A Cytochemical Study on the Pancreas of the Guinea Pig

Abstract

Chymotrypsinogen synthesis in the exocrine cell of the guinea pig pancreas was studied under the following conditions: Animals fed after a fast of ~48 hours received H I hour after feeding an intravenous injection of nL-leucine-l-C 14. At various time intervals (1 to 45 minutes) after the injection, the glands were removed and fractionated into a series of cell fractions of known cytological significance. Ten to twelve animalswere used for each timepoint. From each cell fraction, the chymotrypsinogen was isolated by acid extraction and purified by (NH4)2SO~ fractionation, isoelectric precipitation, and chromatography. Because of the minuteness of the quantities involved, chymotrypsinogen amounts were calculated from enzymatic activity figures, and a carrier method was used to precipitate and count the enzyme. The chymotrypsinogen isolated from the attached ribonucleoprotein particles of the microsomal fraction had the highest specific radioactivity at the early time points (1 to 3 minutes). After long intervals (at 15 to 45 minutes), the specific radioactivity of the enzyme increased in the microsomal contents and finally in the zymogen granules. The results are compatible with the view that the chymotrypsinogen is synthesized in or on the attached RNP particles and subsequently transported to other cell compartments.

Protein Synthesis, Storage, and Discharge in the Pancreatic Exocrine Cell

“…Our results agree in general with those obtained by cell fractionation studies (4,6). The timing of the sequence of events has been, of course, modified by the introduction of an intermediate step between microsomes and zymogen granules, i.e.…”

“…of the intracellular pulse (too long when compared to the speed of the events under study) and by the probable presence of some non-specific binding of free leucine during fixation (as evidenced by the discrepancy between the amounts of label removed by cold TCA and by fixation). The transfer of newly synthesized proteins from the rough surfaced endoplasmic reticulum to the Golgi complex remains, however, a reasonable assumption: it explains satisfactorily our results, as well as those of Siekevitz and Palade (4,6); fits well currently accepted notions on protein synthesis (35); and is process. They demonstrate convincingly the accumulation of secretory products and their progressive concentration, possibly by water withdrawal, in the large vacuoles centrally located in the Golgi complex, and show that this concentration leads to the formation of zymogen granules.…”

“…It seems, therefore, that, in spite of the known ketogenic activity of leucine, most of it is incorporated into proreins, at least in the pancreas. In this gland, especially after stimulation by a cycle of fasting and feeding, the rate of synthesis of exportable proteins (i.e., digestive enzymes) is considerably higher than that of structural or other non-exportable proteins (6,8). We expect, therefore, that, in our autoradiographic experiments, the distribution of radioactivity will reflect that of exportable proteins.…”

“…This system has been the object of a number of recent studies involving the biochemical analysis of cell fractions separated by differential centrifugation (1)(2)(3)(4)(5)(6)(7)(8). Such studies have provided a sizable amount of data on the secretory cycle but have left several points in doubt and could not provide any information on important steps of the process.…”

Alterations in Polyribosomes During Erythroid Cell Maturation

“…Ribosomes, either membrane bound (1)(2)(3) or free (4--6) have been established as sites of protein biosynthesis. In mammalian reticulocytes, protein synthesis has been shown to proceed predominantly on ribosomes with sedimentation coefficients in excess of 110S (7)(8)(9).…”