Showing papers on "Gel electrophoresis published in 1978"

Disc Electrophoresis and Related Techniques of Polyacrylamide Gel Electrophoresis

Abstract: We may not be able to make you love reading, but disc electrophoresis and related techniques of polyacrylamide gel electrophoresis will lead you to love reading starting from now. Book is the window to open the new world. The world that you want is in the better stage and level. World will always guide you to even the prestige stage of the life. You know, this is some of how reading will give you the kindness. In this case, more books you read more knowledge you know, but it can mean also the bore is full.

Resolution of histones by polyacrylamide gel electrophoresis in presence of nonionic detergents.

Abstract: Publisher Summary This chapter describes the technique for the resolution of histones polyacrylamide gel electrophoresis in presence of nonionic detergents. Histones are difficult to resolve by classical biochemical fractionation techniques because of their similarity in size and charge, their tendency to aggregate, and the high frequency of post-transcriptional charge modification. The most widely used analytical system for histones; polyacrylamide gel electrophoresis at low pH resolves five major histone species and some of their modified forms. The resolution of the histones can be improved by the addition of nonionic detergents to the gels that results in a differential reduction in the electrophoretic mobility of different histones. This effect is because of the formation of mixed micelles between the detergent and the hydrophobic regions of protein molecules and is extremely sensitive to small differences in the hydrophobic properties of the proteins. For qualitative comparison of complex protein mixtures, the electrophoresis system can be combined with a simple and effective two-dimensional electrophoresis technique that uses the same buffer system in both directions and therefore avoids the precipitation problems and the possibility of differential loss of proteins during the transition from one buffer system to another.

The Regulation of Glycogen Metaabolism

Abstract: Inhibitor-1 is a protein which inhibits phosphorylase phosphatase only when it has been phosphorvlated by cyclic-AMP-dependent protein kinase [Huang, F. L. and Glinsmann. W. H. (1976) Eur. J. Biochem. 70, 419–426]. Inhibitor-1 was purified by a heat treatment at 90°C, precipitation with ammonium sulphate, chromatography on DEAE-cellulose, gel filtration on Sephadex G-100, and finally recromatography of the phosp0horylated protein on DEAE-cellulose. The protein was purified 4000-fold and 1.5 mg per 1000 g muscle was obtained in seveven days corresponding to an overall yield of 15–20%. The purified protein was in a state approaching homogeneity as judged by the criteria of polyacrylamide-gel electrophoresis and ultracentrifugal analysis. The concentration of inhibitor-1 in vivo was calculated to be 1.5 μMM, which is at least as high as the concentrationof phosphorylase phosphatase. The amino acid composition of inhibitor-1 showed several unusual features. Glutamic acid and proline accounted for nearly one third of the residues, tyrosine, tryptophan and cysteine were absent, and the content of aromatic amino acids was very low. The molecular weight measured by sedimentation equilibraium centrifugation was 19 200 and by amino acid analysis was 20 800. These values were lower than the mol. wt of 26000 determined empirically by gel electrophoresis in the presence of sodium dodecyl sulphate, and much lower than the apparent molecular weitht of 60000 estimated by filtraation on Sephadex G-100. The gel filtration behaviour, stability to heating at 100°C and amino acid composition suggest than inhibitor-1 may possess little ordered structure. The phosphorylated form of inhibitor-1contained close to one molecule of covalently bound phosphate per mole of protein, which is consistent with previous finding of a unique decapeptide sequence at the site of phosphorylation, Ile-Arg-Arg-Arg-Arg-Pro-Thr(p-Pro-Ala-Thr-[Cohen, P., Rylatt, D. B. and Nimmo, G. A. (1977) FEEBS Lett. 76, 182–186]. The phosphorylated form of inhibitor-1 inhibited phosphorylase activity (0.02 U) by 50% at a concentration of only 7.0 nM in the standard assay, but the phosphorylated decapeptide was 1000–2000 times less effective as an inhibitor.

Synaptic proteins. Characterization of tubulin and actin and identification of a distinct postsynaptic density polypeptide.

Abstract: The major proteins in isolated synaptic junctions (SJs) and postsynaptic densities (PSDs) have been compared to actin, tubulin, and the major neurofilament (NF) protein by two-dimensional gel electrophoresis and tryptic peptide map analysis. These studies show: (a) tubulin is present in SJ and PSD fractions and is identical to cytoplasmic tubulin, (b) actin in these fractions is very similar to the gamma- and beta-actin found predominantly in nonmuscle cells, and (c) the major PSD protein is distinct from all other known fibrous proteins.

Characterization of Tubulin and Actin and Identification of a Distinct Postsynaptic Density Polypeptide

Abstract: The major proteins in isolated synaptic junctions (SJs) and postsynaptic densities (PSDs) have been compared to actin, tubulin, and the major neurofilament (NF) protein by two-dimensional gel electrophoresis and tryptic peptide map analysis. These studies show: (a) tubulin is present in SJ and PSD fractions and is identical to cytoplasmic tubulin, (b) actin in these fractions is very similar to the y- and/3actin found predominantly in nonmuscle ceils, and (c) the major PSD protein is distinct from all other known fibrous proteins.

