Biochemical Characterization of Molybdenum-Cofactor in a Cyanobacterium, Nostoc muscorum

Cell-free extracts of nitrate-grown Nostoc muscorum containnitrate reductase and molybdenum-cofactor activities. Whilenitrate reductase activity is associated with the paniculatefraction, cofactor activity is found predominantly in the solublefraction. This activity was distributed between two pools. Inone pool, the molybdenumcofactor is associated with a carrier(protein) of approximately 30,000 Da with an S_{20, w} between2.3 and 2.5. The carrier-bound cofactor is non-dialyzable andis found along with the major proteins during filtration inSephadex G-25 and G-100. The second pool contains free or unboundcofactor. It is separated from soluble proteins by dialysiswith a membrane with a pore-size of 10 to 15 kDa. However, itis retained with a membrane with a pore size of 1 kDa. It isin the included volume during chromatography through SephadexG-25. Its molecular mass is estimated to be between 1,000 and5,000 Da. The molybdenum content was proportional to cofactoractivity in both pools. Reducing agents increased cofactor activity.However, activity in both pools was sensitive to heat, acid,and oxidative treatments. The carrier protein appears to givesome protection. ¹Fulbright Scholar from Department of Biological Sciences, R.D. University, Jabalpur-482001, India. To whom reprint requestsshould be addressed. (Received June 22, 1987; Accepted August 21, 1987)     

Light-induced changes in chlorophyll a fluorescence and cytochrome f in intact spinach chloroplasts: The site of light-dependent regulation of electron transport

Dark-adapted intact spinach chloroplasts exhibited two peaks,P and M₁, at the early phase of fluorescence induction and atransient reduction of cytochrome f shortly after its initialphotooxidation and in parallel to the appearance of P. Analysisof the peak P and the transient reduction of cytochrome f indicatedthat electron transport in intact spinach chloroplasts was regulatedby light: electron transport was inactivated at the reducingside of photosystem I in the dark-adapted chloroplasts but rapidlyreactivated by illumination. The fluorescence peak M₁ was correlatedto the proton gradient formed across the thylakoid membrane. Effects on P and transient reduction of cytochromef of NO₂^–,3-phosphoglycerate (PGA) and oxalacetate (OAA), which can penetrateinto intact chloroplasts and accept electrons at different sitesafter photosystem I, were studied to determine the site of thelight regulation. NC₂^–, which receives electrons fromreduced ferredoxin, markedly diminished both P and the transientreduction of cytochrome.f, whereas PGA and OAA, the reductionsof which are NADP-dependent, failed to affect the two transients.The ineffectiveness of PGA and OAA could not be attributed tothe dark inactivation of glyceraldehyde-3-phosphate and malicdehydrogenases, because dark-adapted chloroplasts still retainedsufficiently high levels of the enzyme activities. The resultsindicate that electron transport in intact spinach chloroplastsis regulated by light after ferredoxin but before NADP, i.e.,at the reducing terminal of the electron transport chain. (Received May 29, 1980; )     

Alterations in Protein Synthesis in vivo in Chilling Sensitive Mung Bean Hypocotyls Caused by Chilling Stress

The effects of chilling on protein synthesis in vivo in etiolatedhypocotyls of Vigna radiata L. were investigated. After exposureof the tissues to 0?C for various periods of time, proteinswere labeled with [³⁵S]-methionine at 26?C. The total amountof ³⁵S incorporated into soluble and membrane proteins was reversiblyreduced by chilling for 24 h, during which time the tissuessuffered no injury. Further prolonged chilling produced an irreversibledecline both in the incorporation of radioactivity and in cellviability as assessed by the extent of leakage of electrolyte.The ³⁵S-labeled proteins in the soluble and the total membranefractions were analyzed quantitatively. Chilling of etiolatedhypocotyls for one or two days induced the syntheses of twosoluble proteins (82 and 74 kDa) and one membrane protein (80kDa). Moreover, three heat-shock proteins (HSPs) that were inducedby heat stress (41 ?C, 4h) had the same electrophoretic mobilitiesas those of the proteins induced directly or indirectly by thechilling treatment. ¹Contribution No. 3170 from the Institute of Low TemperatureScience. (Received April 22, 1988; Accepted September 30, 1988)     

A Comparative Study of the Biological Activities of Two Molecular Species of Chloroplast-type Ferredoxins

