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1.
M. Torras-Llort R. Ferrer J.F. Soriano-García M. Moretó 《The Journal of membrane biology》1996,152(3):183-193
The properties of l-lysine transport in chicken jejunum have been studied in brush border membrane vesicles isolated from 6-wk-old birds. l-lysine uptake was found to occur within an osmotically active space with significant binding to the membrane. The vesicles
can accumulate l-lysine against a concentration gradient, by a membrane potential-sensitive mechanism. The kinetics of l-lysine transport were described by two saturable processes: first, a high affinity-transport system (K
mA= 2.4 ± 0.7 μmol/L) which recognizes cationic and also neutral amino acids with similar affinity in the presence or absence
of Na+ (l-methionine inhibition constant KiA, NaSCN = 21.0 ± 8.7 μmol/L and KSCN = 55.0 ± 8.4 μmol/L); second, a low-affinity transport mechanism (KmB= 164.0 ± 13.0 μmol/L) which also recognizes neutral amino acids. This latter system shows a higher affinity in the presence
of Na+ (KiB for l-methionine, NaSCN = 1.7 ± 0.3 and KSCN = 3.4 ± 0.9 mmol/L). l-lysine influx was significantly reduced with N-ethylmaleimide (0.5 mmol/L) treatment. Accelerative exchange of extravesicular labeled l-lysine was demonstrated in vesicles preloaded with 1 mmol/L l-lysine, l-arginine or l-methionine. Results support the view that l-lysine is transported in the chicken jejunum by two transport systems, A and B, with properties similar to those described
for systems b
0,+ and y+, respectively.
Received: 14 August 1995/Revised: 2 April 1996 相似文献
2.
The rBAT protein, when expressed in Xenopus oocytes, was previously shown to reproduce the selectivity of the Na+-independent neutral and basic amino acid transport system called bo,+. More recently, the capacity of rBAT to generate a transmembrane current was demonstrated when addition of neutral amino
acids stimulated the efflux of cations (presumably basic amino acids) in rBAT-injected oocytes. In the present paper, aminoisobutyric
acid (AIB), a neutral amino acid analogue, was shown to induce outward currents (efflux of basic amino acids) through rBAT
similar to those caused by alanine in terms of affinity, maximal currents and I-V curves. Despite generating similar currents, the AIB transport rate was more than 30 times lower than that of alanine, thus
challenging the assumption that rBAT functions as a classical exchanger. Experiments using a cut-open oocyte voltage clamp
demonstrated that AIB was capable of stimulating rBAT-mediated currents from either side of the membrane. AIB, like alanine,
was able to stimulate the efflux of radiolabeled alanine and arginine while no rBAT-mediated efflux was measurable in the
absence of external rBAT substrates. These results demonstrate that (i) the presence of amino acids is required on both sides
of the membrane for rBAT to mediate amino acid flux and thus rBAT must be some type of exchanger but (ii) rBAT-mediated amino
acid influx is not stoichiometrically related to the efflux. A model of a ``double gated pore' is proposed to account for
these properties of rBAT, which contravene standard models of exchangers and other transporters.
Received: 15 June 1995/Revised: 21 September 1995 相似文献
3.
4.
In studies of molecular evolution, the assumption that protein evolution is reversible has often been made, but rarely tested.
Here we use a large set of orthologous murid protein coding sequences to perform a simple test of reversibility, and find
no evidence to reject the assumption of reversibility in protein evolution.
Received: 10 October 2000 / Accepted: 18 January 2001 相似文献
5.
