盐和干旱胁迫对燕子掌(Crassula agenten Thunb.)叶片液泡膜H+-ATPase活性的影响

专性CAM植物燕子掌离体叶片的盐胁迫处理和失水干旱处理后,液泡膜H+-ATP酶对低浓度(200、400mmol/L)的盐胁迫(NaCl胁迫48h)不敏感,而当盐浓度达到600mmol/L时,ATP水解活性和H+转运活性较对照上升55%～65%,而干旱胁迫(48h,失水12%)使酶活上升约30%,但是上述各种胁迫均不影响ATP水解与H+转运的耦联比率,仍旧维持在12。用westernblot证实在逆境下,H+-ATP酶的3种主要亚基A、B、c在膜上的蛋白含量均有所增加,且以调节亚基(B)的变化最为显著。     

Mg~(2 )对巯基修饰类囊体膜H~ -ATP酶的质子传导途径的调节效应

镁离子为巯基修饰类囊体还原型H~ -ATP酶光活化所必需。介质中MgCl_2浓度由2mmol/L增加到10mmol/L时,解联剂NH_4Cl对H~ -ATP酶光活化的抑制作用明显减弱,而对跨膜⊿pH的消除效应并未减轻。介质中Mg~(2 )浓度的增加不影响DCMU对H~ -ATP酶光活化的抑制作用。 介质中含40μmol/LADP时,低浓度NH_4Cl对H~ -ATP酶催化的刺激效应被消除,仅呈现抑制作用。这种NH_4Cl对H~ -ATP酶催化活性的抑制作用随着反应介质中Mg~(2 )浓度的增加而降低,因此认为Mg~(2 )参与质子传导途径的调节。     

“非双层脂结构形成的倾向性”对猪心线粒体H~+-ATP酶活性的影响

1、PC+PE重组的线粒体H~+-ATP酶,ATP水解活力及其对寡酶素的敏感性,ATP诱导电位随非双层脂PE含量增加显著增大,当PE含量为60%-80%时ATP诱导电位达最高值.2、用PC+DOPE和PC+DEPE重组的脂酶体,前者的ATP诱导电位显著高于后者,但是两种脂酶体的ATP水解活性和寡霉素敏感性没有明显差别.3、降低PH可以促进PA形式非双层结构,PC+20%PA或大豆磷脂重组的脂酶体H~+-ATP酶活性随pH降低显著增高.4、PC+20%PA和大豆磷脂重组的脂酶体膜表层流动性随透析液PH降低而降低,5、综合上述结果.我们认为“非双层脂结构形成的倾向性”的加强,可能导致一种不稳定的或柔性的膜结构的出现,使得H~+-ATP酶呈现发挥其活性的最适构象.     

菠菜能量转化复合体的人工组装

从菠菜叶绿体中分离了H~+-ATP酶复合体,其SDS-聚丙烯酰胺凝胶电泳呈九条亚单位带,为纯度较高的酶复合体。将H~+-ATP酶复合体重组于人工膜(脂质体)上,表现出PiATP交换活力。Mg~(++)-ATP酶活力也明显提高。并表现对DCCD,寡霉素的敏感性。 重组H~+-ATP酶复合体表现有ATP诱导的H~+转移。但脂质体也出现类似现象,为此对以pH改变为标准的检测方法提出商榷。     

甲基紫精对水稻幼苗根细胞膜微囊Ca2+-ATP酶活性的影响

10μmool/L甲基紫精(MV)预处理水稻幼苗可明显提高其抗冷力,但这种功效可被钙的螯合剂EGTA(10 mmol/L)和钙调素(CaM)的抑制剂氯丙嗪(CPZ,0.5 mmol/L)所抑制.MV预处理提高了幼苗质膜、液泡膜Ca2+-ATP酶活性,同时也有提高质膜Fe(CN)3-6还原速率和这些活性的冷适应性,但这些效果均可被EGTA和CPZ所抑制.离体条件下,膜微囊的Ca2+-ATP酶活性对H2O2、O-2、-OH敏感.结果显示,MV预处理提高幼苗的抗冷力可能是通过钙信使介导起作用的,钙信使或CaM可能刺激了质膜、液泡膜Ca2+-ATP酶活性;而该预处理有增加质膜、液泡膜Ca2+-ATP酶的冷稳定性则可能与该处理有提高细胞抗氧化能力、稳定冷胁迫下细胞膜系统结构有关.     

