Strain induced peculiarities in transport properties of Bi nanowires.

We report results on the effect of strain on the thermopower and electrical resistance of glass-coated individual Bi nanowires. Here, we show that there is a critical diameter of wires below which the contribution of holes to the charge transport in pure Bi nanowires is more significant than that of electrons. The properties of Bi nanowires are examined in the light of a strain induced electronic topological transition. At low temperatures, the thermopower dependences on strain exhibit a non-monotonic behavior inherent in thinner wires, where the thermopower is dominated by the diffusion transport mechanism of holes. The hole-dominated transport can be transformed into electron-dominated transport through a smooth manipulation with the phonon spectrum and Fermi surface by applying a uniaxial strain. A fairly high value of the thermoelectric power factor (S(2)/ρ = 89 µW cm(-1) K(-2)) was found in the temperature range of 80-300 K, where the dominant mechanism contributing to the thermopower is diffusive thermoelectric generation with electrons as the majority carrier.

Exposure of phosphatidylcholine and phosphatidylinositol in plasma membranes from rat brain synaptosomes treated with phospholipase A2 toxins (beta-bungarotoxin, notexin) and enzymes (Naja nigricollis, Naja naja atra).

Phospholipase A2 (PLA2) toxins act presynaptically to block acetylcholine release and are much more potent and specific in their actions than PLA2 enzymes even though they have lower enzymatic activity. Since their mechanism of action is not completely understood, it was of interest to examine the toxins' effects on phospholipid asymmetry as changes in asymmetry are associated with changes in membrane functioning. Rat brain synaptosomes were treated with the PLA2 toxins beta-bungarotoxin (beta-BuTx) and notexin and with the PLA2 enzymes Naja nigricollis and Naja naja atra under relatively non-disruptive conditions as judged by leakage of lactate dehydrogenase (LDH) and levels of phospholipid hydrolysis. The exposure of phosphatidylcholine (PC) and phosphatidylinositol (PI) on the synaptosomal surface was investigated by means of a specific PC-exchange protein (PCEP) and a PI-specific phospholipase C (PI-PLC), respectively. Treatment of the synaptosomes with N. nigricollis PLA2, beta-BuTx and notexin did not affect the availability of PC to exchange by PCEP, but significantly increased the exposure of PI to hydrolysis by PI-PLC. In contrast, N. n. atra PLA2 slightly decreased the exposure of PC and did not affect that of PI. The differences between N. n. atra PLA2, on the one hand, and N. nigricollis PLA2, beta-BuTx and notexin, on the other hand, parallel differences in their pharmacological activities. Our earlier studies showed that PLA2 enzymes, and possibly PLA2 toxins, have a pharmacological site separate from the enzymatic site. Since in the present study the effect on PI was abolished by EDTA, the presence of an enzymatic site in addition to the pharmacological site may be required or alternatively divalent cations may be required for the effects on PI asymmetry independent of the inhibition of PLA2 by EDTA.

The asymmetric distribution of phosphatidylcholine in rat brain synaptic plasma membranes.

The distribution of phosphatidylcholine between inner and outer monolayers of rat brain synaptic plasma membrane was investigated by means of a phosphatidylcholine specific exchange protein. About 70% of the total membranal phosphatidylcholine was in the outer leaflet, 33% of which was exposed and readily exchanged in intact synaptosomes while the remainder was exchangeable following osmotic shock. Permeabilization of the synaptic plasma membranes by overnight incubation in buffer or by saponin (< 0.08%) exposed an additional 30% of phosphatidylcholine to exchange, presumably from the inner cytoplasmic leaflet. Phosphatidylcholine is therefore asymmetrically distributed in the synaptosomal plasma membrane, as it is in other plasma membranes.

An EDTA.Ca2+ complex inhibits the enzymatic activity but not the lethality of beta-bungarotoxin.

An EDTA.Ca2+ complex inhibits the phospholipase A2 activity of the presynaptic neurotoxin beta-bungarotoxin without affecting its lethal potency. The EDTA.Ca2+ complex induces a conformational change in the enzymatic active site region of beta-BuTx, as indicated by the suppression of the 340 nm tryptophan fluorescence peak. Modification of the enzymatic site without loss of toxicity supports the presence of separate loci for the two activities.

Inhibitory effect of EDTA.Ca2+ on the hydrolysis of synaptosomal phospholipids by phospholipase A2 toxins and enzymes.

