Lumen pH Research Articles

Sequence and overexpression of GPP130/GIMPc: evidence for saturable pH-sensitive targeting of a type II early Golgi membrane protein.

It is thought that residents of the Golgi stack are localized by a retention mechanism that prevents their forward progress. Nevertheless, some early Golgi proteins acquire late Golgi modifications. Herein, we describe GPP130 (Golgi phosphoprotein of 130 kDa), a 130-kDa phosphorylated and glycosylated integral membrane protein localized to the cis/medial Golgi. GPP130 appears to be the human counterpart of rat Golgi integral membrane protein, cis (GIMPc), a previously identified early Golgi antigen that acquires late Golgi carbohydrate modifications. The sequence of cDNAs encoding GPP130 indicate that it is a type II membrane protein with a predicted molecular weight of 81,880 and an unusually acidic lumenal domain. On the basis of the alignment with several rod-shaped proteins and the presence of multiple predicted coiled-coil regions, GPP130 may form a flexible rod in the Golgi lumen. In contrast to the behavior of previously studied type II Golgi proteins, overexpression of GPP130 led to a pronounced accumulation in endocytotic vesicles, and endogenous GPP130 reversibly redistributed to endocytotic vesicles after chloroquine treatment. Thus, localization of GPP130 to the early Golgi involves steps that are saturable and sensitive to lumenal pH, and GPP130 contains targeting information that specifies its return to the Golgi after chloroquine washout. Given that GIMPc acquires late Golgi modifications in untreated cells, it seems likely that GPP130/GIMPc continuously cycles between the early Golgi and distal compartments and that an unidentified retrieval mechanism is important for its targeting.

Interactions of ANP and ANG II in tubular nephron acidification.

In order to examine the effects and the interaction of angiotensin II (ANG II, 1 pM) and atrial natriuretic peptide (ANP, 1 microM) on the kinetics of bicarbonate reabsorption in the rat middle proximal tubule, we performed in vivo experiments using a stopped-flow microperfusion technique with the determination of lumen pH by Sb microelectrodes. These studies confirmed that ANG II added to the luminal or peritubular capillary perfusion fluid stimulates proximal bicarbonate reabsorption and showed that ANP alone does not affect this process, but impairs the stimulation caused by ANG II. We also studied the effects and the interaction of these hormones in cortical distal nephron acidification. Bicarbonate reabsorption was evaluated by the acidification kinetic technique in early (ED) and late (LD) distal tubules in rats during in vivo stopped-flow microperfusion experiments. The intratubular pH was measured with a double-barreled microelectrode with H(+)-sensitive resin. The results indicate that ANG II acted by stimulating Na+/H+ exchange in ED (81%) and LD (54%) segments via activation of AT1 receptors, as well as vacuolar H(+)-ATPase in LD segments (33%). ANP did not affect bicarbonate reabsorption in either segment and, as opposed to what was seen in the proximal tubule, did not impair the stimulation caused by ANG II. To investigate the mechanism of action of these hormones in more detail, we studied cell pH dependence on ANG II and ANP in MDCK cells using the fluorescent probe BCECF. We showed that the velocity of cell pH recovery was almost abolished in the absence of Na+, indicating that it is dependent on Na+/H+ exchange. ANP (1 microM) alone had no effect on this recovery but reversed both the acceleration of H+ extrusion at low ANG II levels (1 pM and 1 nM), and inhibition of H+ extrusion at higher ANG II levels (100 nM). To obtain more information on the mechanism of interaction of these hormones, we also studied their effects on the regulation of intracellular free calcium concentration, [Ca2+]i, monitored with the fluorescent probe Fura-2 in MDCK cells in suspension. The data indicate that the addition of increasing concentrations of ANG II (1 pM to 1 microM) to the cell suspension led to a progressive increase in [Ca2+]i to 2-3 times the basal level. In contrast, the addition of ANP (1 microM) to the cell suspension led to a very rapid 60% decrease in [Ca2+]i and reduced the increase elicited by ANG II, thus modulating the effect of ANG II on [Ca2+]i. These results may indicate a role of [Ca2+]i in the regulation of the H+ extrusion process mediated by Na+/H+ exchange and stimulated/impaired by ANG II. The data are compatible with stimulation of Na+/H+ exchange by increases of [Ca2+]i in the lower range, and inhibition at high [Ca2+]i levels.

