scholarly journals The effects of iron-limited growth on the reduced nicotinamide–adenine dinucleotide dehydrogenase activity and the membrane proteins of Candida utilis mitochondria

1973 ◽  
Vol 136 (4) ◽  
pp. 1029-1037 ◽  
Author(s):  
Roger A. Clegg ◽  
Jean E. Skyrme
Keyword(s):  
Membrane Proteins ◽  
Mitochondrial Membrane ◽  
Nadh Dehydrogenase ◽  
Candida Utilis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Iron Limitation ◽  
Nicotinamide Adenine Dinucleotide Dehydrogenase ◽  
Reduced Nicotinamide Adenine Dinucleotide ◽  
Haem Iron

1. The mitochondrial NADH dehydrogenase (EC 1.6.99.3) of Candida utilis exhibited altered properties when the organism was grown under iron-limited conditions. No suitable acceptor was found for assay of this enzyme from iron-limited cells. 2. Mitochondrial membrane proteins from C. utilis were analysed by polyacrylamide-gel electrophoresis. Compared with glycerol-limited cells, iron limitation resulted in the loss of at least two polypeptides from the mitochondrial membrane. 3. Neither of the polypeptides affected by iron limitation was part of a cytochrome, although one of them was part of the mitochondrial NADH dehydrogenase. 4. Non-haem iron of mitochondrial membranes was released in the presence of sodium dodecyl sulphate, and electrophoresis in solutions of this detergent cannot be used directly to identify iron–sulphur proteins. Non-ionic detergents do not release non-haem iron but nor do they provide a satisfactory system for electrophoretic separation.

A novel Bicine running buffer system for doubled sodium dodecyl sulfate – polyacrylamide gel electrophoresis of membrane proteins

2006 ◽  
Vol 27 (14) ◽  
pp. 2984-2995 ◽  
Author(s):  
Taufika Islam Williams ◽  
Jennifer C. Combs ◽  
Anup P. Thakur ◽  
Herbert J. Strobel ◽  
Bert C. Lynn
Keyword(s):  
Membrane Proteins ◽  
Sodium Dodecyl Sulfate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Buffer System ◽  
Dodecyl Sulfate

scholarly journals Identification of Polymorphic Outer Membrane Proteins of Chlamydia psittaci 6BC

2001 ◽  
Vol 69 (4) ◽  
pp. 2428-2434 ◽  
Author(s):  
Regina J. Tanzer ◽  
David Longbottom ◽  
Thomas P. Hatch
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Disulfide Bonds ◽  
Outer Membrane Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Late Phase ◽  
Sodium Dodecyl ◽  
Chlamydia Psittaci ◽  
Developmental Cycle ◽  
Lauryl Sarcosine

ABSTRACT The genomes of Chlamydia spp. encode a family of putative outer membrane proteins, referred to as polymorphic outer membrane proteins (POMPs), which may play a role in the avoidance of host immune defenses. We analyzed avian strain 6BC of Chlamydia psittaci by polyacrylamide gel electrophoresis for the expression of POMPs. At least six putative POMPs were identified on the basis of their size (90 to 110 kDa) and labeling with an outer membrane-specific probe, 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine. Three of the putative POMPs reacted with antiserum raised against a recombinant ovine C. psittaci strain POMP, and two possessed surface-exposed, trypsin-sensitive sites. The POMPs were dependent on disulfide bonds for their maintenance in sodium lauryl sarcosine- and sodium dodecyl sulfate-insoluble complexes but did not appear to be interpeptide disulfide bond cross-linked. The putative POMPs were found to be synthesized during the late phase of the chlamydial developmental cycle, cotemporally with the cysteine-rich doublet periplasmic proteins.

scholarly journals Specific and azurophilic granules from rabbit polymorphonuclear leukocytes. I. Isolation and characterization of membrane and content subfractions.

