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FEBS Letters Oct 1983A large amount of specific high affinity binding sites for tumor promoting phorbol esters as well as of a Ca2+- and phospholipid-dependent protein kinase is present in...
A large amount of specific high affinity binding sites for tumor promoting phorbol esters as well as of a Ca2+- and phospholipid-dependent protein kinase is present in cytosol of chick oviduct. 12-O-Tetradecanoyl-phorbol-13-acetate (TPA) is able to replace either Ca2+ or the phospholipid phosphatidylserine as activators of the kinase to some extent. The maximum activity of the enzyme in the presence of Ca2+ and phosphatidylserine, however, cannot be increased further by TPA. Various second stage tumor promoters also exhibit the capacity to stimulate the protein kinase, whereas the non-promoting phorbol ester 4-O-methyl-TPA, as well as the non-promoting, but with respect to other responses TPA-like, calcium ionophore A23187, do not affect the kinase.
Topics: Animals; Binding Sites; Calcium; Chickens; Cytosol; Enzyme Activation; Female; Oviducts; Phorbols; Phosphatidylserines; Phospholipids; Protein Kinases; Tetradecanoylphorbol Acetate
PubMed: 6617887
DOI: 10.1016/0014-5793(83)81067-9 -
Biopolymers Feb 2015Amelogenin protein has the potential to interact with other enamel matrix proteins, mineral, and cell surfaces. We investigated the interactions of recombinant...
Amelogenin protein has the potential to interact with other enamel matrix proteins, mineral, and cell surfaces. We investigated the interactions of recombinant amelogenin rP172 with small unilamellar vesicles as model membranes, toward the goal of understanding the mechanisms of amelogenin-cell interactions during amelogenesis. Dynamic light scattering (DLS), fluorescence spectroscopy, circular dichroism (CD), and nuclear magnetic resonance (NMR) were used. In the presence of phospholipid vesicles, a blue shift in the Trp fluorescence emission maxima of rP172 was observed (∼334 nm) and the Trp residues of rP172 were inaccessible to the aqueous quencher acrylamide. DLS studies indicated complexation of rP172 and phospholipids, although the possibility of fusion of phospholipids following amelogenin addition cannot be ruled out. NMR and CD studies revealed a disorder-order transition of rP172 in a model membrane environment. Strong fluorescence resonance energy transfer from Trp in rP172 to DNS-bound-phospholipid was observed, and fluorescence polarization studies indicated that rP172 interacted with the hydrophobic core region of model membranes. Our data suggest that amelogenin has ability to interact with phospholipids and that such interactions may play key roles in enamel biomineralization as well as reported amelogenin signaling activities.
Topics: Amelogenin; Circular Dichroism; Hydrogen-Ion Concentration; Phospholipids; Protein Binding; Protein Conformation; Scattering, Radiation; Spectrometry, Fluorescence
PubMed: 25298002
DOI: 10.1002/bip.22573 -
The Journal of Biological Chemistry Feb 1991Phospholipids are the major components of pulmonary surfactant. Dipalmitoylphosphatidylcholine is believed to be especially essential for the surfactant function of...
Phospholipids are the major components of pulmonary surfactant. Dipalmitoylphosphatidylcholine is believed to be especially essential for the surfactant function of reducing the surface tension at the air-liquid interface. Surfactant protein A (SP-A) with a reduced denatured molecular mass of 26-38 kDa, characterized by a collagen-like structure and N-linked glycosylation, interacts strongly with a mixture of surfactant-like phospholipids. In the present study the direct binding of SP-A to phospholipids on a thin layer chromatogram was visualized using 125I-SP-A as a probe, so that the phospholipid specificities of SP-A binding and the structural requirements of SP-A and phospholipids for the binding could be examined. Although 125I-SP-A bound phosphatidylcholine and sphingomyeline, it was especially strong in binding dipalmitoylphosphatidylcholine, but failed to bind phosphatidylglycerol, phosphatidylinositol, phosphatidylethanolamine, and phosphatidylserine. Labeled SP-A also exhibited strong binding to distearoylphosphatidylcholine, but weak binding to dimyristoyl-, 1-palmitoyl-2-linoleoyl-, and dilinoleoylphosphatidylcholine. Unlabeled SP-A readily competed with labeled SP-A for phospholipid binding. SP-A strongly bound dipalmitoylglycerol produced by phospholipase C treatment of dipalmitoylphosphatidylcholine, but not palmitic acid. This protein also failed to bind lysophosphatidylcholine produced by phospholipase A2 treatment of dipalmitoylphosphatidylcholine. 125I-SP-A shows almost no binding to dipalmitoylphosphatidylglycerol and dipalmitoylphosphatidylethanolamine. The addition of 10 mM EGTA into the binding buffer reduced much of the 125I-SP-A binding to phospholipids. Excess deglycosylated SP-A competed with labeled SP-A for binding to dipalmitoylphosphatidylcholine, but the excess collagenase-resistant fragment of SP-A failed. From these data we conclude that 1) SP-A specifically and strongly binds dipalmitoylphosphatidylcholine, 2) SP-A binds the nonpolar group of phospholipids, 3) the second positioned palmitate is involved in dipalmitoylphosphatidylcholine binding, and 4) the specificities of polar groups of dipalmitoylglycerophospholipids also appear to be important for SP-A binding, 5) the phospholipid binding activity of SP-A is dependent upon calcium ions and the integrity of the collagenous domain of SP-A, but not on the oligosaccharide moiety of SP-A. SP-A may play an important role in the regulation of recycling and intra- and extracellular movement of dipalmitoylphosphatidylcholine.
