment with Pronase E. Chemical analysis showed that the crude extracts of surface polymers removed by sonication or LiCl solution contained neutral sugars, uronic acids and proteins. Gel chromatography of the extracts revealed that the major carbohydrate-containing polymer(s) had a molecular mass of at least 100 kDa, containing neutral sugars (75% by weight, including glucose, mannose and galactose), uronic acids (6%) and proteins (19%). There was more polymer in mycelium containing trap-bearing cells than in vegetative hyphae. SDS-PAGE of the extracted polymers showed that the trap-forming cells contained at least one protein, with a molecular mass of approx. 32 kDa, not present on vegetative hyphae. Examining the capture of nematodes by traps of A. oligospora in which the layer of surface polymers was modified, or removed by chemical or enzymic treatments, showed that both proteins and carbohydrate surface polymers were involved in the adhesion process.     

Composition of major outer membrane proteins of Vibrio vulnificus isolates: effect of different growth media and iron deficiency

The outer membrane proteins of Vibrio vulnificus including isolates from humans, seawater and an asari clam were examined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. A major outer membrane protein with an apparent molecular weight of 48,000 (48K protein) was common to all the strains grown in 3% NaCl-nutrient broth; however this 48K protein was not produced in any of the strains grown in chemically defined medium. Other major outer membrane proteins with molecular weights ranging from 33,000 to 40,000 varied in number, relative amount and molecular weight depending on the strain. One to three new outer membrane proteins with molecular weights ranging from 74,000 to 85,000 were produced in the cells grown in iron-deficient medium. The 48K protein and one or two major proteins with molecular weights ranging from 35,000 to 37,000 in the cells grown in 3% NaCl-nutrient broth were not solubilized by 2% SDS at 60 C for 30 min and were resistant to trypsin, indicating that they are porins. On the other hand, in cells grown in chemically defined medium, one or two major outer membrane proteins with molecular weights ranging from 33,000 to 40,000 might be porins.     

Isolation and characterization of a hydroxyproline-containing protein from soluble extracts of the leaves of sandal (Santalum album L.)

1. A hydroxyproline-containing protein was isolated from the soluble fraction of sandal leaves (Santalum album L.) and the purified protein was homogeneous by disc electrophoresis. 2. It is a glycoprotein containing 16% carbohydrate, the components of which were mainly arabinose, with only small amounts (about 5%) of galactose. The principal amino acids were glutamic acid, aspartic acid, glycine, alanine, arginine, lysine, proline and hydroxyproline, which together comprised 60% of the total. The number of acidic amino acids exceeds the number of basic amino acids. By Sephadex gel filtration, the approximate molecular weight was found to be about 63000. The ratio of residues of hydroxyproline to those of arabinose was 1:2. 3. The native protein is resistant to the action of several proteolytic enzymes. After partial hydrolysis with 0.1m-HCl, the protein became susceptible to attack by Pronase but remained resistant to collagenase.     

Thyroid microsomal membrane proteins. Effects of solubilization on molecular size.

The molecular size of microsomal membrane proteins from frozen porcine thyroids before and after solubilization by proteolytic and non-proteolytic techniques has been investigated by means of polyacrylamide-gel electrophoresis in the presence of 1% sodium dodecylsulfate. When thyroid microsomal membrane proteins are solubilized by non-proteolytic methods such as high pH, n-butanol, or deoxycholate, no major change in the electrophoretic pattern compared to untreated microsomes has been observed, thereby suggesting that these non-proteolytic methods are capable of extracting membrane proteins from thyroid microsomes without altering their molecular size. However, treatment of microsomes with protein-solubilizing levels of trypsin (1-5 mug trypsin per mg thyroid protein) results in degradation of all major proteins with a molecular weight greater than 30 000. The high-molecular-weight proteins are particularly susceptible to attack by trypsin. Thus, these experiments indicate that the use of trypsin to solubilize thyroid microsomal membrane proteins, particularly thyroid peroxidase, will result in fragmented proteins and should be avoided if intact membrane proteins are desired.     

