External Mg2+ is required for hyperpolarization to occur in ovulated oocytes of the prawn Palaemon serratus

Immediately after dissection, the ovulated oocyte of the prawn Palaemon serratus had a resting potential E_m of −42 ± 2 mV and a membrane resistance R_m of 15 ± 5 MΩ; the membrane was more permeable to Cl⁻ than to K⁺. The oocyte spontaneously hyperpolarized and E_m gradually reached −70 mV 20–30 min after removal of the oocyte from the female, due to increased membrane permeability to K⁺. However, the hyperpolarization occured only if Mg²⁺ was present in the seawater; external Ca²⁺ was not required. Long-term incubation without external Mg²⁺ depolarized the membrane and increased membrane resistance. After preincubation in Mg²⁺-free ASW, oocytes transferred to standard artificial seawater (ASW) transiently hyperpolarized and then repolarized, before gradually hyperpolarizing to a sustained value of −62 ± mV. The respective roles of external Mg²⁺ and fertilization in eliciting the electrical response of the prawn egg at natural spawning are discussed.     

Developmental changes in membrane transport and permeability in the early mouse embryo

Electrophysiological and isotope tracer flux experiments were performed to measure the membrane ion permeability and transport properties of the two-cell mouse embryo. The results show that the internal exchangeable Na and K are 151 and 130 mM, respectively, and their membrane permeabilities are (P_Na) 16 × 10^?8 cm sec^?1 and (P_K) 21 × 10^?8 cm sec^?1. These values predict a membrane potential of ?24 mV (inside negative) which agrees well with ?19 mV measured with microelectrodes. Ouabain-sensitive isotope fluxes demonstrate a Na/K pump mechanism with a stoichiometry of 1.7:1 (Na:K). An external-Na-dependent Na efflux is demonstrated by the reduction of unidirectional Na efflux in Na-free medium, but there is no evidence for a similar mechanism of K efflux at this stage of development. These results are compared with the values reported for the mouse oocyte [Powers, R. D., and Tupper, J. T. (1974). Develop. Biol.38, 320–331; (1975). Exp. Cell Res.91, 413–421]. The hyperpolarization of the membrane potential as compared with the oocyte (?13 mV) results primarily from the increased $\text{P}{}_{\mathrm{<mtext>K</mtext>}}\text{P}{}_{\mathrm{<mtext>Na</mtext>}}$ ratio. A similar phenomenon has been noted in other developing embryos. The increase in pump-mediated K influx at the two-cell stage is accompanied by a decrease of similar magnitude in the external-K-dependent K efflux which is found in the oocyte. This suggests that the KK exchange mechanism may be converted to an active pump. Because of the changes in ion concentrations and movements and the unusual metabolic requirements of the mouse embryo, the effect of external Na on the uptake of glucose and pyruvate in the oocyte and two- and eight-cell stage was examined. No Na-dependent carbohydrate transport could be found at these stages.     

Maturation and fertilization of the sea urchin oocyte: An electrophysiological study

Some electrical properties of the sea urchin oocyte during germinal vesicle breakdown (GVBD) and fertilization have been studied using two intracellular electrodes. Oocytes with distinct germinal vesicles have resting potentials of ?70 to ?90 mV and the specific membrane resistance may range from 3 to 10 kΩ·cm². Around rest the I–V relationship is concave toward the axis origin and the membrane is K⁺ selective. A second electrical state, of lower potential and higher resistance, preexists in the membrane. Following GVBD, the K⁺-selective system is lost and the oocyte attains the characteristics of the second state with a resting potential of ?10 to ?50 mV and specific membrane resistance of 10–50 kΩ·cm². At rest the I–V relationship tends to be convex toward the axis origin. The majority of sea urchin eggs (which have undergone GVBD and completed meiosis) have a resting state of ?10 to ?30 mV; 10–50 kΩ·cm². The I–V relationship around rest is convex toward the axis origin and the resting potential is sensitive to changes of Na⁺, Cl^?, and K⁺ in the external medium. There is probably no major change in the electrical properties of the oocyte during the completion of meiosis. A small percentage of eggs from suboptimal animals have high resting potentials of ?70 to ?90 mV and specific membrane resistance of 5–50 kΩ·cm². Such eggs have predominantly K⁺-selective membranes and we suggest that they are either underripe or aged. The first electrical event across the egg plasma membrane during fertilization is a step-like depolarization which occurs about 2 sec after the attachment of the fertilizing spermatozoon to the vitelline layer. There is no change—at the level of the light microscope—either in the egg surface or in the behavior of the spermatozoon until the second event, the fertilization potential (FP), is initiated 11 sec later. The cortical reaction occurs simultaneously with the FP and during the rising phase of the FP the spermatozoon stops gyrating around its point of attachment. Oocytes, which do not have cortical granules, upon insemination exhibit step events but no FP; in contrast artificially activated eggs, either spontaneous or induced by the ionophore A23187, give rise to only the FP. We suggest that the FP is the electrical result of the modification of the egg plasma membrane during cortical exocytosis.     