New Glycoprotein-Associated Amino Acid Transporters 总被引:2,自引:0,他引:2
Verrey F Jack DL Paulsen IT Saier MH Pfeiffer R 《The Journal of membrane biology》1999,172(3):181-192
The L-type amino acid transporter LAT1 has recently been identified as being a disulfide-linked ``light chain' of the ubiquitously
expressed glycoprotein 4F2hc/CD98. Several LAT1-related transporters have been identified, which share the same putative 12-transmembrane
segment topology and also associate with the single transmembrane domain 4F2hc protein. They display differing amino acid
substrate specificities, transport kinetics and localizations such as, for instance, y+LAT1 which is localized at the basolateral membrane of transporting epithelia, and the defect of which causes lysinuric protein
intolerance. The b0,+AT transporter which associates with the 4F2hc-related rBAT protein to form the luminal high-affinity diamino acid transporter
defective in cystinuria, belongs to the same family of glycoprotein-associated amino acid transporters (gpaATs). These glycoprotein-associated
transporters function as amino acid exchangers. They extend the specificity range of vectorial amino acid transport when located
in the same membrane as carriers that unidirectionally transport one of the exchanged substrates. gpaATs belong to a phylogenetic
cluster within the amino acid/polyamine/choline (APC) superfamily of transporters. This cluster, which we designate the LAT
family (named after its first vertebrate member), includes some members from nematodes, yeast and bacteria. The latter of
these proteins presumably lack association with a second subunit. In this review, we focus on the animal members of the LAT
cluster that form, together with some of the nematode members, the family of glycoprotein-associated amino acid transporters
(gpaAT family).
Received: 20 July 1999/Revised: 7 September 1999 相似文献
6.
The functional properties of the transport of lysine across the chicken erythrocyte membrane were investigated. The animal
population studied (male Leghorn chickens, 6–14 weeks old) was found to consist of two groups presenting either low (LT, 19
individuals) or high transport rates (HT, 20 individuals). The rates of influx in the two groups, measured at a concentration
of l-lysine of 1 μm, differed by a factor of 34. The transport activities observed in LT and HT erythrocytes were compatible with the general
features of system y+L, but showed some differences in specificity. The transporter in the LT group was found to bind l-lysine, l-leucine, l-methionine and l-glutamine with high affinity, in the presence of sodium, as described for system y+L in human erythrocytes. The activity present in HT erythrocytes exhibited a much lower affinity for l-leucine, but was able to interact strongly with l-glutamine and l-methionine. The specificity pattern of the HT transporter, has not been described in other cell types. In other respects,
the properties of the two systems were similar. Sodium replacement with potassium, drastically reduced the affinity for l-leucine, without affecting lysine transport. Both transporters function as tightly coupled exchangers, are inactivated by
p-chloromercuribenzene sulfonate and resistant to N-ethylmaleimide. These findings explain previous results obtained in selective
breeding experiments of chicken with high and low amino-acid transport activity.
Received: 12 February 2001/Revised: 11 June 2001 相似文献
7.
System y+L is a broad-scope amino acid transporter which binds and translocates cationic and neutral amino acids. Na+ replacement with K+ does not affect lysine transport, but markedly decreases the affinity of the transporter for l-leucine and l-glutamine. This observation suggests that the specificity of system y+L varies depending on the ionic composition of the medium. Here we have studied the interaction of the carrier with various
amino acids in the presence of Na+, K+, Li+ and guanidinium ion. In agreement with the prediction, the specificity of system y+L was altered by the monovalent cations. In the presence of Na+, l-leucine was the neutral amino acid that interacted more powerfully. Elongation of the side chain (glycine - l-norleucine) strengthened binding. In contrast, bulkiness at the level of the β carbon was detrimental. In K+, the carrier behaved as a cationic amino acid specific carrier, interacting weakly with neutral amino acids. Li+ was found to potentiate neutral amino acid binding and in general the apparent affinities were higher than in Na+; elongation of the nonpolar side chain made a more important contribution to binding and the carrier was more tolerant towards
β carbon substitution. Guanidinium stimulated the interaction of the carrier with neutral amino acids, but the effect was
restricted to certain analogues (e.g., l-leucine, l-glutamine, l-methionine). Thus, in the presence of guanidinium, the carrier discriminates sharply among different neutral amino acids.
The results suggest that the monovalent cations stabilize different carrier conformations.
Received: 22 January 1996/Revised: 26 April 1996 相似文献
8.
