scholarly journals Using antibodies against Dictyostelium membranes to identify an actin-binding membrane protein.

1987 ◽  
Vol 104 (3) ◽  
pp. 513-518 ◽  
Author(s):  
S S Brown ◽  
A S Petzold
Keyword(s):  
Membrane Protein ◽  
Polyclonal Antibodies ◽  
Internal Surface ◽  
Protein S ◽  
Actin Binding ◽  
Blocking Antibody ◽  
Whole Cells ◽  
Peripheral Membrane Proteins ◽  
Blocking Activity ◽  
A Minor

Polyclonal antibodies made against Dictyostelium discoideum membranes were used to block the interaction of those membranes with actin. As expected, actin interacted mostly with the internal surface of the membrane, demonstrated by the fact that whole cells could only absorb out a minor fraction of the blocking antibody. The antibody was used to show that the membrane component(s) which interacted with actin were probably integral; they could be extracted with detergent but not with solutions designed to extract peripheral membrane proteins. To identify the responsible protein(s), Western transfers of membranes were cut into fractions which were tested for their ability to absorb out the blocking activity of the antibody. We observed a single peak at a molecular weight of approximately 20,000, and thus conclude that a 20,000-mol-wt protein is a major integral membrane actin-binding protein in Dictyostelium. This approach to the identification of proteins involved in actin-membrane interaction has allowed us to make the first identification of an actin-binding membrane protein which is based on its activity in native membranes.

LOW TOTAL PROTEIN S ANTIGEN BUT HIGH PROTEIN S ACTIVITY DUE TO DECREASED C4b-BINDING PROTEIN (C4b-BP) LEVELS IN NEWBORNS

1987 ◽  
Author(s):  
H P Schwarz ◽  
W Muntean
Keyword(s):  
Vitamin K ◽  
Limit Of Detection ◽  
Polyclonal Antibodies ◽  
Regulatory Protein ◽  
Activated Protein C ◽  
Protein S ◽  
Bleeding Risk ◽  
Free Form ◽  
Classical Complement Pathway ◽  
Coagulation Proteins

Vitamin K-dependent coagulation proteins are known to be decreased in the neonatal period. So far no data have been published on protein S (PS), the vitamin K-dependent cofactor for the antithrombotic enzyme, activated protein C (APC) in this period. We determined, therefore, PS antigen, PS activity and C4b-BP,a regulatory protein of the classical complement pathway to which PS is complexed, in 36 neonates. Total PS antigen in newborns was below the range associated with thromboembolism in patients congenitally deficient in this protein (22±9.6%, mean±SD). None of these infants had clinical or laboratory evidence of thromboembolism or DIC. In contrast to the PS antigen level PS activity measured by the ability of APC to prolong the clotting time of a modified APTT assay using PS-immunodep1eted plasma was significantly higher (77.6±14%, mean±SD, p< 0,001), suggesting a shift in PS to the free form. In fact two dimensional immunoe1ectrophoresis studies revealed the absence of protein S-C4b-BP complexes and only one precipitation indicating free PS was seen in 15 out of the 36 infants. In these 15 neonates C4b-BP was below the limit of detection by sensitive quantitative immunob1otting techniques using monoclonal or polyclonal antibodies. In the remaining 21 infants PS-C4b-BP complexes were detected, but in contrast to adult normal plasma approximately 80% of PS was found in the free form. Mixing experiments with normal human plasma and newborn’s plasma indicate that PS in neonate deficient of C4b-BP can bind normally to C4bp. Absence of C4b-BP did not correlate to gestational age. If an equilibrium distribution of PS between bound and free form regulates the cofactor activity of PS for the anticoagulant and profibrino 1ytic properties of APC in normal adults, our study demonstrates that the absence of PS-C4b-BP complexes in newborns and the presence of free PS only may contribute to the increased bleeding risk of premature infants.

scholarly journals Structure of the monotopic membrane protein (S)-mandelate dehydrogenase at 2.2 Å resolution

Biochimie ◽  
2018 ◽  
Vol 154 ◽  
pp. 45-54
Author(s):  
N. Sukumar ◽  
S. Liu ◽  
W. Li ◽  
F.S. Mathews ◽  
B. Mitra ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Protein S

scholarly journals Inhibition of acanthamoeba actomyosin-II ATPase activity and mechanochemical function by specific monoclonal antibodies.