A single point mutation (E166Q) prevents dicyclohexylcarbodiimide binding to the photosystem II subunit CP29

Energy-dependent quenching of chlorophyll fluorescence (qE) reflects the action of a powerful mechanism of protection from photoinhibition in which the low pH in the chloroplast lumen induces dissipation of excess excitation energy. Dicyclohexylcarbodiimide (DCCD), a protein-modifying agent, is a powerful inhibitor of qE and has been shown to react with acidic residues, in a hydrophobic environment, involved in proton translocation. The CP29 subunit of photosystem II has been proposed to be the site of qE quenching and shown to bind DCCD. We have hypothesised, on the basis of the CP29 protein sequence and of the structure of light-harvesting complex II protein, that glutamic acid 166 is the DCCD binding site. In this study, we have produced recombinant proteins either with wild-type sequence or carrying a mutation on the 166 position. We show that the mutant protein does not bind DCCD. This identifies E166 as the site whose protonation may lead to a conformational change triggering qE.

Regulation of cardiac Ca2+ release channel (ryanodine receptor) by Ca2+, H+, Mg2+, and adenine nucleotides under normal and simulated ischemic conditions.

In myocardial ischemia, pHi and [ATP] fall, whereas the free [Ca2+] and [Mg2+] rise. The effects of these changes on cardiac Ca2+ release channel (ryanodine receptor) activity were investigated in [3H]ryanodine binding and single-channel measurements, using isolated membrane and purified channel preparations. In the absence of the two channel ligands Mg2+ and ATP, cardiac Ca2+ release channels were half-maximally activated at pH 7.4 by approximately 4 mumol/L cytosolic Ca2+ and half-maximally inhibited by approximately 9 mmol/L cytosolic Ca2+. Regulation of channel activity by Ca2+ was modulated by Mg2+ and ATP. Single-channel activities were more sensitive to a change of cytosolic pH than SR lumenal pH. Reduction in lumenal and/or cytosolic pH from 7.3 to 6.5 and 6.0 resulted in decreased single-channel activities without a change in single-channel conductance. [3H]Ryanodine binding measurements also indicated that acidosis impairs cardiac Ca2+ release channel activity. Mg2+ and adenine nucleotide concentrations regulated the extent of inhibition and the Ca2+ dependence of binding. In the presence of 5 mmol/L Mg2+ and 5 mmol/L beta, gamma-methyleneadenosine 5'-triphosphate (AMPPCP, a nonhydrolyzable ATP analogue), the free [Ca2+] for half-maximal [3H]ryanodine binding was increased from 1.9 mumol/L at pH 7.3 to 36 mumol/L at pH 6.5 and to 89 mumol/L at pH 6.2. These results suggest that ionic and metabolic changes that might be expected to affect sarcoplasmic reticulum Ca2+ release channel activity in ischemic myocardium include an altered Ca2+ sensitivity of the channel, a fall in pH, and a loss of the high-energy adenine nucleotide pool, leading to an increased inhibition by Mg2+.

Suppression of flash‐induced PSII‐dependent electrogenesis caused by proton pumping in chloroplasts

