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1.
The observed endogenous circadian rhythm in plants performing Crassulacean acid metabolism is effected by malate transport
at the tonoplast membrane. Experimental and theoretical work asks for a hysteresis switch, regulating this transport via the
ordering state of the membrane. We apply a schematic molecular model to calculate the thermally averaged order parameter of
the membrane lipid structure in its dependence on external parameters temperature and area per molecule. The model shows a
first order structural phase transition in a biologically relevant temperature range. Osmotic consequences of malate accumulation
can trigger a transition between the two phases by changing the surface area of the cell vacuole. Estimation of the energy
needed to expand the vacuole under turgor pressure because of osmotic changes while acidifying shows that energy needed as
latent heat for the calculated change between phases can easily be afforded by the cell. Thus, malate content and the coexisting
two phases of lipid order, showing hysteretic behavior, can serve as a feedback system in an oscillatory model of Crassulacean
acid metabolism, establishing the circadian clock needed for endogenous rhythmicity.
Received: 2 September 1997/Revised: 24 April 1998 相似文献
2.
E.V. Sviderskaya E. Jazrawi S.A. Baldwin C.C. Widnell C.A. Pasternak 《The Journal of membrane biology》1996,149(2):133-140
The stimulation of glucose transport in response to various types of stress has been studied. There is no relationship between
effects of stress-inducing agents on glucose transport and their effects on cellular protein synthesis. Although the effect
of stress on glucose transport appears analogous to its stimulation by insulin, cells that are slightly insulin-sensitive
in terms of glucose transport (BHK cells) show a similar degree of stimulation as highly insulin-sensitive cells (differentiated
3T3-L1 cells). External labeling of the transporter protein with a photoactivatable derivative of mannose, 2-N-4-(1-azi-2,2,2-trifluoroethyl)
benzoyl-1, 3-bis-(D-mannos-4-yloxy)-propylamine, shows that most of the increased glucose transport activity correlates with
an increase in the amount of the transporter on the cell surface. Cells subjected to K+-depletion, which inhibits endocytosis and results in an accumulation of receptors at the cell surface, show the same increase
in glucose transport as cells exposed to stress; stressed cells show no further increase in glucose transport when subjected
to K+ depletion. These results support the view (Widnell, C.C., Baldwin, S.A., Davies, A., Martin, S., Pasternak, C.A. 1990. FASEB J
4:1634–1637) that cellular stress increases glucose transport by promoting the accumulation of glucose transporter molecules
at the cell surface.
Received: 20 June 1995/Revised: 29 September 1995 相似文献
3.
The AAA proteins (ATPases Associated with a variety of cellular Activities) are found in eubacterial, archaebacterial, and eukaryotic species and participate in a large number of cellular
processes, including protein degradation, vesicle fusion, cell cycle control, and cellular secretory processes. The AAA proteins
are characterized by the presence of a 230 to 250-amino acid ATPase domain referred to as the Conserved ATPase Domain or CAD. Phylogenetic analysis of 133 CAD sequences from 38 species reveal that AAA CADs are organized into discrete
groups that are related not only in structure but in cellular function. Evolutionary analyses also indicate that the CAD was
present in the last common ancestor of eubacteria, archaebacteria, and eukaryotes. The eubacterial CADs are found in metalloproteases,
while CAD-containing proteins in the archaebacterial and eukaryotic lineages appear to have diversified by a series of gene
duplication events that lead to the establishment of different functional AAA proteins, including proteasomal regulatory,
NSF/Sec, and Pas proteins. The phylogeny of the CADs provides the basis for establishing the patterns of evolutionary change
that characterize the AAA proteins.
Received: 28 January 1997 / Accepted: 8 May 1997 相似文献
4.
Our understanding of cell structure and function derives from applications of a variety of physical and life science disciplines,
methods and models to an important physiological process, namely, the exchange and transport of ions and molecules across
biological membranes. We know that ion transport through membranes arises from a diversity of interrelated and interactive
physical and chemical phenomena over a wide range of spatial and temporal scales. Among these phenomena common to all cellular
structure and function include metabolism, kinetics of molecules, chemically mediated alteration of cell membrane electrical
potential, membrane ion conductance, electrical signal propagation, and modulation by chemo- and mechanoreceptive mechanisms.
