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A new siliconized glass fibre as support to protein chemical analysis of electro-blotted protein

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Abstract

A new hydrophobic glass-fiber support is presented, which is well suited to the electrophoretic transfer of proteins from polyacrylamide gels and subsequent protein-chemical analysis. Modified glass-fiber sheets are easily prepared by chemical reaction of the surface with poly(methyl-3,3,3-trifluoropropylsiloxane) in trifluoroacetic acid. The modification is stable during electroblotting, amino acid sequence analysis and hydrolysis. The siliconized glass fiber exhibits a high protein-binding capacity, allows the application of well-established staining procedures, and does not interfere with the analytical methods of modern protein chemistry at the low picomole level. Samples separated by electrophoresis and immobilized on hydrophobic supports fail to exhibit any detectable contamination in amino acid sequence analysis hence allowing the high performance of the available protein-chemical methods to be exploited.

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IntroductionRemoval of Salts or Polar ComponentsRemoval of Detergents and Apolar Contaminants“Golden Rules” for Protein and Peptide HandlingReferences
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Chapter
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Chapter
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Chapter
IntroductionFrom Protein Analysis to Proteome AnalysisMethods of Proteome AnalysisSummary and Outlook
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Chapter
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Chapter
Regulation of energy metabolism in mitochondria is essential for survival of animals, since variations of work load, speed of reactivity, amount of thermogenesis, and adaptation to starvation require specific adjustments of the rate of oxygen consumption and efficiency of energy transduction in the respiratory chain. The various aspects of regulation of oxygen consumption in animals have been reviewed by Skulachev (1997). For warm-blooded animals, regulation of thermogenesis is of particular importance. Apart from thermogenesis in brown adipose tissue of rodents via the uncoupling protein (Nedergaard and Cannon, 1984; Nicholls and Locke, 1984), the mechanism of nonshivering thermogenesis in mammals and birds is largely unknown (Block, 1994).
Chapter
Sialidases (N-acetylneuraminosyl-glycohydrolases; EC 3.2.1.18) regularly occur in higher animals of the deuterostomate lineage from the echinoderms to the vertebrates but have not been found in lower animals and plants (Corfield et al., 1981; Corfield and Schauer, 1982b). They also occur in myxoviruses (Drzeniek et al., 1974), some Protozoa (Pereira, 1983; Reuter et al., 1987), fungi (Corfield et al., 1981), and in many different bacterial species (Müller, 1974; Rosenberg and Schengrund, 1976). Strikingly, most of these microorganisms have close contact to higher animals and are commensals or pathogens. Such pathogenic bacteria, for instance, are Clostridia, a few species of which were described to secrete sialidase (Fraser, 1978; Clostridium perfringens, C. septicum, C. chauvoei, C. sordellii, C. tertium). Popoff and Dodin (1985) also detected sialidase in a few strains (8%) of C. butyricum.
Chapter
This chapter focuses on the internal amino acid sequencing of proteins recovered from one- or two-dimensional gels. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis, combined with electroblotting and automated Edman degradation is routinely used for protein purification and N-terminal amino acid sequence analysis. In addition, development of the amino acid sequencing technology toward higher sensitivities unexpectedly leads to cases where artifactual N-terminal blocking is noted. Internal amino acid sequences are obtained by either in-gel or on-membrane cleavage, followed by separation and sequencing of the peptides generated. These methods can be carried out successfully only when the protein concentration in the gel or on the membrane is sufficiently high. For low-abundance proteins, it is therefore necessary to use methods that allow concentration of proteins into small volumes or onto small membrane surfaces.
Chapter
This chapter focuses on amino acid analysis on microscale from electroblotted proteins. Amino acid analysis is routinely used to determine the amount and the composition of proteins, peptides, and free amino acids. Proteins at present often are isolated by sodium dodecyl sulfate-PAGE and electroblotting onto a chemically inert membrane like polyvinylidenfluoride. Amino acid analysis involves two major steps, complete hydrolysis of proteins and peptides, followed by quantification of the amino acids liberated. Hydrolysis is the most crucial step, especially susceptible to contamination and to loss of sample. With submicrogram amounts of samples, it has to be performed in gas phase, because of reduced contamination compared with liquid hydrolysis. However, all reverse-phase HPLC systems used for amino acid analysis are very sensitive to minimal changes in buffer concentrations, gradient shape, and temperature.