Pre‐illumination of the thylakoid membrane of Peperomia metallica chloroplasts leads to a reversible suppression of the flash‐induced electrical potential as measured either with the electrochromic bandshift (P515), microelectrode impalement or patch‐clamp technique. The energization‐dependent potential suppression was not observed in the presence of 1 μM nigericin suggesting the involvement of proton and/or cation gradients. Energization in the presence of 3‐(3,4‐dichlorophenyl)‐1,1‐dimethylurea (DCMU) and N,N,N′,N′‐tetramethylphenylenediamine (TMPD), i.e. cyclic electron flow around photosystem (PS) I, results in the accumulation of TMPD+ in the thylakoid lumen. The reversible suppression of the flash‐induced membrane potential was not observed in these conditions indicating that it is not a general cation‐induced increase of membrane capacitance. Cyclic electron flow around PSI in the presence of DCMU and phenazine methosulfate (PMS) results in the accumulation of PMS+ and H+ in the thylakoid lumen. The absence of reversible suppression of the flash‐induced membrane potential for this condition shows that accumulation of protons does not lead to (1) a reversible increase of membrane capacitance and (2) a reversible suppression of PSI‐dependent electrogenesis. Reversible inactivation of PSII by a low pH in the thylakoid lumen is therefore proposed to be the cause for the temporary suppression of the flash‐induced electrical potential. The flash‐induced PSII‐dependent membrane potential, as measured after major oxidation of P700 in far‐red background light, was indeed found to be suppressed at low assay pH (pH 5) in isolated spinach (Spinacia oleracea) chloroplasts.

Relationship between ventilator-associated pneumonia and intramucosal gastric pHi: A case-control study

Purpose: Prior investigations have suggested a clear relationship between nosocomial pneumonia and intramucosal gastric pH (pHi), a probable marker of bacterial translocation. Methods: We studied 33 patients (18 with pneumonia and 15 without) admitted to an intensive care unit and hospitalized longer than 72 hours with the aim of assessing the relationship between nosocomial pneumonia, pHi, and outcome. pHi was estimated at the time of inclusion of patients into the study. Arterial pH (pHa) and bicarbonate and stomach pH and tonometer Ptco 2 were also recorded. Values of <7.32 or ΔpHa - pHi of >+0.06 were used to differentiate between normal and low pHi. Quantitative cultures of pharyngeal swabs, gastric lumen, and protected specimen brush from lower airways were also done. Results: The mean pHi values were 7.397 ± 0.105 (range, 7.14 to 7.53) and 7.452 ± 0.059 (range, 7.37 to 7.56) for patients with and without pneumonia, respectively ( P = .073). Five patients, all with pneumonia, had pHi < 7.32. No patients without pneumonia had pHi < 7.32 ( P = .04). The mean ΔpHa - pHi was 0.04 ± 0.07 (range, −0.11 to 0.13) and 0.05 ± 0.09 (range, −0.09 to 0.28; P = .72) for patients with and without pneumonia, respectively. However, there were significant differences when tonometer Ptco 2 values of both groups were compared (38.9 ± 8.3 and 30.6 ± 4.7 mm Hg, respectively; P = .025). Patients with pneumonia had higher alkaline gastric lumen pH (5.2 ± 1.0) than those without pneumonia (3.8 ± 1.4; P = .006). Nonsurvivors (n = 7) had more acidic pHi (7.33 ± 0.11) than survivors (7.44 ± 0.06; P = .045). The mean gastric lumen bacterial concentration was 4.14 ± 1.01 Log 10 CFU/mL in patients with pneumonia and 4.28 ± 1.22 Log 10 CFU/mL in patients without pneumonia ( P = NS). When patients with and without intramucosal gastric acidosis (pHi < 7.32) were compared, the gastric bacterial burden was 4.42 ± 0.82 Log 10 CFU/mL and 4.32 ± 1.03 Log 10 CFU/mL ( P = .08), respectively. Conclusions: Most patients with nosocomial pneumonia had no associated intramucosal gastric acidosis. However, low pHi was associated with increased mortality.

In vitro fusion of endocytic vesicles: effects of reagents that alter endosomal pH.

