Showing papers on "Gel electrophoresis published in 1983"

Cell surface P-glycoprotein associated with multidrug resistance in mammalian cell lines.

Abstract: The plasma membranes of hamster, mouse, and human tumor cell lines that display multiple resistance to drugs were examined by gel electrophoresis and immunoblotting. In every case, increased expression of a 170,000-dalton surface antigen was found to be correlated with multidrug resistance. This membrane component is of identical molecular size and shares some immunogenic homology with the previously characterized P-glycoprotein of colchicine-resistant Chinese hamster ovary cells. This finding may have application to cancer therapy.

Enzyme-linked immunoelectrotransfer blot techniques (EITB) for studying the specificities of antigens and antibodies separated by gel electrophoresis

Abstract: Publisher Summary This chapter describes the enzyme-linked immunoelectrotransfer blot techniques (EITB) for studying the specificities of antigens and antibodies separated by gel electrophoresis. The enzyme-linked immunoelectrotransfer blot technique (EITB) combines the high resolving power of gradient sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) and the high sensitivity of the enzyme-linked immunosorbent assay (ELISA) to produce an extremely powerful qualitative tool for studying antigen–antibody pairs. Using this procedure, antigens electrophoretically resolved on SDS–PAGE are transferred onto nitrocellulose or diazo sheets and identified by ELISA methods. The chapter discusses the general comments about the technique. The EITB is conducted in three stages: (a) the antigen mixture that can be extremely complex, such as solubilized whole cells, is first resolved by gel electrophoresis (two-dimensional gels can also be used); (b) the resolved gel is then electrophoretically blotted onto nitrocellulose sheets; and (c) the blotted nitrocellulose is then developed by ELISA.

Immunochemical identification of membrane proteins after sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Abstract: Publisher Summary Electrophoresis in the presence of sodium dodecyl sulfate (SDS) is a key method for analyzing membrane proteins, and provides information on the size and the amount of these molecules. This chapter describes a method, immunochemical identification of membrane proteins after sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and discusses its various possible applications in the study of membrane proteins. These procedures are based on the transfer of proteins from the polyacrylamide gel slab to an insoluble matrix, such as chemically reactive cellulose or, more conveniently, commercially available nitrocellulose sheets. The immobilized proteins on the replica are readily accessible to antibodies or other specific probes; this makes possible the in situ localization and characterization of individual proteins in complex mixtures. Polypeptides transferred to nitrocellulose sheets can also be identified with specific probes other than antibodies—for example, glycoproteins can be visualized by reaction with radio-labeled lectins, such as concanavalin A or wheat germ agglutinin.

Purification and characterization of a Shigella dysenteriae 1-like toxin produced by Escherichia coli.

Abstract: A toxin from an enteropathogenic strain of Escherichia coli (E. coli H30) was purified to apparent homogeneity from cell lysates. The steps used to isolate the E. coli H30 toxin included French pressure-cell disruption of bacteria grown in iron-depleted media. Affi-Gel Blue chromatography, chromatofocusing, and anti-Shiga toxin affinity chromatography. The mobilities of the subunits of radioiodinated E. coli H30 toxin and Shiga toxin observed after the two toxins were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis were identical. In the absence of 2-mercaptoethanol, a narrow band was seen at Mr 31,500 (+/- 1,000), and a wide heavy band was observed between Mr 4,000 and 15,000. In the presence of 2-mercaptoethanol, bands were seen at Mr 31,500 (+/- 1,000), 27,000, and 4,000 to 15,000. Other similarities between purified E. coli H30 and Shiga 60R toxins included identical isoelectric points (7.03 +/- 0.02); comparable biological activities, i.e., cytotoxicity, lethality for mice, and enterotoxicity; and the same relative heat stabilities (up to 65 degrees C for 30 min). Nevertheless, the two toxins had apparently different molecular weights as determined by sucrose gradient analysis, by gel filtration, and by cross-linking experiments with dimethyl suberimidate. The Mr of native E. coli H30 toxin estimated from cross-linking studies was 48,000, whereas the estimated Mr of Shiga 60R toxin was 58,000. These results suggest that like the cholera-E. coli-heat-labile toxin family, a family of Shiga-like toxins exists.

