An Investigation into the Roles of Photosynthesis and Respiration in H<sup>+</sup> Efflux from Aerated Suspensions of <i>Asparagus</i> Mesophyll Cells

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Mechanically isolated Asparagus sprengeri Regel mesophyll cells were suspended in 1 millimolar CaSO4. Immediate alkalinization of the medium occured on the addition of 1 millimolar concentrations of Lglutamate (Glu) and its analog L-methionine-D,L-sulfoximine (L-MSO). D-Glu and the L isomers of the protein amino acids did not elicit alkalinization. L-Glu dependent alkaliniation was transient and acidification resumed after approximately 30 to 45 minutes. At pH 6.0, 5 millimolar L-Glu stimulated initial rates of alkainization that varied between 13 to 4.1 nmol H/10' cells-minute. L-Glu dependent alkalinization was saturable, increased with decreasing pH, was inhibited by carbonyl cyanide-ptrichloromethoxyphenyl hydrazone (CCCP), and was not stimulated by light. Uptake of L4U-"Ckglutamate increased as the pH decreased from 6.5 to 5.5, and was inhibited by L-MSO. L-Glu had no influence on K efflux. Although evidence for multiple anino acid/proton cotransport systems has been found in other tissues, the present report indicates that a highly specific L-Glu/proton uptake process is present in Asparagus mesophyll cells.passive K+ efflux represented the repolarizing current (10, 13). Evidence for a repolarizing flux of K+ was obtained when cultured sugarcane cells were used to obtain rates of amino acid uptake and simultaneous measurements of amino acid dependent proton influx and K+ efflux. The presence of three distinct proton co-transport systems for neutral, basic, and acidic amino acids was indicated and a role of K+ efflux in charge compensation was suggested in each case (23).Although the leaf cells of many plants receive their nitrogen via an apoplastic route in amino acid form (12) there is little published information on amino acid/proton uptake processes in photosynthetic leaf cells. Mechanically isolated photosynthetically competent mesophyll cells from Asparagus sprengeri (5), can be maintained in a stirred, aerated, and defined suspension medium and have been used to study energy dependent proton effilux (2, 3). This system does not suffer from diffusion problems inherent in studies conducted with fragile nonaerated leaf protoplasts or bulky leaf slices. The objective of the present study was to determine whether amin;o acid proton cotransport processes could be detected and characterized in these cells.Recent data suggest that the immediate source of energy driving amino acid uptake is a proton electrochemical gradient across the plasma membrane (13). A plasma membrane located ATPase pumps protons out ofthe cell establishing both electrical and pH gradients which favor the reentry of protons (16,17). Influx of the amino acid is believed to be coupled to and driven by the downhill influx of protons. Models of amino acid/proton cotransport suggest that a plasma membrane carrier binds exogenous amino acids and protons prior to entry into the cell (13). (8).Between 10 to 15 x 106 aerated (500 ml/min) and tirred cells in 10 ml of 1 mm CaSO4 were maintained at 30°C in a water jacketed glass vessel. Ne...

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confidence: 99%

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confidence: 99%

Evidence for a Specific Glutamate/H⁺ Cotransport in Isolated Mesophyll Cells

McCutcheon¹,

Bown²

1987

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“…The reduction of exogenous Fe(CN)63-by Elodea leaf cells and an associated decline in membrane potentials has been reported (1 1). Active ATP driven proton efflux and related transport processes are currently being investigated using isolated Asparagus mesophyll cells (3,5,20 (6). Cell numbers were measured using a light microscope and hemocytometer.…”

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confidence: 99%

“…Net H4 efflux was normally measured using a recording pH meter as described previously (5,20 (Fig. 3).…”

