Urea-sodium Dodecyl Sulfate-polyacrylamide Gel Electrophoresis Research Articles

Mechanisms of ertapenem resistance in Enterobacteriaceae isolates in a tertiary university hospital

The aim of this study was to investigate the molecular mechanisms of ertapenem resistance among Enterobacteriaceae isolates in a clinical microbiology laboratory at a tertiary university hospital. A total of...

Detection of plasmid-mediated IMP-1 metallo-β-lactamase and quinolone resistance determinants in an ertapenem-resistant Enterobacter cloacae isolate

To investigate the mechanism of carbapenem resistance and the occurrence of plasmid-mediated quinolone resistance determinants qnr and aac(6')-Ib-cr in a clinical isolate of Enterobacter cloacae. An ertapenem-resistant E. cloacae ZY106, which was isolated from liquor puris of a female gastric cancer patient in a Chinese hospital, was investigated. Antibiotic susceptibilities were determined by agar dilution method. Conjugation experiments, isoelectric focusing, polymerase chain reaction (PCR), and DNA sequence analyses of plasmid-mediated carbapenemases and quinolone resistance determinants were preformed to confirm the genotype. Outer membrane proteins (OMPs) were examined by urea-sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Urea-SDS-PAGE). Minimum inhibitory concentrations (MICs) of imipenem, meropenem, and ertapenem for ZY106 were 2, 4, and 16 microg/ml, respectively. Conjugation studies with Escherichia coli resulted in the transfer of significantly reduced carbapenem susceptibility. ZY106 produced IMP-1 metallo-beta-lactamase and CTX-M-3 extended-spectrum beta-lactamase, and E. coli transconjugant produced IMP-1. Plasmid-mediated quinolone resistance determinant qnrS1 was detected in ZY106. Transfer of the qnrS1-encoding-plasmid into E. coli by conjugation resulted in intermediate resistance to ciprofloxacin in E. coli transconjugant. Urea-SDS-PAGE analysis of OMPs showed that ZY106 lacked an OMP of approximately 38 kDa. It is the first IMP-1-producing Enterobacteriaceae in China and the first report of a clinical isolate that harbors both blaIMP and qnrS genes as well. The blaIMP-1, blaCTX-M-3, and qnrS1 are encoded at three different plasmids. IMP-1 combined with the loss of an OMP possibly resulted in ertapenem resistance and reduced imipenem and meropenem susceptibility in E. cloacae.

Electrophoretic mobility of Alzheimer’s amyloid-β peptides in urea–sodium dodecyl sulfate–polyacrylamide gel electrophoresis

The 43-amino acid Alzheimer’s amyloid-β peptide (Aβ peptide) retains a predominantly α-helix and β-strand structure in sodium dodecyl sulfate (SDS) solution. This conformer has a high tendency to aggregate during conventional SDS–polyacrylamide gel electrophoresis (PAGE). Both the secondary structure and the proclivity for aggregation are obviated by the use of urea–SDS–PAGE: In 8 M urea—with or without SDS—the Aβ peptide becomes 100% random coil and remains monomeric. However, during electrophoresis in this medium, the peptide and its truncated variants do not obey the law of mass/mobility relationship that most proteins—including Aβ peptides—follow in conventional SDS–PAGE. Rather, the smaller carboxy-terminally truncated peptides migrate slower than the larger full-length peptide, while the amino terminally truncated peptide does migrate faster than the full-length Aβ peptide. Thus, despite their small size (2–4 kDa) and minor differences between their lengths, the Aβ peptides display a wide separation in this low-porosity (12% acrylamide) gel. We found that this unusual electrophoretic mobility in 8 M urea is due to the fact that the quantity of [ 35S]SDS bound to the Aβ peptides, instead of being proportional to the total number of amino acids, is rather proportional to the sum of the hydrophobicity consensus indices of the constituent amino acids. It is then their hydrophobicity and, hence, the net negative charges contributed by the peptide-bound SDS that plays a major role in determining the mobility of Aβ peptides in 8 M urea–SDS–PAGE. The high selectivity of the 8 M urea–SDS–PAGE method allowed us to detect the presence of hitherto unknown Aβ peptide variants that were secreted in the conditioned medium by cultured HeLa cells.