Pairs of two molecular species of soluble chloroplast-type ferredoxins(Fd I and Fd II) from Nostoc muscorum and Aphanothece sacrumwere used to examine and compare the abilities of ferredoxinto substitute for spinach ferredoxin in the photoreduction ofNADP⁺ by spinach chloroplasts or N. muscorum membrane fragmentsand to link the reducing power of illuminated spinach chloroplaststo the Bacillus polymyxa nitrogenase system. Ferredoxins II of Nostoc and Aphanothece showed rather low activitiesin NADP⁺ photoreduction and nitrogenase system with spinachchloroplasts as the photosensitizer, compared to other ferredoxins.However, there was no difference between two ferredoxins (FdI and Fd II) from Nostoc in NADP⁺ photoreduction by photosyntheticmembrane fragments prepared from the same organism, N. muscorum. The biological significance of two molecular species of ferredoxinsin one organism could be ascribed to the different contributionof each ferredoxin to certain biological reactions in whichferredoxin functioned as an electron carrier. (Received November 4, 1980; Accepted January 9, 1981)     

Photosynthetic Characteristics of Chloroplasts Isolated from Euglena gracilis Z Grown Photoautotrophically

Photosynthetically competent chloroplasts were isolated fromcells of Euglena gracilis Z grown photoautotrophically in 1.5%CO₂. The isolated chloroplasts were intact and substantiallyfree from cytosolic, mitochondrial and microbody materials.The effects of some compounds on the activity of photosynthetic¹⁴CO₂ fixation were examined. The optimal pH and sorbitol concentrationwere 8.0 and 0.33 M, respectively. The chloroplasts requireda high level of P, (5 to 20 mM) for the maximal rate of photosynthesis.They were insusceptible to 10 mM of free Mg²⁺. ATP, ADP andAMP at 1 to 5 mM notably stimulated photosynthesis, althoughhigh concentrations of AMP were unfavorable. In the assay mediumdeveloped for this study, the chloroplasts exhibited photosyntheticactivity of 120µmoles-mg^–1 Chl-h^–1 at 30?C. Chloroplasts could also be isolated from cells grown under ordinaryair. The rate of photosynthetic ¹⁴CO₂ fixation at 1 mM NaH^l4CO₃was higher in these chloroplasts than in those isolated fromcells grown in 1.5% CO₂, whereas at 10 mM NaH^l4CO₃, the ratesof the two types of chloroplasts were nearly the same. Theseresults suggest that the CO₂ concentration given during growthof the algal cells affects the affinity for dissolved inorganiccarbon at the chloroplast level. (Received March 30, 1987; Accepted August 17, 1987)     

Identification of the Product of ndhA Gene as a Thylakoid Protein Synthesized in Response to Photooxidative Treatment

A 76 amino acid sequence of NDH-A (the protein encoded by plastidndhA gene) from barley (Hordeum vulgare L.) was expressed asa fusion protein with rß-galactosidase in E. coli.The corresponding antibody generated in rabbits was used toinvestigate localization, expression and synthesis in vitroof NDH-A. NDH-A was identified as a 35 kDa polypeptide localizedin thylakoid membrane. Western blots shows a large increasein NDH-A levels when barley leaves were incubated under photooxidativeconditions, which was more pronounced in mature-senescent leavesthan in young leaves. Immunoprecipitation of the [³⁵S]methioninelabelled proteins, synthesized in vitro by isolated chloroplasts,demonstrated the synthesis in chloroplasts of the NDH-A 35 kDapolypeptide when barley leaves had been incubated under photooxidativeconditions. The results indicate that ndh genes may be involvedin the protection of chloroplasts against photooxidative stress,particularly in mature-senescent leaves. (Received November 13, 1995; Accepted February 5, 1996)     

Isolation of Chloroplasts from Poteriochromonas malhamensis with the Capacity for Translation

An improved method for the isolation of chloroplasts from Poteriochromonasmalhamensis is described. Poteriochromonas cells were brokenby passage through a nylon mesh with pores of 6µ in diameterat a flow rate of about 5 ml/15 s. After centrifugation thecrude chloroplast fraction was purified by centrifugation ina step gradient of Percoll. The isolated chloroplasts were enclosedby envelope membranes and were still surrounded in part by cytoplasmicresidues. The chloroplasts had the capacity for translation,which was both chloramphenicol-sensitive and cycloheximide-insensitive.The properties of these isolated chloroplasts from Poteriochromonasare discussed in relation to experiments on the transport intothe chloroplasts of nucleus-encoded proteins. ² Present address: Bundesgesundheitsamt, Zulassungsstelle furGentechnologie, Columbiadamm 3, D-1000 Berlin, F.R.G. (Received July 24, 1990; Accepted March 15, 1991)     

