Glycoprotein synthesis in the adult rat pancreas: IV. Subcellular distribution of membrane glycoproteins

Zymogen granule membranes from the rat exocrine pancreas displays distinctive, simple protein and glycoprotein compositions when compared to other intracellular membranes. The carbohydrate content of zymogen granule membrane protein was 5–10-fold greater than that of membrane fractions isolated from smooth and rough microsomes, mitochondria and a preparation containing plasma membranes, and 50–100-fold greater than the zymogen granule content and the postmicrosomal supernate. The granule membrane glycoprotein contained primarily sialic acid, fucose, mannose, galactose and $\text{N-}\text{acetylglucosamine}$. The levels of galactose, fucose and sialic acid increased in membranes in the following order: rough microsomes < smooth microsomes < zymogen granules.Membrane polypeptides were analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The profile of zymogen granule membrane polypeptide was characterized by GP-2, a species with an apparent molecular weight of 74 000. Radioactivity profiles of membranes labeled with [³H]glucosamine or [³H]leucine, as well as periodic acid-Schiff stain profiles, indicated that GP-2 accounted for approx. 40% of the firmly bound granule membrane protein. Low levels of a species similar to GP-2 were detected in membranes of smooth microsomes and the preparation enriched in plasma membranes but not in other subcellular fractions. These results suggest that GP-2 is a biochemical marker for zymogen granules.Membrane glycoproteins of intact zymogen granules were resistant to neuraminidase treatment, while those in isolated granule membranes were readily degraded by neuraminidase. GP-2 of intact granules was not labeled by exposure to galactose oxidase followed by reduction with NaB³H₄. In contrast, GP-2 in purified granule membranes was readily labeled by this procedure. Therefore GP-2 appears to be located on the zymogen granule interior.     

Glycoprotein synthesis in the adult rat pancreas. IV. Subcellular distribution of membrane glycoproteins

Zymogen granule membranes from the rat exocrine pancreas displays distinctive, simple protein and glycoprotein compositions when compared to other intracellular membranes. The carbohydrate content of zymogen granule membrane protein was 5-10-fold greater than that of membrane fractions isolated from smooth and rough microsomes, mitochondria and a preparation containing plasma membranes, and 50-100-fold greater than the zymogen granule content and the postmicrosomal supernate. The granule membrane glycoprotein contained primarily sialic acid, fucose, mannose, galactose and N-acetylglucosamine. The levels of galactose, fucose and sialic acid increased in membranes in the following order: rough microsomes less than smooth microsomes less than zymogen granules. Membrane polypeptides were analyzed by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The profile of zymogen granule membrane polypeptides was characterized by GP-2, a species with an apparent molecular weight of 74 000. Radioactivity profiles of membranes labeled with [3H]glucosamine or [3H]leucine, as well as periodic acid-Schiff stain profiles, indicated that GP-2 accounted for approx. 40% of the firmly bound granule membrane protein. Low levels of a species similar to GP-2 were detected in membranes of smooth microsomes and the preparation enriched in plasma membranes but not in other subcellular fractions. These results suggest that GP-2 is a biochemical marker for zymogen granules. Membrane glycoproteins of intact zymogen granules were resistant to neuraminidase treatment, while those in isolated granule membranes were readily degraded by neuraminidase. GP-2 of intact granules was not labeled by exposure to galactose oxidase followed by reduction with NaB3H4. In contrast, GP-2 in purified granule membranes was readily labeled by this procedure. Therefore GP-2 appears to be located on the zymogen granule interior.     

