Gel electrophoretic isolation, in the hundred microgram range, of recombinant SDS-syntaxin from sea urchin egg cortical vesicles.

Recombinant urchin syntaxin [Xa cut], electrophoresed at pH 9.0 (25 degrees C) or 10.2 (0 degrees C) in a discontinuous Tris-chloride-glycinate buffer system in the presence of 0.03% SDS in the catholyte, exhibits a multicomponent pattern in gels of a polyacrylamide concentration of 12% and 3% crosslinking. The position in the pattern of the syntaxin band was identified by reference to electropherograms of a previous study (P. Backlund, pers. comm.). The complexity of the protein composition of the preparation was reduced by selective stacking of proteins with mobilities greater than that of syntaxin. This provides a gel pattern consisting of two bands with mobilities close to that identified as syntaxin, as well as a minor, more slowly migrating, contaminant. The two major components are designated as S1 and S2, the latter being the larger species. In the absence of SDS, the preparation exhibits two pairs of protein components. Three of the proteins are charge isomers, i.e., of equal size, differing only in net charge, assumed to be forms of S1, while the fourth component is larger and is assumed to be S2. Aliquots of the preparation, containing 150 microg of protein were loaded on a cylindrical polyacrylamide gel of 18 mm diameter, and separated S1 and S2 were excised in a position defined by their characteristic values of relative mobility (Rf). Two or three gel slices, corresponding in Rf to S1 or S2, were pooled and loaded onto a Stacking Gel (5% polyacrylamide, 20% cross-linked) of 18 mm diameter, equipped with a collection chamber of 200 microL volume. The protein was electroeluted from the gel slices and concentrated into a stack by electrophoresis. The stack, marked by bromphenolblue, was allowed to migrate into the collection chamber, was collected and analyzed by protein assay and re-electrophoresis. Re-electrophoresis of S1 shows that it consists of at least three components. Recovered S1 constitutes 47% of the preparation, based on protein assay, S2 4%. S1, isolated from SDS-PAGE, exhibits an apparent Mr of 22.7 kDa, S2 one of 34.5 kDa, similar to the value of 32.6 kDa expected from the structure of syntaxin. The absence of S2 from the electroeluate re-electrophoresed at 0 degrees C and their molecular weight relationship suggest a proteolytic transformation of S2 to S1.     

The ribosomal proteins of Drosophila melanogaster. I. Characterization in polyacrylamide gel of proteins from larval, adult, and ammonium chloride-treated ribosomes

Summary Ribosomes were isolated from larvae and adult flies, and the purity of the preparation was checked by electron microscopy. The ribosomal proteins were extracted with cold dilute hydrochloric acid, and precipitated with cold acetone. The proteins were characterized by polyacrylamide gel electrophoresis. At pH 3.0 at least 25 bands of different color intensities were resolved, forming a complex pattern.On the basis of electrophoretic mobilities, it was shown that some ribosomal proteins are species-specific, and that larval ribosomes have three protein components more than ribosomes from adult flies.Incubation of the ribosomes with 0.75 M NH₄Cl at a low Mg⁺⁺ concentration lead to detachment of 64% of the ribosomal protein. This detachment of protein molecules was considerably reduced by a five-fold increase of Mg⁺⁺ ions.     

The preparation of calmodulins from barley (Hordeum sp.) and basidiomycete fungi.

1. Calmodulin-like proteins were purified from the fruiting bodies of higher (basidiomycete) fungi and barley (Hordeum sp.) shoots. 2. These calmodulins have electrophoretic mobilities on 10% (w/v) polyacrylamide gels at pH 8.3 in the presence of 6 M-urea and at pH 8.3 in the presence of 0.1% sodium dodecyl sulphate similar to that of bovine brain calmodulin. They interacted with rabbit skeletal-muscle troponin I in the presence of Ca2+. 3. Barley and fungal calmodulins activated myosin light-chain kinase and phosphodiesterase in the presence of Ca2+, although the amounts needed were at least an order of magnitude greater than is required to produce the same effect with mammalian calmodulin. 4. Amino acid analyses indicated a number of differences from the mammalian protein, most notably the absence of trimethyl-lysine. 5. By using 125I-labelled calmodulin, a small amount of calmodulin-binding protein was detected in homogenates of barley and fungi. 6. No protein corresponding to calmodulin could be found in Escherichia coli or yeast, although a relatively high concentration of a protein that bound calmodulin was detected in E. coli by this technique.     