The mammalian defensin molecule is a short, highly cationic peptide cytotoxic to both microbial and mammalian cells which
is cleaved from a precursor including a signal peptide and a highly anionic propiece. A phylogenetic analysis of 28 complete
sequences from five mammalian species (mouse, rat, guinea pig, rabbit, and human) showed species-specific clusters of sequences,
indicating that the genes duplicated after divergence of these species. Comparison of rates of synonymous and nonsynonymous
nucleotide substitution suggested that gene duplication has often been followed by a period in which diversification of the
mature defensins at the amino acid level has been selectively favored. In some comparisons, it appeared that amino acid differences
in this region have appeared in a nonrandom fashion so as to change the pattern of residue charges. Because it has been hypothesized
that the negative charge in the propiece serves to balance the positive charge in the mature defensin and thus to prevent
cytotoxicity prior to cleavage, we used a maximum likelihood method of reconstructing ancestral states in order to test whether
this balance has been maintained over evolutionary time in spite of rapid diversification of the mature defensin at the amino
acid level. Reconstructed ancestral sequences always maintained a charge balance between mature defensin and propiece, and
changes in the net positive charge of the mature defensin were balanced by corresponding changes in the propiece. The results
support the hypothesis that, in the evolution of these proteins, amino acid changes have occurred in a coordinated fashion
so as to preserve an adaptive phenotype.
Received: 23 October 1996 / Accepted: 7 January 1997 相似文献
9.
The usage of synonymous codons and the frequencies of amino acids were investigated in the complete genome of the bacterium
Thermotoga maritima using a multivariate statistical approach. The GC3 content of each gene was the most prominent source of variation of codon
usage. Surprisingly the usage of UGU and UGC (synonymous triplets coding for Cys, the least frequent amino acid in this species)
was detected as the second most prominent source of variation. However, this result is probably an artifact due to the very
low frequency of Cys together with the nonbiased composition of this genome. The third trend was related to the preferential
usage of a subset of codons among highly expressed genes, and these triplets are presumed to be translationally optimal. Concerning
the amino acid usage, the hydropathy level of each protein (and therefore the frequency of charged residues) was the main
trend, while the second factor was related to the frequency of usage of the smaller residues, suggesting that the cell economy
strongly influences the architecture of the proteins. The third axis of the analysis discriminated the usage of Phe, Tyr,
Trp (aromatic residues) plus Cys, Met, and His. These six residues have in common the property of being the preferential targets
of reactive oxygen species, and therefore the anaerobic condition of T. maritima is an important factor for the amino acid frequencies. Finally, the Cys content of each protein was the fourth trend.
Received: 22 June 2001 / Accepted: 1 October 2001 相似文献
10.
R. Cermak A. Evelgünne C. Lawnitzak E. Scharrer 《The Journal of membrane biology》1999,171(3):235-243
The Ussing chamber technique was used to measure unidirectional Rb+ fluxes under short-circuit conditions across tissue sheets from proximal, central, and distal jejunum of rats.
Whereas the proximal and central parts of the jejunum did not show any net transport of Rb+, there was a net secretion of around 0.2 μmol hr−1 cm−2 in the distal segment. This secretion could not be influenced significantly by mucosal application of K+ channel blockers such as Ba2+ (5 mm), tetraethylammonium (20 mm) or quinine (1 mm). Serosal ouabain (1 mm) blocked net secretion by increasing mucoserosal flux. Blockers of H+/K+ ATPases could not alter net fluxes of Rb+. Stimulation of Cl− secretion by forskolin (10 μm) or of Na+ absorption by serine (10 mm) failed to influence the observed secretion of Rb+. Adrenaline (10 μm) also had no effect on Rb+ fluxes. Blocking Na+/H+ exchange by 5-(N-Ethyl-N-isopropyl)-amilorid (100 μm) blocked net secretion by increasing mucoserosal flux, as did the addition of Na+ acetate (30 mm) to the mucosal solution.
We conclude that the distal jejunum of the rat secretes K+ under short-circuit conditions. This secretion does not seem to occur via K+ channels, but through a pH dependent mechanism.
Received: 16 February 1999/Revised: 29 June 1999 相似文献
11.