A method for the recovery of DNA from agarose gels

Abstract: We describe a quick and versatile method for the isolation of DNA from agarose gels. The DNA is electrophoresed into a trough containing hydroxyapatite, where it is bound. The hydroxyapatite is taken out and the DNA eluted with phosphate buffer. By putting the hydroxyapatite on a small column of Sephadex G50, elution and subsequent removal of phosphate can be performed in one step. The DNA recovered can be used equally well in enzymatic incubations as DNA not purified through agarose gel electrophoresis. Several applications of this technique are described.

The relationship between molecular structure and transformation efficiency of some S. aureus plasmids isolated from B. subtilis.

Abstract: DNA preparations of the chloramphenicol resistance determining S. aureas plasmids pC194, pC223, and PUB112 can be fractionated by gel electrophoresis into various bands. Electromicroscopic investigations of these various molecular species obtained with pC194 indicated that, depending on the preparations, 70 to 80% of the molecules were monomers, while the rest consisted of various classes of concatemeric and/or interlocked multimers. Measurements of the specific transforming activity of the various molecular classes indicated that the monomers had less than one thousandth the activity of the multimeric plasmid DNA. pC194 DNa of high specific transforming activity could also be obtained by ligation of HindIII generated monomers into concatemeric DNA.

Degradation of serum amyloid A protein by surface-associated enzymes of human blood monocytes.

Abstract: Peripheral blood monocytes incubated in a serum-free medium degraded serum amyloid A (SAA) protein along three pathways. Of 20 normal subjects, 8 degraded SAA completely with no detectable intermediates. Eight subjects transiently produced an amyloid A (AA)-like intermediate which comigrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) with tissue AA protein and reacted with antisera to AA, whereas four subjects yielded a persistent AA-like intermediate on PAGE. This group also failed to degrade tissue AA protein. Cells from 10 patients with amyloidosis fell into the second group. The responsible enzymes appear to be serine proteases because they are inhibited by disopropyl fluorophosphate. They were not affected by epsilon-amino caproic acid, L-1-tosylamide-2-phenylethyl chloromethyl ketone, or N-alpha-p-tosyl-L-lysine chlormethyl ketone. It appears possible that the enzymes are associated with the outer membrane of the cell because only a small fraction of the activity is secreted into the medium and because enzyme activity remains after fixation of the cells with glutaraldehyde which completely stops phagocytosis. Perhaps differences in patterns of proteolysis may play a role in the predisposition to amyloidosis.

Separation and characterization of the outer membrane of Pseudomonas aeruginosa.

Abstract: A method is described for the preparation of outer and cytoplasmic membranes of Pseudomonas aeruginosa, and the outer membrane proteins characterized. Isolated outer and cytoplasmic membranes differed markedly in the content of 2-keto-3-deoxyoctonate (lipopolysaccharide) and phospholipid as well as in the localization of certain enzymes (NADH oxidase, succinate dehydrogenase, D-lactate dehydrogenase, malate dehydrogenase, and phospholipase), and also in the microscopic morphology. The outer membrane preparation showed activity neutralizing a certain bacteriocin or bacteriophages, whereas the cytoplasmic membrane preparation showed no neutralizing activity. The protein composition of membrane preparations from five different strains of P. aeruginosa [P14, M92 (PAO1), PAC1, P15, and M2008 (PAT)] were determined by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. More than 50 protein bands were detected in the cytoplasmic membrane preparation. The protein compositions of outer membranes from the five different strains were very similar: at least 6 major bands were found (apparent molecular weights: Band D, 50,000; band E, 45,000; band F, 33,000; bands G and H, 21,000; and band I, 8,000). The protein composition of outer membranes was affected by some physiological growth conditions. Some features of major outer membrane proteins were also studied. Band F showed anomalous migration on SDS polyacrylamide gel electrophoresis depending on the solubilizing conditions or pretreatment with TCA. Band I seemed to be a protein analogous to the lipoprotein which had been found in the outer membrane of Escherichia coli.

Purification of mouse interferon by sequential affinity chromatography on poly(U)--and antibody--agarose columns.

Abstract: Mouse interferon has been purified to homogeneity by two-step affinity chromatography. Two polypeptide bands were obtained on sodium dodecyl sulphate–polyacrylamide gel electrophoresis migrating at molecular weights 35,000 and 22,000, both having antiviral activity. The 35,000 but not the 22,000 band, also stained with periodic acid–Schiff. The specific activity was 8×109 of our laboratory units, corresponding to 2.4×109 NIH reference units.

Extracellular nucleases of pseudomonas BAL 31. III. Use of the double-strand deoxyriboexonuclease activity as the basis of a convenient method for the mapping of fragments of DNA produced by cleavage with restriction enzymes.

