The subunit interface of the Escherichia coli ribosome. Crosslinking of 30 S protein S9 to proteins of the 50 S subunit.

Purified 50 S ribosomal subunits were found to contain significant amounts of protein coincident with the 30 S proteins S9 and/or S11 on two-dimensional polyacrylamide/urea electropherographs. Peptide mapping established that the protein was largely S9 with smaller amounts of S11. Proteins S5 and L6 were nearly coincident on the two-dimensional polyacrylamide/urea electropherographs. Peptide maps of material from the L6 spot obtained from purified 50 S subunits showed the presence of significant amounts of the peptides corresponding to S5. Experiments in which ³⁵S-labelled 30 S subunits and non-radioactive 50 S subunits were reassociated to form 70 S ribosomes showed that some radioactive 30 S protein was transferred to the 50 S subunit. Most of the transferred radioactivity was associated with two proteins, S9 and S5. Sulfhydryl groups were added to the 50 S subunit by amidination with 2-iminothiolane (methyl 4-mercaptobutyrimidate). These were oxidized to form disulfide linkages, some of which crosslinked different proteins of the intact 50 S ribosomal subunit. Protein dimers were partially fractionated by sequential salt extraction and then by electrophoresis of each fraction in polyacrylamide gels containing urea. Slices of the gel were analysed by two-dimensional polyacrylamide/sodium dodecyl sulfate diagonal gel electrophoresis. Final identification of the constituent proteins in each dimer by two-dimensional polyacrylamide/urea gel electrophoresis showed that 50 S proteins L5 and L27 were crosslinked to S9. The evidence suggests that proteins S5, S9, S11, L5 and L27 are located at the interface region of the 70 S ribosome.     

Identification of neighbor relationships among proteins in the 30 S ribosome: intermolecular cross-linkage of three proteins induced by tetranitromethane

Earlier studies have indicated that the reaction of tetranitromethane with the 30 S riboaome from Escherichia coli results in the disappearance of two protein bands from the polyacrylamide gel electrophoresis pattern (Craven et al., 1969b). As tetranitromethane is known to induce intermolecular cross-linkage in other protein systems, we studied further this reaction with the view that it might yield knowledge of protein-protein neighbor relationships within the ribosome.The use of two-dimensional polyacrylamide gel electrophoresis showed that the reaction with tetranitromethane caused the disappearance of four proteins from the pattern of 30 S ribosomal proteins. It was shown that this alteration in electrophoretic behavior was not due to simple protein modification (e.g. production of 3-nitrotyrosine), as reaction with extracted protein in 8 M-urea resulted in no observable change in the electrophoretic pattern.It was also shown that three of these proteins could be uniquely labeled with [¹⁴C]iodoacetate without changing their reactivity with tetranitromethane. Thus, ribosomes were labeled with [¹⁴C]iodoacetate, reacted with tetranitromethane and the radioactive reaction products were isolated by column chromatography and preparative gel electrophoresis. The radioactive peptide patterns of the three proteins digested by trypsin were compared with the three major reaction products. One of these products was shown to contain the radioactive tryptic peptides of all three proteins. We believe that this reaction product is an intermolecular cross-linked aggregate of these three proteins, identified as S11, S18 and S21. We suggest that these three proteins are clustered closely together in the 30 S ribosome. The fourth protein, S12, may also be involved in this aggregate.     

Sites of phosphorylation and mutation in regulatory subunit of cyclic AMP-dependent protein kinase from S49 mouse lymphoma cells: mapping to structural domains

