Synthetic copoly(Lys/Phe) and poly(Lys) translocate through lipid bilayer membranes

Several membrane-transporting peptides (MTP) containing basic amino acid residues such as Lys and Arg that carry macromolecules such as DNA and proteins across cell plasma membranes by an unknown mechanism have been actively studied. On the basis of these results, we have been investigating the translocation ability of synthetic polypeptides, copoly(Lys/Phe) and poly(Lys), through negatively charged phospholipid (soybean phospholipid (SBPL)) bilayer membranes by zeta potential analysis, circular dichroism (CD) spectroscopy, fluorescence spectroscopy, an electrophysiology technique, and confocal laser scanning microscopy (CLSM). The binding of these polypeptides to the membrane, which is the first step for translocation across the membrane, resulted in the conformational transition of the polypeptide from a random coil form or helix-poor form to a helix-rich form. The fluorescence studies demonstrated that the time-dependent decrease in the fluorescence intensities of the FITC-labeled polypeptides bound to the SBPL liposome reflected translocation of the polypeptide across the lipid bilayer with the low dielectric constant. Both the rate constant and the efficiency of the polypeptide translocation across the lipid bilayer were greater for copoly(Lys/Phe) than for poly(Lys). These results suggest that the random incorporation of the hydrophobic Phe residue into the positively charged Lys chain results in a lowering of the potential barrier for passage of the polypeptide in the hydrophobic core portion of the lipid bilayer. We presented the first direct observation that the positively charged polypeptides, copoly(Lys/Phe) (MW: 41,500) and poly(Lys) (MW: 23,400), could translocate across the lipid bilayer membrane.     

Translocation of positively charged copoly(Lys/Tyr) across phospholipid membranes

Much attention has recently been paid to the study of positively charged polypeptides as a possible carrier for therapeutic protein or DNA delivery to cells. In this study, we have investigated the translocation of positively charged copoly(Lys/Tyr) (MW=72000, DP=385) across lipid membranes constituted from egg-phosphatidylcholine (EPC), dioleoyl-phosphatidylethanolamine (DOPE), as well as soybean phospholipids (SBPL) using zeta potential method, circular dichroism spectroscopy (CD), electrophysiology technique, fluorescence spectroscopy, and confocal laser scanning microscopy. Results of zeta potentials show that copoly(Lys/Tyr) associate with lipid membranes and become gradually saturated on the membranes either hydrophobically or electrostatically or both. CD studies demonstrate that the copoly(Lys/Tyr) takes and remains beta-sheet conformation during its interaction with liposome membranes, indicating that the translocation process should be carpet-mode like. Data from the electrophysiology technique reveal that positively charged copoly(Lys/Tyr) can cause transmembrane currents under an applied voltage, confirming its transfer across lipid membranes. Fluorescence spectroscopy results display a three-step mechanism of translocation across membrane: adsorption, transportation, and desorption, which has been verified by results from confocal laser scanning microscopy. We provided the first direct observation that the positively charged polypeptides, copoly(Lys/Tyr), can translocate through SBPL and EPC/DOPE lipid bilayer membranes. In addition, we found that the translocation efficiency of copoly(Lys/Tyr) was higher on the EPC/DOPE lipid membrane than on the SBPL lipid membrane.     

Proton/hydroxide conductance through lipid bilayer membranes

Summary A simple method of measuring proton/hydroxide conductance (G _H/OH) through planar lipid bilayer membranes is described. First the total conductance (G _m ) is measured electrically. Then the H⁺/OH^– transference number (T _H/OH) is estimated from the diffusion potential (V _m ) produced by a transmembrane pH gradient. The pH gradient is produced by a pair of buffered solutions which have identical concentrations of all ions except H⁺ and OH^–. Thus,V _m is due entirely to H⁺/OH^– diffusion andG _H/OH can be calculated from the relations,V _m =T _H/OH E _H/OH andG _H/OH=T _H/OH G _m , whereE _H/OH is the equilibrium potential for H⁺ and OH^–. In bilayers made from bacterial phosphatidylethanolamine (PE) inn-decane,G _H/OH is nearly independent of pH, ranging from about 10^–9 S cm^–2 at pH 1.6 to 10^–8 S cm^–2 at pH 10.5. BecauseG _H/OH is nearly independent of pH, the calculated permeability coefficients to H⁺ and/or OH^– are extremely pH dependent, which partly explains the wide range of values reported for phospholipid vesicles and biological membranes.G _H/OH appears to be independent of the membrane surface charge, because titrating either the phosphate or the amino group of PE has little effect onG _H/OH.G _H/OH is reduced about 10-fold when the water activity is reduced 33% by replacement with glycerol. Although the mechanism of H⁺/OH^– conductance is not known, the relation betweenG _H/OH and water activity suggests that several water molecules are involved in the H⁺/OH^– transport process.     

