Changing homecare practice to support patients who are terminally ill.
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Consumer Behavior↗
Biomedical subjects
S McLaughlin
Publications and source records attributed to S McLaughlin.
Kinetics of interaction of the myristoylated alanine-rich C kinase substrate, membranes, and calmodulin.
Membrane binding of the myristoylated alanine-rich C kinase substrate (MARCKS) requires both its myristate chain and basic "effector" region. Previous studies with a peptide corresponding to the effector region, MARCKS-(151-175), showed that the 13 basic residues interact electrostatically with acidic lipids and that the 5 hydrophobic phenylalanine residues penetrate the polar head group region of the bilayer. Here we describe the kinetics of the membrane binding of fluorescent (acrylodan-labeled) peptides measured with a stopped-flow technique. Even though the peptide penetrates the polar head group region, the association of MARCKS-(151-175) with membranes is extremely rapid; association occurs with a diffusion-limited association rate constant. For example, kon = 10(11) M-1 s-1 for the peptide binding to 100-nm diameter phospholipid vesicles. As expected theoretically, kon is independent of factors that affect the molar partition coefficient, such as the mole fraction of acidic lipid in the vesicle and the salt concentration. The dissociation rate constant (koff) is approximately 10 s-1 (lifetime = 0.1 s) for vesicles with 10% acidic lipid in 100 mM KCl. Ca2+-calmodulin (Ca2+.CaM) decreases markedly the lifetime of the peptide on vesicles, e.g. from 0.1 to 0.01 s in the presence of 5 micrM Ca2+.CaM. Our results suggest that Ca2+.CaM collides with the membrane-bound MARCKS-(151-175) peptide and pulls the peptide off rapidly. We discuss the biological implications of this switch mechanism, speculating that an increase in the level of Ca2+-calmodulin could rapidly release phosphatidylinositol 4, 5-bisphosphate that previous work has suggested is sequestered in lateral domains formed by MARCKS and MARCKS-(151-175).
Amino Acid Sequence↗
Electrostatic interaction of myristoylated proteins with membranes: simple physics, complicated biology.
Cell membrane association by several important peripheral proteins, such as Src, MARCKS, HIV-1 Gag, and K-Ras, requires nonspecific electrostatic interactions between a cluster of basic residues on the protein and acidic phospholipids in the plasma membrane. A simple theoretical model based on the nonlinear Poisson-Boltzmann equation describes well the experimentally measured electrostatic association between such proteins and the cell membrane.
Amino Acid Sequence↗
Translation of the diabetes nutrition recommendations for health care institutions: technical review.
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Diabetes Mellitus↗
Electrostatic binding of proteins to membranes. Theoretical predictions and experimental results with charybdotoxin and phospholipid vesicles.
We previously applied the Poisson-Boltzmann equation to atomic models of phospholipid bilayers and basic peptides to calculate their electrostatic interactions from first principles (Ben-Tal, N., B. Honig, R. M. Peitzsch, G. Denisov, and S. McLaughlan. 1996. Binding of small basic peptides to membranes containing acidic lipids. Theoretical models and experimental results. Biophys. J. 71:561-575). Specifically, we calculated the molar partition coefficient, K (the reciprocal of the lipid concentration at which 1/2 the peptide is bound), of simple basic peptides (e.g., pentalysine) with phospholipid vesicles. The theoretical predictions agreed well with experimental measurements of the binding, but the agreement could have been fortuitous because the structure(s) of these flexible peptides is not known. Here we use the same theoretical approach to calculate the membrane binding of two small proteins of known structure: charybdotoxin (CTx) and iberiotoxin (IbTx); we also measure the binding of these proteins to phospholipid vesicles. The theoretical model describes accurately the dependence of K on the ionic strength and mol % acidic lipid in the membrane for both CTx (net charge +4) and IbTx (net charge +2). For example, the theory correctly predicts that the value of K for the binding of CTx to a membrane containing 33% acidic lipid should decrease by a factor of 10(5) when the salt concentration increases from 10 to 200 mM. We discuss the limitations of the theoretical approach and also consider a simple extension of the theory that incorporates nonpolar interactions.