恒态光合磷酸化与△pH和△ψ的关系

在恒态下推动ATP形成的△μH~+中,除ApH外,△ψ的贡献也很明显:进行PSP反应,耗用H~+,降低了⊿pH幅度。NH_4Cl,尼日利亚菌素(Nig)对⊿pH和PSP的抑制作用是同步的。CCCP影响膜对质子的透性,加速内部质子外流,降低内部质子浓度,减少⊿pH幅度,抑制了PSP反应。PSP更依赖于类囊体内部质子浓度。在短杆菌环肽(Gram)(～5×10~(-8)mol/L)和缬氨霉素(Val)(～5×10~(-7)mol/L,+K~+)的作用下,⊿ψ几乎被完全消去,并对⊿pH无明显作用,但PSP分别被抑制了～30％(Gram)和～50％(Val),表明在恒态PSP反应中有30％～50％的能量来源可由⊿ψ提供。     

NaCl胁迫下大麦根系液泡膜H+-ATP酶活性受ATP和焦磷酸含量的调节

选用两个耐盐性强弱不同的大麦(Hordeumvulgare L.)品种,研究了NaCl胁迫下其幼苗根中ATP和焦磷酸(PPi)含量的变化以及PPi对液泡膜H -ATP酶活性的影响.结果表明:在含NaCl 200mmol/L的1/2 Hoagland溶液中处理2 d,耐盐品种(滩引2号)根中液泡膜H -ATP酶活性增加,然后逐渐下降,而H -PPi酶活性在NaCl处理9 d中'直下降.盐敏感品种(科品7号)在NaCl胁迫下根中H -ATP酶和H -PPi酶活性都下降(图1).与对照相比较,NaCl胁迫下耐盐品种根中ATP含量2 d时增加,4 d后下降;盐敏感品种根中ATP积累受NaCl胁迫的抑制(图2).NaCl胁迫下,两品种的PPi含量皆略有增加(图3).PPi对液泡膜H -ATP酶活性有竞争性抑制作用(图4).结果表明:ATP积累是NaCl胁迫下液泡膜H -ATP酶活性增加的原因之一,NaCl胁迫下大麦品种根中ATP含量下降和PPi对液泡膜H -ATP酶的抑制使该酶活性下降.     

海洋酸化对泥蚶精子运动的影响及作用机理初探

研究分析了未来海洋酸化情景(pH7.8和pH7.4)对典型滩涂贝类泥蚶(Tegillarca granosa)精子运动活力的影响, 并从精子运动供能角度探究了其作用机理。研究结果表明, 海洋酸化可以显著削弱泥蚶的精子运动速度。由于精子的三磷酸腺苷(ATP)水平与其活力呈显著的正相关, 研究检测了不同酸化条件下泥蚶精子的ATP含量及其合成关键酶酶活, 实验结果显示精子的ATP含量以及磷酸果糖激酶和丙酮酸激酶活力在酸化条件下均显著下降。此外, 精子内的Ca2+-ATP酶(Ca2+-ATPase)调节了精子的运动能力, 因此实验也探究了海洋酸化对泥蚶精子Ca2+-ATPase酶活的影响, 结果证实该酶活力在海水pH为7.4时被显著抑制。综合本研究结果可以得出, 海洋酸化很可能会通过干扰精子细胞内ATP的合成及Ca2+的调控进而削弱精子的运动速度。     

水稻幼苗根细胞质膜和液泡膜微囊Ca2+-ATP酶的特性

水稻幼苗根质膜和液泡膜Ca2 -ATP酶对ATP的Km值分别为7.1和4.5 μ mol·L-1;反应的最适pH分别为8.0和7.0.两者活性均受Na3VO4和曙红B(EB)抑制;CPZ抑制质膜Ca2 -ATP酶活性,但促进液泡膜Ca2 -ATP酶活性.30mmol·L-1CaCl2浸种和CaCl2浸种结合低温锻炼预处理,均可提高此酶的活性和冷稳定性.     

