CONICET | Buscador de Institutos y Recursos Humanos

Studies on the putative symbiotic nature of C and K corpuscles of the midgut gland of several Pomacea species may be benefited by the obtainment of these corpuscles from gland homogenates in fairly enriched preparations. A method involving osmotic lysis of the gland cells and their organelles, and which is followed by sequential sedimentations of the osmotic resistant corpuscles, was developed. The procedure involved the following steps at 4 ºC: (1) tissue homogenization in ClNa 0.1% and 0.1% sodium azide; (2) centrifugation at 750 g for 10 min; (3) the precipitate was processed through a series of five resuspensions in saline-sodium azide solution, each followed by a period of benchtop sedimentation of both the resuspended precipitates and their corresponding supernatants; this gave, finally, 8 precipitates and 8 supernantants; (4) the precipitates were pooled and washed repeatedly to obtain fraction K; (5) the supernatants were pooled and centrifuged at 750 g for 5 min to obtain fraction C. Finally, both fractions were washed (thrice) in 10mM Tris and 1mM EDTA solution. Both fractions K and C were composed of corpuscles similar to those observed microscopically in tissue sections and in the feces. These resistant corpuscles may be lysed by different procedures, to solubilize and quantitate their contents. A 25 min incubation in 0.2 N sodium hydroxide gave a supernatant extract suitable for measuring DNA (bis-benzymide method of Labarca & Paigen) and proteins (method of Lowry). Such determinations made on lysates of C corpuscles from paper fed animals yielded a DNA concentration of 50.8 ± 9.2 mg/mg of protein (mean ± SEM; N=12) which is consistent with the putative symbiotic nature of C corpuscles. Similar measurements on K corpuscles yielded only 1.6 ± 0.4 mg/mg of protein (N=12), presumably explained by contamination of the K fraction with C corpuscles.

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