Function of the "quantasome" in photosynthesis: structure and properties of membrane-bound particle active in the dark reactions of photophosphorylation.

Membrane-bound particles called "quantasomes"' are found in shadowed2 or negatively stained3 chloroplast preparations examined in the electron microscope. The membrane-bound particles were considered to be integral components of the chloroplast electron transport system4 and were interpreted as morphological counterparts of photosynthetic units.5 Recently, it was demonstrated that the quantasome particles do not participate in photoreduction reactioins. ' This statement is based on the observation that chloroplast membrane preparations treated with dilute ethylenediaminetetraacetate (EDTA) solutions gave particle-free membrane fractions showing undiminished rates of photoreduction relative to unextracted controls. In addition, the EDTA treatment of the chloroplast membranes solubilized a noinpigmented protein species, morphologically identical to the quantasome. Several laboratories6-8 have already reported that EDTA extraction of chloroplast membranes causes an uncoupling of photophosphorylation from electron transport, and that the supernatant solutions obtained in these extractions contain a "coupling factor" and a Ca++-dependent adenosine triphosphatase (ATPase) activity. It was, therefore, the purpose of this study to determine if the isolated particle, separated from the membranes with EDTA, and complying with the description of the quantasome,4 was capable of ATPase activity and of reconstituting photophosphorylation in uncoupled chloroplast mnembrane preparations. To this end, the isolated quantasomes were separated and morphologically distinguished from the more abundant particulate species, which contains carboxydismutase. The quantasome particle was purified from other chloroplast phosphorylation activities and characterizedl in terms of its sedimentation coefficient, substructure, and enzymatic activities. The evidence presented below demonstrates that the purified quantasome particle functions in the dark stage of photophosphorylatiilg events. Materials and Methods. -Exceptwhere indicated, all procedures were carried out at 1 ? C. Chloroplast membrane preparations were handled under dim light conditions. Isolation of chloroplast fractions: Chloroplasts were isolated from 750 gm of spinach leaves by a method modified from that of Hind and Jagendorf.9 The chloroplasts were extracted with distilled water and subsequently with 1 mM EDTA, as described elsewhere. " 18 Water and EDTA-wash supernatant solutions were adjusted to 5 mM dithiothreitol (DTT) and 10 mM Tris-SO4 (pH 8.0) by dropwise addition of 50 mM buffer. The supernatant solutions were cleared of membranes by centrifugation at 105,000 g for 30 min. Each supernatant solution was brought to 60% saturation by slowly adding solid (NH4)2SO4 and centrifuged at 105,000 g for 15 min. The pellets were suspended in and dialyzed against several changes of fresh DTT, Tris-SO4 buffer. Unsuspended material was removed by centrifugation at 144,000 g for 15 min and discarded. Approximately 2-mg aliquots of the dialyzed protein samples were layered on 25 ml sucrose gradients (5-20%) in DTT, Tris-S04 buffer. The gradients were centrifuged in a SW-25 rotor at