This review focuses on the unique information contained in fluctuations in electrical properties associated with cell membrane
ion transport.
Received: 19 May 2000/Revised: 10 July 2000 相似文献
5.
We present a novel hypothesis for the origin of the eukaryotic cell, or eukaryogenesis, based on a metabolic symbiosis (syntrophy)
between a methanogenic archaeon (methanobacterial-like) and a δ-proteobacterium (an ancestral sulfate-reducing myxobacterium).
This syntrophic symbiosis was originally mediated by interspecies H2 transfer in anaerobic, possibly moderately thermophilic, environments. During eukaryogenesis, progressive cellular and genomic
cointegration of both types of prokaryotic partners occurred. Initially, the establishment of permanent consortia, accompanied
by extensive membrane development and close cell–cell interactions, led to a highly evolved symbiotic structure already endowed
with some primitive eukaryotic features, such as a complex membrane system defining a protonuclear space (corresponding to
the archaeal cytoplasm), and a protoplasmic region (derived from fusion of the surrounding bacterial cells). Simultaneously,
bacterial-to-archaeal preferential gene transfer and eventual replacement took place. Bacterial genome extinction was thus
accomplished by gradual transfer to the archaeal host, where genes adapted to a new genetic environment. Emerging eukaryotes
would have inherited archaeal genome organization and dynamics and, consequently, most DNA-processing information systems.
Conversely, primordial genes for social and developmental behavior would have been provided by the ancient myxobacterial symbiont.
Metabolism would have been issued mainly from the versatile bacterial organotrophy, and progressively, methanogenesis was
lost.
Received: 5 January 1998 / Accepted: 18 March 1998 相似文献
6.
The charge-pulse relaxation spectrum of nonperfused and perfused (turgescent) cells of the giant marine alga Ventricaria ventricosa showed two main exponential decays with time constants of approximately 0.1 msec and 10 msec, respectively, when the cells were bathed in artificial sea water (pH 8). Variation of the external pH did not change the relaxation pattern (in contrast to other giant marine algae). Addition of nystatin (a membrane-impermeable and pore-forming antibiotic) to the vacuolar perfusion solution resulted in the disappearance of the slow exponential, whereas external nystatin decreased dramatically the time constant of the fast one. This indicated (by analogy to corresponding experiments with Valonia utricularis, J. Wang, I. Spiess, C. Ryser, U. Zimmermann, J. Membrane Biol. 157: 311-321, 1997) that the fast relaxation must be assigned to the RC-properties of the plasmalemma and the slow one to those of the tonoplast. Consistent with this, external variation of [K+]o or of [Cl-]o as well as external addition of K+- or Cl--channel/carrier inhibitors (TEA, Ba2+, DIDS) affected only the fast relaxation, but not the slow one. In contrast, addition of these inhibitors to the vacuolar perfusion solution had no measurable effect on the charge-pulse relaxation spectrum. The analysis of the data in terms of the "two membrane model" showed that K+- and (to a smaller extent) Cl--conducting elements dominated the plasmalemma conductance. The analysis of the charge-pulse relaxation spectra also yielded the following area-specific data for the capacitance and the conductance for the plasmalemma and tonoplast (by assuming that both membranes have a planar surface): (plasmalemma) Cp = 0.82 * 10(-2) F m-2, Rp = 1.69 * 10(-2) Omega m2, Gp = 5.9 * 10(4) mS m-2, (tonoplast) Ct = 7. 1 * 10(-2) F m-2, Rt = 14.9 * 10(-2) Omega m2 and Gt = 0.67 * 10(4) mS m-2. The electrical data for the tonoplast show that (in contrast to the literature) the area-specific membrane resistance of the tonoplast of these marine giant algal cells is apparently very high as reported already for V. utricularis. The exceptionally high value of the area-specific capacitance could be explained - among other interpretations - by assuming a 9-fold enlargement of the tonoplast surface. The hypothesis of a multifolded tonoplast was supported by transmission electronmicroscopy of cells fixed under maintenance of turgor pressure and of the electrical parameters of the membranes. This finding indicates that the tonoplast of this species exhibited a sponge-like appearance. Taking this result into account, it can be easily shown that the tonoplast exhibits a high-resistance (1.1 Omega m2). Vacuolar membrane potential measurements (performed in parallel with charge-pulse relaxation studies) showed that the potential difference across the plasmalemma was mainly controlled by the external K+-concentration which suggested that the resting membrane potential of the plasmalemma is largely a K+-diffusion potential. After permeabilization of the tonoplast with nystatin the potential of the intact membrane barrier dropped from about slightly negative or positive (-5.1 to +18 mV, n = 13) to negative values (-15 up to -68 mV; n = 8). This indicated that the cytoplasm of V. ventricosa was apparently negatively charged relative to the external medium. Permeabilization of the plasmalemma by addition of external nystatin resulted generally in an increase in the potential to slightly more positive values (-0.8 to +4.3 mV; n = 5), indicating that the vacuole is positively charged relative to the cytoplasm. These findings apparently end the long-term debate about the electrical properties of V. ventricosa. The results presented here support the findings of Davis (Plant Physiol. 67: 825-831, 1981), but are contrary to the results of Lainson and Field (J. Membrane Biol. 29: 81-94, 1976). 相似文献
7.