Phospholipases A2 (PLA2) are Ca2(+)-dependent enzymes that are inhibited by EDTA; this inhibition would be expected to be reversed by restoring the Ca2+ concentration. By examining the hydrolysis of synaptosomal phospholipids by PLA2 enzymes, Naja naja atra and Naja nigricollis, and by toxins with PLA2 activity, beta-bungarotoxin (beta-BuTX) and notexin, we demonstrated a novel inhibitory action of EDTA manifested in the presence of excess Ca2+. We postulate the formation of an EDTA.Ca2+ complex which inhibits PLA2 activity in a concentration-dependent manner. Synaptosomes in which phospholipids are hydrolyzed by PLA2 have membranal damage expressed by increased acetylcholine (ACh) release and decreased osmotic activity. Addition of EDTA.Ca2+, which inhibits phospholipid hydrolysis, also reversed the PLA2 effect on ACh release, but not its effect on osmotic activity. The inhibition of PLA2 was observed on membranal phospholipids as well as on an artificial substrate of phospholipid-Triton mixed micelles. Moreover, we found that another enzyme, lactate dehydrogenase, was also inhibited. Our results indicate a non-specific inhibition exerted on the enzyme rather than on the substrate.

Phospholipid hydrolysis and loss of membrane integrity following treatment of rat brain synaptosomes with beta-bungarotoxin, notexin, and Naja naja atra and Naja nigricollis phospholipase A2.

The effects of the phospholipase A2 (PLA2) toxins, beta-bungarotoxin and notexin, and the PLA2 enzymes from Naja naja atra and Naja nigricollis snake venoms on the plasma membrane integrity of synaptosomes were examined. Synaptosomes were isolated from rat brain cerebral cortex, corpus striatum and hippocampus. Osmotic activity, lactate dehydrogenase leakage, and leakage of 2-deoxy-D-(1-3H)-glucose-6-phosphate were monitored (37 degrees C, 10-120 min) following incubation with 0.5, 5 and 50 nM concentrations of toxins and enzymes. Damage to the synaptosomal plasma membrane was time and concentration but not tissue dependent. The potencies of the treatments were as follows: N. n. atra PLA2 greater than or equal to N. nigricollis PLA2 greater than notexin greater than beta-bungarotoxin. Chelation of Ca2+ with 5 mM EDTA completely inhibited plasma membrane disruption caused by beta-bungarotoxin and N. n. atra PLA2. One mg/ml of bovine serum albumin also blocked the disruptive action of N. n. atra PLA2, while 8 mg/ml was required to antagonize beta-bungarotoxin. A correlation between phospholipid hydrolysis and loss of membrane integrity was also observed. The generation of phospholipid hydrolytic products may be critical in the permeabilization of synaptic plasma membranes by these toxins and enzymes, however, they do not explain the presynaptic specificity and potency of beta-bungarotoxin and notexin.

Do chemical modifications dissociate between the enzymatic and pharmacological activities of beta bungarotoxin and notexin?

We have measured enzymatic, hemolytic and anticoagulant activities, lethal potencies and effects on contractions of the phrenic nerve-diaphragm preparation, by chemically modified derivatives of beta bungarotoxin (beta BuTX) and notexin, two presynaptically acting toxins which have PLA2 activity. The following chemical modifications of beta BuTX were tested: alkylation and methylation of histidine 48, alkylation of tryptophan 19, sulfonylation of tyrosine 68, oxidation of methionines 6 and 8, semicarbazide addition under varied conditions to carboxyl groups, varied extents of carbamylation or trinitrophenylation of lysines and guanidination of all lysines with or without trinitrophenylation of the N-terminal asparagine. Only the histidine, tryptophan and tyrosine residues were modified in notexin. The results obtained were compared with those previously obtained using chemically modified derivatives of Naja nigricollis and Naja naja atra PLA2 enzymes which do not have a specific presynaptic site of action. The results with oxidized methionine and lysine-modified derivatives of beta BuTX are supportive of the suggestions of others that the N-terminal region and basic residues away from the enzymatic active region contribute towards the beta type presynaptic neurotoxicity of the PLA2 toxins. Using modified derivatives of beta BuTX and notexin, the dissociations between enzymatic activities and pharmacological properties were not as marked as previously observed with N. nigricollis and N. n. atra PLA2; nevertheless, several dissociations were noted. We conclude that, just as with non-presynaptically acting PLA2 enzymes, some pharmacological actions of presynaptically acting PLA2 toxins may occur independently of phospholipid hydrolysis.

