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1.
The export of proOmpA, the precursor of outer membrane protein A from Escherichia coli, requires preprotein translocase, which is comprised of SecA, SecY/E, and acidic phospholipids. Previous studies of proOmpA translocation intermediates (Schiebel, E., Driessen, A. J. M., Hartl, F.-U., and Wickner, W. (1991) Cell 64, 927-939) suggested that the "slippage" of the translocating polypeptide chain and the high level of ATP hydrolysis, characteristic of the "translocation ATPase," were part of a futile cycle. To examine the role of the mature domain of proOmpA in its translocation-dependent ATP hydrolysis, we used chemical cleavage to generate NH2-terminal fragments of this preprotein. Each fragment contained the 21-residue leader region and either 53 or 228 residues of the mature domain (preproteins P74 and P249, respectively). As observed with full-length proOmpA, the translocation of each fragment requires ATP and both the SecA and SecY/E domains of translocase and is stimulated by the transmembrane proton electrochemical gradient. The apparent maximal velocities of P74 and proOmpA translocation are similar. While the translocation of P74 and of proOmpA show the same apparent Km for ATP, far less ATP is hydrolyzed during the translocation of P74. Thus, the mature carboxyl-terminal domain of proOmpA has a major role in supporting the translocation ATPase. 相似文献
2.
The introduction of positive charges at the amino terminus of the mature domain of secretory proteins resulted in strong inhibition of their translocation across the cytoplasmic membrane of Escherichia coli, both in vitro and in vivo. The model secretory proteins used were OmpF-Lpp chimeric proteins possessing a cleavable or uncleavable signal peptide, beta-lactamase (Bla) and Bla-Lpp chimeric proteins. It is suggested that positively charged residues preceding the hydrophobic domain of the signal peptide have a positive effect, and ones following the hydrophobic domain, a negative effect on the translocation. These findings are discussed in relation to the orientation of membrane proteins, of which positive charges are predominant on the cytoplasmic surface. 相似文献
3.
The electrochemical potential drives the translocation of the precursor form of outer membrane protein A (proOmpA) and other proteins across the plasma membrane of Escherichia coli. We have measured the electrical potential, delta psi, across inverted membrane vesicles during proOmpA translocation. delta psi, generated by the electron transport chain, is substantially dissipated by proOmpA translocation. delta psi dissipation requires SecA, ATP, and proOmpA. proOmpA which, due to the covalent addition of a folded protein to a cysteinyl side chain, is arrested during its translocation, can nevertheless cause the loss of delta psi. Thus the movement of charged amino acyl residues is not dissipating the potential. This translocation-specific reduction in delta psi is only seen in the presence of halide anions, although halide anions are not needed for proOmpA translocation per se. We therefore propose that translocation intermediates directly increase the membrane permeability to halide anions. 相似文献
4.
A variety of model presecretory proteins, proOmpF-Lpps, possessing different numbers of lysine residues (0, 2, and 4) as positively charged amino acid residues and different numbers of leucine residues (7, 8, and 9) as hydrophobic amino acid residues in their signal peptides were constructed. The effect of positive charges on the in vitro translocation efficiency markedly differed with the number of leucine residues. Positive charges were strongly required for translocation when the hydrophobic region comprised 7 or 8 leucine residues, whereas the translocation of proOmpF-Lpps possessing 9 leucine residues took place efficiently even in the absence of positive charges and the introduction of positive charges did not significantly enhance the translocation efficiency. The translocation of all the proOmpF-Lpps, including one possessing no positive charge, was ATP-, protonmotive force-, and SecA-dependent and accompanied by signal peptide cleavage, indicating that they are translocated via the usual secretory pathway. It is likely that the requirement of positive charges can be compensated for by a longer hydrophobic stretch in the functioning of the signal peptide. 相似文献
5.