The effect of cross-links on the mobility of proteins in dodecyl sulphate–polyacrylamide gels

The effect of reduction of intramolecular disulphide bridges on the mobility of proteins in 5% (w/v) polyacrylamide gels in the presence of sodium dodecyl sulphate was investigated. A series of polypeptide polymers, containing up to 68 intramolecular disulphide bridges, was prepared by cross-linking proteins of known structure with glutaraldehyde. These model polypeptides were denatured with heat, sodium dodecyl sulphate and urea, and their mobilities in sodium dodecyl sulphate-polyacrylamide gels compared before and after reduction with dithiothreitol. The mobilities of polypeptides containing no cystine were unaffected by reduction. However, reduction generally decreased the mobilities of polypeptides containing cystine; the extent of this decrease depended on the number of cystine residues originally present in the polypeptide polymer, and on the protein from which the latter was derived. In contrast with their higher oligomers, the monomer of lysozyme and the dimer of ribonuclease increased in mobility after reduction. The reduced polypeptide oligomers formed by reaction with glutaraldehyde were generally found to migrate at a rate significantly faster than was expected from their calculated molecular weights. It was concluded that the use of unreduced proteins and protein aggregates for molecular-weight measurements by the sodium dodecyl sulphate-polyacrylamide-gel method may give erroneous estimates of the molecular weight of any protein being investigated.     

Structural Changes in Thylakoid Proteins during Cold Acclimation and Freezing of Winter Rye (Secale cereale L. cv. Puma)

Thylakoids were isolated from nonhardened and cold-hardened winter rye (Secale cereale L. cv. Puma), and subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the presence and absence of sulfhydryl reagents. Electrophoresis of cold-hardened rye thylakoid proteins revealed the presence of a 35 kilodalton polypeptide and the absence of a 51 kilodalton polypeptide found in nonhardened rye thylakoid proteins. The 35 kilodalton band could be induced by adding β-mercaptoethanol to nonhardened rye thylakoid proteins, whereas the 51 kilodalton band could be formed by adding cupric phenanthroline to these same proteins. Sulfhydryl group titration showed that cold-hardened rye thylakoid proteins contained more free sulfhydryls than nonhardened rye proteins. Although amino acid analysis of thylakoid proteins revealed quantitative differences in several amino acid residues, the polarity of thylakoid proteins did not change during cold acclimation. No significant changes in sodium dodecyl sulfate-polyacrylamide gels of thylakoid proteins appeared when either nonhardened or cold-hardened plants were frozen in vivo or in vitro. However, thylakoid proteins did aggregate when frozen in the presence of β-mercaptoethanol. Although thylakoid proteins isolated from cold-hardened rye contained more reduced thiols, a general state of reduction did not act as a cryoprotectant. It is hypothesized that conformational changes of specific proteins may be important for low temperature growth of rye.     

Detection of disulphide bonds and localization of interchain linkages in the third (C3) and the fourth (C4) components of human complement.

Disulphide bonds contribute significantly to the maintenance of structural/functional integrity of many proteins. Therefore it was of interest to study the distribution and the effect of disulphides on conformation of complement components C3 and C4. These proteins are precursors of several fragments with various binding sites and distinct physiological functions. The constituents of C3c (beta, alpha 27, alpha 43) and those of C4c (beta, alpha 27, alpha 16, gamma) were investigated, since other fragments of C3 or C4 do not participate in interchain linkages. Inter-and intra-chain disulphide bonds in C3c and C4c were localized by using a modification of conventional SDS (sodium dodecyl sulphate)/polyacrylamide-gel electrophoresis such that the change in mobility of disulphide-bond-containing proteins can be detected throughout the transition from a non-reduced to a fully reduced state. Several forms of the alpha 43 fragment from C3, and of the gamma-chain of C4, with different mobilities can exist, depending on the number of intra-chain disulphide bonds reduced. The intermediates (heterodimers) generated by a partial reduction of C3c or C4c were characterized by two-dimensional SDS/polyacrylamide-gel electrophoresis performed in the absence, then in the presence, of beta-mercaptoethanol. The inter-chain linkages in C3c were determined to be beta-alpha 27 and alpha 27- alpha 43, thus indicating the presence of only one interchain bond in C3. The two interchain bonds in C4c are beta-alpha 27 and alpha 16-gamma. The third interchain bond in C4 (alpha 27-gamma, tentative) remains to be determined.     