1983 ◽  
Vol 96 (4) ◽  
pp. 1030-1039 ◽  
Author(s):  
W J Brown ◽  
W A Shannon ◽  
W J Snell
Keyword(s):  
Membrane Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Composition ◽  
Granule Protein ◽  
Isolation And Characterization ◽  
Cytoplasmic Face ◽  
Sds Page ◽  
Granule Membrane ◽  
Granule Membrane Proteins

The specific and azurophilic granules of rabbit polymorphonuclear heterophils (PMNs) have been isolated and fractionated into membrane and extractable subfractions. Analysis by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS PAGE) revealed several features of the protein composition of the two granules: (a) Whereas each type of granule had 40-60 proteins separable on one-dimensional gradient gels, few of the proteins were common to both granules. (b) The proteins of the extractable fractions (which comprised approximately 98% of the total granule protein) of each granule were distinct from the proteins of the membrane fractions (which comprised approximately 2% of the total granule protein). (c) The extractable proteins co-migrated with those collected from the medium of ionophore-treated, degranulating PMNs and therefore were defined as content proteins. These results were confirmed by radiolabeling studies. Lactoperoxidase-catalyzed iodination of intact granules did not label the content proteins but did label proteins that co-migrated with major granule membrane proteins. Moreover, disruption of the granules before iodination led to labeling of both content and membrane proteins. We conclude that the membranes of specific and azurophilic granules, which arise from different faces of the Golgi complex, are composed of unique sets of membrane proteins some of which are exposed on the cytoplasmic face of the granules.

scholarly journals Partial purification and properties of the external NADH dehydrogenase from cuckoo-pint (Arum maculatum) mitochondria

1984 ◽  
Vol 224 (1) ◽  
pp. 171-179 ◽  
Author(s):  
I R Cottingham ◽  
A L Moore
Keyword(s):  
Nadh Dehydrogenase ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Partial Purification ◽  
Purification And Properties ◽  
Nadh Dehydrogenases ◽  
Arum Maculatum ◽  
A Minor ◽  
Considerable Loss

The external NADH dehydrogenase has been purified from Arum maculatum (cuckoo-pint) mitochondria by phosphate washing, extraction with deoxycholate, ion-exchange and gel-filtration chromatography. Sodium dodecyl sulphate/polyacrylamide-gel electrophoresis shows, when the gel is silver-stained, that the purified enzyme contains two major bands of Mr 78 000 and 65 000 and a minor one of Mr about 76 000. It is not possible at present to determine which of these, or which combination, constitutes the dehydrogenase. The enzyme contains non-covalently bound FAD and a small amount of FMN. Since the conditions of purification lead to considerable loss of flavin and possibly iron-sulphur centres, it is not possible to decide with certainty whether the enzyme is a flavo- or ferroflavo-protein. The enzyme has been distinguished from the other NADH dehydrogenases on the basis of its substrate specificity, its capability of reducing electron acceptors such as ubiquinone-1 and 2,6-dichlorophenol-indophenol and its sensitivity towards Ca2+, EGTA and dicoumarol.

Outer membrane proteins from Serratia marcescens

1993 ◽  
Vol 39 (1) ◽  
pp. 108-111 ◽  
Author(s):  
Marta Puig ◽  
Carme Fusté ◽  
Miquel Viñas
Keyword(s):  
Membrane Proteins ◽  
Serratia Marcescens ◽  
Outer Membrane ◽  
Nutrient Availability ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Outer Membrane Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Cultural Conditions

The outer membrane proteins (OMPs) of several strains of Serratia marcescens have been studied by sodium dodecyl sulphate – urea – polyacrylamide gel electrophoresis. Four major OMPs, named Omp1, Omp2, Omp3, and OmpA (42, 40, 39, and 37 kDa, respectively), have been visualized. The relative proportions of Omp2 and Omp3 depend on cultural conditions (temperature of incubation, osmolarity, and nutrient availability).Key words: Serratia marcescens, outer membrane proteins, porin.

