Spontaneous and promoted association of linear oligoglycines

Linear oligoglycines of various lengths bearing a carboxyl or an amide group at their C-termini and also their poly(acrylamide) conjugates were synthesized. No self-assembly into supramolecular structures was observed for free oligoglycines H-(Gly)m-OH(m = 3-5). At the same time, oligoglycylamides H-(Gly)m-NH2 (m = 3-5) demonstrated ability for both self-assembly in aqueous solution and assembly promoted by an additional interaction with surface. In the case of polymer-bound oligoglycines (and their amides), no intramolecular clustering of peptide chains, as expected, was observed. This means that the presence of several oligoglycine chains bound to each other in one center is not a necessary prerequisite for polyglycine II-type association.     

Kinetic studies on the chymotrypsin Aα-catalyzed hydrolysis of a series of N-acetyl-l-phenylalanyl peptides

A series of N-acetyl-l-phenylalanyl peptides of general formula Ac-Phe-(Gly)_n-NH₂ (n = 0–2) has been synthesized to study the effect of leaving group chain length on the efficiency of chymotrypsin A_α amidase and peptidase activities. The effect upon catalysis of hydrophobic side chains on the leaving group was investigated using similar substrates with one of the glycine residues selectively substituted by an alanine residue as in AcPheAlaNH₂, AcPheAlaGlyNH₂, and AcPheGlyAlaNH₂. Values of k_cat and K_m have been obtained from kinetic measurements at pH 8.00 and 25 °C. The results are shown to be consistent with binding schemes postulated from published model building studies. The catalytic reactions were studied over a range of temperature (15–35 °C) and in each case the Arrhenius law was obeyed. It was thus possible to obtain meaningful values for the thermodynamic functions of activation for the acylation step of the catalytic reaction. The results are shown to confirm the findings of postulated binding schemes but indicate that conclusions drawn from kinetic measurements at a single temperature may sometimes be misleading.     

Diketopiperazine-mediated peptide formation in aqueous solution II. Catalytic effect of phosphate

The previous paper (I) reported that DKP (glycine anhydride) spontaneously reacts with glycine (Gly) or oligoglycines (Gly _n ) to produce longer oligoglycines (Gly _n+2). This paper presents that phosphate catalyzes the condensation reaction quite effectively.Formation of Gly₄ from DKP (0.1 M) and Gly₂ (0.1 M) in phosphate solution of various concentrations was investigated at a neutral pH at 41 °C. The yields of Gly₄ increased almost linearly with the concentration of phosphate from 0.06 M to 0.24 M. The yield in 0.24 M phosphate solution was approximately one hundred times as high as that in the absence of the phosphate, whereas in the case of Gly₃ formation from DKP and Gly the effect of the phosphate was of ten times lower than in the former case. Orthophosphate was the most effective catalyst among the various kind of chemicals tried in the present investigation including polyphosphates.     

Crystal structures of two cyclic pseudopentapeptides containing ψ[CH2S] and ψ[CH2SO] backbone surrogates

The solid state conformations of cyclo[Gly–Proψ[CH₂S]Gly–D –Phe–Pro] and cyclo[Gly–Proψ[CH₂–(S)–SO]Gly–D –Phe–Pro] have been characterized by X-ray diffraction analysis. Crystals of the sulfide trihydrate are orthorhombic, P2₁2₁2₁, with a = 10.156(3) Å, b = 11.704(3) Å, c = 21.913(4) Å, and Z = 4. Crystals of the sulfoxide are monoclinic, P2₁, with a = 10.662(1) Å, b = 8.552(3) Å, c = 12.947(2) Å, β = 94.28(2), and Z = 2. Unlike their all-amide parent, which adopts an all-trans backbone conformation and a type II β-turn encompassing Gly-Pro-Gly-D -Phe, both of these peptides contain a cis Gly¹-Pro² bond and form a novel turn structure, i.e., a type II′ β-turn consisting of Gly–D –Phe–Pro–Gly. The turn structure in each of these peptides is stabilized by an intramolecular H bond between the carbonyl oxygen of Gly¹ and the amide proton of D -Phe⁴. In the cyclic sulfoxide, the sulfinyl group is not involved in H bonding despite its strong potential as a hydrogen-bond acceptor. The crystal structure made it possible to establish the absolute configuration of the sulfinyl group in this peptide. The two crystal structures also helped identify a type II′ β-turn in the DMSO-d₆ solution conformers of these peptides. © 1993 John Wiley & Sons, Inc.     

