Showing papers on "Gel electrophoresis published in 1974"

Actin Is the Naturally Occurring Inhibitor of Deoxyribonuclease I

Abstract: Various tissues and cells in culture contain a specific inhibitor of DNase I (EC 3145) In this paper evidence is presented that this inhibitor is actin, one of the major structural proteins of muscle and nonmuscle cells (a) The inhibitor is a major cellular component constituting 5-10% of the soluble protein (b) It migrates with actin on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, having a characteristic molecular weight of 42,000 (c) It has an amino-acid composition closely similar to that of actin (d) The peptide maps of the two proteins are nearly identical (e) Skeletal muscle actin inhibits the enzymatic activity of DNase I (f) DNase I-agarose affinity chromatography quantitatively retains purified skeletal muscle actin, and actin, specifically, from high-speed supernatants of whole cell extracts (g) An antibody to purified inhibitor protein from calf thymus, used in indirect immunofluorescence on cells grown in culture, stains a two-dimensional network of fibers similar to that seen with an actin-specific antibody The observation that actin can be isolated by DNase-agarose affinity chromatography provides a useful tool for the biochemical study of actin under different physiological conditions

The Amino-Acid Sequence of the Alkali Light Chains of Rabbit Skeletal-Muscle Myosin

Abstract: The amino-acid sequence of the alkali light chain 1 (Al) of rabbit skeletal muscle myosin has been determined and compared with the sequence of the smaller but related alkali light chain 2 (A2). The molecular weights of the two proteins calculated from these sequence determinations are 20700 and 16500, respectively. The results show that the two proteins have identical sequences over their C-terminal141 residues. There is an additional sequence of 41 residues at the N-terminal end of A1 which accounts for the extra 4000 molecular weight. Between this additional sequence and the sequence common to both proteins are eight amino-acid residues. Comparison of the sequences of these eight residues in A1 and A2 reveals five amino-acid substitutions. Thus in spite of the very extensive homology between the two proteins, these substitutions indicate that there must be two RNA coding sequences for these light chains in rabbit fast muscle myosin. Rabbit skeletal muscle myosin contains two heavy chains of molecular weight about 200000 [l 21 and four light chains of molecular weight about 20000 [3]. Two chemical classes of light chains have been characterised by their thiol peptides [4] and reaction of myosin with 5,5’-dithiobis(2-nitrobenzoic acid) (Nbs,) selectively dissociated a substantial proportion of one of these light chains without significant effect on the myosin ATPase activity. This light chain has been termed the “Nbs, light chain” l. The remaining light chains cannot be dissociated without total loss of ATPase activity and these were termed the “alkali light chains”, since they were first shown to be released under alkaline conditions [5]. Gel electrophoresis of myosin in the presence of sodium dodecylsulphate shows the presence of three light chain components with apparent molecular weights of 25000, 18000 and 16000 [3]. The 18000-M, component corresponds to the Nbs, light chain and the other two components are classified as alkali light chains since they both contains the single thiol sequence characteristic of this class of light chains in fast muscle myosin [6,7]. The extent of sequence homology between alkali light chain 1 (Al, Mr = 25000) and alkali light chain 2 (A2, Abbreviation. Nbs,, 5,5’-dithiobis(2-nitrobenzoic acid). Nomenclature. The term “alkali light chain” is based on the observation that these proteins are dissociated from myosin in alkali [5]. Other terminology used elsewhere includes LC1(= Al) and LC3 (= A2) [26] and OL and y chains [29]. 1 The “Nbs, light chain” was previously called the “DTNB light chain” [4]. Mr = 16000) was examined by peptide mapping of tryptic digests of the two proteins. Peptides from both proteins stained selectively for arginine, tyrosine and histidine had identical mobilities in the two-dimensional maps, and all the peptides present in A2 could be found in corresponding positions in Al. Such differences as there were could be accounted for by additional peptide material in Al, though the amino-acid compositions indicated minimum molecular weights of 21000 for A1 and 17000 for A2, a difference of 4000 instead of the 8000 estimated from apparent molecular weights determined by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate. From these results it was concluded that either A2 was a degraded fragment of A1 or the two proteins showed an exceptionally high degree of homology. The relative constancy in the amounts of A1 and A2 in rabbit myosin prepared under a variety of conditions together with observations that both components are found in myosin from chicken breast and leg muscles [S], and the fast skeletal muscle of pig, sheep and cat, make it unlikely that A2 arises as a result of adventitious proteolytic activity. Nevertheless we could not rule out a specific cleavage occurring in the polypeptide chains as a consequence of post-translational modification or during the myosin assembly process. For this reason detailed sequence analysis of both A1 and A2 has been carried out to investigate the relationship between them and show whether they represent products of different genes. Part of the results of this work have been published previously [9].