1984 ◽  
Vol 99 (3) ◽  
pp. 1024-1033 ◽  
Author(s):  
D P Kiehart ◽  
T D Pollard
Keyword(s):  
Monoclonal Antibodies ◽  
Atpase Activity ◽  
Conformational Changes ◽  
Binding Sites ◽  
Polyclonal Antibodies ◽  
Myosin Ii ◽  
Antigenic Site ◽  
Actin Binding ◽  
Filamentous Form ◽  
Antibody Effects

Monoclonal and polyclonal antibodies that bind to myosin-II were tested for their ability to inhibit myosin ATPase activity, actomyosin ATPase activity, and contraction of cytoplasmic extracts. Numerous antibodies specifically inhibit the actin activated Mg++-ATPase activity of myosin-II in a dose-dependent fashion, but none blocked the ATPase activity of myosin alone. Control antibodies that do not bind to myosin-II and several specific antibodies that do bind have no effect on the actomyosin-II ATPase activity. In most cases, the saturation of a single antigenic site on the myosin-II heavy chain is sufficient for maximal inhibition of function. Numerous monoclonal antibodies also block the contraction of gelled extracts of Acanthamoeba cytoplasm. No polyclonal antibodies tested inhibited ATPase activity or gel contraction. As expected, most antibodies that block actin-activated ATPase activity also block gel contraction. Exceptions were three antibodies M2.2, -15, and -17, that appear to uncouple the ATPase activity from gel contraction: they block gel contraction without influencing ATPase activity. The mechanisms of inhibition of myosin function depends on the location of the antibody-binding sites. Those inhibitory antibodies that bind to the myosin-II heads presumably block actin binding or essential conformational changes in the myosin heads. A subset of the antibodies that bind to the proximal end of the myosin-II tail inhibit actomyosin-II ATPase activity and gel contraction. Although this part of the molecule is presumably some distance from the ATP and actin-binding sites, these antibody effects suggest that structural domains in this region are directly involved with or coupled to catalysis and energy transduction. A subset of the antibodies that bind to the tip of the myosin-II tail appear to inhibit ATPase activity and contraction through their inhibition of filament formation. They provide strong evidence for a substantial enhancement of the ATPase activity of myosin molecules in filamentous form and suggest that the myosin filaments may be required for cell motility.

scholarly journals Immunoglobulin G antibodies directed against protein III block killing of serum-resistant Neisseria gonorrhoeae by immune serum.

1986 ◽  
Vol 164 (5) ◽  
pp. 1735-1748 ◽  
Author(s):  
P A Rice ◽  
H E Vayo ◽  
M R Tam ◽  
M S Blake
Keyword(s):  
Membrane Protein ◽  
Neisseria Gonorrhoeae ◽  
Human Serum ◽  
Outer Membrane ◽  
Outer Membrane Protein ◽  
Immune Serum ◽  
Igg Antibodies ◽  
Porin Protein ◽  
Blocking Activity ◽  
Normal Human

Neisseria gonorrhoeae that resist complement-dependent killing by normal human serum (NHS) are sometimes killed by immune convalescent serum from patients recovering from disseminated gonococcal infection (DGI). In these studies, killing by immune serum was prevented or blocked by IgG isolated from NHS. Purified human IgG antibodies directed against gonococcal protein III, an antigenically conserved outer membrane protein, contained most of the blocking activity in IgG. Antibodies specific for gonococcal porin (protein I), the major outer membrane protein, displayed no blocking function. In separate experiments, immune convalescent DGI serum which did not exhibit bactericidal activity was restored to killing by selective depletion of protein III antibodies by immunoabsorption. These studies indicate that protein III antibodies in normal and immune human serum play a role in serum resistance of N. gonorrhoeae.

scholarly journals Porphyromonas gingivalis Fimbriae Bind to Cytokeratin of Epithelial Cells

2002 ◽  
Vol 70 (1) ◽  
pp. 96-101 ◽  
Author(s):  
Hakimuddin T. Sojar ◽  
Ashu Sharma ◽  
Robert J. Genco
Keyword(s):  
Epithelial Cell ◽  
Epithelial Cells ◽  
Porphyromonas Gingivalis ◽  
Polyclonal Antibodies ◽  
Cell Extract ◽  
Host Cells ◽  
Amino Terminal ◽  
A Minor ◽  
Sepharose 4B ◽  
Overlay Assay

