Kinetic analysis of guanosine 5'-O-(3-thiotriphosphate) effects on phosphatidylinositol turnover in NRK cell homogenates
Addition of the guanine nucleotide analogue guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S) to (/sup 3/H)inositol-labeled NRK cell homogenates resulted in rapid breakdown of cellular polyphosphoinositides. GTP gamma S stimulated phospholipase C, resulting in a more than 4-fold increase in the hydrolysis rates of phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bis(phosphate) (PIP2). No significant effect of GTP gamma S on direct phosphatidylinositol (PI) hydrolysis was detected. There was an increase in water-soluble inositols, with inositol tris(phosphate) (IP3) levels increasing at least 10 times over the decrease seen in PIP2, indicating that PIP kinase activity was also accelerated following GTP gamma S addition. Inositol 1,4,5-tris(phosphate) peaked rapidly after GTP gamma S addition (less than 2 min) while inositol 1,3,4-tris-(phosphate) was produced more slowly and leveled off after approximately 10 min. The differential equations describing conversion between intermediates in the PI turnover pathway were solved and fitted to data obtained from both (/sup 3/H)inositol and (/sup 32/P)phosphate fluxes by nonlinear least-squares analysis. GTP gamma S effects on the pseudo-first-order rate constants for the lipase, kinase, and phosphatase steps were determined from the analysis. From these measurements it can be estimated that, in the presence of GTP gamma S and calcium buffered to 130 nM, hydrolysis of PIP2 accounts for at least 10 times as much diacylglycerol as direct PI breakdown despite the 100-fold excess of PI over PIP2. From the kinetic model it is predicted that small changes in the activities of PI and PIP kinases can have large but different effects on the level of IP3 and diacylglycerol following GTP gamma S addition. These results argue that regulation of PI and PIP kinases may be important for determining both cellular IP3 and diacylglycerol levels.
- Research Organization:
- Tufts Univ. School of Medicine, Boston, MA
- OSTI ID:
- 6525965
- Journal Information:
- Biochemistry; (United States), Journal Name: Biochemistry; (United States) Vol. 2; ISSN BICHA
- Country of Publication:
- United States
- Language:
- English
Similar Records
Plasma membrane associated phospholipase C from human platelets: Synergistic stimulation of phosphatidylinositol 4,5-bisphosphate hydrolysis by thrombin and guanosine 5 prime -O-(3-thiotriphosphate)
Rapid changes in polyphosphoinositide metabolism associated with the response of Dunaliella salina to hypoosmotic shock
Related Subjects
59 BASIC BIOLOGICAL SCIENCES
AMINES
ANIMAL CELLS
ANIMALS
AROMATICS
ATP
AZAARENES
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
BIOCHEMICAL REACTION KINETICS
BODY
CARBOHYDRATES
CHEMICAL REACTIONS
DAYS LIVING RADIOISOTOPES
ENZYME ACTIVITY
GUANINE
GUANOSINE
HETEROCYCLIC COMPOUNDS
HOMOGENATES
HYDROXY COMPOUNDS
INOSITOLS
ISOTOPES
KIDNEYS
KINETICS
LABELLED COMPOUNDS
LIGHT NUCLEI
MAMMALS
MATHEMATICS
MONOSACCHARIDES
NUCLEI
NUCLEOSIDES
NUCLEOTIDES
ODD-ODD NUCLEI
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
ORGANS
PHOSPHORUS 32
PHOSPHORUS COMPOUNDS
PHOSPHORUS ISOTOPES
PHOSPHORYLATION
PURINES
RADIOISOTOPES
RATS
REACTION KINETICS
RIBOSIDES
RODENTS
SACCHARIDES
TRITIUM COMPOUNDS
VERTEBRATES