A novel peptide mapping approach has been used to map sites of charge modification to major structural domains of regulatory subunit (R) of type I cAMP-dependent protein kinase from S49 mouse lymphoma cells. Proteolytic fragments of crude, radiolabeled R were purified by cAMP affinity chromatography and displayed by two-dimensional polyacrylamide gel electrophoresis. [35S]methionine-labeled peptides containing sites of mutation or phosphorylation exhibited charge heterogeneity attributable to the modification. Phosphate-containing fragments were also labeled with [32P]orthophosphate to confirm their phosphorylation. Major fragments from [35S]methionine-labeled S49 cell R corresponded in size to carboxyterminal cAMP-binding fragments reported from proteolysis of purified type I Rs from various mammalian species; additional fragments were also visualized. End-specific markers in Rs from some mutant S49 sublines confirmed that cAMP-binding fragments extended to the carboxyterminus of R. Aminoterminal endpoints of fragments could be deduced, therefore, from peptide molecular weights. Clustering of proteolytic cleavage sites within the "hinge-region" separating aminoterminal and carboxyterminal domains of R permitted high resolution mapping in this region: the endogenous phosphate and a "phenotypically-silent" electrophoretic marker mutation fell within a 2.5-kdalton interval at its aminoterminal end. On the other hand, Ka mutations that increase the apparent constant for activation of kinase by cAMP mapped within the large cAMP-binding region of R. A map of charge density distribution within the hinge-region of R was constructed to facilitate structural comparisons between Rs from S49 cells and from other mammalian sources.     

Two Acidic Proteins Associated with Brain Development in Chick Embryo

The developmental changes in protein composition of the chick optic tectum were analyzed by two-dimensional gel electrophoresis. Staining with Coomassie Brilliant Blue R revealed 54 major proteins, eight of which remarkably changed their abundance during development: Four of these proteins (S8, S14, S30, and S54) increased and two of them (S7 and S37) decreased in the course of the brain development. The other two proteins (S5 and S6) appeared at specific embryonic stages and were not detected in the adult. The abundance of S5 protein was highest at day 7, and that of S6 protein at days 9-18. The two proteins were present in other regions of the embryonic brain but were not detected in the embryonic liver. The proteins were purified from the soluble fraction of embryonic chick brains by pH 5.5 precipitation, DEAE-Sepharose column chromatography, ammonium sulfate precipitation, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The molecular weights of S5 and S6 proteins were 95,000 and 100,000, respectively, and their isoelectric points were about 4.5. They were compared by peptide mapping using V8 protease and found to share 11 common peptides out of 17 distinct ones. This indicates a strong degree of structural homology between these two proteins.     

In vivo acylation of Dictyostelium actin with palmitic acid

Cells of Dictyostelium discoideum were incubated with [³H]palmitic acid during development, and recovery of the fatty acid label in soluble and membrane-associated proteins was investigated. One of the major labeled proteins was found exclusively in the soluble fraction. This protein, with an apparent mol. wt. of 44 kd, was identified as actin based on its labeling with a monoclonal anti-actin antibody, its coincidence with the major [³⁵S]methionine-labeled protein after two-dimensional electrophoresis and its binding to a DNase I affinity column. The ³H-label was resistant to chloroform-methanol extraction and boiling in SDS-containing buffer. After partial purification by preparative SDS-polyacrylamide gel electrophoresis, the 44-kd protein was treated with KOH, the fatty acids released were derivatized to methyl esters and palmitic acid methylester was identified by gas-liquid chromatography.     

Surface peptides on intact Ehrlich ascites tumor cells : Identification by a method not requiring subcellular fractionation

Lactoperoxidase catalysed iodination of tyrosyl residues was used to label the exposed plasma membrane proteins in intact Ehrlich ascites tumor cells. Autoradiography of ¹²⁵I-labeled intact cells revealed that the label was predominantly associated with the plasma membrane. When whole cells were solubilized and subjected to gel electrophoresis, two major labeled peptide classes of 100 000 and 80 000 D along with 4 minor labeled classes were found. An identical labeling pattern was obtained when plasma membranes isolated from labeled cells were solubilized and subjected to gel electrophoresis. These results demonstrate that the number of exposed plasma membrane peptides and their molecular weights can be determined without first isolating the membrane by subcellular fractionation procedures, a standard approach in most studies.     

