Ca binding to the human red cell membrane: Characterization of membrane preparations and binding sites

Summary Inside out and right side out vesicles were used to study the sidedness of Ca binding to the human red cell membrane. It was shown that these vesicles exhibited only a limited permeability to Ca, enabling the independent characterization of Ca binding to the extracellular and cytoplasmic membrane surfaces. Ca binding was studied in 10 mM Tris HCl at pH 7.4, 22±2°C and was shown to be complete in under 5 min. Scatchard plots were made from Ca binding data obtained at free Ca concentrations in the range of 10^–6 to 10^–3M. Under these conditions inside out vesicles exhibit two independent binding sites for Ca with association constants of 1×10⁵ and 6×10³ M^–1, and right side out vesicles exhibit three independent binding sites with association constants of 2×10⁵, 1.4×10⁴ and 3×10²M^–1. Upon the addition of 0.1M KCl a third, high affinity site was found on inside out vesicles with an association constant of 3×10⁵, (in 0.1 M KCl). Ca binding to inside out vesicles increased nearly linearly with pH in the, range of pH 4 to pH 11, while binding to right side out vesicles remained practically unchanged in the range of pH 7 to pH 9. Progressive increase of the ionic strength of the medium by the addition of K, Mg or Tris decreased Ca binding to inside out vesicles as did the addition of ATP. Comparison of a series of cation competitors for Ca binding sites on inside out vesicles at 0.003 mM Ca showed that La was the most effective competitor of all while Cd was the most effective divalent cation competitor of those tested. Our findings suggest that the effects of low concentrations of Ca at the inner surface of the red cell membrane are mediated primarily through Ca binding to site 1 (and, possibly site 2) of inside out vesicles of which there are approximately 1.6×10⁵ per equivalent cell.     

Relationships between membrane binding, affinity and cell internalization efficacy of a cell-penetrating peptide: penetratin as a case study

Background

Penetratin is a positively charged cell-penetrating peptide (CPP) that has the ability to bind negatively charged membrane components, such as glycosaminoglycans and anionic lipids. Whether this primary interaction of penetratin with these cell surface components implies that the peptide will be further internalized is not clear.

Methodology

Using mass spectrometry, the amount of internalized and membrane bound penetratin remaining after washings, were quantified in three different cell lines: wild type (WT), glycosaminoglycans- (GAG^neg) and sialic acid-deficient (SA^neg) cells. Additionally, the affinity and kinetics of the interaction of penetratin to membrane models composed of pure lipids and membrane fragments from the referred cell lines was investigated, as well as the thermodynamics of such interactions using plasmon resonance and calorimetry.

Principal Findings

Penetratin internalized with the same efficacy in the three cell lines at 1 µM, but was better internalized at 10 µM in SA^neg>WT>GAG^neg. The heat released by the interaction of penetratin with these cells followed the ranking order of internalization efficiency. Penetratin had an affinity of 10 nM for WT cells and µM for SA^neg and GAG^neg cells and model membrane of phospholipids. The remaining membrane-bound penetratin after cells washings was similar in WT and GAG^neg cells, which suggested that these binding sites relied on membrane phospholipids. The interaction of penetratin with carbohydrates was more superficial and reversible while it was stronger with phospholipids, likely because the peptide can intercalate between the fatty acid chains.

Conclusion/Significance

These results show that accumulation and high-affinity binding of penetratin at the cell-surface do not reflect the internalization efficacy of the peptide. Altogether, these data further support translocation (membrane phospholipids interaction) as being the internalization pathway used by penetratin at low micromolecular concentration, while endocytosis is activated at higher concentration and requires accumulation of the peptide on GAG and GAG clustering.     