Acyltransferase activities in adult rat type II pneumocyte-derived subcellular fractions

Acyl-CoA: lysophosphatidylcholine, acyl-CoA: lysophosphatidylethanolamine, and lysophosphatidylcholine:lysophosphatidylcholine acyltransferases were investigated using subcellular fractions derived from adult rat type II pneumocytes in primary culture. Acyl-CoA:lysophospholipid acyltransferase activities were determined to be microsomal, while lysophosphatidylcholine:lysophosphatidylcholine acyltransferase activity was found to be cytosolic. Total palmitoyl CoA:lysophosphatidylcholine acyltransferase activity was 30-fold greater than lysophosphatidylcholine:lysophosphatidylcholine acyltransferase activity, indicating that the former enzyme is more important in the synthesis of dipalmitoyl phosphatidylcholine. Palmitoyl-CoA and oleoyl-CoA lysophosphatidylcholine acyltransferase activities were approximately equal under optimal substrate conditions. Specific activities of the enzyme using arachidoyl-CoA and arachidonoyl-CoA were 46% and 18%, respectively, of those with palmitoyl-CoA. Acyl-CoA:lysophosphatidylethanolamine acyltransferase showed a preference for palmitoyl-CoA as opposed to oleoyl-CoA under optimal conditions. However, when equimolar concentrations of either palmitoyl-CoA and oleoyl-CoA or palmitoyl-CoA and arachidoyl-CoA were assayed together, the relative utilization of the two substrates was found to be dependent on total acyl-CoA concentration. At higher concentrations, the incorporation of palmitoyl-CoA into phosphatidylcholine was less than other acyl-CoAs. However, at lower concentrations palmitoyl-CoA was utilized quite selectively. Whole lung microsomes did not show as marked a preference for palmitoyl-CoA as did type II pneumocyte microsomes under these same conditions. In similar experiments, low total acyl-CoA concentrations produced greater incorporation of oleoyl-CoA into phosphatidylethanolamine. For both enzymes total activity at the lowest concentrations used was at least 45% that at optimal conditions. This demonstrates that the type II pneumocyte acyltransferase system(s) can selectively utilize palmitoyl-CoA. No evidence for direct exchange of palmitoyl-CoA with 1-saturated-2-unsaturated phosphatidylcholine in subcellular fractions from type II pneumocytes was found.     

Glycoprotein synthesis in the mucous cells of the vascularly perfused rat stomach. II. Differentiating mucous cells

Labeled leucine, serine, galactose, glucosamine and sulphate were administered to rat stomachs in a perfusion system. Sections of the gastric fundus were studied by light microscopic autoradiography. Five categories of mucous cells were distinguished and their glycoprotein synthetic activity was measured in autoradiographs by counting silver grains over each category. During their differentiation, while migrating from the isthmus of the fundic glands to the free luminal surface, the surface mucous cells (SMC) showed an increase in incorporation of all precursors used. Differences between the incorporation patterns of the various precursors, in cells of different ages, suggest that structural development runs ahead of functional activity, and that the latter continues up to the very moment the cell is shed from the surface. Sulphate was incorporated at a considerably lower rate by the SMC of the free surface than by the foveolar SMC, in which by cytochemical staining strongly acidic glycoproteins were shown. Since the mucous neck cells incorporated all precursors at a low rate, these cells apparently do not play an important role in gastric mucus synthesis. They did not incorporate sulphate, which is consistent with histochemical observations.     

The phospholipid contents in rat liver Golgi-rich membrane fractions after streptozotocin and/or prostaglandin treatment

After dmPGE2 treatment, both alone or together with streptozotocin, the lower, statistically significant total PL contents (in mumoles P per mg of protein) in rat liver Golgi-rich membrane fractions were found. In these groups of investigated rats, the percentage of PE+PA were statistically significantly lower than in control. For the present it is impossible to certify, if there is a causal-result dependence between these two facts. The administration of streptozotocin alone (both after 6 or 11 days) caused the increase of PE+PA percentage, however not statistically significant.     

Characterization of alpha-melanocyte-stimulating hormone in rat pancreas

Reverse-phase high performance liquid chromatography and radioimmunoassay were used to characterize alpha-melanocyte-stimulating hormone (alpha-MSH)-like peptides in rat pancreas. Relative to synthetic alpha-MSH standards, serial dilutions of pancreas extracts showed parallel and concentration dependent displacement of (125I) alpha-MSH from alpha-MSH antibody. Chromatographic separation revealed immunoreactive material coeluting with synthetic N,O-diacetyl alpha-MSH, which accounted for 78% of total alpha-MSH materials in this tissue. The remainder of immunoreactive alpha-MSH coeluted with synthetic alpha-MSH, desacetyl alpha-MSH, or their methionine sulfoxides. In contrast with anterior pituitary, it appears that biosynthetic processing of alpha-MSH from pro-opiomelanocortin (POMC) may be similar in rat pancreas and pituitary intermediate lobe, since their relative alpha-MSH immunoreactive elution profiles were similar. These findings support the hypothesis of tissue specific regulation of biosynthetic processing of POMC.     