Mg~(2+)影响嵌有线粒体H~+-ATP酶脂酶体的表面电荷密度和流动性

<正> 我们实验室曾报道,用胆酸盐透析法将猪心线粒体H~+-ATP酶嵌入大豆磷脂脂质体形成脂酶体时,透析液中Mg~(2+)的存在会降低脂酶体的膜脂流动性,并明显提高重建H~+-ATP酶的活性以及对寡霉素或DCCD的敏感性,因而推论Mg~(2+)的作用很可能是通过改变膜脂的物理状态,形成了维持H~+-ATP酶较高活性的合适构象。但共确切的作用机制仍     

Functional colocalization of water channels and proton pumps in endosomes from kidney proximal tubule

The apical membrane of mammalian proximal tubule undergoes rapid membrane cycling by exocytosis and endocytosis. Osmotic water and ATP- driven proton transport were measured in endocytic vesicles from rabbit and rat proximal tubule apical membrane labeled in vivo with the fluid phase marker fluorescein-dextran. Osmotic water permeability (Pf) was determined from the time course of fluorescein-dextran fluorescence after exposure of endosomes to an inward osmotic gradient in a stopped- flow apparatus. Pf was 0.009 (rabbit) and 0.029 cm/s (rat) (23 degrees C) and independent of osmotic gradient size. Pf in rabbit endosomes was inhibited reversibly by HgCl2 (KI = 0.2 mM) and had an activation energy of 6.4 +/- 0.5 kcal/mol (15-35 degrees C). Endosomal proton ATPase activity was measured from the time course of internal pH, measured by fluorescein-dextran fluorescence, after the addition of external ATP. Endosomes contained an ATP-driven proton pump that was sensitive to N-ethylmaleimide and insensitive to vanadate and oligomycin. In response to saturating [ATP] the pump acidified the endosomal compartment at a rate of 0.17 (rat) and 0.029 pH unit/s (rabbit); at an external pH of 7.4, the steady-state pH was 6.4 (rat) and 6.5 (rabbit). To examine whether water channels and the proton ATPase were present in the same endosome, the time course of fluorescein-dextran fluorescence was measured in response to an osmotic gradient in the presence and absence of ATP. ATP did not alter endosome Pf, but decreased the amplitude of the fluorescence signal by 43 +/- 3% (rabbit) and 47 +/- 4% (rat).(ABSTRACT TRUNCATED AT 250 WORDS)     

Heterogeneity in ATP-dependent acidification in endocytic vesicles from kidney proximal tubule. Measurement of pH in individual endocytic vesicles in a cell-free system.

Measurement of membrane transport in suspensions of isolated membrane vesicles provides averaged information over a potentially very heterogeneous vesicle population. To examine the regulatory mechanisms for ATP-dependent acidification, methodology was developed to measure pH in individual endocytic vesicles. Endocytic vesicles from proximal tubule apical membrane of rat kidney were labeled in vivo by intravenous infusion of FITC-dextran (9 kD); a microsomal fraction was obtained from dissected renal cortex by homogenization and differential centrifugation. Vesicles were immobilized on a polylysine coated coverglass and imaged at high magnification by a silicon intensified target camera. ATP-dependent acidification was not influenced by endosome immobilization. Endosome pH was determined from the integrated fluorescence intensity of individual labeled vesicles after background subtraction. Calibration studies with high K and nigericin showed nearly identical fluorescence vs. pH curves for different endosomes with a standard deviation for a single pH measurement in a single endosome of approximately 0.2 pH units. In response to addition of 1 mM MgATP in the presence of K and valinomycin, endosome pH decreased from 7.2 to a mean of 6.4 with a unimodal distribution with width at half-maximum of approximately 1 pH unit. The drop in endosome pH increased and the shape of the distribution changed when the time between FITC-dextran infusion and kidney removal was increased from 5 to 20 min. Differences in ATP-dependent acidification could not be attributed to heterogeneity in passive proton conductance. These results establish a direct method to measure pH in single endocytic vesicles and demonstrate remarkable heterogeneity in ATP-dependent acidification which was interpreted in terms of heterogeneity in the number and/or activity of proton pumps at serial stages of endocytosis.     