Synthesis and properties of cationic oligopeptides with different side chain lengths that bind to RNA duplexes

A series of artificial peptides bearing cationic functional groups with different side chain lengths were designed, and their ability to increase the thermal stability of nucleic acid duplexes was investigated. The peptides with amino groups selectively increased the stability of RNA/RNA duplexes, and a relationship between the side chain length and the melting temperature (T_m) of the peptide–RNA complexes was observed. On the other hand, while peptides with guanidino groups exhibited a similar tendency with respect to the peptide structure and thermal stability of RNA/RNA duplexes, those with longer side chain lengths, such as l-2-amino-4-guanidinobutyric acid (Agb) or l-arginine (Arg) oligomers, stabilized both RNA/RNA and DNA/DNA duplexes, and those with shorter side chain lengths exhibited a higher ability to selectively stabilize RNA/RNA duplexes. In addition, peptides were designed with different levels of flexibility by introducing glycine (Gly) residues into the l-2-amino-3-guanidinopropionic acid (Agp) oligomers. It was found that insertion of Gly did not affect the thermal stability of the peptide–RNA complexes, but an alternate arrangement of Gly and Agp apparently decreased the thermal stability. Therefore, in the Agp oligomer, consecutive Agp sequences are essential for increasing the stability of RNA/RNA duplexes.     

Nanoscale mono- and multi-layer cylinder structures formed by recombinant S-layer proteins of mosquitocidal Bacillus sphaericus C3-41

The mature surface layer (S-layer) protein SlpC of mosquitocidal Bacillus sphaericus C3-41 comprises amino acids 31–1,176 and could recrystallize in vitro. The N-terminal SLH domain is responsible for binding function. Deletion of this part, S-layer proteins could not bind to the cell wall sacculi. To investigate the self-assembly ability of SlpC from B. sphaericus, nine truncations were constructed and their self-assembly properties were compared with the recombinant mature S-layer protein rSlpC_31–1,176. The results showed that rSbsC_31–1,176 and truncations rSlpC_211–1,176, rSlpC_278–1,176, rSlpC_31–1,100, and rSlpC_31–1,050 could assemble into multilayer cylinder structures, while N-terminal truncations rSlpC_338–1,176, rSlpC_438–1,176, and rSlpC_498–1,176 mainly showed monolayer cylinders in recombinant Escherichia coli BL21 (DE3) cells. Growth phase analysis of the self-assembly process revealed that rSlpC_498–1,176 mainly formed monolayer cylinders in the early stage (0.5 and 1 h induction of expression), but few double-layer or multilayer cylinders were also found with the cells growing, while rSlpC_31–1,176 could formed multilayer cylinders in all the growth stage in the E. coli cells. It is concluded that the deletion of the C-terminal 126 aa or the N-terminal 497 aa did not interfere with the self-assembly process, the fragment (amino acids 278 to 337) is essential for the multilayer cylinder formation in E. coli BL21 (DE3) cells in the early stage and the fragment (amino acids 338 to 497) is related to monolayer cylinder formation. The information is important for further studies on the assembly mechanism of S-layer proteins and forms a basis for further studies concerning surface display and nanobiotechnology.     

Raman spectra and conformations of n-hexadecynoic fatty acids and their potassium salts

Raman spectra of n-hexadecynoic fatty acids, CH₃(CH₂)_m?2C  C(CH₂)_n?2COOH (with n = 4, 6, 7, 8 and 12, m + n = 16) and their potassium salts were recorded and assigned. The skeletal optical mode (SOM) regions were analysed on using the dispersion curve of the ν₄ (stretch) vibrations of saturated fatty acids. The localised vibrations of the carboxyl- and methyl-terminated sections of the hydrocarbon chains were assigned to the phase differences δ = kπ/m and kπ/n (k = 1, 2, …), respectively. Evidence for a ν₄ vibration with δ = π/2m was found. The materials were found to be resistant to chemical decomposition under laser illumination.     

