scholarly journals Dibutylchloromethyltin chloride, a covalent inhibitor of the adenosine triphosphate synthase complex

1977 ◽  
Vol 166 (3) ◽  
pp. 593-602 ◽  
Author(s):  
Kelvin Cain ◽  
Michael D. Partis ◽  
David E. Griffiths
Keyword(s):  
Oxidative Phosphorylation ◽  
Polyacrylamide Gel Electrophoresis ◽  
Specific Interaction ◽  
Sodium Dodecyl ◽  
Mitochondrial Atpase ◽  
Protein Subunit ◽  
Binding Studies ◽  
Mitochondrial Atp Synthase ◽  
Chloroform Methanol ◽  
Covalent Inhibitor

1. The synthesis of dibutylchloromethyltin chloride, a new covalent inhibitor of the mitochondrial ATP synthase [oligomycin-sensitive ATPase (adenosine triphosphatase)] complex is described, together with a method for preparing dibutylchloro[3H]methyltin chloride. 2. Studies with the yeast mitochondrial oligomycin-sensitive ATPase complex show that dibutylchloromethyltin chloride inhibits both the membrane-bound enzyme and also the purified Triton X-100-dispersed preparation. 3. F1-ATPase is not inhibited even at 500nmol of dibutylchloromethyltin chloride/mg of protein, and the general inhibitory properties are similar to those of triethyltin, oligomycin and dicyclohexylcarbodi-imide, known energy-transfer inhibitors of oxidative phosphorylation. 4. Binding studies with yeast submitochondrial particles show that dibutylchloromethyltin chloride antagonizes the binding of triethyl[113Sn]tin, indicating that there is an interaction between the two inhibitor-binding sites. 5. Unlike triethyltin, inhibition by dibutylchloromethyltin chloride is due to a covalent interaction which titrates a component of the inner mitochondrial membrane present at a concentration of 8–9nmol/mg of protein. 6. All of the labelled component can be extracted with chloroform/methanol (2:1, v/v), and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis of the chloroform/methanol extract indicates that the labelled component has an apparent mol.wt. of 6000–8000. However, t.l.c. reveals the presence of only one labelled component which is lipophilic and non-protein and is distinct from the free inhibitor, mitochondrial phospholipids and the dicyclohexylcarbodi-imide-binding protein (subunit 9). 7. Inhibition of mitochondrial ATPase and oxidative phosphorylation is correlated with specific interaction with a non-protein lipophilic component of the mitochondrial inner membrane which is proposed to be a co-factor or intermediate of oxidative phosphorylation.

scholarly journals Time-dependent association between platelet-bound fibrinogen and the Triton X-100 insoluble cytoskeleton

Blood ◽  
1991 ◽  
Vol 77 (3) ◽  
pp. 508-514 ◽  
Author(s):  
EI Peerschke
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Adenosine Diphosphate ◽  
Time Dependent ◽  
Binding Studies ◽  
Fibrinogen Binding ◽  
Human Thrombin ◽  
Membrane Bound ◽  
Independent Association ◽  
Triton X