J.D.H. Bursell J. Kirk S.T. Hall A.M. Gero K. Kirk 《The Journal of membrane biology》1996,154(2):131-141
The unicellular protozoan parasite, Crithidia luciliae, responded to osmotic swelling by undergoing a regulatory volume decrease. This process was accompanied by the efflux of amino
acids (predominantly alanine, proline and glycine). The relative loss of the electroneutral amino acids proline, valine, alanine
and glycine was greater than that for the anionic amino acid, glutamate; there was negligible loss of the cationic amino acids,
lysine, arginine and ornithine. The characteristics of amino acid release were investigated using a radiolabeled form of the
nonmetabolized alanine analogue α-aminoisobutyrate. α-Aminoisobutyrate efflux was activated within a few seconds of a reduction
of the osmolality, and inactivated rapidly (again within a few seconds) on restoration of isotonicity. The initial rate of
efflux of α-aminoisobutyrate from cells in hypotonic medium was unaffected by the extracellular amino acid concentration.
Hypotonically activated α-aminoisobutyrate efflux (as well as the associated regulatory volume decrease) was inhibited by
the sulfhydryl reagent N-ethylmaleimide but was not inhibited by a range of anion transport blockers. As in the efflux experiments, unidirectional
influx rates for α-aminoisobutyrate increased markedly following reduction of the osmolality, consistent with the swelling-activated
amino acid release mechanism allowing the flux of solutes in both directions. Hypotonically activated α-aminoisobutyrate influx
showed no tendency to saturate up to an extracellular concentration of 50 mm. The functional characteristics of the amino acid release mechanism are those of a channel, with a preference for electroneutral
and anionic amino acids over cationic amino acids. However, the pharmacology of the system differs from that of the anion-selective
channels that are thought to mediate the volume-regulatory efflux of organic osmolytes from vertebrate cells.
Received: 13 May 1996/Revised: 9 July 1996 相似文献
12.
C. Bookstein M.W. Musch Y. Xie M.C. Rao E.B. Chang 《The Journal of membrane biology》1999,171(1):87-95
Until recently, studies to characterize the intestinal epithelial Na+/H+ exchangers had to be done in nonepithelial, mutated fibroblasts. In these cells, detection of any Na+/H+ exchange activity requires prior acid loading. Furthermore, most of these experiments used intracellular pH changes to measure
NHE activity. Because changes in pH
i
only approximate Na+/H+ exchange activity, and may be confounded by alterations in buffering capacity and/or non-NHE contributions to pH regulation,
we have used 22[Na] unidirectional apical to cell uptake to measure activities specific to NHE2 or NHE3. Furthermore, we performed these
measurements under basal, nonacid-stimulated conditions to avoid bias from this nonphysiological experimental precondition. Both brush border NHEs, when expressed in
the well-differentiated, intestinal villuslike Caco-2 subclone, C2bbe (C2), localize to the C2 apical domain and are regulated
by second messengers in the same way they are regulated in vivo. Increases in intracellular calcium and cAMP inhibit both isoforms, while phorbol ester affects only NHE3. NHE2 inhibition
by cAMP and Ca++ involves changes to both K
Na
and V
max
. In contrast, the same two second messengers inhibit NHE3 by a decrease in V
max
exclusively. Phorbol ester activation of protein kinase C alters both V
max
and K
Na
of NHE3, suggesting a multilevel regulatory mechanism. We conclude that NHE2 and NHE3, in epithelial cells, are basally active
and are differentially regulated by signal transduction pathways.
Received: 28 January 1999/Revised: 18 May 1999 相似文献
13.
Takehiro Kusakabe Isato Araki Noriyuki Satoh William R. Jeffery 《Journal of molecular evolution》1997,44(3):289-298
The origin and evolutionary relationship of actin isoforms was investigated in chordates by isolating and characterizing
two new ascidian cytoplasmic and muscle actin genes. The exon–intron organization and sequences of these genes were compared
with those of other invertebrate and vertebrate actin genes. The gene HrCA1 encodes a cytoplasmic (nonmuscle)-type actin, whereas the MocuMA2 gene encodes an adult muscle-type actin. Our analysis of these genes showed that intron positions are conserved among the
deuterostome actin genes. This suggests that actin gene families evolved from a single actin gene in the ancestral deuterostome.
