Both Class I (intact) and Class II (without the outer plastid membrane) chloroplasts of Spinacea oleracea exhibit a shrinkage of the thylakoid volume under conditions which lead to the well known light‐induced light scattering increases. In the present report this shrinkage has been measured on micrographs prepared by the freeze‐etch technique. In cloroplasts kept in darkness through the freezing or in those treated with DCMU prior to exposure to red light, the thylakoids are in a slightly swollen condition: in plastids exposed to red light and no inhibitor, the thylakoid membranes are closely appressed, giving the thylakoid a shrunken appearance relative to the control. It is further shown that Class I chloroplasts which are actively fixing CO2 do not give appreciable light scattering changes, but lowering the pH away from the optimum for ATP formation (and CO2 fixation) or adding the uncoupler quinacrine restores the light‐induced scattering increases.
Abstract. The photochemical activities of various species of unicellular algae (Anacystis nidulans, Chlorella pyrenoidosa, and Porphyridium cruentum) were studied following chemical fixation. Fixation with formaldehyde and glutaraldehyde yielded cells which retained their ability to perform photosystem I and photosystem II reactions. The photochemical efficiencies of some fixed algae are as great as those of unfixed spinach chloroplasts. Fixed algae containing accessory pigments appear to be useful models for further studies of the light reactions of photosynthesis.In 1966 Park et al. (14) reported that glutaraldehyde fixed Chlorella cells and chloroplasts isolated from glutaraldehyde fixed spinach leaves performed light dependent O2 production in 'the presence of suitajble electron acceptors. The quantum yields for photosystem II reactions of spinach chloroplasts from fixed leaves were about 25 % those of chloroplasts from unfixed leaves. At that time we fores!aw that this method might be a useful technique for studying photosystems I and II in algae containing accessory pigments. Such algal cells are generally impermeable to the components of reaction mixtures used to study photosystems I and II. We predicted that aldehyde fixation should remove this permeability barrier and circumvent many of the problems caused by cell breakage in these algae. Cell breakage is usually -accompanied by considerable loss of accessory pigment from thylakoid membranes (4,17).However in our initial efforts we failed to demonstrate Hill reaction either in glutaraldehyde fixed algae which contained accessory pigments or in spinach chloroplasts which were fixed after isolation.Both these obstacles are now removed. In this paper we report retention of both photosystems I and II
In 1969, Hallier and Park (5) showed that glutaraldehydefixed spinach thylakoids retained appreciable Hill reaction activity after extraction with 1% Triton X-100. The resistance of the fixed membranes to detergent destruction led them to suggest that such membranes might be favorable starting material for subsequent detergent fractionation studies. They postulated that cross-linking of membrane proteins would allow isolation of the larger membrane subunits identified by electron microscopy (8, 9). This paper reports an exploration of this suggestion. We investigated whether fixed membranes fragment differently from unfixed membranes when treated with various detergents and whether the fragments so liberated could be related morphologically to structures observed in the intact thylakoid.MATERUILS AND METHODS Preparation of Thylakoids. All experiments were performed on EDTA-washed thylakoids prepared from market spinach according to the method of Howell and Moudrianakis (6). The thylakoids collected from the final step of the Howell-Moudrianakis procedure were washed once with 0.01 M Na-borate buffer, pH 8.0, and once with 0.05 M borate and were finally suspended in 0.05 M borate. The thylakoids were examined by electron microscopy after negative staining with ammonium molybdate to ensure that they were devoid of ATPase particles (6). Freeze fracturing and deep etching of these thylakoids showed characteristic faces and surfaces (9).Fixation with Glutaraldehyde. The thylakoids washed with EDTA were fixed by passage through a sucrose density gradient containing 5 % glutaraldehyde. Solutions of sucrose in borate buffer were made to obtain densities of 1.28 g/cc and 1.1 g/cc. Another solution of sucrose in borate buffer containing 5% glutaraldehyde, purified as described by Hallier and Park (5), was made to a density of 1.05. The solutions were layered on the top of each other in a 32-ml centrifuge tube so that the tube contained 15 ml of solution with density 1.28, 10 ml of solution with density 1.10, and 5 ml of 5% glutaraldehyde solution with density 1.05. A suspension of thylakoids in 0.05 M borate was layered on top. This preparation was then centrifuged at 24,000 rpm for 20 min in an SW 25-1 rotor. The fixed thylakoids sediment to the boundary between the two sucrose layers. The green material was removed, washed once with 0.01 M borate and then with 0.05 M borate, pH 8.0, and was finally taken up in 0.05 M borate. The unfixed thylakoids were subjected to a similar density gradient centrifugation except that 5% glutaraldehyde was excluded from the 1.05 density solution.1 Supported by National Institute of General Medical Sciences Grant GM-13943-05.Treatment with Detergents. Fixed or unfixed thylakoids were taken up in 0.05 M borate. An equal volume of detergent solution was added to obtain a desired final detergent concentration. The solutions were incubated at 1 to 2 C for 30 min and then centrifuged at 10,000g or 20,000g for 30 min. The precipitate and supernatant fractions from such a separation were furthe...
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