Chapter
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The monoclonal antibody C215 (IgG2a) was obtained by the immunization of BALB/c mice with the human colon adenocarcinoma cell line COLO 205 and used in the targeting of colorectal carcinomas. The partial characterization and purification of the C215 target molecule from solubilized COLO 205 membranes indicated that it is an integral membrane glycoprotein of the non-mucin type. The denatured antigen appeared as a major 40-kDa form in Western blots after SDS-polyacrylamide gel electrophoresis and migrated as a monomeric 36-kDa species after the reductive cleavage of intramolecular disulfide bridges. Using a five-step procedure, the antigen was purified 4,300-fold from COLO 205 tumors raised in nude mice to a homogeneity of 95% when assessed by capillary electrophoresis. Removal of N-linked carbohydrate by peptide: N-glycosidase treatment did not affect the visualization of the purified antigen in immunoblots but resulted in a faster migration in the SDS gels. The amino acid sequence was partially determined. Seventeen contiguous NH2-terminal amino acids were identified and coincided exactly with residues 82-98 of the GA733-2 protein cloned by Szala et al. (Szala, S., Froehlich, M., Scollon, M., Kasai, Y., Steplewske, Z., Koprowski, H., and Linnenback, A. J. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 3542-3546). Therefore, the predicted amino acid sequence of this protein was used to prepare overlapping synthetic peptides that cover the entire extracellular domain ih order to identify the C215 epitope. A likely epitope, close to the NH2 terminus and corresponding to the first distinct hydrophilic stretch after the putative signal sequence, was identified in a peptide enzyme-linked immunosorbent assay. Moreover, GA733-2 cDNA was used for the cloning of the C215 protein from COLO 205 cells and the subsequent transfection to K36.16 mouse T cell leukemia cells. The transfected cells were C215 reactive in fluorescence-activated cell sorter analysis, and a 42 kDa band was visualized in Western blots under both non-reducing and reducing conditions. Our findings indicate a close relationship between the C215 antigen and other members of the GA-733 family, some of which are currently being used as targets in clinical trials with monoclonal antibodies. The mammalian expression system described here will enable further studies into the biological role of this protein and the construction of animal models in order to develop optimal therapeutic strategies.
Chapter
One of the most fascinating aspects of life is its innumerable diversification in shape and function. Nevertheless all living species are constructed by the same chemical principle, due to a common evolutionary origin. In fact the basic reactions of cellular energy synthesis by oxidative phosphorylation in mitochondria, once established early during evolution, remained unchanged up to the appearance of men.
Chapter
2D-gel electrophoresis is routinely used to study the integral protein composition of a given cell type, often revealing more than 2,000 individual spots in a single gel. Due to its high resolution power, the technique has been employed to study alterations in cellular protein expression in response to various stimuli or as a result of differentiation and development (Celis and Bravo, 1984). Identification of the separated proteins can be achieved by comigration electrophoresis or by immunological techniques. Proteins can also be recognized by their amino acid composition, their exact molecular weight as determined by mass spectrometry or by partial amino acid sequencing. The latter approach further allows cDNA cloning from the resultant sequences.
Chapter
Proteins, which are characteristic for a specific state of differentiation, the transformed phenotype or pathological conditions of human cells and tissues were identified by computer analyzed two-dimensional gel electrophoresis. Sequenceable amounts of protein were collected from multiple gels with a gel-concentration device, enabling the elution and concentration of more than twenty protein spots, suspended in 1 ml of sample buffer. The eluted protein was concentrated in a new gel in a very small spot and then electroblotted onto polybase-coated glass-fiber or polyvinylidene-difluoride membranes and in situ digested. The released peptides were separated by micro-bore or narrow-bore reversed phase HPLC and immediately collected on polyethylenimine-coated glass-fiber discs for sequencing. These variations of previously developed methods allowed us to work at higher sensitivity. The procedure is currently being used to try out a systematic analysis of human proteins recovered from two-dimensional gels.