Functional water channels and proton pumps are in separate populations of endocytic vesicles in toad bladder granular cells

Functional water channels are retrieved by endocytosis from the apical membrane of toad bladder granular cells in response to vasopressin [Shi, L.-B., & Verkman, A.S. (1989) J. Gen. Physiol. 94, 1101-1115]. To examine whether endocytic vesicles which contain the vasopressin-sensitive water channel fuse with acidic vesicles for entry into a lysosomal pathway, ATP-dependent acidification and osmotic water permeability were measured in endosomes from control bladders and bladders treated with vasopressin (VP) and/or phorbol myristate acetate (PMA). Endosomes were labeled with the fluid-phase markers 6-carboxyfluorescein or fluorescein-dextran. Osmotic water permeability (Pf) was measured by stopped-flow fluorescence quenching and proton ATPase activity by ATP-dependent, N-ethylmaleimide-inhibitable acidification. In a microsomal pellet, Pf was low (less than 0.002 cm/s, 20 degrees C) in labeled endocytic vesicles from control bladders but high (0.05-0.1 cm/s) in a subpopulation (50-70%) of vesicles from VP- and PMA-treated bladders. Following ATP addition, the average drop in pH was 0.1 (control), 0.3 (VP), and 0.2 (PMA) unit. Measurement of pH in individual endocytic vesicles by quantitative image analysis showed that less than 20% of vesicles from VP-treated bladders acidified by greater than 0.5 pH unit. To examine whether water channels and proton pumps were present in the same endocytic vesicles, the pH of endosomes with high and low water permeability was measured from the effect of ATP on the amplitude of the fluorescence quenching signal in response to an osmotic gradient.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endocytic vesicles from renal papilla which retrieve the vasopressin- sensitive water channel do not contain a functional H+ ATPase

The water permeability of the kidney collecting duct epithelium is regulated by vasopressin (VP)-induced recycling of water channels between an intracellular vesicular compartment and the plasma membrane of principal cells. To test whether the water channels pass through an acidic endosomal compartment during the endocytic portion of this pathway, we measured ATP-dependent acidification of FITC-dextran-labeled endosomes in isolated microsomal fractions from different regions of Brattleboro rat kidneys. Both VP-deficient controls and rat treated with exogenous VP were examined. ATP-dependent acidification was not detectable in endosomes containing water channels from distal papilla (osmotic water permeability Pf = 0.038 +/- 0.004 cm/s). In contrast, the addition of ATP resulted in a strong acidification of renal cortical endosomes (pHmin = 5.8, initial rate = 0.18-0.25 pH U/s). Acidification of cortical endosomes was reversed with nigericin and strongly inhibited by N-ethyl-maleimide. Passive proton permeability was similar and low in both cortical and papillary endosomes from rats treated or not treated with VP. The fraction of labeled endosomes present in microsomal preparations was determined by fluorescence imaging microscopy of microsomes nonspecifically bound to poly-l-lysine-coated coverslips and was 25% in cortical preparations compared to 14% (+VP) and 9% (-VP) in papillary preparations. The fraction of cortical endosomes was enriched 1.5-fold by immunoabsorption to coverslips coated with mAbs against the bovine vacuolar proton pump. In contrast, the fraction of papillary endosomes was depleted more than twofold by immunoabsorption to identical coverslips. Finally, sections of distal papilla stained with antibodies against the lysosomal glycoprotein LGP120 showed that most of the entrapped FITC-dextran did not colocalize with this lysosomal protein. These results demonstrate that vesicles which internalize water channels in kidney collecting duct principal cells lack functional proton pumps, and do not deliver the bulk of their FITC-dextran content to lysosomes. The data suggest that the principal cell contains a specialized nonacidic apical endocytic compartment which functions primarily to recycle membrane components, including water channels, to the plasma membrane.     