Synthesis of two proteins in chloroplasts and mRNA distribution between thylakoids and stroma during the cell cycle of Chlamydomonas reinhardii

Chloroplasts contain thylakoid-bound and free ribosomes and polysomes. Whether binding of polysomes plays an immediate role in the regulation of chloroplast protein synthesis is not yet clear. In the present work, variations of protein synthesis and of mRNA content were measured not in greening, but in fully differentiated chloroplasts during the cell cycle of synchronized cultures of Chlamydomonas reinhardii. At different times of the vegetative cell cycle, the RNA was extracted from free and thylakoid-bound chloroplast polysomes and the partition of mRNAs between stroma and thylakoids was measured for two proteins, i.e. the 32-kDa herbicide-binding membrane protein and the soluble large subunit of the ribulose-1,5-bisphosphate carboxylase. At the same time the rates of synthesis of these two proteins were also determined. At 2 h after the onset of light, the content of both mRNAs in chloroplasts had doubled and 75-90% of each of these mRNAs were found to be bound to the thylakoids. The rate of protein synthesis, however, increased 10-fold, but reached its maximum only after about 6 h in the light. The differences in the time courses, in the stimulation of the rate of protein synthesis, and in the mRNA-binding to thylakoids point to a translational regulation of protein synthesis. Furthermore, since a very high proportion of polysomes were bound to thylakoids, containing mRNA for both a membrane and a soluble protein, this light-induced binding of polysomes to thylakoids seems to be an essential, but not the only, prerequisite for protein synthesis in chloroplasts.     

Chlorophyll a forms in mesophyll and bundle sheath chloroplasts of Zea mays leaves grown at low light intensity

Mesophyll and bundle sheath chloroplasts were prepared fromleaves of Zea mays grown at light intensities of 1.1 and 240µW/cm², respectively. The mesophyll chloroplasts thatdeveloped at the low intensity and bundle sheath chloroplatsthat developed at both low and high intensities showed higherratios of chlorophyll a/b and P700/chlorophylls compared withthe normal ratios found for the mesophyll chloroplasts thathad developed at the high intensity. Derivative absorption spectrophotometryat 77?K revealed that the low intensity mesophyll chloroplastscontained more of chlorophyll a forms with longer wavelengthred bands than high intensity mesophyll chloroplasts. More ofthe longer wavelength forms of chlorophyll a were also presentin the bundle sheath chloroplasts that had developed at lowand high intensities. All these four types of chloroplasts showedtwo peaks of fluorescence, one at 687 hra and the other at 733or 738 nm. In addition to these peaks, the high intensity mesophyllchloroplasts showed a shoulder at 697 nm, and the two typesof bundle sheath chloroplasts showed a shoulder at 680 nm. (Received June 17, 1974; )     

Translatable mRNAs for Chloroplast-Targeted Proteins in Detached Radish Cotyledons during Senescence in Darkness

A protein-import system prepared with isolated chloroplastswas used to monitor changes in levels of mRNAs for chloroplast-targetedproteins during dark-induced leaf senescence. Biologically activechloroplasts were isolated from young (9-day-old) and aged (14-day-old)radish cotyledons. Poly(A)⁺-RNA was prepared from radish cotyledonsthat had been detached from seedlings and placed in darknessto accelerate senescence. The RNA was translated in a wheatgerm system, and the products were added to an import systemprepared with chloroplasts from young cotyledons. Electrophoreticanalysis of the imported proteins suggested that most chloroplast-targeted proteins decreased in abundance during dark treatmentof cotyledons. However, the relative abundance of 38 stromaland three thylakoid proteins increased transiently or continuouslyamong the products of translation of RNA isolated during thecourse of senescence. The efficiency of the uptake of precursorproteins by chloroplasts isolated from aged cotyledons was lowerthan that by chloroplasts from young tissue. The chloroplastsfrom aged cotyledons more efficiently imported at least onestromal protein and one thylakoid protein than chloroplastsfrom the young tissue. The relative abundance of these two proteinsincreased among the products of translation of RNA from senescingcotyledons when tested in the uptake system with chloroplastsfrom young cotyledons. These results suggest that some nucleargenes for chloroplast-targeted proteins are expressed in senescingcotyledons more efficiently than in young tissue, and that themachinery for import of proteins into chloroplasts changes duringaging of the tissue to allow more efficient import of certainproteins that may be responsible for the senescence of the chloroplasts. ¹Present address: Kihara Institute for Biological Research,Yokohama City University, Mutsukawa 3-122-20, Minami-ku, Yokohama,232 Japan     