Human Immunodeficiency Virus Type 1 Protease Triggers a Myristoyl Switch That Modulates Membrane Binding of Pr55gag and p17MA

The human immunodeficiency virus type 1 (HIV-1) Pr55^gag gene product directs the assembly of virions at the inner surface of the cell plasma membrane. The specificity of plasma membrane binding by Pr55^gag is conferred by a combination of an N-terminal myristoyl moiety and a basic residue-rich domain. Although the myristate plus basic domain is also present in the p17MA proteolytic product formed upon Pr55^gag maturation, the ability of p17MA to bind to membranes is significantly reduced. It was previously reported that the reduced membrane binding of p17MA was due to sequestration of the myristate moiety by a myristoyl switch (W. Zhou and M. D. Resh, J. Virol. 70:8540–8548, 1996). Here we demonstrate directly that treatment of membrane-bound Pr55^gag in situ with HIV-1 protease generates p17MA, which is then released from the membrane. Pr55^gag was synthesized in reticulocyte lysates, bound to membranes, and incubated with purified HIV-1 protease. The p17MA product in the membrane-bound and soluble fractions was analyzed following proteolysis. Newly generated p17MA initially was membrane bound but then displayed a slow, time-dependent dissociation resulting in 65% solubilization. Residual p17MA could be extracted from the membranes with either high pH or high salt. Treatment of membranes from transfected COS-1 cells with protease revealed that Pr55^gag was present within sealed membrane vesicles and that the release of p17MA occurred only when detergent and salt were added. We present a model proposing that the HIV-1 protease is the “trigger” for a myristoyl switch mechanism that modulates the membrane associations of Pr55^gag and p17MA in virions and membranes.     

Inhibition of Catalase by Tea Catechins in Free and Cellular State: A Biophysical Approach

Tea flavonoids bind to variety of enzymes and inhibit their activities. In the present study, binding and inhibition of catalase activity by catechins with respect to their structure-affinity relationship has been elucidated. Fluorimetrically determined binding constants for (−)-epigallocatechin gallate (EGCG) and (−)-epicatechin gallate (ECG) with catalase were observed to be 2.27×10⁶ M⁻¹ and 1.66×10⁶ M⁻¹, respectively. Thermodynamic parameters evidence exothermic and spontaneous interaction between catechins and catalase. Major forces of interaction are suggested to be through hydrogen bonding along with electrostatic contributions and conformational changes. Distinct loss of α-helical structure of catalase by interaction with EGCG was captured in circular dichroism (CD) spectra. Gallated catechins demonstrated higher binding constants and inhibition efficacy than non-gallated catechins. EGCG exhibited maximum inhibition of pure catalase. It also inhibited cellular catalase in K562 cancer cells with significant increase in cellular ROS and suppression of cell viability (IC₅₀ 54.5 µM). These results decipher the molecular mechanism by which tea catechins interact with catalase and highlight the potential of gallated catechin like EGCG as an anticancer drug. EGCG may have other non-specific targets in the cell, but its anticancer property is mainly defined by ROS accumulation due to catalase inhibition.     

Differences in the mechanism of NADPH- and cumene hydroperoxide-supported reactions of cytochrome P-450

A study has been carried out on the association of aldolase with the human erythrocyte membrane. It has been shown that the conditions employed during hypotonic hemolysis affect the amount of aldolase that remains bound to the cell membrane. Thus, the in vivo nature of this binding cannot be ascertained by this technique. Therefore, a method has been developed in which aldolase is crosslinked with glutaraldehyde to the inner surface of the membrane in intact red blood cells. Under the specified conditions, over 90% of the intracellular aldolase can be crosslinked to the membrane with less than 10% of the hemoglobin becoming bound. These results suggest that the localization of aldolase in situ is on or near the inner surface of the membrane. The amount of aldolase bound to the membrane following crosslinking can be decreased by preincubating the cells with cytoskeletal agents such as cytochalasin B, colchicine, and vinblastine sulfate. The in vitro binding of aldolase to the purified spectrin-actin and F-actin complexes was studied. Aldolase bound both complexes very tightly (K_D ? 10^?9m) and this binding could be inhibited by cytochalasin B, but not by colchicine. A competition binding study was carried out to determine if the binding of aldolase to F-actin involved specific interactions. Neither bovine serum albumin nor cytochrome c significantly inhibited the binding of aldolase to F-actin when each was present at equimolar concentrations with aldolase. However, glyceraldehyde 3-phosphate dehydrogenase inhibited aldolase binding to F-actin and when present at equimolar concentrations with aldolase completely blocked the association. The association of aldolase and other glycolytic enzymes with the erythrocyte membrane is discussed and it is postulated that aldolase could be localized in vivo on the inner surface of the membrane by attachment to actin or a spectrin-actin complex.     

