Multiple binding sites for local anesthetics in membranes: characterization of the sites and their equilibria by deuterium NMR of specifically deuterated procaine and tetracaine

Anesthetics bound to model membranes were observed directly by means of deuterium nuclear magnetic resonance (NMR). The specifically deuterated local anesthetics procaine and tetracaine were synthesized, and their partition coefficients (water:phosphatidylcholine) and pKa values determined. The interaction of these anesthetics with lamellar dispersions of egg phosphatidylcholine was studied by 2H nuclear magnetic resonance and by electron spin resonance (ESR) of a spin-labelled phospholipid at low (5.5) and high (9.5) pH. The ESR experiments suggest that tetracaine intercalates in the membrane and that it equilibrates between water and the phospholipid bilayers of the multilamellar system. The NMR results are consistent with a model where the anesthetic is (1) free in water, (2) weakly bound, and (3) strongly bound to the membrane. A fast exchange exists between the two first sites, but exchange is slow with the third site. Binding of type 3 is observed only at high pH for procaine, whereas it is found both at low and high pH for tetracaine. Calculations of the partition coefficients for the charged and uncharged forms of tetracaine indicate that both sites, 2 and 3, are occupied by the charged form at low pH and by the uncharged form at high pH. The partition coefficient for the weakly bound species was estimated from an analysis of the dependence of line width on the lipid to water ratio. The NMR data suggest that the binding sites for the strongly bound charged and uncharged species are different, the former probably being closer to the membrane-water interface. Estimates of molecular order parameters for the strongly bound species indicate that it is located with its long molecular axis approximately parallel to the director for ordering of the fatty acyl chains. A small increase in lipid ordering by tetracaine is observed at low pH, as evidenced by 2H NMR of the deuterated N-methyl groups of phosphatidylcholine; the reverse occurs at high pH.     

Interactions of the local anesthetic tetracaine with membranes containing phosphatidylcholine and cholesterol: a 2H NMR study

The interactions of the local anesthetic tetracaine with multilamellar dispersions of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and cholesterol have been investigated by deuterium nuclear magnetic resonance of specifically deuteriated tetracaines, DMPC and cholesterol. Experiments were performed at pH 5.5, when the anesthetic is primarily charged, and at pH 9.5, when it is primarily uncharged. The partition coefficients of the anesthetic in the membrane have been measured at both pH values for phosphatidylcholine bilayers with and without cholesterol. The higher partition coefficients obtained at pH 9.5 reflect the hydrophobic interactions between the uncharged form of the anesthetic and the hydrocarbon region of the bilayer. The lower partition coefficients for the DMPC/cholesterol system at both pH values suggest that cholesterol, which increases the order of the lipid chains, decreases the solubility of tetracaine into the bilayer. For phosphatidylcholine bilayers, it has been proposed [Boulanger, Y., Schreier, S., & Smith, I. C. P. (1981) Biochemistry 20, 6824-6830] that the charged tetracaine at low pH is located mostly at the phospholipid headgroup level while the uncharged tetracaine intercalates more deeply into the bilayer. The present study suggests that the location of tetracaine in the cholesterol-containing system is different from that in pure phosphatidylcholine bilayers: the anesthetic sits higher in the membrane. An increase in temperature results in a deeper penetration of the anesthetic into the bilayer. Moreover, the incorporation of the anesthetic into DMPC bilayers with or without cholesterol results in a reduction of the lipid order parameters both in the plateau and in the tail regions of the acyl chains, this effect being greater with the charged form of the anesthetic.     