Design and synthesis of cholecystokinin-4 dipeptide analogues with anxiolytic and anxiogenic activities

A new series of dipeptide analogues of the general formula Ph(CH₂) _n CO-NH(CH₂) _m CO-Trp-NH₂ (n = 1, 3–5; m = 1–3) was designed based on the structure of the endogenous tetrapeptide cholescystokinin-4 (CCK-4) and the topochemical Shemyakin-Ovchinnikov-Ivanov principle. The L-tryptophan derivatives exhibited anxiolytic properties and the D-tryptophan derivatives, anxiogenic properties. The dipeptide Ph(CH₂)₅CO-Gly-L-Trp-NH₂ (GB-115) with the activity in rats of 0.05–0.2 mg/kg after oral and intraperitoneal administration was chosen for further studies as a promising anxiolytic agent.     

Stereoselectivity in the nucleophilic addition-type polymerization of α-amino acid N-caboxyanhydride initiated by diastereomeric dipeptide amines

In order to investigate the effect of the chiral penultimate unit on the stereoselection of α-amino acid N-carboxyanhydride (NCA) by the terminal unit of a growing chain in the nucleophilic addition-type polymerization, the diastereomers of dipeptide amines, H-(R)-Phe-(S)-Phe-Mo and H-(S)-Phe-(S)-Phe-Mo, in which Mo represents a morpholine residue, were synthesized, and the stereoselectivity in their nucleophilic addition reactions to NCA was investigated and compared with that of a monopeptide amine H-(S)-Phe-OEt. In the reaction with Phe NCA in nitrobenzene, either of the dipeptide amines reacted preferentially with an enantiomer of NCAs having a configuration opposite to the N-terminal unit of the dipeptide amine. The preference of enantiomeric NCA and the extent of stereoselectivity were nearly the same as those found with H-(S)-PheOEt. The opposite-enantiomer selectivity of the dipeptide amines was also observed in the reaction with N-MePhe NCA, and the extent of stereoselectivity was found to increase very much in the reaction of H-(R)-PHe-(S)-Phe-Mo compared with that of H-(S)-Phe-OEt. Therefore, the enhancement of the stereoselectivity of the N-terminal unit by the penultimate unit was shown experimentally. On the other hand, the stereoselectivity of H-(S)-Phe-(S)-Phe-Mo was not very different from that of H-(S)-Phe-OEt. These results were obtained either in nitrobenze or in m-dimethoxybenzene. H-(S)-Phe-(S)-Phe-OEt tends to aggregate by an intermolecular hydrogen bond in aqueous and tetrahydrofuran solutions. Its pK_a value and nucleophilicity towards NCA were much lower than H-(R)-Phe-(S)-Phe-Mo, which was free from the aggregation under similar conditions. These experimental results suggest that the major product in the polymerization of (RS)-Phe NCA by amine should be an alternating copolymer. However, this prediction was not verified experimentally, and the important contributions from the aggregation and the molecular weight distribution of growing chains were suggested.     

Metal complexes of polycyclic tertiary amines. Part VII. Preparation and crystal structure determination of polymeric hexamethylenetetramine—mercury(II) thiocyanate (12), (CH2)6N4·2Hg(SCN)2

A 1:2 complex of hexamethylenetetramine with mercury(II) thiocyanate, (CH₂)₆N₄·2Hg(SCN)₂, was prepared and shown by X-ray crystallography to be polymeric. The mixed-ligand complex crystallizes in the space group P2₁/m, with a = 6.059(2), b = 19.710(5), c = 7.895(2) Å, β = 105.63(2)°, and Z = 2. The structure was refined to R_F = 0.060 for 1634 observed MoKα diffractometer data. Mercury(II) atoms in a row are linked pairwise by two thiocyanato groups in an end-to-end bridging mode, to give an infinite chain running in the a direction. Two neighboring chains are further laterally connected, successively by bidentate organic ligands which lie on a crystallographic mirror plane. The coordination geometry about Hg(II) is distorted tetragonal pyramidal, the metal atom binding strongly to two S atoms and a tertiary amino N atom (apex), and weakly to two thiocyanato N atoms.     