Ricin, a plant toxin that binds to galactose-terminated glycoproteins and glycolipids on the cell surface, is internalized into endosomes before reaching the cytosol where it exerts its toxic activity. Fusion of early endosomes containing ricin or transferrin was demonstrated by using postnuclear supernatant fractions from K-562 cells. For both ligands, fusion depended on time, temperature, and ATP and was blocked by preincubation with N-ethylmaleimide. Some reagents that increase endosomal pH, the ionophores monensin and nigericin and the weak base chloroquine, stimulated the rate of fusion. However, bafilomycin A1, a specific inhibitor of vacuolar H(+)-ATPases, did not alter the rate of fusion. Moreover, it reduced or eliminated stimulation caused by monensin, nigericin, or chloroquine. Thus, the increased rate of fusion did not correlate with the higher lumenal pH of the endosome. The results suggest instead that fusion was stimulated by reagents that promoted accumulation of cations within the vesicles.

An endosomal beta COP is involved in the pH-dependent formation of transport vesicles destined for late endosomes.

In this paper, we show that beta COP is present on endosomes and is required for the formation of vesicles which mediate transport from early to late endosomes. Both the association of beta COP to endosomal membranes as well as transport vesicle formation depend on the lumenal pH. We find that epsilon COP, but not gamma COP, is also associated to endosomes, and that this association is also lumenal pH dependent. Our data, thus, indicate that a subset of COPs is part of the mechanism regulating endosomal membrane transport, and that membrane association of these COPs is controlled by the acidic properties of early endosomes, presumably via a trans-membrane pH sensor.

Intrathylakoid pH in Isolated Pea Chloroplasts as Probed by Violaxanthin Deepoxidation.

Light-driven violaxanthin deepoxidation was measured in isolated pea (Pisum sativum) chloroplasts without ATP synthesis (basal conditions) and with ATP synthesis (coupled conditions). Thylakoids stored in high salt (HS) or low salt (LS) storage medium were tested. In previous experiments, HS thylakoids and LS thylakoids were related to delocalized and localized proton coupling, respectively.Light-driven deepoxidase activity was compared to the pH dependence of deepoxidase activity established in dark reactions. At an external pH of 8, light-driven deepoxidation indicated effective pH values close to pH 6 for all reaction conditions. Parallel to deepoxidation, the thylakoid lumen pH was estimated by the fluorescent dye pyranine.In LS thylakoids under coupled conditions the lumen pH did not drop below pH 6.7. At pH 6.7, no deepoxidase activity is expected based on the pH dependence of enzyme activity. The results suggest that deepoxidation activity is controlled by the pH in sequestered membrane domains, which, under localized proton coupling, can be maintained at pH 6.0 when the lumen pH is far above pH 6.0. The extent of violaxanthin conversion (availability), however, appeared to be regulated by lumenal pH. Dithiothreitol-sensitive nonphotochemical quenching of chlorophyll fluorescence was dependent on zeaxanthin and not related to lumenal pH. Thus, zeaxanthin-dependent quenching[mdash]known to be pH dependent[mdash]appeared to be triggered by the pH of localized membrane domains.

Higher plant light-harvesting complexes LHCIIa and LHCIIc are bound by dicyclohexylcarbodiimide during inhibition of energy dissipation.

We have investigated the binding to proteins of the photosynthetic apparatus of the carboxy-modifying agent dicyclohexylcarbodiimide, (cHxN)2C; this inhibits the protective dissipation of excess absorbed light energy (qE) by the light-harvesting apparatus of photosystem II (LHCII), suggesting that carboxyl amino-acid side chains within hydrophobic protein domains may be involved in qE. (cHxN)2(14)C was used to label thylakoids and photosystem II particles, so as to identify proteins which may be involved in the detection of lumen pH during qE induction. Of six thylakoid proteins labelled with (cHxN)2C under conditions where qE is efficiently induced, three are associated with photosystem I, and none with the bulk LHCII. PSII-associated label is bound to three minor components of LHCII, identified as LHCIIa (two species) and LHCIIc, as shown by protein sequencing of tryptic fragments of purified complexes. pH titration of qE formation and protein labelling in coupled thylakoids showed that both qE and labelling of LHCIIa increased at pH 7-8.

