Density gradient separation of two populations of lysosomes from rat parotid acinar cells.

Abstract

Exocrine acinar cells possess two cytochemically distinct populations of secondary lysosomes. One population is Golgi associated and has demonstrable acid phosphatase (AcPase) activity, whereas the second is basally located and lacks AcPase activity but has trimetaphosphatase (TMPase) activity. The basal lysosomes are tubular in shape and rapidly label with horseradish peroxidase (HRP) after intravenous injection. In the present study using isolated rat parotid acinar cells, the two lysosomal populations were separated by cell fractionation on Percoll density gradients and were analyzed biochemically and by EM cytochemistry. On 35% Percoll gradients, two peaks of AcPase and beta-hexosaminidase, both lysosomal marker enzymes, and succinic dehydrogenase, an enzyme marker for mitochondria, could be resolved. The major peaks of beta-hexosaminidase and succinic dehydrogenase and the minor peak of AcPase corresponded with the dense lysosome fraction. The major peak of AcPase and the minor peaks for beta-hexosaminidase and succinic dehydrogenase coincided with the light membrane fraction. Galactosyl transferase (a marker enzyme for Golgi saccules) and 5'-nucleotidase (a plasma membrane marker) were also associated with this fraction. By electron microscopy, the light membrane fraction was seen to contain tubular elements, multivesicular bodies (MVB), Golgi saccules, GERL, immature secretory granules, and some mitochondria. Electron microscopic cytochemical examination showed that these tubular structures were lysosomes. The dense lysosome fraction contained lysosomes positive for both AcPase and TMPase. After continuous incubation of isolated acinar cells with HRP, reaction product was rapidly localized to the light membrane fraction (greater than 2 min), where it was found in vesicles and tubular lysosomes. By 10 min it was present in MVB and tubular lysosomes, but by 60 min no HRP reaction product had appeared in the dense lysosomes. These results demonstrate that the tubular lysosomes are separable from dense lysosomes, typical secondary lysosomes, and are involved in the initial stages of endocytosis.

Tuning the Cellular Trafficking of the Lysosomal Peptide Transporter TAPL by its N-terminal Domain

“…The presence of wt‐TAPL, TMD0 or core‐TAPL in complex with TMD0 in the light fraction does not seem to reflect a mistargeting of these constructs but may be mainly due to lysosomes with low density, because these fractions contained also the lysosomal marker LAMP‐2. As minor population, low density lysosomes with a different morphology are found in several tissue preparations (19). However, this lower density can also result from leakiness of the lysosomes as this fraction showed very low peptide transport activity (see below).…”

Isolation of Lysosomes from Tissues and Cells by Differential and Density Gradient Centrifugation

“…In the case of cultured cells, a postnuclear supernatant (PNS) is often used as the source of crude lysosomes for gradient centrifugation. This was also used by Oliver et al (1989) for rat parotid acinar cells. If a LMF is used, then it is normally sedimented from the PNS by centrifuging 10 to 15 min at 18,000 to 20,000 × g; slightly lower than that used for the purification of peroxisomes (UNIT 3.5) which is routinely 25,000 × g for 10 to 15 min.…”

“…That is, lower RCFs and shorter centrifugation times will tend to produce sigmoidal gradients which are very shallow in the middle, while higher RCFs and times cause the gradient to become more linear (although a truly linear gradient is difficult to achieve with Percoll). Usually a fixed-angle rotor is used to create the gradient, but there are examples which use a vertical rotor (Kelly et al, 1989;Oliver et al, 1989). Oliver et al (1989) included a cushion of 2.5 M sucrose, presumably to prevent any silica particles pelleting on to the vertical wall of the tube.…”

Structure of serous cells in salivary glands

“…In the rat parotid gland, there are two varieties of lysosomes, one located in the basal regions of the serous cells, the other in the apical cytoplasm (Oliver, 1983a). The basal lysosomes, which have a highly pleomorphic form, frequently appearing as a system of anastomosing tubules, contain trimetaphosphatase but lack acid phosphatase (Oliver et al, 1989). In contrast, the apical lysosomes are of typical morphology and contain acid phosphatase (Oliver, 1983b).…”