Photo-induced H+ Transport between Chloroplasts and the Cytoplasm in a Protoplasmic Droplet of Characeae

Svintitskikh, V. A., Andrianov, V. K. and Bulychev, A. A. 1985.Photo-induced H⁺ transport between chloroplasts and the cytoplasmin a protoplasmic droplet of Characeae.—J. exp. Bot. 36:1414–1429. The effects of light on the membrane potential and cytoplasmicpH of isolated droplets of protoplasm from Nitella have beenstudied using microcapillary electrodes and pH-sensitive antimonymicro-electrodes. Illumination of chloroplast-containing dropletscaused a change of the membrane potential with a concomitantacidification of both the cytoplasm and the outer medium, butit had no effect on the electrical resistance of the surfacemembrane. Treatment of protoplasmic droplets with uncouplers(NH₄Cl and CCCP) resulted in a complete inhibition of the light-inducedacidification of the cytoplasm, whereas the energy transferinhibitor DCCD had no effect. A correlation between the formationof a pH gradient across the thylakoid membrane and the acidificationof the cytoplasm was explicable in terms of the assumption ofrestricted spatial communication between the intra-thylakoidvolume and the cytoplasm in intact chloroplast. The photo-inducedacidification of the boundary layer of an external medium wasmarkedly stimulated under the action of inhibitors of H⁺-ATPaseDCCD and DES. These findings suggest that the active extrusionof H⁺ from the cytoplasm into the external medium is not drivenby an ATPase, although H+-conducting channels of membrane ATPaseprovide a pathway for a passive diffusion of protons from outsideinto the cytoplasm Key words: Transport of protons, protoplasmic droplet, intact chloroplasts, Characeae     

Gene Activity during Germination of Spores of the Fern, Onoclea sensibilis: RNA and Protein Synthesis and the Role of Stored mRNA

Pattern of ³H-uridine incorporation into RNA of spores of Onocleasensibilis imbibed in complete darkness (non-germinating conditions)and induced to germinate in red light was followed by oligo-dTcellulose chromatography, gel electrophoresis coupled with fluorographyand autoradiography. In dark-imbibed spores, RNA synthesis wasinitiated about 24 h after sowing, with most of the label accumulatingin the high mol. wt. poly(A)^–RNA fraction. There was noincorporation of the label into poly(A) ⁺ RNA until 48 h aftersowing. In contrast, photo-induced spores began to synthesizeall fractions of RNA within 12 h after sowing and by 24 h, incorporationof ³H-uridine into RNA of irradiated spores was nearly 70-foldhigher than that into dark-imbibed spores. Protein synthesis,as monitored by ³H-arginine incorporation into the acid-insolublefraction and by autoradiography, was initiated in spores within1–2 h after sowing under both conditions. Autoradiographicexperiments also showed that the onset of protein synthesisin the cytoplasm of the germinating spore is independent ofthe transport of newly synthesized nuclear RNA. One-dimensionalsodium dodecyl sulphate-polyacrylamide gel electrophoresis of³⁵S-methionine-labelled proteins revealed a good correspondencebetween proteins synthesized in a cell-free translation systemdirected by poly(A) ⁺RNA of dormant spores and those synthesizedin vivo by dark-imbibed and photo-induced spores. These resultsindicate that stored mRNAs of O. sensibilis spores are functionallycompetent and provide templates for the synthesis of proteinsduring dark-imbibition and germination. Key words: Onoclea sensibilis, fern spore germination, gene expression, protein synthesis, sensitive fern, stored mRNA     

Pyruvate Uptake by Mesophyll and Bundle Sheath Chloroplasts of a C4 Plant, Panicum miliaceum L.

