Synthesis and secretion of rat pancreatic proteins by Xenopus laevis oocytes.
An in vivo translation system, the Xenopus laevis oocyte, was employed to study the synthesis and secretion of pancreatic proteins. RNA was purified from normal and diabetic rat pancreas and normal rat liver by use of guanidine isothiocyanate lysis and cesium chloride gradient centrifugation. The presence of functional mRNA was documented by translation in a reticulocyte lysate that yielded precursors of all major secretory proteins, i.e., slightly higher Mr than proteins synthesized in situ by pancreatic acini. Mature X. laevis oocytes were then microinjected with either total RNA or purified mRNA. When oocytes were subsequently incubated with 35S-methionine, pancreatic secretory proteins or hepatic albumin could be immunoprecipitated from oocyte lysate with specific polyclonal antibodies against amylase, trypsin, ribonuclease, and albumin. Amylase was shown to be enzymatically active. Moreover, oocytes released pancreatic secretory proteins into the medium when injected with pancreatic RNA in a time-dependent manner. Only the mature form of amylase was secreted and secretion was not regulated by secretagogues. When a comparison was made after injection of RNA from diabetic pancreas known to contain altered amounts of individual mRNAs, there was a decrease in amylase and an increase in trypsinogen synthesis in oocytes that was comparable to the results of cell free translation. The oocyte expression system, therefore, should be useful not only for studies of protein synthesis but also for processing and secretion.
Moessner
J
J
Department of Physiology, University of California, San Francisco.
Okabayashi
Y
Y
Perara
E
E
Logsdon
C D
CD
Lingappa
V R
VR
Williams
J A
JA
D000418:Albumins / Q000096:biosynthesis; D000818:Animals; D008297:Male; D009865:Oocytes / Q000378:metabolism*; D010179:Pancreas / Q000201:enzymology*; D014176:Protein Biosynthesis; D011506:Proteins / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D014982:Xenopus laevis
D000418:Albumins; D011506:Proteins