The effect of charges existing on the mature domain of secretory proteins on the efficiency and protonmotive force dependence of translocation into everted membrane vesicles of Escherichia coli was studied. Model secretory proteins devoid of charges on the mature domain were constructed at the DNA level using proOmpF-Lpp as the starting protein. The chargeless presecretory proteins thus constructed were translocated and processed for the signal peptide much faster than proOmpF-Lpp and the rate of translocation was appreciably enhanced by imposition of the protonmotive force. Not only the membrane potential but also delta pH were effective in stimulating the rate of translocation of the chargeless proteins. The results indicate that the mature domain does not have to be charged for the secretory translocation and that the major requirement of the protonmotive force for the secretory translocation is not for the movement, including an electrophoretic one, of charged regions of the mature domain. All of the proOmpF-Lpp derivatives thus constructed were translocated efficiently into everted membrane vesicles in a SecA-dependent manner, irrespective of their size. The mature domain of the smallest one was 45 amino acid residues in length. Contrary to the views previously presented by other workers, these results suggest that there is no sharp boundary at the reported regions for the translocation of presecretory proteins across the cytoplasmic membrane or for the requirement of SecA. 相似文献
6.
The role of the voltage sensor positive charges in the activation and deactivation gating of the rat brain IIA sodium channel was investigated by mutating the second and fourth conserved positive charges in the S4 segments of all four homologous domains. Both charge-neutralizing (by glutamine substitution) and -conserving mutations were constructed in a cDNA encoding the sodium channel α subunit that had fast inactivation removed by the incorporation of the IFMQ3 mutation in the III–IV linker (West, J.W., D.E. Patton, T. Scheuer, Y. Wang, A.L. Goldin, and W.A. Catterall. 1992. Proc. Natl. Acad. Sci. USA. 89:10910–10914.). A total of 16 single and 2 double mutants were constructed and analyzed with respect to voltage dependence and kinetics of activation and deactivation. The most significant effects were observed with substitutions of the fourth positive charge in each domain. Neutralization of the fourth positive charge in domain I or II produced the largest shifts in the voltage dependence of activation, both in the positive direction. This change was accompanied by positive shifts in the voltage dependence of activation and deactivation kinetics. Combining the two mutations resulted in an even larger positive shift in half-maximal activation and a significantly reduced gating valence, together with larger positive shifts in the voltage dependence of activation and deactivation kinetics. In contrast, neutralization of the fourth positive charge in domain III caused a negative shift in the voltage of half-maximal activation, while the charge-conserving mutation resulted in a positive shift. Neutralization of the fourth charge in domain IV did not shift the half-maximal voltage of activation, but the conservative substitution produced a positive shift. These data support the idea that both charge and structure are determinants of function in S4 voltage sensors. Overall, the data supports a working model in which all four S4 segments contribute to voltage-dependent activation of the sodium channel. 相似文献
7.
Preprotein transfer to the Escherichia coli translocase requires the co-operative binding of SecB and the signal sequence to SecA 总被引:2,自引:2,他引:0
Peter Fekkes Janny G. de Wit Jeroen P. W. van der Wolk Harvey H. Kimsey Carol A. Kumamoto & Arnold J. M. Driessen 《Molecular microbiology》1998,29(5):1179-1190
In Escherichia coli , precursor proteins are targeted to the membrane-bound translocase by the cytosolic chaperone SecB. SecB binds to the extreme carboxy-terminus of the SecA ATPase translocase subunit, and this interaction is promoted by preproteins. The mutant SecB proteins, L75Q and E77K, which interfere with preprotein translocation in vivo , are unable to stimulate in vitro translocation. Both mutants bind proOmpA but fail to support the SecA-dependent membrane binding of proOmpA because of a marked reduction in their binding affinities for SecA. The stimulatory effect of preproteins on the interaction between SecB and SecA exclusively involves the signal sequence domain of the preprotein, as it can be mimicked by a synthetic signal peptide and is not observed with a mutant preprotein (Δ8proOmpA) bearing a non-functional signal sequence. Δ8proOmpA is not translocated across wild-type membranes, but the translocation defect is suppressed in inner membrane vesicles derived from a prlA4 strain. SecB reduces the translocation of Δ8proOmpA into these vesicles and almost completely prevents translocation when, in addition, the SecB binding site on SecA is removed. These data demonstrate that efficient targeting of preproteins by SecB requires both a functional signal sequence and a SecB binding domain on SecA. It is concluded that the SecB–SecA interaction is needed to dissociate the mature preprotein domain from SecB and that binding of the signal sequence domain to SecA is required to ensure efficient transfer of the preprotein to the translocase. 相似文献
8.