The role of 3′,5′-cyclic adenosine monophosphate (cAMP), protein kinase A (PKA), protein kinase C (PKC) and phosphatases
in the regulation of the taurine influx via the β-system in Ehrlich ascites tumor cells has been investigated. The taurine
uptake by the β-system in Ehrlich cells is inhibited when PKC is activated by phorbol 12-myristate 13-acetate (PMA) and when
protein phosphatases are inhibited by calyculin A (CLA). On the other hand, taurine uptake by the β-system is stimulated by
an increased level of cAMP or following addition of N6,2′-O-dibutyryl-3′,5′-cyclic adenosine monophosphate (dbcAMP). The effect of dbcAMP is partially blocked by addition of the
protein kinase inhibitor H-89, and suppressed in the presence of CLA. It is proposed that the β-system in the Ehrlich cells
exists in three states of activity: State I, where a PKC phosphorylation site on the transporter or on a regulator is phosphorylated and transport activity is low. State II, where the PKC phosphorylation site is dephosphorylated and transport activity is normal. State III, representing a state with high transport activity, induced by an elevated cellular cAMP level. Apparently, cAMP preferentially
stimulates taurine transport when the β-system is in State II.
Received: 8 September/Revised: 9 November 1995 相似文献
8.
P.R. Andjus M.R. Djurišić Z. Žujović N. Begović R. Srejić D. Vučelić 《The Journal of membrane biology》1999,167(3):267-274
The NMR (nuclear magnetic resonance) method of Conlon and Outhred (1972) was used to measure diffusional water permeability
of the nodal cells of the green alga Chara gymnophylla. Two local minima at 15 and 30°C of diffusional water permeability (P
d
) were observed delimiting a region of low activation energy (E
a
around 20 kJ/mol) indicative of an optimal temperature region for membrane transport processes. Above and below this region
water transport was of a different type with high E
a
(about 70 kJ/mol). The triphasic temperature dependence of the water transport suggested a channel-mediated transport at
15–30°C and lipid matrix-mediated transport beyond this region. The K+ channel inhibitor, tetraethylammonium as well as the Cl− channel inhibitor, ethacrynic acid, diminished P
d
in the intermediate temperature region by 54 and 40%, respectively. The sulfhydryl agent p-(chloromercuri-benzensulfonate)
the water transport inhibitor in erythrocytes also known to affect K+ transport in Chara, only increased P
d
below 15°C. In high external potassium (`K-state') water transport minima were pronounced. The role of K+ channels as sensors of the optimal temperature limits was further emphasized by showing a similar triphasic temperature dependence
of the conductance of a single K+ channel also known to cotransport water, which originated from cytoplasmic droplets (putatively tonoplast) of C. gymnophylla. The minimum of K+ single channel conductance at around 15°C, unlike the one at 30°C, was sensitive to changes of growth temperature underlining
membrane lipid involvement. The additional role of intracellular (membrane?) water in the generation of discontinuities in
the above thermal functions was suggested by an Arrhenius plot of the cellular water relaxation rate which showed breaks at
13 and 29°C.
Received: 12 August 1998/Revised: 13 November 1998 相似文献
9.
