Solubilization and partial characterization of a microsomal high affinity GTPase

Conference · Fri May 01 04:00:00 EDT 1987 · Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States)

OSTI ID:6029582

Nicchitta, C; Williamson, J R

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-
• Conference Information: Journal Name: Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States) Journal Volume: 46:6
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ACID ANHYDRASES
AFFINITY
ANIMALS
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BODY
CELL CONSTITUENTS
CELL MEMBRANES
CHEMICAL COMPOSITION
DAYS LIVING RADIOISOTOPES
DIGESTIVE SYSTEM
ENZYME ACTIVITY
ENZYMES
GLANDS
GUANYLIC ACID
HYDROLASES
ISOTOPE APPLICATIONS
ISOTOPES
LIGHT NUCLEI
LIVER
MAMMALS
MEMBRANES
MICROSOMES
NUCLEI
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANOIDS
ORGANS
PHOSPHOHYDROLASES
PHOSPHORUS 32
PHOSPHORUS ISOTOPES
RADIOISOTOPES
RATS
RESPONSE MODIFYING FACTORS
RODENTS
SOLUBILITY
TRACER TECHNIQUES
VERTEBRATES