A study of Li+-selective permeation through lipid bilayer membranes mediated by a new ionophore (AS701)

The neutral, noncyclic, imide and ether containing ionophore AS701, has been developed as Li⁺-selective molecule, to be used potentially as an aid in the Li⁺-therapy of manic-depressive illness. The present report is a characterization of this molecule in neutral lipid bilayer membranes. This ionophore was found to the bilayers Li⁺-selective, acting as a selective carrier of monovalent cations. In addition, this molecule was found to be capable of acting as a selective carrier of monovalent anions. For both types of ions, the rate-limitting step in the process of permeation was found to be the diffusion of the carrier-ion complex through the membrane. The membrane-permeating species were found to be 2 : 1 carrier-ion complexes, carrying either a monovalent cation or a monovalent anion. The selectivity sequences among the ions studied being: Li⁺(1) > ClO₄^?(0.7) > Na⁺(0.07) > K⁺(0.016) > Rb⁺(0.0095) > Cs⁺(0.0083) > Cl^?(0.001). Mg²⁺ and SO₄^2? were found to be impermeant (under present experimental conditions). This sequence shows that the AS701 molecule has low selectivity for ions present in biological media, among those studied (i.e. Na⁺, K⁺, Mg²⁺, Cl^2? and SO₄^2?). This indicates that these ions will not interfere in the Li⁺ permeability induced by this carrier in vivo, and that the carrier will not interfere in the normal transport processes of these ions.     

Inorganic mercury (Hg2+) transport through lipid bilayer membranes

Summary Diffusion of inorganic mercury (Hg²⁺) through planar lipid bilayer membranes was studied as a function of chloride concentration and pH. Membranes were made from egg lecithin plus cholesterol in tetradecane. Tracer (²⁰³Hg) flux and conductance measurements were used to estimate the permeabilities to ionic and nonionic forms of Hg. At pH 7.0 and [Cl^–] ranging from 10–1000mm, only the dichloride complex of mercury (HgCl₂) crosses the membrane at a significant rate. However, several other Hg complexes (HgOHCl, HgCl ₃ ^– and HgCl ₄ ^2– ) contribute to diffusion through the aqueous unstirred layer adjacent to the membrane. The relation between the total mercury flux (J _Hg), Hg concentrations, and permeabilities is: 1/J _Hg=1/P ^ul[Hg ^t ]+1/P ^m [HgCl₂], where [Hg ^t ] is the total concentration of all forms of Hg,P ^ul is the unstirred layer permeability, andP ^m is the membrane permeability to HgCl₂. By fitting this equation to the data we find thatP ^m =1.3×10^–2 cm sec^–1. At Cl^– concentrations ranging from 1–100mm, diffusion of Hg ^t through the unstirred layer is rate limiting. At Cl^– concentrations ranging from 500–1000mm, the membrane permeability to HgCl₂ becomes rate limiting because HgCl₂ comprises only about 1% of the total Hg. Under all conditions, chemical reactions among Hg²⁺, Cl^– and/or OH^– near the membrane surface play an important role in the transport process. Other important metals, e.g., Zn²⁺, Cd²⁺, Ag⁺ and CH₃Hg⁺, form neutral chloride complexes under physiological conditions. Thus, it is likely that chloride can facilitate the diffusion of a variety of metals through lipid bilayer and biological membranes.     

