Actin-Based Gelation of Cytoplasmic Extracts from Brain Tissue

Incubation of rat brain cytoplasmic extracts under the conditions described in this paper results in the formation of three-dimensional gels. Ultrastructurally, these gels correspond to complex supramolecular structures formed by single microfilaments and by microfilament bundles. Analysis of protein composition indicates that cytoplasmic gels are composed of actin and several associated proteins. Among the latter class of components, we have identified polypeptides with molecular weights of 55,000 (55K), 140,000 (140K), and a set of two or three polypeptides with molecular weights in the range of 235,000-245,000 (235-245K). The 55K and 140K components do not seem to correspond to any previously identified actin-associated proteins, while the 235-245K polypeptides may correspond to the protein known as fodrin.     

Rhythmic chloroplast migration in the green alga Ulva: dissection of movement mechanism by differential inhibitor effects

Rhodopseudomonas viridis thylakoid membrane polypeptides were characterised by SDS gels, 2 D gels and surface-specific iodination. Four polypeptides with apparent molecular weights of 38 000, 33 000, 27 000, and 24 000 (reaction centre) and three low molecular weight polypeptides 11 000, 8000 and 6000 (probably light harvesting polypeptides) were identified. Antibodies were produced against the polypeptides eluted from SDS gels and tested for specificity by an immunoblotting assay. The antibodies were bound to the membranes and viewed by electron microscopy using a modification of the ferritin labelling technique. It is suggested that antigenic determinants for the 38 000, 33 000, and 27 000 reaction centre polypeptides and the 11 000 and 8000 low molecular weight polypeptides are present on the cytoplasmic membrane surface. The 33 000, 27 000, 11 000 and 6000 polypeptides appear to have surface-located residues which can be iodinated. The photosynthetic membrane of Rps. viridis appears to be a highly asymmetrical membrane.     

Schistosoma mansoni: identification and characterization of schistosomula polypeptides

Schistosomula proteins separated by a two-dimensional (NEPHGE) gel system identify 94 major silver-stained polypeptides. When compared to polypeptides similarly separated from cercariae and adult worms; cercariae share the same polypeptides as schistosomula, adult worms share ca. 60% of the polypeptides. A group of five schistosomula polypeptides 15-31 kDa (apparent pI 8.2-8.9) was not found in adult worm extracts. To identify which polypeptides were immunogens, Western blots of the NEPHGE gels were probed with sera either from humans with chronic schistosomiasis or from mice vaccinated with irradiated cercariae. For characterization studies, polyclonal antibodies were made against the five schistosomula-specific and selected immunogenic polypeptides by immunizing mice with silver-stained spots removed from NEPHGE gels. We show that the polyclonal serum against a polypeptide of 12.5 kDa and an apparent pI of 6.70 mediated complement and eosinophil-dependent killing of schistosomula in an in vitro assay. Epitopes recognized by antibody against the 12.5-kDa polypeptide show a diffuse distribution and are found on flame cells of the excretory system of the schistosomula.     

Gel Electrophoresis of Chloroplast Polypeptides: Comparison of One-Dimensional and Two-Dimensional Gel Analyses of Chloroplast Polypeptides From Euglena gracilis