Chapter
Polyacrylamide is a very good matrix for the separation of proteins, but it is a poor matrix for their characterization, with the exceptions of molecular mass and isoelectric point estimations. For most proteins elution from gels is accompanied with losses of up to 90%. The introduction of nitrocellulose as a matrix onto which proteins can be blotted and on which the proteins are immobilized has allowed a wide range of characterization reactions. The first membrane-immobilized proteins were characterized by immunostaining (Towbin et al. 1979; Burnette 1981). Other overlay techniques are lectin staining for the identification of glycoproteins (Clegg 1982), radioactive calcium overlays (Maru-yama et al. 1984), or GTP overlays for the identification of GTP-binding proteins (McGrath et al. 1984). However, protein-chemical investigations cannot be performed within an acryla-mide gel and nitrocellulose membrane is not compatible with automated NH2-terminal sequencing. Therefore, an important advance in the combination of two-dimensional electrophoresis (2-DE) and protein-chemical methods was the development of inert membranes onto which proteins can be blotted with high efficiency. The first sequencer-stable membranes were glass fiber membranes modified either with positively charged (Vande-kerckhove et al. 1985; Aebersold et al. 1986) or hydrophobic, uncharged (Eckerskorn et al. 1988a) groups, or pure organic polymers such as polyvinylidene difluoride (PVDF) (Matsudaira 1987) or polypropylene (PP) (Eckerskorn and Lottspeich 1990; Jungblut et al. 1990). For different hydrophobic membranes and proteins the blotting efficiency has been tested, the blotting parameters optimized and a blotting mechanism proposed (Jungblut et al. 1990).
Chapter
The feasibility of using matrix-assisted laser desorption mass spectrometry (MALDI-MS) for analysis of proteins electroblotted onto polymer substrates after a one dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis (1D-SDS-PAGE) separation of a protein mixture has been reported recently [1–3]. In this paper, the current state-of-the-art is summarized and exemplified by focusing on some fundamental analytical aspects, such as the mass range accessible, the application to hydrophilic membranes, the sample consumption per laser shot, the compatibility with different matrices and different staining materials as well as the accuracy of mass determination.
Chapter
This chapter presents a study focusing on peptide mapping of 2-D gel proteins by capillary HPLC. E. coli were lysed by sonication in lysis buffer containing 8 M urea, 2% Triton X-100, 2% 2-mercaptoethanol, and 8 mM phenylmethylsulfonylfluoride (PMSF). The solubilized proteins were centrifuged at 12,000 g in an Eppendorf microfuge, and the supernatant was diluted 1:1 with the sample buffer recommended by Pharmacia. Samples were centrifuged prior to application to minimize streaking. Spot #1 was sequenced in a Applied Biosystem 470A sequencer utilizing a blot cartridge. The initial yield indicated a total of 2–4 pmol of protein. The sequence yield combined with the Coomassie blue staining intensity of the sequenced spot can be used as a convenient estimate of the amount of protein in other spots on the electroblot. A computer search of the protein database identified spot #1 as alkyl hydroperoxide reductase.
Article
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An MS/MS based analytical strategy was followed to solve the complete sequence of two new peptides from frog (Odorrana schmackeri) skin secretion. This involved reduction and alkylation with two different alkylating agents followed by high resolution tandem mass spectrometry. De novo sequencing was achieved by complementary CID and ETD fragmentations of full-length peptides and of selected tryptic fragments. Heavy and light isotope dimethyl labeling assisted with annotation of sequence ion series. The identified primary structures are GCD[I/L]STCATHN[I/L]VNE[I/L]NKFDKSKPSSGGVGPESP-NH2 and SCNLSTCATHNLVNELNKFDKSKPSSGGVGPESF-NH2, i.e. two carboxyamidated 34 residue peptides with an aminoterminal intramolecular ring structure formed by a disulfide bridge between Cys2 and Cys7. Edman degradation analysis of the second peptide positively confirmed the exact sequence, resolving I/L discriminations. Both peptide sequences are novel and share homology with calcitonin, calcitonin gene related peptide (CGRP) and adrenomedullin from other vertebrates. Detailed sequence analysis as well as the 34 residue length of both O. schmackeri peptides, suggest they do not fully qualify as either calcitonins (32 residues) or CGRPs (37 amino acids) and may justify their classification in a novel peptide family within the calcitonin gene related peptide superfamily. Smooth muscle contractility assays with synthetic replicas of the S–S linked peptides on rat tail artery, uterus, bladder and ileum did not reveal myotropic activity.