Fluorescence-quenching and resonance energy transfer studies of lipid microdomains in model and biological membranes (Review)

Measurements of contact-dependent fluorescence quenching and of fluorescence resonance energy transfer (FRET) within bilayers provide information concerning the spatial relationships between molecules on distance scales of a few nm or up a few tens of nm, respectively, and are therefore well suited to detect the presence and composition of membrane microdomains. As described in this review, techniques based on fluorescence quenching and FRET have been used to demonstrate the formation of nanoscale liquid-ordered domains in cholesterol-containing model membranes under physiological conditions, and to investigate the structural features of lipids and proteins that influence their partitioning between liquid-ordered and liquid-disordered domains. FRET-based methods have also been used to test for the presence of ‘raft’ microdomains in the plasma membranes of mammalian cells. We discuss the sometimes divergent findings of these studies, possible modifications to the ‘raft hypothesis’ suggested by studies using FRET and other techniques, and the further potential of FRET-based methods to test and to refine current models of the nature and organization of membrane microdomains.     

A novel composite nanofiltration (NF) membrane prepared from graft copolymer of trimethylallyl ammonium chloride onto chitosan (GCTACC)/poly(acrylonitrile) (PAN) by epichlorohydrin cross-linking

A novel composite nanofiltration (NF) membrane was prepared by over-coating the PAN ultrafiltration (UF) membrane with a GCTACC thin layer. The effects of membrane preparation techniques and operating conditions on the performance of the composite membrane were studied. The results indicate that a composite NF membrane from 1.0wt% GCTACC casting solution, vaporized for 2h at 50 degrees C, cross-linked for 20h at 50 degrees C and pH approximately 12 with ethanol/epichlorohydrin (50/0.45 wt/wt) had optimum performance. The resultant GCTACC/PAN composite membrane was positively charged. Scanning electron microscopy showed its asymmetric and composite features. At 25 degrees C and 30L/h of cycling flow, the permeability of pure water through this membrane is 6.3L/hm(2)MPa. At 25 degrees C, 1.2MPa and 30L/h of cycling flow, the rejection of 1000mg/L MgCl(2), CaCl(2), MgSO(4), Na(2)SO(4), and NaCl solutions is 0.976, 0.972, 0.897, 0.65, and 0.407, respectively, with fluxes of 6.8, 6.12, 6.12, 5.57, and 5.51L/hm(2), respectively. The order of rejection of different salts follows the decreasing order of MgCl(2), CaCl(2), MgSO(4), NaCl, KCl, Na(2)SO(4), and K(2)SO(4), which reveals the characteristics of the positively charged NF membrane. In addition, the curve for the streaming potential also illustrates the positively charged characteristics of this membrane, with a pressure osmotic coefficient of 11.7mVMPa(-1).     

Transbilayer (flip-flop) lipid motion and lipid scrambling in membranes

This paper reviews the current knowledge on the various mechanisms for transbilayer, or flip-flop, lipid motion in model and cell membranes, enzyme-assisted lipid transfer by flippases, floppases and scramblases is briefly discussed, while non-catalyzed lipid flip-flop is reviewed in more detail. Transbilayer lipid motion may occur as a result of the insertion of foreign molecules (detergents, lipids, or even proteins) in one of the membrane leaflets. It may also be the result of the enzymatic generation of lipids, e.g. diacylglycerol or ceramide, at one side of the membrane. Transbilayer motion rates decrease in the order diacylglycerol ? ceramide ? phospholipids. Ceramide, but not diacylglycerol, can induce transbilayer motion of other lipids, and bilayer scrambling. Transbilayer lipid diffusion and bilayer scrambling are defined as two conceptually and mechanistically different processes. The mechanism of scrambling appears to be related to local instabilities caused by the non-lamellar ceramide molecule, or by other molecules that exhibit a relatively slow flip-flop rate, when asymmetrically inserted or generated in one of the monolayers in a cell or model membrane.     

Properties of the large ion-permeable pores formed from protein F of Pseudomonas aeruginosa in lipid bilayer membranes

The incorporation of porin protein F from the outer membrane of Pseudomonas aeruginosa into artificial lipid bilayers results in an increase of the membrane conductance by many orders of magnitude. The membrane conductance is caused by the formation of large ion-permeable channels with a single-channel conductance in the order of 5 nS for 1 M alkali chlorides. The conductance has an ohmic current vs. voltage relationship. Further information on the structure of the pore formed by protein F was obtained by determining the single-channel conductance for various species differing in charge and size, and from zero-current potential measurements. The channel was found to be permeable for large organic ions (Tris⁺, N(C₂H₅)₄⁺, Hepes^?) and a channel diameter of 2.2 nm could be estimated from the conductance data (pore length of 7.5 nm). At neutral pH the pore is about two times more permeable for cations than for anions, possibly caused by negative charges in the pore. The consistent observation of large water filled pores formed by porin protein F in model membrane systems is discussed in the light of the known low permeability of the Ps. aeruginosa outer membrane towards antibiotics. It is suggested that this results from a relatively low proportion of open functional porin protein F pores in vivo.     

