B.S. Prabhananda

Monensin-mediated transports of H+, Na+, K+ and Li+ ions across vesicular membranes: T-jump studies

Theoretical expression for the rate of decay of delta pH across vesicular membrane due to carrier-mediated ion transports, 1/tau, has been modified taking note of carrier states (such as mon- and mon-H-M+) for which the translocation rate constants in the membrane are small. The rates of delta pH decay due to monensin-mediated H+ and M+ transports (M+ = Na+, K+, Li+) observed in our experiments in the pH range 6-8, and [M+] range 50-250 mM at 25 degrees C have been analysed with the help of this expression. delta pH across soybean phospholipid vesicular membranes were created by temperature jump in our experiments. The following could be inferred from our studies. (a) At low pH (approximately 6) 1/tau in a medium of Na+ is greater than that in a medium of K+. In contrast with this, at higher pH (approximately 7.5) 1/tau is greater in a medium of K+. Such contradictory observations could be understood with the help of our equation and the parameters determined in this work. The relative concentrations of the rate-limiting species (mon-H, mon-K, and mon-Li at Ph approximately 7 in vesicle solutions having Na+, K+ and Li+, respectively) can explain such behaviours. (b) The proton dissociation constant KH for mon-H in the lipid medium (pKH approximately 6.55) is larger than the reported KH in methanol. (c) The concentrations of mon- and mon-H-Na+ are not negligible under the conditions of our experiments. The latter species cause a [Na+]-dependent inhibition of ion transports. (d) The relative magnitudes of metal ion dissociation constants KHM (approximately 0.05 M) for mon-H-Na+ and KM (approximately 0.03 M) for mon-Na suggest that the carboxyl group involved in the protonation may not be dominantly involved in the metal ion complexation. (e) The estimates of KM (approximately 0.03 M for Na+, 0.5 M for K+ and 2.2 M for Li+) follow the ionophore selectivity order. (f) The rate constants k1 and k2 for the translocations of mon-H and mon-M (M+ = Na+, K+ and Li+) are similar in magnitude (approximately 9 x 10(3) s-1) and are higher than that for nig-H and nig-M (approximately 6 x 10(3) s-1) which can be expected from the relative molecular sizes of the ion carriers.

Nigericin-mediated H+, K+ and Na+ transports across vesicular membrane: T-jump studies.

The decay of delta pH across vesicular membranes by nigericin-mediated H+ and metal ion (M+) transports has been studied at 25 degrees C after creating delta pH by temperature jump (T-jump). In these experiments K+ or Na+ were chosen as M+ for the compensating flux. Theoretical expressions derived to analyse these data suggest a method for estimating the intrinsic rate constants for the translocation of nig-H (k1) and for the translocation of nig-M (k2) across membrane, from the pH dependence of the delta pH decay. The following could be inferred from the analysis of data. (a) At pH approximately 7.5 and 250 mM ion concentrations, nigericin-mediated H+ and M+ transport rates are lower in a medium of K+ than in a medium of Na+, although ionophore selectivity of nigericin towards K+ is 25-45-times higher than that towards Na+. However, at lower [M+] (approximately 50 mM) the transport rates are higher in a medium of K+ than in a medium of Na+. Such behaviours can be understood with the help of parameters determined in this work. (b) The intrinsic rate constants k1 and k2 associated with the translocations of nig-H and nig-K or nig-Na across membrane are similar in magnitude. (c) At pH approximately 7.5 translocation of nig-H is the dominant rate-limiting step in a medium containing K+. In contrast with this, at this pH, translocation of nig-M is the dominant rate-limiting step when metal ion is Na+. (d)k1 approximately k2 approximately 6.10(3) s-1 could be estimated at 25 degrees C in vesicles prepared from soyabean phospholipid, and lipid mixtures of 80% phosphatidylcholine (PC) + 20% phosphatidylethanolamine and 92% PC + 8% phosphatidic acid. (e) The apparent dissociation constants of nig-M in vesicles were estimated to be approximately 1.5.10(-3) M for K+ and 6.4.10(-2) M for Na+ (at 50 mM ion concentrations) using approximately 10(-8.45) M for the apparent dissociation constant of nig-H.

Effect of phloretin on ionophore mediated electroneutral transmembrane translocations of H(+), K(+) and Na(+) in phospholipid vesicles.