Chapter
Publisher Summary This chapter describes problems and technical solutions in protein biochemistry. The vast majority of biological processes and pathways are tightly controlled. This applies equally to well-understood and relatively simple processes such as oxygen transport and storage and two as yet molecularly poorly understood phenomena of extreme complexity such as development, cell differentiation, and signal transduction pathways that serve to elicit the appropriate intracellular responses to extracellular stimuli. Large-scale DNA mapping and sequencing efforts, aimed at deciphering the complete genomic sequences of a number of species or the expressed sequences represented by cDNAs of differentiated tissues and cells, are the most widely publicized and discussed global programs in biology. The analysis of myristoylated and palmitylated proteins follows the same principles as that of prenylated proteins, except that there is no necessity to block an endogenous enzyme to allow efficient incorporation of the radiolabel into the target proteins. It is found that depending on structural requirements of the binding site, specific protein, ligand interactions may require refolding of the polypeptide after denaturing gel electrophoresis.
Chapter
IntroductionProgram and AlgorithmSearching by Total Molecular WeightSearching by the Molecular Weight of a Set of Peptides Generated by Sequence-Specific Cleavage of a ProteinSearching by the Molecular Weight of a Peptide and Its Partial SequenceConclusion References
Chapter
StrategyDenaturation, Reduction and AlkylationEnzymatic FragmentationChemical FragmentationReferences
Article
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A method has been devised for the electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets. The method results in quantitative transfer of ribosomal proteins from gels containing urea. For sodium dodecyl sulfate gels, the original band pattern was obtained with no loss of resolution, but the transfer was not quantitative. The method allows detection of proteins by autoradiography and is simpler than conventional procedures. The immobilized proteins were detectable by immunological procedures. All additional binding capacity on the nitrocellulose was blocked with excess protein; then a specific antibody was bound and, finally, a second antibody directed against the first antibody. The second antibody was either radioactively labeled or conjugated to fluorescein or to peroxidase. The specific protein was then detected by either autoradiography, under UV light, or by the peroxidase reaction product, respectively. In the latter case, as little as 100 pg of protein was clearly detectable. It is anticipated that the procedure will be applicable to analysis of a wide variety of proteins with specific reactions or ligands.
Article
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An expression system was developed that allows the production of a completely functional antigen-binding fragment of an antibody in Escherichia coli. The variable domains of the phosphorylcholine-binding antibody McPC603 were secreted together into the periplasmic space, where protein folding as well as heterodimer association occurred correctly. Thus, the assembly pathway for the Fv fragment in E. coli is similar to that of a whole antibody in the eukaryotic cell. The Fv fragment of McPC603 was purified to homogeneity with an antigen-affinity column in a single step. The correct processing of both signal sequences was confirmed by amino-terminal protein sequencing. The functionality of the recombinant Fv fragment was demonstrated by equilibrium dialysis. These experiments showed that the affinity constant of the Fv fragment is identical to that of the native antibody McPC603, that there is one binding site for phosphorylcholine in the Fv fragment, and that there is no inactive protein in the preparation. This expression system should facilitate future protein engineering experiments on antibodies.