A 2H-NMR study on the interactions of the local anesthetic tetracaine with membranes containing phosphatidylserine

The interaction of the local anesthetic tetracaine with phosphatidylserine-containing model membranes has been studied by 2H-NMR. Charged tetracaine exhibited an unusually large partition coefficient into multilamellar dispersions of phosphatidylserine. The 2H-NMR spectra consisted of a Pake doublet and a narrow line, with the former corresponding to tetracaine in the bilayer and the latter to tetracaine free in solution. A strong pH dependence of the quadrupole splittings indicated different membrane locations for charged and uncharged tetracaine. In equimolar mixtures of phosphatidylserine and phosphatidylcholine the partition coefficients and 2H-NMR spectra were much more like those observed in neat phosphatidylcholine than in neat phosphatidylserine. Dilution studies at pH 5.5 indicated that in phosphatidylserine/phosphatidylcholine mixtures tetracaine experiences a three-site exchange similar to that found earlier for tetracaine in phosphatidylcholine. Tetracaine is in fast exchange between sites weakly bound to membrane and free in solution, and in slow exchange with a strongly bound site in the membrane.     

The application of pH-sensitive spin labels to studies of surface potential and polarity of phospholipid membranes and proteins.

The effects of pH titration on the EPR spectra of imidazolidine nitroxides located at the surface of mixed bilayers composed of dimyristoylphosphatidylglycerol (DMPG) and dimyristoylphosphatidylcholine (DMPC), and at the surface of the protein, human serum albumin (HSA), have been investigated. It is found that the shift in pKa of the amino group of the imidazolidine radical from its value of 4.6 in water depends both on the interfacial polarity (delta pKapol) and on the electrostatic surface potential (delta pKael) when it is positioned at the bilayer/water interface by an anchoring hydrocarbon tail. The polarity shift is determined to be: delta pKapol = -1.3 units at the surface of DMPC bilayers at 17 degrees C, corresponding to an effective interfacial dielectric constant of epsilon approximately 37, and depends on the temperature with a coefficient of d delta pKapol/dT approximately -0.01 per degree. The electrostatic shift at the surface of DMPG bilayers is delta pKael = +1.6 units in 0.1 M KCl, which corresponds to an electrostatic surface potential of -95 mV. This electrostatic shift depends strongly both on ionic strength and on the fraction of charged lipid in the DMPC/DMPG mixtures, in a manner that agrees with the predictions of electrostatic double-layer theory. It is found that the shift in pKa of an imidazolidine radical covalently bound at the surface of HSA is determined mainly by the surface electrostatics (delta pKapol approximately 0) and corresponds to an electrostatic potential of +33 mV in 0.01 M KCl at a pH below the isoelectric point of the protein.     

Influence of electrostatic interactions on partitioning in aqueous polyethylene glycol/dextran biphasic systems: Part I

To study the influence of charges on the partition of solutes in aqueous two-phase systems of polyethylene glycol and dextran, partition coefficients of dimethylaminoethyl-dextran, trimethylamino-dextran, and bis (alpha,omega)-amino-poly(ethylene glycol) were determined as a function of pH (range 2 to 12) and ionic strength. These polymers are derivatives of the phase forming components and carry ionizable groups that are charged or uncharged depending on the pH. Unexpectedly, the largest differences in the partition coefficients were found at high pH, where the modified polymers are uncharged. In addition, the partitioning of low-molecular-weight model compounds, ethylenediamine and iminodiacetic acid, as well as poly-L-lysine and poly(allylamine) was analyzed. A consistent pattern was observed in the partition of polyelectrolytes reflecting the influence of charge, but another property of aqueous phase systems unrelated to charge and changing with pH seems to be superimposed. (c) 1995 John Wiley & Sons, Inc.     

Effect of sodium octanoate and sodium perfluorooctanoate on gel-to-liquid-crystalline phase transition of dipalmitoylphosphatidylcholine vesicle membrane

