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J Gierow, SV Andersson, EC Sjögren; Regulated Secretion and Subcellular Distribution of Rabbit Lacrimal Gland alpha-Fucosidases . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3125.
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© ARVO (1962-2015); The Authors (2016-present)
Purpose: Our previous results have indicated the presence of two forms of alpha-fucosidase, one acidic and one neutral, that are expressed and secreted by rabbit lacrimal acinar cells in primary culture (IOVS 42: s259, 2001). The purpose of the present study was to further characterize the two forms of alpha-fucosidase regarding their subcellular distribution and regulated secretion Methods: Single acinar cells were isolated from female NZW rabbit lacrimal glands and cultured for 40 h on Matrigel, thus allowing the cells to re-organize into acinar-like structures. The cells were then rinsed and incubated at 37C for 1 h in the absence or presence of 0.1 mM carbachol. Media were collected and analyzed for alpha-fucosidase catalytic activity, at pH 4.0 and pH 7.0, using a 4-methyl umbelliferyl conjugate as substrate.Tissue and cultured cells were homogenized and fractionated by centrifugation yielding a soluble, a nuclear and a microsomal fraction. The membrane fractions were further fractionated by density gradient centrifugation on 10-30% Iodioxanol (Optiprep) gradients, and the resulting fractions were analyzed for fucosidase activity. Results: Secretion of the acidic and the neutral form were both stimulated 5-fold by carbachol. Subcellular fractionation of tissue resulted in 60% recovery of both the acidic and the neutral fucosidase activity in the soluble fraction and 15% in the nuclear (low-speed) pellet, whereas fractionation of cultured cells resulted in a reversed distribution, the difference probably a result of the harsher homogenization procedures used for tissue. Density gradient centrifugation of the membrane fractions revealed the presence of at least three fucosidase-containing membrane populations: One fraction of low density of unknown origin, one high-density, potentially lysosomal population and one intermediate fraction, possibly Golgi-related. Conclusion: No sigificant differences were observed between the distribution and secretion of the acidic and neutral forms of alpha-fucosidase, indicating that they are sequestered in the same endomembrane compartments and that they are under the same secretory control.
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