Article
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Small amounts (7-250 pmol) of myoglobin, beta-lactoglobulin, and other proteins and peptides can be spotted or electroblotted onto polyvinylidene difluoride (PVDF) membranes, stained with Coomassie Blue, and sequenced directly. The membranes are not chemically activated or pretreated with Polybrene before usage. The average repetitive yields and initial coupling of proteins spotted or blotted into PVDF membranes ranged between 84-98% and 30-108% respectively, and were comparable with the yields measured for proteins spotted onto Polybrene-coated glass fiber discs. The results suggest that PVDF membranes are superior supports for sequence analysis of picomole quantities of proteins purified by gel electrophoresis.
Article
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We have developed a new method for the isolation of proteins for microsequencing. It consists of electrophoretic transfer (electroblotting) of proteins or their cleavage fragments onto activated glass filter paper sheets immediately after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The proteins are immobilized on the glass fiber sheets by ionic interactions or by covalent attachment. A wide range of proteins can be prepared in this fashion with no apparent restriction due to solubility, size, charge, or other intrinsic properties of the proteins. As little as 50 ng of the transferred proteins can be detected using Coomassie Blue or fluorescent dye staining procedures and even smaller amounts of radiolabeled proteins by autoradiography. After detection, the protein-containing bands or spots are cut out and inserted directly into a gas-phase sequenator. The piece of glass fiber sheet acts as a support for the protein during the sequencing. Amounts of protein in the 5- to 150-pmol range can be sequenced, and extended runs can be obtained from the blotted samples because of improved stepwise yields and lower backgrounds. The method has been successfully applied to the sequencing of a variety of proteins and peptides isolated from one-dimensional and two-dimensional polyacrylamide gels.
Book
When a vacancy is formed in one of the inner shells of an atom, it may be filled by either a radiative (x ray) or nonradiative (Auger) process. In most instances nature chooses the Auger process. Only when the transition energy exceeds roughly 10 keV is x-ray emission predominant. Thus, the fluorescence yield is greater than 0.5 only for vacancies in the K shells of atoms whose Z > 30, and in a few cases in the L shell for very heavy elements. (The fluorescence yield ω is defined as equal to the number of times a vacancy in a given shell is filled by a radiative process divided by the total number of times that hole is filled.) Why, then, are x rays well known even to the man in the street, while the understanding of the Auger processes has been until recently restricted to the specialist? The answer lies simply in the ease of measurement. X rays are a highly penetrating radiation, while Auger electrons have only a small mean free path in solids.
Article
Proteolytic fragments were obtained by limited proteolysis of 124-kDa (kilodalton) phytochrome from etiolatedAvena sativa using trypsin, endoproteinase-Lys-C, endoproteinase-Glu-C and subtilisin. The fragments were separated by sodium dodecyl sulfate gel electrophoresis, blotted onto activated glass-fiber sheets and investigated by amino-acid sequencing in a gas-phase sequencer. Determination of N-terminal sequences in three to six Edman degradation steps allowed the exact localization of the fragments within the published entire amino-acid sequence of 124-kDaAvena phytochrome (H.P. Hershey, R.F. Barker, K.B. Idler, J.L. Lissemore, P.H. Quail (1985), Nucleic Acids Res.13, 8543-8559). From the knowledge of the exact sites for preferred proteolytic cleavage of undenatured phytochrome, conclusions on the conformation of the phytochrome protein were drawn. Sites of preferred cleavage are considered to be freely exposed to the environment whereas potential cleavage sites which are resistant to proteolysis over a long time are considered to be localized in the interior of the native phytochrome. Two different sites which are exposed in the far-red-absorbing form but not in the red-absorbing form of phytochrome are localized at amino-acid residues 354 and 753, respectively. The N-terminal region which is exposed only in the red-absorbing form stretches only as far as amino-acid residue 60.
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High-performance liquid chromatography is the most common method used for the identification of phenylthiohydantoin amino acid derivatives of amino acids. Isocratic separation conditions offer advantages over gradient conditions in baseline stability and reproducibility. The isocratic separation of the common phenylthiohydantoin amino acid derivatives on two columns, suitable for routine analyses, is described. In microsequencing, identification and quantification of the phenylthiohydantoin amino acids at the femtomole level is necessary. With normal analytical columns the detection limit is about 4 pmol. Therefore, microbore columns were used. With the usual equipment, the 2 mm I.D. columns seem to be most effective for the highly sensitive and reliable identification of the phenylthiohydantoin amino acid derivatives.