The gel-to-liquid-crystalline phase transition of dipalmitoylphosphatidylcholine (DPPC) vesicle membrane was measured in the presence of sodium octanoate (SO) (pH 3 and 10) and sodium perfluorooctanoate (SPFO) (pH uncontrolled) by monitoring the scattered light intensity of the vesicle suspension. The phase transition temperature, Tm, decreased linearly with the concentration of added SO within the measured concentration range; the uncharged form of SO (pH 3) was much more effective for the depression of Tm than the charged form (pH 10). On the other hand, with increasing SPFO concentration, levelling off of Tm was observed after depression at an initial stage. From the depression of Tm, the partition coefficients, K, of these surfactants between bulk solution and DPPC vesicle membrane were estimated and compared with those obtained previously for other surfactant systems. The value of K for charged SO fell on the straight line of log K vs. Nc plot for anionic surfactants, where Nc is the carbon number of the hydrocarbon chain of surfactants, whereas K for uncharged SO showed a large positive deviation from the straight line of the plot for non-ionic surfactants. The latter suggested that some specific interaction, presumably hydrogen bond formation, may act between the protonated carboxyl group of SO and the lipid head group. The K value estimated for SPFO was much larger than that for charged SO. This difference in the affinity for the lipid bilayer between fluorocarbon surfactant and hydrocarbon surfactant may be attributed to the difference in their hydrophobicity.     

On the mechanism of substrate binding to the purine-transport system of Saccharomyces cerevisiae.

The yeast Saccharomyces cerevisiae takes up adenine, guanine, hypoxanthine, and cytosine via a common energy-dependent transport system. The apparent affinity of the transport system to these and other purines and pyrimidines is correlated with their capability to be protonated to the positively charged form. Further organic molecules are competitive inhibitors when they are cationic, e.g. guanidine and octylguanidine in contrast to urea, or hexadecyltrimethylammonium in contrast to dodecylsulfate and Triton X-100. The influence of the pH on the kinetic constants of hypoxanthine transport points to a stoichiometry of one proton being associated to the transport system together with one substrate molecule. The pKa values of two ionizable groups that are involved in substrate binding are revealed; one of which (pKa = 1.8) may be attributed to the substrate, the other (pKa = 5.1) to an amino acid residue in the recognition site of the transport system. Studies with group-specific inhibitors indicate that this amino acid residue contains a carboxyl group. The results are in accordance with the assumption that a carboxyl group of the transport system, a proton and a substrate molecule arrange to an uncharged ternary complex.     

EPR studies on the influence of chain length on the segmental motion of spin-labelled fatty acids in dimyristoylphosphatidylcholine bilayers.

The rotational dynamics of spin-labelled fatty acids of different chainlengths (9, 10, 12, 14, 16 and 18 C-atoms) and different positions of labelling (5-C, 6-C and 7-C) have been studied in dimyristoylphosphatidylcholine bilayers using EPR spectroscopy. The segmental flexibility at a given label position is found to vary considerably with the length of the lipid chain, when this is less than that of the dimyristoylphosphatidylcholine host lipid. For both the charged and protonated forms of labelled fatty acids with chainlengths of 9, 10, and 12 C-atoms, the spectral anisotropy decreases steadily with decreasing chainlength in fluid phase bilayers. The differences become especially pronounced at the 7-C position of caprylic acid and the 6-C position of nonanoic acid, where the label is located close to the terminal methyl end of the chain. An unusually high degree of motional freedom is found for both these spin-labels, even in gel phase bilayers. There is relatively little effect of chainlength of the labelled fatty acid when this is longer or comparable to that of the host lipid (i.e., for fatty acid chainlengths of 18, 16 and 14 C-atoms), except if the label position is close to the terminal methyl end of the chain. The implications for the heterogeneous lipid chain composition in biological membranes are discussed.     

Clustering of fatty acids in phospholipid bilayers.

From electrophoresis experiments it is concluded that acidic phospholipids incorporated in liquid crystalline phosphatidylcholine bilayers at neutral pH are randomly distributed. The same is true for spin-labelled fatty acids. In contrast, long chain fatty acids are not fully ionized at neutral pH and appear to be clustered, i.e. they segregate out into patches. Only at pH greater than 11 is the fatty acid-COOH group fully ionized and charge repulsion leads to a random distribution of the fatty acid within the plane of the bilayer.     

