The biosynthesis of GP-2, the major glycoprotein associated with zymogen-granule membranes in the pancreas, was studied in acinar cell suspensions from rat pancreas. Pulse-chase experiments, using [35S]methionine, were performed and the processing of GP-2 was analyzed by immunoprecipitation and sodium dodecyl sulfate/ polyacrylamide gel electrophoresis.GP-2 is synthesized as a precursor glycoprotein with apparent molecular weight M , = 73 000. Within 60 min after synthesis it is almost completely converted to the mature form ( M , = 78000-80000). Only the precursor form of GP-2 is sensitive to digestion with the glycosidase endo-P-N-acetylglucosaminidase H, indicating that the observed conversion reflects the processing of 'high-mannose' oligosaccharides into complex type oligosaccharides.Acinar cells cultured in the presence of increasing concentrations of the N-glycosylation inhibitor tunicamycin synthesize 5 -6 distinct precursor GP-2 species with apparent molecular weights decreasing from 73 000 -61 000. We conclude that GP-2 contains five or six N-linked carbohydrate chains.From cell fractionation studies it was established that the precursor GP-2 is present in a microsomal fraction with high density (> 1.169 g/ml) presumably derived from the rough endoplasmic reticulum; mature GP-2 is localized in low density microsomes (< 1.130 g/ml) probably Golgi vesicles. The GP-2 in zymogen granule membranes is also in the mature form.Although a considerable amount of knowledge has accumulated concerning the secretory process in mammalian cells, the basic mechanisms involved in formation and maturation of secretory granules are still poorly understood [I]. In our laboratory granule formation has been investigated in relation to (g1yco)protein synthesis, intracellular transport and secretion in a number of different cell types [2-41.In recent years we have studied the peculiar behaviour of a glycoprotein, characteristically associated with zymogen granule membranes in the pancreas [5, 61. This glycoprotein was first described by MacDonald and Ronzio [7] and provisionally named GP-2. In the rat pancreas, like that of several other mammals, GP-2 is a major component of purified zymogen granule membranes [7,8], accounting for about one third of the proteins in these membranes. It has an apparent molecular weight in SDS gels of approximatcly 80000. Immunocytochemically it has a membrane localization (in cndoplasmic reticulum, Golgi apparatus, zymogen granules and plasma membrane), but it is also present in the granule interior and acinar lumen [5, 61. Sarras et al. [9] recently suggested, on the basis of their observations of the effect of tunicamycin on developing pancreas, that the 80000-M, glycoprotein of zymogen granule membranes could be involved in granule formation during pancreatic development.Ahhreviufions. GP-2, major glycoprotein of zymogen granule membranes from pancreas; SDS, sodium dodecyl sulfate. Considering the available information and suggestions in the literature about GP-2 we anticipate that it may b...

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1. The uptake and incorporation in vitro of radioactive leucine and lysine in rat pancreas fragments have been studied.2. The uptake and incorporation of both leucine and lysine were found t o be influenced by the concentration of leucine in the medium.3. Extracellular and intracellular free amino acids can be distinguished by washing the tissue fragments a t 0 "C.4. Though it takes some 20 min for each of both radioactive amino acids to reach maximum concentration in the intracellular soluble amino acid pool, their incorporation into protein becomes linear shortly after the start of incubation in a medium with labelled amino acids.5. I n chase-type experiments the incorporation of each of both radioactive amino acids stops immediately when the labelled medium is replaced by unlabelled medium, though the free, intracellular amino-acid radioactivity declines only slowly.6. These results indicate that in the pancreas of the fasting rat in vitro the amino acid molecules incorporated into protein are derived from the cxtracellular pool. Other molecules enter and leave the intracellular amino acid pool but do not contribute to incorporation.I n earlier studies from our laboratory it was found that the rate of protein synthesis as measured by the rate of incorporation of radioactive amino acid in the rat pancreas was not influenced by the extent to which pancreatic cells are filled with secretory granules nor by the actual process of secretion. These studies [ 1,21 were performed on fasting animals, fed animals and animals which were stimulated to secrete by pilocarpine. The possible influence of an altered specific radioactivity of the precursor pool in these studies was discussed but not actually measured. The role of the intracellular amino acid pool in cellular metabolism has not been defined, but one of its functions has been assumed to be that of an obligatory precursor pool for protein synthesis. This assumption has been seriously questioned as a result of studies with rat diaphragm I n our studies in vitro with rat pancreas fragments we measured the influx of radioactive lysine and leucine from the medium into the intracellular amino acid pool and into cellular protein. Our results suggest that the amino acids used for protein synthesis are utilized directly from the extracellular fluid and not from the intracellular pool. Unlabelled amino acids were from Sigma Chem. Co. and Fluka; cycloheximide was obtained from Sigma Chem. Co.Young adult male rats (about 200g) from the Wistar strain and obtained from TNO (Holland) were fed ad lib. until 24 h before the expcriment. IncubationAfter decapitat,ion the pancreas was cut out and fragmented with the TC-2 Sorvall Tissue Sectioner into pieces of 1 mm3 each. About 50 mg fragmented pancreas was incubated a t 37 "C for 10 min in a conical flask containing 2 ml Krebs-Ringer bicarbonate buffer in which the KC1 concentration was raised to 13 ml. I n addition, the complete medium (pH 7.6) contained all amino acids in about the same concentrations as in rat plasma [7], the fo...

Frog pancreatic tissue was pulse-labelled in vitro with 3H-leucine and protein transport was studied in exocrine cells by electron microscope autoradiography. The proteins appeared to be synthesized in the RER and transported to the secretory granules along a similar route and with the same velocity as previously described under in vitro conditions. Evidence was obtained for the involvement of the vesicular and tubular elements at the periphery of the Golgi system in transferring protein from the RER to the Golgi cisternae. Kinetics of the release of newly synthesized proteins from the RER and their appearance in the condensing vacuoles are discussed and related to results reported from other tissues. The transport velocity in this poikilothermic system was studied in relation to the incubation temperature and compared with results reported from its mammalian counterpart. At temperatures between 20 and 30 degrees C intracellular protein transport occurs faster in the frog than in the Guinea pig pancreas. At higher temperature the transport process was severely disturbed in the frog.