Loss of sensitivity to diuron after trypsin digestion of chloroplast photosystem II particles

Croze, Ed; Kelly, Mark; Horton, Peter

doi:10.1016/0014-5793(79)81242-9

Cited by 34 publications

(15 citation statements)

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“…It was inhibited by 0.05% Triton X-100 which is in accord with our previous results con- 47 25 cerning the effect of Triton X-100 on the photochemistry of PSII ferricyanide (24). In addition, Horton and Croze (5) found that trypsin completely abolished DCMU inhibition in PSII membrane fragments (TSF II particles). We find that our PSII particles as SF-IIa particles on SDS gels isolated have a biphasic response to DCMU (Fig.…”

Section: Resultssupporting

confidence: 80%

“…As can be seen, the PSII particles are remarkably stable to proteolytic digestion. At a concentration of 5 ,ug/ml (Chl:protease ratio of 10), Even at higher concentrations of the proteases, up to 100 ,jg/ml (Chl:protease ratio of 0.5), the activity was inhibited only 40% compared to an untreated sample. We also determined the activity as a function of time of incubation (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Proteolytic enzymes have been used extensively to study the localization of thylakoid membrane proteins, particularly the Chlprotein complexes (3,5,18,(21)(22)(23) tochemical properties altered. These authors suggest that the Chl present in the complexes stabilizes the structure of the complex.…”

Section: Introductionmentioning

confidence: 99%

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Effects of Proteolysis on the Photochemical Activity and Polypeptide Composition of the Photosystem II Core Complex Isolated from Spinach Chloroplasts

Laszlo¹,

Gross²

1981

Plant Physiol.

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The photosystem II core complex (TSF-IIa) is composed of pobpeptides of molecular weight 54-, 47-, 42-, and 30 kiodaltons (kD) and cytochrome b-559. After treatment with trypin or a-chymotrypsin for 20 hours, the TSF-IIa particles stfll retained their photochemical activity and the Lighti d cytochrome b-559 siga, althugh all of the polypeptides of the complexes, except the 30 kD unit were extensively degraded. Proteolytic treatment decreased the apparent molecular weight of the complex fom 250,000 to 100,000 daltons as determined by gel filtratdon, and also decreased the protein to chloophyll ratio by 40%. Chlorophyll a appeared to be assocated with the 47-and 42 kD pobpeptides. Proteolysis of the complex produced a single chlophyll a band with a slightly higher electrophoretic mobility. This band was not eqivalent to the 30 kD polypeptide. Proteolysis also reduced the sensitivity of the TSF-IIa particles to 3-(Y,-4khlorophenyl)-1,1-dmethylurea (DCMU), but did not completely abolish it.It was conchlded that altbough the polypeptides of the photosystem II core complex were cleaved by the proteases, many of the peptide fragments do not diciate from the complex, particularly the chlorophyll a-containing portions, leaving the complex photochemically active. This supports the concept that chlorophyll has a structural function in chlorophyll-protein complexes.Proteolytic enzymes have been used extensively to study the localization of thylakoid membrane proteins, particularly the Chlprotein complexes (3,5,18,(21)(22)(23) tochemical properties altered. These authors suggest that the Chl present in the complexes stabilizes the structure of the complex. Unfortunately, their observations were not extended to include the effects of proteolysis on PSII. The present study describes the effects of trypsin and a-chymotrypsin on the photochemical activity and structure of the isolated PSII core complex. At low concentrations of either protease, the photochemical activity ofthe complex was not inhibited; however, the component polypeptides were extensively degraded. This suggests that the reaction center polypeptide of PSII was cleaved during proteolysis but the peptide fragments, possibly held in place by their associated Chl, did not dissociate from the complex.MATERIALS AND METHODS Preparation of TSF-IIa Particles. The initial purification procedures followed those of Vernon et al. (26) with some modifications. Spinach thylakoids prepared in 50 mm Tris-Cl (pH 7.6) containing 350 mm sucrose, were twice treated with 2.0 M NaBr as described by Nelson (12) to remove coupling factor, and then washed with the above buffer. The lamellae were resuspended in 50 mm Tris-Cl (pH 8.0) and 0.5 M sucrose at a concentration of 2.5 mg Chl/ml. Triton X-100 was added to give a final ratio of 40 mg Chl:l g Triton X-100. The solution was stirred on ice for 1 h after which it was subjected to a low speed centrifugation (5,000g, 5 min.) to remove debris. The supernatant was centrifuged at 144,000g for 1 h (2 C) and the resultant supernatan...

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Section: Resultssupporting

confidence: 80%

Section: Resultsmentioning

confidence: 99%

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Effects of Proteolysis on the Photochemical Activity and Polypeptide Composition of the Photosystem II Core Complex Isolated from Spinach Chloroplasts

Laszlo¹,

Gross²

1981

Plant Physiol.

