Dataset Information

PH-dependent processing of secretogranin II by the endopeptidase PC2 in isolated immature secretory granules.

ABSTRACT: We have previously characterized the processing of secretogranin II (SgII) in PC12 cells that were stably transfected with the endopeptidase PC2. Here we show that processing of SgII can be observed in isolated immature secretory granules (ISGs) derived from this cell line in a temperature- and ATP-dependent manner. The stimulatory effect of ATP on processing can be attributed to the activation of the vacuolar H(+)-ATPase and a concomitant decrease in intragranular pH. The immature secretory granule therefore provides an adequate environment for correct processing of SgII by PC2. The rate of SgII processing was strongly dependent on the intragranular pH, suggesting that processing of SgII can be used as a pH indicator for the granule interior. A standard curve was prepared using SgII processing in ISGs equilibrated at a range of pH values. The extent of processing in ISGs incubated in the presence of ATP at physiological pH was compared with the standard curve, and the intragranular pH was determined. From these observations, we propose an intragranular pH of 6.3 +/- 0.1 for ISGs in a physiological buffer in the presence of ATP. Hence, the pH of ISGs seems to be similar to the pH of the trans-Golgi network (TGN) and is clearly higher than the pH of mature secretory granules (pH 5.0-5.5). Interestingly, no processing of SgII could be observed in a membrane fraction that is highly enriched in TGN under conditions for which processing was readily obtained in isolated ISGs.

SUBMITTER: Urbe S

PROVIDER: S-EPMC1218037 | biostudies-other | 1997 Jan

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:We investigated the basis for previous observations that fluorescent-labeled neuropeptide Y (NPY) is usually released within 200 ms after fusion, whereas labeled tissue plasminogen activator (tPA) is often discharged over many seconds. We found that tPA and NPY are endogenously expressed in small and different subpopulations of bovine chromaffin cells in culture. We measured the mobility of these proteins (tagged with fluorophore) within the lumen of individual secretory granules in living chromaffin cells, and related their mobilities to postfusion release kinetics. A method was developed that is not limited by standard optical resolution, in which a bright flash of strongly decaying evanescent field (∼64 nm exponential decay constant) produced by total internal reflection (TIR) selectively bleaches cerulean-labeled protein proximal to the glass coverslip within individual granules. Fluorescence recovery occurred as unbleached protein from distal regions within the 300 nm granule diffused into the bleached proximal regions. The fractional bleaching of tPA-cerulean (tPA-cer) was greater when subsequently probed with TIR excitation than with epifluorescence, indicating that tPA-cer mobility was low. The almost equal NPY-cer bleaching when probed with TIR and epifluorescence indicated that NPY-cer equilibrated within the 300 ms bleach pulse, and therefore had a greater mobility than tPA-cer. TIR-fluorescence recovery after photobleaching revealed a significant recovery of tPA-cer (but not NPY-cer) fluorescence within several hundred milliseconds after bleaching. Numerical simulations, which take into account bleach duration, granule diameter, and the limited number of fluorophores in a granule, are consistent with tPA-cer being 100% mobile, with a diffusion coefficient of 2 × 10(-10) cm(2)/s (∼1/3000 of that for a protein of similar size in aqueous solution). However, the low diffusive mobility of tPA cannot alone explain its slow postfusion release. In the accompanying study, we suggest that, additionally, tPA itself stabilizes the fusion pore with dimensions that restrict its own exit.

Project description:Insulin-secretory granules isolated from a pancreatic islet-cell tumour by centrifugation on Percoll density gradients exhibited a membrane-associated Mg(2+)-dependent ATPase activity. In granule suspensions incubated in iso-osmotic media, activity was increased 2-3-fold by carbonyl cyanide p-trifluoromethoxyphenylhydrazone, the combination of valinomycin, nigericin and K(2)SO(4) or by the addition of a detergent. Permeant anions also increased Mg(2+)-dependent ATPase activity under iso-osmotic conditions when combined with K(+) and nigericin, or NH(4) (+). It was deduced that a major component of the activity was coupled to the translocation of protons into the granule interior. The granule membrane appeared poorly permeable to H(+), K(+), NH(4) (+) and SO(4) (2-) but permeable, in increasing order, to phosphate or acetate, Cl(-), I(-) and SCN(-). Like the proton-translocating ATPase of mammalian mitochondria the granule enzyme when membrane-bound was inhibited by up to 85% by tributyltin or NN'-dicyclohexylcarbodi-imide and was solubilized in a tributyltin-insensitive form after extraction with dichloromethane. It was clearly not a mitochondrial contaminant as evidence by the distribution of marker proteins on density gradients. Unlike mitochondrial activity it was insensitive to oligomycin, efrapeptin, atractyloside, azide and oxyanions. Its properties, however, were indistinguishable from those of the proton-translocating ATPase found in the chromaffin granules of the adrenal medulla. Moreover, insulin granules and chromaffin granules exhibited similar levels of activity. This indicated that in spite of the differences in their internal composition, granules from tissues involved in polypeptide and amine hormone secretion possess catalytic components in common. Only a minor role for the ATPase in amine transport in insulin granules was apparent. Rather, its presence here may relate to the process of secretory vesicle morphogenesis or to the exocytotic mechanism.

Dataset Information

PH-dependent processing of secretogranin II by the endopeptidase PC2 in isolated immature secretory granules.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets