Alkaline urea solubilization, two-dimensional electrophoresis and lectin staining of mammalian cell plasma membrane and plant seed proteins

Plasma membranes, isolated from Chinese hamster ovary cells and seed proteins from Arachis hypogaea (L.) were analyzed by two-dimensional electrophoresis. Polypeptides were solubilized without employing sodium dodecyl sulfate (SDS), using in its place 5 mm K₂CO₃ and 9.5 m urea. After addition of dithiothreitol and the nonionic detergent Nonidet P-40, more than 95% of the total protein remained in the supernatant fraction after the preparation was centrifuged at 100,000 g. The solubilization was comparable to that achieved with boiling SDS solution. This soluble material could be used directly for either isoelectric focusing or nonequilibrium pH gradient electrophoresis in narrow bore, tubular, polyacrylamide gels crosslinked by means of N,N′-diallyltartardiamide. Up to 750 μg of protein could be analyzed in one such 3 mm gel. Electrophoresis in polyacrylamide slab gels containing SDS was used for separations in the second dimension. The method allows large amounts of both basic and acidic insoluble proteins to be solubilized and then analyzed without employing SDS as a solubilizing agent. Classes of glycoproteins on the gels were detected by incubating with small volumes of ¹²⁵I-lectins in heat-sealed plastic bags. CHO cells contain several high molecular weight acidic glycoproteins that bind wheat germ agglutinin, but which do not stain with Coomassie blue. Several of the storage polypeptides in peanut seeds were also shown to bind wheat germ agglutinin and are probably, therefore, glycoproteins containing N-acetyl d-glucosamine.     

Solubilization of a guanine nucleotide-sensitive form of vasopressin V2 receptors from porcine kidney

Vasopressin (V2) receptors were solubilized from porcine kidney membranes with the detergent egg lysolecithin. Binding of [3H]vasopressin to the solubilized fraction was rapid, specific, and saturable. The agonist dissociation constants observed in membranes and solubilized fractions were 1.7 +/- 0.3 and 2.3 +/- 0.2 nM, respectively. In competition binding experiments, the solubilized fraction exhibited the same pharmacological profile as the membranes. Chemical crosslinking of [125I]vasopressin to the solubilized fraction followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis demonstrated a 62-kDa band which was specifically labeled with [125I]vasopressin. Vasopressin binding sites from the solubilized fractions were resolved by gel filtration and ultracentrifugation on a sucrose gradient. In addition, agonist high affinity binding to V2 receptors and its sensitivity to guanine nucleotides were preserved even after solubilization in the absence of prebound agonist prior to solubilization. Addition of guanine nucleotides such as GTP gamma S decreased the specific binding of [3H]arginine vasopressin to these solubilized fractions in a dose-dependent manner, suggesting the solubilization of a V2 receptor-G protein complex. [32P]ADP ribosylation of the solubilized fraction by cholera and pertussis toxins revealed specifically labeled proteins with molecular weights of 42,000-43,000 and 39,000-41,000, respectively, on sodium dodecyl sulfate polyacrylamide gels. Furthermore [35S]GTP gamma S binding to these solubilized fractions was enhanced by vasopressin, confirming that a significant proportion of the vasopressin receptors must be closely coupled to G proteins even when these receptors are solubilized in the absence of agonist. These results are in contrast with those reported for beta, alpha 2 adrenergic and D2 dopaminergic receptor systems, but in agreement with D1 dopaminergic and A1 adenosine receptors. The molecular mechanism responsible for this difference remains to be determined.     

Homogeneity of Envelope Proteins of Escherichia coli Separated by Gel Electrophoresis in Sodium Dodecyl Sulfate

Some envelope proteins of Escherichia coli show variable behavior in acrylamide gel electrophoresis in 1% sodium dodecyl sulfate, depending upon the conditions of the solubilization. When solubilized in 1% sodium dodecyl sulfate at 70 C for 20 min, three distinct peaks (peaks 4, 6, and 7) are seen at molecular weights of 57,800, 44,300, and 38,400, respectively. However, when the envelope fractions are solubilized in 1% sodium dodecyl sulfate at 100 C for 5 min, or when they are treated with N, N-dimethylformamide at acidic pH before solubilization by our method, only a single peak at 48,000 molecular weight is observed in the molecular weight range mentioned above. That is, peaks 4 and 7 disappear and a new peak appears at the position overlapping with peak 6. Proteins isolated from peaks 4 and 7 show the similar molecular weight shifts to the new peak by the treatment at 100 C. No other peaks show any change by the heat treatment. The increase at the new peak is completely accounted for by the decrease at peaks 4 and 7, indicating that the new peak is composed of proteins from peaks 4, 6, and 7. However, it is concluded that these three peaks consist of distinctly different proteins for the following reasons: (i) they have different amino acid compositions, (ii) they show different solubilities in the nonionic detergent, Nonidet P-40, and as shown previously, (iii) peak 6 (protein Y) is related to deoxyribonucleic acid synthesis, and (iv) proteins in peaks 4, 6, and 7 have different resistance to proteolytic enzymes. Although the reasons for the anomalous molecular weight shifts of these peaks are not well understood at present, it is important to solubilize the E. coli envelope proteins by the standard method in order to investigate their properties and functions of the envelope proteins.     