Enhancement by Artificial Electron Acceptors of Thylakoid Lumen Acidification and Zeaxanthin Formation

In chloroplasts the enzyme violaxanthin de-epoxidase is responsible for the transformation of violaxanthin (Vio) to zeaxanthin (Zea) in the presence of ascorbate (Asc). The enzyme is mobile within the thylakoid lumen and becomes bound to the membrane in its active state. It is characterized by a narrow pH optimum at 5.2 and is presumed to be activated by the photosynthetic electron transport via the acidification of the lumenal pH of the thylakoid vesicles. Correlations between the pH of the lumen and the formation of Zea were studied using isolated thylakoids and leaf disks from Spinacia oleracea L. The alterations of the lumenal pH were measured by a photometric method using neutral red as indicator. A so far not documented bathochromic spectral shift of the protonated dye in the thylakoid lumen was taken into consideration. The xanthophylls were quantified by HPLC. In vitro , when using thylakoids that had been illuminated by red light at pH 7.5, artificial electron acceptors (such as methyl viologen (MV) or menadione (MD)) increased both the light-dependent H + -accumulation within the thylakoid lumen and the transformation of Vio to Zea. In vivo , after infiltration in leaf disks, MV was also effective. In its presence a 5-min illumination of these disks increased the Zea content by about 100 % in comparison with the MV-free control. Moreover, in isolated preilluminated Zea-containing thylakoids MD strongly inhibited the epoxidation of Zea to Vio, which is catalyzed by a mixed-function oxygenase in the presence of the cosubstrates O 2 and NAD(P)H at pH 7.5 in the dark. This inhibition was the result of an oxidation of NAD(P)H by MD catalyzed by an undefined thylakoid enzyme. The decreased level of (reduced) NAD(P)H and the fact that the reduced MD cannot substitute for NAD(P)H in the epoxidation reaction caused the diminished Zea epoxidation. The results demonstrate that under the influence of artificial electron acceptors an increased formation of Zea in the thylakoid membrane is strictly correlated with an increased light-dependent H + concentration in the thylakoid lumen.

A molecular mechanism for q E-quenching

We discuss energy-dependent fluorescence lowering (q E-quenching), and suggest a model to explain the experimental data currently available. The main elements of the model are: (a) the q E-quenching reflects a mechanism associated with a component of the light-harvesting antenna rather than the reaction center of photosystem (PS) II — we suggest that it occurs through formation of an efficient quencher in one of the minor chlorophyll protein (CP) complexes; (b) the minor CPs have glutamate residues instead of glutamines at positions shown in light-harvesting complex II (LHCII) to be ligands to chlorophylls near the lumenal interface. We suggest that the quenching reflects a change in ligation of chlorophyll on protonation of these glutamate residues leading to formation of an exciton coupled dimer with a neighboring pigment, in which additional energy levels allow vibrational relaxation of the excited singlet. The model accounts for the dependence on low lumenal pH, the ligand residue changes between LHCII and the minor CPs, the preferential distribution of components of the xanthophyll cycle in the minor CPs, the inhibition of q E-quenching by DCCD, and the specific binding of DCCD to the minor CPs.

Hydrogen ion concentration in the mucus layer on top of acid-stimulated and -inhibited rat gastric mucosa

Background/Aims: The gastric mucosa is covered by a continuous layer of bicarbonate-containing mucus gel; the question arises how acid, formed in the gastric glands, moves into the lumen. Methods: The pH in the gastric mucus gel and gel thickness were measured in anesthetized rats with pH-sensitive microelectrodes (tip diameter, 1–5 μm). Results: During pentagastrin (40 μg · kg−1 · h−1) stimulation of acid secretion, the pH was higher in the gel than in the lumen (pH 2) up to a distance of 115 ± 18 μm from the epithelial surface and maximal (pH 7.2 ± 0.1) at the surface. A similar pH gradient was recorded at luminal pH 3. After omeprazole (10 μmol/kg) inhibition of endogenous acid secretion and with exogenous acid in the lumen, the pH profile was broader: 204 ± 26 μm at luminal pH 2 and 231 ± 63 μm at luminal pH 3. In contrast, the pH at the epithelial surface was lower (pH 6.8–6.9). The gel thickness (200–300 μm) was similar in all groups. Conclusions: The significantly higher surface pH in acid-secreting stomachs probably reflects better availability of interstitial mucosal bicarbonate. Bulk transport of secreted acid in channels created by the gland luminal hydrostatic pressure may additionally act to limit acidification of the mucus gel.