Intact chloroplasts were isolated from mesophyll and bundlesheath protoplasts of a C₄ plant, Panicum miliaceum L., to measurethe uptake of [1-¹⁴C]pyruvate into their sorbitol-impermeablespaces at 4?C by the silicone oil filtering centrifugation method.When incubated in the dark, both chloroplasts showed similarslow kinetics of pyruvate uptake, and the equilibrium internalconcentrations were almost equal to the external levels. Whenincubated in the light, only mesophyll chloroplasts showed remarkableenhancement of the uptake, the internal concentration reaching10–30 times of the external level after 5 min incubation.The initial uptake rate of the mesophyll chloroplasts was enhancedabout ten fold by light and was saturated with increasing pyruvateconcentration; K_m and V_max were 0.2–0.4 mM and 20–40µmol(mg Chl)^–1 h^–1, respectively. The lightenhancement was abolished by DCMU and uncoupling reagents suchas carbonylcyanide-m-chlorophenylhydrazone and nigericin. Theseresults indicate the existence of a light-dependent pyruvatetransport system in the envelope of mesophyll chloroplasts ofP. miliaceum. The uptake activity of mesophyll chloroplastsboth in the light and the dark was inhibited by sulfhydryl reagentssuch as mersalyl and p-chloromercuriphenylsulfonate, but thebundle sheath activity was insensitive to the reagents. Thesefindings are further evidence for the differentiation of mesophylland bundle sheath chloroplasts of a C₄ plant with respect tometabolite transport. (Received July 3, 1986; Accepted October 8, 1986)     

Temperature dependence of chlorophyll {alpha} fluorescence in lettuce and spinach chloroplasts at sub-zero temperatures

The temperature dependence of chlorophyll fluorescence wasmeasured in spinach and lettuce chloroplasts at sub-zero temperaturesin the presence of 50% ethylene glycol. In the presence of 5mM Mg²⁺, a fluorescence maximum appeared at –31?C in boththe spinach and lettuce chloroplasts, while in the presenceof only 5 mM Na⁺ as cations the maximum shifted to –20?Cin the spinach chloroplasts and to –11?C in the lettucechloroplasts. Since the occurrence of a maximum in the temperatureversus fluorescence curve is an indication for the transitionof the physical phase of thylakoid membrane lipids between theliquid crystalline and the phase-separation state (16, 18),these findings suggest that the (major) phase transition ofmembrane lipids occurs at these low temperatures in chloroplastsof higher plants and also that the phase transition temperatureis markedly lowered by the presence of divalent cations. Ethylene glycol at a concentration of 50% had almost no effecton the temperature dependence of chlorophyll fluorescence ina lamellar membrane preparation of Anabaena variabilis. In awater suspension of dimyristoylphosphatidylcholine, the additionof ethylene glycol to 50% did not alter the characteristic featureof the temperature dependence of fluorescence of 1-anilinonaphthalene-8-sulfonate.These findings suggest that 50% ethylene glycol does not affectthe temperature of the transition of the physical phase of membranelipids. ¹ C.I.W.-D.P.B. Publication No. 592. ² Present Address: Department of Biophysics and Biochemistry,Faculty of Science, University of Tokyo, Hongo 113, Tokyo, Japan. (Received June 22, 1977; )     

Syntaxin 1A has a specific binding site in the H3 domain that is critical for targeting of H+-ATPase to apical membrane of renal epithelial cells

H⁺ transport in the collecting duct is regulated by exocytic insertion of H⁺-ATPase-laden vesicles into the apical membrane. The soluble N-ethylmaleimide-sensitive fusion protein attachment protein (SNAP) receptor (SNARE) proteins are critical for exocytosis. Syntaxin 1A contains three main domains, SNARE N, H3, and carboxy-terminal transmembrane domain. Several syntaxin isoforms form SNARE fusion complexes through the H3 domain; only syntaxin 1A, through its H3 domain, also binds H⁺-ATPase. This raised the possibility that there are separate binding sites within the H3 domain of syntaxin 1A for H⁺-ATPase and for SNARE proteins. A series of truncations in the H3 domain of syntaxin 1A were made and expressed as glutathione S-transferase (GST) fusion proteins. We determined the amount of H⁺-ATPase and SNARE proteins in rat kidney homogenate that complexed with GST-syntaxin molecules. Full-length syntaxin isoforms and syntaxin-1AC [amino acids (aa) 1–264] formed complexes with H⁺-ATPase and SNAP23 and vesicle-associated membrane polypeptide (VAMP). A cassette within the H3 portion was found that bound H⁺-ATPase (aa 235–264) and another that bound SNAP23 and VAMP (aa 190–234) to an equivalent degree as full-length syntaxin. However, the aa 235–264 cassette alone without the SNARE N (aa 1–160) does not bind but requires ligation to the SNARE N to bind H⁺-ATPase. When this chimerical construct was transected into inner medullary collecting duct cells it inhibited intracellular pH recovery, an index of H⁺-ATPase mediated secretion. We conclude that within the H3 domain of syntaxin 1A is a unique cassette that participates in the binding of the H⁺-ATPase to the apical membrane and confers specificity of syntaxin 1A in the process of H⁺-ATPase exocytosis. soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor proteins; exocytosis; H⁺⁺ transport     

