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M Eisenbach

Publications and source records attributed to M Eisenbach.

At least 19 recordsLinked to original sources
PubMed1992

Acetyladenylate or its derivative acetylates the chemotaxis protein CheY in vitro and increases its activity at the flagellar switch.

CheY, a key protein in the mechanism of bacterial chemotaxis, is known to interact with the flagellar switch and thereby cause clockwise rotation. This activity of CheY was significantly increased by producing acetyladenylate (AcAMP) within cytoplasm-free bacterial envelopes containing purified CheY. This was achieved by including in the envelopes the enzyme acetyl-CoA synthetase (ACS) and ATP, and adding acetate externally. The fraction of clockwise-rotating envelopes, tethered to glass by their flagella, increased from 14% to 58% by the presence of AcAMP (or its derivative). In parallel experiments carried out with [14C]acetate under similar conditions, CheY became acetylated: [1-14C]acetate was as effective as [2-14C]acetate in labeling CheY, and ACS-dependent labeling of CheY by [alpha-32P]ATP was not detected. The switch proteins, FliG, FliM, and FliN, isolated to purity, were not acetylated. The acetylation was specific for CheY and dependent on its native conformation. The acetylated form the CheY was estimated to be more active than its nonacetylated form by 4-5 orders of magnitude. Acetylated CheY was stable in the presence of the strong nucleophiles hydroxylamine or ethanolamine, indicative of N-acetylation. There was a correlation between the activity of CheY in vivo and its ability to be acetylated in vitro. Thus, proteins with a single substitution at their active site, CheY57DE and CheY109KR, are not active in vivo and accordingly were not acetylated in vitro; in contrast, the protein CheY13DK is active in vivo and was normally acetylated in vitro. The possibility that CheY acetylation plays a role in bacterial chemotaxis is discussed.

Acetylation↗
PubMed1992

Correlation between phosphorylation of the chemotaxis protein CheY and its activity at the flagellar motor.

Phosphorylation of the chemotaxis protein CheY by its kinase CheA appears to play a central role in the process of signal transduction in bacterial chemotaxis. It is presumed that the role is activation of CheY which results in clockwise (CW) flagellar rotation. The aim of this study was to determine whether this activity of CheY indeed depends on the protein being phosphorylated. Since the phosphorylation of CheY can be detected only in vitro, we studied the ability of CheY to cause CW rotation in an in vitro system, consisting of cytoplasm-free envelopes of Salmonella typhimurium or Escherichia coli having functional flagella. Envelopes containing just buffer rotated only counterclockwise. Inclusion of CheY caused 14% of the rotating envelopes to go CW. This fraction of CW-rotating envelopes was not altered when the phosphate potential in the envelopes was lowered by inclusion of ADP together with CheY in them, indicating that CheY has a certain degree of activity even without being phosphorylated. Attempts to increase the activity of CheY in the envelopes by phosphorylation were not successful. However, when CheY was inserted into partially-lysed cells (semienvelopes) under phosphorylating conditions, the number of CW-rotating cells increased 3-fold. This corresponds to more than a 100-fold increase in the activity of a single CheY molecule upon phosphorylation. It is concluded that nonphosphorylated CheY can interact with the flagellar switch and cause CW rotation, but that this activity is increased by at least 2 orders of magnitude by phosphorylation. This increase in activity requires additional cytoplasmic constituents, the identity of which is not yet known.

Adenosine Diphosphate↗
PubMed1992

Effect of mechanical removal of pili on gliding motility of Myxococcus xanthus.

Gliding motility of Myxococcus xanthus is governed by both the adventurous (A) and the social (S) motility gene systems. The presence of pili has previously been shown to be correlated with a genetically intact S-motility system (D. Kaiser, Proc. Natl. Acad. Sci. USA 76:5952-5956, 1979). The purpose of the present work was to study the direct effect of mechanical removal of pill on the social motility of M. xanthus. Depiliation resulted in (i) a loss of streaming motility of A- S+ mutants, i.e., strains which are able to move by virtue of the S-motility system only, (ii) no effect on motility in A+ S- mutants, i.e., strains capable of movement by the A-motility system only, and (iii) a retardation of streaming speed in the wild-type strain (A+ S+). Cell-cell cohesion, another characteristic of social behavior, was not affected by mechanical removal of pill. The observation that mechanical depiliation perturbed the motility of strains which rely on the S-motility system strongly supports a role for pili in social motility of M. xanthus.

Bacterial Adhesion↗
PubMed1992

Fumarate or a fumarate metabolite restores switching ability to rotating flagella of bacterial envelopes.

Flagella of cytoplasm-free envelopes of Escherichia coli or Salmonella typhimurium can rotate in either the counterclockwise or clockwise direction, but they never switch from one direction of rotation to another. Exogenous fumarate, in the intracellular presence of the chemotaxis protein CheY, restored switching ability to envelopes, with a concomitant increase in clockwise rotation. An increase in clockwise rotation was also observed after fumarate was added to partially lysed cells of E. coli, but the proportion of switching cells remained unchanged.