Biophysical Phenomena↗
Translation of the diabetes nutrition recommendations for health care institutions.
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Diabetes Mellitus↗
Myristoylated alanine-rich C kinase substrate (MARCKS) produces reversible inhibition of phospholipase C by sequestering phosphatidylinositol 4,5-bisphosphate in lateral domains.
The myristoylated alanine-rich protein kinase C substrate (MARCKS) is a major protein kinase C (PKC) substrate in many different cell types. MARCKS is bound to the plasma membrane, and several recent studies suggest that this binding requires both hydrophobic insertion of its myristate chain into the bilayer and electrostatic interaction of its cluster of basic residues with acidic lipids. Phosphorylation of MARCKS by PKC introduces negative charges into the basic cluster, reducing its electrostatic interaction with acidic lipids and producing translocation of MARCKS from membrane to cytoplasm. The present study shows that physiological concentrations of MARCKS (<10 microM) inhibit phospholipase C (PLC)-catalyzed hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) in phospholipid vesicles. A peptide corresponding to the basic cluster, MARCKS(151-175), produces a similar inhibition, which was observed with both PLC-delta1 and -beta1. Direct fluorescence microscopy observations demonstrate that the MARCKS peptide forms lateral domains enriched in the acidic lipids phosphatidylserine and PIP2 but not PLC, which accounts for the observed inhibition of PIP2 hydrolysis. Phosphorylation of MARCKS(151-175) by PKC releases the inhibition and allows PLC to produce a burst of inositol 1,4, 5-trisphosphate and diacylglycerol.
Animals↗
Binding of small basic peptides to membranes containing acidic lipids: theoretical models and experimental results.
We measured directly the binding of Lys3, Lys5, and Lys7 to vesicles containing acidic phospholipids. When the vesicles contain 33% acidic lipids and the aqueous solution contains 100 mM monovalent salt, the standard Gibbs free energy for the binding of these peptides is 3, 5, and 7 kcal/mol, respectively. The binding energies decrease as the mol% of acidic lipids in the membrane decreases and/or as the salt concentration increases. Several lines of evidence suggest that these hydrophilic peptides do not penetrate the polar headgroup region of the membrane and that the binding is mainly due to electrostatic interactions. To calculate the binding energies from classical electrostatics, we applied the nonlinear Poisson-Boltzmann equation to atomic models of the phospholipid bilayers and the basic peptides in aqueous solution. The electrostatic free energy of interaction, which arises from both a long-range coulombic attraction between the positively charged peptide and the negatively charged lipid bilayer, and a short-range Born or image charge repulsion, is a minimum when approximately 2.5 A (i.e., one layer of water) exists between the van der Waals surfaces of the peptide and the lipid bilayer. The calculated molar association constants, K, agree well with the measured values: K is typically about 10-fold smaller than the experimental value (i.e., a difference of about 1.5 kcal/mol in the free energy of binding). The predicted dependence of K (or the binding free energies) on the ionic strength of the solution, the mol% of acidic lipids in the membrane, and the number of basic residues in the peptide agree very well with the experimental measurements. These calculations are relevant to the membrane binding of a number of important proteins that contain clusters of basic residues.
Amino Acid Sequence↗
The pleckstrin homology domain of phospholipase C-delta 1 binds with high affinity to phosphatidylinositol 4,5-bisphosphate in bilayer membranes.
The pleckstrin homology (PH) domain of phospholipase C-delta 1 (PLC-delta 1) binds to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in phospholipid membranes with an affinity (Ka approximately 10(6) M-1) and specificity comparable to those of the native enzyme. PLC-delta 1 and its PH domain also bind inositol 1,4,5-trisphosphate, the polar head group of PI(4,5)P2, with comparable affinity and approximately 1:1 stoichiometry. A peptide corresponding to amino acids 30-43 of the PLC-delta 1 PH domain contains several basic residues predicted to bind PI(4,5)P2, but binds weakly and with little specificity for PI(4,5)P2; hence the tertiary structure of the isolated PH domain is required for high affinity PI(4,5)P2 binding. Our PI-(4,5)P2 binding results support the hypothesis that the intact PH domain, serving as a specific tether, directs PLC-delta 1 to membranes enriched in PI(4,5)P2 and permits the active site, located elsewhere in the protein, to hydrolyze multiple substrate molecules before this enzyme dissociates from the membrane surface.
