Evidence for a membrane-bound form of a bacteriocin of Bacteroides uniformis Tl-1

1984 ◽  
Vol 30 (2) ◽  
pp. 268-272 ◽  
Author(s):  
Sandra L. Austin-Prather ◽  
S. James Booth
Keyword(s):  
Molecular Weight ◽  
Outer Surface ◽  
Guanidine Hydrochloride ◽  
Membrane Vesicles ◽  
Apparent Molecular Weight ◽  
Whole Cells ◽  
Membrane Bound ◽  
Bleb Formation

The bacteriocin of Bacteroides uniformis Tl-1 had previously been reported to have a molecular weight of ≥ 300 000. Reexamination of the B. uniformis bacteriocin revealed that the bacteriocin was found in association with membrane vesicles which had been released by bleb formation from the outer surface of the B. uniformis cells. The bacteriocin could be released from whole cells or purified membrane vesicles by treatment with 6 M guanidine hydrochloride or 7 M urea and had an apparent molecular weight of 5000–6200.

Localization and Functional Characterization of Three Thylakoid Membrane Polypeptides of the Molecular Weight 66000

1977 ◽  
Vol 32 (9-10) ◽  
pp. 817-827 ◽  
Author(s):  
Friederike Koenig ◽  
Wilhelm Menke ◽  
Alfons Radunz ◽  
Georg H. Schmid
Keyword(s):  
Molecular Weight ◽  
Electron Transport ◽  
Outer Surface ◽  
Thylakoid Membrane ◽  
Functional Characterization ◽  
Apparent Molecular Weight ◽  
Photochemical Reactions ◽  
Antigenic Determinants ◽  
Reaction Centre

Abstract Three polypeptide fractions with the apparent molecular weight 66 000 were isolated from stroma-freed Antirrhinum chloroplasts which were solubilized with dodecyl sulfate. Antisera to these fractions affect electron transport in distinctly different ways. For the characterization of the three antisera photochemical reactions of chloroplast preparations with artificial electron donors and acceptors as well the analysis of fluorescence rise curves were used. Antiserum 66 000 PSI-96 inhibits electron transport apparently on the acceptor side of photosystem I, provided the antibodies are adsorbed onto the outer surface of the thylakoid membrane. Antiserum 66 000 PSI-88 probably acts directly on the reaction centre I or on its immediate vicinity, if the antibodies are adsorbed at the inner surface of the thylakoid membrane. Antiserum 66 000 PSII-42 inhibits electron trans­ port in the region of photosystem II. The antigen towards which the antiserum is directed appears to belong to the reaction centre II, as also in the condition of high inhibition degrees, the fluorescence intensity remains unchanged. The antigenic determinants are located at the outer surface of the thylakoid membrane.

Physikalische, chemische und biologische Charakterisierung von menschlichem Choriongonadotropin

1968 ◽  
Vol 23 (11) ◽  
pp. 1412-1426 ◽  
Author(s):  
H. D. Schlumberger
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Biological Activities ◽  
Guanidine Hydrochloride ◽  
Group Analysis ◽  
Residual Activity ◽  
Apparent Molecular Weight ◽  
Terminal Amino Acid ◽  
Original Activity ◽  
Terminal Amino

Purification of commercially available HCG preparations with DEAE Sephadex A 50 and Sephadex G 100 column chromatography gave homogeneous fractions having specific biological activities four fold those of the starting materials. The purified HCG has ICSH characteristics and, in high doses, a definite FSH effect is present.Chemical analysis of HCG showed it to contain 29% carbohydrates and 69% peptides. The C-terminal amino acid of the peptide chain was found to be serine, but the N-terminal amino acid could not be determined with normal methods. A molecular weight of 22000 — 27000 daltons was obtained by quantitative end group analysis. Ultracentrifugation experiments in 4 m guanidine hydrochloride gave a molecular weight of 27200 daltons, but in neutral saline solutions at HCG concentrations above 2 mg/ml the apparent molecular weight was higher and indicated dimer formation. A dissociation constant of 10-5 mol/l was estimated for the monomer-dimer equilibrium. Since biological activity is found with 0.1 to 0.5 µg, it was concluded that the HCG monomer is the active entity.The purified HCG is stable from pH 4.5 to pH 10 for 6 hours at 37 °C. At pH 2.5 only 5 to 10% of the original activity is retained. HCG is rapidly inactivated at 100 °C, but a residual activity of 6 — 10% remained after 30 minutes at 80 °C. No activity was lost after 30 minutes incubation at 60 °C.