Molecular dynamics simulations of local anesthetic articaine in a lipid bilayer

In order to investigate structural and dynamical properties of local anesthetic articaine in a model lipid bilayer, a series of molecular dynamics simulations have been performed. Simulations were carried out for neutral and charged (protonated) forms of articaine inserted in fully hydrated dimyristoylphosphatidylcholine (DMPC) lipid bilayer. For comparison purpose, a fully hydrated DMPC bilayer without articaine was also simulated. The length of each simulation was 200 ns. Various properties of the lipid bilayer systems in the presence of both charged and uncharged forms of articaine taken at two different concentrations have been examined: membrane area per lipid, mass density distributions, order parameters, radial distribution functions, head group tilt, diffusion coefficients, electrostatic potential, etc, and compared with results of previous simulations of DMPC bilayer in the presence of lidocaine. It was shown that addition of both charged and neutral forms of articaine causes increase of the dipole electrostatic potential in the membrane interior.     

Penetration of ascorbic acid into bilayer phospholipid membranes

Using the EPR method, the temperature dependencies of the rates of ascorbic acid-induced reduction of nitroxyl radicals carrying the nitroxyl fragment in different positions of the fatty acid chain [N(4-methylidene++-1-oxyl-2,2,5,5-tetramethyl-3-imidazolidine hydrazine)]myristic acid (I) and 1-oxyl-2,2-dimethyloxazolidine derivatives of 5-ketostearic (II) and 12-ketostearic (III) acids incorporated into egg phosphatidylcholine liposomal membranes were studied. The reduction rates, activation energy and shape of kinetic curves were found to be dependent on the mode of liposome preparation (ultrasonication or reverse phase evaporation), label type and chemical composition of the membrane (with regard to the presence or absence of stearic acid). The coefficients of partition and diffusion of ascorbic acid through the membrane lipid bilayer were calculated from the rates of transbilayer (flip-flop) diffusion of I and ascorbate penetration inside the liposomes containing Fremi salt nitroxyl radical. The experimental results formed the basis for a hypothesis on the dependence of the rate of membrane-embedded spin probe reduction on the ascorbate distribution pattern inside the lipid bilayer.     

Local anesthetic-induced microscopic and mesoscopic effects in micelles. A fluorescence,spin label and SAXS study

The interaction of the local anesthetic tetracaine (TTC) with anionic sodium lauryl sulfate (SLS) and zwitterionic 3-(N-hexadecyl-N,N-dimethylammonio)propanesulfonate (HPS) micelles was investigated by fluorescence, spin labeling EPR and small angle X-ray scattering (SAXS). Fluorescence pH titrations allowed the choice of adequate pHs for the EPR and SAXS experiments, where either charged or uncharged TTC would be present. The data also indicated that the anesthetic is located in a less polar environment than its charged counterpart in both micellar systems. EPR spectra evidenced that both anesthetic forms increased molecular organization within the SLS micelle, the cationic form exerting a more pronounced effect. The SAXS data showed that protonated TTC causes an increase in the SLS polar shell thickness, hydration number, and aggregation number, whereas the micellar features are not altered upon incorporation of the uncharged drug. The combined results suggest that the electrostatic interaction between charged TTC and SLS, and the intercalation of the drug in the micellar polar region induce a change in molecular packing with a decrease in the mean cross-sectional area, not observed when the neutral drug sinks more deeply into the micellar hydrophobic domain. In the case of HPS micelles, the EPR spectral changes were small for the charged anesthetic and the SAXS data did not evidence any change in micellar structure, suggesting that this species protrudes more into the aqueous phase due to the lack of electrostatic attractive forces in this system.     

Local anesthetic-induced microscopic and mesoscopic effects in micelles. A fluorescence, spin label and SAXS study. Single angle X-ray scattering

