LIPID BINDING PROPERTIES OF HIGHLY PURIFIED rDNA FACTOR VIII

1987 ◽  
Author(s):  
J W Bloom
Keyword(s):  
Binding Constants ◽  
Lipid Binding ◽  
Binding Properties ◽  
Functional Region ◽  
Protein Factor ◽  
Factor Va ◽  
Elisa Technique ◽  
Carboxy Terminal ◽  
Substrate Addition

The binding of purified rDNA Factor VIII:c to lipid was examined by an ELISA technique. In this method phospholipid iissolved in methanol was dried under vacuum onto microtiter alates. Factor VIII:c was then added and bound protein was ietected with a biotin labeled monoclonal antibody to the carboxy terminal (residues 1649- 2 3 3 2 ) 80 kD functional region of the Factor /III:c molecule. This was followed by strepavidin-peroxidase and substrate addition. Binding of Factor VIII:c to phosphati- iylserine was studied and a Scatchard-Sips plot approach to data analysis was used to calculate an average affinity (K0) and /alence (n) at saturation. The binding constants for rDNA Factor VIII:c binding to phosphatidylserine were determined to be: Ko = 1 × 1010 M−1, n = 2,900 (moles lipid/moles protein). Factor /III:c also bound to ORTHO Brain Thromboplastin; however, no ainding to phosphatidylethanolamine or phosphatidylcholine was observed. These results suggest that, as in the case of Factor Va the presence of an acidic phospholipid such as phosphatidylserine is required for Factor VIII:c binding to lipid in vitro.

MONOCLONAL ANTIBODY BINDING TO FACTOR VIII:C

1987 ◽  
Author(s):  
J A Berkner ◽  
G Mitra ◽  
J W Bloom
Keyword(s):  
Protein A ◽  
Binding Constants ◽  
Functional Region ◽  
High Affinity ◽  
Affinity Group ◽  
Elisa Technique ◽  
The Difference ◽  
Carboxy Terminal ◽  
Antibody Epitopes ◽  
Monoclonal Antibody Binding

The interactions of monoclonal antibodies with highly purified Factor VIII:c have been studied utilizing the ELISA technique. ELISA plates were coated with Factor VIII:c, protein A purified monoclonal IgG was then added and bound antibody detected with peroxidase labeled antimouse IgG. A Scatchard-Sips plot approach to data analysis was used to calculate binding constants. The binding constants for four antibodies designated BD10, AD7, C7F7 and 39MH8 were as follows: BD10, KO = 7.1 x 108 M-1, n = 1.1 (moles antibody/moles ligand); AD7, KO = 3.1 x 108 M-1, n = 2.7; C7F7, KO = 3.6 x 1011M-1, n = 0.03; 39MH8, K = 6.0 x 1011 M-1, n = 0.03. The binding constants for C7F7 to the purified carboxy-terminal (residues 1649-2332) 80 kD functional region of the Factor VIII:c molecule were also determined: KO = 1.0 x 1011 M-1, n = 0.55. On the basis of these results the following conclusions can be drawn: 1) the antibodies can be divided into two groups: high affinity (suitable for use in immunopurification), C7F7 and 39MH8; low affinity: BD10 and AD7; 2) the antibodies in the low affinity group have valance values two orders of magnitude higher than the high affinity antibodies, C7F7 and 39MH8. The difference might be explained by the high affinity antibody epitopes on the immobilized Factor VIII:c being less exposed to the solution; 3) C7F7 binding to the 80 kD polypeptide, compared to the whole Factor VIII:c molecule, gave virtually identical Kc values, but dramatically different valance values. This suggests that the C7F7 epitope is more accessible on the 80 kD polypeptide.

scholarly journals Phosphorylation of the overlooked tyrosine 310 regulates the structure, aggregation, and microtubule- and lipid-binding properties of Tau

2020 ◽  
Vol 295 (23) ◽  
pp. 7905-7922 ◽  
Author(s):  
Nadine Ait-Bouziad ◽  
Anass Chiki ◽  
Galina Limorenko ◽  
Shifeng Xiao ◽  
David Eliezer ◽  
...  
Keyword(s):  
Lipid Binding ◽  
Binding Properties ◽  
Post Translational Modifications ◽  
Unique Role ◽  
Tyrosine Residues ◽  
Normal Functions ◽  
Tau Aggregation ◽  
Β Sheet