A calcium/calmodulin-dependent protein kinase from mammalian brain that phosphorylates Synapsin I: partial purification and characterization

Abstract: A calcium/calmodulin-dependent protein kinase, which phosphorylates a synaptic vesicle-associated protein designated Synapsin I, has been shown to be present in both soluble and particulate fractions of rat brain homogenates. In the present study, the particulate activity was solubilized by washing with a low ionic strength solution, and the enzymes from the two fractions were partially purified by ion exchange chromatography and calmodulin-Sepharose affinity chromatography. By each of several criteria, the partially purified enzymes from the two sources were indistinguishable. These criteria included specificity for various substrate proteins, concentration dependence of activation by calcium and calmodulin, pH dependence, and apparent affinities for the substrates Synapsin I and ATP. The mild conditions that released the particulate enzyme indicated that it was not tightly bound to the membrane and suggested that it may exist in a dynamic equilibrium between soluble and particulate-bound states. The partially purified enzyme preparations from both the soluble and particulate fractions contained three proteins that were phosphorylated in the presence of calcium and calmodulin, a 50-kilodalton (Kd) protein and two proteins in the 60-Kd region. When compared by phosphopeptide mapping and two-dimensional gel electrophoresis, the proteins were indistinguishable from three proteins of corresponding molecular weights that were shown by Schulman and Greengard (Schulman, H., and P. Greengard (1978) Nature 271: 478-479) to be prominent substrates for calcium/calmodulin-dependent protein kinase in a crude particulate preparation from rat brain. The 50-Kd substrate was the major Coomassie blue staining protein in both partially purified enzyme preparations. The peak of this protein coincided with that of enzyme activity during DEAE-cellulose and calmodulin-Sepharose chromatography. These results suggest that the 50-Kd phosphoprotein may be an autophosphorylatable subunit of the Synapsin I Kinase or may exist in a complex with it.

Increased transport of 44,000- to 49,000-dalton acidic proteins during regeneration of the goldfish optic nerve: a two-dimensional gel analysis

Abstract: Proteins synthesized in goldfish retinal ganglion cells and rapidly transported to the terminals of regenerating optic nerves were analyzed by two-dimensional (2-D) gel electrophoresis. Among the rapidly transported components, the most dramatic change observed during regeneration was for a family of polypeptides having molecular weights between 44,000 and 49,000 (44-49K) and isoelectric points of 4.6 to 4.9. Studies using [35S]methionine as a metabolic precursor in the eye showed that these proteins are present in both membranous and soluble fractions of the optic tectum, particularly during early stages of regeneration. Contralateral visual pathways, left intact to serve as controls, showed only very low levels of the proteins. These labeling changes were quantified in double-isotope studies, in which proteins from intact and regenerating sides were differentially labeled with [3H]proline and [14C]proline, comigrated on 2-D gels, and then counted for 3H/14C ratios. The labeling change for the 44-49K acidic proteins relative to the intact state was found to be over 100-fold in some day 19 regeneration samples and about 30-fold on day 40. Silver-stained gels of a tectal membrane fraction also revealed increased levels of the 44-49K acidic proteins during regeneration, indicating that the observed synthetic changes are accompanied by a net accumulation of the proteins.

Purification of a novel calmodulin binding protein from bovine cerebral cortex membranes.