The interaction of the local anesthetic tetracaine (TTC) with anionic sodium lauryl sulfate (SLS) and zwitterionic 3-(N-hexadecyl-N,N-dimethylammonio)propanesulfonate (HPS) micelles was investigated by fluorescence, spin labeling EPR and small angle X-ray scattering (SAXS). Fluorescence pH titrations allowed the choice of adequate pHs for the EPR and SAXS experiments, where either charged or uncharged TTC would be present. The data also indicated that the anesthetic is located in a less polar environment than its charged counterpart in both micellar systems. EPR spectra evidenced that both anesthetic forms increased molecular organization within the SLS micelle, the cationic form exerting a more pronounced effect. The SAXS data showed that protonated TTC causes an increase in the SLS polar shell thickness, hydration number, and aggregation number, whereas the micellar features are not altered upon incorporation of the uncharged drug. The combined results suggest that the electrostatic interaction between charged TTC and SLS, and the intercalation of the drug in the micellar polar region induce a change in molecular packing with a decrease in the mean cross-sectional area, not observed when the neutral drug sinks more deeply into the micellar hydrophobic domain. In the case of HPS micelles, the EPR spectral changes were small for the charged anesthetic and the SAXS data did not evidence any change in micellar structure, suggesting that this species protrudes more into the aqueous phase due to the lack of electrostatic attractive forces in this system.     

Clustering of fatty acids in phospholipid bilayers

From electrophoresis experiments it is concluded that acidic phospholipids incorporated in liquid crystalline phosphatidylcholine bilayers at neutral pH are randomly distributed. The same is true for spin-labelled fatty acids. In contrast, long chain fatty acids are not fully ionized at neutral pH and appear to be clustered, i.e. they segregate out into patches. Only at $\text{pH}\text{>11}$ is the fatty acid-COOH group fully ionized and charge repulsion leads to a random distribution of the fatty acid within the plane of the bilayer.     

Partitional and motional properties of a spin-labeled daunomycin in lipid bilayers. An ESR study.

The partition coefficient of a spin-labeled daunomycin (DAU-SL) in dimyristoylphosphatidylcholine membrane has been determined using the electron spin resonance (ESR) method. The experiment was carried out as a function of temperature between 5 degrees C and 35 degrees C, giving partition coefficients between 2 and 6 without abrupt change at the phase transition. The thermodynamic parameters on transferring the DAU-SL from the aqueous phase to the lipid bilayer were also calculated. The calculated values are: delta H = 6.11 kcal/mol and delta S = 23 cal/K mol. The partitioning of the DAU-SL and its motion in the membrane were investigated in a wide range of pH (4-10.3). The data show that pH has no effect on partitioning of the DAU-SL which suggest that the drug exists in the uncharged form in the bilayer.     

Spin-labeled extracellular loop from a seven-transmembrane helix receptor: studies in solution and interaction with model membranes

A spin-labeled pentadecapeptide was synthesized containing 2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid (TOAC) as the N-terminal amino acid and residues 253-266 (EYWSTFGNLHHISL) of the mass oncogene receptor, a membrane-bound protein from the G-protein coupled receptors family. According to predictions, this protein folds into seven transmembrane helices connected by three extra- and three intracellular loops, and the peptide encompasses part of the third extracellular loop and part of the seventh helix. Electron paramagnetic resonance (EPR) spectra of the spin-labeled peptide (TOAC-14) were obtained in aqueous solution as a function of pH and temperature, in a secondary structure-inducing solvent [trifluoroethanol (TFE)], and in the presence of detergent micelles and phospholipid bilayers. The charged and uncharged amino groups of TOAC and TOAC-14 yielded spectra with different isotropic hyperfine splittings (aN). The slow exchange between protonated and unprotonated forms in the EPR time scale gave rise to composite spectra weighted by the Henderson-Hasselbalch equation. Plots of aN vs pH allowed the determination of the amino group pK values (8.4 and 4.5, for TOAC and TOAC-14, respectively). A small change in aN centered at pH 6.5 was ascribed to the titration of the histidines. Values of calculated rotational correlation times were indicative of a pH-induced conformational change. A conformational change was also observed in TFE. TOAC-14 bound to micelles irrespective of peptide and detergent head group charge. In contrast, the peptide bound to phospholipid bilayers only when both carried opposite charges. The slow exchange (in the EPR time scale) between membrane-bound and free TOAC-14 allowed the calculation of the peptide's partition coefficient. The spectral line shapes were affected by aggregate size and degree of packing of the constituent molecules. It is proposed that pH, polarity, and lipid environment can affect the conformation of water-exposed regions of membrane-bound receptors, thereby playing a role in the mechanism of signal transduction.     

