Protein–protein and protein–DNA interactions in calf thymus nuclear matrix using cross-linking by ultraviolet irradiation

1986 ◽  
Vol 64 (5) ◽  
pp. 474-484 ◽  
Author(s):  
Teni Boulikas
Keyword(s):  
Uv Irradiation ◽  
Uv Light ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Micrococcal Nuclease ◽  
Cross Linking ◽  
Repeated Treatment ◽  
Major Protein ◽  
Protein Species ◽  
Calf Thymus

Nuclear matrices from calf thymus contained 30–50 protein species with one prominent band at 70 kilodaltons tentatively identified by its isoelectric point, apparent molecular weight, charge modification, and abundance as bovine lamin. The amount of DNA present in the matrix fraction was strongly dependent on the extent of digestion of the nuclei by micrococcal nuclease. The size of the DNA was higher than two kilobase pairs, although the chromatin DNA had been digested down to short oligonucleosomes. The lamin band was preferentially dissociated from isolated matrices during repeated treatment by 2 M NaCl or 5 M urea. Irradiation of calf thymus nuclear matrices at 313 nm induced protein–protein and protein–DNA cross-linking, as well as double-strand breaking of DNA, presumably at unprotected, protein-free regions. Lamin protein was more dramatically affected than other protein species by ultraviolet (UV) irradiation. In situ DNA hydrolysis, after the separation of the cross-linked matrix components on polyacrylamide – sodium dodecyl sulfate gels, followed by two-dimensional electrophoresis, showed lamin to be the major protein that was cross-linked to the DNA. Lamin molecules were also cross-linked by UV light to each other to form lamin homooligomers. A discrete size DNA fragment of approximately 450 base pairs is protected by lamin homooligomers from breakdown during UV irradiation. It is proposed that the direct contact between lamin and DNA found in this study is responsible for anchoring chromatin loops (domains) to a stable, immobile matrix structure.

DNA affinity labeling of adenovirus type 2 upstream promoter sequence-binding factors identifies two distinct proteins

1988 ◽  
Vol 8 (1) ◽  
pp. 105-113
Author(s):  
B Safer ◽  
R B Cohen ◽  
S Garfinkel ◽  
J A Thompson
Keyword(s):  
Uv Light ◽  
Promoter Sequence ◽  
Adenovirus Type ◽  
Micrococcal Nuclease ◽  
Cross Linking ◽  
Affinity Labeling ◽  
Upstream Promoter ◽  
Upstream Promoter Sequence ◽  
Adenovirus Type 2

A rapid affinity labeling procedure with enhanced specificity was developed to identify DNA-binding proteins. 32P was first introduced at unique phosphodiester bonds within the DNA recognition sequence. UV light-dependent cross-linking of pyrimidines to amino acid residues in direct contact at the binding site, followed by micrococcal nuclease digestion, resulted in the transfer of 32P to only those specific protein(s) which recognized the binding sequence. This method was applied to the detection and characterization of proteins that bound to the upstream promoter sequence (-50 to -66) of the human adenovirus type 2 major late promoter. We detected two distinct proteins with molecular weights of 45,000 and 116,000 that interacted with this promoter element. The two proteins differed significantly in their chromatographic and cross-linking behaviors.

Dual cross-linking ribonucleoprotein immunoprecipitation assay

2014 ◽  
Vol 92 (4) ◽  
pp. 317-319 ◽  
Author(s):  
Dilshad H. Khan ◽  
James R. Davie
Keyword(s):  
Uv Irradiation ◽  
Protein Interactions ◽  
Uv Light ◽  
Target Identification ◽  
Cross Linking ◽  
Immunoprecipitation Assay ◽  
Cross Linker ◽  
Subsequent Target ◽  
Protein Cross Linking

Ribonucleoprotein immunoprecipitation (RIP) is an antibody-based method to detect RNA–protein interactions in situ. In the assay, UV cross-linking is commonly used to preserve RNA–protein interactions for subsequent target identification. UV light is a zero-length cross linker and thus identifies proteins directly bound to RNAs. Here, we describe a dual cross-linking RIP method that involves sequential protein–protein cross-linking step with a protein–protein cross-linker, followed by protein–RNA fixation by UV irradiation. In this way, proteins that indirectly bound to RNA can be analyzed.

scholarly journals DNA affinity labeling of adenovirus type 2 upstream promoter sequence-binding factors identifies two distinct proteins.