Euglena chloroplast polypeptides are resolved by an adaptation of the two-dimensional gel electrophoretic technique of O'Farrell (1975 J Biol Chem 250: 4007-4021). The present results are compared with those obtained by our earlier two-dimensional gel analyses as well as those obtained by one-dimensional gel analyses. Up to 75 micrograms of Euglena chloroplast polypeptides are resolved on one-dimensional sodium dodecylsulfate linear gradient 7.5 to 15% polyacrylamide gels into 43 stained polypeptide bands compared to only 33 bands resolved on a similar gel containing only 10% polyacrylamide. In contrast, two-dimensional gel electrophoresis (isoelectric focusing for the first dimension, sodium dodecylsulfate gel electrophoresis for the second dimension) further improves the resolution of the chloroplast polypeptides and especially so when a linear gradient gel is used for the second dimension. Delipidation of Euglena chloroplasts with acetone-ether and subsequent solubilization of polypeptides with Triton X-100 followed by sonication are all necessary for successful resolution of chloroplast polypeptides on two-dimensional gels. Up to 300 micrograms of chloroplast polypeptides can be clearly resolved into 56 to 59 stainable spots by the present two-dimensional gel technique when a linear gradient gel is used for the second dimension. Thus, about 30% of the polypeptide bands on a one-dimensional gel are separated into multiple polypeptides on a two-dimensional gel. The use of two-dimensional gels to separate labeled polypeptides with subsequent detection of labeled spots by autoradiography or fluorography again improves the resolution of the chloroplast polypeptides. For example, when ³⁵S-labeled chloroplast polypeptides are separated by the present two-dimensional gel technique with a linear gradient polyacrylamide gel in the second dimension, autoradiography or fluorography detects over 80 individual polypeptide spots. This is about twice the number resolved by our previous analyses which used a 10% polyacrylamide gel in the second dimension. Polypeptides detected range in molecular weight from about 8.5 to about 145 kilodaltons with apparent isoelectric points from pH 4.5 to 8.0. Fluorography provides rapid detection of labeled polypeptides and is 10 times more sensitive than autoradiography.     

The sites of synthesis of the principal thylakoid membrane polypeptides in Chlamydomonas reinhardtii

The sites of synthesis of the major thylakoid membrane polypeptides have been studied in the green alga Chlamydomonas reinhardtii by pulse labeling of cells with [14C]acetate in the presence of inhibitors specific for chloroplast and cytoplasmic protein synthesis. The labeled membrane polypeptides were separated by an improved method of sodium dodecyl sulfate (SDS) gradient gel electrophoresis, and autoradiographs were made of the dried gels. The results demonstrate that of the 33 polypeptides resolved in the gels, at least nine are made on chloroplast ribosomes. Two of these (polypeptides 2 and 6) are associated with the reaction centers of photosystems I and II. Another polypeptide (polypeptide 5) appears from genetic data to be coded by chloroplast DNA. Experiments with a mutant whose chloroplast ribosomes are resistant to spectinomycyn (spr-u-1-6-2) show that polypeptides whose synthesis takes place on chloroplast ribosomes are made in the presence of spectinomycin in the mutant although their synthesis is blocked by this antibiotic in wild type cells.     

Use of avidin-sepharose to isolate and idenify biotin polypeptides from crude extracts

A method of isolating and identifying biotin polypeptides from crude cellular extracts is described. Protein samples are run on small avidin-Sepharose columns. After washing away nonspecifically bound protein, the biotin enzymes are eluted using an SDS-urea solution. The inactive polypeptides are then electrophoresed on SDS-polyacrylamide gels. The biotin polypeptides in the gels are identified by using fluorescent avidin or by analyzing for radioactive biotin-labeled polypeptides. A sensitive method for assaying biotin using avidin-Sepharose is also described.     

Properties and topography of the major integral plasma membrane protein of a unicellular organism

The cellular distribution, membrane orientation, and biochemical properties of the two major NaOH-insoluble (integral) plasma membrane proteins of Euglena are detailed. We present evidence which suggests that these two polypeptides (Mr 68 and 39 kD) are dimer and monomer of the same protein: (a) Antibodies directed against either the 68- or the 39-kD polypeptide bind to both 68- and 39-kD bands in Western blots. (b) Trypsin digests of the 68- and 39-kD polypeptides yield similar peptide fragments. (c) The 68- and 39-kD polypeptides interconvert during successive electrophoresis runs in the presence of SDS and beta- mercaptoethanol. (d) The 39-kD band is the only major integral membrane protein evident after isoelectric focusing in acrylamide gels. The apparent shift from 68 to 39 kD in focusing gels has been duplicated in denaturing SDS gels by adding ampholyte solutions directly to the protein samples. The membrane orientation of the 39-kD protein and its 68-kD dimer has been assessed by radioiodination in situ using intact cells or purified plasma membranes. Putative monomers and dimers are labeled only when the cytoplasmic side of the membrane is exposed. These results together with trypsin digestion data suggest that the 39- kD protein and its dimer have an asymmetric membrane orientation with a substantial cytoplasmic domain but with no detectable extracellular region. Immunolabeling of sectioned cells indicates that the plasma membrane is the only cellular membrane with significant amounts of 39- kD protein. No major 68- or 39-kD polypeptide bands are evident in SDS acrylamide gels or immunoblots of electrophoresed whole flagella or preparations enriched in flagellar membrane vesicles, nor is there a detectable shift in any flagellar polypeptide in the presence of ampholyte solutions. These findings are considered with respect to the well-known internal crystalline organization of the euglenoid plasma membrane and to the potential for these proteins to serve as anchors for membrane skeletal proteins.     