The role of membrane-bound magnesium in the permeability of ghosts to K+

Mg²⁺ is required for restoring a low cation permeability of erythrocyte membranes after osmotic lysis. These ions promote binding of haemoglobin to the ghost membrane. Because the optimal binding occurs at a pH where the K⁺ retention by ghosts is maximal, the possibility exists that Mg²⁺ exerts an indirect effect by facilitating the adsorption of haemoglobin, which may be essential for the maintenance of a low permeability. In order to investigate this possibility, a systematic study was done of the effects of pH and hypotonic washing on the retention of haemoglobin, Mg²⁺ and K⁺ by human erythrocyte ghosts.Between pH 5.5 and 7.5, the retention of haemoglobin paralleled that of K⁺ and Mg²⁺ on resealing immediately after lysis. Such a parallelism was not observed if ghosts were both washed with fresh haemolytic media and resuspended in a medium of lower osmolarity before reversal.No correspondence was found between ghost K⁺ content and membrane-bound haemoglobin, indicating that the latter is not involved in the maintenance of a low permeability. By contrast, Mg²⁺ binding was altered by pH in the same way as ghost K⁺ and a strict relationship between membrane-bound Mg²⁺ and K⁺ retention was obtained.The results suggest that K⁺ permeability is mainly determined by the amount of Mg²⁺ associated with the ghost membrane.     

Adenosine 3',5' monophosphate binds only to the inner surface of human erythrocyte membranes

The binding of adenosine 3′,5′ monophosphate (cyclic AMP) to each surface of the isolated human erythrocyte membrane was measured. Unsealed ghosts, in which both membrane faces are accessible, and sealed inside-out vesicles, which expose only the cytoplasmic side of the membrane, both bound approximately 6,000 cyclic AMP molecules per cell membrane equivalent with a dissociation constant, K ? 2.5 × 10^?9. The binding of this nucleotide by preparations rich in sealed ghosts and right-side-out vesicles, which sequester the inner surface, was limited and could be correlated precisely with small amounts of exposed cytoplasmic surface. We conclude that these binding sites for cyclic AMP are confined to the cytoplasmic side of the erythrocyte membrane.     

The characterisation of inducible dehydrogenases specific for the oxidation of d-alanine,allohydroxy-d-proline,choline and sarcosine as peripheral membrane proteins in Pseudomonas aeruginosa

The interaction with the cytoplasmic membrane of the inducible, membrane-bound, cytochrome-linked dehydrogenases specific for the oxidation of d-alanine, allohydroxy-d-proline, choline and sarcosine in Pseudomonas aeruginosa was investigated. The susceptibility of d-alanine dehydrogenase to solubilisation by cation depletion or by washing with high ionic strength buffers indicated that it was a peripheral membrane protein. The effect of various divalent cations in reducing the amount of enzyme released by cation depletion suggests a requirement for Mg²⁺ in the binding of d-alanine dehydrogenase to the cytoplasmic membrane. The peripheral nature of all four dehydrogenases was confirmed by examination of the molecular properties and phospholipid content of preparations of the enzymes solubilised with 1 M phosphate buffer (pH 7.0). Additional confirmatory evidence was provided by Arrhenius plots of membrane-bound activity of d-alanine and allohydroxy-d-proline dehydrogenases which were monophasic and independent of the discontinuities attributable to membrane lipid phase separations which characterise such plots of the activity of integral membrane-bound enzymes. The shape of the Arrhenius plots obtained for the activities of known integral respiratory proteins of P. aeruginosa suggests that these enzymes may remain in a fluid environment throughout the course of the phase separation.     

Naphthylphthalamic acid-binding sites in cultured cells from Nicotiana tabacum

Naphthylphthalamic acid (NPA), an inhibitor of polar auxin transport, binds with high affinity to membrane preparations from callus and cell suspension cultures derived from Nicotiana tabacum (K _d approx. 2·10^–9 M). The concentration of membrane-bound binding sites is higher in cell suspension than in callus cultures. The binding of NPA to these sites seems to be a simple process, in contrast to the binding of the synthetic auxin naphthylacetic acid (1-NAA) to membrane preparations from callus cultures, which is more complex (A.C. Maan et al., 1983, Planta 158, 10–15). Naphthylacetic acid, a number of structurally related compounds and the auxin-transport inhibitor triiodobenzoic acid were all able to compete with NPA for the same binding site with K _d values ranging from 10^–6 to 10^–4 M. On the other hand, NPA was not able to displace detectable amounts of NAA from the NAA-binding site. A possible explantation is the existence of two different membrane-bound binding sites, one exclusively for auxins and one for NPA as well as auxins, that differ in concentration. The NPA-binding site is probably an auxin carrier.Abbreviations 1-NAA 1-Naphthylacetic acid - 2-NAA 2-Naphthylacetic acid - NPA N-1-Naphthylphthalamic acid     