ABSTRACT The adherence of Porphyromonas gingivalis to host cells is likely a prerequisite step in the pathogenesis of P. gingivalis-induced periodontal disease. P. gingivalis binds to and invades epithelial cells, and fimbriae are shown to be involved in this process. Little is known regarding epithelial receptor(s) involved in binding of P. gingivalis fimbriae. Using an overlay assay with purified P. gingivalis fimbriae as a probe, two major epithelial cell proteins with masses of 50 and 40 kDa were identified by immunoblotting with fimbria-specific antibodies. Iodinated purified fimbriae also bound to the same two epithelial cell proteins. An affinity chromatography technique was utilized to isolate and purify the epithelial components to which P. gingivalis fimbriae bind. Purified fimbriae were coupled to CNBr-activated Sepharose-4B, and the solubilized epithelial cell extract proteins bound to the immobilized fimbriae were isolated from the column. A major 50-kDa component and a minor 40-kDa component were purified and could be digested with trypsin, suggesting that they were proteins. These affinity-eluted 50- and 40-kDa proteins were then subjected to amino-terminal sequencing, and no sequence could be determined, suggesting that these proteins have blocked amino-terminal residues. CNBr digestion of the 50-kDa component resulted in an internal sequence homologous to that of Keratin I molecules. Further evidence that P. gingivalis fimbriae bind to cytokeratin molecule(s) comes from studies showing that multicytokeratin rabbit polyclonal antibodies cross-react with the affinity-purified 50-kDa epithelial cell surface component. Also, binding of purified P. gingivalis fimbriae to epithelial components can be inhibited in an overlay assay by multicytokeratin rabbit polyclonal antibodies. Furthermore, we showed that biotinylated purified fimbriae bind to purified human epidermal keratin in an overlay assay. These studies suggest that the surface-accessible epithelial cytokeratins may act as receptor(s) for P. gingivalis fimbriae. We hypothesize that adherence of P. gingivalis fimbriae to cytokeratin may be important for colonization of oral mucous membranes and possibly also for activation of epithelial cells.

Quantitation and topography of membrane proteins in highly water-permeable vesicles from ADH-stimulated toad bladder

1991 ◽  
Vol 261 (1) ◽  
pp. C143-C153 ◽  
Author(s):  
H. W. Harris ◽  
M. L. Zeidel ◽  
C. Hosselet
Keyword(s):  
Membrane Proteins ◽  
Membrane Protein ◽  
Lipid Bilayer ◽  
Apical Membrane ◽  
Water Channel ◽  
Protein S ◽  
Water Channels ◽  
Toad Bladder ◽  
Western Blots ◽  
Vesicle Protein

Antidiuretic hormone (ADH) stimulation of toad bladder granular cells rapidly increases the osmotic water permeability (Pf) of their apical membranes by insertion of highly selective water channels. Before ADH stimulation, these water channels are stored in large cytoplasmic vesicles called aggrephores. ADH causes aggrephores to fuse with the apical membrane. Termination of ADH stimulation results in prompt endocytosis of water channel-containing membranes via retrieval of these specialized regions of apical membrane. Protein components of the ADH water channel contained within these retrieved vesicles would be expected to be integral membrane protein(s) that span the vesicle's lipid bilayer to create narrow aqueous channels. Our previous work has identified proteins of 55 (actually a 55/53-kDa doublet), 17, 15, and 7 kDa as candidate ADH water channel components. We now have investigated these candidate ADH water channel proteins in purified retrieved vesicles. These vesicles do not contain a functional proton pump as assayed by Western blots of purified vesicle protein probed with anti-H(+)-ATPase antisera. Approximately 60% of vesicle protein is accounted for by three protein bands of 55, 53, and 46 kDa. Smaller contributions to vesicle protein are made by the 17- and 15-kDa proteins. Triton X-114-partitioning analysis shows that the 55, 53, 46, and 17 kDa are integral membrane proteins. Vectorial labeling analysis with two membrane-impermeant reagents shows that the 55-, 53-, and 46-kDa protein species span the lipid bilayer of these vesicles. Thus the 55-, 53-, and 46-kDa proteins possess characteristics expected for ADH water channel components. These data show that the 55- and 53- and perhaps the 46-, 17-, and 15-kDa proteins are likely components of aqueous transmembrane pores that constitute ADH water channels contained within these vesicles.

PLATELETS EXPRESS A MEMBRANE PROTEIN COMPLEX IMMUNOLOGICALLY RELATED TO THE FIBROBLAST FIBRONECTIN RECEPTOR

1987 ◽  
Author(s):  
F G Giancotti ◽  
L R Languino ◽  
A Zanetti ◽  
G Grignani ◽  
G Tarone ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Western Blot ◽  
Protein Complex ◽  
Polyclonal Antibodies ◽  
Flow Cytometric Analysis ◽  
Integral Membrane Protein ◽  
Human Platelets ◽  
Mouse Fibroblast ◽  
Platelet Surface ◽  
Fibronectin Receptor

The heterodimer complex GpIIb-IIIa on human platelets can specifically bind fibronectin (FN) only when platelets are activated by thrombin. However unstimulated platelets can adhere and spread on a FN substratum. This suggests the existence of a second binding site for FN on the platelet surface that does not require activation for its expression. We have previously identified and characterized a membrane glycoprotein complex (Gp 150/135) that functions as fibronectin receptor (FN-R) in mouse fibroblast adhesion. To investigate whether this molecule was also present in platelets we have produced an affinity purified polyclonal antibodies monospecific for the lower subunit of the fibroblast FN-R. These antibodies specifically stained human and rat platelet surface as determined by fluorescence flow cytometric analysis and reacted with a component of 138 Kd m w in Western blot of platelet membranes. Experiments of differential extraction revealed that the 138 Kd component is an integral membrane protein. Moreover the antibodies precipitated the 138 Kd component together with a 160 Kd protein suggesting that the two molecules are associated in a supramolecular complex. A comparative analysis indicated that this protein complex is clearly distinct from the GpIIb-IIIa. In addition platelets from a thrombastenic patient reacted normally with 138 Kd but not with GpIIb-IIIa antibodies by Western blot analysis. These data indicate that normal human platelets express both GpIIb-IIIa and FN-R on their membrane and that these receptors are composed of structurally and antigenically distinct proteins.