Cell Membrane↗
PubMed1992

Precontact mammalian sperm-egg communication and role in fertilization.

In creatures with external fertilization, e.g., metazoa, it is well established that there is precontact sperm-egg communication in the form of chemotaxis. An intriguing question is whether also in mammals, where fertilization is internal and the need for precontact sperm-egg communication is not self-evident, such a process occurs and what its physiological significance may be. Here we review the evidence related to such a process in mammals, evidence which suggests that sperm attraction to the ovulated egg may indeed occur. On the basis of the available data we propose a hypothesis, according to which a sperm population is heterogeneous with respect to its physiological state; some spermatozoa are at a physiological state ready for fertilizing an egg, while others are premature or overmature. According to the hypothesis this is a dynamic state; the population of fertilizing spermatozoa gradually loses its potency and, at the same time, other spermatozoa mature and acquire fertilizing ability. After ovulation, only the fertilizing spermatozoa are attracted to the egg, while the rest are either repelled or inhibited and thus prevented from reaching the egg. The potential significance of sperm-egg communication is discussed.

Animals↗
PubMed1991

Sperm attraction to a follicular factor(s) correlates with human egg fertilizability.

Spermatozoa normally encounter the egg at the fertilization site (in the Fallopian tube) within 24 hr after ovulation. A considerable fraction of the spermatozoa ejaculated into the female reproductive tract of mammals remains motionless in storage sites until ovulation, when the spermatozoa resume maximal motility and reach the fertilization site within minutes. The nature of the signal for sperm movement is not known, but one possible mechanism is attraction of spermatozoa to a factor(s) released from the egg. We have obtained evidence in favor of such a possibility by showing that human spermatozoa accumulate in follicular fluid in vitro. This accumulation into follicular fluid was higher by 30-260% than that observed with buffer alone and was highly significant (P less than 10(-8)). Not all of the follicular fluids caused sperm accumulation; however, there was a remarkably strong correlation (P less than 0.0001) between the ability of follicular fluid from a particular follicle to cause sperm accumulation and the ability of the egg, obtained from the same follicle, to be fertilized. These findings suggest that attraction may be a key event in the fertilization process and may give an insight into the mechanism underlying early egg-sperm communication.

Chemotaxis↗
PubMed1979

Interaction of purple membrane with solvents. I. Applicability of solubility parameter mapping.

We carried out spectral studies on the interaction between purple membrane fragments (isolated from Halobacterium halobium) and a series of different solvents, classified quantitatively according to their solubility parameters delta d, delta p, delta h. These represent the contribution of dispersion forces, polar forces, and hydrogen bonding, respectively, to the cohesive energy density of the solvent. Purple membrane fragments, kept in the dark, were suspended in each of the solvents as well as in binary mixtures of solvents, and the spectrum of the resulting suspension was recorded in the wavelength region 250-700 nm. The interaction of each solvent with the membrane fragments can be represented by a point on either a ternary diagram, where each of the three axes represents one of the solubility parameters, or a binary diagram, where one of the two axes is a combination of two of the solubility parameters (delta v = square root of delta d2 + delta p2 or delta a = square root of delta p2 + delta h2). In the former type of solvent map the contribution of each of the parameters is distinct but only their relative contributions are expressed. In the latter the absolute values of delta i are considered. In each of these modes of presentation an inner closed region is observed. The solvents inside its borders interact with bacteriorhodopsin with a resultant spectral change. Mixtures of solvents fit the maps according to their calculated delta values. Thus, a mixture of an apolar solvent with a highly polar solvent interacts with bacteriorhodopsin, even though each of these solvents alone does not.

Bacteriorhodopsins↗
PubMed1979

Interaction of purple membrane with solvents. II. Mode of interaction.

Using the solubility parameter mapping technique (Eisenbach, M., Caplan, S.R. and Tanny, G (1979) Biochim. Biophys. Acta 554, 269-280) we studied spectroscopically the mode of interaction between the purple membrane of Halobacterium halobium and pure organic solvents or solvent mixtures. Although the interacting solvents formed a well-defined closed region in the interaction maps, mapping the modes of interaction did not reveal a closed region for each spectrally classifiable type. A suggested interpretation for this is that interaction with the purple membrane chromophore requires that a solvent (or solvent mixture) possess apolar groups in order to obtain access to the chromophore, together with a polar character and hydrogen-bonding capacity. The mode of interaction, however, is dependent on the specificity of the reactive group of the solvent for retinal, and this has nothing to do with membrane properties. We also examined the influence of the duration of the interaction and of illumination. Some solvents appeared to react more sluggishly than others, but no generalization in terms of the solubility parameter mapping was found, probably because the map describes thermodynamic rather than kinetic phenomena. The only effect of illumination was to enhance the reaction of some of these solvents. It did not change the solubility parameters of purple membrane.

Bacteriorhodopsins↗
PubMed1979

Light-induced pH changes in sub-bacterial particles of Halobacterium halobium. Effects of ionophores.