Expression of the murine CB 2 cannabinoid receptor using a recombinant Semliki Forest virus

A recombinant Semliki Forest virus (SFV) RNA construct, SFV1-mCB 2 RNA, was employed for the high-level expression of the murine CB 2 (mCB 2) cannabinoid receptor in baby hamster kidney cells. Biosynthetic radiolabel incorporation studies in concert with urea-sodium dodecylsulfate–polyacrylamide gel electrophoresis (urea-SDS–PAGE) and western immunoblotting revealed that two major proteins of approximately 26 and 40 kDa were produced by the construct. The 40 kDa product, but not the 26 kDa product, was glycosylated as determined by 2-deoxy- d-glucose incorporation and peptide- N-glycosidase F digestion analysis. Assessment of [ 3 H ]CP55940 ([ 3 H ]-(−)- cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]- trans-4-(3-hydroxypropyl)cyclohexanol) binding data for membranes of cells transfected with SFV1-mCB 2 RNA indicated a K d of 0.35±0.04 nM and a B max of 24.4±2.7 pmol/mg. A rank order of binding affinities for cannabinoids, which paralleled that reported for native mCB 2 receptors, was observed. The CB 2 receptor-specific antagonist SR144528 ( N-[(1 S)-endo-1,3,3-trimethyl bicyclo[2.2.1]heptan-2-yl]-5-(4-chloro-3-methylphenyl)-1-(4-methylbenzyl)-pyrazole-3-carboxamide) blocked binding of [ 3 H ]CP55940, while the CB 1 receptor-specific antagonist SR141716A [ N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1 H-pyrazole-3-carboxamide hydrochloride] had a minimal effect. These results indicate that the recombinant receptor expressed from SFV1-mCB 2 RNA exhibits properties, including ligand binding features, that are consistent with those for the native mCB 2 receptor. However, the presence of both 26 and 40 kDa receptor species is consistent with alternative translation from two AUG start sites using the SFV1-mCB 2 RNA expression system.

The Tumor Suppressor p53 Is Bound to RNA by a Stable Covalent Linkage

We have previously shown that the carboxyl-terminal tryptic peptide of the tumor suppressor p53 coeluted from reverse-phase high-performance liquid chromatography (HPLC) with ribonucleotides, suggesting the possible linkage of RNA to p53. In this report, we establish that p53 is covalently linked to RNA, using biochemical criteria at the levels of both tryptic peptide and intact protein: the electrophoretic properties of a tryptic peptide containing phosphorylated Ser-389 and the HPLC chromatographic properties of p53 depend on the linked RNA, p53, purified through urea-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and HPLC, copurifies with RNA, and Ser-389 liberates ribonucleotides upon RNase or alkali treatment. Wild-type and mutant p53s from both simian virus 40 (SV40)-transformed and SV40-nontransformed cells are RNA linked, indicating that RNA linkage may be a general property of p53. The RNA is labeled in vivo with 3H-uridine and in vitro by RNA ligase, suggesting that the RNA is bound by a 5' linkage. The RNA is a long-lived, integral component of p53 rather than a transient reaction intermediate. RNA linkage occurs at an evolutionarily conserved site on p53. We propose that RNA-linked p53 is a major biologically active form of p53 and that its interaction with RNA-linked SV40 T antigen reflects a role in RNA metabolism.

The tumor suppressor p53 is bound to RNA by a stable covalent linkage.

We have previously shown that the carboxyl-terminal tryptic peptide of the tumor suppressor p53 coeluted from reverse-phase high-performance liquid chromatography (HPLC) with ribonucleotides, suggesting the possible linkage of RNA to p53. In this report, we establish that p53 is covalently linked to RNA, using biochemical criteria at the levels of both tryptic peptide and intact protein: the electrophoretic properties of a tryptic peptide containing phosphorylated Ser-389 and the HPLC chromatographic properties of p53 depend on the linked RNA, p53, purified through urea-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and HPLC, copurifies with RNA, and Ser-389 liberates ribonucleotides upon RNase or alkali treatment. Wild-type and mutant p53s from both simian virus 40 (SV40)-transformed and SV40-nontransformed cells are RNA linked, indicating that RNA linkage may be a general property of p53. The RNA is labeled in vivo with 3H-uridine and in vitro by RNA ligase, suggesting that the RNA is bound by a 5' linkage. The RNA is a long-lived, integral component of p53 rather than a transient reaction intermediate. RNA linkage occurs at an evolutionarily conserved site on p53. We propose that RNA-linked p53 is a major biologically active form of p53 and that its interaction with RNA-linked SV40 T antigen reflects a role in RNA metabolism.

Identification and purification of truncated insulin-like growth factor I from porcine uterus. Evidence for high biological potency.

