Solubilization and partial characterization of a microsomal high affinity GTPase
Isolated rat liver microsomes release sequestered Ca/sup 2 +/ following addition of GTP. In contrast to permeabilized cells, GTP dependent microsomal Ca/sup 2 +/ release requires low concentrations of polyethylene glycol (PEG). They have identified a microsomal, PEG-sensitive high affinity GTPase which shares a number of characteristics with the GTP-dependent Ca/sup 2 +/ release system. To aid in further characterization of this activity they have initiated studies on the solubilization and purification of the microsomal GTPases. When microsomes are solubilized under the following conditions (150 mM NaCl, 5 mg protein/ml, 1% Triton X-114) PEG sensitive GTPase activity selectively partitions into the detergent rich phase of the Triton X-114 extract. As observed in intact microsomal membranes the Triton X-114 soluble GTPase is maximally stimulated by 3% PEG. Half maximal stimulation is observed at 1% PEG. PEG increases the Vmax of this activity; no effects on Km were observed. The Km for GTP of the detergent soluble GTPase is 5 ..mu..M. This GTPase is sensitive to inhibition by sulfhydryl reagents. PEG-sensitive GTPase activity was completely inhibited in the presence of 25 ..mu..M p-hydroxymercuribenzoate (PHMB); half maximal inhibition was observed at 5 ..mu..M. Labeling of the Triton X-114 extract with the photosensitive compound (/sup 32/P) 8-azido GTP indicated the presence of two prominent GTP binding proteins of approximate molecular weights 17 and 54 kD.
- Research Organization:
- Univ. of Pennsylvania, Philadelphia
- OSTI ID:
- 6029582
- Report Number(s):
- CONF-870644-; TRN: 87-037137
- Journal Information:
- Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States), Vol. 46:6; Conference: 78. annual meeting of the American Society of Biological Chemists conference, Philadelphia, PA, USA, 7 Jun 1987
- Country of Publication:
- United States
- Language:
- English
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PHOSPHOHYDROLASES
CHEMICAL COMPOSITION
ENZYME ACTIVITY
SOLUBILITY
AFFINITY
CELL MEMBRANES
GUANYLIC ACID
LIVER
MICROSOMES
PHOSPHORUS 32
RATS
RESPONSE MODIFYING FACTORS
TRACER TECHNIQUES
ACID ANHYDRASES
ANIMALS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BODY
CELL CONSTITUENTS
DAYS LIVING RADIOISOTOPES
DIGESTIVE SYSTEM
ENZYMES
GLANDS
HYDROLASES
ISOTOPE APPLICATIONS
ISOTOPES
LIGHT NUCLEI
MAMMALS
MEMBRANES
NUCLEI
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANOIDS
ORGANS
PHOSPHORUS ISOTOPES
RADIOISOTOPES
RODENTS
VERTEBRATES
550201* - Biochemistry- Tracer Techniques