1988 ◽  
Vol 8 (1) ◽  
pp. 105-113 ◽  
Author(s):  
B Safer ◽  
R B Cohen ◽  
S Garfinkel ◽  
J A Thompson
Keyword(s):  
Uv Light ◽  
Promoter Sequence ◽  
Adenovirus Type ◽  
Micrococcal Nuclease ◽  
Cross Linking ◽  
Affinity Labeling ◽  
Upstream Promoter ◽  
Upstream Promoter Sequence ◽  
Adenovirus Type 2

A rapid affinity labeling procedure with enhanced specificity was developed to identify DNA-binding proteins. 32P was first introduced at unique phosphodiester bonds within the DNA recognition sequence. UV light-dependent cross-linking of pyrimidines to amino acid residues in direct contact at the binding site, followed by micrococcal nuclease digestion, resulted in the transfer of 32P to only those specific protein(s) which recognized the binding sequence. This method was applied to the detection and characterization of proteins that bound to the upstream promoter sequence (-50 to -66) of the human adenovirus type 2 major late promoter. We detected two distinct proteins with molecular weights of 45,000 and 116,000 that interacted with this promoter element. The two proteins differed significantly in their chromatographic and cross-linking behaviors.

scholarly journals Purification of sphingomyelinase to apparent homogeneity by using hydrophobic chromatography

1981 ◽  
Vol 195 (2) ◽  
pp. 373-382 ◽  
Author(s):  
C S Jones ◽  
P Shankaran ◽  
J W Callahan
Keyword(s):  
Molecular Weight ◽  
Large Scale ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Hydrophobic Interaction Chromatography ◽  
Apparent Molecular Weight ◽  
Kinetic Properties ◽  
Major Protein ◽  
Apparent Homogeneity ◽  
Nitrophenyl Phosphate

Placental sphingomyelinase has been purified to apparent homogeneity by a procedure that makes extensive use of hydrophobic interaction chromatography on sphingosylphosphocholine-CH-, octyl-, hexyl- and Blue-Sepharoses. Enzyme purification is about 10000- 14000-fold over starting extract with excellent yield (usually greater than 28%). Purification of bis-4-methylumbelliferyl phosphate phosphodiesterase activity generally paralleled that of sphingomyelinase during the final stages of the procedure. The enzyme also hydrolysed bis-p-nitrophenyl phosphate, but at a lower rate compared with bis-4-methylumbelliferyl phosphate. A single major protein was observed under non-denaturing conditions. Sphingomyelinase, denatured by reduction and alkylation, is composed of a major polypeptide chain with an apparent molecular weight of 89 100 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Two minor lower-molecular-weight components were consistently obtained at 47 500 and 30 700. These results were also obtained after maleoylation of the reduced and alkylated sample. The enzyme contains a blocked-N-terminal amino acid. An extensive search for contaminating enzymes revealed the presence of minor amounts of acid phosphatase, which were removed from the final enzyme sample. The highly purified enzyme is stable for several weeks when stored with Triton X-100 at 4 degrees C. The pure enzyme aggregates under denaturing and electrophoretic conditions and special care must be taken to ensure that hydrophobic bonding of the protein is decreased as much as possible. The reproducibility and large scale of this procedure should facilitate further study on the structure and kinetic properties of the enzyme.