Examination of the Protein Composition of the Cell Envelope of Escherichia coli by Polyacrylamide Gel Electrophoresis

An envelope preparation containing the cell wall and cytoplasmic membrane of Escherichia coli was obtained by breaking the cells with a French pressure cell and sedimentating the envelope fraction by ultracentrifugation. This fraction was prepared for polyacrylamide gel electrophoresis by dissolving the protein in an acidified N,N'-dimethylformamide, removing lipids by gel filtration in the same organic solvent and removing the solvent by dialysis against aqueous urea solutions. More than 80% of the total protein of the envelope fraction was recovered in soluble form. Electrophoresis on sodium dodecyl sulfate-containing gels yielded from 20 to 30 well-resolved bands of protein. One major protein band was observed on the gels. This protein had a molecular weight of 44,000 and accounted for as much as 40% of the total protein of the envelope fraction. A double-labeling technique was used to examine the protein composition of the envelope fraction from cells grown under different sets of conditions which result in large changes in the levels of membrane-bound oxidative enzymes. These changes in growth conditions resulted in only minor alterations in the protein profiles observed on the gels, suggesting that this organism is able to adapt to changes in growth environment with only minor modifications of the major proteins of the cell envelope.     

Effect of Enzymes on the Composition and Structure of Chromobacterium violaceum Cell Envelopes

Cell envelopes of Chromobacterium violaceum were isolated and treated under controlled conditions with trypsin, Pronase, lipase, phospholipase C, lysozyme, and a mixture of enzymes produced by a bacteriolytic Pseudomonas sp. After each enzyme treatment, losses in dry weight, protein, lipid, carbohydrate, 2,6-diaminopimelic acid, and total phosphorus were determined. Electron-microscopic examination of the enzyme-treated envelopes indicated complete or partial loss of envelope rigidity or some envelope fragmentation, or both. Each enzyme hydrolyzed at least one envelope component and liberated several others into the supernatant fluid, where they appeared as nondialyzable particulate components, identified by means of electron microscopy. Unlike the other enzymes, the Pseudomonas sp. enzyme mixture partially liberated all major envelope components except phosphorus, heptose, and 2-keto-3-deoxy octonic acid. In spite of these large losses, the envelopes preserved some features of their integrity and elongated shape.     

Surface properties of sendai virus envelope

Surface properties of Sendai virus envelope membrane have been measured, using both biological and biophysical techniques. Both normal and trypsin-treated virus were studied. SDS gel electrophoresis showed cleavage of the F protein exclusively by trypsin. The major activity change was observed in the hemolysing activity which is an expression of F protein. Hemolysis was reduced to less than 10% of its value for intact virus. ³¹P nuclear magnetic resonance studies of the envelope surface of the native virus showed a highly restricted phospholipid headgroup environment. Interestingly, this restriction was relieved by treatment with trypsin. Thus these data suggest a role of the F protein of Sendai virus in tightly organizing the surface of the viral envelope membrane.     

Polypeptide composition of extracellular enveloped vaccinia virus.

Extracellular enveloped vaccinia (EEV) virus grown in SIRC and in HeLa cells was purified by consecutive equilibrium centrifugations in sucrose and cesium chloride gradients. A higher degree of purity was obtained with virus material prepared in SIRC cells. The polypeptides of purified EEV and INV (intracellular naked vaccinia) virus were compared in polyacrylamide slab gel electrophoresis. Three proteins (200,000 molecular weight [200K], 95K, and 13K) detected in HeLa-derived INV were absent in EEV. In addition, two INV proteins (65K and 30K) occurred in reduced concentrations in EEV, white another INV protein (27K) was increased in EEV. INV from SIRC cells showed similar alterations of these proteins (with the exception of the 30K and 13K proteins). Detergent treatment, ether extraction, and Pronase treatment showed that these six proteins are located at the surface of INV and are not cecessary for infectivity. Eight proteins (210K, 110K, 89K, 42K, 37K, 21.5K, 21K, and 20K) were detected in EEV that were absent from inv. Brij-58 treatment was employed to remove the envelope from EEV, resulting in the formation of naked particles and an envelope fraction which were separated on cesium chloride gradients. The envelope fractions contained all eight proteins. Seven of the eight proteins were glycoproteins, with the 37K protein being the only unglycosylated protein. It is concluded that a processing of surface INV particle proteins occurs during evelopment. The resultant EEV particle is comprised of an INV particle with a modified surface composition enclosed in an envelope containing virus-specific proteins unique to EEV.