We report the completion of the purification of uterine-derived growth factors (UDGF) described previously by this laboratory [Ikeda, T., & Sirbasku, D.A. (1984) J. Biol. Chem. 259, 4049-4064]. During isolation, the mitogenic activity was monitored by using the human MCF-7 breast cancer cells in serum-free Ham's F12 and Dulbecco's modified Eagle's medium (1:1, v/v) containing 15 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (pH 7.2), 200 micrograms/mL bovine serum albumin, and 10 micrograms/mL human transferrin. This medium sustained growth for several days in response to a single addition of growth factor. The isolation of UDGF began with acetic acid extraction followed by sulfopropyl-Sephadex chromatography, Bio-Gel P-10 molecular sieve fractionation, and a series of reverse-phase high-pressure liquid chromatography separations. Purifications [[(1.0-8.5) X 10(6)]-fold] of three mitogens (5-20 ng each) were achieved. The mitogens were shown by protein microsequencing to be DES 1----3 to DES 1----6 forms of insulin-like growth factor I (truncated IGF-I). An Mr estimated by 125I labeling, urea-sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and autoradiography was consistent with a DES 1----3(4) N alpha truncation. Immunoadsorption and radioimmunoassay confirmed immunological properties equivalent to IGF-I. Radioreceptor assays showed truncated IGF-I was functionally equivalent to recombinant IGF-I. The ED50 values of DES 1----3 IGF-I and recombinant IGF-I for MCF-7 cell growth were 0.8-6.0 and 30-150 pg/mL, respectively. With Balb/c 3T3 mouse fibroblasts, the ED50 of DES 1----3 IGF-I was 100 times lower than that of IGF-I. We conclude that the major acid-stable low-Mr mitogenic activities isolated from uterus are very potent forms of truncated IGF-I capable of stimulating growth of epithelial and mesenchymal cells.

Purification of a major sialoglycoprotein (SGP140) on P12/Ichikawa cells and its expression on differentiated HL-60 cells.

SGP140 glycoprotein, a major cell surface sialoglycoprotein with an apparent m.w. of 140,000, was detected on the human T lymphoblastoid cell line P12/Ichikawa by labeling with periodate-tritiated sodium borohydride, followed by urea-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and fluorography. Then SGP140 was purified from P12/Ichikawa cells for study of its biochemical character and its distribution in various cell lines. The purification was performed by 0.2% Triton X-100 solubilization from crude membranes, DEAE-Sephacel column chromatography, WGA-agarose column chromatography, Blue-Sepharose 6MB column chromatography, and Sephadex G-150 gel filtration. Antiserum raised against SGP140 was then prepared, and immunoprecipitation and membrane immunofluorescence assay were performed on various cell lines. SGP140 was detected on P12/Ichikawa, Raji, P3HR-1, Daudi, Namalva, BALL-1, MOLT-4B, TALL-1, NALL-1, and K562 cells, but was not detected on HL-60 cells. When HL-60 cells were treated with dimethyl sulfoxide, retinoic acid, or 12-O-tetradecanoylphorbol-13-acetate, SGP140 was detected on cell surfaces. We discuss the possibility that SGP140 may be a differentiation antigen.

Comparative studies on the chemical and immunochemical properties of human milk, human pancreatic juice and bovine milk lactoferrin

1. 1. Lactoferrin from human milk, pancreatic juice and bovine milk were purified by heparin-Sepharose affinity chromatography procedure. 2. 2. Urea-sodium dodecylsulfate-polyacrylamide gel electrophoresis of these lactoferrin preparations indicated similar molecular weights (80,000). 3. 3. Metal analyses showed that lactoferrin of bovine milk contained the highest amount of iron while lactoferrin of human milk and human pancreatic juice were similar in content of iron, approximately four-fold lower than bovine milk. All these lactoferrin preparations were also found to contain minor amounts of copper and manganese. 4. 4. Double diffusion analyses indicated that lactoferrin of human milk was immunochemically identical to lactoferrin of human pancreatic juice. On the other hand, immunochemically, bovine milk lactoferrin was not identical to human milk lactoferrin. 5. 5. Sequence analyses of human milk and pancreatic juice lactoferrin indicated that they shared the same N-terminal sequence for the 16 residues analyzed. Although human milk and bovine milk lactoferrin had sequence homologies, bovine milk lactoferrin had a closer homology to ovotransferrin than to human milk lactoferrin.

