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E Bamberg

Publications and source records attributed to E Bamberg.

At least 73 records · Page 4Linked to original sources
PubMed1993

Kinetics of transient pump currents generated by the (H,K)-ATPase after an ATP concentration jump.

(H,K)-ATPase containing membranes from hog stomach were attached to black lipid membranes. Currents induced by an ATP concentration jump were recorded and analyzed. A sum of three exponentials (tau 1(-1) approximately 400 sec-1, tau 2(-1) approximately 100 sec-1, tau 3(-1) approximately 10 sec-1; T = 300 K, pH 6, MgCl2 3 mM, no K+) was fitted to the transient signal. The dependence of the resulting time constants and the peak current on electrolyte composition, ATP conversion rate, temperature, and membrane conductivity was recorded. The results are consistent with a reaction scheme similar to that proposed by Albers and Post for the NaK-ATPase. Based on this model the following assignments were made: tau 2 corresponds to ATP binding and exchange with caged ATP. tau 1 describes the phosphorylation reaction E1 x ATP-->E1P. The third, slowest time constant tau 3 is tentatively assigned to the E1P-->E2P transition. This is the first electrogenic step and is accelerated at high pH and by ATP via a low affinity binding site. The second electrogenic step is the transition from E2K to E1H. The E2K<==>E1H equilibrium is influenced by potassium with an apparent K0.5 of 3 mM and by the pH. Low pH and low potassium concentration stabilize the E1 conformation.

Adenosine Diphosphate↗
PubMed1993

Charge transport by ion translocating membrane proteins on solid supported membranes.

A new method for the investigation of ion translocating membrane proteins is presented. Protein containing membrane fragments or vesicles are adsorbed to a solid supported membrane. The solid supported membrane consists of a lipid monolayer on a gold evaporated or gold sputtered glass substrate which is coated with a long chained mercaptan (CH3(CH2)mSH, m = 15, 17). Specific conductance and specific capacitance of the solid supported membrane are comparable to those of a black lipid membrane. However, the solid supported membrane has the advantage of a much higher mechanical stability. The electrical activity of bacteriorhodopsin, Na,K-ATPase, H,K-ATPase, and Ca-ATPase on the solid supported membrane is measured and compared to signals obtained on a conventionally prepared black lipid membrane. It is shown that both methods yield similar results. The solid supported membrane therefore represents an alternative method for the investigation of electrical properties of ion translocating transmembrane proteins.

Adenosine Triphosphatases↗
PubMed1992

A unifying concept for ion translocation by retinal proteins.

First, halorhodopsin is capable of pumping protons after illumination with green and blue light in the same direction as chloride. Second, mutated bacteriorhodopsin where the proton acceptor Asp85 and the proton donor Asp96 are replaced by Asn showed proton pump activity after illumination with blue light in the same direction as wildtype after green light illumination. These results can be explained by and are discussed in light of our new hypothesis: structural changes in either molecule lead to a change in ion affinity and accessibility for determining the vectoriality of the transport through the two proteins.

Amino Acid Sequence↗
PubMed1991

Ion channel formation by synthetic transmembrane segments of the inhibitory glycine receptor--a model study.

The inhibitory glycine receptor (GlyR) of rat spinal cord contains an intrinsic transmembrane channel mediating agonist-gated anion flux. Here, synthetic peptides modelled after the predicted transmembrane domains M2 and M4 of its ligand-binding subunit were incorporated into lipid vesicle membranes and black lipid bilayers to analyze their channel forming capabilities. Both types of peptides prohibited the establishment of, or dissipated, preexisting transmembrane potentials in the vesicle system. Incorporation of peptide M2 into the black lipid bilayer elicited randomly gated single channel events with various conductance states and life-times. Peptide M4 increased the conductance of the bilayer without producing single channels. Exchange of the terminal arginine residues of peptide M2 by glutamate resulted in a significant shift towards cation selectivity of the respective channels as compared to peptide M2. In conclusion, the peptide channels observed differed significantly from native GlyR in both conductivity and ion-selectivity indicating that individual synthetic transmembrane segments are not sufficient to mimic a channel protein composed of subunits with multiple transmembrane segments.

Amino Acid Sequence↗
PubMed1991

Concentrations of progestagens and oestrogens in the faeces of pregnant Lipizzan, trotter and thoroughbred mares.