Comparison of enzymatic and pharmacological activities of lysine-49 and aspartate-49 phospholipases A2 from Agkistrodon piscivorus piscivorus snake venom.

The basic Lys-49 phospholipase A2 (PLA2) from Agkistrodon piscivorus piscivorus venom is homologous to the basic Asp-49 PLA2 from the same venom as well as other snake venom PLA2 enzymes. It differs, however, in several respects, most important being replacement of the previously invariant Asp-49 at the calcium binding site by Lys, resulting in a reversed order of addition of calcium and phospholipid, phospholipid binding first. Although the preferences for phospholipid substrates of the two enzymes are identical, the apparent Vmax of the Lys-49 PLA2 was only 1.4 to 3% that of the Asp-49 enzyme. Similarly, the Lys-49 PLA2, compared to the Asp-49 PLA2 had less than 3% of the intraventricular lethal potency and 4% of the anticoagulant activity. The intravenous lethal potency of the Lys-49 enzyme was 20% that of the Asp-49 PLA2 and both had little direct hemolytic activity. In contrast, both enzymes were approximately equipotent on the phrenic nerve-diaphragm preparation and on the isolated ventricle strip of the heart. On the cardiac and neuromuscular preparations, the effects of the Asp-49 PLA2 were accompanied by hydrolysis of phosphatidylcholine and phosphatidylethanolamine, whereas no phospholipid hydrolysis was observed with the Lys-49 PLA2. Evaluation of the present results, along with earlier findings using Asp-49 PLA2 enzymes from Naja nigricollis, Hemachatus haemachatus and Naja naja atra venoms, allows us to conclude that: The A. p. piscivorus Asp-49 PLA2 enzyme resembles the Asp-49 enzymes from N. n. atra and H. haemachatus. In contrast, the A. p. piscivorus Lys-49 PLA2 has much lower enzymatic and anticoagulant activities than the Asp-49 enzymes, but equal cardiotoxic and junctional effects. In contrast to some previous suggestions, basic PLA2 enzymes are not necessarily more toxic than neutral or acidic enzymes. Pharmacological effects upon the heart and phrenic nerve-diaphragm preparation correlate neither with in vitro measurements of PLA2 activity nor with actual levels of phospholipid hydrolysis in the heart or diaphragm. This suggests that PLA2 enzymes exert effects independent of phospholipid hydrolysis.

Effects of modification of tyrosines 3 and 62 (63) on enzymatic and toxicological properties of phospholipases A2 from Naja nigricollis and Naja naja atra snake venoms.

Previously we selectively modified His (48), Arg, Lys, Asp, Glu and Trp residues in the basic phospholipase A2 from Naja nigricollis and the acidic phospholipase A2 from N. n. atra snake venoms. Evidence was obtained for the existence of separate but perhaps overlapping sites responsible, respectively, for their enzymatic and pharmacological properties. We have now modified one or two (Tyr 3, Tyr 62 [63], Tyr 3 + 62 [63]) out of the nine tyrosine residues in these enzymes using p-nitrobenzenesulfonyl fluoride. The derivatives were separated by HPLC, and modified residues determined by amino acid analysis. Enzymatic activity was tested on lecithin--Triton mixed micelles, egg yolk and heart and diaphragm homogenates. The N. nigricollis modified derivatives retained a greater percentage of their enzymatic activities than did the N. n. atra derivatives and also a greater percentage of their activity on natural substrates than on lecithin--Triton mixed micelles. The greatest loss in activity resulted when both tyrosines were modified and the least when tyrosine 3 was modified. Modification of tyrosine 62 of N. nigricollis caused a much greater loss of intraventricular lethal potency than of enzymatic activity, whereas modification of tyrosine 3 of N. n. atra increased lethal potency over six-fold while enzymatic activity decreased about 60%. Examples of dissociation between enzymatic and pharmacological potencies were also noted when hemolytic, anticoagulant and cardiotoxicity on isolated ventricular muscle were measured. The extents of phospholipid hydrolysis were relatively low in brain homogenates, synaptic plasma membranes and heart ventricular muscle. However, they were similar for the native enzymes and all of the tyrosine modified derivatives. These tyrosines do not appear to be part of the enzymatic active site, even though they are thought to be associated with substrate and calcium binding. These results strengthen our earlier conclusion that some pharmacological effects of phospholipase A2 are not due to enzymatic hydrolysis, and that there are separate but perhaps partly overlapping sites for enzymatic and pharmacological activities.