Na+ and K+ uptake and exchange by the amphibian oocyte during the first meiotic division

The uptake and efflux of ²²Na and ⁴²K were studied in denuded Rana pipiens oocytes following progesterone induction of the resumption of meiotic maturation. Coincident with the breakdown of the large nucleus, or germinal vesicle, there is a virtual disappearance of K⁺ permeability of the oocyte plasma membrane. Only about 1–2% of the total [K⁺]_i is exchanged by completion of nuclear breakdown (8–10 hr) and accounts for the finding that there is no detectable change in total [K⁺]_i during the first meiotic division (20–24 hr). In the case of Na⁺, influx, exchange, and efflux kinetics were unchanged during the first meiotic division, with 20 and 35% of the total oocyte Na⁺ exchanging by the completion of nuclear breakdown and first meiotic division, respectively. Removal of Na⁺ from the incubation medium produced and earlier nuclear breakdown, whereas a K-free medium delayed breakdown. There was no effect of 10 μm/ml tetrodotoxin or 10^?5M strophanthidin on the time course of nuclear breakdown. Thus one action of progesterone appears to be a selective turning off of “K channels” in the oocyte plasma membrane. The disappearance of K selectivity of the oocyte plasma membrane coincides with plasma membrane depolarization, as well as nuclear swelling and breakdown.     

Voltage dependent potassium fluxes and the significance of action potentials in Acetabularia

Membrane potential, V_m, and K⁺(⁸⁶Rb⁺) fluxes have been measured simultaneously on individual cells of Acetabularia mediterranea. During resting state (resting potential approx. ?170 mV) the K⁺ influx amounts to 0.24–0.6 pmol · cm^?2 · s^?1 and the K⁺ efflux to 0.2–1.5 pmol · cm^?2 s^?1. According to the K⁺ concentrations inside and outside the cell (40 : 1) the voltage dependent K⁺ flux (zero at V_m = E_K = ?90 mV) is stimulated approx. 40-fold for V_m more positive than E_K.It is calculated that during one action potential (temporary depolarization to V_m more positive than E_K) a cell looses the same amount of K⁺, which leaks in during 10–20 min in the resting state (V_m = ?170 mV). Since action potentials occur spontaneously in Acetabularia, they are therefore suggested to have a significant function for the K⁺ balance of this alga.     

Biochemical correlates of progesterone-induced plasma membrane depolarization during the first meiotic division inRana oocytes

Summary Changes in protein synthesis, protein phosphorylation and lipid phosphorylation in the amphibian oocyte plasma membrane have been correlated with electrical changes following steroid induction of the completion of the first meiotic division. The oocyte first depolarizes from about –60 mV (inside negative) to about –25 mV 1 to 2 hr before breakdown of the large nucleus followed by a further depolarization beginning 3 to 6 hr after nuclear breakdown. The initial depolarization is associated with appearance of previously described cycloheximide-sensitive cytoplasmic factor(s) which induce both nuclear breakdown and plasma membrane depolarization. We found a similar ED₅₀ (0.4 m) for cycloheximide inhibition of nuclear breakdown, membrane depolarization, and [³H]-leucine incorporation. Emetine (1nm to 1mm) was inactive. The period of cycloheximide sensitivity (first 5 hr) is essentially the same for plasma membrane depolarization phase following nuclear breakdown is associated with a marked increase in the rate of [³H]-leucine and [³²PO₄] incorporation into membrane protein and lipid. Polyacrylamide gel electrophoresis of membrane protein and lipoprotein indicated that a major newly synthesized membrane component is proteolipid. An increase in [³²PO₄] incorporation into membrane phosphatidylserine and phosphatidylethanolamine (with a decrease in phosphatidylcholine [³²PO₄] begins during the second depolarization phase and coincides with the appearance of excitability in the oocyte plasma membrane. In toto, the bulk of the biochemical changes (proteins, phosphoproteins, proteolipids, phospholipids) appear to be associated with plasma membrane components and coincide with stepwise changes in membrane permeability to specific ions (e.g. Cl^–).     