Topics: 1,2-Dipalmitoylphosphatidylcholine; Animals; Binding, Competitive; Electrophoresis, Polyacrylamide Gel; Fatty Acids; Humans; Iodine Radioisotopes; Phospholipids; Proteolipids; Pulmonary Surfactant-Associated Protein A; Pulmonary Surfactant-Associated Proteins; Pulmonary Surfactants; Rats
PubMed: 1993679
DOI: No ID Found -
The Journal of Biological Chemistry May 2023Decarboxylation of phosphatidylserine (PS) to form phosphatidylethanolamine by PS decarboxylases (PSDs) is an essential process in most eukaryotes. Processing of a...
Decarboxylation of phosphatidylserine (PS) to form phosphatidylethanolamine by PS decarboxylases (PSDs) is an essential process in most eukaryotes. Processing of a malarial PSD proenzyme into its active alpha and beta subunits is by an autoendoproteolytic mechanism regulated by anionic phospholipids, with PS serving as an activator and phosphatidylglycerol (PG), phosphatidylinositol, and phosphatidic acid acting as inhibitors. The biophysical mechanism underlying this regulation remains unknown. We used solid phase lipid binding, liposome-binding assays, and surface plasmon resonance to examine the binding specificity of a processing-deficient Plasmodium PSD (PkPSDS308A) mutant enzyme and demonstrated that the PSD proenzyme binds strongly to PS and PG but not to phosphatidylethanolamine and phosphatidylcholine. The equilibrium dissociation constants (K) of PkPSD with PS and PG were 80.4 nM and 66.4 nM, respectively. The interaction of PSD with PS is inhibited by calcium, suggesting that the binding mechanism involves ionic interactions. In vitro processing of WT PkPSD proenzyme was also inhibited by calcium, consistent with the conclusion that PS binding to PkPSD through ionic interactions is required for the proenzyme processing. Peptide mapping identified polybasic amino acid motifs in the proenzyme responsible for binding to PS. Altogether, the data demonstrate that malarial PSD maturation is regulated through a strong physical association between PkPSD proenzyme and anionic lipids. Inhibition of the specific interaction between the proenzyme and the lipids can provide a novel mechanism to disrupt PSD enzyme activity, which has been suggested as a target for antimicrobials, and anticancer therapies.
Topics: Amino Acid Motifs; Calcium; Carboxy-Lyases; Enzyme Precursors; Liposomes; Phosphatidic Acids; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Phosphatidylinositols; Phosphatidylserines; Phospholipids; Protein Binding; Malaria; Proteolysis; Surface Plasmon Resonance; Plasmodium
PubMed: 36997087
DOI: 10.1016/j.jbc.2023.104659 -
The Journal of Biological Chemistry May 1990Photoaffinity labeling of calcineurin by 1,2-distearoyl-sn-glycero-3-phospho-N-(4-azido-3-[125I]iodo-2- hydroxybenzoyl)ethanolamine resulted in preferential labeling of...
Photoaffinity labeling of calcineurin by 1,2-distearoyl-sn-glycero-3-phospho-N-(4-azido-3-[125I]iodo-2- hydroxybenzoyl)ethanolamine resulted in preferential labeling of its regulatory B subunit. Photolabeling of B was greatly enhanced by Ca2+ which further supports the hypothesis that the phospholipid-binding site of calcineurin is located on this Ca2(+)-binding subunit. Extending the time of incubation of calcineurin with the photoprobe prior to photolysis also elevated labeling of the B subunit, probably as a result of time-dependent changes in protein conformation. Support for these conformational changes was obtained when time-dependent preincubation of calcineurin with acidic phospholipids enhanced subsequent tryptic degradation of its B subunit. Activity measurements and analyses of the reversibility of phospholipid-binding provided evidence for a two-stage mechanism of calcineurin-phospholipid interactions. Initial binding of calcineurin to phospholipids is rapid, Ca2(+)-sensitive, reversible, and leads to stimulation of the phosphatase toward a number of its substrates. A subsequent slow phase strengthens the association and appears to correlate with the phospholipid-promoted conformational change of the B subunit; the corresponding time-dependent effects on enzymatic activity are, again, substrate-dependent.
