Functional intracellular glutaminase activity in intact astrocytes.
Numerous cellular metabolites such as glutamine, glutamate, phosphate, calcium, ammonia and acetyl derivatives are known to affect the phosphate-activated glutaminase activity in whole cell homogenates or extracts. Since measurements in extracts under non-physiological conditions may obscure the actual intracellular metabolic flux, the "functional" intracellular phosphate-activated glutaminase activity was measured by the formation of 3H2O from L-[2-3H]glutamine (Anal. Biochem. 127:134-142, 1982) in cultures of intact astrocytes, untreated and treated with dibutyryl c-AMP (DiBcAMP), in the presence of several potential effectors. These values were compared with enzyme levels determined in extracts from identical cells. The rate of 14CO2 release from L-[1-14C]glutamine was also measured in both untreated and DiBcAMP treated astrocytes. The intracellular activity of glutaminase for untreated cells assayed in MEM medium with 1 mM radioactive glutamine was 88 nmol/mg protein/h and in DiBcAMP treated cells the rate was 153 nmol/mg protein/h. However, the enzymatic activity measured under optimal conditions in extracts from both untreated and treated cells was much higher, but essentially the same, about 1,750 nmol/mg protein/h. The rate of 14CO2 release from L-[1-14C]glutamine was 74 and 133 nmol/mg protein/h in untreated and DiBcAMP treated cells, respectively. This represents approximately 85% of the intracellular glutaminase activity. Furthermore, increasing the concentration of glutamine in the medium from 1 to 6.4 mM increased glutaminase intracellular activity about 3 fold in both untreated and treated cells. Addition of 250 microM glutamate to the medium inhibited intracellular glutaminase activity by 70% under both treatment conditions. Deletion of glucose stimulated glutaminase activity. In contrast the removal of fetal bovine serum decreased activity by 35%.(ABSTRACT TRUNCATED AT 250 WORDS)
Zielke
H R
HR
Department of Pediatrics, University of Maryland School of Medicine, Baltimore 21201.
Tildon
J T
JT
Zielke
C L
CL
Baab
P J
PJ
Landry
M E
ME
D000818:Animals; D001253:Astrocytes / Q000166:cytology / Q000201:enzymology*; D005972:Glutaminase / Q000378:metabolism*; D005973:Glutamine / Q000378:metabolism; D051381:Rats
D005973:Glutamine; D005972:Glutaminase