ISOLATION OF ZYMOGEN GRANULES FROM RAT PANCREAS AND CHARACTERIZATION OF THEIR MEMBRANE-PROTEINS

  • 1 January 1982
    • journal article
    • research article
    • Vol. 28  (1) , 20-26
Abstract
A zymogen granule fraction was isolated from rat pancreas and its purity was assessed by biochemical and morphological criteria. Specific activities of 2 marker enzymes, amylase and chymotrypsin, were increased by 4.6 and 5.4-fold, respectively, as compared to the homogenate. The purified fraction was devoid of detectable RNA, DNA and 5''-nucleotidase, glucose-6-phosphatase and cytochrome c oxidase activities. EM confirm the absence of mitochondria, lysosomes and rough endoplasmic reticulum fragments. Zymogen granule membranes were isolated from this fraction on a sucrose gradient following lysis in alkaline buffer. Secretory contaminants were efficiently removed from the membranes as indicated by experiments in which labeled secretory proteins were added during the isolation procedure and secondly by measuring residual levels of amylase and chymotrypsin. Three enzyme activities were found in the membranes: thiamine pyrophosphatase, ATP-diphosphohydrolase and low levels of acid phosphatase. Membrane proteins were solubilized by urea- Triton X-100 and separated in double-dimension (isoelectric focusing and sodium dodecylsulfate-polyacrylamide gel electrophoresis). Isoelectric point and MW of each protein band were determined.