Cell homogenates of Pleurochrysis sp. (CCMP299) were fractionated by means of sucrose gradients. Ca2+‐stimulated ATPase (EC 3.6.1.3., ATP phosphohydrolase) was associated primarily with the plasma membrane, Golgi, and high density (1.21 g·cm−3) membranous structures. Ca2+‐stimulated ATPase was highly enriched in the latter. Based on treatments with Triton X‐100 and NBD ceramide, we conclude that the high‐density structures were membrane‐delimited organelles. These vesicle‐like organelles contained complex polysaccharides, a high concentration of calcium, and, upon microscopic examination, structures resembling coccoliths. These findings are consistent with observations on the known composition of coccoliths and the presumed mineralizing function of the sub‐cellular coccolith‐producing compartment. The high‐density vesicles were linked to the Golgi by means of colchicine‐sensitive materials, presumably microtubules. These data and prior ultrastructural observations by other investigators indicating vectorial assembly and secretion suggest that the subcellular movement of the newly formed coccoliths may be directed and/or powered by colchicine‐sensitive cytoskeletal elements. We interpret the data to mean that the high‐density vesicles represent the coccolith‐producing compartment previously observed by others in electron micrographs.
Cells of the marine alga Coccolithus pelagicus (Wal‐lich)J. Schiller grown in axenic cultures were homogenized and fractionated. The distribution of organelle markers was assessed enzymatically after centrifugation through zonal, density, and flotation gradients made with sucrose, sorbitol, or Percoll. Mitochondria (1.19 g·cm‐3) and chloroplasts (1.15 g·cm‐3) were recovered in sucrose gradients at densities similar to those observed for higher plants and most algae. The position of endoplasmic reticulum and plasma membrane in the gradients was monitored by NADPH cytochrome c reductase and vanadate‐sensitive Mg2+‐ATPase, respectively. Higher plant Golgi markers, latent undine diphosphatase (UDPase) and glucan synthase I, were colocalized at a density range including two peaks of activity at 1.13–1.15 g·cm‐3. Bound calcium was associated with high density (1.15 g·cm‐3) membranes. Ca2+‐stimulated ATPase was found at high levels on membranes that did not coisolate with the latent UDPase‐containing membranes. The Ca2+‐stimulated ATPase, a possible participant during calcification, was associated with a chloroplast‐enriched fraction in all the organelle separation systems. However, about 30% of the total activity was separated from both the chloroplasts and Golgi on 0–70% Percoll gradients containing 0.4 M sucrose. The possible relationship of the Golgi and the high‐density organelle exhibiting Ca2+‐stimulated ATPase to coccolithogenesis and the process of calcification and crystal formation is discussed.
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