The unique lipid composition of gecko (Gekko Gekko) photoreceptor outer segment membranes

This study investigated the lipid and fatty acid composition of gecko photoreceptor outer segment membranes which contain the P521 cone-type pigment. The lipids of gecko photoreceptor outer segment membranes were first extracted and separated by thin layer chromatography (TLC) and then analyzed by gas chromatography (GC). Our results show that gecko photoreceptor outer segment membranes contain less phosphatidylethanolamine (PE) and more phosphatidylcholine (PC) and phosphatidylserine (PS) compared with those of bovine and frog. The content of the polyunsaturated fatty acid, docosahexaenoic acid (DHA), in PC and PS is also the highest yet reported (55 and 63%, respectively). These lipid differences may provide some insight into the specific lipid requirements of cone-type pigments.     

Electrogenic and selective transport of biogenic amines across lipid bilayer membranes mediated by a neutral ionophore (AS701)

The neutral noncyclic imide and ether containing ionophore (AS701), a selective carrier for Li⁺ among alkali cations, was found to be capable of mediating the transport of NH₄⁺ and of biogenic amines (catechols and indoles) across lipid bilayer membranes also. Ionophore-mediated electrical properties of planar lipid bilayers were studied under experimental conditions where the positively-charged amine species was dominant. The ionophore was found to act as a selective carrier of the biogenic amines, mediating their electrogenic transport across the membrane, forming 2:1 carrier-amine permeant complexes, carrying a net-charge of +1. Selectively among the amines corresponding to the following sequence: tryptamine (35) > Li⁺ (1) > serotonin (0.60) > dopamine (0.19) > norepinephrine (0.13) > epinephrine (0.05) > NH₄⁺ (0.05). The molecular factors involved in determining these selectivities are assessed.     

Permeability of bilayer lipid membranes for superoxide (O2) radicals

Egg yolk phosphatidylcholine monolamellar liposomes (1000 Å in diameter) loaded with cytochrome c were placed into an external solution, in which superoxide radicals, O₂⁻, were generated by a xanthine-xanthine oxidase system. The penetration of the superoxide radicals across the liposomal membrane was detected by cytochrome c reduction in the inner liposome compartment. The effects of modifiers and temperature on this process were studied. The permeability of liposomal membrane for O₂⁻(P′_O2⁻ = (7.6 ± 0.3) · 10^-8 cm/s), or HO₂^• (P′_HO2⁻ = 4.9 · 10^-4 cm/s) were determined. The effect of the transmembrane electric potential (K⁺ concentration gradient, valinomycin) on the permeability of liposomal membranes for O₂⁻ were investigated. It was found that O₂⁻ can penetrate across liposomal membrane in an uncharged form. The feasibility of penetration of superoxide radicals through liposomal membrane, predominantly via anionic channels, was demonstrated by the use of an intramolecular cholesterol-amphotericin B complex.     

A hydroxide ion carrier in planar phospholipid bilayer membranes: (C6F5)2Hg (dipentafluorophenylmercury)

Although a number of molecules are known to function as current-carrying proton carriers across lipid bilayer membranes, no such hydroxide ion carriers have been found to date. We report that (C₆F₅)₂ Hg, which can function as a chloride ion carrier, can also carry a hydroxide ion. In 100 mM Na₂SO₄ solutions, membranes treated with (C₆F₅)₂Hg are almost ideally selective for H⁺/OH⁻ between pH 6.0 and 9.5. Membrane conductance varies linearly with [OH⁻] over this pH range and with the square of the (C₆F₅)₂Hg concentration. The presumed current-carrying species is the dimer [(C₆F₅)₂Hg]₂OH⁻, which, along with the neutral molecule (C₆F₅)₂Hg, shuttles back and forth within the bilayer. In 0.2 M NaCl at pH 9.5, the OH⁻ and Cl⁻ conductances are approximately equal. Thus, the carrier displays an approximately 10⁴-fold preference for OH⁻ over Cl⁻.     