Effect of alkylation of tryptophan residues on the enzymatic and pharmacological properties of snake venom phospholipase A2.

Snake venom phospholipases A2 show a remarkable degree of amino acid sequence homology yet differ markedly in enzymatic and pharmacological activities. The basic phospholipase A2 from Naja nigricollis venom has much greater lethal potency, cardiotoxicity, hemolytic and anticoagulant activity than the acidic or neutral enzymes from Naja naja atra or Hemachatus haemachatus venoms, respectively, even though it has lower enzymatic activity than the latter two enzymes. Previous studies in which we selectively modified lysine and free carboxyl groups suggested that the pharmacological and enzymatic active sites are not identical. Tryptophan residues have been suggested as being involved in substrate binding although some phospholipases have no tryptophan. We investigated the effect of alkylating the tryptophans in N. nigricollis, N. n. atra, and H. haemachatus phospholipases A2 with 2-hydroxy-5-nitrobenzyl bromide. Chemical modification caused decreases in enzymatic activity, although the extent of inactivation varied with the enzyme and with the substrate (lecithin micelles, egg yolk, heart homogenates). The specificity of the enzymes for individual phospholipid substrates was not affected. Alkylation of the tryptophans also caused decreases in lethal, hemolytic, anticoagulant, and cardiotoxic potencies, which were similar to the extents of decrease in enzymatic activity. Our results suggest that tryptophans are not specifically associated with either the enzymatic or the pharmacological active site nor are essential for either activity.

Effect of carboxylate group modification on enzymatic and cardiotoxic properties of snake venom phospholipases A2.

By treating Naja nigricollis and Naja naja atra phospholipase A2 with carbodiimide and semicarbazide, we obtained derivatives having varied numbers of modified carboxylate groups. When tested on artificial and natural substrates, derivatives of both enzymes with a modified carboxylate group at the active site (Asp-49) retained little enzymatic activity (1/41 to 10%). However, the derivatives of N. nigricollis also lost most of their lethal potency (5% of native), while those of N. n. atra retained considerable lethality (29%). Carboxyl modification with protection of Asp-49 in N. n. atra enzyme resulted in a derivative with lethal potency equal to or greater than the native enzyme and enzymatic activity which was low on all substrates (12-17% of native). Similar protection of Asp-49 at the active site in N. nigricollis enzyme produced a derivative with decreased enzymatic activity on artificial substrate (22% of native) and decreased lethality (17-33% of native), but with full enzymatic activity on natural substrates. When tested on electrical and mechanical properties of the isolated perfused heart and the isolated ventricle muscle wall, the derivatives of both enzymes retained considerably more of the cardiotoxic activity than would have been expected based on their residual enzymatic activity. The one exception occurred with the least modified N. nigricollis derivative which had an unaltered Asp-49, this enzyme retained both cardiotoxic activity and full enzymatic activity on natural substrates. The extent of phospholipid hydrolysis following treatment was measured in the isolated heart preparation and in hearts removed from mice following i.v. injection of the phospholipases. Very low levels of phospholipid hydrolysis were observed and no correlation could be made between the extent of hydrolysis and the pharmacological potencies of these enzymes. Modification of the enzymatic active site, whether of Asp-49 in this study of His-48 in prior studies, leads to a large decrease in both enzymatic activity and lethal potency. Asp and Glu residues outside of the enzymatic site contribute significantly to the lethal potency of the N. nigricollis enzyme and to the enzymatic activity of the N. n. atra enzyme. Based on these and previous data we conclude that changes in isoelectric points are not responsible for altered lethal potencies following chemical modification and that some pharmacological effects of snake venom phospholipases A2 are due to a non-enzymatic action, suggesting two distinct but perhaps overlapping active sites.

Dissociation of pharmacological and enzymatic activities of snake venom phospholipases A2 by modification of carboxylate groups.

