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J Meldolesi

Publications and source records attributed to J Meldolesi.

At least 235 records · Page 13Linked to original sources
PubMed1972

Composition of cellular membranes in the pancreas of the guinea pig. IV. Polyacrylamide gel electrophoresis and amino acid composition of membrane proteins.

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 approximately 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 method. 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.

Amino Acids↗
PubMed1971

Composition of cellular membranes in the pancreas of the guinea pig. I. Isolation of membrane fractions.

The subcellular components involved in the synthesis, transport, and discharge of secretory proteins in the guinea pig pancreatic exocrine cell have been isolated from gland homogenates by differential and gradient centrifugation. They include rough and smooth microsomes derived respectively from the rough endoplasmic reticulum and Golgi periphery, a zymogen granule fraction consisting mainly of mature zymogen granules and a smaller population of condensing vacuoles, and a plasmalemmal fraction. Membrane subfractions were obtained from the particulate components by treatment with mild (pH 7.8) alkaline buffers which extract the majority (>95%) of the content of secretory proteins, allowing the membranes to be recovered from the extracting fluid by centrifugation. The purity of the fractions was assessed by electron microscopy and by assaying marker enzymes for cross-contaminants. The rough and smooth microsomes were essentially free of mitochondrial contamination; the smooth microsomes contained <15% rough contaminants. The zymogen granule fraction and its derived membranes were free of rough microsomes and contained <3% contaminant mitochondria. The plasmalemmal fraction was heterogeneous as to origin (deriving from basal, lateral, and apical poles of the cell) and contained varying amounts of adherent fibrillar material arising from the basement membrane and terminal web. The lipid and enzymatic composition of the membrane fractions are described in the following reports.

Amylases↗
PubMed1971

Composition of cellular membranes in the pancreas of the guinea pig. II. Lipids.

The lipid composition of rough and smooth microsomal membranes, zymogen granule membranes, and a plasmalemmal fraction from the guinea pig pancreatic exocrine cell has been determined. As a group, membranes of the smooth variety (i.e., smooth microsomes, zymogen granule membranes, and the plasmalemma) were similar in their content of phospholipids, cholesterol and neutral lipids, and in the ratio of total lipids to membrane proteins. In contrast, rough microsomal membranes contained much less sphingomyelin and cholesterol and possessed a smaller lipid/protein ratio. All membrane fractions were unusually high in their content of lysolecithin (up to approximately 20% of the total phospholipids) and of neutral lipids, especially fatty acids. The lysolecithin content was shown to be due to the hydrolysis of membrane lecithin by pancreatic lipase; the fatty acids, liberated by the action of lipase on endogenous triglyceride stores, are apparently scavenged by the membranes from the suspending media. Similar artifactually high levels of lysolecithin and fatty acids were noted in hepatic microsomes incubated with pancreatic postmicrosomal supernatant. E 600, an inhibitor of lipase, largely prevented the appearance of lysolecithin and fatty acids in pancreatic microsomes and in liver microsomes treated with pancreatic supernatant.

Amino Alcohols↗
PubMed1971

Composition of cellular membranes in the pancreas of the guinea pig. 3. Enzymatic activities.

A COMPARATIVE STUDY OF THE ENZYMIC ACTIVITIES OF MEMBRANE FRACTIONS DERIVED FROM GUINEA PIG PANCREATIC HOMOGENATES HAS YIELDED THE FOLLOWING RESULTS: Rough microsomal membranes (derived from the rough ER) have the reductase activities of the two microsomal electron transport systems but lack enzyme activities of Golgi-type (TPPase) and plasmalemmal-type (5'-nucleotidase, beta-leucyl naphthylamidase, Mg-ATPase). Smooth microsomal membranes (derived primarily from the Golgi complex), zymogen granule membranes, and plasmalemmal fractions possess overlapping enzyme activities of plasmalemmal type, in different relative concentrations for each fraction. In addition, the smooth microsomal membranes exhibit TPPase and ADPase activity and share with rough microsomes the reductase activities of the two electron transport chains. Taken together with recent data on the lipid composition of the same fractions (2), these results indicate that the membranes of the pancreatic exocrine cell are chemically and functionally distinct, and hence do not mix with one another during the transport of secretory products.

Adenosine Triphosphatases↗
PubMed1971

In vitro stimulation of enzyme secretion and the synthesis of microsomal membranes in the pancreas of the guinea pig.

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 degrees 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 NaHCO(3). 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.

Amylases↗
PubMed1970

Effect of caerulein on protein synthesis and secretion in the guinea-pig pancreas.

1. Caerulein produced a striking increase in the rate of secretion of the guinea-pig pancreas. When tested in vivo with pancreatic slices its optimal concentration was found to be 2.5 ng/ml. In vivo the intravenous injection of 400 ng/kg and the subcutaneous injection of 4 mug/kg of the drug produced marked secretory responses.2. Neither incubation of pancreatic slices in vitro for various lengths of time in the presence of caerulein nor the intravenous injection of the drug brought about any detectable changes in the rate of (14)C-L-leucine incorporation into protein of pancreatic slices incubated in vitro. By contrast, a two-fold increase in the incorporation of leucine was found in fasting animals injected subcutaneously with caerulein twice a day for 4 days. The same treatment in fed animals, or one single dose of caerulein injected subcutaneously 4 h before killing in guinea-pigs starved for 4 days did not affect the rate of incorporation. Moreover the long term treatment with the cholinergic segretagogue bethanechol in fasted guinea-pigs was also ineffective.3. It is concluded (a) that in the guinea-pig pancreas, stimulation of enzyme secretion does not affect per se the rate of synthesis of digestive enzymes; and (b) that the increase of protein synthesis elicited by caerulein is critically dependent both on the nature of the segretagogue (that is, it is elicited by the polypeptide but not by a cholinergic drug) and on the experimental conditions (it is evident only in fasted animals after prolonged treatment).

Animals↗