Two methods of polyacrylamide gel electrophoresis (the acid method of Eytan and Ohad and the Na dodecylsulfate (SDS) disc method of Maizel) have been used for analyzing the proteins of gel fractions isolated from the guinea pig pancreatic exocrine cells and in particular the proteins bound to the membranes involved in the synthesis, intracellular transport, and discharge of secretory enzymes : rough (RM) and smooth microsome (SM) membranes, zymogen granule (ZG) membranes, and plasma membranes (PM) . Since in the two systems the electrophoretic mobility of proteins depends on different factors (size, shape, and net charge of molecules in the acid system ; size only in the SDS system) a deeper insight into the protein composition of the fractions could be obtained . The gel patterns of RM, SM, and ZG membranes turned out to be accounted for mainly by segregated secretory enzymes (in rough microsomes also by ribosome proteins) and thus were found to share most of the bands . In contrast, with highly purified membrane fractions different patterns were obtained : RM and SM membrane proteins turn out to contain a large number of different proteins with molecular weights varying between 150,000 and 15,000 daltons . The pattern of ZG membranes was greatly different in the two systems : only two bands were separated by the acid method and as many as 23 by the SDS m ethod. PM gave a rather complex pattern in either system . Both ZG membranes and PM were found to contain a large proportion of low molecular weight proteins . Nothing appears in common between the proteins of SM membranes (primarily of Golgi origin) and those of ZG membranes, while the latter and PM exhibit a certain degree of similarity . By amino acid analysis we found only slight differences : relative to the other fractions : RM membranes were higher in basic amino acids and ZG membranes contained a larger amount of methionine . Taken together with recent data on lipid composition and enzyme activities of the same fractions, these results indicate that the membranes of the pancreatic exocrine cells are chemically and functionally distinct, and hence do not mix randomly with one another during the transport of secretory products .It is well known that in pancreatic acinar cells functionally interconnected membrane-bound secretory proteins, following their synthesis in the compartments : in sequence, the RER, the Golgi attached ribosomes, are segregated within the complex (GC), and the zymogen granules (ZG) . cisternae of the rough endoplasmic reticulum (RER)' and then transported through a series of ' Abbreviations used : RER, rough-surfaced endo-
The suspension of catalyst in a gas-agitated 1.8-inch i.d. reactor was observed for the cases of Raney nickel catalyst in water and in acetone.A model was developed which predicts the catalyst profile in a reactor as a function of physical properties and operating conditions, and was applied with success to both laboratory and plant scale reactors.The effects of gas and liquid flow rates were investigated.
Previous investigations, performed on isolated rat atria, showed that the lipophylic spin-trapping agent N-tert-butyl- alpha-phenylnitrone (PBN) is able to prevent the acute cardiotoxic effects produced by doxorubicin (DXR), whereas the hydrophylic compound 5,5-dimethyl-pyrroline-N-oxide (DMPO) is inactive. The present study was designed to ascertain whether differences in the pharmacological effects of the two spin traps are related to their different subcellular distribution. Langendorff rat hearts were perfused for 60 minutes with [14C]-DXR and either PBN or DMPO. The subcellular mapping of the three compounds was performed by measuring DXR by liquid scintillation counting, PBN by GC/MS, and DMPO by HPLC in the following isolated fractions: nuclei, mitochondria, sarcoplasmic reticulum, sarcolemma, cytosol. DMPO was shown to accumulate in the cytosolic compartment; both PBM and DXR are taken up by nuclei and mitochondria, while only trace amounts of DXR were detected in the sarcoplasmic reticulum. These results suggest that mitochondrial (and not sarcoplasmic) enzymes are mainly involved in DXR-induced free radical production, which is thought to cause the acute cardiotoxic effects of DXR. An involvement of DXR-induced free radical generation in the nuclear compartment seems unlikely in the short-term "in vitro" effects observed with the experimental model adopted for these studies, although it may play a role in the delayed pathology.
Several mechanisms have been suggested to explain how secretory cells remove from the plasmalemma the excess membrane resulting from the insertion of granule membrane during exocytosis : intact patches of membrane may be internalized and then reutilized within the cell ; alternatively these membranes may be either disassembled to subunits or degraded . In the latter case new membranes should be synthetized at other sites of the cell, probably in the rough-surfaced endoplasmic reticulum (RER) and the Golgi complex . In the present research, membrane subfractions were obtained from rough microsomes (derived from fragmented and resealed RER cisternae) and from smooth microsomes (primarily contributed by Golgi stacks and vesicles) of the guinea pig pancreas by incubation at 4°C for 4 hr in 0 .0005 M puromycin at high ionic strength followed by mild (pH 7 .8) alkaline extraction with 0 .2 M NaHCO3 . Such treatments release the majority of nonmembrane components of both microsomal fractions (i .e., contained secretory enzymes, ribosomes, and absorbed proteins of the cell sap) and allow the membranes to be recovered by centrifugation . The effect of in vitro stimulation of enzyme secretion (brought about in pancreas slices by 0 .0001 M carbamoyl choline) on the rate of synthesis of the phospholipid (PLP) and protein of these membranes was then investigated . In agreement with previous data, we observed that in stimulated slices the synthesis of microsomal PLP was greatly increased . In contrast, the synthesis of microsomal membrane proteins was unchanged . These results suggest that exocytosis is not coupled with an increased rate of synthesis of complete ER and Golgi membranes and are, therefore, consistent with the view that excess plasma membrane is preserved and reutilized, either as discrete membrane patches or as membrane macromolecules, throughout the secretory cycle . 6It is now firmly established that, in the acinar cells of the pancreas, stimulation of secretion ultimately results in the discharge of digestive enzymes stored within zymogen granules by a process akin to exocytosis (1-5) . Such a process consists of fusion of the limiting membrane of the granule with the apical portion of the plasmalemma and therefore results, at least temporarily, in the incorporation of the first membrane into the latter. The mechanism of removal of excess membrane from the acinar lumen is still unknown . As proposed by Palade (1), intact patches of membranes may be internalized from the cell surface and reutilized in the packaging of new zymogen granules . Alternatively these membranes may be degraded as suggested originally by Fawcett (6), new membranes being synthesized at other sites in the cell .The results of a series of papers by the Hokins
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
customersupport@researchsolutions.com
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.