Placement of loci for avenins and resistance to Puccinia coronata to a common linkage group in Avena strigosa.

Alcohol-soluble seed storage proteins of oat (avenins) were extracted from two diploid accessions representing the A genome and separated by high-resolution acid polyacrylamide gel electrophoresis. Polymorphisms were detected for three clearly resolved protein bands. Linkage analysis of 88 F2:3 families mapped the three bands to a single locus. Integration of avenin segregation data with an RFLP linkage map constructed from the same population, mapped the avenin locus to a linkage group containing a locus conferring resistance to nine isolates of Puccinia coronata. Linkage between genes encoding alcohol-soluble seed proteins and genes for resistance to Puccinia species was also observed for the homoeologous group 1 chromosomes of barley (1H), rye (1R), wheat (1A, 1B, 1D), and chromosomes 4 and 10 of maize.     

The identification of a heat-shock protein complex in chloroplasts of barley leaves

In vivo radiolabeling of chloroplast proteins in barley (Hordeum vulgare L. cv Corvette) leaves and their separation by one-dimensional electrophoresis revealed at least seven heat-shock proteins between 24 and 94 kD, of which most have not been previously identified in this C₃ species. Fractionation into stromal and thylakoid membrane components showed that all chloroplast heat-shock proteins were synthesized on cytoplasmic ribosomes, translocated into the chloroplast, and located in the stroma. Examination of stromal preparations by native (nondissociating) polyacrylamide gel electrophoresis revealed the presence of a high-molecular mass heat-shock protein complex in barley. This complex was estimated to be 250 to 265 kD in size. Dissociation by denaturing polyacrylamide gel electrophoresis revealed a single protein component, a 32-kD heat-shock protein. The synthesis of this protein and the formation of the heat-shock protein complex were dependent on functional cytoplasmic ribosomes. Immunological studies showed that the heat-shock protein complex did not contain any proteins homologous to the α-subunit of ribulose bisphosphate carboxylase oxygenase subunit-binding protein. Other features about the complex included the absence of nucleic acid (RNA or DNA) and its nondissociation in the presence of Mg²⁺/ATP. These results suggest that the heat-shock protein complex in barley chloroplasts is a homogeneous octamer of 32-kD subunits.     

The purification and characterization of homologous high molecular weight storage proteins from grain of wheat,rye and barley

Summary Homologous high molecular weight storage prolamins were purified from grain of wheat, rye and barley using combinations of gel filtration, ion-exchange chromatography and preparative isoelectric focusing. Sodium dodecylsulphate polyacrylamide gel electrophoresis showed that the components were single bands with apparent mol.wts. of above 100,000. Molecular weights determined by sedimentation equilibrium ultracentrifugation were considerably lower; 54,700, 67,600 and 69,600 for the components from barley, rye and wheat respectively. Amino acid analysis showed the presence of 13.6 to 16.5 mol% glycine, 29.6 to 34.0 mol% glutamate + glutamine, 11.4 to 13.7 mol% proline and a total of 4.0 to 5.7 mol% basic amino acids. Automated N-terminal amino acid sequencing of the component from wheat showed the presence of cysteine residues at positions 5 and 10, and this is discussed in relation to the possible role of these proteins in the visco-elastic gluten network.     

