Interaction of Nitroxide Spin Labels with Chloroplasts

Briggs, Steven P.; Haug, A.; Scheffer, R. P.

doi:10.1104/pp.70.3.668

Cited by 3 publications

(3 citation statements)

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“…However, ascorbate caused no significant reduction of PC (5,10). The results with PC(5, 10) are consistent with those of Rousselet et al (21) who found that 1 h was required to completely reduce PC (10,3) in the outer leaflet of erythrocyte membranes, but that 4 h were required for comparable reduction of PC (7,6). PC(m,n) spin labels are probably more resistant to ascorbate reduction than are I(m,n) spin labels because PC(m,n) labels have greater stability in the membrane; I(m,n) spin labels may orient in bilayers such that their nitroxyl and carboxyl groups are at the membrane surface (9) facilitating interaction with ascorbate.…”

Section: Discussionsupporting

confidence: 82%

“…Oxidation seems to take place within chloroplast membranes but only if the protoplasts are intact; isolated chloroplasts reduce spin label in the light (6).…”

Section: Discussionmentioning

confidence: 99%

“…Protoplasts which were precooled to 0°C were resistant to mechanical lysis. Therefore, pro- plasts may be the site of light-mediated oxidation of spin label within protoplasts (6). Therefore, oxidation of spin label was used as an assay of spin label location.…”

mentioning

confidence: 99%

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Localization of Spin Labels in Oat Leaf Protoplasts

Briggs

Haug²,

Scheffer³

1982

Plant Physiol.

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An assay based on light-mediated oxidation was used to determine whether specific spin labels were partitioned throughout the protoplast or retained in the plasmalemma of Avena sativa L. cv. Garry and Park. Many classes of spin label were tested, including phospholipids, fatty acid, fatty acid methyl ester, maleimide, iodoacetamide, short chain hydrocarbon, androstane, 2,2,6,6-tetramethyl-4-aminopiperidinooxyl (TEMPAMINE) and 2,2,6,6-tetramethylpiperdinooxyl (TEMPO). All except the phosphotidylcholine spin label were found to partition throughout the cell. The phosphotidylcholine spin label may have been selectdvely retained in the plasmalemma.Changes in the plasma membranes of higher plants have been proposed as the basis of many cellular phenomena. Examples are development of cold tolerance, salt tolerance, and senescence. Also, changes in membrane structure may result from exposure to hormones, pathogens, radiation, and pressure. Usually, membrane structure has been studied with fixed tissues or with isolated membranes, precluding direct measurement of membrane changes in living cells.Spin labels have been used to study the plasma membrane of living cells. However, to selectively probe the plasmalemma of an intact protoplast, the probe must be reporting only from the plasmalemma. This condition can be met if the probe is only present in the plasmalemma or is spectroscopically inactive everywhere except in the plasmalemma. Kaplan et al. (14) claimed that by adding K3Fe(CN)6 (a spin label-activating agent) to a labeled cell suspension, only the spin-label in the plasma membrane gave an ESR signal. The assumptions that spin labels cannot move quickly between membranes and that different membranes would create different spin label spectra are also used to support the contention that fatty acid spin labels are good probes for the plasma membrane (2-4, 7, 10, 13, 24, 25). However, some investigators dispute these assumptions (12, 16), arguing that most spin labels report an average from every membrane in the cell.The objectives of this report were to: (a) evaluate whether or not fatty acid spin labels are selective for the plasmalemma of plant protoplasts; (b) develop criteria for ascertaining the selectivity of a spin label for the plasmalemma; and (c) test various classes of spin label and identify those which may have selectivity for the plasmalemma. MATERIALS AND METHODSAvena sativa L. cv. Garry and Park were grown in the laboratory at 22°C in vermiculite irrigated with White's nutrient solution (8). Fluorescent and incandescent bulbs provided a 16-h photoperiod. Primary leaves were harvested from 1-to 2-week-old seedlings.Protoplasts were isolated by peeling away the lower epidermis of the leaves with forceps and floating the leaves (peeled surface down) on a solution composed 6f 0.5% Cellulysin (CalbiochemBehring Corp.) and 0.6 M sorbitol, adjusted to pH 5.6 with KOH. The preparation was incubated at 28°C for 3 h in the light, then was swirled gently to release protoplasts. The protoplasts were fi...

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Section: Discussionsupporting

confidence: 82%

“…Oxidation seems to take place within chloroplast membranes but only if the protoplasts are intact; isolated chloroplasts reduce spin label in the light (6).…”

Section: Discussionmentioning

confidence: 99%