Cell surface protein of Salmonella typhimurium

1975 ◽  
Vol 21 (7) ◽  
pp. 1132-1136 ◽  
Author(s):  
S. Kabir
Keyword(s):  
Cell Surface ◽  
Salmonella Typhimurium ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Surface Protein ◽  
Apparent Molecular Weight ◽  
Major Protein ◽  
Cell Surface Protein ◽  
Exterior Surface

Lactoperoxidase-catalyzed radioiodination with Na125I was performed both on intact Salmonella typhimurium 1195 and on ghost membrane isolated from the same bacterial strain. Ghost membrane was also prepared from radioiodinated whole bacteria. The labelled proteins from both these ghost membrane preparations were compared by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate to identify the cell surface protein. From the results obtained it was concluded that one major protein with an apparent molecular weight of 12 000 – 13 000 was exposed on the exterior surface of the ghost membrane.

scholarly journals Exocrine pancreas transcription factor 1 binds to a bipartite enhancer element and activates transcription of acinar genes.

1991 ◽  
Vol 11 (10) ◽  
pp. 4985-4997 ◽  
Author(s):  
S L Weinrich ◽  
A Meister ◽  
W J Rutter
Keyword(s):  
Transcription Factor ◽  
Exocrine Pancreas ◽  
Sodium Dodecyl ◽  
Binding Activity ◽  
Cross Linking ◽  
Major Protein ◽  
Enhancer Element ◽  
Transcription Factor 1

Exocrine pancreas (XP) enhancers, which contain a conserved core sequence, are active only in XP cells. A core enhancer-binding activity also appears to be restricted to XP nuclei. Here we describe the properties of a factor, purified approximately 100,000-fold from pancreas nuclei, which displays core enhancer-binding activity. It is not identical to previously characterized factors and is termed exocrine pancreas transcription factor 1 (XPF-1). In the highly purified preparation, only a single major protein of 60 kDa was detected by silver staining on sodium dodecyl sulfate-gels and by UV cross-linking. XPF-1 binds to the core enhancer of all tested XP genes and not to a mutant sequence which is inactive in vivo. High-affinity binding sites are bipartite. The results of competition binding and UV-cross-linking assays suggest that XPF-1 interacts with both motifs. XPF-1 selectively stimulates transcription of core enhancer templates in an in vitro transcription system. We hypothesize that XPF-1 plays a role in activation of the transcription of XP-specific genes.

Reconstitution of isolated Ca(2+)-activated K+ channel proteins from basolateral membranes of rabbit colonocytes

1991 ◽  
Vol 261 (4) ◽  
pp. C713-C717 ◽  
Author(s):  
S. Liu ◽  
W. P. Dubinsky ◽  
M. K. Haddox ◽  
S. G. Schultz
Keyword(s):  
High Selectivity ◽  
Sodium Dodecyl ◽  
Membrane Vesicles ◽  
Voltage Gating ◽  
K Channel ◽  
Major Protein ◽  
Protein Species ◽  
Basolateral Membranes ◽  
Molecular Masses ◽  
35 Kda Protein

Using calmodulin-affinity chromatography, we have isolated a fraction of proteins from solubilized basolateral membranes of rabbit colonocytes which when reconstituted into planar phospholipid bilayers disclosed Ca(2+)-activated single K+ channel activities. The properties of the reconstituted channels are identical to those of native membrane vesicles incorporated into these bilayers with respect to their high selectivity for K+ over C-, high ("maxi") conductance, voltage gating, and inhibition by trifluoperazine. Two-dimensional sodium dodecyl sulfate gel electrophoresis of these proteins revealed three major protein species with molecular masses of 120, 60, and 35 kDa, which constituted 70, 10, and 20%, respectively, of the total protein. The results of other studies strongly suggest that the 35-kDa protein may be the Ca(2+)-activated K+ channel protein in these membranes.