Segregation of histone fractions from purified rat pancreas nuclei by isoelectric focusing.

The present report describes the purification of nuclei from rat pancreas and the use of isoelectric focusing for the segregation of histone fractions, a heterogeneous mixture with respect to charge. This procedure employs gels of 5% polyacrylamide as support matrices, in 6.25 M urea, and spans a pH range of 7.5-10.8. It was, therefore, found adequate for study of the microheterogeneity of histones. The banding pattern of histone fractions in pancreas development using electrofocusing is presented.     

Glycoprotein synthesis in the mucous cells of the vascularly perfused rat stomach. I. Surface mucous cells

We developed a constant-pressure vascular perfusion system of the isolated rat stomach, utilizing an artificial, fluorocarbon (FC-75)-containing medium. Perfusion could be maintained for at least six hours, as demonstrated by the ultrastructure of the mucosal cells and by the constant incorporation of [3H]-galactose in the surface mucous cells. Moreover all mucous cell types in tissue fixed after six hours of perfusion showed the same histochemical reactions for glycoproteins as in tissue fixed shortly after decapitation of the animal. The surface mucous cells of the antrum incorporated 30% less [3H]-galactose, [3H]-serine and [35S]-sulphate than those of the fundus. The amount of radioactivity incorporated per cell did not decrease during a subsequent 2 hour chase.     

Synthesis of protein in the pancreas. II. The role of ribonucleoprotein in protein synthesis

1. The ribonucleoprotein of the microsome fraction which sediments at 40,000 R.P.M. as a pellet (and which is referred to as the pellet material) has been studied with reference to its role in protein synthesis in the pancreas. 2. In pellet material nucleic acid and protein form a definite complex as shown by its electrophoretic behavior and unchanging composition under various conditions. 3. Protein of pellet material is not especially rich in the diamino acids. 4. Evidence is brought forward indicating that the protein component of pellet material takes part in the general process of protein synthesis in the cell. (a) The well known correlation between quantity of RNA and rate of protein synthesis in a tissue implicates the protein of the pellet material, for most of the RNA in the pancreas and other tissues is in this material. (b) Uptake of isotopically labelled glycine by the pellet material, confirming results of previous workers, is for short periods greater than in other protein fractions. (c) Comparing the pellet materials of pancreas, liver, and kidney-three tissues with vastly different rates of protein synthesis, in the sequence given-there is a correlation between the quantity of RNA in the pellet and the rate of protein synthesis in the tissue; a similar correlation between quantity of RNA in the pellet material and rate of N(15)-glycine uptake by the protein component of the pellet; and finally, the level of uptake by total protein varies with the tissue and is related to the uptake of N(15)-glycine by protein of the pellet. 5. In the pancreas a distinction can be made between proteins synthesized for secretion and the nucleoprotein of the pellet (not found in the secretion) which, however, takes part in the synthetic process, as shown by the fact that the N(15) uptake by protein of the pellet is increased when the synthesis of digestive enzymes is stimulated by secretion. 6. The time course of N(15) uptake by proteins of the pancreas indicates that pellet protein serves as precursor material in the synthesis of the secretory proteins. 7. Rate of uptake of N(15)-glycine by the purines of RNA of the pellet material is not correlated with uptake by the protein. 8. The uptake of C(14)-alanine by an in vitro system of microsomes + mitochondria is impaired by preincubation of the microsomes with ribonuclease. This is direct experimental evidence for the dependence of protein synthesis upon the presence or intactness of ribonucleic acid in the microsomes.