MOLECULAR-FORM AND KINETIC-PROPERTIES OF PHOSPHATE-DEPENDENT GLUTAMINASE IN THE MITOCHONDRIA ISOLATED FROM THE KIDNEYS OF NORMAL AND ACIDOTIC RATS

1. 1. Investigation of the mechanism of renal ammoniagenesis revealed that the Km of phosphate-dependent glutaminase (l-glutamine amidohydrolase, EC 3.5.1.2) for glutamine was significantly decreased, whereas V was increased if the mitochondria from both normal and acidotic rats were suspended in a hypotonic medium. These and other data indicate that the inner membrane regulates glutaminase activity and that in metabolic acidosis the rate of glutamine influx is increased. 2. 2. The study of molecular form and kinetic properties of glutaminase contained in the mitochondria showed that only in the case of acidotic Wistar rats the enzyme exhibits unusual stability if heated 10 min at 50°C. Furthermore, 3 min heating at 50°C resulted in a strong activation of the enzyme. This suggests that in acidosis the enzyme is in a different molecular form or in a different conformational state. 3. 3. The enzyme is equally sensitive to the inhibition by glutamate and NaCl in both kinds of mitochondria, whereas the value of the Hill coefficient for phosphate is 2. This indicates that glutaminase is in the form of a monomer. It is most probable that in vivo there is an equilibrium between monomer and dimer forms of the enzyme. 4. 4. Determination of the activation energy of glutaminase showed that in the case of normal and acidotic Wistar rats a break in the Arrhenius plot at 28°C and 24°C occurs. This difference results probably from changes in the enzyme microenvironment during adaptation to acidosis. © 1979.