Ca- and Mg-dependent association of phosphorylase kinase with human erythrocyte membranes

The interaction of rabbit muscle phosphorylase kinase (EC 2.7.1.38) with human erythrocyte membranes was investigated. It was found that at pH 7.0 the kinase binds to the inner face of the erythrocyte membrane (inside-out vesicles) and that this binding is Ca²⁺- and Mg²⁺-dependent. The sharpest increase in the binding reaction occurs at concentrations between 70 and 550 nM free Ca²⁺. Erythrocyte ghost or right-side out erythrocyte vesicles showed a significantly lower capacity to interact with phosphorylase kinase. Autophosphorylated phosphorylase kinase shows a similar Ca²⁺-dependent binding profile, while trypsin activation of the kinase and calmodulin decrease the original binding capacity by about 50%. Heparin (200 μg/ml) and high ionic strength (50 mM NaCl) almost completely blocks enzyme-membrane interaction; glycogen does not affect the interaction.     

The Cratylia mollis Seed Lectin Induces Membrane Permeability Transition in Isolated Rat Liver Mitochondria and a Cyclosporine A‐Insensitive Permeability Transition in Trypanosoma cruzi Mitochondria

Previous results provided evidence that Cratylia mollis seed lectin (Cramoll 1,4) promotes Trypanosoma cruzi epimastigotes death by necrosis via a mechanism involving plasma membrane permeabilization to Ca²⁺ and mitochondrial dysfunction due to matrix Ca²⁺ overload. In order to investigate the mechanism of Ca²⁺‐induced mitochondrial impairment, experiments were performed analyzing the effects of this lectin on T. cruzi mitochondrial fraction and in isolated rat liver mitochondria (RLM), as a control. Confocal microscopy of T. cruzi whole cell revealed that Cramoll 1,4 binding to the plasma membrane glycoconjugates is followed by its internalization and binding to the mitochondrion. Electrical membrane potential (?Ψ_m) of T. cruzi mitochondrial fraction suspended in a reaction medium containing 10 μM Ca²⁺ was significantly decreased by 50 μg/ml Cramoll 1,4 via a mechanism insensitive to cyclosporine A (CsA, membrane permeability transition (MPT) inhibitor), but sensitive to catalase or 125 mM glucose. In RLM suspended in a medium containing 10 μM Ca²⁺ this lectin, at 50 μg/ml, induced increase in the rate of hydrogen peroxide release, mitochondrial swelling, and ?Ψ_m disruption. All these mitochondrial alterations were sensitive to CsA, catalase, and EGTA. These results indicate that Cramoll 1, 4 leads to inner mitochondrial membrane permeabilization through Ca²⁺ dependent mechanisms in both mitochondria. The sensitivity to CsA in RLM characterizes this lectin as a MPT inducer and the lack of CsA effect identifies a CsA‐insensitive MPT in T. cruzi mitochondria.     

The effect of solubilisation on the character of an ethylene-binding site from Phaseolus vulgaris L. cotyledons