Abstract Previous studies indicated a correlation between the formation of EDTA- resistant (irreversible) platelet-fibrinogen interactions and platelet cytoskeleton formation. The present study explored the direct association of membrane-bound fibrinogen with the Triton X-100 (Sigma Chemical Co, St Louis, MO) insoluble cytoskeleton of aspirin-treated, gel-filtered platelets, activated but not aggregated with 20 mumol/L adenosine diphosphate (ADP) or 150 mU/mL human thrombin (THR) when bound fibrinogen had become resistant to dissociation by EDTA. Conversion of exogenous 125I-fibrinogen to fibrin was prevented by adding Gly-Pro-Arg and neutralizing THR with hirudin before initiating binding studies. After 60 minutes at 22 degrees C, the cytoskeleton of ADP-treated platelets contained 20% +/- 12% (mean +/- SD, n = 14) of membrane-bound 125I-fibrinogen, representing 10% to 50% of EDTA- resistant fibrinogen binding. The THR-activated cytoskeleton contained 45% +/- 15% of platelet bound fibrinogen, comprising 80% to 100% of EDTA-resistant fibrinogen binding. 125I-fibrinogen was not recovered with platelet cytoskeletons if binding was inhibited by the RGDS peptide, excess unlabeled fibrinogen, or disruption of the glycoprotein (GP) IIb-IIIa complex by EDTA-treatment. Both development of EDTA- resistant fibrinogen binding and fibrinogen association with the cytoskeleton were time dependent and reached maxima 45 to 60 minutes after fibrinogen binding to stimulated platelets. Although a larger cytoskeleton formed after platelet stimulation with thrombin as compared with ADP, no change in cytoskeleton composition was noted with development of EDTA-resistant fibrinogen binding. Examination of platelet cytoskeletons using monoclonal antibodies, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and Western blotting showed the presence of only traces of GP IIb-IIIa in the cytoskeletons of resting platelets, with no detectable increases after platelet activation or development of EDTA-resistant fibrinogen binding. These data suggest that GP IIb-IIIa-mediated fibrinogen binding to activated platelets is accompanied by time-dependent alterations in platelet- fibrinogen interactions leading to the GP IIb-IIIa independent association between bound fibrinogen and the platelet cytoskeleton.

scholarly journals Purified mu EBP-E binds to immunoglobulin enhancers and promoters.

1988 ◽  
Vol 8 (11) ◽  
pp. 4972-4980 ◽  
Author(s):  
C L Peterson ◽  
S Eaton ◽  
K Calame
Keyword(s):  
Heavy Chain ◽  
Binding Sites ◽  
Dissociation Constants ◽  
Sequence Similarity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Binding Studies ◽  
Apparent Homogeneity ◽  
Enhancer Binding Protein ◽  
Chemical Nuclease

We describe the purification to apparent homogeneity of the murine immunoglobulin heavy-chain (IgH) enhancer-binding protein mu EBP-E from murine plasmacytoma cells by ion exchange and affinity chromatography. Glycerol gradient sedimentation, UV cross-linking, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis confirm that mu EBP-E is a 45-kilodalton molecular mass protein. Orthophenanthroline-copper chemical nuclease footprinting with purified protein has identified high-affinity binding sites for mu EBP-E within the IgH enhancer at the previously identified site E and at sites within IgH promoters and in the kappa light-chain enhancer. Equilibrium binding studies indicate that the dissociation constants for mu EBP-E binding to site E within the enhancer and to a binding site within the V1 heavy-chain promoter are quite low, about 2 x 10(-11) M. Comparison of four mu EBP-E recognition sequences detects only limited sequence similarity among binding sites.

An osteonectinlike protein in porcine periodontal ligament and its synthesis by periodontal ligament fibroblasts

1984 ◽  
Vol 62 (6) ◽  
pp. 470-478 ◽  
Author(s):  
Safia Wasi ◽  
Kichibee Otsuka ◽  
Kam-Ling Yao ◽  
Pierre S. Tung ◽  
Jane E. Aubin ◽  
...  
Keyword(s):  
Periodontal Ligament ◽  
Culture Medium ◽  
Alveolar Bone ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Relative Mass ◽  
Bovine Bone ◽  
Binding Studies ◽  
Periodontal Ligament Fibroblasts ◽  
Sds Page