Transfer of long-chain fluorescent free fatty acids between unilamellar vesicles

Movement of free fatty acids (ffa) between small unilamellar vesicles (SUV) was studied by measuring the transfer of fluorescent n-(9-anthroyloxy)-labeled analogues (AOffa) between donor and acceptor vesicles. Donors were composed of egg phosphatidylcholine (PC) loaded with 1-2 mol % AOffa, and acceptors were egg PC containing 10-12 mol % N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine (NBD-PE). The fluorescence of AO added directly to acceptor SUV was greater than 98% quenched by energy transfer to NBD. Thus, AOffa movement from donor to acceptor was monitored by the time-dependent decrease in AO fluorescence. The transfer of the short-chain AOffa, although too fast to be resolved by the methods used here, is consistent with studies that find transfer rates on the order of milliseconds and kinetics which are first order. In contrast, transfer rates for the long-chain AOffa are more than 2 orders of magnitude slower, and the kinetics of the transfer process are best described by the sum of two exponentials plus a constant. The ffa ionization state was also found to be an important determinant of transfer rate. The charged species transferred an average of 10-fold faster than the protonated ffa. The ffa pKa in the membrane is 9, as calculated from the pH dependence of transfer. Similar to results found for other lipids, long-chain AOffa are transferred via water rather than a collision-mediated process. The aqueous phase route of AOffa intermembrane transfer is indicated by the lack of effect on transfer of large alterations in the product of donor and acceptor phospholipid concentrations. Moreover, the transfer rate is decreased as [NaCl] is increased from 0.1 to 4 M. This effect of ionic strength is probably due not only to a decrease in the aqueous phase partition of the AOffa but also to an alteration in bilayer structure, as measured by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene. The observed kinetics are consistent with a model in which the transfer involves two steps: transbilayer movement between the inner and outer bilayer leaflets, followed by transfer from the outer leaflet to the aqueous phase (off rate). Within the framework of this model, the observed slow rate is primarily determined by the rate of transbilayer movement, and the observed fast rate is approximately equal to the off rate. The off rate is about 10-fold faster than the rate of transbilayer movement.     

Ionization states of the complex formed between 2-benzyl-3-phosphonopropionic acid and carboxypeptidase A.

The binding to carboxypeptidase A of two phosphonic acid analogues of 2-benzylsuccinate, 2-DL-2-benzyl-3-phosphonopropionic acid (inhibitor I) and 2-DL-2-benzyl-3-(-O-ethylphosphono)propionic acid (inhibitor II) was studied by observing their 31P resonances when free and bound to the enzyme in the range of pH from 5 to 10. The binding of I by co-ordination to the active-site Zn(II) lowered the highest pKa of I from a value of 7.66(+/- 0.10) to a value of 6.71(+/- 0.17). No titration of any protons on II occurred over the pH range studied. The enzyme-bound inhibitor II also did not titrate over the pH range 6.17-7.60. The pH-dependencies of the apparent inhibition constants for I and II were also investigated by using N-(-2-(furanacryloyl)-L-phenylalanyl-L-phenylalanine as substrate. Two enzymic functional groups with pKa values of 5.90(+/- 0.06) and 9.79(+/- 0.14) must be protonated for binding of inhibitor I, and two groups with pKa values of 6.29(+/- 0.10) and 9.19(+/- 0.15) for binding of inhibitor II. Over the pH range from 6.71 to 7.66, inhibitor I binds to the enzyme in a complex of the enzyme in a more protonated form, and the inhibitor in a less protonated form than the predominant unligated forms at this pH. Mock & Tsay [(1986) Biochemistry 25, 2920-2927] made a similar finding for the binding of L-2-(1-carboxy-2-phenylethyl)-4-phenylazophenol over a pH range of nearly 4 units. The true inhibition constant for the dianionic form of inhibitor I (racemic) was calculated to be 54.0(+/- 5.9) nM and that of the trianionic form to be 5.92(+/- 0.65) nM. The true inhibition constant of the fully ionized II (racemic) was calculated to be 79.8(+/- 6.4) nM.     