the stability in various detergents of transferrin-transferrin receptor complexes from reticulocyte plasma membranes

Transferrin-membrane protein complexes were solubilized either with 0.4% sodium dodecyl sulfate (SDS), 1% Triton X-100 or 0.5% sulfobetaine 3-14 from the plasma membranes of rabbit reticulocytes previously labeled with 125I and then incubated with 131-labeled transferrin. When the solubilized membranes were analyzed by gel filtration fractionation, marked variation in the preservation of transferrin-transferrin receptor interaction was noted between the three detergents. After SDS solubilization, more than 80% of the 131I-labeled transferrin remained associated with membrane proteins with apparent molecular weight of the transferrin-receptor complexes of 1400 000 and 240 000. In contrast, after Triton X-100 solubilization only 40% of the transferrin was still complexed to membrane proteins with an apparent molecular weight of the complex of 450 000. Dissociation of transferrin from its receptor was most marked following sulfobetaine solubilization, with less than 30% of the transferrin still complexed. Following gel filtration 131I-labeled transferrin-125I-labeled membrane protein complexes were immunoprecipitated with goat specific anti-rabbit transferrin antibodies. The immunoprecipitates were analyzed under stringent dissociating conditions by two SDS-polyacrylamide gel electrophoretic techniques. In a linear 5-25% polyacrylamide gradient the 125I-labeled receptor obtained after membrane solubilization with all three detergents had an apparent molecular weight of 80 000. In contrast, in a different system using 10% polyacrylamide gel two 125I-labeled receptor components were detected wih apparent molecular weights of 90 000 and 80 000. These results demonstrate that estimates of the molecular weight of the transferrin receptor depended on the conditions of electrophoresis and suggest that the transferrin receptor is partially modified, perhaps by glycosylation.     

Studies on the brush border membrane of the mouse duodenum

Summary Brush border membranes have been isolated from villus epithelial cells of the adult Swiss mouse duodenum. Preparations of these membranes are not contaminated by other organelles as judged from electron-micrographs of sectioned pellets of brush borders. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins from brush borders solubilized in Tris-sodium dodecyl sulfate buffer reveals a reproducible Coomassie Brilliant Blue pattern of 17 bands. By comparing the brush border protein band positions with those of standard proteins run concurrently on sodium dodecyl sulfate-polyacrylamide gel slabs it is estimated that the 17 brush border proteins and subunits have molecular weights ranging from over 250,000 to around 16,000. Periodate-fuchsin sulfite staining shows that the five more slowly migrating, high molecular weight proteins are glycoproteins. The two proteins of smallest molecular size react positively with Oil Red O but have very small amounts of lipophilic amino acid residues, which indicates that the lipid extractable from the gels in these areas is a contaminant and is not bound to the proteins.     

Studies on the brush border membrane of the mouse duodenum. I. Membrane isolation and analysis of protein components.

Brush border membranes have been isolated from villus epithelial cells of the adult Swiss mouse duodenum. Preparations of these membranes are not contaminated by other organelles as judged from electron-micrographs of sectioned pellets of brush borders. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of proteins from brush borders solubilized in Tris-sodium dodecyl sulfate buffer reveals a reproducible Coomassie Brilliant Blue pattern of 17 bands. By comparing the brush border protein band positions with those of standard proteins run concurrently on sodium dodecyl sulfate-polyacrylamide gel slabs it is estimated that the 17 brush border proteins and subunits have molecular weights ranging from over 250,000 to around 16,000. Periodate-fuchsin sulfite staining shows that the five more slowly migrating, high molecular weight proteins are glycoproteins. The two proteins of smallest molecular size react positively with Oil Red O but have very small amounts of lipophilic amino acid residues, which indicates that the lipid extractable from the gels in these areas is a contaminant and is not bound to the proteins.     