Polypeptides of the oxygen-evolving photosystem II complex. Immunological detection and biogenesis

Oxygen-evolving photosystem II complex was isolated from spinach chloroplasts. The individual polypeptides of the complex were isolated from sodium dodecyl sulfate (SDS)-polyacrylamide gels and antibodies were raised in rabbits against these polypeptides. After washing of the isolation complex by 0.8 M Tris to release the extrinsic proteins, a distinct diffused protein band was revealed at the position of 33 kDa in SDS gels containing 4 M urea. When this band was electroeluted from the gel and subsequently electrophoresed on SDS gels, three distinct protein bands became apparent. Antibodies raised against each one of these polypeptides cross-reacted with the other two polypeptides to varying degrees but not with the other subunits of the complex. The three polypeptides were denoted as "34," "33," and "32" kDa and the 33 being the herbicide-binding protein. Using the antibodies, the relative amounts of the photosystem II polypeptides were followed during greening of etiolated spinach seedlings. While all three extrinsic polypeptides were present in etiolated leaves at relatively high amounts, the other polypeptides could not be detected prior to an approximate 6-h illumination period. Further illumination induced the appearance of all of the rest of the subunits in a relatively similar rate. The oxygen evolution activity was developed parallel to the increase in the amounts of these polypeptides. Therefore, the assembly of the active photosystem II during greening is a two-step process in contrast with the photosystem I reaction center, which is assembled step by step, and the rest of the chloroplast protein complexes, which are assembled by a concerted mechanism.     

Proteins Specified by Herpes Simplex Virus XII. The Virion Polypeptides of Type 1 Strains

The polypeptides from purified virions of a herpes simplex 1 (human herpes-virus 1) strain, F1, which had been passaged a limited number of times in cell culture after isolation, formed 33 bands on electrophoretic separation in polyacrylamide gels cross-linked with N, N'-diallyltartardiamide in contrast to a maximum resolution of only 24 to 25 bands in gels cross-linked with N, N'-methylenebisacrylamide. This increase in the number of bands was due chiefly to an improved separation of glycosylated polypeptides from nonglycosylated polypeptides with which they co-electrophoresed on methylenebisacrylamide cross-linked gels. Purified virions of HSV-1 [F1] had a protein/DNA mass ratio of 10.7 +/- 0.96, and based on a DNA molecular mass of 85 x 10(6) to 100 x 10(6) the estimated weight of virion polypeptides ranges from 16.4 to 19.4 x 10(-16) g. The number of molecules of each polypeptide per virion ranged from less than 50 to 1,500. Comparison of the virion polypeptides of two HSV-1 strains with similar isolation and limited passage history with those of four HSV-1 strains with histories of numerous passages outside the human host showed a number of nonrandom variations in virion polypeptides. Thus, although the virion polypeptides of two strains with similar isolation and limited passage history could not be differentiated, strains with extended passage histories differed markedly from each other and from the limited passage strains in the number and electrophoretic mobility of noncapsid polypeptides and notably in those of the envelope.     