Lipid-protein interactions in frog rod outer segment disc membranes. Characterization by spin labels

Freely-diffusing phospholipid spin labels have been employed to study rhodopsin-lipid interactions in frog rod outer segment disc membranes. Examination of the ESR spectra leads us to the conclusion that there are two motionally distinguishable populations of lipid existing in frog rod outer segment membranes over a wide physiological temperature range. Each of the spin probes used shows a two-component electron spin resonance (ESR) spectrum, one component of which is motionally restricted on the ESR timescale, and represents between 33 and 40% of the total integrated spectral intensity. The second spectral component which accounts for the remainder of the spectral intensity possesses a lineshape characteristic of anisotropic motion in a lipid bilayer, very similar in shape to that observed from the same spin labels in dispersions of whole extracted frog rod outer segment lipid. The motionally restricted spectral component is attributed to those spin labels in contact with the surface of rhodospin, while the major component is believed to originate from spin labels in the fluid lipid bilayer region of the membranes. Calculations indicate that the motionally restricted lipid is sufficient to cover the protein surface. This population of lipids is shown here and elsewhere (Watts, A., Volotovski, I.D. and Marsh, D. (1979) Biochemistry 18, 5006-5013) to be by no means rigidly immobilized, having motion in the 20 ns time regime as opposed to motions in the one nanosecond time regime found in the fluid bilayer. Little selectivity for the motionally restricted population is observed between the different spin-labelled phospholipid classes nor with a spin-labelled fatty acid or sterol.     

Studies on the aminopeptidase activity of rat cathepsin H.

Three synthetic substrates H-Arg-NH-Mec, Bz-Arg-NH-Mec and H-Cit-NH-Mec (Bz, Benzoyl; NH-Mec, 4-methylcoumaryl-7-amide; Cit, citrulline) were used to characterize specificity requirements for the P1-S1 interaction of cathepsin H from rat liver. From rapid equilibrium kinetic studies it was shown that Km, kcat and the specificity constants kcat/Km are quite similar for substrates with a free alpha-amino group. In contrast, a 25-fold decrease of kcat/Km was observed for the N-terminal-blocked substrate Bz-Arg-NH-Mec. The activation energies for H-Arg-NH-Mec and Bz-Arg-NH-Mec were determined to be 37 kJ/mol and 55 kJ/mol, respectively, and the incremental binding energy delta delta Gb of the charged alpha-amino group was estimated to -8.1 kJ/mol at pH 6.8. The shown preference of cathepsin H for the unblocked substrates H-Arg-NH-Mec and H-Cit-NH-Mec was further investigated by inspection of the pH dependence of kcat/Km. The curves of the two substrates with a charged alpha-amino group showed identical bell-shaped profiles which both exhibit pKa1 and pKa2 values of 5.5 and 7.4, respectively, at 30 degrees C. The residue with a pKa1 of 5.5 in the acid limb of the activity profile of H-Arg-NH-Mec was identified by its ionization enthalpy delta Hion = 21 kJ/mol as a beta-carboxylate or gamma-carboxylate of the enzyme, whereas the residue with a pKa2 of 7.4 was assigned to the free alpha-amino group of the substrate with a delta Hion of 59 kJ/mol. Bz-Arg-NH-Mec showed a different pH-activity profile with a pKa1 of 5.4 and a pKa2 of 6.6 at 30 degrees C. Cathepsin H exhibits no preference for a basic P1 side chain as has been shown by the similar kinetics of H-Arg-NH-Mec and the uncharged, isosteric substrate H-Cit-NH-Mec. In summary, specific interactions of an anionic cathepsin H active site residue with the charged alpha-amino group of substrates caused transition state stabilization which proves the enzyme to act preferentially as an aminopeptidase.