A pK change of acidic residues contributes to cation countertransport in the Ca-ATPase of sarcoplasmic reticulum. Role of H+ in Ca(2+)-ATPase countertransport

Proteoliposomal vesicles reconstituted with sarcoplasmic reticulum ATPase and exogenous lipids sustain ATP-dependent Ca2+ uptake and H+ ejection, as well as net charge displacement by Ca2+. We have studied the effect of lumenal (inner) and medium (extravesicular) pH variations on the countertransport ratios of H+ and Ca2+. We find that the Ca2+/H+ molar ratio is approximately 1 when the lumenal and medium pH is near neutrality, but changes with a specific pattern when the medium pH is varied in the presence of a constant lumenal pH and when the lumenal pH is varied in the presence of a constant medium pH. Empirical analysis of the experimental data shows that the apparent pK of the residue(s) releasing H+ into the medium is approximately 6.1, whereas the apparent pK of the residue(s) binding lumenal H+ is approximately 7.7. Assuming that the same acidic residues are involved in H+ and Ca2+ countertransport, our findings suggest a lower affinity for H+ in their outward orientation (prevalent in the ground state of the enzyme) and a higher affinity for H+ in lumenal orientation (prevalent in the phosphorylated state of the enzyme). Cyclic pK changes, coupled to ATP utilization, promote cation exchange, Ca2+ uptake, and H+ ejection by the vesicles. The stoichiometry of countertransport and net charge displacement is matched by a corresponding electrogenic behavior. A calculation of voltage development related to initial rates of charge transfer (dV/dt = (dQ/dt)/Cm) is given as a corrective replacement of a previous steady state calculation.

Vacuolar ATPase inactivation blocks recycling to the trans-Golgi network from the plasma membrane

TGN38/41 is an integral membrane protein which recycles between the trans-Golgi network (TGN) and the cell surface but is predominantly located in the TGN of rat (NRK) cells at steady state. As part of our studies on the mechanism and route of recycling between the TGN and the cell surface we have used chloroquine or Bafilomycin A 1 to modulate the lumenal pH of endocytic organelles. The data we present demonstrate that inactivation of the proton pump which maintains the acidic environment within the lumen of endocytic organelles leads to an accumulation of TGN38/41 in early endosomes. These data confirm the observation that TGN38/41 recycles between the plasma membrane and the TGN and identifies a specific block in that recycling pathway.

Ca2+/H+ countertransport and electrogenicity in proteoliposomes containing erythrocyte plasma membrane Ca-ATPase and exogenous lipids.

A reconstituted proteoliposomal system was obtained with Ca-ATPase purified from human erythrocyte membrane (plasma membrane, PM ATPase), and liposomes prepared by reverse-phase evaporation. The reconstituted PM ATPase behaved as an electrogenic Ca2+/H+ exchanger and, under optimal conditions, utilization of 1 mol of ATP was accompanied by uptake of one Ca2+ by the vesicles, and ejection of one H+ from the lumen of the vesicles. Ca2+ uptake was greatly (5-fold) stimulated by the addition of calmodulin, and by collapsing the H+ gradient with the ionophore carbonyl cyanide p-trifluoromethoxyphenylhydrazone. In the presence of calmodulin and p-trifluoromethoxyphenylhydrazone, the reconstituted system sustained transport rates of 1.00 +/- 0.12 mumol of Ca2+/mg of protein min-1 (30 degrees C), reaching asymptotic levels of 8.05 +/- 0.41 mumol of Ca2+/mg of protein (i.e. 20 mM lumenal Ca2+). The corresponding net charge transfer produced a maximal electrical gradient of 40.5 +/- 1.8 mV at steady state. Demonstration of the electrogenic behavior of the PM ATPase, obtained for the first time with these experiments, was critically dependent on the detergent used in the reconstitution procedure. The lumenal pH rise had a much greater rate-limiting effect on the pump, than the electrical potential developed by the pump.