Schwann Cell Surface Proteins and Glycoproteins

Abstract: To identify surface sialoglycoproteins of rat Schwann cells and to compare molecular weights of these sialoglycoproteins with those present in rat peripheral nervous system myelin, we prepared Schwann cells from sciatic nerves of 1–3-day-old rats and cultured them in monolayer. Surface sialoglycoproteins of the cultured cells were tritium-labeled by the periodateborohydride procedure and compared with sialoglycoproteins of adult rat peripheral nervous system myelin by fluorography following polyacrylamide slab gel electrophoresis in sodium dodecyl sulfate. Three radioactive bands with apparent molecular weights of 114,000–132,000, 105,000–115,000, and 44,000–56,000 were observed in both the Schwann cell and myelin preparations. Bands of similar apparent molecular weights were noted in Schwann cells metabolically radiolabeled with d -[1,6-³H]glucosamine. A band co-migrating with myelin P₀ glycoprotein was the most intensely radiolabeled of all peptides in periodate-B³H₄^?treated myelin, but was present in only trace amounts in periodate-B³H₄^? or d -[1,6-³H]glucosamine radiolabeled Schwann cells. Many presumably non-myelin glycoproteins were identified in the cultured Schwann cells by the periodate-borohydride procedure and by incubation of the cells with d -[1,6-³H]glucosamine. An immunoprecipitation technique was used to detect radiolabeled peptides in a nonionic detergent extract of freshly prepared, surface-radioiodinated Schwann cells that were bound by a rabbit anti-Schwann cell serum preabsorbed with rat fibroblasts. Many radioactive peptides were detected in the immunoprecipitate, but the two most intensely radiolabeled had apparent molecular weights of 105,000–115,000 and 95,000–106,000. This study has identified a number of glycoproteins synthesized by cultured rat Schwann cells which resemble in apparent molecular weight the glycoproteins expressed in rat peripheral nervous system myelin and has defined Schwann cell surface proteins recognized by a specific anti-rat Schwann cell antiserum.     

Differential Protein Composition and Gene Expression in Leaf Mesophyll Cells and Bundle Sheath Cells of the C(4) Plant Digitaria sanguinalis (L.) Scop

The distribution and molecular weights of cellular proteins in soluble and membrane-associated locations were analyzed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Coomassie blue staining of leaf (Digitaria sanguinalis L. Scop.) extracts and isolated cell extracts. Leaf polypeptides also were pulse-labeled, followed by isolation of the labeled leaf cell types and analysis of the newly synthesized polypeptides in each cell type by electrophoresis and fluorography.

Comparison of the electrophoretic patterns of crabgrass whole leaf polypeptides with isolated cell-type polypeptides indicated a difference in protein distribution patterns for the two cell types. The mesophyll cells exhibited a greater allocation of total cellular protein into membrane-associated proteins relative to soluble proteins. In contrast, the bundle sheath cells exhibited a higher percentage of total cellular protein in soluble proteins. Phosphoenolpyruvate carboxylase was the major soluble protein in the mesophyll cell and ribulose bisphosphate carboxylase was the major soluble protein in the bundle sheath cell. The majority of in vivo³⁵S-pulse-labeled proteins synthesized by the two crabgrass cell types corresponded in molecular weight to the proteins present in the cell types which were detected by conventional staining techniques. The bundle sheath cell and mesophyll cell fluorograph profiles each had 15 major ³⁵S-labeled proteins. The major incorporation of ³⁵S by bundle sheath cells was into products which co-electrophoresed with the large and small subunits of ribulose bisphosphate carboxylase. In contrast, a major ³⁵S-labeled product in mesophyll cell extracts co-electrophoresed with the subunit of phosphoenolpyruvate carboxylase. Both cell types exhibited equivalent in vivo labeling of a polypeptide with one- and two-dimensional electrophoretic behavior similar to the major apoprotein of the light-harvesting chlorophyll a/b protein. Results from the use of protein synthesis inhibitors during pulse-labeling experiments indicated intercellular differences in both organelle and cytoplasmic protein synthesis. A majority of the ³⁵S incorporation by crabgrass mesophyll cell 70S ribosomes was associated with a pair of membrane-associated polypeptides of molecular weight 32,000 and 34,500; a comparison of fluorograph and stained gel profiles suggests these products resemble the precursor and mature forms of the maize chloroplast 32,000 dalton protein reported by Grebanier et al. (1978 J. Cell Biol. 28:734-746). In contrast, crabgrass bundle sheath cell organelle translation was directed predominantly into a product which co-electrophoresed with the large subunit of ribulose bisphosphate carboxylase.