Comparative analysis of proteins from the fibrous sheath and outer dense fibers of rat spermatozoa

The protein composition of the fibrous sheath (FS) and the outer dense fibers (ODF), two cytoskeletal components of the tail of spermatozoa, was compared by using polyacrylamide gel electrophoresis and immunochemistry applied to Western blots and to spermatozoa. Isolated FS and ODF, the purity of which were verified by electron microscopy (EM), were denatured and either run on sodium dodecyl sulfate-polyacrylamide gels or used to raise antibodies. The gels revealed at least 18 and 14 polypeptide bands for the FS and ODF, respectively. The major bands of the FS had molecular masses of 75, 27.5, and 14.4 kDa, whereas the major bands of the ODF-connecting piece had molecular masses of 32-26, 20, 14.4, 84, and 80 kDa. Several prominent FS and ODF bands were found to comigrate on gels, and the 14.4 kDa polypeptides had similar electrophoretic properties. Anti-FS serum reacted with the majority of Western blot-transferred FS polypeptides, but also cross-reacted strongly with a major 14.4 kDa ODF polypeptide and with less affinity to other major ODF polypeptides. Anti-ODF serum reacted with the majority of ODF polypeptides, but also cross-reacted strongly with a major 14.4 kDa FS polypeptide, and with less affinity to several other FS polypeptides including the 75 kDa band. Antibodies affinity-purified from the 14.4 kDa FS polypeptide only cross-reacted with the 14.4 kDa ODF polypeptide, whereas antibodies purified from the 14.4 kDa ODF polypeptide cross-reacted with 14.4, 27.5, 57, and 63 kDa FS polypeptides. The immunocross-reactions observed on Western blots were confirmed by immunocytochemical methods applied to spermatozoa. This study demonstrates that the FS and ODF, both composed of many polypeptides, several having similar molecular weights, are related cytoskeletal structures as they have epitopes in common, and both contain 14.4 kDa polypeptides with common antigenic and electrophoretic properties.     

Detection of basic proteins and low molecular weight peptides in polyacrylamide gels by formaldehyde fixation

Certain basic and low molecular weight proteins are not retained in gels by standard acid fixation procedures. Formaldehyde has been used to covalently link proteins and polypeptides in polyacrylamide gels. The method has been tested with a variety of gel systems and proteins. In all the systems tested the new method retained all the proteins seen in the standard method and also additional low molecular weight and basic proteins and polypeptides and ampholines which were lost in the standard systems. Therefore, the method is suitable for the detection of these substances.     

Characterisation of size variants of type I DNA topoisomerase isolated from calf thymus

Calf thymus DNA topoisomerase I, which belongs to the eukaryotic type I topoisomerases, is in a typical preparation purified as a set of five major polypeptides with Mr between 70000 and 100000. At least four of these proteins have binding affinity for DNA as was shown by incubating them with radioactive single-stranded DNA after separation in dodecylsulfate polyacrylamide gels and blotting onto nitrocellulose filters. That these polypeptides have DNA relaxing activity was directly demonstrated with protein extracted from single bands of dodecylsulfate/polyacrylamide gels. We consider the 100000-Mr protein to be the native enzyme. The smaller components are catalytically active fragments of the native topoisomerase most probably arising from limited proteolysis either within the nucleus or during the purification of the enzyme. In two-dimensional non-equilibrium pH-gradient electrophoresis gels the topoisomerase size variants exhibit apparent pI values between 8.1 and 8.3, with small but distinct differences between the components. The calf thymus topoisomerase I, upon binding to phage fd-DNA, protects a stretch of 15-25 nucleotides against digestion with DNase I.     

Radiolabelling of DNA/polypeptide complexes in isolated bulk DNA and in residual nuclear matrix DNA by nick-translation

Conditions are described that allow 32P-radiolabelling and detection of tight complexes between DNA and polypeptides by nick-translation. Prolonged nick-translation of purified bulk DNA results in radiolabelled complexes migrating on SDS-polyacrylamide gels with apparent molecular weights of 68 kd and 54 kd respectively. Residual nuclear matrix DNA which is not accessible to DNase I on the nuclear level becomes accessible to radiolabelling by nick-translation on the nuclear matrix level. In this case the in situ radiolabelled complexes migrate on SDS-polyacrylamide gels with apparent molecular weights of 68 kd and 100 kd. The DNA/polypeptide complexes are stable during treatments with SDS, beta-mercapto ethanol and alkali which points to covalent bonds between the polypeptides and DNA strands.     