Rates of M(+)/H(+) exchange (M(+)=K(+), Na(+)) across phospholipid membranes by ionophore mediated electroneutral translocations and transports through channels could either increase or decrease or change negligibly on adding the polar molecule phloretin to the membrane. The changes depend on pH, the concentration and choice of M(+) and choice of ionophore/channel. Such diverse behaviours have been inferred from studies on the decay of the pH difference across soybean phospholipid vesicular membrane (=Delta pH). The transporters used in this study are (a) the exchange ionophores: nigericin, monensin; (b) combinations of alkali metal ion carriers, valinomycin or nonactin with weak acids carbonyl cyanide m-chlorophenylhydrazone or 2,4-dinitrophenol and (c) channels formed by gramicidin A. All the diverse results can be rationally explained if we take note of the following. (i) The rate limiting steps are associated with the transmembrane translocations involving the rate limiting species identified in the literature. (ii) Phloretin in the membrane decreases the apparent M(+) dissociation constant, K(M), of the M(+) bound ionophores/channels which has the effect of increasing the concentration of these species. (iii) The concentrations of H(+) bound ionophores/channels decrease on adding phloretin. (iv) Phloretin inhibits ternary complex formation (involving valinomycin or nonactin, M(+) and an anion) by forming 1:2 complexes with valinomycin-M(+) or nonactin-M(+). (v) On adding 6-ketocholestanol to the membrane (instead of phloretin) K(M) increases. The decreases/increases in K(M) mentioned above are consistent with the consequences of a hypothesis in which phloretin decreases and 6-ketocholestanol increases the positive internal membrane dipole potential.

Enhancement of rates of H+, Na+ and K+ transport across phospholipid vesicular membrane by the combined action of carbonyl cyanide m-chlorophenylhydrazone and valinomycin: temperature-jump studies.

Enhancement of delta pH relaxation rate by the combined action of valinomycin (VAL) and carbonyl cyanide m-chlorophenylhydrazone (CCCP) has been studied under a variety of concentration conditions in soyabean phospholipid (SBPL) vesicles after creating a pH gradient across the vesicular membrane delta pH by temperature jump. After taking note of the changes by VAL and CCCP induced membrane disorder (using nigericin and monensin mediated delta pH decay as probes) the following could be inferred about the mechanism of enhancement of delta pH decay rate: (i) in solutions containing KCl, the rate limiting species have been identified to be (a) Val-K(+)-CCCP-, at low [Val]0 and [CCCP]0 (with translocation rate constant k2 approximatley 3.2 x 10(3) s-1); (b) CCCPH, at high [Val]0 (with translocation rate constant k1 approximately 2 x 10(5) s-1); (c) the neutral valinomycin species Val, at high [CCCP]0. (ii) In solutions containing NaCl, in our concentration range, the rate limiting species are Val-Na(+)-CCCP-. (iii) The apparent dissociation constant K*M of Val-M+ decreases with pH in SBPL vesicles but is independent of pH in vesicles prepared from PC + 6% PA. (iv) The differences in the ionic strength dependencies of kinetic data shows that the environments of Na+ and K+ binding sites on VAL are different. (v) In vesicle solutions containing 100 mM MCl, the cation selectivity of VAL (towards K+ in preference to Na+) is reduced when CCCP- is already bound to it in the membrane. The CCCP- dissociation constant of Val-M(+)-CCCP- is smaller with M+ = Na+ (approximatley 0.22 mM at 100 mM NaCl) when compared to that with M+ = K+ (approximately 2 mM at 100 mM KCl). Attributing these differences to the differences in electrostatic interaction between CCCP- and M+ in Val-M(+)-CCCP-, we can say that CCCP- binds closer to the Na+ binding site than to the K+ binding site on VAL.

H+, K+, and Na+ transport across phospholipid vesicular membrane by the combined action of proton uncoupler 2,4-dinitrophenol and valinomycin.

The decay of the pH difference (delta pH) across soyabean phospholipid vesicular membrane (created by temperature jump), by the combined action of valinomycin and 2,4-dinitrophenol (DNP) has been monitored with the help of fluorescence from pyranine entrapped inside the vesicles under a variety of concentration conditions. The results suggest the following for the pH region of our interest (pH approximately 6 to pH approximately 8): (i) The rate limiting step in the proton transport cycle is not the transport of proton as DNPH, but the back transport of DNP- and the alkali metal ion M+ as Val-M(+)-DNP- across the membrane. The rate constant associated with the transport of the ternary complex has been estimated to be approximately 1.5 x 10(3) s-1. (ii) The dissociation constant of the ternary complex Val-M(+)-DNP- in the membrane are approximately 1 mM for M+ = K+ and approximately 0.001 mM for M+ = Na+. (iii) The reduction in the cation selectivity of valinomycin on complexing with DNP- is much more than that observed with the anionic form of carbonyl cyanide m-chlorophenylhydrazone (CCCP). The results also provide a verification of a corollary of Mitchells hypothesis: an experimental strategy which enhances the delta pH decay rate should also be a strategy for the efficient uncoupling of oxidative and photophosphorylation.

Evidence for dimer participation and evidence against channel mechanism in A23187-mediated monovalent metal ion transport across phospholipid vesicular membrane.