The ethylene-binding site (EBS) from Phaseolus vulgaris cv. Canadian Wonder cotyledons can be solubilised from 96,000 g pelleted material by Triton X-100 or sodium cholate. Extraction of 96,000 g pellets with acetone, butanol or butanol and ether results in a total loss of ethylene-binding activity. Like the membrane-bound form, the solubilised EBS has an apparent K_D(liquid) of 10^-10 M at a concentration of 32 pmol EBS per gram tissue fresh weight. Propylene and acetylene act as competitive inhibitors, carbon dioxide appears to promote ethylene binding and ethane has no significant effect. The solubilised EBS is completely denatured affect. The solubilised EBS is completely denatured after 10 min at 70°C, by 1 mM mercaptoethanol and 0.1 mM dithiothreitol, but not by trypsin or chymotrypsin. However, solubilisation decreases the rate constant of association from 10³ M^-1 s^-1 to 10¹–10² M^-1 s^-1 and hence does not permit experimental determination of the rate constant of dissociation. The pH optimum for ethylene binding is altered from the range pH 7–10 in the membrane-bound form to the pH range 4–7 in the solubilised form. The EBS appears to be a hydrophobic, intergral membrane protein, which requires a hydrophobic environment to retain its activity. Partitioning of the EBS into polymer phases is determined by the detergent used for solubilisation indicating that when solubilised, the EBS forms a complex with detergent molecules.Abbreviations EBS ethylene-binding site - PEG polyethylene glycol     

Detailed characterization of Synechocystis PCC 6803 ferredoxin:NADP+ oxidoreductase interaction with model membranes

Direct interaction of ferredoxin:NADP⁺ oxidoreductase (FNR) with thylakoid membranes was postulated as a part of the cyclic electron flow mechanism. In vitro binding of FNR to digalactosyldiacylglycerol and monogalactosyldiacylglycerol membranes was also shown. In this paper we deal with the latter interaction in more detail describing the effect for two FNR forms of Synechocystis PCC 6803. The so-called short FNR (sFNR) is homologous to FNR from higher plant chloroplasts. The long FNR (lFNR) form contains an additional domain, responsible for the interaction with phycobilisomes. We compare the binding of both sFNR and lFNR forms to native and non-native lipids. We also include factors which could modulate this process: pH change, temperature change, presence of ferredoxin, NADP⁺ and NADPH and heavy metals. For the lFNR, we also include phycobilisomes as a modulating factor. The membrane binding is generally faster at lower pH. The sFNR was binding faster than lFNR. Ferredoxin isoforms with higher midpoint potential, as well as NADPH and NADP⁺, weakened the binding. Charged lipids and high phosphate promoted the binding. Heavy metal ions decreased the rate of membrane binding only when FNR was preincubated with them before injection beneath the monolayer. FNR binding was limited to surface lipid groups and did not influence hydrophobic chain packing. Taken together, FNR interaction with lipids appears to be non-specific, with an electrostatic component. This suggests that the direct FNR interaction with lipids is most likely not a factor in directing electron transfer, but should be taken into account during in vitro studies.     

ACTH binding to phospholipid bilayers: A 1H-NMR study of the 5-14, 1-14, and 1-24 derivatives

The interaction of the 5-14, 1-14, and 1-24 fragments of ACTH with sonicated phospholipid bilayers containing egg yolk phosphatidylcholine (EPC) either pure or mixed with 10 mole % phosphatidic acid (EPA), was investigated by proton nuclear magnetic resonance (¹H-nmr). The effects observed with zwitterionic EPC vesicles were small, indicating a low binding of the ACTH derivatives. The N-terminal aromatic resonances of the ACTH peptides were markedly broadened in the presence of negatively charged vesicles (EPC/EPA 9:1 M/M), while those of the C-terminal end were barely affected, showing that ACTH interacts with its N-terminal fragment. The choline resonance of the EPC molecules of the outer monolayer was shifted and broadened upon ACTH binding to the lipid vesicles, while that of the inner layer was not affected, suggesting that the peptide molecules interact only with the external leaflet of the lipid bilayer. The C²H and C⁴H resonances of the histidine-6 side chain were both shifted downfield upon peptide binding to the negatively charged lipid interface. In the case of the 1–24 derivative, these resonances were also split into two signals reflecting two different species of membrane-bound ACTH 1–24. Analysis of the line width and chemical shift variations of the ACTH and lipid resonances observed upon peptide binding shows that the membrane-binding potency of the shorter 5–14⁺¹ fragment, which presents a +1 net charge, is roughly similar to that of the highly cationic 1–24⁺⁶ (net charge +6) derivative, implying that the 15–24⁺⁵ segment is not essential for membrane binding. The nmr measurements at a fixed lipid-to-peptide ratio in the presence of increasing amounts of spin-labeled lipids demonstrate that the N-terminal fragment of ACTH does not penetrate the hydrophobic core of the bilayer, and should lie parallel to the membrane surface. © 1997 John Wiley & Sons, Inc. Biopoly 42: 731–744, 1997     