     

Levels of ribosomal protein S1 and elongation factor G in the growth cycle of Escherichia coli.

The relative levels of ribosomes, ribosomal protein S1, and elongation factor G in the growth cycle of Escherichia coli were examined with two-dimensional polyacrylamide gel electrophoresis. Nonequilibrium pH gradient polyacrylamide gel electrophoresis was used in the first dimension, and polyacrylamide gradient-sodium dodecyl sulfate gel electrophoresis was used in the second dimension. The identities of protein spots containing S1 and elongation factor G were confirmed by radioiodination of the proteins and peptide mapping of the radiolabeled peptides. The levels of ribosomes and ribosomal protein S1 were coordinately reduced during transition from exponential phase to stationary phase. There was no accumulation of S1 in the stationary phase. In marked contrast, the level of elongation factor G showed no significant change from exponential phase to stationary phase. The relative level of elongation factor G compared with ribosomes or S1 increased by about 2.5-fold during transition from exponential phase to stationary phase. The results show that there are differences between the regulation of the levels of elongation factor G and of ribosomal proteins, including S1, apparent during the transition from exponential to stationary phase.     

Influence of Iron Deprivation on the Membrane Composition of Anacystis nidulans

Cultures of the cyanobacterium Anacystis nidulans were grown under iron-deficient conditions and then restored by the addition of iron. Membrane proteins from iron-deficient and iron-restored cells were analyzed by lithium dodecyl sulfate-polyacrylamide gradient gel electrophoresis. The incorporation of [³⁵S]sulfate into membrane proteins and lactoperoxidase-catalyzed ¹²⁵I iodination were used to monitor the rates of polypeptide biosynthesis and surface exposure of membrane proteins, respectively. These polypeptide profiles revealed major differences in the membrane composition of iron-deficient and normal cells. Iron deficiency caused a decrease in the amount of certain important membrane proteins, reflecting a decreased rate of biosynthesis of these peptides. Several photosystem II peptides also showed an increase in surface exposure after iron stress. In addition, iron deficiency led to the synthesis of proteins at 34 and 52 kilodaltons which were not present in normal cells. When iron was restored to a deficient culture, a metabolic sequence was initiated within the first 12 h after the addition of iron which led to phenotypically normal cells. Pulse labeling with [³⁵S]sulfate during this period demonstrated that iron addition initiates a coordinated pattern of synthesis that leads to the assembly of normal membranes.     

Altered chloroplast ribosomal proteins in a yellow mutant of Chlamydomonas reinhardii.

Summary Ribosomes and ribosomal proteins from wild-type and a yellow mutant of Chlamydomonas reinhardii were analysed and compared by two-dimensional gel electrophoresis.Mixothrophycally grown yellow-27 mutant differs from wild-type cells in lowered chlorophyll content and grana fromation of the chloroplast.Analytical ultracentrifuge analyses of cell extracts show a reduced amount of free 70S ribosomes and increased level of 50S subunits in the mutant cells. Similar results were obtained by electronmicroscopical method.Two-dimensional gel electrophoresis shows alterations in protein composition of 70S ribosomes of the mutant. Two proteins of 70S ribosomes have been altered. One of them with high molecular weight is practically absent while there is an additional, intensively stained spot in the mutant.Since the mutation is inherited in a non-Mendelian manner it is possible that the protein alterations in 70S ribosome are localized in the chloroplast DNA.     

p-Azidophenylglyoxal-Epidermal Growth Factor: A Photoactivatable Affinity Crosslinking Reagent