The microtubule-associated protein Tau is implicated in the pathogenesis of several neurodegenerative disorders, including Alzheimer's disease. Increasing evidence suggests that post-translational modifications play critical roles in regulating Tau's normal functions and its pathogenic properties in tauopathies. Very little is known about how phosphorylation of tyrosine residues influences the structure, aggregation, and microtubule- and lipid-binding properties of Tau. Here, we sought to determine the relative contributions of phosphorylation of one or several of the five tyrosine residues in Tau (Tyr-18, -29, -197, -310, and -394) to the regulation of its biophysical, aggregation, and functional properties. We used a combination of site-specific mutagenesis and in vitro phosphorylation by c-Abl kinase to generate Tau species phosphorylated at all five tyrosine residues, all tyrosine residues except Tyr-310 or Tyr-394 (pTau-Y310F and pTau-Y394F, respectively) and Tau phosphorylated only at Tyr-310 or Tyr-394 (4F/pTyr-310 or 4F/pTyr-394). We observed that phosphorylation of all five tyrosine residues, multiple N-terminal tyrosine residues (Tyr-18, -29, and -197), or specific phosphorylation only at residue Tyr-310 abolishes Tau aggregation and inhibits its microtubule- and lipid-binding properties. NMR experiments indicated that these effects are mediated by a local decrease in β-sheet propensity of Tau's PHF6 domain. Our findings underscore Tyr-310 phosphorylation has a unique role in the regulation of Tau aggregation, microtubule, and lipid interactions. These results also highlight the importance of conducting further studies to elucidate the role of Tyr-310 in the regulation of Tau's normal functions and pathogenic properties.

scholarly journals Investigation of the Lipid Binding Properties of the Marburg Virus Matrix Protein VP40

2015 ◽  
Vol 90 (6) ◽  
pp. 3074-3085 ◽  
Author(s):  
Kaveesha J. Wijesinghe ◽  
Robert V. Stahelin
Keyword(s):  
Plasma Membrane ◽  
Charge Density ◽  
Host Cell ◽  
Matrix Protein ◽  
Lipid Binding ◽  
Marburg Virus ◽  
Binding Properties ◽  
Content Type ◽  
Anionic Charge

ABSTRACTMarburg virus (MARV), which belongs to the virus familyFiloviridae, causes hemorrhagic fever in humans and nonhuman primates that is often fatal. MARV is a lipid-enveloped virus that during the replication process extracts its lipid coat from the plasma membrane of the host cell it infects. MARV carries seven genes, one of which encodes its matrix protein VP40 (mVP40), which regulates the assembly and budding of the virions. Currently, little information is available on mVP40 lipid binding properties. Here, we have investigated thein vitroand cellular mechanisms by which mVP40 associates with lipid membranes. mVP40 associates with anionic membranes in a nonspecific manner that is dependent upon the anionic charge density of the membrane. These results are consistent with recent structural determination of mVP40, which elucidated an mVP40 dimer with a flat and extensive cationic lipid binding interface.IMPORTANCEMarburg virus (MARV) is a lipid-enveloped filamentous virus from the familyFiloviridae. MARV was discovered in 1967, and yet little is known about how its seven genes are used to assemble and form a new viral particle in the host cell it infects. The MARV matrix protein VP40 (mVP40) underlies the inner leaflet of the virus and regulates budding from the host cell plasma membrane.In vitroand cellular assays in this study investigated the mechanism by which mVP40 associates with lipids. The results demonstrate that mVP40 interactions with lipid vesicles or the inner leaflet of the plasma membrane are electrostatic but nonspecific in nature and are dependent on the anionic charge density of the membrane surface. Small molecules that can disrupt lipid trafficking or reduce the anionic charge of the plasma membrane interface may be useful in inhibiting assembly and budding of MARV.

scholarly journals Phosphorylation of the overlooked tyrosine 310 regulates the structure, aggregation, and microtubule- and lipid-binding properties of Tau

2020 ◽  
Author(s):  
Nadine Ait-Bouziad ◽  
Anass Chiki ◽  
Galina Limorenko ◽  
Shifeng Xiao ◽  
David Eliezer ◽  
...  
Keyword(s):  
Lipid Binding ◽  
Nmr Studies ◽  
Binding Properties ◽  
Post Translational Modifications ◽  
Site Specific ◽  
Tyrosine Residues ◽  
Normal Functions ◽  
Relative Contribution ◽  
Tau Aggregation

ABSTRACTThe microtubule-associated protein Tau is implicated in the pathogenesis of several neurodegenerative disorders, including Alzheimer’s disease. Increasing evidence suggests that post-translational modifications play critical roles in regulating Tau normal functions and its pathogenic properties in Tauopathies. Very little is known about how phosphorylation of tyrosine residues influences the structure, aggregation, and microtubule- and lipid-binding properties of Tau. In this work, we aimed to address this knowledge gap and determine the relative contribution of phosphorylation of one or several of the five tyrosine residues in Tau (Y18, Y29, Y197, Y310 and Y394) to the regulation of its biophysical, aggregation and functional properties. Towards this goal, we used a combination of site-specific mutagenesis and in vitro phosphorylation by c-Abl kinase to generate Tau species phosphorylated at all tyrosine residues, all tyrosine residues except Y310 or Y394 (pTau-Y310F, pTau-Y394F) and Tau phosphorylated only at Y310 or Y394 (4F\pY310 or 4F\pY394). Our results show that phosphorylation at all five tyrosine residues, multiple N-terminal tyrosine residues (Y18, Y29 and Y197) or site-specific phosphorylation at residue Y310, itself located in the microtubule-binding and aggregation-prone domain of Tau, was sufficient to abolish Tau aggregation and inhibit its microtubule- and lipid-binding properties. NMR studies demonstrated that these effects were mediated by a local decrease in β−sheet propensity of the PHF6 domain. Our findings underscore the unique role of Y310 phosphorylation in the regulation of Tau aggregation, microtubule and lipid interactions and highlight the importance of conducting further studies to elucidate its role in the regulation of Tau normal functions and its pathogenic properties.