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confidence: 99%

A Plasmamembrane Redox System and Proton Transport in Isolated Mesophyll Cells

Neufeld¹,

Bown²

1987

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Substantial evidence indicates that a plasma membrane located, ATP requiring outwardly directed proton pump establishes a proton electrochemical gradient that is used to drive the transport of solutes into and out of plant cells ( 17,23,24). However, a proton electrochemical gradient across biological membranes may also be established by redox reactions involving the sequential operation of hydrogen and electron carriers (7,14 (12,21). A third model has recently been proposed in which plasma membrane redox activity transports only electrons to an exogenous acceptor. The resulting depolarization then activates the proton ATPase and accounts for the increase in H4 efflux observed on addition of Fe(CN)631 (18).Very little is known concerning the existence of plasma membrane redox activities in photosynthetic cells. The reduction of exogenous Fe(CN)63-by Elodea leaf cells and an associated decline in membrane potentials has been reported (1 1). Active ATP driven proton efflux and related transport processes are currently being investigated using isolated Asparagus mesophyll cells (3,5,20

“…Asparagus sprengeri Regel was grown and mesophyll cells isolated using previously described methods (3,4). Chl content of cell suspensions was determined by the method of Amon (2) DIECA in the suspending medium was determined by measuring the absorbance of a copper (II) DIECA complex at 433 nm (1).…”

Section: Methodsmentioning

confidence: 99%

Disulfiram Metabolism in Isolated Mesophyll Cells and Inhibition of Photosynthesis and Cyanide-Resistant Respiration

Bown¹,

Pullen²,

Shadeed³

1984

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Disulfiram (tetraethylthiuram disulfide) is a potent inhibitor of cyanide-resistant respiration in isolated mitochondria (7) or intact mesophyll cells of Asparagus sprengeri Regel (17). In addition, disulfiram and other thiuram disulfides inhibit photosynthesis (13,14). The mechanisms by which disulfiram inhibits these processes are not understood. Inhibition in both cases may result from a reaction between the disulfide component of disulfiram and enzyme thiol groups essential for the cyanide-resistant pathway or photosynthesis (7,14,16). Another possible mechanism of inhibition involves chelation by disulfiram of transition metals located in enzyme systems (14). Inhibition may also be mediated by disulfiram's ability to function as a free radical scavenger and react with fatty acid peroxy radicals (18). In the present paper, the ability of isolated Asparagus mesophyll cells to rapidly reduce disulfiram to DIECA2 is demonstrated. The possible involvement of DIECA production in inhibition of either process is investigated. MATERIALS AND METHODSAsparagus sprengeri Regel was grown and mesophyll cells isolated using previously described methods (3,4). Chl content of cell suspensions was determined by the method of Amon (2) DIECA in the suspending medium was determined by measuring the absorbance of a copper (II) DIECA complex at 433 nm (1).Rates of 02 production in the light or 02 consumption in the dark were determined with a calibrated 02 electrode (YSI 4004) using cells suspended in 7.5 ml of a 30'C buffered medium contained witin a closed system (3). A concentration of NaHCO3 saturating for photosynthesis (4) was added to the cell suspension immediately after illumination with a 300-w reflector lamp (Sylvania) began. Irradiance at the surface of the vessel was 1200 x 10-5 w cm-2. Rates were corrected for consumption by the 02 electrode and expressed as Mmol 02/mg Chl -h or Mmol 02/106 cells h.Stock solutions of DIECA and disulfiram were made using 80% (v/v) ethanol and volumes of KCN, ethanol, DIECA, or disulfiram were dispensed with Hamilton syringes to give the concentrations indicated. RESULTS Measurements of net rates of H' efflux were initiated by the addition of 9 to 12 x 106 cells suspended in 3 ml unbuffered salt solution to 7 ml of similar medium. The resulting 10 ml cell suspension was stirred and aerated. The initial value for the net rate of H' efflux varied between 0.3 and 1.8 nmol H'/I06 cellsmin and the mean value expressed on a Chl basis was 31.6 nmol H'/mg Chl min. Disulfiram stimulated acidification with no apparent lag (Fig. 1). In the light, 0.4 mM disulfiram stimulated acidification to a mean value of 18 nmol H'/I06 cells min and the overall decline in pH was equivalent to 396 + 83 (SD) nmol H'. The molar ratio ofH+ appearing in the medium to disulfiram added was 0.1. In the dark, 0.4 mm disulfiram stimulated the acidification rate to 8.6 nmol H+/I06 cells-min and the pH decrease was equivalent to 210 + 64 (SD) nmol H+ (Fig. 1)