Binding of Antibodies onto the Thylakoid Membrane III. Proteins in the Outer Surface of the Thylakoid Membrane

1978 ◽  
Vol 33 (9-10) ◽  
pp. 731-734 ◽  
Author(s):  
Alfons Radunz
Keyword(s):  
Molecular Weight ◽  
Electron Transport ◽  
Outer Surface ◽  
Thylakoid Membrane ◽  
Apparent Molecular Weight ◽  
Photosynthetic Electron Transport ◽  
Coupling Factor ◽  
Cytochrome F ◽  
Threefold Increase ◽  
Close Relationship

Abstract The maximal binding of antibodies to ferredoxin-NADP+ -reductase, cytochrome f, plastocyanin, coupling factor of photophosphorylation, carboxydismutase and to a polypeptide with the apparent molecular weight 24 000 onto stroma-freed chloroplasts of Antirrhinum majus was determined. The three proteins involved in photosynthetic electron transport bind approximately 0.05 to 0.07 g antibodies per g chloroplasts. The chloroplast preparation itself binds maximally about 1 gantibodies. From an antiserum to carboxydismutase and to a membrane polypeptide with the apparent molecular weight 24 000 approximately double the amount of antibodies namely 0.1 to 0.14 g antibodies per g chloroplasts are bound. Extraction of stroma-freed chloroplasts with 0.02 ᴍ Tris buffer pH 7.8 containing 0.7 mᴍ EDTA caused a threefold increase of the amount of bound anti­ bodies in the case of the membrane protein. 40% of the amount of antibodies which can be maxi­mally bound by this chloroplast preparation is adsorbed out of an antiserum to the coupling factor.Out of an antiserum which contains equal concentrations of antibodies to ferredoxin-NADP+ -reductase, cytochrome f and plastocyanin the same amount of antibodies is bound as out of an antiserum directed to only one of these components. This shows that the proteins involved in electron transport are located in a very close relationship to each other in the outer surface of the thylakoid membrane.

scholarly journals Incorporation of l-[3H]fucose and d-[3H]glucosamine into cell-surface-associated glycoconjugates in epidermis of cultured pig skin slices

1980 ◽  
Vol 190 (1) ◽  
pp. 65-77 ◽  
Author(s):  
I A King ◽  
A Tabiowo ◽  
R H Williams
Keyword(s):  
Molecular Weight ◽  
Cell Surface ◽  
Hydrophobic Interactions ◽  
Sodium Dodecyl ◽  
Apparent Molecular Weight ◽  
Membrane Glycoproteins ◽  
Molecular Weight Range ◽  
Electron Microscope Autoradiography ◽  
Pig Skin ◽  
Membrane Bound

1. Electron microscope autoradiography indicated that L-[3H]fucose and D-[3H]glucosamine were both incorporated into cell-surface-associated glycoconjugates in the epidermis of cultured pig skin slices. 2. Acid hydrolysis and paper chromatography of skin homogenates confirmed that there was little metabolic conversion of the labeled precursors to other sugars. 3. Epidermis was separated from dermis using CaCl2, and was extracted with 8 M-urea/5% (w/v) sodium dodecyl sulphate and was then analysed by gel electrophoresis. The major component labelled with D-[3H]glucosamine had an apparent molecular weight in excess of 200 000. This material was not labelled with L-[3H]fucose. Lower molecular-weight components were labelled to a similar extent with both L-[3H]fucose and D-[3H]glucosamine. 4. The high molecular-weight material labelled with D-[3H]glucosamine was released into the medium when the epidermal cells were dispersed with trypsin, indicating that it was either surface-associated or was extracellular. It was also labelled with D-[14C]glucuronic acid, 35SO4(2-) and to a small extent with 14C-labelled amino acids indicating that it contained glycosaminoglycans derived from epidermal proteoglycans. This was confirmed by the fact that it was degraded by testicular hyaluronoglucosidase. It was not present in isolated membranes but was recovered in the soluble fraction from epidermal homogenates. It is therefore only very loosely bound at the cell surface or is present in the extracellular spaces. 5. Membrane-bound [3H]glycoproteins were identified after differential centrifugation of epidermal homogenates. The radioactivity profiles of membrane glycoproteins were similar whether L-[3H]fucose or D-[3H]glucosamine were used and both consisted of a major heterogeneous peak in the apparent mol.wt. range 70 000–150 000. [3H]Glycoproteins in this molecular-weight range were also major components of a plasma-membrane-enriched fraction. These glycoproteins were probably bound to the membrane by hydrophobic interactions, since they were only solubilized by treatment with detergent or organic solvent. They contained terminal sialic acid residues, since they were degraded by neuraminidase.