Article
Phytochrome from etiolated oat seedlings was digested with subtilisin. A 16 kDa fragment was isolated and investigated by microsequencing. Its amino-terminal sequence SLPGGSMEV/ML revealed a heterogeneity (valine and methionine) at position 9. This proves expression of several phytochrome genes on the protein level. One can conclude from the ratio valine/methionine that the isophytochrome derived from AP5 (containing methionine) is a major gene product besides isophytochromes derived from genes AP4 and AP3 (containing valine).
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Quantitative information from electron spectroscopy for chemical analysis requires the use of suitable atomic sensitivity factors. An empirical set has been developed, based upon data from 135 compounds of 62 elements. Data upon which the factors are based are intensity ratios of spectral lines with F1s as a primary standard, value unity, and K2p3/2 as a secondary standard. The data were obtained on two instruments, the Physical Electronics 550 and the Varian IEE-15, two instruments that use electron retardation for scanning, with constant pass energy. The agreement in data from the two instruments on the same compounds is good. How closely the data can apply to instruments with input lens systems is not known. Calculated cross-section data plotted against binding energy on a log-log plot provide curves composed of simple linear segments for the strong lines: 1s, 2p3/2, 3d5/2 and 4f7/2. Similarly, the plots for the secondary lines, 2s, 3p3/2, 4d5/2 and 5d5/2, are shown to be composed of linear segments. Theoretical sensitivity factors relative to F1s should fall on similar curves, with minor correction for the combined energy dependence of instrumental transmission and mean free path. Experimental intensity ratios relative to F1s were plotted similarly, and best fit curves were calculated using the shapes of the theoretical curves as a guide. The intercepts of these best fit curves with appropriate binding energies provide sensitivity factors for the strong lines and the secondary lines for all of the elements except the rare earths and the first series of transition metals. For these elements the sensitivity factors are lower than expected, and variable, because of multi-electron processes that vary with chemical state. From the data it can be shown that many of the commonly-accepted calculated cross-section data must be significantly in error—as much as 40% in some cases for the strong lines, and far more than that for some of the secondary lines.
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A simple method has been adopted for the isolation of the H+-ATPase from cell membranes of Rhodobacter capsulatus that is based on octylglucoside solubilization. The highly purified F0F1 enzyme catalyzes ATP hydrolysis and DCCD-sensitive ATP-Pi exchange. The isolated complex consists of nine different polypeptides with apparent molecular masses of 50, 45, 34, 29, 23, 20, 18.5, 11 and 7.5 kDa. The N-terminal sequence of the eight largest subunits were determined by automated Edman degradation. The five F1 polypeptides could be readily identified by their homology to the F1 subunits α, β, γ, δ and ϵ from other Rhodospirillaceae. In contrast to prokaryotic F0 complexes known today, the membrane portion of H+-ATPase from R. capsulatus was found to be composed of four polypeptides. Three of these correspond to subunit a, bandcof the Escherichia coli enzyme (29, 18.5 and 7.5 kDa). A fourth subunit of 23 kDa may be also related to subunit b. The ATP-synthase of this photosynthetic bacterium is in this respect similar to that of chloroplasts.
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A new method for the acid hydrolysis of protein is presented. Peptide bonds are cleaved by the action of an HCl/trifluoroacetic acid (TFA) vapor mixture. Contamination for the hydrolysis mixture is reduced to low levels (1-3 pmol). Recovery of hydrophobic amino acid is improved. Short reaction times are achieved and rapid removal of acids is facilitated. The reaction temperature is 158 degrees C for reaction times of 22.5 and 45 min with 7 M HCl and 10% TFA containing 0.1% phenol.
