Subcellular distribution of small GTP binding proteins in pancreas: Identification of small GTP binding proteins in the rough endoplasmic reticulum

Journal Article · Thu Feb 01 00:00:00 EST 1990 · Proceedings of the National Academy of Sciences of the United States of America; (USA)

DOI:https://doi.org/10.1073/pnas.87.4.1296· OSTI ID:6172301

Nigam, S K ^[1]

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1. Rockefeller Univ., New York, NY (USA)

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{prime}-({gamma}-({sup 35}S)thio)triphosphate (GTP({gamma}-{sup 35}S)) was assayed in each fraction. Enrichment of GTP({gamma}-{sup 35}S) 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}-{sup 32}P)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}-{sup 35}S) binding, as well as the small GTP binding proteins detected by the ({alpha}-{sup 32}P)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}-{sup 35}S) 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.

• OSTI ID: 6172301
• Journal Information: Proceedings of the National Academy of Sciences of the United States of America; (USA), Vol. 87:4; ISSN 0027-8424
• Country of Publication: United States
• Language: English

Related Subjects

59 BASIC BIOLOGICAL SCIENCES
GUANOSINE
CROSS-LINKING
LABELLED COMPOUNDS
PROTEINS
SUBCELLULAR DISTRIBUTION
CENTRIFUGATION
DOGS
ELECTROPHORESIS
ENDOPLASMIC RETICULUM
NUCLEOTIDES
PANCREAS
RIBOSOMES
SULFUR 35
TRANSMISSION ELECTRON MICROSCOPY
ANIMALS
AROMATICS
AZAARENES
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BODY
CELL CONSTITUENTS
CHEMICAL REACTIONS
DAYS LIVING RADIOISOTOPES
DIGESTIVE SYSTEM
DISTRIBUTION
ELECTRON MICROSCOPY
ENDOCRINE GLANDS
EVEN-ODD NUCLEI
GLANDS
HETEROCYCLIC COMPOUNDS
ISOTOPES
LIGHT NUCLEI
MAMMALS
MICROSCOPY
NUCLEI
NUCLEOSIDES
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANOIDS
ORGANS
POLYMERIZATION
PURINES
RADIOISOTOPES
RIBOSIDES
SEPARATION PROCESSES
SULFUR ISOTOPES
VERTEBRATES
550201* - Biochemistry- Tracer Techniques