Exocrine pancreas transcription factor 1 binds to a bipartite enhancer element and activates transcription of acinar genes

1991 ◽  
Vol 11 (10) ◽  
pp. 4985-4997
Author(s):  
S L Weinrich ◽  
A Meister ◽  
W J Rutter
Keyword(s):  
Transcription Factor ◽  
Exocrine Pancreas ◽  
Sodium Dodecyl ◽  
Binding Activity ◽  
Cross Linking ◽  
Major Protein ◽  
Enhancer Element ◽  
Transcription Factor 1

Exocrine pancreas (XP) enhancers, which contain a conserved core sequence, are active only in XP cells. A core enhancer-binding activity also appears to be restricted to XP nuclei. Here we describe the properties of a factor, purified approximately 100,000-fold from pancreas nuclei, which displays core enhancer-binding activity. It is not identical to previously characterized factors and is termed exocrine pancreas transcription factor 1 (XPF-1). In the highly purified preparation, only a single major protein of 60 kDa was detected by silver staining on sodium dodecyl sulfate-gels and by UV cross-linking. XPF-1 binds to the core enhancer of all tested XP genes and not to a mutant sequence which is inactive in vivo. High-affinity binding sites are bipartite. The results of competition binding and UV-cross-linking assays suggest that XPF-1 interacts with both motifs. XPF-1 selectively stimulates transcription of core enhancer templates in an in vitro transcription system. We hypothesize that XPF-1 plays a role in activation of the transcription of XP-specific genes.

scholarly journals Biochemical study of KB-cell receptor for adenovirus

1977 ◽  
Vol 166 (2) ◽  
pp. 237-247 ◽  
Author(s):  
Bernadette Hennache ◽  
Pierre Boulanger
Keyword(s):  
Specific Activity ◽  
Biochemical Study ◽  
Gradient Centrifugation ◽  
Sodium Dodecyl ◽  
Cell Receptor ◽  
Surface Proteins ◽  
Cross Linking ◽  
Affinity Column ◽  
Major Protein ◽  
Sucrose Density Gradient Centrifugation

Three different approaches were used in an attempt to characterize the KB-cell receptor for adenovirus: affinity chromatography, immunoadsorption and cross-linking with a cleavable bifunctional reagent. The first system used an affinity gel consisting of adenovirus-fibre projection linked to Sepharose matrix by an intermediate bis(aminopropyl)amine arm, the amino groups of the fibre ligand being preserved by prior citraconylation. The second system consisted of adenovirus complete penton capsomere attached to anti-(penton base) antibody and cross-linked to polyacrylamide particles with glutaraldehyde. In this latter affinity model, the penton-fibre projection was appropriately oriented outwards, as in the virus particle. Both affinity systems permitted isolation from a KB-cell plasma-membrane extract of fibre-binding and penton-fibre-binding protein material, which inhibited adenovirus attachment. The penton–immunoadsorbent appeared more efficient and more specific than the affinity column of fibre–bis(aminopropyl)amino-Sepharose gel in specific activity of inhibition of adenovirus attachment. The third method consisted of reversibly cross-linking KB-cell receptor proteins with adenovirus particles by means of a cleavable di-imidoester and isolation of the complexes by sucrose-density-gradient centrifugation. Polypeptide analysis on sodium dodecyl sulphate/polyacrylamide gel of labelled KB-cell surface proteins, selected by the different procedures, showed that three major protein subunits of 78000, 42000 and 34000mol.wt. were common to the three selection systems. A possible model for the structure and function of the KB-cell receptor for adenovirus is discussed.

The Activation of Human Factor IX

1975 ◽  
Vol 33 (03) ◽  
pp. 553-563 ◽  
Author(s):  
B Østerud ◽  
K Laake ◽  
H Prydz
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Factor Ix ◽  
Factor Vii ◽  
Activate Factor ◽  
Human Factor Ix ◽  
Factor Xia ◽  
Tissue Thromboplastin ◽  
Native Factor

SummaryThe activation of factor IX purified from human plasma has been studied. Factor XIa and kallikrein separately activated factor IX to factor IXa. In both cases factor IX a had an apparent molecular weight of about 42–45000 in sodium dodecyl sul-phate-polyacrylamide disc gel electrophoresis compared with a molecular weight of about 70000 for the native factor IX. The activation by XIa required Ca2+-ions whereas Ca2+-ions did not influence the activation by kallikrein. A mixture of tissue thromboplastin and factor VII or RusselPs-viper venom alone did not activate factor IX. Trypsin activated and plasmin inactivated factor IX.

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