The decay of the pH difference (DeltapH) across soybean phospholipid vesicular membrane by ionophore A23187 (CAL)-mediated H+/M+ exchange (M+ = Li+, Na+, K+, and Cs+) has been studied in the pH range 6-7.6. The DeltapH in these experiments were created by temperature jump. The observed dependence of DeltapH relaxation rate 1/tau on the concentration of CAL, pH, and the choice of M+ in vesicle solutions lead to the following conclusions. 1) The concentrations of dimers and other oligomers of A23187 in the membrane are small compared to the total concentration of A23187 in the membrane, similar to that in chloroform solutions reported in the literature. 2) In the H+ transport cycle leading to DeltapH decay, the A23187-mediated H+ translocation across the membrane is a fast step, and the rate-limiting step is the A23187-mediated M+ translocation. 3) Even though the monomeric Cal-H is the dominant species translocating H+, Cal-M is not the dominant species translocating M+ (even at concentrations higher than [Cal-H]), presumably because its dissociation rate is much higher than its translocation rate. 4) The pH dependence of 1/tau shows that the dimeric species Cal2LiLi, Cal2NaNa, Cal2KH, and Cal2CsH are the dominant species translocating M+. The rate constant associated with their translocation has been estimated to be approximately 5 x 10(3) s-1. With this magnitude for the rate constants, the dimer dissociation constants of these species in the membrane have been estimated to be approximately 4, 1, 0.05, and 0.04 M, respectively. 5) Contrary to the claims made in the literature, the data obtained in the DeltapH decay studies do not favor the channel mechanism for the ion transport in this system. 6) However, they support the hypothesis that the dissociation of the divalent metal ion-A23187 complex is the rate limiting step of A23187-mediated divalent metal ion transport.

Phenolsulfophthalein dyes as probes in the study of lysozyme activity towards cell wall substrates

Difference spectra have shown that the dissociation constant associated with the dominant species, formed by the binding of bromophenol blue or bromocresol purple to lysozyme, is not sensitive to pH in the range 6-9.5. This was confirmed from temperature-jump studies. However, the inhibition of lysozyme catalysed cell lysis by these dyes is dependent on pH and ionic strength. In the reaction scheme, which takes note of both these observations, we have to consider the formation of an enzyme-dye substrate complex (EDS) which has the same kcat as does the enzyme-substrate complex (ES). The formation of EDS from ES and free dye (D) is controlled by an ionisable group. Analysis of the data using an equation similar to that of Maurel and Douzou gives a pK of approx. 5.6 for this group and this pK is close to that of histidine-15. The inhibition mainly comes from the difference in the formation constants of EDS and ES. The initial binding site of the substrate (S) in ES is not in the cleft region A-F. The cell lysis takes place after S binds in the cleft, in a subsequent step. The rate constants of this step are included in kcat. (kcat is obtained by analysing activity using the simple Michaelis-Menten kinetics). Inhibition by chitotriose also supports this conclusion. The dye binding site is also suggested to be close to histidine-15. Experimental results support the contention that the electrostatic potential due to the negatively charged cell wall substrate could alter the effective pK of ionisable groups on the enzyme in ES.

Role of metal ion free valinomycin-carbonyl cyanide m-chlorophenylhydrazone complex in the enhancement of the rates of gramicidin facilitated net H+, Li+ and Na+ transport across phospholipid vesicular membrane

The studies on the decay of the pH difference, delta pH, across soyabean phospholipid vesicular membrane have shown that the rates of net proton transport and the associated Li+ and Na+ ion transport across the membrane can be enhanced by the combined action of gramicidin, valinomycin and carbonyl cyanide m-chlorophenylhydrazone (CCCP) in K(+)-free vesicle solutions. The data obtained under different experimental conditions suggest that this enhancement is a consequence of facilitation of CCCP- transport (1) by complexing CCCP- with the highly membrane permeant valinomycin without the metal ion bound to it and (2) by the associated Li+ or Na+ transport through the gramicidin channel such that no net charge is transported across the membrane. The dissociation constant of the weak valinomycin-CCCP- complex has been estimated to be > 200 mM in the membrane. The delta pH in these experiments were created by temperature jump.

Binding site of the dye in Bromophenol blue-lysozyme complex. Proton magnetic resonance study in aqueous solutions.

The forward rate constant for the binding of Bromophenol blue to lysozyme is estimated to be 10(5)M-1 X S-1 at room temperature and ionic strength 0.01, from the line broadening of the 1H-NMR spectrum of Bromophenol blue at 270 MHz. The broadening of the 1H-NMR line corresponding to histidine-15 of lysozyme, in the presence of Bromophenol blue in solution at pH 4.5, suggests that the binding site of Bromophenol blue is not far from this protein residue. The extent of this broadening is pH-dependent and can be interpreted in terms of the average coulombic interaction between the dye and His-15. Since Bromophenol blue is known to inhibit lytic activity of lysozyme towards cell wall substrates, we can say that the region near His-15 may also be important for the efficient catalytic activity of lysozyme.