Tris(2-carboxyethyl)phosphine (TCEP) reduces (cleaves) disulfide bonds of the renal proximal tubule type IIa Na/Pi- cotransporter
(rat NaPi IIa) and thereby inhibits its function. We tested the effect of TCEP on the murine type IIa Na/P
i
-cotransporter and the corresponding IIb intestinal isoform both expressed in Xenopus laevis oocytes. After incubation with TCEP the function of NaPi IIa was inhibited and protein amount was decreased. Injection of
the lysosomal inhibitor leupeptin prevented degradation of the protein. Exposure of oocytes to TCEP at 0°C led to a reduction
in transport function without concomitant loss in Na/Pi IIa protein. In contrast to NaPi type IIa, the type IIb isoform was
neither inhibited, nor degraded after incubation with TCEP. These results suggest that cleavage of disulfide bonds led to
changes within the confirmation of the type IIa transporter that result in (i) inhibition of the transport activity and (ii)
internalization and subsequent lysosomal degradation of transporter protein. Sequence comparisons suggest the involvement/presence
of different disulfide bonds in type IIa and type IIb Na/P
i
-cotransporters.
Received: 13 December 1999/Revised: 31 March 2000 相似文献
10.
The present study deals with photomodification of the electrical properties of the plasma membrane of an epithelial cell
line (opossum kidney (OK) cells). The effect of photofrin II (previously investigated) is compared with that of 5 other membrane-active
sensitizers: sulfonated Zn-phthalocyanine, merocyanine 540, rose bengal, methylene blue and protoporphyrin IX (an endogenous
sensitizer induced by addition of its biosynthetic precursor 5-aminolaevulinic acid). The study was performed in order to
investigate whether photomodification of the ion transport properties of the plasma membrane by membrane-active sensitizers
is a general and early event in cellular photosensitization. The changes in the electrical properties were monitored by application
of the whole-cell and the inside-out configuration of the patch-clamp technique.
Illumination in the presence of the compounds (apart from merocyanine 540) gave rise to similar changes of the electrical
properties of the membrane: depolarization of the membrane potential, inactivation of a large-conductance, Ca2+-dependent K+-channel (maxi-KCa), and a strong increase of the leak conductance of the membrane. This similarity indicates the general character of the functional
photomodifications by membrane-active sensitizers previously reported for photofrin II.
Received: 5 September 2000/Revised: 28 December 2000 相似文献
11.
ATP-Induced Shape Change of Nuclear Pores Visualized with the Atomic Force Microscope 总被引:1,自引:0,他引:1
A. Rakowska T. Danker S.W. Schneider H. Oberleithner 《The Journal of membrane biology》1998,163(2):129-136
Bidirectional transport of molecules between nucleus and cytoplasm through the nuclear pore complexes (NPCs) spanning the
nuclear envelope plays a fundamental role in cell function and metabolism. Nuclear import of macromolecules is a two-step
process involving initial recognition of targeting signals, docking to the pore and energy-driven translocation. ATP depletion
inhibits the translocation step. The mechanism of translocation itself and the conformational changes of the NPC components
that occur during macromolecular transport, are still unclear. The present study investigates the effect of ATP on nuclear
pore conformation in isolated nuclear envelopes from Xenopus laevis oocytes using the atomic force microscope. All experiments were conducted in a saline solution mimicking the cytosol using
unfixed nuclear envelopes. ATP (1 mm) was added during the scanning procedure and the resultant conformational changes of the NPCs were directly monitored. Images
of the same nuclear pores recorded before and during ATP exposure revealed dramatic conformational changes of NPCs subsequent
to the addition of ATP. The height of the pores protruding from the cytoplasmic surface of the nuclear envelope visibly increased
while the diameter of the pore opening decreased. The observed changes occurred within minutes and were transient. The slow-hydrolyzing
ATP analogue, ATP-γ-S, in equimolar concentrations did not exert any effects. The ATP-induced shape change could represent
a nuclear pore ``contraction.'
Received: 10 February 1997/Revised: 10 February 1998 相似文献
12.