Phosphorylation of the (Na,K)-ATPase by a plasma membrane-bound protein kinase in friend erythroleukemia cells

A decrease in the activity of the (Na,K)-ATPase is an early and essential step in commitment of Friend virus-infected murine erythroleukemia cells to terminal erythroid differentiation. Plasma membranes from these cells were purified and shown to contain ouabain-inhibitable (Na,K)-ATPase present as approximately 0.4% of the total membrane protein. Protein kinase activity also co-purified with the plasma membrane and preferentially phosphorylated a Nonidet P-40 detergent-extractable 100,000-Da peptide. The 100,000-Da phosphopeptide migrated with the alpha subunit of dog kidney (Na,K)-ATPase when electrophoresis was carried out in the presence of sodium dodecyl sulfate in either 5 or 10% polyacrylamide gels. In two-dimensional gel electrophoresis, it separated into a series of spots between pH 5.1 and 5.4, while dog kidney alpha subunit appeared as a doublet at pH 5.3-5.4. When Nonidet P-40-solubilized plasma membranes were passed through a ouabain affinity column in the presence of Mg2+, Na+, and ATP, the 100,000-Da phosphopeptide was retained and could be eluted by ouabain. This peptide was also phosphorylated in living murine erythroleukemia cells, and proteolysis patterns of the peptide labeled in vitro, the peptide labeled in vivo, and the purified dog kidney alpha subunit using V8 protease were nearly identical. Phosphothreonine was detected in both the peptides labeled in vivo and in vitro.     

Preliminary characterization of two thymus-dependent xenoantigens from mouse lymphocytes.

Preliminary characterization of two mouse thymus-dependent (T) lymphocyte xenoantigens, T25 and T200, which are selectively labelled by lactoperoxidase-catalysed iodination of T-cells, is described. Both molecules are membrane-bound glycoproteins. Fractionation of membrane vesicles prepared from BW5147 lymphoma cells by sedimentation through sucrose density gradients show that antigens T25 and T200 are in fractions enriched with plasma membrane. Moreover antigen T200 is partially degraded when viable cells are treated briefly with low concentrations of trypsin. Both molecules are efficiently solubilized in buffers containing sodium deoxycholate or Nonidet P-40, as measured by failure to sediment at 100000g for 60min. However, gel filtration on Sepharose 6B showed the presence of aggregated material in Nonidet P-40 extracts which was not found in deoxycholate-solubilized membranes. After solubilization in detergent, antigens T25 and T200 bind to, and may be specifically eluted from, columns of pea lectin--Sepharose or concanavalin A--Sepharose. Both molecules are heterogeneous when examined by polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate. As judged by its binding to columns of pea lectin, at least part of the heterogeneity of mouse thymocyte antigen T25 resides in its carbohydrate moiety.     

Isolation and characterization of gastric microsomal glycoproteins. Evidence for a glycosylated beta-subunit of the H+/K(+)-ATPase.

Detergent-solubilization of hog gastric microsomal membrane proteins followed by affinity chromatography using wheat germ agglutinin or Ricinus communis I agglutinin resulted in the isolation of five glycoproteins with the apparent molecular masses on sodium dodecyl sulfate polyacrylamide gels of (in kDa): 60-80 (two glycoproteins sharing this molecular mass); 125-150; and 190-210. In the nonionic detergent Nonidet P-40 (NP-40), the 94 kDa H+/K(+)-ATPase was recovered exclusively in the lectin-binding fraction; however, in the cationic detergent dodecyltrimethylammonium bromide, most of the ATPase was recovered in the nonbinding fraction. Detection of glycoproteins either by periodic acid-dansyl hydrazine staining of carbohydrate in polyacrylamide gels or by Western blots probed with lectins indicated that the majority of the ATPase molecules are not glycosylated. In addition, in the absence of microsomal glycoproteins, the NP-40-solubilized ATPase does not bind to a lectin column. Taken together, these results suggest that the recovery of NP-40-solubilized ATPase in the lectin-binding fraction is due to its noncovalent interaction with a gastric microsomal glycoprotein. Immunoprecipitation of the ATPase from NP-40-solubilized microsomal membrane proteins resulted in the co-precipitation of a single 60-80 kDa glycoprotein. Characterization of the 60-80 kDa glycoprotein associated with the ATPase revealed that: it is a transmembrane protein; it has an apparent core molecular mass of 32 kDa; and, it has five asparagine-linked oligosaccharide chains. Given its similarity to the glycosylated beta-subunit of the Na+/K(+)-ATPase, this 60-80 kDa gastric microsomal glycoprotein is suggested to be a beta-subunit of the H+/K(+)-ATPase.     