scholarly journals Identification of a platelet dense granule membrane protein that is deficient in a patient with the Hermansky-Pudlak syndrome

Blood ◽  
1991 ◽  
Vol 77 (1) ◽  
pp. 101-112 ◽  
Author(s):  
JM Gerrard ◽  
D Lint ◽  
PJ Sims ◽  
T Wiedmer ◽  
RD Fugate ◽  
...  
Keyword(s):  
Membrane Protein ◽  
Western Blot ◽  
Protein S ◽  
Dense Granule ◽  
Western Blots ◽  
Dense Granules ◽  
Granule Membrane ◽  
Thrombin Activation ◽  
Hermansky Pudlak Syndrome ◽  
Slot Blot Analysis

Abstract Monoclonal antibodies were raised after injecting mice with isolated human dense granules. Several of these monoclonals were found to recognize a 40-Kd dense granule membrane protein. Western blot and immunofluorescent analysis confirmed the dense-granule specificity. After thrombin activation, the protein was found in patches on the external platelet membrane. By Western blot and slot blot analysis, the protein was found to be markedly deficient in a patient with the Hermansky-Pudlak syndrome. Studies of neutrophils and endothelial cells show the presence of immunologically related granule-membrane protein(s). Western blots using four anti-synaptophysin antibodies and three antibodies to the platelet 40-Kd protein suggest that the protein may share some homology with, but is not identical to, the synaptosomal membrane protein synaptophysin.

scholarly journals Interaction between Mitochondria and the Actin Cytoskeleton in Budding Yeast Requires Two Integral Mitochondrial Outer Membrane Proteins, Mmm1p and Mdm10p

1998 ◽  
Vol 141 (6) ◽  
pp. 1371-1381 ◽  
Author(s):  
Istvan Boldogh ◽  
Nikola Vojtov ◽  
Sharon Karmon ◽  
Liza A. Pon
Keyword(s):  
Membrane Proteins ◽  
Actin Cytoskeleton ◽  
Outer Membrane ◽  
Outer Membrane Proteins ◽  
Protein S ◽  
Binding Activity ◽  
Actin Binding ◽  
Mitochondrial Outer Membrane ◽  
Mitochondrial Movement ◽  
Mitochondrial Motility

Transfer of mitochondria to daughter cells during yeast cell division is essential for viable progeny. The actin cytoskeleton is required for this process, potentially as a track to direct mitochondrial movement into the bud. Sedimentation assays reveal two different components required for mitochondria–actin interactions: (1) mitochondrial actin binding protein(s) (mABP), a peripheral mitochondrial outer membrane protein(s) with ATP-sensitive actin binding activity, and (2) a salt-inextractable, presumably integral, membrane protein(s) required for docking of mABP on the organelle. mABP activity is abolished by treatment of mitochondria with high salt. Addition of either the salt-extracted mitochondrial peripheral membrane proteins (SE), or a protein fraction with ATP-sensitive actin-binding activity isolated from SE, to salt-washed mitochondria restores this activity. mABP docking activity is saturable, resistant to high salt, and inhibited by pre-treatment of salt-washed mitochondria with papain. Two integral mitochondrial outer membrane proteins, Mmm1p (Burgess, S.M., M. Delannoy, and R.E. Jensen. 1994. J.Cell Biol. 126:1375–1391) and Mdm10p, (Sogo, L.F., and M.P. Yaffe. 1994. J.Cell Biol. 126:1361– 1373) are required for these actin–mitochondria interactions. Mitochondria isolated from an mmm1-1 temperature-sensitive mutant or from an mdm10 deletion mutant show no mABP activity and no mABP docking activity. Consistent with this, mitochondrial motility in vivo in mmm1-1 and mdm10Δ mutants appears to be actin independent. Depolymerization of F-actin using latrunculin-A results in loss of long-distance, linear movement and a fivefold decrease in the velocity of mitochondrial movement. Mitochondrial motility in mmm1-1 and mdm10Δ mutants is indistinguishable from that in latrunculin-A–treated wild-type cells. We propose that Mmm1p and Mdm10p are required for docking of mABP on the surface of yeast mitochondria and coupling the organelle to the actin cytoskeleton.

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