Amino Acid Sequence↗
Calculations of the electrostatic potential adjacent to model phospholipid bilayers.
We used the nonlinear Poisson-Boltzmann equation to calculate electrostatic potentials in the aqueous phase adjacent to model phospholipid bilayers containing mixtures of zwitterionic lipids (phosphatidylcholine) and acidic lipids (phosphatidylserine or phosphatidylglycerol). The aqueous phase (relative permittivity, epsilon r = 80) contains 0.1 M monovalent salt. When the bilayers contain < 11% acidic lipid, the -25 mV equipotential surfaces are discrete domes centered over the negatively charged lipids and are approximately twice the value calculated using Debye-Hückel theory. When the bilayers contain > 25% acidic lipid, the -25 mV equipotential profiles are essentially flat and agree well with the values calculated using Gouy-Chapman theory. When the bilayers contain 100% acidic lipid, all of the equipotential surfaces are flat and agree with Gouy-Chapman predictions (including the -100 mV surface, which is located only 1 A from the outermost atoms). Even our model bilayers are not simple systems: the charge on each lipid is distributed over several atoms, these partial charges are non-coplanar, there is a 2 A ion-exclusion region (epsilon r = 80) adjacent to the polar headgroups, and the molecular surface is rough. We investigated the effect of these four factors using smooth (or bumpy) epsilon r = 2 slabs with embedded point charges: these factors had only minor effects on the potential in the aqueous phase.
Biophysical Phenomena↗
The myristoyl-electrostatic switch: a modulator of reversible protein-membrane interactions.
Hydrophobic insertion of the acyl chain into the bilayer is necessary but not sufficient for the membrane binding of a myristoylated protein. The myristoylated alanine-rich C kinase substrate (MARCKS), Src, ADP-ribosylation factor and human immunodeficiency virus-1 matrix proteins also contain a cluster of basic residues that bind to acidic phospholipids; the hydrophobic and electrostatic interactions act together to anchor the protein to a membrane. For MARCKS, and perhaps other proteins, phosphorylation of serines within its basic cluster reduces the electrostatic attraction, producing translocation of the protein from the membrane to the cytosol by a simple 'electrostatic switch' mechanism.
Cell Membrane↗
Amino-terminal basic residues of Src mediate membrane binding through electrostatic interaction with acidic phospholipids.
Membrane targeting of pp60src (Src) is mediated by its myristoylated amino terminus. We demonstrate that, in addition to myristate, six basic residues in the amino terminus are essential for high-affinity binding to the lipid bilayer via electrostatic interaction with acidic phospholipids. Specifically, c-Src was shown to bind 2500-fold more strongly to vesicles composed of the physiological ratio of 2:1 phosphatidylcholine (PC)/phosphatidylserine (PS) than to neutral PC bilayer vesicles. The apparent Kd for binding of c-Src to the PC/PS bilayer was 6 x 10(-7) M. This interaction is sufficiently strong to account for c-Src membrane targeting. Mutants of c-Src in which the amino-terminal basic residues were replaced by neutral asparagine residues exhibited binding isotherms approaching that of wild-type binding to neutral bilayers (apparent Kd of 2 x 10(-3) M). The transforming v-Src and activated c-Src (Y527F) proteins also bound more strongly to PC/PS bilayers (apparent Kd of approximately 1 x 10(-5) M) than to neutral PC bilayers. In vivo experiments with Src mutants confirmed the role of positive charge in mediating membrane binding and cellular transformation.
3T3 Cells↗
Phosphorylation, high ionic strength, and calmodulin reverse the binding of MARCKS to phospholipid vesicles.