Female specific proteins in Triatoma infestans haemolymph

The presence of female specific proteins in triatoma infestans haemolymph, as well as the relationship between the female specific proteins and egg proteins, were analysed. At the same time, the presence of specific female proteins in different instars was studied. Cellulose acetate electrophoresis, polyacrylamide gel electrophoresis, and immunochemical methods were used.No differences between immature female and male haemolymph were established. Female haemolymph obtained from insects with ovary development revealed quantitative differences, with cellulose acetate, with respect to the control males. With polyacrylamide gel electrophoresis, one component that is not detected in control males was detected in mature female haemolymph. With immunochemical assays, at least two antigenic components that were not observed in male haemolymph were detected.Egg extract showed, with cellulose acetate, two bands with a mobility similar to that of the proteins increased in the haemolymph of the mature female; with polyacrylamide gel, two major bands with a mobility similar to that of the specific female haemolymph protein were detected. Egg extract contains at least two components demonstrated by double-diffusion assays and three components by immunoelectrophoresis, with immunological identity to specific mature female haemolymph proteins.The extract obtained from recently hatched insects revealed two components with immunological identity to specific female proteins. Haemolymph from first, second, third, fourth and fifth instars do not appear to contain any femalespecific haemolymph protein.     

The influence of the rye genome on expression of heat shock proteins in triticale

Summary The heat shock protein profiles from Secale cereale L. cv Imperial, Triticum aestivum L. cv Chinese Spring, S. cereale x T. aestivum amphiploid, and the seven disomic S. cereale addition lines to T. aestivum were used to compare the wheat, rye, and triticale Heat Shock Protein profiles and to study the influence of the rye genome on heat shock protein expression in triticale. Three-day-old seedlings were heat shocked for 2 h at 40 °C in the presence of ³⁵S-methionine, and polypeptides from root tissues were subjected to one- or two-dimensional gel electrophoresis. The wheat and rye heat shock protein profiles each consisted of > 150 heat shock proteins, of which 94 were sufficiently reproducible to construct a standard map. There were 11 unique rye heat shock proteins compared to 22 unique wheat heat shock proteins. The triticale heat shock protein profile resembled the rye parent more than the wheat parent. There were 22 heat shock proteins expressed uniquely by wheat that were not expressed in triticale. Rye chromosomes 1 and 3 exhibited a substantial repressive influence on the expression of 95% of the unique wheat heat shock proteins in triticale, while rye chromosome 4 appeared to have the least repressive influence on expression of the unique wheat heat shock proteins in triticale.Mention of a trade name or proprietary product does not constitute a guarantee, warranty, or recommendation of the product by the United States Department of Agriculture or the University of Missouri and does not imply its approval to the exclusion of other products that may be suitable     

Unusual features of cereal seed protein structure and evolution

The alcohol-soluble (prolamin) storage proteins of barley, wheat and rye vary in their structures, but all have two features in common: the presence of distinct structural domains differing in amino acid compositions, and of repeats within one of these domains. Detailed comparisons of amino acid sequences show that all appear to have evolved from a single ancestral gene consisting of three short related regions (called A, B and C). Regions related to A, B and C are also present in the minor prolamins of maize and in three other groups of seed proteins: inhibitors of alpha-amylase and/or trypsin from cereals. 25 storage globulins from several dicotyledonous species and a 2S albumin from sunflower. It is suggested that these proteins together constitute a protein superfamily with limited sequence homology.     

Clustering of genes for 20 kd zein subunits in the short arm of maize chromosome 7

Zein is the major storage protein of the endosperm of maize kernels. When this alcohol-soluble protein is subjected to SDS polyacrylamide gel electrophoresis, it is resolved into four fractions of different molecular weight: 10, 14, 20 and 22 kilodaltons (kd). Each fraction is heterogeneous with respect to isoelectric pH. For example, the 20 kd fraction contains at least seven subfractions as revealed by isoelectric focusing in polyacrylamide gels. In this report, we present evidence that the structural genes coding for the 20 kd proteins are clustered on the short arm of chromosome 7, a region that also bears loci regulating endosperm zein biosynthesis [opaque-2 (02) and defective endosperm-B30 (De^*-B30)]. The organization of these zein genes suggests that the evolution of at least some of the maize genome has occurred as the result of repeated duplication and divergence of chromosome segments.     