Lithium-selective permeation through lipid bilayer membranes mediated by a di-imide ionophore with nonsymmetrical imide substituents (ETH1810)

Summary The neutral, noncyclic Li^–-selective ionophore ETH1810, which is a di-imide, differs structurally from previous similar ionophores by removal of the intramolecular symmetry of the N-imide substituents. Properties of this ionophore, as a potential carrier of lithium, were probed through studies of ionophore-induced changes in electrical properties of lipid bilayer membranes. ETH1810 was found capable of transporting lithium and other monovalent cations, across lipid bilayer membranes, forming 21 ionophore: ion membrane-permeating species. It was found to be 10-fold more potent than ETH1644, which was the previous best ionophore of this type. The selectivity sequence among alkali cations was found to be: Li⁺(1)>Na⁺ (0,009)>K⁺ (0.004)>Cs⁺(0.0035). Among the physiological alkali cations, it constitutes a 40 (vs. Na⁺) to 160% (vs. K⁺) improvement over ETH1644. ETH1810 was also found to be capable of acting as a carrier of biogenic amines and related molecules, with the following selectivity sequence: tryptamine (20)>phenylethylamine (7.8)>tyramine (4.3)>serotonin (2.5)>Li⁺ (1)>NH ₄ ⁺ (0.013)>dopamine (0.012). It was found that protons, at physiological concentrations, do not interfere with the lithium transport mediated by ETH1810. The relationship between the improvements in ionic selectivity and potencyvs. the differences in structural features is discussed.     

Plasmodium berghei: Electron spin resonance and lipid analysis of infected mouse erythrocyte membranes

Red blood cells from mice infected with Plasmodium berghei and from uninfected mice were labeled with stable, free radical derivatives of stearic acid. Electron spin resonance spectra of these samples showed that the degree of molecular order in these membranes decreased, and the rate of motion of the probe increased, with increasing levels of parasitemia. Parasitemia increased the ratio of unsaturated to saturated 18-carbon fatty acids, and decreased the percentage of arachidonic acid and of cholesterol. The effects of parasitemia on the membrane properties correlated with decreases in cholesterol/fatty acid ratios.     

The interaction of phospholipid membranes with poly(ethylene glycol). Vesicle aggregation and lipid exchange

(1) The water soluble polymer, poly(ethylene glycol), causes aggregation of sonicated vesicles of dimyristoylphosphatidylcholine in a manner consistent with a steric exclusion mechanism. (2) Poly(ethylene glycol) promotes the exchange of lipids between multilamellar vesicles of dimyristoylphosphatidylcholine and dipalmitoylphosphatidylcholine when the lipids are in the liquid-crystalline state. (3) ³¹P-NMR studies demonstrate that the bilayer configuration of smectic mesophases of dipalmitoylphosphatidylcholine is substantially maintained in the presence of poly(ethylene glycol).     

A study of conformational stability of poly(L-alanine), poly(L-valine), and poly(L-alanine)/poly(L-valine) blends in the solid state by (13)C cross-polarization/magic angle spinning NMR

13C cross-polarization/magic angle spinning (CP/MAS) NMR and (1)H T(1rho) experiments of poly(L-alanine) (PLA), poly(L-valine) (PLV), and PLA/PLV blends have been carried out in order to elucidate the conformational stability of the polypeptides in the solid state. These were prepared by adding a trifluoroacetic acid (TFA) solution of the polymer with a 2.0 wt/wt % of sulfuric acid (H(2)SO(4)) to alkaline water. From these experimental results, it is clarified that the conformations of PLA and PLV in their blends are strongly influenced by intermolecular hydrogen-bonding interactions that cause their miscibility at the molecular level.     