The carboxylate groups in an acidic and in a basic phospholipase A2 (PLA2) enzyme, purified, respectively, from Naja naja atra and Naja nigricollis snake venoms, were modified with carbodiimide and semicarbazide. The derivatives modified at pH 3.5 and pH 5.5 had less than 1% (N. nigricollis) or 2% (N. n. atra) residual enzymatic activity, whereas 12-16% enzymatic activity remained following modification at pH 5.5 in the presence of Ca2+. In marked contrast, these derivatives retained variable, but significantly greater, levels of lethal potency, hemolytic and anticoagulant activities, and abilities to block indirectly and directly induced contractions of the diaphragm. By this modification of aspartic and glutamic acid residues we have, for the first time, obtained derivatives of PLA2 which selectively retain greater pharmacological activity relative to enzymatic activity. Previously, we had found that modification of lysine and arginine residues produced derivatives which retain enzymatic activity but show a loss of pharmacological properties. These findings suggest that some pharmacological effects of snake venom PLA2 enzymes are due to a non-enzymatic action, suggesting two distinct but perhaps overlapping active sites.

Effect of methylation of histidine-48 on some enzymatic and pharmacological activities of snake venom phospholipases A2.

The effects on some pharmacological and enzymatic properties were determined following methylation of histidine at the enzymatic active site of the basic relatively toxic Naja nigricollis and the acidic relatively non-toxic Naja naja atra phospholipases A2. Following methylation a very low residual enzymatic activity (0.4-1% of control) was accompanied by a parallel loss in intraventricular lethality, anticoagulant potency, direct hemolytic action and ability to block directly and indirectly evoked contractions of the mouse phrenic nerve-diaphragm preparation. Since methylation does not impair the enzyme's ability to bind monomeric or micellar substrates or Ca2+, the results suggest that the pharmacologically active region of the molecule is different from the micellular substrate binding site but strongly influenced by the invariant histidine-48 located at the enzymatic active site.

Phospholipid hydrolysis in serum lipoproteins by a basic phospholipase A2 from Naja nigricollis snake venom and an acidic phospholipase A2 from Naja naja atra snake venom.

Apparent Km and Vmax values for PC and PE hydrolysis were determined following exposure of HDL, LDL, and VLDL to a basic phospholipase A2 from N. nigricollis snake venom and an acidic phospholipase A2 from N. nigricollis snake venom and an acidic phospholipase A2 from N. n. atra snake venom. Both enzymes hydrolyzed the lipoprotein phospholipids approximately as fast as they hydrolyzed pure phospholipids in mixed micelles, however, the N. nigricollis enzyme, which has a much stronger anticoagulant effect than the N. n. atra enzyme, had lower apparent Vmax values. These values were highest for phospholipids in VLDL and lowest for HDL, however, the differences between the lipoproteins were relatively small with the N. nigricollis enzyme while the differences were much larger with the N. n. atra enzyme. Fractions of the two enzymes in which varying numbers of lysines were carbamylated showed much larger differences in relative rates of phospholipid hydrolysis in HDL, LDL and VLDL. Triton X-100 eliminates these differences in rates of hydrolysis. These results are discussed in terms of the differences in the organized structure of the lipoprotein classes and in the penetration ability of the phospholipases.

Ethoxyformylation and guanidination of snake venom phospholipases A2: effects on enzymatic activity, lethality and some pharmacological properties.

Lysine residues in the basic and relatively toxic N. nigricollis phospholipase A2 and in the acidic and relatively nontoxic N. n. atra phospholipase A2 were modified by acylation with ethoxyformic anhydride (in the presence or absence of the substrate dihexanoyl lecithin) or guanidination with O-methylisourea. Ethoxyformylation gave rise to some protein fractions in which enzymatic activity was preserved to a greater degree than intraventricular lethality. Guanidination had little effect on the isoelectric point or catalytic activity of either enzyme or on the lethal potency of the N. n. atra enzyme. However, the intraventricular lethality of the N. nigricollis enzyme was decreased much more than was its intravenous lethality, direct hemolytic potency, anticoagulant activity or cardiotoxic action on rat atrium. These results are compared to those previously obtained when the lysines in these two enzymes were carbamylated with potassium cyanate, a procedure which markedly decreased the isoelectric point of the enzymes. It is concluded that charge alone does not account for differences in toxicity. The data also indicate that there are at least two distinct active sites in both enzymes, one being primarily responsible for enzymatic activity and the other(s) associated with lethal and pharmacological effects of the protein. Modification of lysines affects the latter site(s), while having little or no effect on enzymatic activity.