Periodontal ligament, a soft connective tissue that lies between cementum and alveolar bone in the periodontium, has been shown to contain an osteonectinlike protein. The similarity between porcine ligament osteonectin and bovine bone osteonectin was evident from immunochemical studies, from migration characteristics on sodium dodecyl sulfate – polyacrylamide gel electrophoresis (SDS–PAGE) and from binding studies on hydroxyapatite. Using immunotransfer and immunodot analyses, ligament osteonectin was found to be extractable from tissues with 4 M guanidine–HCl (GuHCl) and 4 M GuHCl − 0.5 M EDTA and to comigrate with authentic bovine osteonectin on SDS–PAGE with a relative mass ~ 38 000. Furthermore, osteonectin from guanidine extracts of ligament was bound to hydroxyapatite in the presence of 4 M GuHCl. Immunofluorescence studies showed the osteonectin to be distributed throughout the extracellular matrix of the ligament and to be present within the ligament fibroblasts in a perinuclear, punctate distribution. Biosynthesis of osteonectin by ligament fibroblasts was studied following pulse-chase labelling with [35S]methionine and immunoprecipitation. The labelled osteonectin in the chased culture medium represented ~0.5% of the total labelled proteins secreted. It comigrated on SDS–PAGE with the corresponding labelled protein from pulsed cells and with the protein extracted from the tissue.

scholarly journals Modulation of complement activation on hemodialysis membranes by immobilized heparin.

1992 ◽  
Vol 2 (8) ◽  
pp. 1328-1337
Author(s):  
A K Cheung ◽  
C J Parker ◽  
J Janatova ◽  
E Brynda
Keyword(s):  
Complement Activation ◽  
Alternative Pathway ◽  
Membrane Surface ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Fluid Phase ◽  
Factor H ◽  
Binding Studies ◽  
Factor B ◽  
Pathway Activity

To determine the effects of surface-associated heparin on the capacity of hemodialysis membranes to activate complement, cellulose acetate (CA) membranes that were untreated and CA membranes that had been coated with heparin (HCA) were incubated with C3-depleted serum repleted with radio-labeled C3. Next, the proteins in the supernatant and those eluted from the membranes were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis. C3 activation was quantified by determining the radioactivity of the C3a-containing band in the gel. Total C3a generation (fluid phase C3a plus membrane-associated C3a) was three times greater in the presence of HCA compared with CA. Most (88%) of the C3a generated in the presence of HCA, however, was adsorbed onto the membrane surface. Consequently, there was more C3a in the CA supernatant than in the HCA supernatant. To determine the mechanism by which heparin enhanced alternative pathway activity, binding studies with radiolabeled factor B and factor H were performed. HCA bound 3.4 times more factor B and 20 times more factor H than did CA. The binding of these proteins, however, was not dependent on complement activation. Studies designed to test the functional activity of isolated factor H and factor B that had been adsorbed to the membrane showed that factor H was active on both CA and HCA, whereas factor B was active only on HCA. These data demonstrate that heparin immobilized onto CA hemodialysis membrane enhances C3 activation but produces low levels of C3a in the fluid phase because of high surface adsorption of the anaphylatoxin. Heparin appears to augment alternative pathway activity by favoring the interactions of factor B with other constituents of the amplification C3 convertase of the alternative pathway of complement.

scholarly journals Purification of a Zinc Binding Protein from Xylem of Citrus jambhiri

2002 ◽  
Vol 127 (5) ◽  
pp. 718-723 ◽  
Author(s):  
Kathryn C. Taylor ◽  
Danielle R. Ellis ◽  
Luciano V. Paiva
Keyword(s):  
Total Protein ◽  
Proteinase Inhibitors ◽  
Binding Protein ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sulfhydryl Group ◽  
Size Exclusion ◽  
Binding Studies ◽  
Citrus Jambhiri ◽  
Citrus Rootstock

Zinc in xylem and phloem of the citrus rootstock, rough lemon [Citrus jambhiri (L.)] was associated with a Zn-binding protein, designated citrus vascular Zn-binding protein (CVZBP). The apparent molecular mass of the CVZBP was 19.5 kDa after nondenaturing size exclusion chromatography and 21.8 kDa after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Ion exchange chromatography demonstrated that CVZBP was anionic, requiring 0.43 n NaCl for elution from quaternary aminoethyl Sepharose. Antiserum to the protein cross-reacted more with total protein extracts from leaf midveins than with total protein from the rest of the leaf lamina, further suggesting a vascular location of the Zn-binding protein. Quantitative analysis indicated that ≈2 to 3 mol of Zn were associated with 1 mol of native protein. Binding studies with the partially purified CVZBP demonstrated a capacity to bind several divalent cations: Cd, Ni, Pb, and Zn. Reaction with Ellman's reagent suggested that the protein has significant sulfhydryl group content that may be involved in metal binding. N-terminal sequencing demonstrates identity with papaya latex trypsin inhibitor, sporamin, or other Kunitz soybean proteinase inhibitors.