Secretion in yeast: preprotein binding to a membrane receptor and ATP- dependent translocation are sequential and separable events in vitro 总被引:6,自引:3,他引:3
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We have used a cytosol-free assay in which efficient translocation and signal peptide cleavage is achieved when the affinity-purified precursor of OmpA (proOmpA) is diluted out of 8 M urea into a suspension of yeast rough microsomes. This aspect of protein targeting and transport occurs in two discernible steps: (a) in the absence of ATP and cytosolic factors, the precursor binds to the membranes but is not translocated; (b) addition of ATP results in the translocation of the bound precursor and its processing to the mature form. The binding to microsomes of radiolabeled proOmpA is saturable and inhibited by the addition of unlabeled proOmpA but not by mature OmpA or other proteins. The binding of radiolabeled prepro-alpha-factor is also effectively competed by other preproteins, but not by mature ones. Scatchard analysis showed the Kd of proOmpA to be 7.5 X 10(-9) M. Binding is most likely protein mediated as treatment of the microsomes with the protease papain was found to be inhibitory. These results represent the first functional characterization of secretory protein precursor binding to membranes. Alkylation of the microsomes with NEM, washing the membranes with urea or using membranes from the (translocation) mutant ptll at the nonpermissive temperature, did not affect binding, but did eliminate the subsequent ATP-dependent translocation. The ability to subdivide translocation into individual reactions provides a more precise means of determining the membrane components involved in this process. 相似文献
9.
Differential translocation of protein precursors across SecY-deficient membranes of Escherichia coli: SecY is not obligatorily required for translocation of certain secretory proteins in vitro.
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SecY, a component of the protein translocation system in Escherichia coli, was depleted at a nonpermissive temperature in a strain which had a temperature-sensitive polar effect on the expression of its secY. Membrane vesicles prepared from these cells, when grown at the nonpermissive temperature, contained about 5% SecY and similarly low levels of SecG. As expected, translocation of alkaline phosphatase precursors across these SecY-deficient membranes was severely impaired and appeared to be directly related to the decrease of SecY amounts. However, despite such a dramatic reduction in SecY and SecG levels, these membranes exhibited 50 to 70% of the wild-type translocation activity, including the processing of the signal peptide, of OmpA precursor (proOmpA). This translocation activity in SecY-deficient membranes was still SecA and ATP dependent and was not unique to proOmpA, as lipoprotein and lambda receptor protein precursors were also transported efficiently. Membranes that were reconstituted from these SecY-depleted membranes contained undetectable amounts of SecY yet were also shown to possess substantial translocation activity for proOmpA. These results indicate that the requirement of SecY for translocation is not obligatory for all secretory proteins and may depend on the nature of precursors. Consequently, it is unlikely that SecY is the essential core channel through which all precursors traverse across membranes; rather, SecY probably contributes to efficiency and specificity. 相似文献
10.
Human ether-a-go-go-related gene (HERG) potassium channels contribute to the repolarization of the cardiac action potential and display unique gating properties with slow activation and fast inactivation kinetics. Deletions in the N-terminal 'proximal' domain (residues 135-366) have been shown to induce hyperpolarizing shifts in the voltage dependence of activation, suggesting that it modulates activation. However, we did not observe a hyperpolarizing shift with a subtotal deletion designed to preserve the local charge distribution, and other deletions narrowed the region to the KIKER containing sequence 362-372. Replacing the positively charged residues of this sequence by negative ones (EIEEE) resulted in a -45 mV shift of the voltage dependence of activation. The shifts were intermediate for individual charge reversals, whereas E365R resulted in a positive shift. Furthermore, the shifts in the voltage dependence were strongly correlated with the net charge of the KIKER region. The apparent speeding of the activation was attributable to the shifted voltage dependence of activation. Additionally, the introduction of negative charges accelerated the intermediate voltage-independent forward rate constant. We propose that the modulatory effects of the proximal domain on HERG gating are largely electrostatic, localized to the charged KIKER sequence. 相似文献
11.
Nico Nouwen 《Journal of molecular biology》2009,386(4):1000-1010
Sec translocase catalyzes membrane protein insertion and translocation. We have introduced stretches of charged amino acid residues into the preprotein proOmpA and have analyzed their effect on in vitro protein translocation into Escherichia coli inner membrane vesicles. Both negatively and positively charged amino acid residues inhibit translocation of proOmpA, yielding a partially translocated polypeptide chain that blocks the translocation site and no longer activates preprotein-stimulated SecA ATPase activity. Stretches of positively charged residues are much stronger translocation inhibitors and suppressors of the preprotein-stimulated SecA ATPase activity than negatively charged residues. These results indicate that both clusters of positively and negatively charged amino acids are poor substrates for the Sec translocase and that this is reflected by their inability to stimulate the ATPase activity of SecA. 相似文献
12.