Sequence comparisons and molecular phylogenetic analyses also suggested a close relationship between the ascidian and vertebrate
actin isoforms. It was also found that there are two distinct lineages of muscle actin isoforms in ascidians: the larval muscle
and adult body-wall isoforms. The four muscle isoforms in vertebrates show a closer relationship to each other than to the
ascidian muscle isoforms. Similarly, the two cytoplasmic isoforms in vertebrates show a closer relationship to each other
than to the ascidian and echinoderm cytoplasmic isoforms. In contrast, the two types of ascidian muscle actin diverge from
each other. The close relationship between the ascidian larval muscle actin and the vertebrate muscle isoforms was supported
by both neighbor-joining and maximum parsimony analyses. These results suggest that the chordate ancestor had at least two
muscle actin isoforms and that the vertebrate actin isoforms evolved after the separation of the vertebrates and urochordates.
Received: 20 June 1996 / Accepted: 16 October 1996 相似文献
14.
Nucleotide Composition Bias Affects Amino Acid Content in Proteins Coded by Animal Mitochondria 总被引:16,自引:0,他引:16
We show that in animal mitochondria homologous genes that differ in guanine plus cytosine (G + C) content code for proteins
differing in amino acid content in a manner that relates to the G + C content of the codons. DNA sequences were analyzed using
square plots, a new method that combines graphical visualization and statistical analysis of compositional differences in
both DNA and protein. Square plots divide codons into four groups based on first and second position A + T (adenine plus thymine)
and G + C content and indicate differences in amino acid content when comparing sequences that differ in G + C content. When
sequences are compared using these plots, the amino acid content is shown to correlate with the nucleotide bias of the genes.
This amino acid effect is shown in all protein-coding genes in the mitochondrial genome, including cox I, cox II, and cyt b, mitochondrial genes which are commonly used for phylogenetic studies. Furthermore, nucleotide content differences are shown
to affect the content of all amino acids with A + T- and G + C-rich codons. We speculate that phylogenetic analysis of genes
so affected may tend erroneously to indicate relatedness (or lack thereof) based only on amino acid content.
Received: 3 July 1996 / Accepted: 6 November 1996 相似文献
15.
16.
Outwardly oriented H+ gradients greatly enhanced thiamine transport rate in brush border membrane vesicles from duodenal and jejunal mucosa of
adult Wistar rats. At a gradient pHin5:pHout7.5, thiamine uptake showed an overshoot, which at 15 sec was three times as large as the uptake observed in the absence of
the gradient. Under the same conditions, the binding component of uptake accounted for only 10–13% of intravesicular transport.
At the same gradient, the K
m
and J
max values of the saturable component of the thiamine uptake curve after a 6 sec incubation time were 6.2 ± 1.4 μm and 14.9 ± 3 pmol · mg−1 protein · 6 sec−1 respectively. These values were about 3 and 5 times higher, respectively, than those recorded in the absence of H+ gradient. The saturable component of the thiamine antiport had a stoichiometric thiamine: H+ ratio of 1:1 and was inhibited by thiamine analogues, guanidine, guanidine derivatives, inhibitors of the guanidine/H+ antiport, and imipramine. Conversely, the guanidine/H+ antiport was inhibited by unlabeled thiamine and thiamine analogues; omeprazole caused an approximately fourfold increase
in thiamine transport rate. In the absence of H+ gradient, changes in transmembrane electrical potential did not affect thiamine uptake. At equilibrium, the percentage membrane-bound
thiamine taken up was positively correlated with the pH of the incubation medium, and increased from about 10% at pH 5 to
99% at pH 9.
Received: 17 July 1997/Revised: 16 September 1997 相似文献
17.
Shuji Shigenobu Hidemi Watanabe Yoshiyuki Sakaki Hajime Ishikawa 《Journal of molecular evolution》2001,53(4-5):377-386
Endosymbiotic bacteria live in animal cells and are transmitted vertically at the time of the host's reproduction. In view
of their small and asexual populations with infrequent chances of recombination, these endocellular bacteria are expected
to accumulate mildly deleterious mutations. Previous studies showed that the DNA sequences of these bacteria evolved faster
than those of free-living bacteria. In this study, we compared all the ORFs of Buchnera, an endocellular bacterial symbiont of aphids, with those of 34 other prokaryotic organisms and estimated the effect of the
accelerated evolution of Buchnera on the functions of its proteins. It was revealed that Buchnera proteins contain many mutations at the sites where sequences are conserved in their orthologues in many other organisms.
