Roderic B. Park

Characterization of chloroplast photosystems 1 and 2 separated by a non-detergent method

Abstract Class II spinach chloroplasts were fragmented by passage through the French pressure cell (French press), and the fragments were separated by fractional centrifugation. Fragments sedimenting between 1000 × g and 10000 × g (10K) have a lower chlorophylla/chloropyllb ratio and lower P-700 content than whole chloroplasts. Fragments sedimenting between 40000 × g and 160000 × g (160K) have a much higher chlorophylla/chlorophyllb ratio (6.0) and a much higher P-700 content (1 P-700 per 105 chlorophylls) than whole chloroplasts. The chlorophyll and cytochrome contents of the French press fractions are similar to those found in fractions isolated after digitonin disruption. The 160K fraction performs Photosystem 1 but not Photosystem 2 reactions. The 10K fraction contains both photosystems. Electrophoresis of sodium dodecyl sulfate solubilized 10K and 160K fractions gives further evidence for this distribution of photosystems. Thin sectioning and freeze fracturing show that the 160K fraction originates from stroma lamellae and the end membranes of grana stacks and contains only 110Aparticles. The 10K fraction originates from the partition regions of grana stacks and contains both 110 and 175Aparticles. This distribution of particles on fracture faces of stroma versus grana lamellae is shown to exist in freeze fractured Class I chloroplasts. These data demonstrate that both digitonin and French press treatments of chloroplasts initially break stroma lamellae and end membranes to yield small vesicles which contain only Photosystem 1.

Correlation of structure with function in Spinacea oleracea chloroplasts

Spinach chloroplasts, isolated in isotonic sucrose medium, were sonically ruptured and separated by differential centrifugation into three main fractions: a green precipitate, a colourless supernatant and a yellow, low density lipid-containing layer. These fractions were analyzed for their physical, chemical and biochemical properties. Comparison of a thin section of an isolated osmium-stained chloroplast with a thin section of osmium-stained green precipitate, by electron microscopy, shows that the particles in the green precipitate correspond both in thickness and lateral dimensions to the lamellar structure of the isolated chloroplast. Heavy metal shadowing of both air-dried and lyophilized green precipitate demonstrates the presence of lamellar structures from 10,000 to 20,000 A in diameter. The lamellar structure, totaling 160 A in thickness, is composed of two layers; each layer is made up of granular subunits 100 A thick. Chlorophyll to nitrogen weight ratios are fairly constant for green lamellar structures from 800 to 20,000 A in diameter, indicating that chlorophyll is uniformly distributed throughout the lamellar structure of the chloroplast. The similarity of relative photosynthetic pigment concentrations over the visible region, of the Hill reaction activity and of the carbon dioxide fixation capacities for the various sized particles in the green precipitate suggests that the smallest lamellar fragments used in these experiments are large in comparison with the smallest fragment which converts electromagnetic to chemical energy. A model for chloroplast lamellar structure is proposed on the basis of the results obtained. The supernatant, by electron microscopy, is seen to consist principally of oblately spherical particles, 100 A thick and 200 A in diameter. These particles are indistinguishable from the main water-soluble protein of the chloroplast with sedimentation coefficient 16 (Fraction I protein). About 90% of the total carboxydismutase activity is associated with the supernatant proteins. In addition, these proteins are able to convert fructose-6-phosphate into other photosynthetic carbon cycle intermediates. This supernatant must be added to the green precipitate in order to obtain maximum carbon dioxide fixation rates. Electron micrographs of unfractionated osmium-fixed chloroplast sonicate show large osmium-stained spherical objects corresponding in size and staining properties to the osmiophyllic granules observed in thin sections of osmiumstained chloroplast in the intact leaf. Similar granules, 500 to 1500 A in diameter, were obtained when the yellow, low density lipid layer was fixed with osmium and then shadowed.

Subunits in chloroplast lamellae.

The structure of chlorophyll-containing lamellae has been studied in freeze-etched preparations of isolated and in situ chloroplasts. These lamellae are composed of at least two classes of subunits. The first type of subunit has an average diameter of 175 A and is not less than 90 A high. It corresponds in size and distribution to the quantasome. The second type of subunit has an average diameter of 110 A and appears to form part of an embedding matrix around the larger, 175 A, units. Thus, chloroplast lamellae consist of a matrix within which are densely packed subunits that form the major structural constituents of the photosynthetic membrane.

Quantasome: Size and Composition

The quantasome as seen in a two-dimensional crystalline array is 185 � long, 155 � wide, and 100 � thick. The surface of the quantasome appears to contain four or more subunits. The molecular weight, determined from volume and density measurements, is 2 x 106 This is twice the minimum molecular weight calculated from the manganese content and corresponds to a chlorophyll content of 230 chlorophyll molecules per quantasome.