Steady-state gradient in calcium ion activity across the intercellular bridges connecting oocytes and nurse cells in Hyalophora cecropia

Intracellular activities of K⁺, H⁺, Mg²⁺, Ca²⁺, and Cl^?, measured with ion selective microelectrodes in the oocyte and the nurse cells in ovarian follicles of Hyalophora cecropia, indicated that a Ca²⁺ current is a key component of the electrical potential that is maintained across the intercellular bridges connecting these two cells. In vitellogenic follicles, Ca²⁺ activity averaged 650 nM in the oocyte and 190 nM in the nurse cells, whereas activities of the other ions studied differed between these cells by no more than 6%. Incubation in 200 μM ammonium vanadate caused a reversal of electrical potential from 8.3 mV, nurse cell negative, to 3.0 mV, oocyte negative, and at the same time the Ca²⁺ gradient was reversed: activities rose to an average 3.0 μM in the nurse cells and 1.6 μM in the oocyte, whereas transbridge ratios of the other cations remained at 0–3%. In immature follicles that had not yet initiated their transbridge potentials, Ca²⁺ activities averaged ～? 2 μM in both oocyte and nurse cells. The results suggest that vitellogenic follicles possess a vanadatesensitive Ca²⁺ extrusion mechanism that is more powerful in the nurse cells than in the oocyte. © 1994 Wiley-Liss, Inc.     

An electrophysiological study of the membrane properties of the immature and mature oocyte of the Batstar,Patiria miniata

Immature oocyte membrane properties of a starfish, Patiria miniata, were investigated by microelectrode techniques. The resting membrane potential in artificial seawater (ASW) was ?78.5 ± 6.7 mV (n = 61, inside negative). This was mainly accounted for by a selective permeability to potassium ions. Potassium ion-selective microelectrodes were used to measure intracellular K⁺ ion activity, which was 350 mM. The sodium to potassium permeability ratio was 0.02 ± 0.01 (n = 4). The current-voltage relation was nonlinear. The I–V curve included both areas of inward and outward rectification. The dependence of inward rectification upon the K⁺ ion electrochemical gradient was demonstrated. The membrane was capable of a regenerative action potential due to permeability changes for Ca²⁺ and Na⁺ ions. The Ca and Na components of the action potential were identified. The Ca component was reversibly suppressed by cobalt and irreversibly blocked by D-600. The Na component was tetrodotoxin (TTX) insensitive. The excitable response of P. miniata oocytes is similar to that described by Miyazaki et al. (1975a) for those of the starfish Asterina pectinifera.Immature oocytes were stimulated to mature with 10^?5M 1-methyladenine (1-MA) during continuous monitoring of the membrane potential. The resting potential in ASW became more inside negative during maturation. This change of the passive membrane property of the oocyte may be accounted for by the increased selectivity to K⁺ ions. The specific membrane resistance near the resting potential increased from 4.2 ± 1.4 to 21 ± 8.7 kΩ·cm² (n = 15) during maturation, while the specific membrane capacitance decreased slightly from 2 ± 0.5 to 1.7 ± 0.6 μF/cm² (n = 5). Maturation had little effect upon the active membrane properties.     