scholarly journals Self-association of dermatan sulphate proteoglycans from bovine sclera

1981 ◽  
Vol 197 (2) ◽  
pp. 483-490 ◽  
Author(s):  
L Cöster ◽  
L A Fransson ◽  
J Sheehan ◽  
I A Nieduszynski ◽  
C F Phelps
Keyword(s):  
Molecular Weight ◽  
Light Scattering ◽  
Guanidine Hydrochloride ◽  
Apparent Molecular Weight ◽  
Sedimentation Equilibrium ◽  
Gel Chromatography ◽  
Side Chains ◽  
Dermatan Sulphate ◽  
Molecular Weights ◽  
High Particle

1. Two proteodermatan sulphate fractions (I and II) from bovine sclera were studied by gel chromatography, light-scattering and ultracentrifugation under various conditions. 2. Gel chromatography of proteoglycans in the absence or presence of hyaluronate was performed under associative conditions. No effect on the elution profile was noted. 3. Ultracentrifugation experiments (sedimentation-velocity and sedimentation-equilibrium) with proteoglycan I and II in 6 M-guanidine hydrochloride gave molecular weights (Mw) of 160000-220000 and 70000-100000 respectively. As the protein contents were 45% and 60% respectively, it may be calculated that proteoglycan I contained four to five side chains, whereas proteoglycan II contained one or two. Sedimentation-equilibrium runs performed in 0.15 M-NaCl gave an apparent molecular weight (Mw) of 500000-800000 for proteoglycan I and 90000-110000 for proteoglycan II. 4. In light-scattering experiments both proteoglycans I and II yielded high particle weights in 0.15 M-NaCl (3.1 × 10(6) and 3.4 × 10(6) daltons respectively). In the presence of 6 M-guanidine hydrochloride the molecular weights decreased to 410000 and 130000 respectively. The particle weights in 0.15 M-NaCl were not altered by the addition of hyaluronate or hyaluronate oligosaccharides. 5. The dermatan sulphate side chains of scleral proteoglycans (L-iduronate/D-glucuronate ratio 7:13) gave a particle weight of 100000 daltons in 0.15 M-NaCl. In 1.00 M-KCl/0.02M-EDTA the molecular weight was 24000. Addition of free scleral dermatan sulphate chains to a solution of proteoglycan II promoted further multimerization of the macromolecule.

Phytoene Desaturase, the Essential Target for Bleaching Herbicides

Weed Science ◽  
1991 ◽  
Vol 39 (3) ◽  
pp. 474-479 ◽  
Author(s):  
Gerhard Sandmann ◽  
Arno Schmidt ◽  
Hartmut Linden ◽  
Peter Böger
Keyword(s):  
Molecular Weight ◽  
Fusion Gene ◽  
Higher Plants ◽  
Hydrogen Abstraction ◽  
Apparent Molecular Weight ◽  
Phytoene Desaturase ◽  
Cross Resistance ◽  
Gene Probe ◽  
E Coli ◽  
Membrane Bound

Many bleaching herbicides with different core structures inhibit phytoene desaturase (PD), a membrane-bound enzyme in the carotenogenic pathway catalyzing the hydrogen abstraction step at the first C40precursor of β-carotene. Prospects are good that new PD-active herbicides will be discovered by screening for bleaching activity. Accordingly, interest in PD enzymology and molecular genetics has increased. Although active carotenogenic cell-free systems are available, no isolation of PD has been achieved since the enzyme cannot be detected in its isolated form due to complete loss of activity. A portion of theRhodobacterPD gene was incorporated into an appropriate plasmid which could be expressed inE. coli.This system was used to produce an antibody specific against PD from higher plants as well asRhodobacter.All PDs assayed had an apparent molecular weight of 52 to 55 kDa. ARhodobactergene probe hybridized with a 3.1 kbBamH I fragment fromAphanocapsawhich allowed us to sequence the PD gene from this cyanobacterium. Its DNA sequence matched with the apparent molecular weight of the PD band in the western blot, and a fusion-gene product was found to be immunoreactive with theRhodobacterPD antibody,Anacystismutants were produced exhibiting cross-resistance against norflurazon and fluorochloridone. Apparently, this resistance is due to an altered PD with concurrent decrease of inhibitor binding affinity. Cloning of the resistant gene into the wild type is in progress.

scholarly journals Generation of a Membrane Potential by Lactococcus lactis through Aerobic Electron Transport