Article
An oligonucleotide probe corresponding to nucleotides of a cDNA encoding the T cell-associated proteinase 1 (TSP-1) was chosen to study the induction and expression of TSP-1-specific transcripts in mouse T lymphocytes and tissues. We demonstrate that TSP-1 mRNA is only expressed in activated T lymphocytes and is absent from all mouse tissues tested including those containing resting mature T lymphocytes. Expression of the TSP-1 gene was observed in T lymphocytes in vitro in response to either phorbolester (phorbol 12-myristate 13-acetate), Ca2+ ionophore (A23187), lectin or alloantigen. In general, TSP-1 mRNA appeared and peaked later compared to interleukin 2 transcripts. Furthermore, TSP-1 mRNA was inducible in vitro in both Ly-2+ and L3T4+ lymphocyte populations treated with alloantigen and/or lectin. The transcription of the TSP-1 gene was always accompanied by the expression of proteinase activity. High expression of TSP-1 transcripts was also observed in in vivo derived T effector cells specific for lymphocytic choriomeningitis virus. However, TSP-1 mRNA was predominantly associated with virus-specific Ly-2+ T cells and correlated with their proteinase and cytolytic activities. The data suggest that TSP-1 gene transcription is a useful marker to characterize T effector cells in vitro and in vivo.
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A method has been devised which allowed the isolation of highly purified reaction center from the thermophilic green bacterium, Chloroflexus aurantiacus. The procedure consisted of three chromatography steps. The final step was fast protein liquid chromatography on Mono Q in the presence of nonanoyl-N-methylglucamide (Mega-9). The purified reaction center complex was photochemically active and had an A280/A813 of 1.4 or less. Under non-denaturing conditions, a pigmented protein band having a Mr of 52000–55000 was observed in sodium dodecyl sulfate gels. When the isolated complex was heat-dissociated in the presence of sodium dodecyl sulfate, just two polypeptides having very similar Mr (24000 and 24500) were observed. Two protein bands were also observed in two-dimensional isoelectric focusing/sodium-dodecyl-sulfate polyacrylamide gel electropheresis; the PI values of the two polypeptides were 6.5 and 6.7. Partial peptide mapping of the two isolated subunits, using both enzymatic and chemical cleavage techniques, yielded almost identical patterns which indicated a high degree of sequence homology between the two polypeptides. The N-terminal amino acid sequences of the two polypeptides were identical and did not exhibit any homology to reaction center subunits of purple sulfur bacteria. The Chloroflexus reaction center is believed to be composed of one molecule of each polypeptide, the photoactive bacteriochlorophyll a dimer and, as accessory pigments, an additional bacteriochlorophyll a and three bacteriopheophytins. Hence, it appears to be the smallest photochemically active reaction center isolated to date.
Article
The spike glycoprotein of influenza C/Johannesburg/1/66 was isolated in a soluble form by digestion of MDCK cell-grown virions with bromelain. The whole ectodomain of the glycoprotein could be recovered with an apparent molecular weight of 75,000 daltons determined in SDS-PAGE. Comparison to Triton X-100-isolated glycoprotein revealed that a C-terminal peptide of 3000-4500 daltons must have remained in the viral membrane. When purified by sucrose density gradient centrifugation the glycoprotein sedimented with a sedimentation coefficient of 10 S, indicating a molecular weight of 206,000 daltons, which is consistent with a trimeric structure of the spike molecule. The trimeric form was stabilized in sucrose gradients by Ca2+ ions. Bromelain digestion of virions with uncleaved glycoprotein, grown in MDCK cells without trypsin, produced two disulphide-linked subunits with similar electrophoretic mobilities in SDS-PAGE to the biologically active glycoprotein. The smaller subunit differed from the product cleaved in vivo (gp 30) by the presence of an additional arginine residue at the N-terminus. The soluble glycoprotein appears to possess both receptor-binding and receptor-destroying enzyme activities, as isolated glycoprotein inhibited hemagglutination of intact influenza C virions and showed RDE activity in an in vitro test. Glycoprotein exposed to low pH, which was sensitive to trypsin digestion, also demonstrated both these biological activities. Glycoprotein-mediated hemolysis could not be observed.