Identification of ATP-sensitive Potassium Channel in Frog Ventricular Myocytes

Nuclear magnetic resonance (NMR) microimaging and proton relaxation times were used to monitor differences between the hydration state of the nucleus and cytoplasm in the Rana pipiens oocyte. Individual isolated ovarian oocytes were imaged in a drop of Ringer's solution with an in-plane resolution of 80 μm. Proton spin echo images of oocytes arrested in prophase I indicated a marked difference in contrast between nucleoplasm and cytoplasm with additional intensity gradations between the yolk platelet-rich region of the cytoplasm and regions with little yolk. Neither shortening τ_e (spin echo time) to 9 msec (from 18 msec) nor lengthening τ_r (spin recovery time) to 2 sec (from 0.5 sec) reduced the observed contrast between nucleus and cytoplasm. Water proton T₁ (spin-lattice) relaxation times of oocyte suspensions indicated three water compartments that corresponded to extracellular medium (T₁= 3.0 sec), cytoplasm (T₁= 0.8 sec) and nucleoplasm (T₁= 1.6 sec). The 1.6 sec compartment disappeared at the time of nuclear breakdown. Measurements of plasma and nuclear membrane potentials with KCl-filled glass microelectrodes demonstrated that the prophase I oocyte nucleus was about 25 mV inside positive relative to the extracellular medium. A model for the prophase-arrested oocyte is proposed in which a high concentration of large impermeant ions together with small counter ions set up a Donnan-type equilibrium that results in an increased distribution of water within the nucleus in comparison with the cytosol. This study indicates: (i) a slow exchange between two or more intracellular water compartments on the NMR time-scale, (ii) an increased rotational correlation time for water molecules in both the cytoplasmic and nuclear compartments compared to bulk water, and (iii) a higher water content (per unit dry mass) of the nucleus compared to the cytoplasm, and (iv) the existence of a large (about 75 mV positive) electropotential difference between the nuclear and cytoplasmic compartments. Received: 18 January 1996/Revised: 29 April 1996     

Differential accumulation of two size classes of poly(A) associated with messenger RNA during oogenesis in Xenopus laevis

The RNA of full-grown oocytes of Xenopus laevis contains two distinct size classes of poly(A), designated poly(A)_S and poly(A)_L, which contain 15–30 (mean = 20) and 40–80 (mean = 61) A residues, respectively. Both poly(A)_L and poly(A)_S are associated with RNA which is heterogeneous in size. The two classes of poly(A)⁺ RNA can be separated by affinity chromatography: Only poly(A)_L⁺ RNA binds to oligo(dT)-cellulose under appropriate conditions, but up to 50% of the poly(A)_S⁺ RNA can be isolated from the void fraction by binding to poly(U)-Sepharose. Both classes of poly(A)⁺ RNA are active as messenger RNA in an in vitro system and yield identical patterns of in vitro protein products. Previtellogenic oocytes contain almost exclusively poly(A)_L, which accumulates up to vitellogenesis but remains almost constant in amount (molecules/oocyte) during vitellogenesis and in the full-grown oocyte. Poly(A)_S accumulates (molecules/oocyte) from early vitellogenesis up to the full-grown oocyte. The total number of poly(A)⁺ RNA molecules per oocyte increases throughout oogenesis from 2 × 10¹⁰/previtellogenic oocyte [80–90% poly(A)_L] to 20 × 10¹⁰/full-grown oocyte (25–40% poly(A)_L). It is argued that poly(A)_S is protected from degradation in the oocyte, thus stabilizing the “maternal” poly(A)⁺ mRNA.     

Structural modifications of the nuclear components during lizard oogenesis in relation to the differentiation of the follicular epithelium

This paper deals with an electron microscope study of nucleolar ultrastructural modifications that occur in the oocytes of the lizard Podarcis sicula during ovarian follicle differentiation. In small diplotene oocytes around which a monolayered follicular epithelium forms, the nucleolus appears as a fibrillo-granular structure. Afterwards, simultaneously with the beginning of pyriform cell differentiation inside the granulosa, the nucleolus progressively condenses and breaks into fragments, forming dense spherical bodies. In larger follicles, with well differentiated pyriform cells, a typical nucleolus is no longer detectable in the oocyte nucleus. These ultrastructural modifications suggest a possible impairment of the oocyte nucleolus in ribosome organization. A possible involvement of pyriform cells in supplying ribosomes to the growing oocyte is discussed.     