Calcium Binding to Plasma Membranes from Normal and SV40 Transformed Hamster Lymphocytes

Abstract

The calcium binding properties of isolated plasma membranes from normal and SV40 transformed hamster lymphocytes were compared over the Ca²⁺ concentration range of 10^?5M to 5 × 10^?3M and at physiological ionic strength. At all Ca²⁺ concentrations, normal membranes bound more Ca²⁺ than tumor membranes; at blood Ca²⁺ levels (1–2 mM) plasma membranes of normal cells bind twice as much as membranes from tumor cells. Normal plasma membranes demonstrated positive cooperative Ca²⁺ binding whereas tumor membranes displayed non-interacting Ca²⁺-binding sites. Ca²⁺ binding to both membranes was insensitive to Mg²⁺ (0.1 to 2.5 mM). A pH shift from 7 to 6 resulted in a 70% decrease of normal membrane-bound Ca²⁺ compared to a 40% decrease observed with tumor membranes. Extracellular surface Ca²⁺ binding to intact cells was also studied after a 72-hour equilibration of cells with ⁴⁵Ca²⁺ and with ethylene-glycol-bis-(β-amino-ethyl ether) N, N′-tetraacetate chelation as marker for surface Ca²⁺. Tumor cell surface Ca²⁺ binding was only 10% of that observed with quiescent lymphocytes. Normal lymphocytes stimulated to divide with phytohemagglutinin also showed a decreased level of surface Ca²⁺ (50%). However, plasma membranes isolated from non-dividing and phytohemagglutinin-stimulated lymphocytes exhibited equivalent Ca²⁺ binding.     

Role of membrane-bound Ca in ghost permeability to Na and K

Summary The permeability of red cell ghosts to K is determined by the amount of membrane-bound Mg which, in turn, depends on internal Mg. Contrasting with such effect, an increase in cellular Ca raises K permeability. To test whether this, action is due to a competitive displacement of membrane Mg, the free Ca content of human red cell ghosts was altered by means of Ca-EGTA buffers. Net Na and K movements as well as Ca and Mg bindings, were assessed after incubation in a Na-medium at 37°C. Raising Ca from 3×10^–7 to 1×10^–2M caused a large K efflux with very little Na gain. Under similar conditions, Ca binding was increased without affecting membranebound Mg. Both Ca binding and K loss were markedly diminished by either adding ATP to the hemolytic medium or increasing internal Mg at a fixed Ca concentration. A Scatchard analysis showed three Ca binding sites, two of them having high affinity. It is concluded that Ca action does not arise from a displacement of membrane-bound Mg but from binding to different sites in the membrane. Presumably, high affinity sites are involved in the control of K permeability.     

A spectroscopic study of the membrane interaction of tuberoinfundibular peptide of 39 residues (TIP39)

The membrane interaction of tuberoinfundibular peptide of 39 residues (TIP39), which selectively activates the parathyroid hormone 2 (PTH2) receptor (PTH2-R), has been studied by fluorescence and NMR spectroscopic techniques. Membrane binding would be the first step of a potential membrane-bound activation pathway which has been discussed for a number of neuropeptides and G-protein coupled receptors (GPCRs). Here, the orientation of TIP39 on the surface of membrane mimicking dodecyl-phosphocholine (DPC) micelles was monitored by Photo-CIDNP (chemically-induced dynamic nuclear polarization) NMR which indicates that both Trp25 and Tyr29 face the membrane surface. However, the PTH2 receptor is located in the hypothalamus membrane, for which a more realistic model is required. Therefore, liposomes containing different mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylcholine (POPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphatidylserine (POPS) and cholesterol were used for fluorescence and solid-state NMR spectroscopy. Fluorescence spectroscopy showed that a large proportion of TIP39 added to these liposomes binds to the membrane surface. Proton-decoupled ³¹P-MAS NMR is used to investigate the potential role of individual lipid headgroups in peptide binding. Significant line-broadening in POPC/cholesterol and POPC/POPS liposomes upon TIP39 association supports a surface binding model and indicates an interaction which is slightly mediated by the presence of POPS and cholesterol. Furthermore, smoothed order parameter profiles obtained from ²H powder spectra of liposomes containing POPC-d31 as bulk lipid in addition to POPS and cholesterol show that TIP39 does not penetrate beyond the headgroup region. Spectra of similar bilayers with POPS-d31 show a small increase in segmental chain order parameters which is interpreted as a small but specific interaction between the peptide and POPS. Our data demonstrate that TIP39 belongs to a class of signaling peptides that associate weakly with the membrane surface but do not proceed to insert into the membrane hydrophobic compartment.     