Ruth Meléndez Enrique Meléndez-Hevia Marta Cascante 《Journal of molecular evolution》1997,45(4):446-455
Optimization of molecular design in cellular metabolism is a necessary condition for guaranteeing a good structure–function
relationship. We have studied this feature in the design of glycogen by means of the mathematical model previously presented
that describes glycogen structure and its optimization function [Meléndez-Hevia et al. (1993), Biochem J 295: 477–483]. Our
results demonstrate that the structure of cellular glycogen is in good agreement with these principles. Because the stored
glucose in glycogen must be ready to be used at any phase of its synthesis or degradation, the full optimization of glycogen
structure must also imply the optimization of every intermediate stage in its formation. This case can be viewed as a molecular
instance of the eye problem, a classical paradigm of natural selection which states that every step in the evolutionary formation of a functional structure
must be functional. The glycogen molecule has a highly optimized structure for its metabolic function, but the optimization
of the full molecule has meaning and can be understood only by taking into account the optimization of each intermediate stage
in its formation.
Received: 23 October 1996 / Accepted: 21 April 1997 相似文献
13.
Nuclear magnetic resonance (NMR) microimaging and proton relaxation times were used to monitor differences between the hydration
state of the nucleus and cytoplasm in the Rana pipiens oocyte. Individual isolated ovarian oocytes were imaged in a drop of Ringer's solution with an in-plane resolution of 80
μm. Proton spin echo images of oocytes arrested in prophase I indicated a marked difference in contrast between nucleoplasm
and cytoplasm with additional intensity gradations between the yolk platelet-rich region of the cytoplasm and regions with
little yolk. Neither shortening τe (spin echo time) to 9 msec (from 18 msec) nor lengthening τr (spin recovery time) to 2 sec (from 0.5 sec) reduced the observed contrast between nucleus and cytoplasm. Water proton T1 (spin-lattice) relaxation times of oocyte suspensions indicated three water compartments that corresponded to extracellular
medium (T1= 3.0 sec), cytoplasm (T1= 0.8 sec) and nucleoplasm (T1= 1.6 sec). The 1.6 sec compartment disappeared at the time of nuclear breakdown. Measurements of plasma and nuclear membrane
potentials with KCl-filled glass microelectrodes demonstrated that the prophase I oocyte nucleus was about 25 mV inside positive
relative to the extracellular medium. A model for the prophase-arrested oocyte is proposed in which a high concentration of
large impermeant ions together with small counter ions set up a Donnan-type equilibrium that results in an increased distribution
of water within the nucleus in comparison with the cytosol. This study indicates: (i) a slow exchange between two or more
intracellular water compartments on the NMR time-scale, (ii) an increased rotational correlation time for water molecules
in both the cytoplasmic and nuclear compartments compared to bulk water, and (iii) a higher water content (per unit dry mass)
of the nucleus compared to the cytoplasm, and (iv) the existence of a large (about 75 mV positive) electropotential difference
between the nuclear and cytoplasmic compartments.
Received: 18 January 1996/Revised: 29 April 1996 相似文献
14.
Getting In or Out: Early Segregation Between Importers and Exporters in the Evolution of ATP-Binding Cassette (ABC) Transporters 总被引:17,自引:0,他引:17
ATP-binding cassette (ABC) systems, also called traffic ATPases, are found in eukaryotes and prokaryotes and almost all participate
in the transport of a wide variety of molecules. ABC systems are characterized by a highly conserved ATPase module called
here the ABC module, involved in coupling transport to ATP hydrolysis. We have used the sequence of one of the first representatives
of bacterial ABC transporters, the MalK protein, to collect 250 closely related sequences from a nonredundant protein sequence
database. The sequences collected by this objective method are all known or putative ABC transporters. After having eliminated
short protein sequences and duplicates, the 197 remaining sequences were subjected to a phylogenetic analysis based on a mutational
similarity matrix. An unrooted tree for these modules was found to display two major branches, one grouping all collected
uptake systems and the other all collected export systems. This remarkable disposition strongly suggests that the divergence
between these two functionally different types of ABC systems occurred once in the history of these systems and probably before
the differentiation of prokaryotes and eukaryotes. We discuss the implications of this finding and we propose a model accounting
for the generation and the diversification of ABC systems.
Received: 23 February 1997 / Accepted: 7 April 1998 相似文献
15.