Nuclear pore flow rate of ribosomal RNA and chain growth rate of its precursor during oogenesis of Xenopus laevis

The number of ribosomal RNA molecules which are transferred through an average nuclear pore complex per minute into the cytoplasm (nuclear pore flow rate, NPFR) during oocyte growth of Xenopus laevis is estimated. The NPFR calculations are based on determinations of the increase of cytoplasmic rRNA content during defined time intervals and of the total number of pore complexes in the respective oogenesis stages. In the mid-lampbrush stage (500–700 μm oocyte diameter) the NPFR is maximal with 2.62 rRNA molecules/pore/minute. Then it decreases to zero at the end of oogenesis. The nucleocytoplasmic RNA flow rates determined are compared with corresponding values of other cell types. The molecular weight of the rRNA precursor transcribed in the extrachromosomal nucleoli of Xenopus lampbrush stage oocytes is determined by acrylamide gel electrophoresis to be 2.5 × 10⁶ daltons. From the temporal increase of cytoplasmic rRNA (3.8 μg per oocyte in 38 days) and the known number of simultaneously growing precursor molecules in the nucleus the chain growth rate of the 40 S precursor RNA is estimated to be 34 nucleotides per second.     

Oxidation-reduction properties of the electron acceptors of Photosystem II. II. Redox titration at various pH values of the flash-induced formation of C550 in Chlamydomonas Photosystem II particles lacking the secondary quinone electron acceptor

Redox titrations of the flash-induced formation of C550 (a linear indicator of Q^?) were performed between pH 5.9 and 8.3 in Chlamydomonas Photosystem II particles lacking the secondary electron acceptor, B. One-third of the reaction centers show a pH-dependent midpoint potential (E_m,7.5) = ? 30 mV) for redox couple $\text{Q}\text{Q}{}^{\mathrm{?}}$, which varies by ?60 mV/pH unit. Two-thirds of the centers show a pH-independent midpoint potential (E_mm = + 10 mV) for this couple. The elevated pH-independent E_m suggests that in the latter centers the environment of Q has been modified such as to stabilize the semiquinone anion, Q^?. The midpoint potentials of the centers having a pH-dependent E_m are within 20 mV of those observed in chloroplasts having a secondary electron acceptor. It appears therefore that the secondary electron acceptor exerts little influence on the E_m of $\text{Q}\text{Q}{}^{\mathrm{?}}$. An EPR signal at g 1.82 has recently been attributed to a semiquinone-iron complex which comprises Q^?. The similar redox behavior reported here for C550 and reported by others (Evans, M.C.W., Nugent, J.H.A., Tilling, L.A. and Atkinson, Y.E. (1982) FEBS Lett. 145, 176–178) for the g 1.82 signal in similar Photosystem II particles confirm the assignment of this EPR signal to Q^?. At below ?200 mV, illumination of the Photosystem II particles produces an accumulation of reduced pheophytin (Ph^?). At ?420 mV Ph^? appears with a quantum yield of 0.006–0.01 which in this material implies a lifetime of 30–100 ns for the radical pair P-680⁺Ph^?.     

Properties of the Trp290His variant of Fusarium NRRL 2903 galactose oxidase: interactions of the GOasesemi state with different buffers, its redox activity and ability to bind azide

The indole ring of Trp-290 in galactose oxidase has an important role in restricting entry to the substrate-binding (Cu) site of galactose oxidase via a short ～8?Å access pocket/channel. It also overlays and helps stabilise the radical-forming Cu-coordinated Tyr-272, reduction potential 400?mV. In this paper the effect of replacing Trp-290 by the less bulky His residue is explored at 25??°C, I=0.100?M (NaCl), and different effects are quantified. Interactions with buffers, not observed in the case of wild-type (WT) GOase, have been investigated by UV-Vis spectrophotometry on the non-radical GOase_semi (Cu^II) form of the Trp290His variant. Equilibrium constants K _eq/M^–1 from absorbance changes at 635?nm are for 1?:?1 interactions with the OH-containing buffers H₂PO₄ ^– (231), Hepes (43) and Tris (202), concentrations 0–60?mM. No similar interactions are observed with Mes, Lutidine and Ches, when significantly different UV-Vis spectra with no peak at ～635?nm are obtained. At pH 7.5 the reduction potential for the Trp290His GOase_ox/GOase_semi couple is 730?mV, which compares with 400?mV for the WT GOase couple. Consistent with the 730?mV value the GOase_semi form is not oxidised with [Fe(CN)₆]^3– (410?mV) or [W(CN)₈]^3– (530?mV), and much stronger oxidants such as [Mo(CN)₈]^3– (800?mV) and [IrCl₆]^2– (890?mV) are required. The GOase_ox product is unstable and decays within 20?min with re-formation of GOase_semi. From changes in UV-Vis spectra with pH, Trp290His GOase_semi gives a pK _a of 6.9, and rate constants for the oxidation of GOase_semi with [Mo(CN)₈]^3– are dependent on this same pK _a. The latter compares with 7.9 for WT GOase_semi, and is assigned here also as protonation of Tyr-495. The 1?:?1 binding of azide at the substrate-binding (H₂O) site of Trp290His GOase_semi was studied and gives a formation constant 330?M^–1 at pH 7.5, which is an order of magnitude less than the corresponding value for WT GOase_semi. The trends observed indicate less affinity of Trp290His GOase_semi for the ionic reactants H⁺ and N₃ ^–.     