scholarly journals Establishing a Direct Role for the Bartonella bacilliformis Invasion-Associated Locus B (IalB) Protein in Human Erythrocyte Parasitism

2001 ◽  
Vol 69 (7) ◽  
pp. 4373-4381 ◽  
Author(s):  
Sherry A. Coleman ◽  
Michael F. Minnick
Keyword(s):  
Membrane Proteins ◽  
Outer Membrane ◽  
Protein Localization ◽  
Outer Membrane Proteins ◽  
Polyacrylamide Gel Electrophoresis ◽  
Buoyant Density ◽  
Sodium Dodecyl ◽  
Amino Acid Sequence Similarity ◽  
Bartonella Bacilliformis ◽  
Sds Page

ABSTRACT The invasion-associated locus A and B genes (ialAB) ofBartonella bacilliformis were previously shown to confer an erythrocyte-invasive phenotype upon Escherichia coli, indirectly implicating their role in virulence. We report the first direct demonstration of a role for ialB as a virulence factor in B. bacilliformis. The presence of a secretory signal sequence and amino acid sequence similarity to two known outer membrane proteins involved in virulence suggested that IalB was an outer membrane protein. To develop an antiserum for protein localization, the ialB gene was cloned in frame into an expression vector with a six-histidine tag and under control of thelacZ promoter. The IalB fusion protein was purified by nickel affinity chromatography and used to raise polyclonal antibodies. IalB was initially localized to the bacterial membrane fraction. To further localize IalB, B. bacilliformis inner and outer membranes were fractionated by sucrose density gradient centrifugation and identified by appearance, buoyant density (ρ), and cytochromeb content. Inner and outer membrane proteins were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and IalB was positively identified by Western blot. Contrary to expectations, IalB was localized to the inner membrane of the pathogen. To directly demonstrate a role for IalB in erythrocyte parasitism, the B. bacilliformis ialB gene was disrupted by insertional mutagenesis. The resulting ialB mutant strain was complemented in trans with a replicative plasmid encoding the full-length ialB gene. PCR and high-stringency DNA hybridization confirmed mutagenesis and transcomplementation events. Abrogation and restoration of ialB expression was verified by SDS-PAGE and immunoblotting. In vitro virulence assays showed that mutagenesis of ialB decreased bacterial association and invasion of human erythrocytes by 47 to 53% relative to controls. Transcomplementation of ialB restored erythrocyte association and invasion rates to levels observed in the parental strain. These data provide direct evidence for IalB's role in erythrocyte parasitism and represent the first demonstration of molecular Koch's postulates for a Bartonella species.

Compositional and electrophoretic changes in buffalo milk-fat globule membrane proteins during lactation

1976 ◽  
Vol 43 (3) ◽  
pp. 381-388 ◽  
Author(s):  
Sukhminder Singh ◽  
N. C. Ganguli
Keyword(s):  
Membrane Proteins ◽  
Isoelectric Focusing ◽  
Gel Electrophoresis ◽  
Milk Fat ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Polyacrylamide Gel ◽  
Milk Fat Globule Membrane ◽  
Milk Fat Globule ◽  
Fat Globule

SummaryChemical analyses, polyacrylamide-gel electrophoresis and isoelectric focusing of milk-fat globule membrane proteins (FGMP) obtained from the milk of 2 Murrah buffaloes were done to determine if any change in composition occurred during lactation. Changes in the levels of sialic acid, hexose, hexosamine, N and P were found in the FGMP obtained at different stages of lactation. On the day of parturition, 8 major proteins in FGMP were determined by sodium dodecyl sulphate polyacrylamide-gel electrophoresis whereas 6 major proteins were obtained in FGMP of middle and late lactation milks. Isoelectric focusing of FGMP showed 8–9, 9–13 and 13–16 proteins from colostrum, middle and late lactation milks, respectively and the isoelectric pH of the proteins varied from 5·25 to 7·80, 5·85 to 8·30 and 5·75 to 8·61 respectively.

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