The myristoylated alanine-rich protein kinase C substrate (MARCKS) is a major cellular substrate of protein kinase C (PKC), and PKC phosphorylation produces translocation of MARCKS from membrane to cytoplasm in many cells. Our working hypothesis is that binding of MARCKS to biological membranes requires both hydrophobic insertion of its myristoyl chain into the lipid bilayer and electrostatic interaction of its basic domain with acidic lipids. We tested this hypothesis by measuring the binding of murine MARCKS to large unilamellar phospholipid vesicles (LUVs). We estimated the partition coefficient of the myristoyl moiety of MARCKS (KH) by measuring the binding of MARCKS to electrically neutral LUVs (KH = 3 x 10(3) M-1). We examined the effect of electrostatic interactions by measuring the binding of MARCKS to LUVs containing 20% acidic lipid and obtained four results. First, incorporating 20% acidic lipid into the LUVs increased binding of MARCKS about 100-fold. Second, PKC phosphorylation, which added 3 negatively charged phosphate groups to the basic domain, reduced 20-fold the binding of MARCKS to these negatively charged vesicles. Third, increasing the KCl concentration from 0.1 to 0.5 M reduced the binding 15-fold. Fourth, Ca(2+)-calmodulin reduced the binding 20-fold. We present a simple theoretical model that explains these results, which are all consistent with the working hypothesis.
Animals↗
Membrane binding of myristylated peptides corresponding to the NH2 terminus of Src.
Membrane association is required for cell transformation by pp60v-src (v-Src), the product of the v-src oncogene of Rous sarcoma virus. Previous experiments have identified two NH2-terminal membrane-binding motifs: a myristate (14-carbon acyl chain) attached to the NH2-terminal glycine and three basic residues at positions 5, 7, and 9 of Src. We examined the membrane binding of each motif using myristylated (myr-src) and nonmyristylated (nonmyr-src) peptides corresponding to the NH2 terminus of Src. All myristylated peptides partitioned equally well onto electrically neutral phosphatidylcholine vesicles (K1 = 10(4) M-1). Identical binding has been observed for simple myristylated peptides (e.g., myr-Gly) and arises from the hydrophobic insertion of the myristate into the bilayer. A nonmyristylated peptide corresponding to residues 2-16 of Src [nonmyr-src(2-16), net charge = +5] bound to vesicles containing 33% monovalent acidic phospholipids with K1 = 10(3) M-1. Penta(lysine) (+5 net charge) exhibits the same binding behavior, which is due to the electrostatic interaction between basic residues and acidic lipids. The corresponding myristylated peptide, myr-src(2-16), binds 3 orders of magnitude more strongly to vesicles containing 33% acidic lipids than to neutral vesicles. The resulting apparent association constant, K1 = 10(7) M-1, is approximately equal to the product of the partition coefficients for the two individual interactions. This 10(7) M-1 binding is sufficiently strong to anchor the Src protein to biological membranes. We propose a simple model that explains the observed synergism between the two peptide-membrane interactions.
Amino Acid Sequence↗
Influence of protein disulfide isomerase (PDI) on antibody folding in vitro.
The role of eucaryotic protein disulfide isomerase (PDI) in the folding and reoxidation of proteins in vitro was investigated using an antibody Fab fragment as a model substrate, since PDI is known to participate in the disulfide bond formation of immunoglobulins in vivo. PDI has no effect on the folding of the Fab fragment with intact disulfide bonds, suggesting that, at least in this system, PDI is not able to influence the folding process in a chaperone-like manner. Instead, the role of PDI is limited to disulfide bond formation as demonstrated for the folding of the denatured and reduced Fab fragment. Here, PDI influences the yield of reactivation enormously with a maximum effect at about stoichiometric amounts of PDI and Fab. Furthermore, PDI changes the redox dependence of the reaction. In the presence of PDI, formation of the correct disulfide bonds is possible at higher oxidizing conditions compared to the spontaneous reaction. The requirements both for stoichiometric amounts of PDI and for the presence of PDI during the first seconds of refolding suggest that there is a kinetic competition between rapid structure formation of the antibody domains and interaction of PDI with cysteine residues in the folding protein.