The interaction between a positively charged peptide (poly-L-lysine) and model membranes containing charged lipids has been investigated. Conformational changes of the polypeptide as well as changes in the membrane lipid distribution were observed upon lipid-protein agglutination: 1. The strong binding of polylysine is shown directly by the use of spinlabelled polypeptide. Upon binding to phosphatidic acid a shift in the hyperfine coupling constant from 16.5 to 14.6 Oe is observed. The spectrum of the lipid-bound peptide is superimposed on the spectrum of polylysine in solution. Half of the lysine groups are bound to the charged membranes. A change in the conformation of polylysine from a random coil to a partially ordered configuration is suggested. 2. Spin labelling of the lipid component gives evidence concerning the molecular organization of a lipid mixture containing charged phosphatitid acid. Addition of polylysine induces the formation of crystalline patches of bound phosphatidic acid. 3. Excimer forming pyrene decanoic acid has been employed. Addition of positively charged polylysine (pH 9.0) to phosphatidic acid membranes increases the transition temperature of the lipid from Tt = 50 to Tt = 62 degrees C. Thus, a lipid segregation of lipid into regions of phosphatidic acid bound to the peptide which differ in their microviscosity from the surrounding membrane is induced. One lysine group binds one phosphatidic acid molecule, but only half of the phosphatidic acid is bound. 4. Direct evidence for charge induced domain formation in lipid mixtures containing phosphatidic acid is given by electron microscopy. Addition of polylysine leads to a change in the surface curvature of the bound charged lipid. The domain size is estimated from the electron micrographs. The number of domains present is dependent on both the ratio of charged to uncharged lipids as well as on the amount of polylysine added to the vesicles. The size of the domains is not dependent on membrane composition. However, the size seems to increase in a stepwise manner that is correlated with a multiple of the area covered by one polylysine molecule. 相似文献
13.
Zhang M Liu J Jiang M Wu DM Sonawane K Guy HR Tseng GN 《The Journal of membrane biology》2005,207(3):169-181
Studies on voltage-gated K channels such as Shaker have shown that positive charges in the voltage-sensor (S4) can form salt
bridges with negative charges in the surrounding transmembrane segments in a state-dependent manner, and different charge
pairings can stabilize the channels in closed or open states. The goal of this study is to identify such charge interactions
in the hERG channel. This knowledge can provide constraints on the spatial relationship among transmembrane segments in the
channel’s voltage-sensing domain, which are necessary for modeling its structure. We first study the effects of reversing
S4’s positive charges on channel activation. Reversing positive charges at the outer (K525D) and inner (K538D) ends of S4
markedly accelerates hERG activation, whereas reversing the 4 positive charges in between either has no effect or slows activation.
We then use the ‘mutant cycle analysis’ to test whether D456 (outer end of S2) and D411 (inner end of S1) can pair with K525
and K538, respectively. Other positive charges predicted to be able, or unable, to interact with D456 or D411 are also included
in the analysis. The results are consistent with predictions based on the distribution of these charged residues, and confirm
that there is functional coupling between D456 and K525 and between D411 and K538. 相似文献
14.
SecA is a translocation ATPase that drives protein translocation. D209N SecA, a dominant-negative mutant, binds ATP but is unable to hydrolyze it. This mutant was inactive to proOmpA translocation. However, it generated a translocation intermediate of 18 kDa. Further addition of wild-type SecA caused its translocation into either mature OmpA or another intermediate of 28 kDa that can be translocated into mature by a proton motive force. The addition of excess D209N SecA during translocation caused a topology inversion of SecG. Moreover, an intermediate of SecG inversion was identified when wild-type and D209N SecA were used in the same amounts. These results indicate that multiple SecA molecules drive translocation across a single translocon with SecG inversion. Here, we propose a revised model of proOmpA translocation in which a single catalytic cycle of SecA causes translocation of 10-13 kDa with ATP binding and hydrolysis, and SecG inversion is required when the next SecA cycle begins with additional ATP hydrolysis. 相似文献
15.