Study of human erythrocyte membrane proteins by 2-dimensional electrophoresis

Fractionation of human erythrocyte membrane proteins was performed using a modification of two-dimensional gel electrophoresis described by P. O'Farrel with isoelectric point plotted against molecular mass. All major erythrocyte proteins, including high molecular weight proteins, such as spectrin and band 3 protein, identified by one-dimensional sodium dodecyl sulfate gel electrophoresis, were visualized by silver staining of two-dimensional gels. All in all about 50 polypeptides were distinguished on two-dimensional electrophoretic patterns. Preliminary protein map was developed.     

Solubilization and Partial Characterization of Angiotensin II Receptors from Rat Brain

Rat brain angiotensin II (Ang II) receptors were solubilized with a yield of 30-40% using the synthetic detergent 3[(3-cholamidopropyl)dimethylammonio)]-1-propanesulfonate. Kinetic analysis employing the high-affinity antagonist 125I-Sar1,Ile8-Ang II indicated that the solubilized receptors exhibited the same properties as receptors present within intact brain membranes. Furthermore, there was a positive correlation (r = 0.99) between the respective pIC50 values of a series of agonist and antagonists competing for 125I-Sar1,Ile8-Ang II labeled binding sites in either solubilized or intact membranes. Moreover, covalent labeling of 125I-Ang II to solubilized receptors with the homo-bifunctional cross-linker disuccinimidyl suberate, followed by gel filtration, revealed one major and one minor binding peak with apparent molecular weights of 64,000 and 115,000, respectively. Two binding proteins of comparable molecular weights (i.e., 112,000 and 60,000) were also identified by covalent cross-linking of 125I-Ang II to solubilized brain membranes followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. In contrast, only the smaller molecular mass binding protein was observed when solubilized membranes were labeled with the antagonist 125I-Sar1,Ile8-Ang II prior to gel filtration, and chromatofocusing of antagonist labeled sites revealed only one peak with an isoelectric point of 6.2. The successful solubilization of these binding sites should facilitate continued investigation of Ang II receptors in the brain.     

Comparative evaluation of extraction methods for total proteins from Crocus sativus L. (Saffron)

Broadly speaking proteomic studies are one of the various techniques of utmost importance for understanding complex biological processes that occur under inductive conditions and revealing the multidimensional aspects of Crocus sativus in biological systems. In order to get an insight into the molecular changes and to characterize the variations in protein expression of C. sativus, a detailed proteomic analysis on one-dimensional gel electrophoresis is one of the basic steps to accomplish. We have compared total protein profiles of C. sativus extracted by three different recipes and analyzed on 10% sodium dodecyl sulfate polyacrylamide gels. Gels were subjected to densitometric analysis for further characterization. Among three different protocols NP-40 extraction buffer recipe resulted in the extraction of proteins most efficiently with minimum background and streaking. There was maximum solubilization of proteins with high efficiency. Such a profile can be used for high precision analysis of differential protein expression. This work is an attempt to assist researchers in effective extraction of proteins from C. sativus. As a researcher faces a perplexing array of choices as where to start we describe a method based on our collective analysis of the different protein protocols. This paper presents a method that could be applied at the outset of any proteomic study.     

Protein phosphorylation of plasma membranes from bovine epididymal spermatozoa

Plasma membranes from bovine epididymal spermatozoa possess both cAMP-independent and cAMP-dependent protein kinase activity. With the synthetic peptide, Leu-Arg-Arg-Ala-Ser-Leu-Gly as substrate, the basal activity of the membrane-associated protein kinase(s) was 0.1 nmol phosphate incorporated X min X mg protein. In the presence of 5 microM cAMP, the apparent activity was increased about twofold. The addition of Nonidet P-40 (0.05%) to the assay mixture increased protein kinase activity to 0.4 and 4.0 nmol phosphate incorporated X min X mg protein in the absence or presence of 5 microM cAMP, respectively. Both isozymes of the cAMP-dependent protein kinase were detected in detergent-solubilized membranes but 95% of the activity appeared as a Type II form based on DEAE-Sephacel chromatography. Several polypeptide components of the plasma membrane served as substrates for membrane-associated cAMP-dependent protein kinases, in vitro. In the absence of detergent, two cAMP-dependent phosphoproteins of 41,000 Mr and 60,000 Mr were detected by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. When 0.05% Nonidet P-40 was included in the assay mixture, a cAMP-dependent phosphoprotein of 43,000 Mr appeared. Two-dimensional polyacrylamide gel electrophoresis of membranes phosphorylated in the presence of 5 microM and 0.05% Nonidet P-40 revealed phosphoproteins of the following molecular weights/isoelectric points: 56,000/6.7, 56,000/6.9, 51,000/6.2, 42,000/5.9, 42,000/6.0, 38,000/6.1, 38,000/6.4, 14,000/7.2, 12,000/7.4 and a train of five polypeptides appearing at 14,000/5.4-6.0.     