Acid-base interactions during exocrine pancreatic secretion. Primary role for ductal bicarbonate in acinar lumen function.

The role of acid-base interactions during coordinated acinar and duct cell secretion in the exocrine pancreas is described. The sequence of acid-base events may be summarized as follows: (1) Sorting of secretory proteins and membrane components into the regulated secretory pathway of pancreatic acinar cells is triggered by acid- and calcium-induced aggregation and association mechanisms located in the trans-Golgi network. (2) Cholecystokinin-stimulated exocytosis in acinar cells releases the acidic contents of secretory granules into the acinar lumen. (3) Secretin-stimulated bicarbonate secretion from duct and duct-like cells neutralizes the acidic pH of exocytic contents, which leads to dissociation of protein aggregates and solubilization of (pro)enzymes within the acinar lumen. (4) Stimulated fluid secretion transports solubilized enzymes through the ductal system. (5) Further alkalinization of acinar lumen pH accelerates the enzymatic cleavage of the glycosyl phosphatidyl-inositol anchor associated with GP2 and thus releases the GP2/proteoglycan matrix from lumenal membranes, a process that appears to be required for vesicular retrieval of granule membranes from the apical plasma membrane and their reuse in the secretory process. We conclude that the central function of bicarbonate secretion by centroacinar and duct cells in the pancreas is to neutralize and then alkalinize the pH of the acinar lumen, sequential process that are required for (a) solubilization of secreted proteins and (b) cellular retrieval of granule membranes, respectively.

Localization of the xanthophyll-cycle enzyme violaxanthin de-epoxidase within the thylakoid lumen and abolition of its mobility by a (light-dependent) pH decrease

The formation of zeaxanthin (Zea) from violaxanthin (Vio) in chloroplasts of leaves and algae upon strong illumination is currently suggested to play a role in the photoprotection of plants. Properties and location of the enzyme Vio de-epoxidase, which is responsible for the transformation of Vio to Zea, were studied using thylakoid membrane vesicles isolated from leaves of Spinacia oleracea L. Without using detergents a repeated freeze-thaw treatment of thylakoid vesicles was sufficient to release the enzyme into the medium. With the same procedure the mobile electron carrier plastocyanin, known to occur in the thylakoid lumen, was also released. The enzyme was demonstrated by its activity in the supernatant of the pelleted thylakoid vesicles in the presence of the added substrates Vio and ascorbic acid, as well as by staining of the released proteins after polyacrylamide gel electrophoresis. The release of the deepoxidase from the vesicles was pH-dependent, declined below pH 6.5 and ceased in the pH range around 5, which corresponds to the pH optimum of the enzyme activity. By using thylakoid vesicles isolated from pre-illuminated and therefore Zea-containing leaves the release by freeze-thaw cycles of both the de-epoxidase and plastocyanin was diminished compared with the dark control. However, the reason for this effect was not the Zea content but an unknown effect of the illumination on the thylakoid membrane properties. The de-epoxidase collected at pH 7 was able to re-bind to thylakoid membranes at pH 5.5 and to transform intrinsic Vio to Zea in the presence of ascorbate. The isolated de-epoxidase, as well as the endogenous membrane-bound de-epoxidase, was inhibited by dithiothreitol. From these results it is concluded that Vio de-epoxidase, like plastocyanin, is mobile within the thylakoid lumen at neutral pH values which occur under in-vivo conditions in the dark. However, upon strong illumination, when the lumen pH drops (pH < 6.5) due to the formation of a proton gradient, the properties of the de-epoxidase are altered and the enzyme becomes tightly bound to the membrane (in contrast to plastocyanin) thus gaining access to its substrate Vio. These findings corroborate the assumption of a transmembrane opposite location of the two enzymes of the xanthophyll cycle, the ascorbate-dependent Vio deepoxidase at the lumenal side and the NADPH-dependent Zea epoxidase at the stromal side. Indications in favour of a location of Vio within the lipid bilayer of the thylakoid membrane and of a binding of the active deepoxidase to these areas are discussed.