[9] Molecular weight determination of membrane protein and glycoprotein subunits by discontinuous gel electrophoresis in dodecyl sulfate

Abstract: Publisher Summary This chapter describes an electrophoretic system capable of stacking and fractionating protein and glycoprotein-dodecyl sulfate complexes over a range of 2300–320,000 daltons. The system combines the advantages of electrophoresing protein-SDS complexes pioneered by Shapiro and the advantages of achieving thin starting zones by use of discontinuous buffers discovered by Ornstein and Davis. This SDS discontinuous system was developed to fractionate plasma membrane proteins solubilized in SDS and provides high resolution patterns of membrane protein subunits and reliable estimates of protein subunit molecular weights. The methods described in the chapter have been used in the laboratory for the past two years and give highly reproducible results providing that the essential variables are controlled.

Purification and Properties of the Cholinergic Receptor Protein from Electrophorus electricus Electric Tissue

Abstract: The acetylcholine (nicotinic) receptor from the electric organ of Electrophorus electricus has been purified approx. 300-fold from Triton X-100 extracts of electroplax membrane fragments. The receptor protein was first adsorbed from crude extracts on an affinity column containing 1,3-bis(tri-ethylammonium ethoxy)-4-iodoacetamidobenzene diiodide, an analogue of flaxedil, linked to Sepharose 2B, then eluted with a solution of flaxedil and concentrated. The receptor protein was further purified by centrifugation on sucrose gradients containing 1 Triton X-100, 1% Emulphogene, or 0.5% sodium cholate. If desired, detergent could be removed after centrifugation in the presence of sodium cholate by gel filtration on Sephadex G-75. At all stages the receptor was assayed by incubation with α-[3H]toxin from Naja nigricollis followed by isolation of the toxin · receptor complex by filtration on Millipore filters at low detergent concentration. The best preparations bound 1 mol toxin per 150000 g protein. In the presence of non-denaturing detergents the preparations gave a single major band for both protein and activity upon polyacrylamide disc gel electrophoresis or sucrose gradient centrifugation. The receptor protein appears essentially homogeneous in the electron microscope after negative staining with uranyl acetate. Upon gel electrophoresis in the presence of dodecylsulphate, there were two major bands corresponding to molecular weights of 43000 and 48000. The ultraviolet-absorption spectrum and amino-acid composition of the purified receptor protein are typical of those of globular, water-soluble proteins in general and do not reflect the hydrophobic character of the macromolecule. Furthermore, the purified protein interacts with concanavalin A and other plant agglutinins indicating that it contains a carbohydrate moiety. The purified receptor protein carries a single class of binding sites for [3H]decamethonium (Kd= 0.02 μM) at ligand concentrations up to 1 μM and for acetylcholine (Kd= 0.06 μM) at up to 1 μM. No evidence for cooperativity in binding of cholinergic effectors to the receptor protein, either purified or in crude extracts, has been detected and all effectors tested inhibit the binding of [3H]decamethonium in a competitive manner. The affinity of agonists for the solubilized receptor protein is approx. 20-fold greater than for the membrane-bound protein.

Outer Membrane Proteins of Escherichia coli III. Evidence that the Major Protein of Escherichia coli O111 Outer Membrane Consists of Four Distinct Polypeptide Species

Abstract: Previous studies have shown that the outer membrane of Escherichia coli O111 gives a single, major, 42,000-dalton protein peak when analyzed by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis at neutral pH. Further studies have shown that this peak consists of more than a single polypeptide species, and on alkaline SDS-gel electrophoresis this single peak is resolved into three subcomponents designated as proteins 1, 2, and 3. By chromatography of solubilized, outer membrane protein on diethylaminoethyl-cellulose followed by chromatography on Sephadex G-200 in the presence of SDS, it was possible to separate the 42,000-dalton major protein into four distinct protein fractions. Comparison of cyanogen bromide peptides derived from these fractions indicated that they represented at least four distinct polypeptide species. Two of these proteins migrated as proteins 1 and 2 on alkaline gels. The other two proteins migrated as protein 3 on alkaline gels and cannot be separated by SDS-polyacrylamide gel electrophoresis. In purified form, these major proteins do not contain bound lipopolysaccharide, phospholipid, or phosphate. These proteins may contain a small amount of carbohydrate, as evidenced by the labeling of these proteins by glucosamine, and to a lesser extent by glucose, under conditions where the metabolism of these sugars to amino acids and lipids is blocked. All of the proteins were labeled to the same extent by these sugars. Thus, it was concluded that there are at least four distinct polypeptide species with apparent molecular masses of about 42,000 daltons in the outer membrane of E. coli O111.

