Characterization of three kinetically distinct forms of glutamate decarboxylase from pig brain

Abstract

Pig brain contains three forms of glutamate decarboxylase with pI values of 5.3, 5.5 and 5.8, referred to as the α-, β- and γ-forms respectively. These forms were purified and kinetically characterized. The major synaptic form of glutamate decarboxylase (the β-form) migrated as a single band on electrophoresis in sodium dodecyl sulphate/polyacrylamide gels with an apparent Mr of 60 000. Sodium dodecyl sulphate/polyacrylamide gel electrophoresis followed by immunoblotting with an affinity-purified antibody to the enzyme indicated a subunit Mr of 60 000 for the α- and γ-forms as well. An extensive kinetic analysis, aided by an integrated equation that describes the inactivation and re-activation cycle of the enzyme, revealed that the three forms of the enzyme differ markedly in kinetic properties. The Km values for L-glutamate were 0.17, 0.45 and 1.24 mM respectively for the α-, β- and γ-forms. The Ki for 4-aminobutyrate, the first-order rate constants for inactivation by L-glutamate and 4-aminobutyrate, the rate constant for re-activation of the apoenzyme by pyridoxal 5′-phosphate and the dissociation constant for pyridoxal 5′-phosphate also differed in a similar way among the three forms; the values were in the order α-form less than β-form less than γ-form.