In addition, amino acid replacements at the conserved sites are mostly changes to physicochemically different amino acids.
These results suggest that functions and conformations of Buchnera proteins have been seriously impaired or strongly modified. Indeed, extensive loss of functional motifs was observed in some
Buchnera proteins. In many Buchnera proteins mutations were not detected evenly throughout each molecule but tended to accumulate in some functional units, possibly
leading to loss of specific functions. As Buchnera has an unusual and limited gene repertory, it is conceivable that the manner of interactions among its proteins has been
changed, and thus, functional constraints over their amino acid residues have also been changed during evolution. This may
account for the loss of some functional units only in the Buchnera proteins. We obtained evidence that amino acid replacements in Buchnera were not always deleterious, but neutral or, in some cases, even positively selected.
Received: 14 December 2000 / Accepted: 12 March 2001 相似文献
18.
19.
The apical brush border membrane, the main target site of Bacillus thuringiensis toxins, was isolated from gypsy moth (Lymantria dispar) larval midguts and fused to artificial planar lipid bilayer membranes. Under asymmetrical N-methyl-d-glucamine-HCl conditions (450 mm
cis/150 mm
trans, pH 9.0), which significantly reduce endogenous channel activity, trypsin-activated Cry1Aa, a B. thuringiensis insecticidal protein active against the gypsy moth in vivo, induced a large increase in bilayer membrane conductance at much
lower concentrations (1.1–2.15 nm) than in receptor-free bilayer membranes. At least 5 main single-channel transitions with conductances ranging from 85 to
420 pS were resolved. These Cry1Aa channels share similar ionic selectivity with P
Cl/P
NMDG permeability ratios ranging from 4 to 8. They show no evidence of current rectification. Analysis of the macroscopic current
flowing through the composite bilayer suggested voltage-dependence of several channels. In comparison, the conductance of
the pores formed by 100–500 nm Cry1Aa in receptor-free bilayer membranes was significantly smaller (about 8-fold) and their P
Cl/P
NMDG permeability ratios were also reduced (2- to 4-fold). This study provides a detailed demonstration that the target insect
midgut brush border membrane material promotes considerably pore formation by a B. thuringiensis Cry toxin and that this interaction results in altered channel properties.
Received: 23 February 2001/Revised: 15 June 2001 相似文献
20.
Codon Usage in Plastid Genes Is Correlated with Context, Position Within the Gene, and Amino Acid Content 总被引:5,自引:0,他引:5
Highly expressed plastid genes display codon adaptation, which is defined as a bias toward a set of codons which are complementary
to abundant tRNAs. This type of adaptation is similar to what is observed in highly expressed Escherichia coli genes and is probably the result of selection to increase translation efficiency. In the current work, the codon adaptation
of plastid genes is studied with regard to three specific features that have been observed in E. coli and which may influence translation efficiency. These features are (1) a relatively low codon adaptation at the 5′ end of
highly expressed genes, (2) an influence of neighboring codons on codon usage at a particular site (codon context), and (3)
a correlation between the level of codon adaptation of a gene and its amino acid content. All three features are found in
plastid genes. First, highly expressed plastid genes have a noticeable decrease in codon adaptation over the first 10–20 codons.
Second, for the twofold degenerate NNY codon groups, highly expressed genes have an overall bias toward the NNC codon, but
this is not observed when the 3′ neighboring base is a G. At these sites highly expressed genes are biased toward NNT instead
of NNC. Third, plastid genes that have higher codon adaptations also tend to have an increased usage of amino acids with a
high G + C content at the first two codon positions and GNN codons in particular. The correlation between codon adaptation
and amino acid content exists separately for both cytosolic and membrane proteins and is not related to any obvious functional
property. It is suggested that at certain sites selection discriminates between nonsynonymous codons based on translational,
not functional, differences, with the result that the amino acid sequence of highly expressed proteins is partially influenced
by selection for increased translation efficiency.
Received: 21 July 1999 / Accepted: 5 November 1999 相似文献