Chemical composition and the substructure of lamellae isolated from Spinacea oleracea chloroplasts

Purified chlorophyll-containing lamellae isolated from Spinacea oleracea chloroplasts were active in the Hill reaction. Spectrochemical and chemical analyses established the ratios of protein, lipid, organic phosphorus, iron, manganese, copper and chlorophyll in the lamellae. A minimum molecular weight for a lamellar subunit on the basis of manganese content is 960,000. Electron microscopy reveals particles which correspond to a unit of this size within the lamellae. The particulate nature of the lamellae is more pronounced after lipid removal. This observation supports the view that the lamellae consist of proteins embedded in a lipid matrix.

Further evidence for stroma lamellae as a source of Photosystem 1 fractions from spinach chloroplasts

The mechanism of digitonin action on spinach chloroplasts was investigated by thin sectioning. Evidence is presented which shows that digitonin continues to modify membranes for many minutes after the addition of the fixative glutaraldehyde. However, the action of digitonin can be stopped by simultaneous fixation and dilution of the detergent. Such experiments indicate that the initial action of digitonin is to release stroma lamellae which in turn yield a Photosystem 1 fraction. This interpretation is further supported by a significant correlation between the chlorophyll achlorophyll b ratio and the ratio of stroma to grana lamellae in spinach chloroplasts.

Characterization of three new chlorophyll-protein complexes.

Abstract Three new chlorophyll-proteins with electrophoretic mobilities intermediate between those of the P700 chl a-protein and the light-harvesting chl a,b-protein complexes are reported and their absorption spectra and polypeptide composition are characterized. Two of these chlorophyll-proteins, bands IIb and IIa, contain approximately equal amounts of chl a and b, have polypeptide compositions similar to that of the light-harvesting chl a,b-protein and probably represent oligomers of the latter complex. The third new chlorophyll-protein contains only chl a and its major polypeptide(s) is in the 42 kd region. Indirect evidence indicates this chlorophyll-protein is associated with the reaction-center of photosystem II.

Chlorophyll, glycerolipid and protein ratios in spinach chloroplast grana and stroma lamellae☆

Abstract The relative molar amounts of glycerolipids are similar in grana and stroma lamellae, as are the ratios of total glycerolipid to weight of membrane protein. However the chlorophyll content relative to protein of grana lamellae is about 40% higher than that of stroma lamellae from the same preparation. Previous reports of chemical composition or enzyme activity based on chlorophyll alone can be highly misleading. The large functional and conformational differences between these two membranes may be related to these differences in pigment content, but are likely to result primarily from qualitative protein differences. The data are in accord with a membrane model in which nonpolar regions of membrane protein bind lipid in fairly constant amounts.

EXCITATION TRANSFER BY CHLOROPHYLL a IN MONOLAYERS AND THE INTERACTION WITH CHLOROPLAST GLYCOLIPIDS

In mixed monolayers with purified chloroplast glycolipids and other colorless lipids, chlorophyll a fluorescence exhibits a decrease in quantum efficiency with increasing chlorophyll concentration. The fluorescence, which is strongly polarized in dilute films, becomes progressively depolarized as the area fraction of chlorophyll increases, and it is completely depolarized in a pure chlorophyll a monolayer. The observed behavior is consistent with an inductive resonance mechanism of energy transfer among the chlorophyll molecules with a critical transfer distance of 20–90 Å, depending on the model chosen for the energy transfer mechanism.

Compositional characteristics of a chloroform/methanol soluble protein fraction from spinach chloroplast membranes

Extraction of an aqueous suspension of spinach chloroplast lamellae with a chloroform/methanol mixture leads to solubilization of about 1/3 of the total membrane protein. Amino acid analysis of the chloroform/methanol-soluble protein shows that this fraction is largely enriched in the hydrophobic residues proline, leucine, alanine and phenylalanine and considerably depleted in polar amino acids, namely lysine and arginine. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the solubilized material reveals the presence of a variety of low molecular weight polypeptides (molecular weight less than or equal to 25 000), with more than 50% of the total fraction being contributed by a 25 000 dalton band. This band, which accounts for about 25% of the total chloroplast lamellar protein, has recently been identified as the main component of the light-harvesting chlorophyll-protein complex. The physiological role of most of the chloroform/methanol-soluble protein fraction is not known at present. From its chemical properties and apparent biological inertness, we propose that it plays mainly a structural role in situ, interacting with the lipid moiety of the chloroplast membrane. The material insoluble in the aqueous chloroform/methanol mixture is largely enriched in manganese, iron, cytochrome and water-soluble proteins, such as chloroplast coupling factor and ribulose diphosphate carboxylase.