Electrophoretic characterization of human, equine and bovine transferrins

1. Human, bovine and equine transferrins have been characterized with respect to mol. wt, and behavior on urea-polyacrylamide gels, and isoelectric focussing gels. 2. As shown by SDS-polyacrylamide gel electrophoresis human transferrin has one major polypeptide whereas both bovine and equine transferrins have two polypeptides. 3. The transferrins show multiple banded patterns on urea-polyacrylamide and isoelectric focussing gels, particularly when iron saturated. The various forms are not resolved by neuraminidase treatment.     

Transformation in Bacillus subtilis: a 75,000-dalton protein complex is involved in binding and entry of donor DNA.

A 75,000-dalton protein complex involved in DNA binding during transformation was purified from membranes of competent Bacillus subtilis cells. Previous results (Smith et al., J. Bacteriol. 156:101-108, 1983) showed that the complex contained two polypeptides, polypeptide a (molecular weight, 18,000; isoelectric point, 5.0) and polypeptide b (molecular weight, 17,000; isoelectric point, 4.7) in approximately equal amounts. In the present experiments the two polypeptides were extracted from two-dimensional gels and studied separately and in combination with respect to DNA binding and nuclease activities. For DNA binding the interaction of both polypeptides was required. DNA binding occurred efficiently in the presence of EDTA. Nuclease activity was restricted to polypeptide b. The nucleolytic properties of b were identical to those of the native 75,000-dalton complex. Polypeptide a affected b by reducing its nuclease activity. Analysis of the nuclease subunit b on DNA-containing polyacrylamide gels revealed nuclease activities at four different molecular weight positions. These activities were identical to the major competence-specific nuclease activities which were previously implicated in the entry of donor DNA during transformation (Mulder and Venema, J. Bacteriol. 152:166-174, 1982). These results indicate that the 75,000-dalton protein complex is composed of two different competence-specific polypeptides involved in both binding and entry of donor DNA. The possible roles of the two polypeptides in the transformation of B. subtilis are discussed.     

Immunoblotting of keratin polypeptides extracted from tissues preserved in standard histologic fixatives

Cytoskeletal polypeptides from fresh placental tissue, tissue stored at -30 degrees C, and tissue fixed in 10% buffered formalin, Bouin's solution, and Carnoy's solution were extracted, separated by electrophoresis, and immunoblotted using monoclonal antibodies immunoreactive with keratin polypeptides. Storage of the placental tissue at -30 degrees C, or fixation in Carnoy's solution did not alter the extractability, migration pattern, or immunoreactivity of the keratin polypeptides. Keratin polypeptides could not be adequately demonstrated in extracts prepared from formalin- or Bouin's solution-fixed tissues. Several unmasking procedures used on tissues before extraction and on nitrocellulose blots before application of primary antibodies failed to unmask keratin polypeptides, either in Coomassie blue-stained gels or in immunoblots reacted with anti-keratin antibodies. These data indicate that Carnoy's solution is the fixative of choice for tissues in which electrophoretic and immunoblotting analyses of keratin polypeptides might be required.     

On the electrotransfer of polypeptides from gels to nitrocellulose membranes

The conditions which affect the elution of polypeptides from polyacrylamide gels by electrophoresis and polypeptide-nitrocellulose interactions have been studied. The rate of elution of polypeptides from a 15% sodium dodecyl sulfate-polyacrylamide gel is dependent on the molecular weight of the individual polypeptides, which is in agreement with the results of W. N. Burnette (Anal. Biochem. 112, 195 (1981)). We also observed that current density affects the rate of elution. Polypeptides smaller than 20,000 daltons pass through pores of 0.45 microns, but not through the pores of 0.1-microns nitrocellulose membranes during electrophoresis. The nonionic detergent NP-40 inhibits the binding of polypeptides to nitrocellulose and removes prebound polypeptides from the membranes. Amido black and Coomassie blue staining and destaining processes do not remove the bound polypeptides from the membranes, but may affect the antigenicity of polypeptides. Polypeptides immobilized on nitrocellulose can be stored at -70 degrees C for future use.     