Animals↗
Effect of monolayer surface pressure on the activities of phosphoinositide-specific phospholipase C-beta 1, -gamma 1, and -delta 1.
Three isoforms of phospholipase C, either PLC-beta 1, PLC-gamma 1, or PLC-delta 1, were added to the aqueous subphase beneath phospholipid monolayers formed at an air-solution interface, and the initial rate of hydrolysis of phosphatidylinositol 4,5-bisphosphate was measured after addition of 10 microM free Ca2+. The monolayers were formed from mixtures of phosphatidylcholine (65% PC), phosphatidylserine (33% PS), and phosphatidylinositol 4,5-biphosphate (2% PIP2). Increasing the surface pressure of the monolayer, pi, from 15 to 25 mN/m decreases the rate of hydrolysis 16-, 13-, and 5-fold for PLC-beta 1, PLC-gamma 1, and PLC-delta 1, respectively. The simplest interpretation of these results is that a portion of each of the enzymes of area Ap must insert into the monolayer, doing work pi Ap, prior to hydrolysis of PIP2; binding studies with simple model compounds of known cross-sectional area are consistent with this interpretation. Removing the monovalent acidic lipid PS from the monolayer decreases the initial rates of hydrolysis of PIP2 about 3-fold for each PLC isoform, which suggests that negative electrostatic surface potentials increase the PLC activity.
Animals↗
Phosphorylation reverses the membrane association of peptides that correspond to the basic domains of MARCKS and neuromodulin.
Several groups have observed that phosphorylation causes the MARCKS (Myristoylated Alanine-Rich C Kinase Substrate) protein to move off cell membranes and phospholipid vesicles. Our working hypothesis is that significant membrane binding of MARCKS requires both hydrophobic insertion of the N-terminal myristate into the bilayer and electrostatic association of the single cluster of basic residues in the protein with acidic lipids and that phosphorylation reverses this electrostatic association. Membrane binding measurements with myristoylated peptides and phospholipid vesicles show this hydrophobic moiety could, at best, barely attach proteins to plasma membranes. We report here membrane binding measurements with basic peptides that correspond to the phosphorylation domains of MARCKS and neuromodulin. Binding of these peptides increases sigmoidally with the percent acidic lipid in the phospholipid vesicle and can be described by a Gouy-Chapman/mass action theory that explains how electrostatics and reduction of dimensionality produce apparent cooperativity. The electrostatic affinity of the MARCKS peptide for membranes containing 10% acidic phospholipids (10(4) M-1 = chi/[P], where chi is the mole ratio of peptide bound to the outer monolayer of the vesicles and [P] is the concentration of peptide in the aqueous phase) is the same as the hydrophobic affinity of the myristate moiety for bilayer membranes. Phosphorylation decreases the affinity of the MARCKS peptide for membranes containing 15% acidic lipid about 1000-fold and produces a rapid (t1/2 < 30 s) dissociation of the peptide from phospholipid vesicles.
Amino Acid Sequence↗
Age and body surface area related normal upper and lower limits of M mode echocardiographic measurements and left ventricular volume and mass from infancy to early adulthood.
BACKGROUND: M Mode echocardiograms can be measured by two different conventions. In addition, normal limits of echocardiographic measurements have customarily been stratified according to age or body surface area. There is therefore a need to develop a more easily managed approach to calculating normal limits of measurements for the two conventions, one of which, the Penn convention, has not previously been used for echocardiographic measurements in children. METHODS: M mode echocardiograms were recorded in 127 healthy subjects aged from 7 months to 19.5 years. Measurements were made from paper recordings according to the recommendations of the American Society of Echocardiographers and those of the Penn convention. RESULTS: Age and body surface area were found to be highly correlated; but for completeness separate age dependent and body surface area dependent equations for the normal limits of M mode echocardiographic variables were developed. CONCLUSION: A set of age dependent equations and a set of body surface area dependent equations are presented for easy calculation of upper and lower limits of normal M mode echocardiographic variables in infants and children.
Adolescent↗