Water, proton, and urea transport in toad bladder endosomes that contain the vasopressin-sensitive water channel

Vasopressin (VP) increases the water permeability of the toad urinary bladder epithelium by inducing the cycling of vesicles containing water channels to and from the apical membrane of granular cells. In this study, we have measured several functional characteristics of the endosomal vesicles that participate in this biological response to hormonal stimulation. The water, proton, and urea permeabilities of endosomes labeled in the intact bladder with fluorescent fluid-phase markers were measured. The diameter of isolated endosomes labeled with horse-radish peroxidase was 90-120 nm. Osmotic water permeability (Pf) was measured by a stopped-flow fluorescence quenching assay (Shi, L.-B., and A. S. Verkman. 1989. J. Gen. Physiol. 94:1101-1115). The number of endosomes formed when bladders were labeled in the absence of a transepithelial osmotic gradient increased with serosal [VP] (0-50 mU/ml), and endosome Pf was very high and constant (0.08-0.10 cm/s, 18 degrees C). When bladders were labeled in the presence of serosal-to-mucosal osmotic gradient, the number of functional water channels per endosome decreased (at [VP] = 0.5 mU/ml, Pf = 0.09 cm/s, 0 osmotic gradient; Pf = 0.02 cm/s, 180 mosmol gradient). Passive proton permeability was measured from the rate of pH decrease in voltage-clamped endosomes in response to a 1 pH unit gradient (pHin = 7.5, pHout = 6.5). The proton permeability coefficient (PH) was 0.051 cm/s at 18 degrees C in endosomes containing the VP-sensitive water channel; PH was not different from that measured in vesicles not containing water channels. Measurement of urea transport by the fluorescence quenching assay gave a urea reflection coefficient of 0.97 and a permeability coefficient of less than 10(-6) cm/s. These results demonstrate: (a) VP-induced endosomes from toad urinary bladder have extremely high Pf. (b) In states of submaximal bladder Pf, the density of functional water channels in endosomes in constant in the absence of an osmotic gradient, but decreases in the presence of a serosal-to-mucosal gradient, suggesting that the gradient has a direct effect on the efficiency of packaging of water channels into endosomes. (c) The VP-sensitive water channel does not have a high proton permeability. (d) Endosomes that cycle the water channel do not contain urea transporters. These results establish a labeling procedure in which greater than 85% of labeled vesicles from toad urinary bladder are endosomes that contain the VP-sensitive water channel in a functional form.     