Changes in membrane potential during mouse egg development

The electrical membrane potential (E_m) was measured in the developing mouse egg with intracellular microelectrodes. The oocyte had a low negative E_m of ?8.3 ± 0.8 mV (mean ± SE) when immature, which decreased and reversed polarity to a small positive value (+1.9 ± 0.2 mV) in the mature ovulated oocyte. After fertilization E_m returned to a negative value (?9.2 ± 0.5 mV) similar in magnitude to that observed in immature oocytes and then increased significantly (P < 0.01) at both the two-cell (?10.7 ± 0.3 mV) and morula stage (?12.8 ± 0.7 mV) and leveled out at the blastocyst stage (?12.9 ± 0.7 mV). Average potential difference recorded across the blastocoele wall of not fully expanded blastocysts was ?5.0 ± 0.5 mV. These data represent the first report on membrane potentials of the mammalian egg during development. A striking similarity is seen in the relative changes in E_m throughout development of the mouse egg in comparison to those seen in other invertebrate and vertebrate eggs.     

A comparison of Xenopus laevis oocyte and embryo mRNA

Total RNA, extracted from mature oocytes and tadpoles of Xenopus laevis, was used as a template for in vitro protein synthesis. The oocyte RNA is markedly deficient in abundant mRNA species by comparison to tadpole RNA or other somatic RNAs, in agreement with previous experiments using RNA-cDNA hybridization analysis (S. Perlman and M. Rosbash, 1978, Develop. Biol.63, 197–212). Oocyte pA⁺ RNA is also larger than tadpole pA⁺ RNA or other somatic pA⁺ populations. The larger oocyte pA⁺ RNA and smaller oocyte pA⁺ RNA are equally good templates for in vitro protein synthesis, which implies that much, and perhaps all, of the large oocyte pA⁺ RNA is bona fide mRNA. We suggest that the relatively large size of the oocyte pA⁺ RNA population is due, at least in part, to the relative lack of abundant mRNA species in the population. This suggestion follows from the observation of 0. Meyuhas and R. P. Perry (1979, Cell16, 139–148) that L-cell-abundant mRNAs are preferentially small and rare mRNAs preferentially large. Most of the oocyte pA⁺ sequences are also present in tadpoles and are still adenylated at this stage. Oocyte proteins synthesized in vivo do not appear deficient in abundant proteins, suggesting that a translational control mechanism operates to select certain pA⁺ RNAs at higher frequencies than others.     

Localization of the chromatin-remodeling protein ATRX in the oocyte nucleus of some insects

A comparative study of nuclear distribution of the chromatin-remodeling protein ATRX in the oocytes of three species of insects in which the oocyte nucleus at the diplotene stage differs in structure, has been carried out using fluorescent and immunoelectron microscopy. In tóhe oocyte nucleus of the tenebrionid beetles, Tribolium castaneum and Tenebrio molitor, ATRX preferably associates with the karyosphere (karyosome) that represents a result of concentration of the condensed chromosomes in a limited volume of the nucleus. In the oocyte nucleus of the house cricket, Acheta domesticus, in which a karyosphere does not form, the protein ATRX is distributed in the entire nuclear volume in association with the chromatin. The fact of ATRX presence in the extrachromosomal structures of the insect oocyte nucleus, the karyosphere capsule and specific nuclear bodies, is documented for the first time.     