The crystal structure of 2-deoxy-β-d-arabino-hexopyranose at −150°

2-Deoxy-β-d-arabino-hexopyranose, C₆H₁₂O₅, is orthorhombic, P2₁2₁2₁, with cell dimensions at ?150° [20°], a = 6.484(2) [6.510(3)], b = 10.364(2) [10.427(4)], c = 11.134(3) [11.153(5)] Å, V = 748.2 [757.1] ų, Z = 4, D_x = 1.457 [1.440], and D_m = [1.455] g.cm^?3. The intensities of 1269 reflections were measured by using MoKα radiation. The structure was solved by direct methods, and refined by full-matrix least-squares, with anisotropic, thermal parameters for the carbon and oxygen atoms, and isotropic parameters for the hydrogen atoms. The pyranose has the ⁴C₁(d) conformation, with puckering parameters Q = 0.563 Å, θ = 3.9°, and ? = 350.3°. The departure from ideality is very small, and less than that in β-d-glucopyranose, Q = 0.584 Å and θ = 6.9°. The β-glycosidic, CO bond is short, 1.383(4) Å, and the OCOH torsion angle is ?87°, consistent with the anomeric effect. The hydrogen-bonding scheme consists of infinite chains, with side chains terminating at a ring-oxygen atom.     

Enzymic synthesis of lignin precursors. Purification of properties of the S-adenosyl-l-methionine: caffeic acid 3-O- methyltransferase from soybean cell suspension cultures.

A 3-O-methyltransferase which catalyzes the methylation of caffeic acid to ferulic acid using S-adenosyl-l-methionine as methyl donor has been isolated and purified about 60-fold from cell suspension cultures of soybean (Glycine max L., var. Mandarin). The enzyme utilized, in addition to caffeic acid (K_m = 133 μM), 5-hydroxyferulic acid (K_m = 55 μM), 3,4,5-trihydroxy-cinnamic acid (K_m = 100 μM), and protocatechualdehyde (K_m = 50 μM) as substrates. Methylation proceeded only in the meta position. The enzyme was unable to catalyze the methylation of ferulic acid, of ortho-, meta-, and para-coumaric acids, and of the flavonoid compounds quercetin and luteolin. The methylation of caffeic acid and 5-hydroxyferulic acid showed a pH optimum at 6.5–7.0. No stimulation of the reaction velocity was observed when Mg²⁺ ions were added. EDTA did not inhibit the reaction. The K_m for S-adencsyl-l-methionine was 15 μm. S-Adenosyl-l-homocysteine was a potent competitive inhibitor of S-adenosyl-l-methionine (K_i = 6.9 μM).     

Submitochondrial localization and characteristics of thymidine kinase molecular forms in parental and kinase-deficient hela cells

Although HeLa (BU25) cells are deficient in cytosol dT kinase activity, they contain two mitochondrial dT kinases with disc PAGE mobilities (R _m) of 0.4 and 0.6 and isoelectric points (pI) of 8.4 and 5.6, respectively. Mitochondrial extracts of parental HeLa S3 contain the two HeLa (BU25) activities, but also a cytosol-like enzyme (0.25 R _m, pI 9.8). The 0.6-R _m (pI 5.6) mitochondrial activity utilizes ribonucleoside 5′-triphosphates other than ATP (dATP) as phosphate donors and is sensitive to dCTP inhibition. The predominant HeLa S3 cytosol (0.25 R _m) enzyme and the 0.4 R _m mitochondrial enzymeefficiently utilize only ATP as a phosphate donor and are relatively insensitive to dCTP inhibition. Submitochondrial fractionation studies have shown that (1) 74–98% of the mitochondrial dT kinase is located in the matrix plus inner membrane fractions; (2) the matrix fraction has the highest specific activity, contains all the 0.6-R _m activity, most of the HeLa S3 0.25-R _m activity, and some 0.4-R _m activity; (3) the inner membrane fraction is the major site of the 0.4-R _m activity but the outer membrane fraction also contains the 0.4 R _m activity; and (4) all HeLa S3 submitochondrial fractions contain the 0.25-R _m dT kinase activity.     