Muscle-Like Contractile Proteins and Tubulin in Synaptosomes

Abstract: Material in major bands with molecular weights corresponding to those of actin, brain tropomyosin, and myosin is present in purified rat synaptosomes dissolved in sodium dodecyl sulfate and subjected to electrophoresis on dodecyl sulfate-acrylamide gels. A band corresponding to tubulin appears to be the major constituent of synaptosomes, confirming the work of Feit and his coworkers. We have demonstrated by peptide mapping that the proteins in these bands have strong chemical similarities to actin, brain tropomyosin, myosin, and tubulin. We have prepared synaptic membrane, vesicle, and soluble fractions from synaptosomes. The polypeptide composition of synaptic membranes, as determined by dodecyl sulfate-acrylamide gel electrophoresis, is similar to that of synaptosomes, with tubulin, actin, and tropomyosin being major constituents. Synaptic vesicles have as their major polypeptide an unidentified protein with a molecular weight of 50,000; they also have many bands in common with synaptosomes. The soluble fraction predominantly contains actin and tubulin. The possibility that the muscle-like contractile proteins and tubulin are membrane-associated in various cell types is discussed, as is their possible role in neurotransmitter release.

The Heterogeneity of Mouse‐Chromatin Nonhistone Proteins as Evidenced by Two‐Dimensional Polyacrylamide‐Gel Electrophoresis and Ion‐Exchange Chromatography

Abstract: 1 A two-dimensional gel electrophoresis procedure employing a combination of isoelectric focusing and dodecylsulfate electrophoresis has been used to analyse the components of 32P-labelled nonhistone protein fractions obtained by the chromatography of salt-urea dissociated chromatins on hydroxyapatite. 2 In this way the nonhistone proteins eluted from hydroxyapatite by 0.05 M phosphate (fraction H2) have been found to consist of a heterogeneous mixture of components of a molecular weight range of 15000 to 200000 and to have isoelectric points between pH 4.5 and 9. The components of the fraction eluted from hydroxyapatite by 0.2 M phosphate (fraction H3) appeared to consist of a smaller group of proteins with a molecular weight range similar to that of the H2 proteins but whose isoelectric points lay between pH 2 and 6. Both fractions consisted of a mixture of phoshorylated and nonphosphorylated protein species. 3 Comparison of the components of H2 and H3 protein fractions by this electrophoretic procedure showed that many of the phosphorylated and nonphosphorylated proteins were common to mouse liver, kidney and brain chromatins. Only a few species were found to be tissue specific. 4 It was found that chromatography on QAE-Sephadex did not follow the same separation pattern as that obtained by isoelectric focusing, suggesting that the fractionation on this ion-exchange material is not entirely dependent on the overall charge of the polypeptide. 5 Preparation of individual nonhistone protein species has been attempted using this chromatographic procedure in conjunction with gel filtration in guanidine hydrochloride.

Cytochrome b in Neurospora crassa mitochondria. Site of translation of the heme protein.

Abstract: Cytochrome b was purified from Neurospora crassa mitochondria by means of chromatography on oleyl-polymethacrylic acid resin, chromatography on DEAE-cellulose and recycling gel filtration on Sephadex G-75, all steps being performed in a medium containing bile acids and salts The heme content of the preparation was calculated on the basis of incorporated 59Fe and [3H]-leucine to be 33–40 μmol/g protein, corresponding to a minimum molecular weight of 25000 to 30000 Upon gel filtration on Sephadex G-75 in a bile acid and KCl medium the preparation migrated like a 55000-molecular-weight protein Upon gel electrophoresis on polyacrylamide or upon gel filtration on Sephadex G-100 in a dodecylsulfate medium the protein part of the preparation migrated as one band of the apparent molecular weight of approximately 30000 These findings suggest that the purified cytochrome b is a dimeric heme protein In vivo incorporation of [3H]leucine into the protein part of the purified cytochrome b was insensitive to cycloheximide but sensitive to chloramphenicol This indicates that the apoprotein of cytochrome b is translated on mitochondrial ribosomes

Cell free synthesis of adenovirus 2 proteins programmed by fractionated messenger RNA: a comparison of polypeptide products and messenger RNA lengths

Abstract: Cytoplasmic RNA extracted from human tissue culture cells infected with adenovirus type 2 was used to program protein synthesis in a cell-free system derived from mammalian cells. Analysis of the protein product by polyacrylamide gel electrophoresis revealed ten adenovirus-specific polypeptides. Five of these were further identified by analysis of tryptic peptides. Translation of RNA fractionated by sedimentation through sucrose gradients containing formamide demonstrated seven size classes of RNA, each of which programmed the synthesis of only one or two virus-specific polypeptides. Six of the virus-specific polypeptides were translated from RNAs much larger than expected for the size of the polypeptide.