Schistosoma mansoni: One- and two-dimensional electrophoresis of proteins synthesized in vitro by males,females, and juveniles

Polyacrylamide gel electrophoresis coupled with fluorography is a sensitive method for visualizing individual gene products synthesized in vitro by Schistosoma mansoni (K. Atkinson and B. G. Atkinson 1980, Nature (London)283, 478–479). In vitro labelling with radioactive amino acids ensures that the proteins are of parasite origin and fluorography permits detection of minute amounts of newly synthesized, electrophoretically separated gene products. One-dimensional electrophoretic separation in polyacrylamide gels with sodium dodecyl sulphate and fluorography of juvenile and adult proteins reveal that juveniles produce most adult proteins. Although similar studies with proteins from sexed adults imply that analogous gene products are elaborated by both sexes, a number of sex-specific gene products are resolvable by more rigorous two-dimensional electrophoretic separations. The homogametic male produces 5 polypeptides not produced by the heterogametic female. Three outstanding male-specific gene products include a polypeptide with a molecular weight (MW) of 88 kilodaltons (kd) and an isoelectric point (pI) of 5.65, one with an MW of 66 kd and a pI of 5.25, and one with an MW of 58 kd and a pI of 5.25. Other, readily detectable male-specific polypeptides include one which coelectrophoreses with β-actin and one which coelectrophoreses with β-tropomyosin. The female synthesizes 4 specific polypeptides which have isoelectric points between 4.3 and 4.7, are of low molecular weight, and are resolvable only with 12% acrylamide gels. Two-dimensional electrophoresis resolves 74 major polypeptides synthesized by adult worms, and a code is presented which identifies each polypeptide by sex specificity, isoelectric point, and molecular weight.     

Plastid Development in Pisum sativum Leaves during Greening : I. A Comparison of Plastid Polypeptide Composition and in Organello Translation Characteristics

Changes in plastid polypeptide composition during greening of etiolated peas were investigated by two-dimensional gel electrophoresis. One hundred of the more than 250 polypeptides which could be detected upon silver staining were followed during plastid development. Thirty-nine polypeptides decreased in abundance on a per organelle basis. Twentythree of the 46 polypeptides which increased in abundance upon greening could be identified as proteins of the thylakoid membrane. The changes in proteins observed during greening of etiolated leaves corresponded largely to those observed during normal leaf expansion. The origin of some of the polypeptides was traced back by comparing the two-dimensional gels of plastid proteins with in organello translation products and with polypeptides which had been synthesized in vitro from poly(A⁺) mRNA preparations and posttranslationally imported by chloroplasts. Some polypeptides were specifically identified in two-dimensional gels by Western blot analysis.     

Identification of human sperm surface glycoproteins recognized by autoantisera from immune infertile men, women, and vasectomized men

To identify the surface antigens of human sperm recognized by antisera from immune infertility patients and vasectomized men, we labeled sperm surface proteins with 125I- and used patient antisera for immunoprecipitation. Sera were studied from 27 infertile males, 18 infertile females, and 4 vasectomized males, each possessing anti-sperm antibodies detected by immunobead binding. Sera from different infertile males, different infertile females, and vasectomized males were remarkably similar in their surface antigen recognition. The different sera specifically immunoprecipitated the same small group of 125I-labeled surface proteins, which included polypeptides in the region 90 kDa, 40-45 kDa, and 26 kDa. Treatment with N-glycanase showed that the proteins of 90 kDa, 40-45 kDa, and 26 kDa were glycoproteins with N-linked carbohydrate. The immunoprecipitated 125I-labeled proteins and the total extract of 125I-labeled surface proteins were compared on two-dimensional (2D) gels. The results show the 90 kDa polypeptide is a major sperm surface component, whereas 40-45 kDa and 26 kDa polypeptides are minor components. The 2D gel comparison also indicates that 90 kDa, 40-45 kDa, and 26 kDa are a small subset of the total ensemble of sperm surface proteins. Clinical data suggest antibodies to these few proteins interfere with sperm function.