Phosphatidylcholine requirement for the N-glycosylation of synthetic peptides by detergent-solubilized oligosaccharyltrasferase

The ability of dolichyl-P-P-oligosaccharide:peptide oligosaccharyltransferase to use exogenous substrates (a previously labeled oligosaccharide lipid and an Asn-X-Thr containing heptapeptide) is shown to require phospholipid. The enzyme was extracted from porcine thyroid rough microsomes using NaCl-Nonidet P-40. When measured at low concentration, in a neutral detergent-containing medium, it undergoes a rapid loss of activity, which renders impossible quantitative estimates in the range of 0–50 μg microsomal protein /50 μl assay. We observed that inactivation could be prevented by supplementing the assay with a prevoously heat-treated suspension of microsomes in neutral detergent, or with the corresponding extract. Further investigation revealed that phospholipids are responsible for this enzyme stabilization, since phospholipase A₂ and phospholipase C treatments were both able to abolish this effect. When individual phospholipids were compared for their protective efficiency, egg yolk phosphatidylcholine was found to be by far the most efficient. Phosphatidylglycerol, phosphatidylinositol and phosphatidylserine were only slightly effective, while phosphatidylethanolamine and lysophosphatidylcholine had no effect at all. Of those tested, partly unsaturated phosphatidylcholines with 16–18 carbon atom acyl chains were the most active, at an optimal concentration of 1–2 mM. Under these conditions a K_m of 15 μM was measured for the acceptor, a synthetic ribonuclease heptapeptide, and K_m of 0.55 μM for the donor, dolichyl-P-P-GlcNAc₂-Man₉-Glc_2?3. These findings were confirmed by subjecting a sodium deoxycholate extract to depletion of endogenous lipids by gel filtration. Enzyme activity was totally abolished and then restored (up to now only partially) by addition of phosphatidylcholine.     

Characterization of chondroitin sulfate isolated from trypsin-chymotrypsin digests of cartilage proteoglycans

Proteoglycans from bovine tracheal cartilage were digested with trypsin and chymotrypsin by procedures similar to those described by Mathews (Biochem. J.125, 37 (1971)). Chondroitin sulfate-peptide fragments in the digest were precipitated with cetylpyridinium chloride and subsequently fractionated on a preparative Sepharose 6B column. The fragments, which emerged from the column as a broad peak, were divided into five fractions. Rechromatography of these fractions on an analytical Sepharose 6B column indicated that they had K_av values from 0.17 (fraction 1) to 0.62 (fraction 5). The weight average molecular weight values obtained by meniscus depletion equilibrium centrifugation were 193,000, 126,000, 80,000, 46,000, and 23,000 for fractions 1 to 5, respectively. Values for the molecular weights and for the limiting viscosity numbers, [η], of the fractions were used to determine estimates for α of 0.40–0.46 and for K of 0.43–0.88 in the equation [η] = K·M_v^α. These values for α are consistent with a branched structure for the chondroitin sulfate fractions. Papain digests of each of the fractions were chromatographed on Sephadex G-200. The observed distributions of the monomer chains released by this protease were almost the same for each sample, which indicates that the individual chondroitin sulfate chains in all of the original fractions had nearly the same average molecular weights. The data in sum indicate that peptide fragments which contain from 1 to 8 polysaccharide chains are released when the proteoglycans are digested with trypsin-chymotrypsin.Analytical data indicated that all fractions contained 3–11% of their polysaccharide as keratan sulfate. This indicates either that about 50% of the keratan sulfate chains in the original proteoglycan molecules are located in close proximity to the chondroitin sulfate chains or that some peptides contain large numbers of keratan sulfate chains. Proteoglycan preparations which differed by a factor of about 6 in their ratio of chondroitin sulfate to protein yielded very similar elution patterns on Sepharose 6B after trypsin-chymotrypsin digestion.     

Method for the complete separation of radioactively labeled human γ-globin chains from pre-βA chains on CM-cellulose chromatography: Use of cold carrier globin chains

Carboxymethyl (CM)-cellulose chromatography of globins is the technique used most frequently in analysis of hemoglobin synthesis. However, if this method is to be reliable in cases where only small amounts of fetal hemoglobin (α₂γ₂) compared to adult hemoglobin (α₂β₂^A) are synthesized, it is important to obtain a clean separation of γ chains from pre-β^A chains. In the past, it has been found that small amounts of pre-β^A chains tend to elute with the γ chains. Radioactively labeled γ chains can be completely and reproducibly separated from small amounts of labeled pre-β^A chains by the addition of unlabeled β^J chains (Hb J Baltimore = β16 Gly → Asp) which elute at the same position as the pre β^A chains, thus increasing the quantity of this peak and allowing a clean separation from the γ chains.     

