Twelve new or little used hydrogen ion buf-Two of the zwitterions, TV-tris(hydroxymethyl)fers covering the range pKa = 6.15-8 35 have been methylaminoethanesulfonic acid and TV-2-hydroxyprepared and tested. Ten are zwitterionic amino acids, ethylpiperazine-TV'-2-ethanesuIfonic acid, give partieither TV-substituted taurines or TV-substituted glycines, cularly active and stable mitochondrial preparations, and two are cationic primary aliphatic amines. All of These two also give higher rates of protein synthesis the zwitterionic buffers are better than conventional in cell-free bacterial preparations than do trisbuffers in the Hill reaction and in the phosphorylation-(hydroxymethyl)aminomethane (Tris) or phosphate coupled oxidation of succinate by bean mitochondria.buffers.

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.S'io,;iiarv. Spinach chloroplasts isolate(l in miedia conltaining salits and(l the rare chlorol)lasts which are still within their envelop)es alike retaini grana similar to those seen in clhloroplasts in situi.Chloroplasts isolatedl in lox-salt meldia lose their graila without losinig any chlorophyll. '

Ion movements associated with the pH rise that is observed upon illumination of thylakoid suspensions at low pH have been studied by a multiparameter technique. Light-dependent, dark-reversible fluxes of H+, Cl-, Na +, K + and divalent cations were monitored, together with simultaneous changes in the optical density of the suspension. Extensive uptake of Cl-and efflux of Mg2 + accompany the apparent inward movement of H + in the light. Only minor efflux of K+ is seen and Na+ appears immobile. The Cl-and Mg2+ fluxes together compensate for most of the charge transferred as H+, contributing respectively about 49% and 43% on an equivalent basis. The ratio of Cl-influx to Mg2 + efflux is variable, but usually >1.0., The Mg2 + flux can be supplanted by (1) K +flux, if the K+/Mg2+ activity ratio in the suspension is high, and (2) Ca2+ flux, if the thylakoids are equilibrated with suspending media containing Ca2+. The affinity of the divalentcation-binding sites, or carrier mechanism, is greater for Ca2 + than for Mg2 +. Schemes can be drawn up to account for the observed ion movements on the basis of either a chemical or a chemiosmotic mechanism for energy transduction in chloroplasts. In intact chloroplasts, lightdependent control of Mg2 + distribution between thylakoid and stroma could serve to regulate enzyme activities in the carbon fixation pathway, and hence photosynthesis.Ten years ago, the first description appeared (1) of the lightdriven apparent uptake of hydrogen ions by isolated thylakoids (chloroplast inner membranes). The pH rise in the suspending medium was associated with a reversible increase in sample turbidity, and with the ability of energized thylakoids to phosphorylate ADP, upon relaxation in the dark under conditions conducive to photophosphorylation.These observations have been extensively elaborated, and reviewed in detail (2, 3). The pH rise phenomenon attracts continued attention, since it has seemed to provide the most tangible evidence in favor of the chemiosmotic mechanism (4) of energy transduction. Estimates of up to 3.0 pH units have been made (5-7) for the magnitude of the H+ gradient across illuminated thylakoid membranes, and this has been construed as establishing the thermodynamic feasibility of chemiosmotic coupling.Such massive pH shifts cannot occur without concomitant flux of some ion other than H+, as required by the law of electroneutrality. It is commonly assumed that Cl-serves as a counterion, and Cl-uptake has been observed (8-10). That reported by Deamer and Packer (10) was seen under conditions favoring uptake of the phenazine methosulfate cation (11), in a manner presumably analogous to the uptake of amine cations and Cl-(12). A simultaneous demonstration of stoichiometric H+ and Cl-uptake in the absence of uncoupler has yet to be given. Indeed, Dilley and Vernon (13) Abbreviations: HEPES, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid; PIPES, piperazine-N,N'-bis(2-ethanesulfonic acid).