Faecal samples were collected at weekly intervals from pregnant Lipizzan mares during Weeks 7-16 following mating and from Lipizzan, Trotter and Thoroughbred mares during the last 3 months of gestation. After parturition, samples were taken daily from the Thoroughbred mares for another 6 days. Non-pregnant mares served as controls. The concentrations of unconjugated oestrogens (Eg), 20 alpha-OH-progestagens (20 alpha-G) and 20 beta-OH-progestagens (20 beta-G) were measured by enzyme immunoassay. In the faeces of Lipizzan mares, immunoreactive progestagens were significantly (P less than 0.01) elevated above the levels in non-pregnant mares by Week 11, and Eg by Week 13 of pregnancy onwards. During the last 3 months of gestation, concentrations of Eg were significantly higher in Trotter mares than in Lipizzan and Thoroughbred mares. Concentrations of 20 alpha-G and 20 beta-G increased to maximal values in the last month of gestation. There was no significant difference among the 3 breeds with respect to 20 alpha-G but, during the 10 weeks before parturition, concentrations of 20 beta-G in the Lipizzan mares were significantly lower (P less than 0.05) than those in the Thoroughbred mares. They were also significantly lower than those of the Trotter mares during the last 4 weeks of gestation. After parturition, the concentrations of Eg and progestagens had declined to baseline values by Days 3 and 4 respectively. From these results we conclude that high concentrations of progestagens with 20 alpha- and 20 beta-hydroxyl groups are present in the faeces of pregnant mares, especially during the last month of gestation.

Animals↗
PubMed1990

Voltage-dependent pump currents of the sarcoplasmic reticulum Ca2(+)-ATPase in planar lipid membranes.

Sarcoplasmic reticulum vesicles containing largely Ca2(+)-ATPase were incorporated into planar lipid membranes. The ATPase was activated by a UV flash-induced concentration jump of ATP from a photolabile caged ATP. Under these conditions stationary pump currents were observed. The dependence of these pump currents on applied voltages was investigated. The current-voltage curve of the Ca2(+)-ATPase shows monotonously increasing pump currents with increasing positive potentials of the ATP containing compartment. This indicates the existence of electrogenic steps in the direction of the transported Ca2+ ions. From the extrapolated reversal potentials of the curve is concluded that less than four positive net charges are transported per hydrolyzed ATP.

Adenosine Diphosphate↗
PubMed1990

Demonstration of the electrogenicity of proton translocation during the phosphorylation step in gastric H+K(+)-ATPase.

Membrane fragments containing the H+K(+)-ATPase from parietal cells have been adsorbed to a planar lipid membrane. The transport activity of the enzyme was determined by measuring electrical currents via the capacitive coupling between the membrane sheets and the planar lipid film. To initiate the pump currents by the ATPase a light-driven concentration jump of ATP from caged ATP was applied as demonstrated previously for Na+K(+)-ATPase (Fendler, K., Grell, E., Haubs, M., Bamberg, E. 1985. EMBO J. 4:3079-3085). Since H+K(+)-ATPase is an electroneutrally working enzyme no stationary pump currents were observed in the presence of K+. By separation of the H+ and K+ transport steps of the reaction cycle, however, the electrogenic step of the phosphorylation could be measured. This was achieved in the absence of K+ or at low concentrations of K+. The observed transient current is ATP dependent which can be assigned to the proton movement during the phosphorylation. From this it was concluded that the K+ transport during dephosphorylation is electrogenic, too, in contrast to the Na+K(+)-ATPase where the K+ step is electroneutral. The transient current was measured at different ionic conditions and could be blocked by vanadate and by the H+K(+)-ATPase specific inhibitor omeprazole. An alternative mechanism for activation of this inhibitor is discussed.

Adenosine Diphosphate↗
PubMed1989

Aspartic acids 96 and 85 play a central role in the function of bacteriorhodopsin as a proton pump.

A spectroscopic and functional analysis of two point-mutated bacteriorhodopsins (BRs) from phototrophic negative halobacterial strains is reported. Bacteriorhodopsin from strain 384 contains a glutamic acid instead of an aspartic acid at position 85 and BR from strain 326 contains asparagine instead of aspartic acid at position 96. Compared to wild-type BR, the M formation in BR Asp85---Glu is accwelerated approximately 10-fold, whereas the M decay in BR Asp96---Asn is slowed down approximately 50-fold at pH6. Purple membrane sheets containing the mutated BRs were oriented and immobilized in polyacrylamide gels or adsorbed to planar lipid films. The measured kinetics of the photocurrents under various conditions agree with the observed photocycle kinetics. The ineffectivity of BR Asp85---Glu resides in the dominance of an inactive species absorbing maximally at approximately 610 nm, while BR Asp96---Asn is ineffective due to its slow photocycle. These experimental results suggest that aspartic acid 96 plays a crucial role for the reprotonation of the Schiff base. Both residues are essential for an effective proton pump.

Amino Acid Sequence↗
PubMed1989

A defective proton pump, point-mutated bacteriorhodopsin Asp96----Asn is fully reactivated by azide.