Dodecyl maltoside-sodium dodecyl sulfate two-dimensional polyacrylamide gel electrophoresis of chloroplast thylakoid membrane proteins

A two-dimensional electrophoretic system has been developed for the separation of chloroplast thylakoid membrane proteins. This system incorporates nondenaturing polyacrylamide gel electrophoresis in the presence of the nonionic detergent dodecyl-beta-D-maltoside in the first dimension and sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the second dimension. Thylakoid membranes isolated from Spinacia oleracea were solubilized in 1.0% dodecyl-beta-D-maltoside and separated in 4-7% linear acrylamide gradient tube gels which contained 0.05% dodecyl-beta-D-maltoside. After electrophoresis, the tube gels were equilibrated with a sodium dodecyl sulfate-containing equilibration buffer and applied to a 12.5-20% acrylamide linear gradient gel. The Lammelli buffer system was used in both dimensions. The two-dimensional gels were analyzed by staining sequentially with 3,3',5,5'-tetramethylbenzidine-H2O2, Coomassie blue, and silver staining. A number of protein components were identified on "Western blots" of these two-dimensional gels by immunological localization. Membrane protein complexes such as the light-harvesting chlorophyll a/b protein complex, photosystem I, photosystem II, the cytochrome b6/f complex and ribulose bisphosphate carboxylase appear to migrate as essentially intact complexes in the first dimension and appear as vertical series of resolved subunits in the second dimension. This technique complements isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis in providing additional information concerning the subunit composition of membrane protein complexes and may prove to be of general utility for studying the protein composition of other membrane systems.     

Ribosomal proteins from rabbit reticulocytes: number and molecular weights of proteins from ribosomal subunits.

The ribosomal proteins from 40 S and 60 S subunits of rabbit reticulocytes were separated by two-dimensional polyacrylamide gel electrophoresis. The protein spots stained with Coomassie brilliant blue were cut out and the proteins were extracted. The material extracted from each spot was mixed with proteins of known molecular weight and then analyzed by electrophoresis in polyacrylamide gels containing sodium dodecyl sulfate. Both the total number and the molecular weights of each of the proteins were determined by these procedures. Thirty-two proteins were identified in the 40 S subunits; their molecular weights ranged from 8000 to 39,000 (average mol. wt = 25,000). Thirty-nine proteins were identified in the 60 S subunit; their molecular weights ranged from 9000 to 58,000 (average mol. wt = 31,000). The sum of the molecular weights of the individual proteins from each subunit is in agreement with previous estimations, derived from physico-chemical measurements of the total protein in mammalian ribosomal subunits. The molecular weight distribution obtained for the isolated proteins was nearly identical to that derived from spectrophotometric analysis of polyacrylamide-sodium dodecyl sulfate gels of the total protein mixtures from each subunit stained with Coomassie brilliant blue. The results are consistent with the hypothesis that reticulocyte ribosomes contain one copy of most of their protein constituents.     

Molecular-weight estimates of milk-fat-globule-membrane protein–sodium dodecyl sulphate complexes by electrophoresis in gradient acrylamide gels

The molecular weights of milk-fat-globule-membrane proteins solubilized in sodium dodecyl sulphate were estimated by gradient gel electrophoresis. Standard curves were calibrated from both protein and glycoprotein markers of known molecular weight. Six major proteins were observed with Coomassie Blue staining and six with periodic acid-Schiff staining. The behaviour of the membrane proteins and the marker proteins was compared on several different single strength sodium dodecyl sulphate-polyacrylamide gels between 3 and 12% (w/v). The results were used to calculate the free electrophoretic mobility and retardation coefficient of each protein. Glycoprotein markers had a significantly lower mean free electrophoretic-mobility value than the protein markers. Three of the milk-fat-globule-membrane glycoproteins were shown to be independent of any of the Coomassie Blue-stained bands. On the basis of a comparison of the free electrophoretic-mobility and retardation- coefficient values of markers and unknown proteins the most appropriate standard curve for molecular-weight estimation was chosen.     

