[Detection and characteristics of membrane form of phenylalanine hydroxylase (EC 1.14.16.1) from the rat liver].
The proteins extracted with 0.4% Triton X-100 from the 105000 g homogenate fraction were shown to possess the phenylalanine hydroxylase (EC 1.14.16.1) activity. This phenylalanine hydroxylase fraction was designated as the membrane form of the enzyme. However, immunochemical methods of the antigen analysis performed under non-denaturating conditions and employing monospecific antisera to phenylalanine hydroxylase (double immunodiffusion in agar, racket immunoelectrophoresis, enzyme purification on immunoadsorbents) failed to reveal the antigen among the membrane fraction proteins of the liver. In this fraction the antigen was identified only by immunoblotting performed after electrophoresis of the proteins under denaturating conditions. The molecular mass of the cytoplasmic and membrane forms of the enzyme subunits is identical (52 kD). The Km value of phenylalanine for the cytoplasmic form of phenylalanine hydroxylase is 0.32.10(-3) M, that for the membrane form is 1.66.10(-3) M. Both enzyme forms can bind to phenyl-Sepharose after their activation by the substrate, and they dissociate from the carrier after phenylalanine removal from the incubation mixture, which points to the intactness of the phenylalanine binding allosteric center in the membrane form of the enzyme. This finding allowed for the purification of the membrane form of phenylalanine hydroxylase by affinity chromatography on phenyl-Sepharose.
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002462:Cell Membrane / Q000201:enzymology; D002846:Chromatography, Affinity; D005779:Immunodiffusion; D007122:Immunoelectrophoresis; D007527:Isoenzymes / Q000032:analysis / Q000302:isolation & purification*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D010651:Phenylalanine Hydroxylase / Q000032:analysis / Q000302:isolation & purification*; D051381:Rats
D000596:Amino Acids; D007527:Isoenzymes; D010651:Phenylalanine Hydroxylase
Vyiavlenie i kharakteristika membrannoĭ formy fenilalaningidroksilazy (KF 1.14.16.1) pecheni krys.