R.M. Krupka 《The Journal of membrane biology》1999,167(1):35-41
Control of the coupled reaction sequence in active transport depends on systematic changes in the properties of the carrier
protein as the reaction proceeds. These changes would have to be brought about by specific interactions with the substrate,
the binding forces being used to stabilize either (i) a carrier state with altered properties or (ii) the transition state
in a carrier transformation. In the first case the tightness of coupling (the ratio of the coupled rate to slippage) will
at first rise with the increment in binding energy in the altered state but will approach an upper limit when overly strong
binding forces retard substrate dissociation in a subsequent step in the coupled reaction sequence. Primary and secondary
active transport are subject to this limitation because the coupling mechanism necessarily involves intermediates in which
the substrate is strongly bound. Exchange-only transport is not necessarily subject to the same limitation because the mechanism
can involve only a substrate-catalyzed change in carrier state. The available data, although scant, agree with these conclusions.
Received: 3 June 1998/Revised: 22 September 1998 相似文献
16.
Evidence for Multidrug Resistance-1 P-Glycoprotein-dependent Regulation of Cellular ATP Permeability 总被引:8,自引:0,他引:8
R. M. Roman N. Lomri G. Braunstein A. P. Feranchak L. A. Simeoni A. K. Davison E. Mechetner E. M. Schwiebert J. G. Fitz 《The Journal of membrane biology》2001,183(3):165-173
The mechanisms responsible for regulating epithelial ATP permeability and purinergic signaling are not well defined. Based
on the observations that members of the ATP-binding cassette (ABC)1 family of proteins may contribute to ATP release, the purpose of these studies was to assess whether multidrug resistance-1
(MDR1) proteins are involved in ATP release from HTC hepatoma cells. Using a bioluminescence assay to detect extracellular
ATP, increases in cell volume increased ATP release ∼3-fold. The MDR1 inhibitors cyclosporine A (10 μm) and verapramil (10 μm) inhibited ATP release by 69% and 62%, respectively (p < 0.001). Similarly, in whole-cell patch-clamp recordings, intracellular dialysis with C219 antibodies to inhibit MDR1 decreased
ATP-dependent volume-sensitive Cl− current density from −33.1 ± 12.5 pA/pF to −2.0 ± 0.3 pA/pF (−80 mV, p≤ 0.02). In contrast, overexpression of MDR1 in NIH 3T3 cells increased ATP release rates. Inhibition of ATP release by Gd3+ had no effect on transport of the MDR1 substrate rhodamine-123; and alteration of MDR1-substrate selectivity by mutation
of G185 to V185 had no effect on ATP release. Since the effects of P-glycoproteins on ATP release can be dissociated from
P-glycoprotein substrate transport, MDR1 is not likely to function as an ATP channel, but instead serves as a potent regulator
of other cellular ATP transport pathways.
Received: 20 November 2000/Revised: 25 May 2001 相似文献
17.
D.C. Winter M.F. Schneider G.C. O'Sullivan B.J. Harvey J.P. Geibel 《The Journal of membrane biology》1999,170(1):17-26
Aldosterone plays a central role in the homeostatic regulation of extracellular fluid volume by stimulating transepithelial
electrolyte transport. These effects involve binding to an intracellular receptor, modification of genomic events and protein
synthesis. Rapid cellular responses to steroid hormones have been observed in a variety of nonepithelial tissues. The term
``nongenomic' has been proposed for these fast steroid responses since they are unaffected by inhibitors of protein synthesis.
We hypothesized that colonic crypts, recently demonstrated to absorb fluid, would respond rapidly to aldosterone.
Cytoplasmic pH changes in crypts loaded with a pH-sensitive, fluorescent dye (BCECF) were recorded with confocal laser imaging.
An intracellular alkalization of colonic crypts was observed within one minute of aldosterone application that was inhibited
by ethylisopropylamiloride or the absence of extracellular sodium, yet unaffected by inhibitors of protein synthesis. The
genesis of this rapid and distinct steroid action involves a signal transduction pathway that involves G proteins, protein
kinase C, and prostaglandins.
We have identified, by real-time imaging, a nongenomic upregulation of sodium-hydrogen exchange in colonic crypts by aldosterone
that occurs independent of the traditional receptor. This distinct, rapid onset effect of aldosterone on epithelial ion transport
has major implications for our understanding of fluid and electrolyte homeostasis in health and disease.
Received: 27 October 1998/Revised: 23 March 1999 相似文献
18.