Electrophoretic comparison of polypeptides from enriched plasma membrane fractions from developing soybean roots

The polypeptide complement of enriched soybean (Glycine max [L.] Merr. cult. wells) root plasma membrane fractions was studied by two-dimensional gel electrophoresis. Good resolution was obtained when polypeptides were solubilized in sodium dodecyl sulfate and when butylated hydroxytoluene was included in the vesicle isolation and solubilization media. The pattern obtained on the two-dimensional slab gel for root plasma membrane was characteristic for that membrane. The polypeptide complements from mitochondrial membranes and from enriched fractions of three other endomembrane components were solubilized and electrophoresed for comparison. Each membrane preparation was identifiable on the basis of its characteristic electrophoretogram. Electrophoresis of protein solubilized from plasma membrane fractions isolated from meristematic and mature root tissue revealed both qualitative and quantitative differences in the respective protein complements.     

Actin in ejaculated human sperm cells

Previous studies have identified an "actin-like" protein in human sperm by indirect immunofluorescence microscopy with various probes, but no real biochemical confirmation of actin was made. In this study, two-dimensional (2-D) gels of Nonidet P-40 (NP-40) extracts of purified human sperm cells revealed a protein with appropriate pI and molecular weight coordinates for actin. When excised from a 2-D gel, cleaved with N-chlorosuccinimide, and separated on a sodium dodecyl sulfate slab gel, this putative actin showed a cleavage pattern identical to those from known actin. Furthermore, when sperm were immediately purified from whole semen and extracted with 0.3% NP-40, actin was detected almost entirely in the soluble fraction, indicative of unpolymerized actin. Conclusions from these experiments support those implied by direct immunofluorescence microscopy: actin is present in human sperm and appears to be mainly unpolymerized.     

Tubulin: An Integral Protein of Mammalian Synaptic Vesicle Membranes

Abstract: The major protein in isolated synaptic vesicles from bovine cerebral cortex has been compared to tubulin by sodium dodecyl sulphate-urea polyacrylamide gel electrophoresis, by two-dimensional gel electrophoresis and by peptide mapping following limited proteolysis of the protein by Staphylococcus aureus protease. The results establish in purified synaptic vesicles the presence of tubulin, which is composed of the α and β subunits. In the presence of ethyleneglycol bis (aminoethyl ether)- N, N' -tetraacetic acid (EGTA) or magnesium in the isolation buffers, the synaptic vesicles contained mainly the α-tubulin whereas the β subunit was less abundant. Similarly, synaptosomal plasma membranes that were prepared in the presence of EGTA also contained more of α-tubulin than of the β subunit. Non-ionic detergents such as Triton X-100 or Nonidet P-40 failed to solubilize the tubulin from the synaptic vesicles. Ionic detergents such as deoxycholate and sodium dodecyl sulphate solubilized all the vesicle proteins, including tubulin. The results indicate that α-tubulin is an integral vesicle membrane protein, whereas most of the β sub-unit is peripherally attached and can be easily dissociated from the vesicle membrane with EGTA.     

Structural and polypeptide differences between envelopes of infective and reproductive life cycle forms of Chlamydia spp

Significant differences in cysteine-containing proteins and detergent-related solubility properties were observed between outer membrane protein complexes of reproductive (reticulate body) and infective (elementary body) forms of Chlamydia psittaci (6BC). Elementary bodies harvested at 48 h postinfection possessed a 40-kilodalton major outer membrane protein and three extraordinarily cysteine-rich outer membrane proteins of 62, 59, and 12 kilodaltons, all of which were not solubilized by sodium dodecyl sulfate in the absence of thiol reagents. Intracellularly dividing reticulate bodies harvested at 21 h postinfection were severely deficient in the cysteine-rich proteins but possessed almost as much major outer membrane protein as did the elementary bodies. Most of the major outer membrane protein of reticulate bodies was solubilized by sodium dodecyl sulfate and was present in envelopes as monomers, although a proportion formed disulfide-cross-linked oligomers. By 21 to 24 h postinfection, reticulate bodies commenced synthesis of the cysteine-rich proteins which were found in outer membranes as disulfide-cross-linked complexes. The outer membranes of reticulate bodies of Chlamydia trachomatis (LGV434) also were found to be deficient in cysteine-rich proteins and to be more susceptible to dissociation in sodium dodecyl sulfate than were outer membranes of elementary bodies.