Bovine pancreatic polypeptide (bPP) undergoes significant changes in conformation and dynamics upon binding to DPC micelles

The pancreatic polypeptide (PP), a 36-residue, C-terminally amidated polypeptide hormone is a member of the neuropeptide Y (NPY) family. Here, we have studied the structure and dynamics of bovine pancreatic polypeptide (bPP) when bound to DPC-micelles as a membrane-mimicking model as well as the dynamics of bPP in solution. The comparison of structure and dynamics of bPP in both states reveals remarkable differences. The overall correlation time of 5.08ns derived from the 15N relaxation data proves unambiguously that bPP in solution exists as a dimer. Therein, intermolecular as well as intramolecular hydrophobic interactions from residues of both the amphiphilic helix and of the back-folded N terminus contribute to the stability of the PP fold. The overall rigidity is well-reflected in positive values for the heteronuclear NOE for residues 4-34.The membrane-bound species displays a partitioning into a more flexible N-terminal region and a well-defined alpha-helical region comprising residues 17-31. The average RMSD value for residues 17-31 is 0.22(+/-0.09)A. The flexibility of the N terminus is compatible with negative values of the heteronuclear NOE observed for the N-terminal residues 4-12 and low values of the generalized order parameter S(2). The membrane-peptide interface was investigated by micelle-integrating spin-labels and H,2H exchange measurements. It is formed by those residues which make contacts between the C-terminal alpha-helix and the polyproline helix. In contrast to pNPY, also residues from the N terminus display spatial proximity to the membrane interface. Furthermore, the orientation of the C terminus, that presumably contains residues involved in receptor binding, is different in the two environments. We speculate that this pre-positioning of residues could be an important requirement for receptor activation. Moreover, we doubt that the PP fold is of functional relevance for binding at the Y(4) receptor.     

Lipid and protein composition and thermotropic lipid phase transitions in fatty acid-homogeneous membranes of Acholeplasma laidlawii B

The membrane composition and lipid physical properties have been systematically investigated as a function of fatty acid composition for a series of Acholeplasma laidlawii B membrane preparations made homogeneous in various fatty acids by growing cells on single fatty acids and avidin, a potent fatty acid synthetic inhibitor. The membrane protein molecular weight distribution is essentially constant as a function of fatty acid composition, but the lipid/protein ratio varies over a 2-fold range when different fatty acid growth supplements are used. The membrane lipid head-group composition varies somewhat under these conditions, particularly in the ratio of the two major neutral glycolipids. Differential thermal analytical investigations of the thermotropic phase transitions of various combinations of membrane components suggest that these compositional changes are unlikely to result in qualitative changes in the nature of lipid-protein or lipid-lipid interactions, although lesser changes of a quantitative nature probably do occur. The total lipids of membranes made homogeneous in their lipid fatty acyl chain composition exhibit sharper than normal gel-to-liquid-crystalline phase transitions of which midpoint temperatures correlate very well with the phase transition temperatures of synthetic hydrated phosphatidylcholines with like acyl chains. Our results indicate that using avidin and suitable fatty acids to grow A. laidlawii B, it is possible to manipulate the position and the sharpness of the membrane lipid phase transition widely and independently without causing major modifications in other aspects of the membrane composition. This fact makes the fatty acid-homogeneous A. laidlawii B membrane a very useful biological membrane preparation in which to study lipid physical properties and their functional consequences.     

Interaction between poly(L-lysine) and membranes inhibits proton pumping by corn root tonoplast H+-ATPase

The influence of poly(L-lysine) binding on the coupled activities of nitrate-sensitive H⁺-ATPase in isolated corn ( Zea mays L. cv. FRB73) root tonoplast vesicles was investigated. The addition of membrane-impermeable poly(L-lysine) caused a slow increase in light scattering of the tonoplast suspension. Electron microscopy showed that the increase was the result of an aggregation of the vesicles. In the presence of 75 m M KCl, a concentration sufficient to sustain near optimal ATP hydrolysis, poly(L-lysine) slightly enhanced the hydrolysis activity but significantly inhibited proton pumping of the H⁺-ATPase. Inhibition increased with the average molecular mass of poly(L-lysine) and reached a maximum at 58 kDa. When total osmolarity was kept constant, the replacement of sucrose by KCl enhanced both ATP hydrolysis and proton pumping activities. However, enhancement of proton pumping was significantly greater than that of ATP hydrolysis. An increase in KCl, but not K₂SO₄, significantly relieved poly(L-lysine)-induced inhibition of proton pumping. Kinetic analysis indicated that poly(L-lysine) did not significantly affect the proton leakage of the tonoplast membranes under different energetic conditions. These results suggest that the electrostatic interaction between poly(L-lysine) and the negative charges on the exterior surface of tonoplast vesicles could change the coupling ratio of ATP hydrolysis to proton pumping. Thus, the surface charge of the tonoplast membrane may be involved in the regulation of these two activities.