Article
A procedure has been developed which allows the immobilization on glass-fiber sheets coated with the polyquaternary amine, Polybrene, of proteins and protein fragments previously separated on sodium-dodecylsulfate-containing polyacrylamide gels. The transfer is carried out essentially as has been used for protein blotting on nitrocellulose membranes [Towbin, H., Staehelin, T. and Gordon, J. (1979) Proc. Natl Acad. Sci. USA 76, 4350-4354], but is now used to determine the amino acid composition and partial sequence of the immobilized proteins. Protein transfer could be carried out after staining the proteins in the gels with Coomassie blue, by which immobilized proteins are visible as blue spots, or without previous staining, after which transferred proteins are detected as fluorescent spots following reaction with fluorescamine. The latter procedure was found to be more efficient and yielded binding capacities of +/- 20 micrograms/cm2. Fluorescamine detection was of equal or higher sensitivity than the classical Coomassie staining of proteins in the gel. Immobilized proteins could be hydrolyzed when still present on the glass fiber and reliable amino acid compositions were obtained for various reference proteins immobilized in less than 100 pmol quantities. In addition, and more importantly, glass-fiber-bound proteins could be subjected to the Edman degradation procedure by simply cutting out the area of the sheet carrying the immobilized protein and mounting the disc in the reaction chamber of the gas-phase sequenator. Results of this immobilization-sequencing technique are shown for immobilized myoglobin (1 nmol) and two proteolytic fragments of actin (+/- 80 pmol each) previously separated on a sodium-dodecylsulfate-containing gel.
Article
Using an improved method of gel electrophoresis, many hitherto unknown proteins have been found in bacteriophage T4 and some of these have been identified with specific gene products. Four major components of the head are cleaved during the process of assembly, apparently after the precursor proteins have assembled into some large intermediate structure.
Article
Favourable analytical conditions allowing amino acid analysis in biological fluids, acquired from small human biopsy specimens, were achieved by considering various derivatization methods, the mode of detection and the column used. By using o-phthalaldehyde-3-mercaptopropionic acid as derivatization agent (high sensitivity and stability) and fluorescence detection (excitation at 330 nm, emission at 450 nm), excellent separation of 26 amino acids was obtained in the lower pmol range (1-10 pmol). The reproducibility of the retention times was better than 1.0% for the majority of amino acids and the results from high-performance liquid chromatography (HPLC), compared favourably with those of conventional amino acid analysis (r = 0.97). HPLC technology facilitates amino acid analysis in biopsy specimens of less than 1 mg of tissue.
Article
A simple, horizontal device for rapid electrophoretic transfer of proteins from several polyacrylamide gels simultaneously is described. Up to six 'TRANS-UNITS' consisting of soaked filter paper on either side of polyacrylamide gel/nitrocellulose sheets that are separated by dialysis membranes are stacked between graphite plate electrodes. The only buffer reservoir in the apparatus is that in stacked, soaked filter paper. A special buffer system based on the isotachophoresis theory was developed for this purpose. The electrophoretic transfer was performed with equal efficiency in all TRANS-UNITS of the stack. Only traces of a few proteins remained in the polyacrylamide gel after transfer. With this apparatus, 50 protein bands from a human serum protein sample (diluted 1 : 100) were detected by immunoblotting with the retainment of the high resolution of the SDS-PAGE technique. The apparatus provided a constant current density of 0.8 mA/cm2 during the 1-h transfer time at 21 degrees C, irrespective of the number of TRANS-UNITS. The apparatus generated 1-5 W in joule heat, depending on the number of TRANS-UNITS in the stack.
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Dr H. Engelhart, Dr E. Mehl and Miss M. Tragler (Max-Planck- Institut fur Biochemie, Martinsried) for stimulating and helpful discussions. This work was supported by grant BCT03706 from the Bundesministerium fur Forschung und Technologie. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9.
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Marget, M., Eckardt, A,, Ehret, W., v. Specht, U., Duchene, 17. Formanowski, F. & Meier-Ewert, H. (1988) Virus Rex 10, 177-
  • Swingle R. S.