Isolation, purification and partial characterization of chloragocytes from the earthworm species Lumbricus terrestris.
Chloragocytes were isolated from the earthworm species Lumbricus terrestris. After mechanical dissociation and sedimentation through Percoll, a highly purified fraction of viable chloragocytes was obtained. The isolated chloragocytes accumulated the vital dye neutral red and reduced the tetrazolium dye MTT, thereby indicating cellular integrity. Time of flight flow cytometric analyses revealed a main population of large and highly granulated cells in the 30-33 microm size range. Hydrolase measurements showed that beta-D-N-acetyl-glucosaminidase and acid phosphatase exhibited the highest activities (146.6 and 24.9 mU/mg of protein, respectively), possibly indicating a major role for these 2 hydrolases in the physiological function of chloragocytes. In contrast, other acid hydrolases such as beta-D-galactosidase and beta-D-glucuronidase had specific activities of respectively 26 and 182 times lower than the glucosaminidase. The specific activity of the membrane-bound alkaline phosphatase was comparable to that of its acid counterpart (18.9 vs. 24.9 mU/mg of protein, respectively) and this level of activity may show an important trans-membrane activity in chloragocytes. The cytoplasmic and mitochondrial enzyme isocitrate dehydrogenase had a level of activity comparable to that of the exclusively cytoplasmic enzyme lactate dehydrogenase (6.6 vs. 8.1 mIU/mg of protein, respectively). When L. terrestris chloragocyte homogenates were separated on Percoll, results showed that hydrolases and dehydrogenases were mainly associated with the lighter materials that remained above the Percoll layer. Nonetheless, the detection of significant proportions (15-25%) of the total recovered activity of acid phosphatase and beta-galactosidase in the enriched chloragosome fraction supports the notion that some chloragosomes may be 'lysosome-like' organelles.
Affar
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Health and Environment Unit, CHUL Research Centre, CHUQ, Ste-Foy, Québec, Canada.
Dufour
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Poirier
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GG
Nadeau
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D000818:Animals; D002478:Cells, Cultured; D004064:Digestive System / Q000737:chemistry / Q000166:cytology*; D004798:Enzymes / Q000378:metabolism; D005434:Flow Cytometry; D008247:Lysosomes / Q000201:enzymology; D009835:Oligochaeta / Q000737:chemistry / Q000166:cytology*; D013347:Subcellular Fractions / Q000201:enzymology
D004798:Enzymes