Carbonic Anhydrase of Chlamydomonas reinhardtii. Cellular Distribution and Subunit Constitution in Two Strains

Cellular distribution and subunit constitution of carbonic anhydrase(CA) were compared between the CBSC strain of Chlamydomonasreinhardtii (purchased from Carolina Biological Suuply Co.)and C. reinhardtii C-9. CAs existed exclusively outside theplasma membrane inboth strains. Mol wts of the monomer and theholoenzyme from both strains were 35 kDa and 115–117 kDa,respectively. These results show that the CAs of both strainsare identical or verysimilar. ¹Present address: National Institute for Basic Biology, Myodaiji,Okazaki 444, Japan. (Received June 29, 1987; Accepted October 9, 1987)     

Effects of Light on Degradation of Chlorophyll and Proteins during Senescence of Detached Rice Leaves

Regulatory effects of light on senescence of rice leaves wereinvestigated by measuring degradation of chlorophyll and proteinsin leaf segments which had been kept in the dark or under illuminationwith light of different intensities and colors. When leaveshad been left in total darkness for three days at 30°C,there was an initial long lag that lasted for one whole dayand then chlorophyll was rapidly degraded in the second andthird days. Breakdown of chlorophyll was strongly retarded bycontinuous illumination with white light of intensity as lowas 0.5 µmol photons m^–2 s^–1 but the effectof light decreased at intensities above 10 µmol photonsm^–2 s^–2. The initial lag and subsequent degradationof chlorophyll in the dark were little affected by illuminationwith red or far red light at the beginning of dark treatment.However, a brief illumination with red light at the end of thefirst and/or second day significantly suppressed degradationof chlorophyll during subsequent dark periods and the effectof red light was nullified by a short irradiation with far redlight. Thus, degradation of chlorophyll is regulated by phytochrome.Thylakoid membrane proteins and soluble proteins were also largelydegraded during three days in the dark. Degradation of membraneproteins such as the apoproteins of light-harvesting chlorophylla/b proteins of photosystem II and chlorophyll a-binding proteinsof reaction center complexes showed a long lag and was stronglysuppressed by illumination with weak white light. Thus, theloss of chlorophyll can be correlated with degradation of chlorophyll-carryingmembrane proteins. By contrast, light had only a weak protectingeffect on soluble proteins and ribulose-1,5-bisphosphate carboxylase/oxygenaserapidly disappeared under illumination with weak white light.Thus, breakdown of thylakoid membrane and soluble proteins aredifferently regulated by light. Artifacts which would be introducedby detachment of leaves were also discussed. ¹ Present address: Department of Applied Biology, Faculty ofScience and Technology, Science University of Tokyo, Yamazaki,Noda-shi, Chiba, 278 Japan. ² Present address: Department of Life Science, Faculty of Science,Himeji Institute of Technology, Harima Science Park City, Hyogo,678-12 Japan.     

Analysis of Rapid Light-Induced Potential Change in Cells of Chara corallina

Perfused Chara cells were used to measure the rapid light-inducedpotential change (rapid LPC) caused by activation of a K⁺ channelin the plasma membrane through photosynthesis in the presenceof various photosynthetic inhibitors. The rapid LPC was inhibitedby DCMU but recovered on addition of phenazinemethosulfate (PMS)in the presence of DCMU. Carbonylcyanide m-chlorophenylhydrazone(CCCP) stimulated the rapid LPC. DCCD partially inhibited therapid LPC with a partial inhibition of oxygen evolution. Itis concluded that both cyclic and noncyclic electron flows arecoupled with the rapid LPC. To understand the mechanism of K⁺ channel activation by photosyntheticelectron flow, the rapid LPC was measured under continuous internalperfusion. It was suggested that a diffusible substance wasnot released from chloroplasts, since vigorous continuous perfusiondid not inhibit the rapid LPC. The suggestion that the rapid LPC is caused by changes in surfacecharge density of chloroplasts was supported by the fact thatthe rapid LPC was inhibited by increasing the ionic strengthof the perfusion medium. (Received February 28, 1986; Accepted April 30, 1986)