Effect of Sulfur Supply on the Seed Globulin Composition of Various Species of Lupin

1978 ◽  
Vol 5 (5) ◽  
pp. 641 ◽  
Author(s):  
JM Gillespie ◽  
RJ Blagrove ◽  
PJ Randall
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Large Change ◽  
Subunit Composition ◽  
Sulfur Deficiency ◽  
Protein Subunit ◽  
Disulfide Bonding ◽  
Molecular Weights ◽  
Sulfur Supply ◽  
The Individual

The protein level in seeds of six species of lupin, grown either under sulfur deficiency or with adequate sulfur fertilization, is marginally affected by sulfur supply. However, the ratio of total sulfur to total nitrogen in the seed is greatly decreased under sulfur deficiency. This large change in sulfur-to-nitrogen ratio is accompanied by suppression of the synthesis of conglutins α and γ, which contain a significant amount of cyst(e)ine and methionine. The level of protein is maintained by increased synthesis of conglutin β, which normally contains no methionine and a low proportion of cyst(e)ine. These changes in the proportions of the proteins are reflected in the amino acid analyses for the globulin extracts. The changes in protein subunit composition which accompany the differences in the proportions of the proteins have been studied using sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The results emphasize the differences in subunit composition between lupin species in terms of the number of components, their molecular weights and the importance of disulfide bonding. Two-dimensional electrophoresis, using cellulose acetate and SDS-polyacrylamide gradient gels, has been used to compare the subunit composition of the individual globulins for Lupinus angustifolius and L. elegans at both sulfur levels.

Anomalous aggregation of Clostridium perfringens enterotoxin under dissociating conditions

1976 ◽  
Vol 22 (9) ◽  
pp. 1410-1414 ◽  
Author(s):  
George L. Enders Jr. ◽  
Charles L. Duncan
Keyword(s):  
Molecular Weight ◽  
Clostridium Perfringens ◽  
Gel Electrophoresis ◽  
Cetyltrimethylammonium Bromide ◽  
Equilibrium Dialysis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Binding Studies ◽  
Clostridium Perfringens Enterotoxin ◽  
Subunit Molecular Weight

Polyacrylamide gel electrophoresis of highly purified Clostridium perfringens enterotoxin revealed electrophoretic microheterogeneity of the enterotoxin, apparently because of slight charge differences in the peptides. Detergent gel electrophoresis showed that purified enterotoxin formed high molecular weight aggregates in the presence of both sodium dodecyl sulfate (SDS) and cetyltrimethylammonium bromide. No conditions capable of inhibiting this phenomenon were found. Although a molecular weight of 35 000 daltons has been reported in the literature, the experimentally determined molecular weight values in the presence of detergents corresponded to multiples of a theoretical subunit molecular weight of 17 500 daltons. Binding studies performed by equilibrium dialysis and ultracentrifugation methods revealed that the enterotoxin bound very small amounts of SDS per gram of protein. The evidence presented indicates possible detergent induced structural alterations of the protein.