Predicting functional gene links from phylogenetic-statistical analyses of whole genomes

An important element of the developing field of proteomics is to understand protein-protein interactions and other functional links amongst genes. Across-species correlation methods for detecting functional links work on the premise that functionally linked proteins will tend to show a common pattern of presence and absence across a range of genomes. We describe a maximum likelihood statistical model for predicting functional gene linkages. The method detects independent instances of the correlated gain or loss of pairs of proteins on phylogenetic trees, reducing the high rates of false positives observed in conventional across-species methods that do not explicitly incorporate a phylogeny. We show, in a dataset of 10,551 protein pairs, that the phylogenetic method improves by up to 35% on across-species analyses at identifying known functionally linked proteins. The method shows that protein pairs with at least two to three correlated events of gain or loss are almost certainly functionally linked. Contingent evolution, in which one gene's presence or absence depends upon the presence of another, can also be detected phylogenetically, and may identify genes whose functional significance depends upon its interaction with other genes. Incorporating phylogenetic information improves the prediction of functional linkages. The improvement derives from having a lower rate of false positives and from detecting trends that across-species analyses miss. Phylogenetic methods can easily be incorporated into the screening of large-scale bioinformatics datasets to identify sets of protein links and to characterise gene networks.     

Stable expression of <Emphasis Type="Italic">1Dx5</Emphasis> and <Emphasis Type="Italic">1Dy10</Emphasis> high-molecular-weight glutenin subunit genes in transgenic rye drastically increases the polymeric glutelin fraction in rye flour

We generated and characterized transgenic rye synthesizing substantial amounts of high-molecular-weight glutenin subunits (HMW-GS) from wheat. The unique bread-making characteristic of wheat flour is closely related to the elasticity and extensibility of the gluten proteins stored in the starchy endosperm, particularly the HMW-GS. Rye flour has poor bread-making quality, despite the extensive sequence and structure similarities of wheat and rye HMW-GS. The HMW-GS 1Dx5 and 1Dy10 genes from wheat, known to be associated with good bread-making quality were introduced into a homozygous rye inbred line by the biolistic gene transfer. The transgenic plants, regenerated from immature embryo derived callus cultures were normal, fertile, and transmitted the transgenes stably to the sexual progeny, as shown by Southern blot and SDS-PAGE analysis. Flour proteins were extracted by means of a modified Osborne fractionation from wildtype (L22) as well as transgenic rye expressing 1Dy10 (L26) or 1Dx5 and 1Dy10 (L8) and were quantified by RP-HPLC and GP-HPLC. The amount of transgenic HMW-GS in homozygous rye seeds represented 5.1% (L26) or 16.3% (L8) of the total extracted protein and 17% (L26) or 29% (L8) of the extracted glutelin fraction. The amount of polymerized glutelins was significantly increased in transgenic rye (L26) and more than tripled in transgenic rye (L8) compared to wildtype (L22). Gel permeation HPLC of the un-polymerized fractions revealed that the transgenic rye flours contained a significantly lower proportion of alcohol-soluble oligomeric proteins compared with the non-transgenic flour. The quantitative data indicate that the expression of wheat HMW-GS in rye leads to a high degree of polymerization of transgenic and native storage proteins, probably by formation of intermolecular disulfide bonds. Even -40k secalins, which occur in non-transgenic rye as monomers, are incorporated into these polymeric structures. The combination 1Dx5 + 1Dy10 showed stronger effects than 1Dy10 alone. Our results are the first example of genetic engineering to significantly alter the polymerization and composition of storage proteins in rye. This may be an important step towards improving bread-making properties of rye whilst conserving its superior stress resistance.     

COMPARATIVE STUDIES OF HIGHLY BASIC PROTEINS OF OX BRAIN AND RAT BRAIN. MICROHETEROGENEITY OF BASIC ENCEPHALITOGENIC (MYELIN) PROTEIN

(1) The total amount of highly basic proteins in acid extracts of whole ox brain, ox white matter and ox grey matter was determined quantitatively after electrophoresis on 5% polyacrylamide gels at pH 10-6 in the presence of 8 M-urea. (2) Ox white matter gave 13 mg and ox grey matter 2 mg of highly basic proteins per g fresh tissue on treatment with 0-03 n -HCl. The yield of total basic proteins of ox white matter increased to 17-6 mg/g fresh brain on stepwise extraction at pH 3-0, 2-0 and 1-0; the extract at pH 3.0 accounted for 90 per cent of the total basic proteins. (3) The high encephalitogenic activity of the fraction of highly basic proteins extracted at pH 3.0 from ox white matter indicated that these basic proteins were derived from myelin. It is suggested that the amount of basic proteins in a sample of brain extracted under these conditions is proportional to the amount of white matter in the sample. (4) The encephalitogenic (myelin) basic protein fraction was homogeneous with respect to molecular size but could be resolved into at least six components by electrophoresis at high pH. (5) The myelin basic proteins extracted from ox white matter had lower electrophoretic mobilities at high pH than did those of two basic proteins of rat brain apparently derived from myelin.     