Abstract: A new calmodulin (CaM) binding protein, designated P-57, has been purified to apparent homogeneity from bovine cerebral cortex membranes. In contrast to other calmodulin binding proteins, P-57 has higher affinity for calmodulin in the absence of bound Ca2+ than in its presence. The protein was purified by DEAE-Sephacel chromatography and two CaM-Sepharose affinity column steps. The first CaM-Sepharose column was run in the presence of Ca2+; the second was run in the presence of chelator in excess of Ca2+. P-57 was adsorbed by CaM-Sepharose only in the absence of bound Ca2+ and was eluted from the second column by buffers containing Ca2+. Sodium dodecyl sulfate (SDS)-polyacrylamide gels of the purified protein showed only one band at Mr 57 000. The major form of the protein on Bio-Gel A-1.5m and native polyacrylamide gradient gel electrophoresis ran with an apparent Stokes radius of 41 A. Photoaffinity labeling of P-57 with azido[125I]calmodulin yielded one cross-linked product on SDS gels with an Mr of 70 000. This interaction occurred only when excess ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid was present and was inhibited by the presence of Ca2+ in excess of chelator. It appears that P-57 has novel binding properties for calmodulin distinct from all other calmodulin binding proteins described thus far.

Purification and characterization of six cytochrome P-450 isozymes from human liver microsomes.

Abstract: Six cytochrome P-450 (P-450) isozymes were purified to electrophoretic homogeneity from the livers of four human organ donors, with three of these isozymes purified from a single individual. Differences were noted between all six P-450s for some or all of the parameters determined by the techniques of sodium dodecyl sulfate-polyacrylamide gel electrophoresis, peptide mapping, spectral analysis of ferrous-carbon monoxide complexes, double-diffusion immunoprecipitin analysis or crossed immunoelectrophoresis (sodium dodecyl sulfate-polyacrylamide gel electrophoresis/peroxidase-coupled staining) with rabbit antisera raised to five of the P-450s, or catalytic activity toward d-benzphetamine, benzo[a]pyrene, acetanilide, debrisoquine, (R)- and (S)-warfarin, and 1-naphthylamine. While NADPH-fortified human liver microsomal preparations showed catalytic activity toward trichloroethylene, 7-ethoxycoumarin, 2-naphthylamine, and 2-aminofluorene in addition to the other substrates mentioned, none of the P-450s which we purified from these microsomes catalyzed the oxidation of these compounds in reconstituted enzyme systems containing purified rat liver NADPH-P-450 reductase. Antibodies raised against one of the purified P-450s inhibited d-benzphetamine N-demethylase activity in microsomal incubations but did not inhibit the metabolism of 7-ethoxycoumarin, acetanilide, benzo[a]pyrene, or debrisoquine. The data provide a strong biochemical basis for the view that distinct isozymes of P-450 exist in humans and that these isozymes differ in catalytic activity toward drugs and carcinogens.

Characterization and use of monoclonal antibodies for isolation of phosphotyrosyl proteins from retrovirus-transformed cells and growth factor-stimulated cells.

Abstract: Protein kinases that phosphorylate the hydroxyl group of tyrosine residues of proteins have been implicated in cell transformation by some retroviruses and in regulation of normal cell growth by some polypeptide growth factors. To facilitate the identification of tyrosine kinase substrates, we developed monoclonal antibodies to the hapten azobenzylphosphonate. One of these antibodies, MA-2G8, proved to be especially attractive in that it bound a derivative of aminophenylphosphate, a close phosphotyrosine analog, with higher affinity than it bound the corresponding derivative of aminobenzylphosphonate; however, its affinity for phosphoserine was negligible. In this paper we describe the optimal conditions for using this antibody to isolate phosphotyrosine proteins, emphasizing particularly that its interaction with phosphotyrosyl proteins is sensitive to ionic detergents and to antibody density on the immunosorbent matrix. The antibody also bound ATP citrate lyase; this enzyme lacks phosphotyrosine but contains phosphohistidine, which is similar structurally to phosphotyrosine. By attaching the antibody at high density to Sepharose beads and omitting ionic detergents from the buffers, it was possible by microbatch immunoadsorption (followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis) to isolate the 120,000-dalton transforming protein and several other phosphotyrosyl proteins from cells transformed by Abelson murine leukemia virus. Under the same conditions, phosphotyrosyl proteins were also isolated from human epidermal carcinoma cells (A431) that had been stimulated with epidermal growth factor; most prominent among these proteins was the 170,000-dalton receptor for epidermal growth factor.