Topics: Affinity Labels; Animals; Azides; Brain; Calcineurin; Calcium; Calmodulin; Calmodulin-Binding Proteins; Cattle; Enzyme Activation; Kinetics; Macromolecular Substances; Nickel; Phosphatidylethanolamines; Phospholipids; Phosphoprotein Phosphatases; Protein Binding; Substrate Specificity
PubMed: 2159005
DOI: No ID Found -
Scientific Reports Oct 2017ZipA protein from Escherichia coli is one of the essential components of the division proto-ring that provides membrane tethering to the septation FtsZ protein. A...
ZipA protein from Escherichia coli is one of the essential components of the division proto-ring that provides membrane tethering to the septation FtsZ protein. A sedimentation assay was used to measure the equilibrium binding of FtsZ-GDP and FtsZ-GTP to ZipA immobilized at controlled densities on the surface of microbeads coated with a phospholipid mixture resembling the composition of E. coli membrane. We found that for both nucleotide-bound species, the amount of bound FtsZ exceeds the monolayer capacity of the ZipA immobilized beads at high concentrations of free FtsZ. In the case of FtsZ-GDP, equilibrium binding does not appear to be saturable, whereas in the case of FtsZ-GTP equilibrium binding appears to be saturable. The difference between the two modes of binding is attributed to the difference between the composition of oligomers of free FtsZ-GDP and free FtsZ-GTP formed in solution.
Topics: Adsorption; Bacterial Proteins; Carrier Proteins; Cell Cycle Proteins; Cytoskeletal Proteins; Escherichia coli; Escherichia coli Proteins; Guanosine Diphosphate; Guanosine Triphosphate; Liposomes; Microspheres; Phospholipids; Protein Binding
PubMed: 29057931
DOI: 10.1038/s41598-017-14160-y -
Thrombosis Research 2008Activated coagulation factor V (FVa) is an important cofactor that accelerates thrombin production. In human blood, 25% of the factor V (FV) is stored in platelets,...
Activated coagulation factor V (FVa) is an important cofactor that accelerates thrombin production. In human blood, 25% of the factor V (FV) is stored in platelets, complexed to the polymeric, FV binding protein multimerin 1 (MMRN1). The light chain of FV is required for MMRN1 binding, and its C2 domain contains a MMRN1 binding site that overlaps phospholipid binding residues essential for FVa procoagulant function. The homologous structures and roles of the FVa light chain C1 and C2 domains led us to investigate if the C1 domain also contains a MMRN1 binding site. The MMRN1 binding properties of FV constructs were tested by modified enzyme-linked immunoassays, before and after thrombin activation. The constructs tested included the combined C1 and C2 domain deleted FV, and B-domain deleted forms of FV containing C1 domain point mutations or combined C1 and C2 domain phospholipid binding site mutations. The MMRN1 binding site in FV/FVa was mapped to a large region that included the C1 domain phospholipid binding residues Y1956 and L1957. The FV construct with combined C1 and C2 domain phospholipid binding site mutations had no MMRN1 binding, highlighting the critical role of the FV C1 and C2 domain phospholipid binding residues in MMRN1 binding. Our data update the information on the structural features of FV and FVa important for MMRN1 binding, and suggest that the extended MMRN1 binding site in the C1 and C2 domains is important for the storage of FV-MMRN1 complexes in platelets.
Topics: Animals; Binding Sites; Blood Proteins; COS Cells; Chlorocebus aethiops; Culture Media, Serum-Free; Factor V; Humans; Models, Molecular; Phospholipids; Point Mutation; Protein Binding; Protein Structure, Tertiary; Recombinant Proteins; Transfection
PubMed: 18452976
DOI: 10.1016/j.thromres.2008.03.016 -
The Journal of Biological Chemistry Mar 2002Binding of cytochrome c (cyt c) to fatty acids and acidic phospholipid membranes produces pronounced and essentially identical changes in the spectral properties of cyt...