Sec-dependent membrane protein biogenesis: SecYEG, preprotein hydrophobicity and translocation kinetics control the stop-transfer function. 总被引:4,自引:2,他引:2
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Preprotein translocase catalyzes membrane protein integration as well as complete translocation. Membrane proteins must interrupt their translocation and be laterally released from the translocase into the lipid bilayer. We have analyzed the translocation arrest and lateral release activities of Escherichia coli preprotein translocase with an in vitro reaction and the preprotein proOmpA carrying a synthetic stop-transfer sequence. Membrane protein integration is catalytic, occurs with kinetics similar to those of proOmpA itself and only requires the functions of SecYEG and SecA. Though a strongly hydrophobic segment will direct the protein to leave the translocase and enter the lipid bilayer, a protein with a segment of intermediate hydrophobicity partitions equally between the translocated and membrane-integrated states. Analysis of the effects of PMF, varied ATP concentrations or synthetic translocation arrest show that the stop-translocation efficiency of a mildly hydrophobic segment depends on the translocation kinetics. In contrast, the lateral partitioning from translocase to lipids depends solely on temperature and does not require SecA ATP hydrolysis or SecA membrane cycling. Thus translocation arrest is controlled by the SecYEG translocase activity while lateral release and membrane integration are directed by the hydrophobicity of the segment itself. Our results suggest that a greater hydrophobicity is required for efficient translocation arrest than for lateral release into the membrane. 相似文献
16.
The KdpFABC complex, found in a variety of prokaryotes, is an emergency potassium uptake system which belongs to the family of P-type ATPases. Site-directed mutagenesis of the charged residues aspartate 583 and lysine 586 in the putative transmembrane helix V of subunit KdpB revealed that these charges are involved in the coupling of ATP hydrolysis and ion translocation. Phenotypic characterization of KdpFABC derivatives carrying alterations at either D583 or K586 demonstrated that only restoration of charges at these positions allowed growth on low potassium concentrations. Substitutions, which eliminated the negative charge at position 583, did not allow growth below 15 mM potassium on solid media. In contrast, substitutions of the positive charge at position 586 allowed growth down to 0.3 mM potassium. Purified KdpFABC complexes carrying these substitutions exhibited ATPase activity, which was, however, found to be comparatively resistant to o-vanadate. Furthermore, elimination of the charges led to a complete loss of ion-stimulated ATPase activity, though the rate of hydrolysis was comparable to wild-type activity, indicating an uncoupling between ATP hydrolysis and ion translocation. This fact was substantiated by reconstitution experiments, in which the D583A complex was unable to facilitate ion translocation, whereas the D583E mutant complex still exhibited such activity. On the basis of these results, a new transport model for the Kdp-ATPase is presented here, in which the amino acids D583 and K586 are supposed to play a role in the gating mechanism of the complex. Furthermore, movement of the charged side chains could have a direct influence on the free energy profile within the potassium transporting subunit KdpA, thereby facilitating ion transport against the concentration gradient into the cytosol. 相似文献
17.
Delta mu H+ and ATP function at different steps of the catalytic cycle of preprotein translocase. 总被引:34,自引:0,他引:34
Preprotein translocation in E. coli requires ATP, the membrane electrochemical potential delta mu H+, and translocase, an enzyme with an ATPase domain (SecA) and the membrane-embedded SecY/E. Studies of translocase and proOmpA binds to the SecA domain. Second, SecA binds ATP. Third, ATP-binding energy permits translocation of approximately 20 residues of proOmpA. Fourth, ATP hydrolysis releases proOmpA. ProOmpA may then rebind to SecA and reenter this cycle, allowing progress through a series of transmembrane intermediates. In the absence of delta mu H+ or association with SecA, proOmpA passes backward through the membrane, but moves forward when either ATP and SecA or a membrane electrochemical potential is supplied. However, in the presence of delta mu H+ (fifth step), proOmpA rapidly completes translocation. delta mu H(+)-driven translocation is blocked by SecA plus nonhydrolyzable ATP analogs, indicating that delta mu H+ drives translocation when ATP and proOmpA are not bound to SecA. 相似文献
18.