Evidence that the intrinsic membrance protein LHCII in thylakoids is necessary for maintaining localized $$\Delta \tilde \mu _{H^ + } $$ energy coupling

This work tested the hypothesis that thylakoid localized proton-binding domains, suggested to be involved in localized delta mu H(+)-driven ATP formation, are maintained with the involvement of several membrane proteins, including the LHCII (Laszlo, J.A., Baker, G.M., and Dilley, R.A. (1984) Biochim. Biophys. Acta 764, 160-169), which comprises about 50% of the total thylakoid protein. The concept we have in mind is that several membrane proteins cooperate to shield a localized proton diffusion pathway from direct contact with the lumen, thus providing a physical barrier to H+ equilibration between the sequestered domains and the lumen. A barely mutant, chlorina f2, that lacks Chl b and does not accumulate some of the LHCII proteins, was tested for its capacity to carry out localized-proton gradient-dependent ATP formation. Two previously developed assays permit clear discrimination between localized and delocalized delta mu H+ gradient-driven ATP formation. Those assays include the effect of a permeable buffer, pyridine, on the number of single-turnover flashes needed to reach the energetic threshold for ATP formation and the more recently developed assay for lumen pH using 8-hydroxy-1,3,6-pyrene trisulfonic acid as a lumenally loaded pH-sensitive fluorescent probe. By those two criteria, the wild-type barley thylakoids revealed either a localized or a delocalized energy coupling mode under low- or high-salt storage conditions, respectively. Addition of Ca++ to the high-salt storage medium caused those thylakoids to maintain a localized energy-coupling response, as previously observed for pea thylakoids.(ABSTRACT TRUNCATED AT 250 WORDS)

Regulation of bicarbonate-dependent ductular bile secretion assessed by lumenal micropuncture of isolated rodent intrahepatic bile ducts.

While intrahepatic bile duct epithelial cells secrete bile through transport of ions and water, the physiological mechanisms regulating ductular bile secretion are obscure, in part because of the lack of suitable experimental models. We report here the successful micropuncture of the lumen of isolated intrahepatic bile ducts and direct measurements of ductular ion secretion. Intact, polarized bile duct units (BDUs) were isolated from livers of normal rats by enzymatic digestion and microdissection. BDUs were cultured and mounted on a microscope in bicarbonate-containing buffer, and the lumens were microinjected with 2',7'-bis(2-carboxyethyl)-5-(and -6)carboxyfluorescein (BCECF)-dextran. Lumenal pH was measured by ratio imaging of BCECF fluorescence using digitized video fluorescent microscopy. After 36 hr in culture, the ends of BDUs sealed, forming closed compartments. After lumenal microinjection of BCECF-dextran, fluorescence was stable at the pH-insensitive wavelength, indicating no dye leakage. Serial changes in pH of extralumenal buffers containing pH-gradient collapsing ionophores allowed us to establish reliable standard curves relating fluorescence ratio to lumenal pH (r = 0.99; P < 0.001). By this approach, the basal pH inside the lumen of BDUs was 7.87 +/- 0.08 units (n = 9), 0.47 unit higher (P < 0.001) than the bathing buffer pH. Addition of 100 microM forskolin increased (P = 0.02) the lumenal pH from 7.78 +/- 0.06 to 7.97 +/- 0.06 units (n = 5); the forskolin effect was completely abolished by incubation of BDUs in HCO3-/CO2-free buffer. Moreover, forskolin caused a 50-fold increase in cAMP levels in BDUs. The observations are consistent with cAMP-dependent, active lumenal HCO3- secretion by BDUs. Furthermore, they demonstrate the suitability of the BDU model for studying regulatory and mechanistic aspects of ductular bile secretion.