Subfractionation of a canine pancreatic homogenate was performed by several differential centrifugation steps, which gave rise to fractions with distinct marker profiles. Specific binding of guanosine 5'-[gamma-[35S]thio]triphosphate (GTP[gamma-35S]) was assayed in each fraction. Enrichment of GTP[gamma-35S] binding was greatest in the interfacial "smooth" microsomal fraction, expected to contain Golgi and other smooth vesicles. There was also marked enrichment in the rough microsomal fraction. Electron microscopy and marker protein analysis revealed the rough microsomes (RMs) to be highly purified rough endoplasmic reticulum (RER). The distribution of small (low molecular weight) GTP binding proteins was examined by a [alpha-32P]GTP blot-overlay assay. Several apparent GTP binding proteins of molecular masses 22-25 kDa were detected in various subcellular fractions. In particular, at least two such proteins were found in the Golgi-enriched and RM fractions, suggesting that these small GTP binding proteins were localized to the Golgi and RER. To more precisely localize these proteins to the RER, native RMs and RMs stripped of ribosomes by puromycin/high salt were subjected to isopycnic centrifugation. The total GTP[gamma-35S] binding, as well as the small GTP binding proteins detected by the [alpha-32P]GTP blot overlay, distributed into fractions of high sucrose density, as did the RER marker ribophorin I. Consistent with a RER localization, when the RMs were stripped of ribosomes and subjected to isopycnic centrifugation, the total GTP[gamma-35S] binding and the small GTP binding proteins detected in the blot-overlay assay shifted to fractions of lighter sucrose density along with the RER marker.
Animals Cell Fractionation