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“…Its photoregulated synthesis (2,8), apparent rapid turnover, and continued synthesis throughout all stages ofleafdevelopment (2, 3, 9) suggest a regulatory role for the protein product in PS II function. Purified PS II complexes isolated by detergent fractionation techniques contain a protein component of 32 kDal (10,11). Certain mutants ofZ.…”

mentioning

confidence: 99%

“…nays lacking PSII function are deficient in a 32-kDal membrane polypeptide (12). Trypsin treatment of thylakoid membranes (13) or of isolated PS II complexes (10) results in the degradation of the 32-kDal polypeptide with concomitant loss of PS II activity. These lines of evidence also suggest that the 32-kDal polypeptide plays an integral role in PS II electron transport.…”

mentioning

confidence: 99%

Identification of the triazine receptor protein as a chloroplast gene product

Steinback

McIntosh

Bogorad

et al. 1981

Proc. Natl. Acad. Sci. U.S.A.

161

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The triazine herbicides inhibit photosynthesis by blocking electron transport at the second stable electron acceptor of photosystem H. This electron transport component of chloroplast thylakoid membranes is a protein-plastoquinone complex termed "B." The polypeptide that is believed to be a component of the B complex has recently been identified as a 32-to 34-kllodalton polypeptide by using a photoaffinity labeling probe, azido-[14Clatrazine. A 34-kilodalton polypeptide ofpea chloroplasts rapidly incorporates [3S]methionine in vivo and is also a rapidly labeled product of chloroplast-directed protein synthesis. roplasts results in-identical, sequential alterations of the 34-kldodalton polypeptide to species of 32, then 18 and 16 kldodaltons. From the identical pattern ofsusceptibility to trypsin we conclude that the rapidly synthesized 34-kilodalton polypeptide that is a product ofchloroplast-directed protein synthesis is identical to the triazine herbicide-binding protein ofphotosystem II. Chloroplasts of both triazine-susceptible and triazine-resistant biotypes of Amaranthus hybridus synthesize the 34-kilodalton polypeptide, but that of the resistant biotype does not bind the herbicide. When dark-grown seedlings are transferred to light, a sequence of developmental processes leads to the formation of green, photosynthetically competent chloroplasts (1). Rapid accumulation of a major thylakoid protein of 32 kilodaltons (kDal) parallels the appearance offunctional activity (2). In Zea mays, this 32-kDal protein is structurally related to, and is presumably derived from, a 34.5-kDal polypeptide that is the major membrane-bound product of protein synthesis by isolated chloroplasts (3). It has been shown to be encoded by a chloroplast gene in Z. mays (4). A number ofchloroplast-encoded proteins appear to be synthesized in etioplasts of dark-grown maize seedlings (2), but the transcription of the chloroplast gene coding for this polypeptide is light dependent in developing plastids and has thus been described as a "photogene" (5, 6). Until recently, no function had been determined for the photogene 32 product; it has been identified now as a determinant of the electron transport function of a bound plastoquinone molecule in the photosystem II (PS II) complex (7). Its photoregulated synthesis (2, 8), apparent rapid turnover, and continued synthesis throughout all stages ofleafdevelopment (2, 3, 9) suggest a regulatory role for the protein product in PS II function. Purified PS II complexes isolated by detergent fractionation techniques contain a protein component of 32 kDal (10, 11). Certain mutants ofZ. nays lacking PSII function are deficient in a 32-kDal membrane polypeptide (12). Trypsin treatment of thylakoid membranes (13) or of isolated PS II complexes (10) results in the degradation of the 32-kDal polypeptide with concomitant loss of PS II activity. These lines of evidence also suggest that the 32-kDal polypeptide plays an integral role in PS II electron transport.A broad range of inhibitors of photo...

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Control of Photosynthetic Electron Transfer from the Reaction Center to Electron Carriers of Photosystem II Studied by Fluorescence Induction

Malkin

1981

Israel Journal of Chemistry

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Abstract. The relationships between the primary electron acceptor (0) and the secondary pool (AI) are examined from fluorescence induction curves, at various illumination times and relaxation dark times. The fast-phase in the fluorescence rise curve, obtained after insufficient relaxation time, indicates the occurrence of "unconnected" 0, i.e. the reaction between 0-and AI is blocked. Various treatments bring about similar disconnection: high pH, ethylene glycol, low temperature (all reversible), trypsin and reduction of A I by dithionite. All these treatments are similar to DCMU treatment. A scheme is suggested in which 0 is mostly unconnected in the dark and becomes immediately connected as it is reduced by light. The above treatments inhibit the reconnection reaction in the light. The thermodynamic equilibrium between the 0 populations that can or cannot be reconnected was analyzed from the fluorescence curves at various temperatures. The connection-disconnection switching reaction is probably controlled by conformational changes of an associated protein. This scheme explains the difference of apparent redox equilibria of 0 and A in the light or in the dark, as well as the lOP wave in the fluorescence induction of intact cells.

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Loss of sensitivity to diuron after trypsin digestion of chloroplast photosystem II particles

Cited by 34 publications

References 20 publications

Effects of Proteolysis on the Photochemical Activity and Polypeptide Composition of the Photosystem II Core Complex Isolated from Spinach Chloroplasts

Effects of Proteolysis on the Photochemical Activity and Polypeptide Composition of the Photosystem II Core Complex Isolated from Spinach Chloroplasts

Identification of the triazine receptor protein as a chloroplast gene product

Control of Photosynthetic Electron Transfer from the Reaction Center to Electron Carriers of Photosystem II Studied by Fluorescence Induction

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