An estimation of the light-induced electrochemical potential difference of protons across the membrane of Halobacterium halobium

The light-dependent uptake of triphenylmethylphosphonium (TPMP⁺) and of 5,5-dimethyloxazolidine-2,4-dione (DMO) by starved purple cells of Halobacterium halobium was investigated. DMO uptake was used to calculate the pH difference (ΔpH) across the membrane, and TPMP⁺ was used as an index of the electrical potential difference, Δψ.Under most conditions, both in the light and in the dark, the cells are more alkaline than the medium. In the light at pH 6.6, ΔpH amounts to 0.6–0.8 pH unit. Its value can be increased to 1.5–2.0 by either incubating the cells with TPMP⁺ (10^?3 M) or at low external pH (5.5). — ΔpH can be lowered by uncoupler or by nigericin. The TPMP⁺ uptake by the cells indicates a large Δψ across the membrane, negative inside. It was estimated that in the light, at pH 6.6, Δψ might reach a value of about 100 mV and that consequently the electrical equivalent of the proton electrochemical potential difference, , amounts under these conditions to about 140 mV.The effects of different ionophores on the light-driven proton extrusion by the cells were in agreement with the effects of these compounds on — ΔpH.     

A new solid-state microelectrode for measuring intra-cellular chloride activities

Solid-state microelectrodes for measuring intracellular Cl^? activity (aⁱCl) were made by sealing the tips of tapered glass capillaries (tip diameter 0.3 μm), coating them under vacuum with a 0.2–0.3 μm thick layer of spectroscopic grade silver, and sealing them (except for the terminal 2–5 μm of the tip) inside tapered glass shields. 106 microelectrodes had an average slope of 55.0 ± 0.6 mV (S.E.) per decade change in α_Cl. Tip resistance was (77.1 ± 3.1 × 10⁹ω (n=30). Electrode response was rapid (10–20 s), was unaffected by HCO₃^?, H₂PO₄^2? or protein, and remained essentially unchanged over a 24-h period. αⁱ_Cl in frog sartorius muscle fibers and epithelial cells of bullfrog small intestine was measured in vitro. In both tissues, αⁱ_Cl significantly exceeded the value corresponding to equilibrium distribution of Cl^? across the cell membrane.     

Proton Electrochemical Gradients in Washed Cells of Nitrosomonas europaea and Nitrobacter agilis

The components of the proton motive force (Δp), namely, membrane potential (Δψ) and transmembrane pH gradient (ΔpH), were determined in the nitrifying bacteria Nitrosomonas europaea and Nitrobacter agilis. In these bacteria both Δψ and ΔpH were dependent on external pH. Thus at pH 8.0, Nitrosomonas europaea and Nitrobacter agilis had Δψ values of 173 mV and 125 mV (inside negative), respectively, as determined by the distribution of the lipophilic cation [³H]tetraphenyl phosphonium. Intracellular pH was determined by the distribution of two weak acids, ¹⁴C-benzoic and ¹⁴C-acetyl salicylic, and the weak base [¹⁴C]methylamine. Nitrosomonas europaea accumulated ¹⁴C-benzoic acid and ¹⁴C-acetyl salicylic acid when the external pH was below 7.0 and [¹⁴C]methylamine at alkaline pH. Similarly, Nitrobacter agilis accumulated the two weak acids below an external pH of about 7.5 and [¹⁴C]methylamine above this pH. As these bacteria grow best between pH 7.5 and 8.0, they do not appear to have a ΔpH (inside alkaline). Thus, above pH 7.0 for Nitrosomonas europaea and pH 7.5 for Nitrobacter agilis, Δψ only contributed to Δp. In Nitrosomonas europaea the total Δp remained almost constant (145 to 135 mV) when the external pH was varied from 6 to 8.5. In Nitrobacter agilis, Δp decreased from 178 mV (inside negative) at pH 6.0 to 95 mV at pH 8.5. Intracellular pH in Nitrosomonas europaea varied from 6.3 at an external pH of 6.0 to 7.8 at external pH 8.5. In Nitrobacter agilis, however, intracellular pH was relatively constant (7.3 to 7.8) over an external pH range of 6 to 8.5. In Nitrosomonas europaea, Δp and its components (Δψ and ΔpH) remained constant in cells at various stages of growth, so that the metabolic state of cells did not affect Δp. Such an experiment was not possible with Nitrobacter agilis because of low cell yields. The effects of protonophores and ATPase inhibitors on ΔpH and Δψ in the two nitrifying bacteria are considered.