Distribution of characteristic membrane proteins in granum and stroma thylakoids.

Membrane fractions obtained by ultrasonication of Vicia faba thylakoids were analyzed by sodium dodecyl sulphate gel electrophoresis. The polypeptide patterns revealed a six-times-higher ratio of the apoprotein (1) of P700 chlorophyll a protein to the apoprotein (2) of one of the chlorophyll a proteins in photosystem II in the light (stroma) fraction as compared with the heavy (grana) fraction indicating different distribution of the two photosystems. Additionally, the light fraction was clearly depleted in chlorophyll a/b apoproteins 2a and 2b of the light-harvesting complex and enriched in CF1 alpha and beta subunits. In contrast to the fairly constant ratio of the CF1 subunits the ratio of chlorophyll a/b apoprotein 2a/2b differed significantly between the light and heavy fraction suggesting a different composition for light-harvesting complex in stroma and grana regions of the thylakoids.

Resolution of the light-harvesting chlorophyll a/b-protein of vicia faba chloroplasts into two different chlorophyll-protein complexes.

Thylakoids of Vicia faba chloroplasts disaggregated by sodium dodecyl sulfate were separated by means of different electrophoretic systems. Under the conditions of a high resolving gel system the chlorophyll containing zone previously termed chlorophyll-protein complex II or light-harvesting chlorophyll a/b-protein was found to be inhomogeneous. It represents a mixture of two distinct chlorophyll-proteins characterized by different spectral properties and different apoproteins. One chlorophyll-protein exhibits a chlorophyll a/b ratio of 0.9 and is associated with polypetides of 24,000 and 23,000 daltons. The 24,000 dalton band is proved to bind chlorophyll and has a light-harvesting function. The function of the 23,000 dalton band is unknown. The second chlorophyll-protein has a chlorophyll a/b ratio of 2.1 and an additional absorption maximum in the position of 637 nm. It is associated with only one polypeptide which has an apparent molecular weight of 23,000. The two 23,000 dalton polypeptides occurring in both complexes are not identical.

The action of proteolytic enzymes on chloroplast thylakoid membranes.

Envelope- and stroma-free thylakoid membranes of Vicia faba chloroplasts were incubated with trypsin or pronase for several hours. The indigestible residue was analysed by polyacrylamide gel electrophoresis. Trypsinization resulted in a complete digestion of all proteins with the exception of the pigment-protein complexes as well as a polypeptide not yet characterized. Yet, as compared with untreated material, Complex II was found to have higher electrophoretic mobility. Electron-microscopic studies illustrate that the indigestible residue still has a preserved membrane structure. Disintegration of the thylakoid membranes by sodium dodecyl sulfate followed by trypsinization also resulted in the two complexes while all the other proteins were found to be digested. However, after removal of the lipids the protein moieties of the complexes proved to be easily digestible. From these results it is concluded that pigment-protein interaction may be an important factor in maintaining a conformation rather resistant to perturbants and proteases. In contrast to trypsin, pronase completely digested the polypeptides of the thylakoid membranes including the protein moieties of the pigment-protein complexes leaving an amorphous lipid mass. The results support the assumption that the complexes are necessary to maintain the membrane structure.

On the molecular nature of chloroplast thylakoid membranes.

Envelope- and stroma-free thylakoid membranes of Vicia faba chloroplasts were disintegrated and the electrophoretic behavior of the components studied with special regard to the pigment-protein complexes. The process of denaturation of the complexes was found to differ with respect to the other protein components. As the result of denaturation, the pigment-free protein moieties exhibit altered electrophoretic mobilities in relation to the intact complexes mainly conditioned by two processes contrary in their action, i.e. increase of change and change of the hydrodynamic properties. Exhaustive extraction of the thylakoid membranes with 6 M guanidine - HCl removes the proteins mainly associated by polar and weak hydrophobic interactions. The insoluble residue quantitatively exhibits the pigment-protein complexes including their denatured protein moieties, two extrinsic hydrophobic proteins as well as some protein traces. Electron-microscopic studies demonstrate the material still to have a high degree of order and preserved basic structure. After removing the lipids from the basic membrane, large amounts of the protein moeity of Complex II become soluble in guanidine -HCl. Since all other lamellar proteins are removable either by quanidine -HCl extraction or by trypsin digestion it is assumed the basic membrane of thylakoid to consist only of the pigment-protein complexes embedded into the lipid matrix.