Biomaterial Database

Cyclic AMP-dependent protein kinase in canine pancreatic rough endoplasmic reticulum. 1989

S K Nigam, and G Blobel

Laboratory of Cell Biology, Howard Hughes Medical Institute, Rockefeller University, New York, New York 10021.

A canine pancreas homogenate was subfractionated by several differential centrifugation steps. The distribution of cAMP-dependent protein kinase in the various fractions was monitored by assaying [3H]cAMP binding and photo-cross-linking of the regulatory subunits of the enzyme (RI and RII) with radiolabeled 8-azido-cAMP. The distribution of the kinase was also compared to that of markers for the plasma membrane, the endoplasmic reticulum and the cytosol. While our results confirm previous studies suggesting the presence of cyclic AMP-dependent protein kinase in the cytosol and Golgi, a significant amount of the total [3H] cAMP binding and photolabeled R subunits (both RI and RII) were found in rough microsomes (RM). The association is relatively resistant to extraction with EDTA, low and high ionic strength solutions. These extractions unmasked several new phosphorylation substrates in the "stripped" RM that were inaccessible in the RM, possibly because they were covered by ribosomes or peripheral membrane proteins. RII with a molecular mass of 52 kDa (RII-52 kDa) was the predominant RII found in the cytosolic fraction, whereas RII-52 kDa and RII with a molecular mass of 54 kDa (RII-54 kDa) were approximately equally enriched in the RM fraction. The mobility of the RII-52 kDa-photolabeled band could be shifted to the mobility of the RII-54 kDa band by phosphorylation with purified catalytic subunit and ATP, indicating that they represent "dephospho" and "phospho" forms of RII, respectively. A more precise localization to the rough endoplasmic reticulum was accomplished by isopycnic floatation in sucrose gradients. The enzyme cobanded at the density of rough microsomes and shifted to the lower density of "stripped" microsomes after treatment with puromycin/high salt, which specifically removes ribosomes.

UI	MeSH Term	Description	Entries
D007700	Kinetics	The rate dynamics in chemical or physical systems.
D008970	Molecular Weight	The sum of the weight of all the atoms in a molecule.	Molecular Weights,Weight, Molecular,Weights, Molecular
D010179	Pancreas	A nodular organ in the ABDOMEN that contains a mixture of ENDOCRINE GLANDS and EXOCRINE GLANDS. The small endocrine portion consists of the ISLETS OF LANGERHANS secreting a number of hormones into the blood stream. The large exocrine portion (EXOCRINE PANCREAS) is a compound acinar gland that secretes several digestive enzymes into the pancreatic ductal system that empties into the DUODENUM.
D010766	Phosphorylation	The introduction of a phosphoryl group into a compound through the formation of an ester bond between the compound and a phosphorus moiety.	Phosphorylations
D011494	Protein Kinases	A family of enzymes that catalyze the conversion of ATP and a protein to ADP and a phosphoprotein.	Protein Kinase,Kinase, Protein,Kinases, Protein
D004285	Dogs	The domestic dog, Canis familiaris, comprising about 400 breeds, of the carnivore family CANIDAE. They are worldwide in distribution and live in association with people. (Walker's Mammals of the World, 5th ed, p1065)	Canis familiaris,Dog
D004721	Endoplasmic Reticulum	A system of cisternae in the CYTOPLASM of many cells. In places the endoplasmic reticulum is continuous with the plasma membrane (CELL MEMBRANE) or outer membrane of the nuclear envelope. If the outer surfaces of the endoplasmic reticulum membranes are coated with ribosomes, the endoplasmic reticulum is said to be rough-surfaced (ENDOPLASMIC RETICULUM, ROUGH); otherwise it is said to be smooth-surfaced (ENDOPLASMIC RETICULUM, SMOOTH). (King & Stansfield, A Dictionary of Genetics, 4th ed)	Ergastoplasm,Reticulum, Endoplasmic
D000242	Cyclic AMP	An adenine nucleotide containing one phosphate group which is esterified to both the 3'- and 5'-positions of the sugar moiety. It is a second messenger and a key intracellular regulator, functioning as a mediator of activity for a number of hormones, including epinephrine, glucagon, and ACTH.	Adenosine Cyclic 3',5'-Monophosphate,Adenosine Cyclic 3,5 Monophosphate,Adenosine Cyclic Monophosphate,Adenosine Cyclic-3',5'-Monophosphate,Cyclic AMP, (R)-Isomer,Cyclic AMP, Disodium Salt,Cyclic AMP, Monoammonium Salt,Cyclic AMP, Monopotassium Salt,Cyclic AMP, Monosodium Salt,Cyclic AMP, Sodium Salt,3',5'-Monophosphate, Adenosine Cyclic,AMP, Cyclic,Adenosine Cyclic 3',5' Monophosphate,Cyclic 3',5'-Monophosphate, Adenosine,Cyclic Monophosphate, Adenosine,Cyclic-3',5'-Monophosphate, Adenosine,Monophosphate, Adenosine Cyclic
D000818	Animals	Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA.	Animal,Metazoa,Animalia
D046911	Macromolecular Substances	Compounds and molecular complexes that consist of very large numbers of atoms and are generally over 500 kDa in size. In biological systems macromolecular substances usually can be visualized using ELECTRON MICROSCOPY and are distinguished from ORGANELLES by the lack of a membrane structure.	Macromolecular Complexes,Macromolecular Compounds,Macromolecular Compounds and Complexes,Complexes, Macromolecular,Compounds, Macromolecular,Substances, Macromolecular