Abstract: We have previously characterized an extracellular nuclease from Pseudomonas BAL 31 which, in addition to other activities, displays a double-strand exonuclease activity which progressively shortens both strands of linear duplex DNA molecules from both termini. This degradation is accomplished without the introduction of detectable scissions away from the ends of the duplexes. When this nuclease is used to produce a series of progressively shortened samples from a linear duplex DNA, subsequent digestion of these samples with a site-specific restriction endonuclease and analysis of the resulting fragments by gel electrophoresis permits the rapid establishment of the order of the restriction enzyme fragments through the entire genome. This is accomplished by noting from the electropherograms the order in which the various restriction enzyme fragments become noticeably shortened or disappear. Using this method, the five cleavage sites for the endonuclease Hpa I and the single cleavage sites for the nucleases Hpa II and Pst I have been mapped in PM2 bacteriophage DNA. In a more stringent test of the method, 18 of the 24 fragments produced by cleavage of coliphage lambdab2b5c DNA with the Pst I nuclease have been mapped, and five of the six remaining fragments have been assigned to small regions of the genome.

Separation of very large DNA molecules by gel electrophoresis

Abstract: Very large DNA molecules were separated by electrophoresis in horizontal slab gels of dilute agarose. Conditions of electrophoresis were developed using intact DNA molecules from the bacterial viruses lambda, T4 and G. Their DNAs have molecular weights (M) of 32 million, 120 million, and 500 million, respectively. Several electrophoresis conditions were found which give sufficiently high mobilities and large differences that these DNAs are separated in a short time. Electrophoresis in 0.1% agarose at 2.5 V/cm of gel length separates T4 and lambda DNAs by 2.0 cm, and G and T4 DNAs by 1.0 cm in only 10 hr. With some conditions DNA mobilities are directly proportional to log M for M values from 10 to 500 million. The procedures used will allow rapid molecular weight determination and separation of very large DNA molecules.

Steps involved in the processing of common precursor forms of adrenocorticotropin and endorphin in cultures of mouse pituitary cells.

Abstract: The initial steps in the processing of the common precursor to adrenocorticotropin (ACTH) and endorphin in mouse pituitary tumor cells (AtT-20) have been investigated. Three forms of the precursor have been resolved by sodium dodecyl sulfate (NaDodSO4)-polyacrylamide gel electrophoresis with apparent molecular weights of 29 000 (29K ACTH-endorphin), 32 000 (32K ACTH-endorphin) and 34 000 (34K ACTH-endorphin). These forms have a similar peptide backbone, but their carbohydrate content differs. In particular, a tryptic glycopeptide has been observed in 32K ACTH-endorphin which is not present in 29K ACTH-endorphin and has been identified as the tryptic peptide containing the alpha(22--39) sequence of ACTH. Similar heterogeneity in carbohydrate has been observed in some of the smaller molecular weight forms of ACTH which are resolved by NaDodSO4 gel electrophoresis. Pulse chase and continuous labeling studies using radioactive amino acids and sugars suggest that the 29K ACTH-endorphin is converted to 32K and 34K ACTH-endorphin by the addition of carbohydrate. The glycopeptide and pulse chase studies suggest that 29K ACTH-endorphin is at a branch point in the processing pathways. It can either be converted to 4.5K ACTH by proteolytic processing or to 32K ACTH-endorphin by the further addition of carbohydrate. The 32K ACTH-endorphin can then be converted to 13K ACTH, the glycosylated form of 4.5K ACTH (Eipper, B.A., & Mains,, R.E. (1977) J.Biol. Chem.252, 882), by proteolytic processing. A comparison of the distribution of the different molecular weight forms of ACTH and endorphin in mouse pituitary extracts and in the mouse pituitary tumor cells reveals that the pituitary contains all of the forms of ACTH and endorphin seen in the tumor cells, including the three forms of the ACTH-endorphin precursor. However, the molecular weight distribution of the forms in the anterior lobe is very different from that in the intermediate lobe of mouse pituitary.

Transient regulation of protein synthesis in Escherichia coli upon shift-up of growth temperature.

Abstract: Synthesis of total cellular proteins of Escherichia coli was studied upon transfer of a log-phase culture from 30 (or 37) to 42 degrees C. Cells were pulse-labeled with [3H]leucine, and the labeled proteins were analyzed by gel electrophoresis in the presence of sodium dodecyl sulfate. The rates of synthesis of at least five protein chains were found to increase markedly (5- to 10-fold) within 5 min after temperature shift-up and gradually decrease to the new steady-state levels, in contrast to the majority of proteins which gradually increase to the steady-state levels (about 1.5-fold the rate at 30 degrees C). Temperature shift-down did not cause any appreciable changes in the pattern of protein synthesis as detected by the present method. Among the proteins greatly affected by the temperature shift-up were those with apparent molecular weights fo 87,000 (87K), 76K, 73K, 64K, and 61K. Two of them (64K and 61K) were found to be precipitated with specific antiserum against proteins that had previously been shown to have an adenosine triphosphatase activity. The bearings of these findings on bacterial adaptation to variation in growth temperature are discussed.