Protein constituents of the mouse spermatozoon: I. An electrophoretic characterization

Total protein constituents of the mouse spermatozoon have been fractionated and characterized by polyacrylamide gel electrophoresis. Three spermatozoan fractions were obtained following homogenization with 1% sodium dodecylsulfate (SDS) and sucrose gradient centrifugation: SDS-soluble proteins, SDS-insoluble tail components, and SDS-insoluble head components. Purities of these fractions were assessed at greater than 95% using Nomarksi differential interference microscopy. Subsequently, the SDS-insoluble sperm heads were further fractionated into five protein subclasses by ultracentrifugation and ion-exchange chromatography. SDS-Polyacrylamide gel electrophoresis indicates that each of these spermatozoan fractions contains distinct protein species. Furthermore, the electrophoretic profiles are highly reproducible and show no evidence of cross-contamination or proteolysis. The SDS-soluble fraction, which includes proteins from the plasma membrane, acrosome, axoneme, matrix and cristae of the mitochondria, contains one major 39,000-molecular weight band and numerous minor bands with molecular weights ranging from ～30,000 to greater than 100,000. In contrast, electrophoresis of the SDS-insoluble tail proteins reveals the presence of at least nine prominent bands with apparent molecular weights between 21,000 and 89,000. Ultrastructural analysis suggests that this fraction contains proteins from the outer dense fibers, fibrous sheath, outer mitochondrial membranes, and structural elements of the neck region of the sperm tail. Two subfractions of the SDS-insoluble sperm heads each contain one of the two mouse protamines. In addition, the acidic and moderately basic head fractions each contain a limited number of distinct protein bands with molecular weights ranging from 14,000 to 76,000. These proteins are apparently derived from either the spermatozoan nucleus or the associated perinuclear material, since all other sperm head structures are solubilized during SDS treatment. One- and two-dimensional electrophoresis on acetic acid-urea polyacrylamide gels indicates that the moderately basic fraction may contain minor components that resemble certain histones and/or spermatidal basic nuclear proteins.     

Molecular weights of estrogen and androgen binding proteins in the liver of Xenopus laevis.

[3-H]Estradiol-17beta and [3-H]dihydrotestosterone binding proteins in the cytosol fraction of liver from both male and female Xenopus laevis were characterized by electrophoresis on polyacrylamide gels. These binding proteins, which were indistinguishable based upon their mobilities on gels of different acrylamide concentrations, migrated as single components with a molecular weight of 2.0 x 10-4. Separation of native or sodium dodecyl sulfate denatured specific estrogen-binding components on dodecyl sulfate free acrylamide gels gave similar results, i.e., a single species of molecular weight 2.0-2.5 x 10-4. The same molecular weight also was obtained when cytosol was prepared in the presence of either diisopropyl fluorophosphate or phenylmethanesulfonyl fluoride, protease inhibitors. Evidence that the liver components binding either [3-H]estradiol-17-beta or [3-H]dihydrotestosterone were not plasma contaminants was provided by the observation that the plasma sex-steroid binding globulin of Xenopus had a different mobility when separated by polyacrylamide gel electrophoresis.     

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.     

Expression of Myelin Components in Mouse Schwann Cells in Culture

Mouse Schwann cells cultured in vitro are capable of expressing basal levels of the major myelin components P1, P2, P0, and galactocerebroside. Numerical counts of immunostained cultures indicated that between 22 and 40% of the cells are positive up to 21 days for all of the components indicated. Electrophoretic analysis of Schwann cells labeled with a 14C-amino acid mixture revealed the presence of proteins with relative mobilities identical to those of P0 and P1. Positive identification of the two proteins was indicated by immunoprecipitation of P1 and immunoblotting of P0. These data show that in the absence of neurites, Schwann cells in culture can express low levels of myelin characteristic components even in the absence of myelin assembly.