Acidification and ion permeabilities of highly purified rat liver endosomes

While it is well established that acidic pH in endosomes plays a critical role in mediating the orderly traffic of receptors and ligands during endocytosis, little is known about the bioenergetics or regulation of endosome acidification. Using highly enriched fractions of rat liver endosomes prepared by free flow electrophoresis and sucrose density gradient centrifugation, we have analyzed the mechanism of ATP-dependent acidification and ion permeability properties of the endosomal membrane. This procedure permitted the isolation of endosome fractions which were up to 200-fold enriched as indicated by the increased specific activity of ATP-dependent proton transport. Acidification was monitored using hepatocyte and total liver endosomes selectively labeled with pH-sensitive markers of receptor-mediated endocytosis (fluorescein isothiocyanate asialoorosomucoid) or fluid-phase endocytosis (fluorescein isothiocyanate-dextran). In addition, changes in membrane potential accompanying ATP-dependent acidification were directly measured using the voltage-sensitive fluorescent dye Di-S-C3(5). Our results indicate that ATP-dependent acidification of liver endosomes is electrogenic, with proton transport being accompanied by the generation of an interior-positive membrane potential opposing further acidification. The membrane potential can be dissipated by the influx of permeant external anions or efflux of internal alkali cations. Replacement externally of permeable anions with less permeable anions (e.g. replacing Cl- with gluconate) diminished acidification, as did replacement internally of a more permeant cation K+ with less permeant species (such as Na+ or tetramethylammonium). ATP-dependent H+ transport was not coupled to any specific anion or cation, however. The endosomal membrane was found to be extremely permeable to protons, with protons able to leak out almost as fast as they are pumped in. Thus, the internal pH of endosomes is likely to reflect a dynamic equilibrium of protons regulated by the intrinsic ion permeabilities of the endosomal membrane, in addition to the activity of an ATP-driven proton pump.     

Intra-endosomal pH-sensitive recruitment of the Arf-nucleotide exchange factor ARNO and Arf6 from cytoplasm to proximal tubule endosomes

Kidney proximal tubule epithelial cells have an extensive apical endocytotic apparatus that is critical for the reabsorption and degradation of proteins that traverse the glomerular filtration barrier and that is also involved in the extensive recycling of functionally important apical plasma membrane transporters. We show here that an Arf-nucleotide exchange factor, ARNO (ADP-ribosylation factor nucleotide site opener) as well as Arf6 and Arf1 small GTPases are located in the kidney proximal tubule receptor-mediated endocytosis pathway, and that ARNO and Arf6 recruitment from cytosol to endosomes is pH-dependent. In proximal tubules in situ, ARNO and Arf6 partially co-localized with the V-ATPase in apical endosomes in proximal tubules. Arf1 was localized both at the apical pole of proximal tubule epithelial cells, but also in the Golgi. By Western blot analysis ARNO, Arf6, and Arf1 were detected both in purified endosomes and in proximal tubule cytosol. A translocation assay showed that ATP-driven endosomal acidification triggered the recruitment of ARNO and Arf6 from proximal tubule cytosol to endosomal membranes. The translocation of both ARNO and Arf6 was reversed by V-type ATPase inhibitors and by uncouplers of endosomal intralumenal pH, and was correlated with the magnitude of intra-endosomal acidification. Our data suggest that V-type ATPase-dependent acidification stimulates the selective recruitment of ARNO and Arf6 to proximal tubule early endosomes. This mechanism may play an important role in the pH-dependent regulation of receptor-mediated endocytosis in proximal tubules in situ.     

Kinetics of endosome acidification detected by mutant and wild-type Semliki Forest virus.

The fusogenic properties of Semliki Forest virus (SFV) and its mutants were used to follow the kinetics of acidification during the endocytic uptake of virus by BHK-21 cells. It has previously been shown that the low pH of endocytic vacuoles triggers a conformational change in the SFV spike glycoprotein, activating membrane fusion and initiating virus infection. This conformational alteration was here shown to occur in endosomes and to follow the same time course as the intracellular fusion reaction, demonstrating that fusion occurs rapidly after virus exposure to endosome acidity. The kinetics of endosome acidification were monitored using wild type (wt) SFV and fus-1, an SFV mutant with a lower fusion pH threshold. The results presented here demonstrated that wt and mutant virus were internalized with a t1/2 of 10 min, and that endosomes were acidified to the wt threshold of pH 6.2 with a t1/2 of 15 min. In contrast, endosome pH reached the fus-1 threshold of 5.3 with a much longer t1/2 of 45 min. The subsequent degradation of SFV in lysosomes had a t1/2 of 90 min. It was found that after the initial uptake of virus from the plasma membrane, its transit through the endocytic pathway, exposure to endosome acidity and eventual delivery to lysosomes were markedly asynchronous.     