Varela MF Wilson TH Rodon-Rivera V Shepherd S Dehne TA Rector AC 《The Journal of membrane biology》2000,174(3):199-205
Lactose and melibiose are actively accumulated by the wild-type Escherichia coli lactose carrier, which is an integral membrane protein energized by the proton motive force. Mutants of the E. coli lactose carrier were isolated by their ability to grow on minimal plates with succinate plus IPTG in the presence of the
toxic lactose analog β-thio-o-nitrophenylgalactoside (TONPG). TONPG-resistant mutants were streaked on melibiose MacConkey indicator plates, and red clones
were picked. These melibiose positive mutants were then streaked on lactose MacConkey plates, and white clones were picked.
Transport assays indicated that the mutants had altered sugar recognition and a defect in sugar accumulation. The mutants
had a poor apparent K
m
for both lactose and melibiose in transport. One mutant had almost no ability to take up lactose, but melibiose downhill
transport was 58% (V
max
) of normal. All of the mutants accumulated methyl-α-d-galactopyranoside (TMG) to only 8% or less of normal, and two failed to accumulate. Immunoblot analysis of the mutant lactose
carrier proteins indicated that loss of sugar transport activity was not due to loss of expression in the membrane. Nucleotide
sequencing of the lacY gene from the mutants revealed changes in the following amino acids of the lactose carrier: M23I, W151L, G257D, A295D and
G377V. Two of the mutants (G257D and G377V) are novel in that they represent the first amino acids in periplasmic loops to
be implicated with changes in sugar recognition. We conclude that the amino acids M23, W151, G257, A295 and G377 of the E. coli lactose carrier play either a direct or an indirect role in sugar recognition and accumulation.
Received: 12 October 1999/Revised: 21 December 1999 相似文献
19.
M.L. Chalfant J.M. Civan K. Peterson-Yantorno D.R. DiBona T.G. O'Brien M.M. Civan 《The Journal of membrane biology》1996,152(3):207-215
Protein kinase C (PKC) is a major regulator of a broad range of cellular functions. Activation of PKC has been reported to
stimulate Na+ transport across frog skin epithelium by increasing the apical Na+ permeability. This positive natriferic response has not been observed with other epithelial preparations, and could reflect
the specific experimental conditions of different laboratories, or species or organ specificity of the response to PKC.
In the present study, measurements were conducted with skins and urinary bladders from the same animals of two different species.
The PKC activator TPA uniformly increased the transepithelial Na+ transport (measured as amiloride-sensitive short-circuit current, I
SC, across skins from Rana temporaria and Bufo marinus, and inhibited I
SC across bladders from the same animals. Inhibitors of PKC (staurosporine, H-7 and chelerythrine) partially blocked the TPA-induced
stimulation of I
SC across frog skin. The specificity of the PKC response by amphibian skin could have reflected an induction of moulting, similar
to that observed with aldosterone. However, light micrographs of paired areas of frog skin revealed no evidence of the putative
moulting. Separation of stratum corneum from the underlying stratum granulosum could be detected following application of aldosterone.
We conclude that the effect of PKC on epithelial Na+ channels is organ, and not species specific. The stimulation of Na+ permeability in amphibian skin does not arise from sloughing of the stratum corneum. These observations are consistent with the hypothesis that the natriferic action arises from the calcium-independent isozyme
of PKC previously detected in frog skin.
Received: 19 January 1996/Revised: 10 April 1996 相似文献
20.
Fresh-water plants generate extraordinarily high electric potential differences at the plasma membrane. For a deeper understanding
of the underlying transport processes a mathematical model of the electrogenic plasmalemma ion transport was developed based
on experimental data mainly obtained from Egeria densa. The model uses a general nonlinear network approach and assumes coupling of the transporters via membrane potential. A proton pump, an outward-rectifying K+ channel, an inward-rectifying K+ channel, a Cl− channel and a (2H-Cl)+ symporter are considered to be elements of the system. The model takes into consideration the effects of light, external
pH and ionic content of the bath medium on ion transport. As a result it does not only satisfactorily describe the membrane
potential as a function of these external physiological factors but also succeeds in simulating the effects of specific inhibitors
as well as I-V-curves obtained with the patch-clamp technique in the whole cell mode. The quality of the model was checked by stability and
sensitivity analyses.
Received: 18 March 1996/Revised: 17 July 1996 相似文献