Studies on the Functional Mechanism of System II Herbicides in Isolated Chloroplasts

Abstract

The effect of specific proteolytic enzymes on variable fluorescence, p-benzoquinone-mediated oxygen evolution, PS II herbicide (atrazine and bromoxynil) binding, and protein degradation has been analyzed in isolated class II pea chloroplasts. It was found that: 1. Trypsin and a lysine-specific protease effectively reduce the maximum chlorophyll-a fluorescence yield, whereas the initial fluorescence remains almost constant. At the same number of enzymatic activity units both proteases have practically the same effect. 2. Trypsin and a lysine-specific protease inhibit the p-benzoquinone-mediated flash-induced oxygen evolution with trypsin being markedly more effective at the same number of activity units of both enzymes. Unstacked thylakoids exhibit a higher sensitivity to proteolytic degradation by both enzymes. 3. Trypsin and a lysine-specific protease reduce the binding capacity of [14C]atrazine, but enhance that of [14C]bromoxynil (at long incubation times trypsin treatment also impairs bromoxynil binding). At the same specific activity a markedly longer treatment is required for the lysine-specific protease in order to achieve the same degree of modification as with trypsin. 4. Trypsin was found to attack the rapidly-turned-over 32 kDa-protein severely, whereas the lysine-specific protease does not modify this polypeptide. On the other hand, the lysine-specific protease attacks the light harvesting complex II. 5. Under our experimental conditions an arginine-specific protease did not affect chlorophyll-a fluorescence yield, p-benzoquinone-mediated oxygen evolution, herbicide binding and the poly- peptide pattern. Based on these results a mechanism is proposed in which an as yet unidentified polypeptide with exposable lysine residues, as well as the lysine-free “QB-protein” regulate the electron transfer from Q-A to QB and are involved in herbicide binding.

Changes in the binding and inhibitory properties of urea/triazine-type herbicides upon phospholipid and galactolipid depletion in the outer monolayer of thylakoid membranes

“…3A) is indicative of a positive cooperative process. This may be related: i) to the relatively higher lipophilicity of DCMU compared to that of atrazine (and hence to different local concentration of herbicide), as reported recently (Neumann et al 1987); ii) to the proposal made by Gardner (1989) who suggested a stereochemical model for the active site of Photosystem II herbicides in which the plastoquinone molecule is better matched by DCMU than by atrazine; iii) to a more drastic allosteric conformational change of the 32 kDa-HB polypeptide, induced by atrazine compared to DCMU, that may hide binding sites for subsequent inhibitor binding (Vermaas et al 1984, Renger et al 1984). Such a pattern is expressed, in the double reciprocal plot (Fig.…”

“…3A. Another way of plotting binding data, the double reciprocal representation, has often been used in the literature (Tischer and Strotmann 1977, Oettmeier et al 1982a, Laasch et al 1982, Oettmeier et al 1982b, Vermaas et al 1983, Thiel and B6ger 1984, Renger et al 1984, Neumann et al 1984, Herrmann et al 1984), but has the disadvantage of compressing the data points of high herbicide concentrations into a narrow region and emphasizing the points at lower concentrations (Fersht 1977). The shape of this non-linear phase suggests that below 0.04/xM, both herbicides bind in a cooperative way (i.e., below the threshold of 0.04 ~M, the binding of herbicide molecules increases the affinity for subsequent herbicide binding).…”

“…Whether the reduced affinity of DCMU in the R-biotype is due to a conformational change (Renger et al 1984) or to the absence of an hydroxyl group (Sigematsu et al 1989) induced by the glycine 264 mutation in the 32 kDa-HB polypeptide cannot be decided now. Whether the reduced affinity of DCMU in the R-biotype is due to a conformational change (Renger et al 1984) or to the absence of an hydroxyl group (Sigematsu et al 1989) induced by the glycine 264 mutation in the 32 kDa-HB polypeptide cannot be decided now.…”

EPR measurements on the effects of bicarbonate and triazine resistance on the acceptor side of Photosystem II

“…do not interact with the protein(s) to which QA, Fe'+ and Pheo are bound: herbicides are able to change the EPR signals of Qi -Fe2+ and the split Pheo-signal in the presence of QA -Fe2+ [6,7]. This confirms the notion that more proteins than the ABP-32 only are involved in herbicide binding [19,26].…”

The azido[14C]atrazine photoaffinity technique labels a 34‐kDa protein in Scenedesmus which functions on the oxidizing side of photosystem II

“…For example, Oettmeier et al [12] suggested that a photoaffinity analog of plastoquinone preferentially labels a membrane protein in the 30-34-kDa range which is different from that tagged by azido[14C]atrazine. Renger et al [13] presented data which implicate a protein with exposed lysine residues as affecting herbicide binding (nucleotide sequence data indicate the QBprotein has no lysine residues [4,6,29]). Also it was shown that azidoatrazine labels the L subunit of Rhodopseudomonas sphaeroides reaction centers [9] even though immunological data had indicated that the M and H subunits are involved in Qn binding [33].…”