Crassin acetate was first isolated by Ciereszko, Sifford and Weinheimer (1960), from the whole dried gorgonian Pseudoplexaura porosa (Houttuyn). Ciereszko (1962), later reported that this diterpene lactone was present in very high concentration in the symbiotic zooxanthellae isolated from this gorgonian. We have been studying sesquiterpene hydrocarbon biosynthesis in zooxanthellae isolated from P. porosa. A major difficulty in isolating pure zooxanthellae is in separating them from a large volume of colorless micro-crystals. The objectives of the present study were to check the identity of the crystals with crassin acetate, to eliminate the possibility that their extracellular occurrence is an artifact of the isolation procedure, to determine the histological relationship of the crystals, the zooxanthellae, and the host tissue, and to study the metabolism of crystalline crassin acetate.
MATERIALS AND METHODSIsolation of crassin acetate crystals from Pseudoplexaura porosa P. porosa was collected from the coral patch reefs known as Bache Shoal just off Elliott Key, Florida, transported in sea water, and processed immediately on return to land.Segments of gorgonian 3 to 4 cm long were homogenized in a Waring Blendor at full speed for 10 seconds with approximately twice their volume of sterile 50 per cent sea water, a medium which was used throughout this preparation. The medium was sterilized by passage through a 0.45 //, pore size Millipore filter. Large fragments and gorgonian stipes were removed by a single passage through cheese cloth. After the strained homogenate was centrifuged at 3000 g in 250 ml centrifuge bottles for 15 minutes, the supernatant was discarded and the buffcolored surface layer was removed by gentle swirling with medium. The remainder of the pellet was resuspended in a 20-fold volume of medium and centrimgation and washing was repeated two more times.
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