2007 ◽  
Vol 189 (14) ◽  
pp. 5203-5209 ◽  
Author(s):  
R. J. W. Brooijmans ◽  
B. Poolman ◽  
G. K. Schuurman-Wolters ◽  
W. M. de Vos ◽  
J. Hugenholtz
Keyword(s):  
Membrane Potential ◽  
Lactococcus Lactis ◽  
Growth Yield ◽  
Membrane Vesicles ◽  
Proton Motive Force ◽  
Motive Force ◽  
Atpase Inhibitor ◽  
Whole Cells ◽  
Consumption Rates ◽  
Membrane Bound

ABSTRACT Lactococcus lactis, a facultative anaerobic lactic acid bacterium, is known to have an increased growth yield when grown aerobically in the presence of heme. We have now established the presence of a functional, proton motive force-generating electron transfer chain (ETC) in L. lactis under these conditions. Proton motive force generation in whole cells was measured using a fluorescent probe (3′,3′-dipropylthiadicarbocyanine), which is sensitive to changes in membrane potential (Δψ). Wild-type cells, grown aerobically in the presence of heme, generated a Δψ even in the presence of the F1-Fo ATPase inhibitor N,N′-dicyclohexylcarbodiimide, while a cytochrome bd-negative mutant strain (CydAΔ) did not. We also observed high oxygen consumption rates by membrane vesicles prepared from heme-grown cells, compared to CydAΔ cells, upon the addition of NADH. This demonstrates that NADH is an electron donor for the L. lactis ETC and demonstrates the presence of a membrane-bound NADH-dehydrogenase. Furthermore, we show that the functional respiratory chain is present throughout the exponential and late phases of growth.

Physico-Chemical Properties of Recombinant Desulphatohirudin

1990 ◽  
Vol 63 (03) ◽  
pp. 499-504 ◽  
Author(s):  
A Electricwala ◽  
L Irons ◽  
R Wait ◽  
R J G Carr ◽  
R J Ling ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Gel Filtration ◽  
Chemical Properties ◽  
Alpha Helix ◽  
Apparent Molecular Weight ◽  
Correlation Spectroscopy ◽  
Nmr Studies ◽  
Recombinant Hirudin ◽  
Physico Chemical ◽  
Recombinant Molecule

SummaryPhysico-chemical properties of recombinant desulphatohirudin expressed in yeast (CIBA GEIGY code No. CGP 39393) were reinvestigated. As previously reported for natural hirudin, the recombinant molecule exhibited abnormal behaviour by gel filtration with an apparent molecular weight greater than that based on the primary structure. However, molecular weight estimation by SDS gel electrophoresis, FAB-mass spectrometry and Photon Correlation Spectroscopy were in agreement with the theoretical molecular weight, with little suggestion of dimer or aggregate formation. Circular dichroism studies of the recombinant molecule show similar spectra at different pH values but are markedly different from that reported by Konno et al. (13) for a natural hirudin-variant. Our CD studies indicate the presence of about 60% beta sheet and the absence of alpha helix in the secondary structure of recombinant hirudin, in agreement with the conformation determined by NMR studies (17)

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.

Exterior and Interior Events in Early Stage of Thrombin-induced Platelet Aggregation

1977 ◽  
Vol 38 (03) ◽  
pp. 0630-0639 ◽  
Author(s):  
Shuichi Hashimoto ◽  
Sachiko Shibata ◽  
Bonro Kobayashi
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Plasma Membrane ◽  
Platelet Aggregation ◽  
Early Stage ◽  
Adenyl Cyclase ◽  
Cellular Component ◽  
Primary Event ◽  
Rabbit Platelets ◽  
Membrane Bound

SummaryTreatment of washed rabbit platelets with 1 u/ml of thrombin at 37° C resulted in a disappearance from platelets of a protein with 250,000 dalton molecular weight which was shown to be originated from plasma membrane. Parallel loss of adenyl cyclase was noted, and both reactions were complete within 30 sec. From the patterns of disc electrophoretograms, the importance of quick suppression of thrombin action in demonstrating the primary event was stressed.Thrombin induced an apparent activation of membrane bound phosphodiesterase. This reaction was also complete within 30 sec. The cellular component which contained the enzyme activity was distinct from plasma membrane. Soluble phosphodiesterase was not influenced by thrombin at all.These reactions required intact platelet cells to react with thrombin, and no reaction was detected when subcellular preparations were treated with thrombin.Possibility of collaboration of changes in externally located synthetic enzyme with those in internally located degrading enzyme in the early phase of thrombin action on platelets was suggested.

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