Abstract

A photoaffinity derivative of highly purified ¹²⁵I-labelled epidermal growth factor (¹²⁵I-EGF) has been synthesized. The heterobifunctional crosslinking reagent p-azidophenylglyoxal (PAPG) was bound to arginine residues in ¹²⁵I-EGF. PAPG-¹²⁵I-EGF bound to EGF receptors on rat fibroblasts and human A431 epidermoid carcinoma cells in culture. An apparent decreased affinity of PAPG-¹²⁵I-EGF for the EGF receptor is in accord with at least one arginine being at or near the EGF receptor binding site. The PAPG-¹²⁵I-EGF:EGF receptor complexes on rat cells were internalized to the same extent as control EGF:receptor complexes. A431 cells treated with PAPG-¹²⁵I-EGF were irradiated with ultraviolet light and the labelled proteins were analyzed by SDS-polyacrylamide gel electrophoresis. The 3 major labelled proteins had apparent molecular weights ranging from 75,000 to 200,000. Only the labelling of the 200,000-M_r protein was prevented by the addition of excess unlabelled EGF with the PAPG-¹²⁵I-EGF. This molecular weight is in agreement with the reported size of the EGF receptor plus EGF. A protein with apparent molecular weight of 100,000 was labelled by ¹²⁵I-EGF by an unknown mechanism which was dependent on the dose of UV light and blocked by the addition of excess unlabelled EGF.     

Secreted proteins from rat Sertoli cells.

Sertoli cell cultures were prepared from the testes of 20-day-old rats. The proteins which were secreted by the cells into the culture medium were labeled with [³H]leucine or l-[³H]fucose. The proteins were concentrated by ultrafiltration and analysed by polyacrylamide slab gel electrophoresis (PAGE) in the presence of sodium dodecyl sulfate (SDS). Autofluorography of the gels at ?70 °C showed that the rat Sertoli cells synthesized and secreted at least 7 major polypeptides. The polypeptides had molecular weights ranging from 16 000 to 140 000 D. Proteins which were secreted from cultures of testicular fibroblasts and myoid cells had electrophoretic properties on SDS-PAGE which were different from Sertoli cell secreted proteins. Addition of FSH and testosterone to the Sertoli cell cultures increased the total synthesis and secretion of [³H]leucine-labeled proteins. No qualitative changes in the proteins as a result of hormone application could be detected. However, the synthesis of a polypeptide of molecular weight 48 000 was increased relative to the other secreted peptides if the cells were maintained in FSH and testosterone. The Sertoli cell secreted proteins were shown to be glycoproteins which can bind to ConA-Sepharose and can be labeled with [³H]fucose. Tunicamycin, a specific inhibitor of N-glycosylation, inhibited the secretion of [³H]proteins by 50% but had little effect on the intracellular protein synthesis.     

Rous sarcoma virus-induced changes in the pattern of phosphorylation of non-histone nuclear proteins

We here studied the protein kinase activity and in vitro phosphorylable sites of non-histone nuclear proteins, 0.4 M NaCl extracts (mostly chromosomal proteins) from chick embryo fibroblasts (CEF), infected or not with a Schmidt Ruppin strain subgroup A of Rous sarcoma virus (RSV).The infection and transformation of chick fibroblasts by RSV induced an increase in kinase activity and endogenous phosphorylation of non-histone chromosomal (NHC) proteins. The stimulation, by a change of medium, of the proliferation of dense cultures of normal chick fibroblasts also induced an increase in the kinase activity and endogenous phosphorylation of NHC proteins.However, two-dimensional gel electrophoresis of the ³²P-phosphorylated proteins showed that stimulation due to a change of medium and that due to the expression of transformation were very different. The stimulation by a change of medium increased to a greater or lesser extent the phosphorylation of the different NHC proteins, with no fundamental variations in the pattern of protein phosphorylation. In contrast, RSV infection induced significant changes in the pattern of protein phosphorylation. One of the most striking feature was the large increase of amount and phosphorylation of high molecular weight (HMW) proteins in particular of phosphoproteins having an evaluated molecular weight (MW) of 78 K and 82 K and pI>8.2.The percent of phosphotyrosine residues in NHC proteins was clearly increased when the proteins were extracted from transformed cells instead of normal cells. But the alkaline treatment of two-dimensional gel electrophoresis indicated that the 80 K phosphoproteins did not contain phosphotyrosine residues, and thus cannot be considered as substrates for pp60^src kinase.     