Addition of azide fully restored the proton pump activity of defective bacteriorhodopsin (BR) mutant protein Asp96----Asn. The decay time of M of BR Asp96----Asn, the longest living intermediate, was decreased from 500 ms at pH 7.0 to approximately 1 ms under conditions of saturating azide concentrations. This decay was faster than the decay of M in the wild-type, where no such azide effect was detectable. Stationary photocurrents, measured with purple membranes immobilized and oriented in a polyacrylamide gel, increased upon addition of azide up to the level of the wild-type. Different small anions of weak acids restored the pump activity with decreasing affinity in the order: cyanate greater than azide greater than nitrite greater than formiate greater than acetate. The activation energy of the M decay in the mutant was higher in the presence (48 kJ/mol) than in the absence (27 kJ/mol) of 100 mM azide even though the absolute rate was dramatically increased by azide. This effect of azide is due to the substitution of a carboxamido group for a carboxylic group at position 96 which removes the internal proton donor and causes an increase in the entropy change of activation for proton transfer which is reversed by azide.

Anions↗
PubMed1989

Conversion of steroids in bovine blood in vitro.

Blood samples collected from eight Braunvieh cows between the sixth and eighth month of gestation were allowed to stand with and without anticoagulant at 20 degrees C and 0 degrees C for different time periods. In these samples the degree of in vitro conversion of gestagens, androgens and estrogens was investigated. The concentrations were measured by radioimmunoassay. After 24 h at 20 degrees C, the levels of pregnenolone, progesterone, 17alpha-hydroxyprogesterone, androstenedione, dehydroepiandrosterone and estrone decreased to 62, 29, 25, 10, 34 and 44%, respectively, of the initial value and those of 17alpha, 20beta-dihydroxyprogesterone, epitestosterone and estradiol-17alpha increased to 385, 800 and 852%, respectively. The conversion was slower in clotted blood. The concentrations of testosterone and estradiol-17beta were consistent over the 24 h period. There was no marked decrease of the steroid concentration after 24 h of incubation of whole blood at 0 degrees C and of plasma at 20 degrees C. After the addition of (3)H-steroids, conversion could be demonstrated by thin-layer chromatography and autoradiography. These results demonstrate that all investigated hormones except testosterone and estradiol-17beta were metabolized by bovine blood cells.

Journal Article↗
PubMed1989

Dehydroepiandrosterone and epitestosterone in the blood of cows at term.

From Day 268 of gestation till two days after parturition blood samples were collected from the jugular vein of 5 cows with normal and 4 cows with flumethasone induced parturition and centrifuged immediately after sampling. In another experiment blood samples were taken from the jugular and uterine vein of 3 cows during the last week before normal parturition. The dehydroepiandrosterone (DHA) and epitestosterone (ET) concentrations in plasma were measured using radioimmunoassay. In the control group the DHA concentration was in the range of 2 to 5 nmol/l, ET levels varied from 4 to 8 nmol/l before term. In both groups, there was an increase in DHA concentrations during the last periparturient period whereas ET levels increased only after flumethasone induced parturition. The concentrations of both androgens declined after the expulsion of the placenta and they were higher in the uterine vein than in the jugular vein. It is concluded, that both androgens are secreted by the bovine placenta and that the delta 5-pathway of steroidogenesis is active in vivo.

Animals↗
PubMed1989

Caged-Ca2+: a new agent allowing liberation of free Ca2+ in biological systems by photolysis.

The alkaline earth cation complexes of DM-nitrophen [1-(2-nitro-4,5-dimethoxyphenyl)-N,N,N',N'-tetrakis-[(oxycarbonyl) methyl]- 1,2-ethanediamine)] release the bound cation in the millisecond time range upon irradiation by a short UV-light pulse. This technique allows to generate cation (eg. Ca2+) concentration jumps or pulses in solution or in cellular systems. The physico-chemical properties of DM-nitrophen and its Mg2+, Ca2+ and Ba2+ complexes are investigated by employing spectrophotometric and potentiometric techniques. In case of Ca2+ a stability constant of the complex up to nearly 10(11) M-1 is found. The magnitude of representative Mg2+ and Ca2+ concentration jumps which can be generated under realistic experimental conditions are calculated on the basis of the thermodynamic parameters reported here.

Acetates↗
PubMed1988

Identification of synaptophysin as a hexameric channel protein of the synaptic vesicle membrane.

The quaternary structure and functional properties of synaptophysin, a major integral membrane protein of small presynaptic vesicles, were investigated. Cross-linking and sedimentation studies indicate that synaptophysin is a hexameric homo-oligomer, which in electron micrographs exhibits structural features common to channel-forming proteins. On reconstitution into planar lipid bilayers, purified synaptophysin displays voltage-sensitive channel activity with an average conductance of about 150 picosiemens. Because specific channels and fusion pores have been implicated in vesicular uptake and release of secretory compounds, synaptophysin may have a role in these processes.

Animals↗