Analysis of the 220-MHz,P.M.R. spectra of some products of the amadori and heyns rearrangements

In order to establish whether p.m.r. spectroscopy is useful for identifying Amadori- and Heyns-rearrangement products, the p.m.r. spectra at 220 MHz of 16 rearrangement products derived from d-glucose or d-fructose and amino acids have been investigated. At pH 3, the protons of the NCH₂ group of N-substituted 1-amino-1-deoxy-d-fructose (Amadori-rearrangement products) resonate at δ 3.25–3.60 in D₂O and are shifted upfield by 0.3–0.6 p.p.m. at pH 9. These protons exchange with deuterium. Also, in D₂O there is an equilibrium of the acyclic, furanose, and pyranose structures, the last being favoured. At pH ? 7, the equilibrium is completely shifted to the β-pyranose form, which adopts exclusively the ²C₅ conformation. At pH 3, the equilibrium favours the β-furanose form. At pH 3, H-1e and H-1a of N-substituted 2-amino-2-deoxy-d-glucoses (Heyns-rearrangement products) resonate at δ 5.55 and 5.04, respectively. At pH 9, the signal for H-2 is shifted upfield by 0.2–0.7 p.p.m. In D₂O solution, these compounds exist as an equilibrium of α- and β-pyranose forms in the ⁴C₁ conformation. The α anomer is stabilised by the amino acid group at position 2. At pH 3, the αβ-ratio is 2–4:1, and, at pH 9, 1.0–1.1:1.     

Reconstitution of the influenza virus M2 ion channel in lipid bilayers

M₂, an integral membrane protein of influenza A virus, was purified from either influenza A virus-infected CV-1 cells or from Spodoptera frugiperda (Sf9) cells infected with a recombinant-M₂ baculovirus. The purified protein, when incorporated into phospholipid bilayer membranes, produced ion-permeable channels with the following characteristics: (1) The channels appeared in bursts during which unit conductances of diverse magnitudes (25–500 pS) were observed. (2) The most probable open state was usually the lowest unit conductance (25–90 pS). (3) The channels were selective for cations; t _Na = 0.75 when 150 mm NaCl bathed both sides of the membrane. (4) Amantadine reduced the probability of opening of the high conductance state and also the conductance of the most probable state. (5) Reducing pH increased the mean current through the open channel as well as the conductance of the most probable state. (6) The sequence of selectivity for group IA monovalent cations was Rb > K > Cs ～ Na > Li. The pH activation, amantadine block and ion selectivity of the M₂ protein ion channel in bilayers are consistent with those observed on expression of the M₂ protein in oocytes of Xenopus laevis as well as for those predicted for the proposed role of an ion channel in the uncoating process of influenza virus. The finding that the M₂ protein has intrinsic ion channel activity supports the hypothesis that it has ion channel activity in the influenza virus particle.     

Denaturation of DNA in the presence of chromous (Cr2+) ions

The effect of Cr²⁺ ions on the T_m (melting temperature) of DNA has been investigated under appropriate conditions for the stabilization of DNA by Mg²⁺ ions. A significant lowering of T_m, analogous to that observed for Cu²⁺ under normal conditions, was found, for Cr²⁺ at pH = 4.2 and [Mg²⁺] = 5.3 mol per mole of DNA base pair. Cu²⁺ also lowers T_m under similar conditions. The similarity of the effects of Cr²⁺ and Cu²⁺ under comparable conditions may be related to similarities in their coordination properties. It is proposed that Cr²⁺ and Cu²⁺ ions facilitate denaturation by holding together groups on the DNA chains in such a manner that base pairing and base stacking are inhibited. Comparative results for Cr³⁺ and Co²⁺ are also given for these low pH/Mg²⁺ ion conditions.     

Electrically induced protoplast fusion using pulse electric fields for dielectrophoresis

The formation of protoplast chains in suspensions of isolated pea (Pisum sativum cv Ran 1) mesophyll protoplasts induced by electric fields was studied. The chain formation induced by a sine-wave field (2 V, peak to peak; 500-0.1 kHz) was compared to that induced by an alternating pulse field (1 V, amplitude; 0.1-0.4 kHz). An increased number of dielectrophoretically paired protoplasts, formation of protoplast chains in the presence of CaCl₂ up to 5 mm, and protoplast fusion in the presence of 3 mm CaCl₂ were found when the pulse field was applied. The present results suggest the possibility of electrically induced protoplast fusion at cation concentrations that prevent fusion when sine-wave fields are applied.