Translocation of ProOmpA possessing an intramolecular disulfide bridge into membrane vesicles of Escherichia coli. Effect of membrane energization 总被引:15,自引:0,他引:15
In the absence of delta mu H+, the in vitro translocation of proOmpA resulted in the stable accumulation of a possible translocation intermediate in addition to a transiently accumulating one. The stable intermediate was detected on a polyacrylamide gel as two proteinase K-resistant bands corresponding to a molecular weight of about 28,000. The appearance of the bands was appreciably enhanced when proOmpA was oxidized with ferricyanide. No mature OmpA appeared. When proOmpA reduced with dithiothreitol was used, on the other hand, the bands did not appear at all. Upon the replacement of Cys302 of OmpA with Gly, the intermediate accumulation was abolished. The proOmpA treated with dithiothreitol was labeled with N-[3H]-ethylmaleimide, whereas that treated with ferricyanide was not. The ferricyanide-treated proOmpA was translocated into membrane vesicles in the presence of delta mu H+. The mature OmpA thus translocated and processed was not labeled with N-[3H]ethylmaleimide. It is concluded that proOmpA possessing the Cys290-Cys302 disulfide bridge can be translocated without cleavage of the bridge, when delta mu H+ is imposed. The accumulation of the disulfide bridge-containing intermediate was ATP-dependent, whereas its conversion to the translocated mature form was not blocked in the presence of adenosine 5'-(beta, gamma-imino)triphosphate. It is concluded that the early and late stages of the translocation reaction require ATP and delta mu H+ differently. 相似文献
19.
Structural requirements for interruption of protein translocation across rough endoplasmic reticulum membrane 总被引:5,自引:0,他引:5
Co-translational translocation of proteins across the membrane of rough endoplasmic reticulum (ER) is interrupted by particular amino acid sequences, which are functionally termed "stop-transfer sequence." We analyzed the structural requirements for the interruption of the peptide translocation. By the manipulation of the cDNA of interleukin 2 (IL2), which passes through ER membrane co-translationally, the middle portion of the IL2 molecule was replaced with systematically altered hydrophobic segments, leucine, alanine, or leucine/alanine mixed clusters. Furthermore, charged amino acid residues were introduced just downstream of the hydrophobic segments. These modified IL2 peptides were synthesized with wheat germ cell-free system in the presence of rough microsomes and the topology of the peptides in the microsomes was assessed by post-translational digestion with proteinase K. We obtained the following results. (i) Each modified protein was processed to the mature form but the extent of stop-translocation varied widely. The ratio of the stopped to the translocated products increased as the length and hydrophobicity of the inserted segment increased. (ii) Shorter hydrophobic segments than naturally occurring native transmembrane segment promoted stop-translocation. (iii) Proteins with hydrophobic segments followed by positive charges were more efficiently stop-translocated than those having negative charges. (iv) If the hydrophobicity of the segment was sufficiently high, the positive charges after the segment were not essential for stop-translocation. We also suggest that the stop-transfer process includes protein-protein interaction between the hydrophobic segment and translocation channel. 相似文献
20.
(1) The effect of surface charge upon the kinetics of monovalent cation translocation via a two-site mechanism is investigated theroretically. (2) According to the model dealt with, typical relations are expected for the dependence of the kinetic parameters of the translocation process upon the concentration of a polyvalent cation, differing essentially from those derived for the case in which the membrane carries no excess charge. (3) Even when a polyvalent cation does not compete with the substrate cation for binding to the translocation sites, apparently competitive inhibition may occur when the membrane is negatively charged. (4) The model is tested experimentally by studying the effects of the polyvalent cations Mg2+, Sr2+, Ca2+, Ba2+ and Al3+ upon Rb+ uptake into yeast cells at pH 4.5 A good applicability is found. (5) Equimolar concentrations of polyvalent cations reduce the rate of the Rb+ uptake into yeast cells in the order Mg2+ less than Sr2+ less than Ca2+ less than Ba2+ less than Al3+. (6) The conclusion is reached that the reduction in the rate of Rb+ uptake caused by the polyvalent cations applied results mainly from screening of the negative fixed charges on the membrane surface and binding to these negative sites rather than competition with Rb+ for the transport sites. (7) The results of our investigation indicate the affinity of the alkaline-earth cations for the negative fixed charges on the surface to the yeast cell membrane increases in the orther Mg2+ less than Sr2 less than Ca2+ less than Ba2+. (8) Probably mainly phosphoryl groups determine the net charge on the membrane of the yeast cell at a medium pH of 4.5. 相似文献