Acidification of morphologically distinct endosomes in mutant and wild- type Chinese hamster ovary cells

In the preceding paper (Yamashiro, D. J., and F. R. Maxfield. 1987. J. Cell Biol. 105:2713-2721), we have shown that there is rapid acidification of endosomal compartments to pH 6.3 by 3 min in wild-type Chinese hamster ovary (CHO) cells. In contrast, early acidification of endosomes is markedly reduced in the CHO mutants, DTF 1-5-4 and DTF 1-5- 1. Since these CHO mutants are pleiotropically defective in endocytosis (Robbins, A. R., S. S. Peng, and J. L. Marshall. 1983. J. Cell Biol. 96:1064-1071; Robbins, A. R., C. Oliver, J. L. Bateman, S. S. Krag, C. J. Galloway, and I. Mellman. 1984. J. Cell Biol. 99:1296-1308), our results are consistent with a requirement for proper acidification of early endocytic compartments in many pH-regulated endocytic processes. In this paper, by measuring the pH of morphologically distinct endosomes using fluorescence microscopy and digital image analysis, we have determined in which of the endocytic compartments the defective acidification occurs. We found that the acidification of both the para- Golgi recycling endosomes and lysosomes was normal in the CHO mutants DTG 1-5-4 and DTF 1-5-1. The mean pH of large endosomes containing either fluorescein-labeled alpha 2-macroglobulin or fluorescein- isothiocyanate dextran was only slightly less acidic in the mutant cells than in wild-type cells. However, when we examined the pH of individual large (150-250 nm) endosomes, we found that there was an increased number of endosomes with a pH greater than 6.5 in the CHO mutants when compared with wild-type cells. Heterogeneity in the acidification of large endosomes was also seen in DTF 1-5-1 by a combined null point pH method and digital image analysis technique. In addition, both CHO mutants showed a marked decrease in the acidification of the earliest endosomal compartment, a diffusely fluorescent compartment comprised of small vesicles and tubules. We suggest that the defect in endosome acidification is most pronounced in the early, small vesicular, and tubular endosomes and that this defect partially carries over to the large endosomes that are involved in the sorting and processing of ligands. The proper step-wise acidification of the different endosomes along the endocytic pathway may have an important role in the regulation of endocytic processes.     

Chloride accumulation and swelling in endosomes enhances DNA transfer by polyamine-DNA polyplexes

The "proton sponge hypothesis" postulates enhanced transgene delivery by cationic polymer-DNA complexes (polyplexes) containing H+ buffering polyamines by enhanced endosomal Cl- accumulation and osmotic swelling/lysis. To test this hypothesis, we measured endosomal Cl- concentration, pH, and volume after internalization of polyplexes composed of plasmid DNA and polylysine (POL), a non-buffering polyamine, or the strongly buffering polyamines polyethylenimine (PEI) or polyamidoamine (PAM). [Cl-] and pH were measured by ratio imaging of fluorescently labeled polyplexes containing Cl- or pH indicators. [Cl-] increased from 41 to 80 mM over 60 min in endosomes-contained POL-polyplexes, whereas pH decreased from 6.8 to 5.3. Endosomal Cl- accumulation was enhanced (115 mM at 60 min) and acidification was slowed (pH 5.9 at 60 min) for PEI and PAM-polyplexes. Relative endosome volume increased 20% over 75 min for POL-polyplexes versus 140% for PEI-polyplexes. Endosome lysis was seen at >45 min for PEI but not POL-containing endosomes, and PEI-containing endosomes showed increased osmotic fragility in vitro. The slowed endosomal acidification and enhanced Cl- accumulation and swelling/lysis were accounted for by the greater H+ buffering capacity of endosomes containing PEI or PAM versus POL (>90 mM versus 46 H+/pH unit). Our results provide direct support for the proton sponge hypothesis and thus a rational basis for the design of improved non-viral vectors for gene delivery.