Binding of cytochrome c (cyt c) to fatty acids and acidic phospholipid membranes produces pronounced and essentially identical changes in the spectral properties of cyt c, revealing conformational changes in the protein. The exact mechanism of the interaction of fatty acids and acidic phospholipids with cyt c is unknown. Binding of cyt c to liposomes with high contents (mole fraction X > 0.7) of acidic phospholipids caused spectral changes identical to those due to binding of oleic acid. Fluorescence spectroscopy of a cyt c analog containing a Zn(2+) substituted heme moiety and brominated lipid derivatives (9,10)-dibromostearate and 1-palmitoyl-2-(9,10)-dibromo-sn-glycero-3-phospho-rac-glycerol demonstrated a direct contact between the fluorescent [Zn(2+)-heme] group and the brominated acyl chain. These data constitute direct evidence for interaction between an acyl chain of a membrane phospholipid and the inside of the protein containing the heme moiety and provide direct evidence for the so-called extended-lipid anchorage of cyt c to phospholipid membranes. In this mechanism, one of the phospholipid acyl chains protrudes out of the membrane and intercalates into a hydrophobic channel in cyt c while the other chain remains in the bilayer.
Topics: Apoptosis; Cytochrome c Group; Membrane Lipids; Phospholipids; Spectrometry, Fluorescence
PubMed: 11781329
DOI: 10.1074/jbc.M200056200 -
The Biochemical Journal Oct 1997We have studied the binding of two G-protein-regulated phospholipase C (PLC) enzymes, PLCs-beta1 and -beta2, to membrane surfaces using sucrose-loaded bilayer...
We have studied the binding of two G-protein-regulated phospholipase C (PLC) enzymes, PLCs-beta1 and -beta2, to membrane surfaces using sucrose-loaded bilayer phospholipid vesicles of varying compositions. Neither enzyme binds appreciably to pure phosphatidylcholine vesicles at lipid concentrations up to 10(-3) M. PLC-beta1 and PLC-beta2 bind vesicles composed of phosphatidylcholine, phosphatidylserine and phosphatidylethanolamine (molar ratio 1:1:1) with an approximate Kd of 10(-5) M. Inclusion of 2% PtdIns(4,5)P2 in these vesicles had no effect on the affinity of this interaction. As reported by others, removal of the C-terminus of PLC-beta1 and PLC-beta2 produces catalytically active fragments. The affinity of these truncated proteins for phospholipid vesicles is dramatically reduced suggesting that this region of the proteins contains residues important for membrane binding. Inclusion of G-protein alpha- and betagamma-subunit activators in the phospholipid vesicles does not increase the binding of PLC-beta1 or PLC-beta2, and the magnitude of G-protein-mediated PLC activation observed at low phospholipid concentrations (10(-6) M) is comparable to that observed at concentrations at which the enzymes are predominantly membrane-bound (10(-3) M). PLC-beta1 and -beta2 contain C2 domains but Ca2+ does not enhance binding to the vesicles. Our results indicate that binding of these enzymes to membranes involves the C-temini of the proteins and suggest that activation of these enzymes by G-proteins results from a regulated interaction between the membrane-bound proteins rather than G-protein-dependent recruitment of soluble enzymes to a substrate-containing phospholipid surface.
Topics: Animals; Calcium; Cattle; GTP-Binding Proteins; Humans; Isoenzymes; Kinetics; Liposomes; Macromolecular Substances; Phosphatidylinositol Phosphates; Phospholipase C beta; Phospholipids; Recombinant Proteins; Structure-Activity Relationship; Sucrose; Type C Phospholipases
PubMed: 9359412
DOI: 10.1042/bj3270431 -
Journal of Colloid and Interface Science Oct 2010The interaction between a hydrophobically modified 5,10,15,20-tetrakis(4-N-tetradecyl-pyridyl) porphyrin and three phospholipids: two negatively charged, DMPA (the...
The interaction between a hydrophobically modified 5,10,15,20-tetrakis(4-N-tetradecyl-pyridyl) porphyrin and three phospholipids: two negatively charged, DMPA (the sodium salt of dimyristoyl-sn-glycero-phosphatidyl acid) and DMPG (the sodium salt of 1,2-dimyristoyl-sn-glycero-3-[phospho-rac-(1-glycerol)]) and a zwitterionic DMPC (dimyristoyl-sn-glycero-phosphatidylcholine), were studied by means of surface pressure isotherms and spectroscopic methods. The interaction results in partial or total metallation of the porphyrin with zinc ions in the presence of negatively charged phospholipids, as attested by UV-vis and luminescence spectroscopy of the transferred films. In the presence of the zwitterionic phospholipid no insertion of zinc ion in the porphyrin ring is detected. These results are relevant for the understanding of photosensitizer-lipid-carrier binding for use in photodynamic therapy.
Topics: Ions; Microscopy, Fluorescence; Models, Chemical; Molecular Structure; Phospholipids; Porphyrins; Surface Properties; Zinc
PubMed: 20598704
DOI: 10.1016/j.jcis.2010.06.021