Products of mitochondrial protein synthesis in Tetrahymena

1979 ◽  
Vol 57 (4) ◽  
pp. 314-320 ◽  
Author(s):  
Paul G. Young ◽  
Neil P. Hunter
Keyword(s):  
Protein Synthesis ◽  
Gel Electrophoresis ◽  
Trichloroacetic Acid ◽  
Polyacrylamide Gel Electrophoresis ◽  
Mitochondrial Protein ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Mitochondrial Proteins ◽  
Mitochondrial Protein Synthesis ◽  
Chloroform Methanol

The products of mitochondrial protein synthesis have been investigated in Tetrahymena after labelling with [35S]methionine in the presence of cycloheximide. The labelled proteins were analyzed by sodium dodecyl sulfate slab polyacrylamide gel electrophoresis. We have identified 13 electrophoretically discrete bands as well as 4 other bands with a more variable occurrence. These proteins ranged in apparent molecular weight from 8100 to 57 500. The cycloheximide-resistant incorporation could be blocked with chloramphenicol. The mitochondrial proteins appeared to be in a disaggregated state and were stable to agents such as trichloroacetic acid (hot or cold) and chloroform–methanol. The pattern of proteins was similar following labelling times ranging from 30 min to 3 h.

scholarly journals Construction and Characterization ofMoraxella catarrhalis Mutants Defective in Expression of Transferrin Receptors

1999 ◽  
Vol 67 (11) ◽  
pp. 5815-5819 ◽  
Author(s):  
Nicole R. Luke ◽  
Anthony A. Campagnari
Keyword(s):  
Solid Phase ◽  
Iron Acquisition ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Sole Source ◽  
Pcr Analysis ◽  
Binding Studies ◽  
Binding Characteristics ◽  
Isogenic Mutants

ABSTRACT We have previously reported the construction of an isogenic mutant defective in expression of OmpB1, the TbpB homologue, inMoraxella catarrhalis 7169. In this report, we have extended these studies by constructing and characterizing two new isogenic mutants in this clinical isolate. One mutant is defective in expression of TbpA, and the other mutant is defective in expression of both TbpA and TbpB. These isogenic mutants were confirmed by using PCR analysis, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and sequencing. In vitro growth studies, comparing all three mutants, demonstrated that the tbpA mutant and the tbpABmutant were severely limited in their ability to grow with human holotransferrin as the sole source of iron. In contrast, theompB1 (tbpB) mutant was capable of utilizing iron from human transferrin, although not to the extent of the parental strain. While affinity chromatography with human holotransferrin showed that each Tbp was capable of binding independently to transferrin, solid-phase transferrin binding studies using whole cells demonstrated that the tbpA mutant exhibited binding characteristics similar to those seen with the wild-type bacteria. However, theompB1 (tbpB) mutant exhibited a diminished capacity for binding transferrin, and no binding was detected with the double mutant. These data suggest that the M. catarrhalisTbpA is necessary for the acquisition of iron from transferrin. In contrast, TbpB is not essential but may serve as a facilitory protein that functions to optimize this process. Together these mutants are essential to provide a more thorough understanding of iron acquisition mechanisms utilized by M. catarrhalis.

Purified mu EBP-E binds to immunoglobulin enhancers and promoters

1988 ◽  
Vol 8 (11) ◽  
pp. 4972-4980
Author(s):  
C L Peterson ◽  
S Eaton ◽  
K Calame
Keyword(s):  
Heavy Chain ◽  
Binding Sites ◽  
Dissociation Constants ◽  
Sequence Similarity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Binding Studies ◽  
Apparent Homogeneity ◽  
Enhancer Binding Protein ◽  
Chemical Nuclease

We describe the purification to apparent homogeneity of the murine immunoglobulin heavy-chain (IgH) enhancer-binding protein mu EBP-E from murine plasmacytoma cells by ion exchange and affinity chromatography. Glycerol gradient sedimentation, UV cross-linking, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis confirm that mu EBP-E is a 45-kilodalton molecular mass protein. Orthophenanthroline-copper chemical nuclease footprinting with purified protein has identified high-affinity binding sites for mu EBP-E within the IgH enhancer at the previously identified site E and at sites within IgH promoters and in the kappa light-chain enhancer. Equilibrium binding studies indicate that the dissociation constants for mu EBP-E binding to site E within the enhancer and to a binding site within the V1 heavy-chain promoter are quite low, about 2 x 10(-11) M. Comparison of four mu EBP-E recognition sequences detects only limited sequence similarity among binding sites.

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