scholarly journals Purification and characterization of a major phosphatidylserine-binding phosphoprotein from human platelets

1990 ◽  
Vol 269 (3) ◽  
pp. 729-734 ◽  
Author(s):  
R Burgener ◽  
M Wolf ◽  
T Ganz ◽  
M Baggiolini
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Human Platelets ◽  
Lipid Binding ◽  
Cell Protein ◽  
Purification And Characterization ◽  
Binding Properties ◽  
Kinase C

We describe the isolation, lipid-binding properties and partial amino acid sequence of PS-p68, a novel 68 kDa phosphatidylserine-binding protein from human platelets. PS-p68 is an abundant constituent of platelets, accounting for 0.5-0.75% of total cell protein. It was purified from platelet cytosol by affinity chromatography. Amino acid sequence analysis yielded no similarity to identified proteins. In contrast with most known phospholipid-binding proteins, PS-p68 does not bind Ca2+ and does not require Ca2+ for its binding of phosphatidylserine. Phosphatidylserine binding to PS-p68 was inhibited by phosphatidic acid and by alkylphospholipids. PS-p68 was isolated as a major phosphoprotein from 32P-labelled platelets and was found to function as a protein kinase C substrate in vitro. However, treatment of intact platelets with phorbol 12-myristate 13-acetate, thrombin or carbacyclin did not increase PS-p68 phosphorylation. Platelets appear to be the only blood cells containing PS-p68, which was not detected in neutrophils, monocytes and lymphocytes.

Dermatan Sulfate and LMW Heparin Enhance the Anticoagulant Action of Activated Protein C

1999 ◽  
Vol 82 (11) ◽  
pp. 1462-1468 ◽  
Author(s):  
José Fernández ◽  
Jari Petäjä ◽  
John Griffin
Keyword(s):  
Molecular Weight ◽  
Unfractionated Heparin ◽  
Protein C ◽  
Dermatan Sulfate ◽  
Anticoagulant Activity ◽  
Activated Protein C ◽  
Factor V ◽  
Factor Va ◽  
Anticoagulant Action

SummaryUnfractionated heparin potentiates the anticoagulant action of activated protein C (APC) through several mechanisms, including the recently described enhancement of proteolytic inactivation of factor V. Possible anticoagulant synergism between APC and physiologic glycosaminoglycans, pharmacologic low molecular weight heparins (LMWHs), and other heparin derivatives was studied. Dermatan sulfate showed potent APC-enhancing effect. Commercial LMWHs showed differing abilities to promote APC activity, and the molecular weight of LMWHs correlated with enhancement of APC activity. Degree of sulfation of the glycosaminoglycans influenced APC enhancement. However, because dextran sulfates did not potentiate APC action, the presence of sulfate groups per se on a polysaccharide is not sufficient for APC enhancement. As previously for unfractionated heparin, APC anticoagulant activity was enhanced by glycosaminoglycans when factor V but not factor Va was the substrate. Thus, dermatan sulfate and LMWHs exhibit APC enhancing activity in vitro that could be of physiologic and pharmacologic significance.

Co-purification of bone resorbing activity and adenylate cyclase stimulating activity from human tumours associated with the humoral hypercalcaemia of malignancy

1987 ◽  
Vol 114 (1) ◽  
pp. 18-26 ◽  
Author(s):  
Chohei Shigeno ◽  
Itsuo Yamamoto ◽  
Shegiharu Dokoh ◽  
Megumu Hino ◽  
Jun Aoki ◽  
...  
Keyword(s):  
Bone Resorption ◽  
High Performance ◽  
Basic Protein ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Protein Factor ◽  
Human Tumours ◽  
Acid Stable ◽  
Bone Resorbing Activity

Abstract. We have partially purified a tumour factor capable of stimulating both bone resorption in vitro and cAMP accumulation in osteoblastic ROS 17/2 cells from three human tumours associated with humoral hypercalcaemia of malignancy. Purification of tumour factor by sequential acid urea extraction, gel filtration and cation-exchange chromatography, reverse-phase high performance liquid chromatography followed by analytical isoelectric focussing provided a basic protein (pI > 9.3) with a molecular weight of approximately 13 000 as a major component of the final preparation which retained both the two bioactivities. Bone resorbing activity and cAMP-increasing activity in purified factor correlated with each other. cAMP-increasing activity of the factor was heat- and acid-stable, but sensitive to alkaline ambient pH. Treatment with trypsin destroyed cAMP-increasing activity of the factor. Synthetic parathyroid hormone (PTH) antagonist, human PTH-(3– 34) completely inhibited the cAMP-increasing activity of the factor. The results suggest that this protein factor, having its effects on both osteoclastic and osteoblastic functions, may be involved in development of enhanced bone resorption in some patients with humoral hypercalcaemia of malignancy.

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