The kinetics of light-induced acidification and of the subsequent dark-induced alkalization in suspensions of sub-bacterial particles of Halobacterium halobium may be expressed as the sum of two exponentials, indicating two processes (Eisenbach, M., Bakker, E.P., Korenstein, R. and Caplan, S.R. (1976) FEBS Lett. 71, 228--232). We studied the effects of carbonyl cyanide p-trifluoromethyoxy phenyl-hydrazone, nigericin, gramicidin D, valinomycin, and monactin on the extents and the rate constants of the two processes. The various ionophores affected the two processes differently and in general the slower process was more sensitive to their presence. Valinomycin and monactin had relatively minor effects, apparently due to the high ionic strength of the suspension. When an artificial membrane potential was created in the dark, the light-induced acidification was preceded by a transient alkalization as is usually observed in intact cells. These results are discussed in the light of a suggested model accounting for the two processes (Caplan, S.R., Eisenbach, M., Cooper, S., Garty, H., Klemperer, G. and Bakker, E.P. (1977) in Bioenergetics of Membranes (Packer, L., Papageorgiou, G.C. and Trebst, A., eds.), pp. 101--114, Elsevier/North-Holland Biomedical Press, Amsterdam), taking into account the different selectivities of the ionophores applied.

Anti-Bacterial Agents↗
PubMed1978

Bacteriorhodopsin: lipid environment and conformational changes.

The polar lipids of the purple membrane were exchanged for different phosphatidylcholine species. The resulting complexes had the same protein to lipid-phosphorus ratio as the natural membrane, but only about 0.5-1.0 mole of original lipid was still present per mole of bacteriorhodopsin. In such complexes the bacteriorhodopsin photocycle is slowed down 10-20 times, but the strong protein-protein interaction is not abolished. Due to the slow rate of the photocycle we were able to measure in the light the ratio between net proton release and net accumulation of the last intermediate of the photocycle, the unprotonated M412. This ratio was not constant and equal to 1.0, as expected for a single deprotonation reaction, but varied with pH from 1.5 to 0.4. The variable ratio suggests that light-induced conformational changes occur in the nonchromophore part of the protein, which shift the pKa values of unidentified groups so as to cause binding or release of additional protons. A similar conclusion was drawn from experiments on the kinetics of proton transfer by bacteriorhodopsin in subbacterial particles of Halobacterium halobium and in reconstituted bacteriorhodopsin proteoliposomes. However, in this case light-induced association and dissociation of additional protons occurs simultaneously on different sides of the membrane.

Bacteriorhodopsins↗
PubMed1977

Light-driven sodium transport in sub-bacterial particles of Halobacterium halobium.

Light-induced Na+ efflux was observed in sub-bacterial particles of Halobacterium halobium loaded and suspended in 4 M NaCl solution. The Na+ efflux was not ATP driven, since ATPase inhibitors were without effect or even enhanced efflux at low light intensity. Uncouplers, on the other hand, inhibited Na+ efflux, the inhibition being complete at low light intensity. The Na+ efflux was accompanied by proton influx. Both processes were dependent on light intensity, unaffected or enhanced by ATPase inhibitors and similarly affected by uncouplers. Proton influx was not observed in particles loaded with 4 M KCl instead of 4 M NaCl. Na+ transport in the dark could be induced by artificial formation of a pH difference across the membrane; changing the sign of the pH difference reversed the direction of the Na+ transport. Proton influx in the dark followed the artificial formation of a sodium gradient [Na+]in less than [Na+]out). These results may be explained by a Na+/H+ antiport mechanism. The fluxes of Na+ and H+ were of comparable magnitude, but the initial rate of Cl- efflux in the same experiment was one-third of the initial rate of Na+ efflux. Consequently Cl- is not regarded as a participant in the Na+ efflux mechanism.

Arsenates↗
PubMed1975

Characterization of the component, which controls the transformation between the kinetic forms of the b cytochromes.

1. In the presence of KCN and a saturating concentration of antimycin the reduction of the b-type cytochromes in submitochondrial particles is biphasic. This phenomenon was explained by suggesting the existence of two kinetic forms of cytochrome b:bA-the active form which was reduced in the rapid phase, and bS-the sluggish form which was reduced in the slow phase. The ratio between these forms and the transformation from one to other was controlled by the redox state of an unknown component, names "y", located between cytochromes b and c1. Pre-treatment with ascorbate plus N,N,N1,N1-tetramethyl-p-phenylenediamine transforms all the b-type cytochromes to their sluggish form, and the reduction by succinate follows slow monophasic kinetics. The name "dynamic control mechanism" was given to this mechanism [Eisenbach, M. & Gutman, M. (1975) Eur. J. Biochem. 52, 107-116] 2. Increasing concentrations of antimycin (0-2 nmol/mg) in the presence of KCN increased the fraction of the rapid phase of the reduction but did not affect the calculated absolute rates of the reduction. It is concluded that antimycin delays the reduction of "y" and thus permits the observation of the biphasic phenomen, but that it is not essential for the operation of this dynamic control mechanism.

Animals↗