Interaction of lipophorin with Rhodnius prolixus oocytes: biochemical properties and the importance of blood feeding

Lipophorin (Lp) is the main haemolymphatic lipoprotein in insects and transports lipids between different organs. In adult females, lipophorin delivers lipids to growing oocytes. In this study, the interaction of this lipoprotein with the ovaries of Rhodnius prolixus was characterised using an oocyte membrane preparation and purified radiolabelled Lp ⁽¹²⁵I-Lp). Lp-specific binding to the oocyte membrane reached equilibrium after 40-60 min and when ¹²⁵I-Lp was incubated with increasing amounts of membrane protein, corresponding increases in Lp binding were observed. The specific binding of Lp to the membrane preparation was a saturable process, with a K_dof 7.1 ± 0.9 x 10^-8M and a maximal binding capacity of 430 ± 40 ng ¹²⁵I-Lp/µg of membrane protein. The binding was calcium independent and pH sensitive, reaching its maximum at pH 5.2-5.7. Suramin inhibited the binding interaction between Lp and the oocyte membranes, which was completely abolished at 0.5 mM suramin. The oocyte membrane preparation from R. prolixus also showed binding to Lp from Manduca sexta. When Lp was fluorescently labelled and injected into vitellogenic females, the level of Lp-oocyte binding was much higher in females that were fed whole blood than in those fed blood plasma.     

Changes in allosteric properties of phosphofructokinase bound to erythrocyte membranes.

Human and rabbit erythrocyte membranes prepared by hypotonic hemolysis contained 5 to 15% of the phosphofructokinase in the erythrocytes. The membrane-bound phosphofructokinase can be eluted by a saline wash. Human erythrocyte and rabbit muscle phosphofructokinase bind to the saline-washed membranes. This binding is specific for the inner surface of the membrane. The amount of phosphofructokinase bound is dependent on pH; at pH 7, 6 times more enzyme is bound than at pH 7.5. Unlike free phosphofructokinase, the membrane-bound phosphofructokinase is not inhibited by ATP or 2,3-diphosphoglycerate, and its fructose-6-P saturation curve is nonsigmoidal.     

Characterization of the mycoplasma membrane proteins: V. Release and localization of membrane-bound enzymes in Acholeplasma laidlawii

The peripheral membrane protein fraction released by washing Acholeplasma laidlawii membranes with low-ionic strength buffers contained about 50 % of the total membrane-bound ribonuclease and deoxyribonuclease activities. The ATPase, NADH oxidase and p-nitrophenylphosphatase activities remained bound to the membrane even when EDTA was added to the wash fluids, and thus appear to belong to the integral membrane protein group.Serving as a marker for peripheral membrane proteins, the membrane-bound ribonuclease activity was solubilized by bile salts much more effectively than the integral membrane-bound enzymes. On the other hand, the solubilized ribonuclease showed a much lower capacity to reaggregate with other solubilized membrane components to membranous structures. Yet, most of the ribonuclease molecules which were bound to the reaggregated membranes could not be released by low-ionic strength buffer. The reaggregated membranes differed from the native membranes in the absence of particles on their fracture faces obtained by freeze cleaving, and by their much higher labeling by the [¹²⁵I]lactoperoxidase iodination system. These results suggest that most of the proteins are exposed on the reaggregated membrane surfaces, with very little, if any, protein embedded in its lipid bilayer core.Enzyme disposition in the A. laidlawii membrane was studied by comparing the activity of isolated membranes with that of membranes of intact cells after treatment with pronase or with an antiserum to membranes. The data indicate the asymmetrical disposition of these activities, the ATPase and NADH oxidase being localized on the inner membrane surface, while the nucleases are exposed on the external membrane surface.