Studies on potato tuber phosphorylase catalyzed reaction in the absence of an exogenous acceptor: II. Characterization of the reaction product

Incubation of potato tuber phophorylase II with [ 14C]glucose 1-phosphate in the absence of an exogenous acceptor results in the synthesis of a radioactive product, presumably a protein-bound glucan. The carbohydrate moiety of this product was shown to consist of long α-1,4-glucosidic chains. The enzyme itself was originally assumed to be the first glucosyl acceptor in the unprimed reaction. However, as judged by urea-sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the radioactive product is rather resistant to proteolysis and is smaller in size than phosphorylase II. These data are in agreement with those obtained by sucrose density gradient centrifugation and molecular-weight estimation of native products. Thus, some kind of processing postglucosylation has to be proposed to account for the observed decrease in molecular weight. One cannot overlook the probable presence of a low-molecular-weight protein acceptor which copurifies along with phosphorylase II and whose separation from the enzyme can only be achieved upon glucosylation. On the other hand, protein was also found to be present in amylose from potato starch grains. It is therefore suggested that this finding might become an additional evidence of a common biosynthetic pathway for α-1,4-glucans from a precursor protein.

Human milk bile salt-activated lipase. Further characterization and kinetic studies.

Further studies on human milk bile salt-activated lipase were performed to provide kinetic and additional chemical characterizations of this enzyme. The enzyme was homogeneous by urea-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing with an isoelectric point of 3.7. A unique feature of the amino acid composition of this enzyme was a high proline content (13 mol %). Results of carbohydrate analyses indicated that the enzyme was a glycoprotein containing fucose, galactose, glucosamine, galactosamine, and sialic acid. Kinetic studies were performed with various water-soluble esters (p-nitrophenyl acetate, 1-monoacetin, 1-monobutyrin, and 1-monocaprylin) as substrate and taurocholate as activator. In the presence of a saturating level of taurocholate, the enzyme reaction was demonstrated to follow a rapid equilibrium random uni bi mechanism. Also, these kinetic studies indicated the formation of an enzyme-activator-substrate ternary complex through a random pathway. The mechanism of the activation by taurocholate was due to its enhancement of the binding of the enzyme to the substrate (6.2-fold) and its enhancement of the rate of conversion from enzyme-substrate transitory complex to the products (1.57-fold) when examined with p-nitrophenyl acetate as substrate.

Purification of human milk bile salt-activated lipase by cholic acid-coupled sepharose 4B affinity chromatography

Sequential chromatography of human milk whey on concanavalin A—Sepharose 4B followed by cholate—Sepharose 4B yielded a bile salt-activated lipase with 150-fold purification. The lipase was not retained by concanavalin A—Sepharose 4B but was retained by the cholate—Sepharose 4B, from which it was eluted with 2% sodium cholate. The affinity chromatography procedure on cholate—Sepharose 4B was based on the specific structural requirement of the enzyme for a 7-hydroxyl group of bile salt. Sodium deoxycholate, which lacks the 7-hydroxyl group, was effective in removing the nonspecifically bound proteins without affecting the binding of the enzyme. Bile salt-activated lipase showed a single band on urea-sodium dodecyl sulfate—polyacrylamide gel electrophoresis with an apparent molecular weight of 125,000, and based on densitometric measurement accounted for 0.5–1.0% of the protein mass of human whole milk. A rabbit antiserum to the purified bile salt-activated lipase caused no inhibition of human milk lipoprotein lipase activity but completely inhibited bile salt-activated lipase activity.

Isolation and partial characterization of human erythrocyte membrane NADH: (acceptor) oxidoreductase.

The NADH: (acceptor) oxidoreductase (EC 1.6.99.3) was isolated from human erythrocyte ghosts by a procedure including Triton X-100 solubilization, affinity chromatography on an NAD+-Sepharose 4B column, ammonium sulfate precipitation, and isoelectric focusing. This enzyme preparation was characterized by a single band on the urea-sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by a single precipitin line with its corresponding antiserum on double diffusion and immunoelectrophoresis. A 103-fold purification indicates that the oxidoreductase represents approximately 1% of the ghost protein mass. The specific activity of the purified enzyme was 112 units/mg protein. The pH optimum was 6.8 and the isoelectric point, pI, was 6.6 The oxidoreductase has a specificity for NADH as a cofactor. The NADPH was ineffective as a reducing agent. The enzyme activity was strongly temperature-dependent, displaying maximal activity between 35 and 40 degrees C. The energy of activation was 4.9 kcal. The enzyme activity was inhibited by sulfhydryl reagents, anionic detergents, and divalent ions. The amino acid composition of the purified enzyme is characterized by the presence of all common amino acids including half-cystine and tryptophan. The results of carbohydrate and lipid analyses indicated that the oxidoreductase is a glycolipoprotein with fucose, galactose, mannose, and glucosamine as the sugar components and cholesterol and sphingomyelin as the lipid constituents. The apparent subunit molecular weight estimated by urea-sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the absence and presence of 2-mercaptoethanol was 40,000. The antiserum completely inhibited the enzymic activity at the equivalence point. We suggest that the membrane-bound NADH: (acceptor) oxidoreductase might be a transmembrane protein.