Identification of the major membrane and core proteins of vaccinia virus by two-dimensional electrophoresis.

Vaccinia virus assembly has been well studied at the ultrastructural level, but little is known about the molecular events that occur during that process. Towards this goal, we have identified the major membrane and core proteins of the intracellular mature virus (IMV). Pure IMV preparations were subjected to Nonidet P-40 (NP-40) and dithiothreitol (DTT) treatment to separate the core proteins from the membrane proteins. These proteins were subsequently separated by two-dimensional (2D) gel electrophoresis, and the major polypeptide spots, as detected by silver staining and 35S labeling, were identified by either matrix-assisted laser desorption/ionization mass spectrometry, N-terminal amino acid sequencing, or immunoprecipitation with defined antibodies. Sixteen major spots that partitioned into the NP-40-DTT-soluble fraction were identified; 11 of these were previously described virally encoded proteins and 5 were cellular proteins, mostly of mitochondrial origin. The core fraction revealed four major spots of previously described core proteins, two of which were also detected in the membrane fraction. Subsequently, the NP-40-DTT-soluble and -insoluble fractions from purified virus preparations, separated by 2D gels, were compared with postnuclear supernatants of infected cells that had been metabolically labeled at late times (6 to 8 h) postinfection. This relatively short labeling period as well as the apparent shutoff of host protein synthesis allowed the selective detection in such postnuclear supernatants of virus-encoded proteins. These postnuclear supernatants were subsequently treated with Triton X-114 or with sodium carbonate to distinguish the membrane proteins from the soluble proteins. We have identified the major late membrane and nonmembrane proteins of the IMV as they occur in the virus as well as in infected cells. This 2D gel map should provide an important reference for future molecular studies of vaccinia virus morphogenesis.     

A nuclear protein associated with cell divisions in plants

A nuclear protein, present in carrot meristems and rapidly proliferating cultured cells of carrot (Daucus carota L.) has been identified by the use of a monoclonal antibody (MAb 21D7). By combining the techniques of two-dimensional polyacrylamide gel analysis and blotting separated proteins onto nitrocellulose sheets, it was shown that the antibody detected a single polypeptide of apparent molecular mass (M _r) of 45000 and an isoelectric focusing point (pI) of 6.7. This protein was found by subcellular fractionation and immunofluorescence to be highly concentrated in the nucleoli of somatic and zygotic embryos of a wide range of plants. It was not detectable in logarthmically growing cells ofEscherichia coli, yeast, embryos ofDrosophila melanogaster or cultured C3H mouse cells. These data indicate that this protein is a highly conserved non-histone protein associated with nuclei of rapidly dividing plant cells.Abbreviations M _r apparent molecular mass - Da dalton - Ig immunoglobulins - MAb monoclonal antibody - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - 2-D gel two-dimensional gel electrophoresis - 2,4-D 2,4-dichlorophenoxyacetic acid     

Isolation and characterization of the plasma membrane of L-1210 cells. Iodination as a marker for the plasma membrane

Mouse leukemia L-1210 cells were iodinated with ¹²⁵I; this permitted the development of a method for the isolation of the plasma membranes. These show a 10- to 16-fold increase in the specific activity of ¹²⁵I over that of the cell homogenate and a 20-fold increase in the specific activities of 5′-nucleotidase and alkaline phosphate; no mitochondrial or microsomal marker enzyme activities were detected. Sodium dodecyl sulfate gel electrophoresis of the plasma membranes shows approx. 40 peptides with molecular weights ranging from 10 000 to over 200 000; a polypeptide ($\text{M}{}_{\mathrm{<mtext>m</mtext>}}$ 50 000) predominates. Of 13 iodinated surface membrane proteins, the major radioactive peptide has a molecular weight of 85 000. The importance of the selection of the appropriate gel sytem for the analysis of membrane proteins is emphasized.     

