ABSTRACT
L-Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) of the Rhodotorula aurantiaca strain KM-1 deaminates L-phenylalanine according to the Michaelis-Menten kinetics with K(M) = 1.75 +/- 0.44 mM and V(max) = 3.01 +/- 0.43 units/mg. The enzyme is competitively inhibited by D-phenylalanine with K(in) = 3.38 +/- 0.32 mM. The Michaelis-Menten kinetics was analyzed, the inhibition type (competitive, noncompetitive, and mixed) was identified, and corresponding kinetic parameters were calculated using the computer programs written in Gauss 4.0. PAL was most stable at pH 6.55 and lacked approximately 50% of its activity after incubation at 57 degrees C for 15 min. The yield of L-phenylalanine increased in the presence of mercaptoethanol, sodium ethylenediaminetetraacetate (EDTA), and ascorbic acid. The effects of EDTA and ascorbic acid were additive.
Subject(s)
Phenylalanine Ammonia-Lyase/metabolism , Rhodotorula/enzymology , Ascorbic Acid/chemistry , Ascorbic Acid/pharmacology , Catalysis , Edetic Acid/chemistry , Edetic Acid/pharmacology , Enzyme Stability , Hydrogen-Ion Concentration , Kinetics , Mercaptoethanol/chemistry , Mercaptoethanol/pharmacology , Phenylalanine/biosynthesis , Phenylalanine/pharmacology , Phenylalanine Ammonia-Lyase/antagonists & inhibitors , Phenylalanine Ammonia-Lyase/chemistry , Rhodotorula/drug effects , Structure-Activity RelationshipABSTRACT
K+ fluxes of rat cortical synaptosomes were monitored by a K+ sensitive ion-selective membrane electrode. The uptake phases of K+curves from control and experimental measurements were compared and statistically evaluated. GABA significantly reduced K+ uptake but it was able to reverse the decrease of K+ uptake caused by protoveratrine. In the absence of Ca2+ the K+ uptake was diminished; GABA had no effect on this inhibition. The effect of GABA on K+ take was partially reduced by bicuculline and abolished by diamino-butyric acid. The K+ content of synaptosomes decreased, while the NA+ content increased in the presence of GABA. The results are discussed in association with presynaptic depolarisation and with modulatory effects of GABA on the release of other transmitters.