Genetic analysis of human lymphocyte proteins by two-dimensional gel electrophoresis:

Summary Approximately 250 phytohemagglutinin (PHA)-stimulated peripheral blood lymphocyte polypeptides from three unrelated healthy males were compared by high-resolution two-dimensional gel electrophoresis and double-label autoradiography. Comparisons by all possible pairwise combinations of [¹⁴C]leucine-labeled proteins from an individual and [³H]leucine-labeled proteins from another revealed that only three polypeptides differed qualitatively among the three individuals. The degree of variation in lymphocyte polypeptides between different individuals was similar to that in fibroblast polypeptides reported previously. Among the three variant polypeptides, two polypeptides with mol.wt. 64,000 and mol. wt. 37,000 coexisted with a polypeptide with the same molecular weight, and they showed the behavior expected of two allelic gene products separated in the isoelectric focusing dimension by charge differences. Analysis of [¹⁴C]leucine labeled peripheral blood lymphocyte proteints, from the parents of each individual, by two-dimensional gel electrophoresis indicated that the variant polypeptides with mol. wt. 64,000 and mol. wt. 37,000 in the propositus were inherited from one of his parents. The data indicate that genetic analysis of PHA-stimulated peripheral blood lymphocyte proteins is feasible by high-resolution two-dimensional gel electrophoresis in combination with double-label autoradiography and pedigree analysis.     

Five structural classes of major outer membrane proteins in Neisseria meningitidis

Group B Neisseria meningitidis is thus far subdivided into 15 protein serotypes based on antigenically different major outer membrane proteins. Most serotypes have three or four major proteins in their outer membranes. Comparative structural analysis by chymotryptic 125I-peptide mapping was performed on these major proteins from the prototype strains as well as from six non-serotypable strains. The major outer membrane proteins from each of the serotypes were first separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis using the Laemmli system. Individual proteins within the gel slices were radioiodinated and digested with chymotrypsin, and then their 125I-peptides were separated by electrophoresis and chromatography on cellulose thin-layer plates. The peptide maps obtained by autoradiography were categorized into five different structural classes which correlated with the apparent molecular weights of proteins, i.e., 46 +/- 1K, 41 +/- 1K, 38 +/- 1K, 33 +/- 1K, and 28 +/- 1K. Each of the major outer membrane proteins within a strain had a distinctly different chymotryptic peptide map, indicating significant differences in the primary structure of these proteins. In contrast, outer membrane proteins of the same or very similar molecular weight from different serotype strains had similar, occasionally identical peptide maps, indicating a high degree of structural homology. The unique peptides from proteins of the same structural classes were often hydrophilic, whereas common peptides were often hydrophobic, suggesting that the serotype determinants reside within the variable hydrophilic regions of major outer membrane proteins.     

Characterization of a membrane-associated glycoprotein complex implicated in cell adhesion to fibronectin

We have characterized a 140-kDa glycoprotein complex purified by a monoclonal antibody and implicated in cell adhesion to the extracellular molecule fibronectin. Three major polypeptide components were purified by monoclonal antibody JG22E, which had apparent molecular weights of 155,000 (band 1), 135,000 (band 2), and 120,000 (band 3). In two-dimensional gel electrophoresis, each subunit migrated as either a broad band or a series of spots at acidic isoelectric points. After treatment with neuraminidase, the spots became focused around pH 6.2 (band 1), pH 5.6 (band 2), and pH 5.3 (band 3). These three major bands were compared by two-dimensional peptide mapping in a series of pairwise combinations and were found to be distinct proteins. In sucrose gradients, these proteins co-migrated as a complex sedimenting at approximately 8.4 S either before or after affinity purification, whereas separated subunits migrated at 4.7 to 5.8 S. Amino acid analysis revealed no detectable hydroxyproline and a composition characterized by a substantial number of cysteine residues compared to the average protein. Our results suggest that a noncovalent complex of structurally distinct glycoproteins is involved in adhesive interactions of fibronectin with cells.