JBC, Vol. 251, Issue 1, 88-97, Jan, 1976
Biochemical studies of tast sensation. Binding of L-[3H]alanine to a sedimentable fraction from catfish barbel epithelium
J. M. Krueger and R. H. Cagan
Large numbers of taste buds are distributed over the body surface of the
channel catfish ictalurus punctatus, with the barbels having an especially
high density. L-Alanine, as well as certain other amino acids, are taste
stimuli in this animal. Epithelial tissue obtained by gentle scraping of
the barbel surface was fractionated by differential centrifugation. A
sedimentable fraction (P2) was prepared that was enriched in L[OH]alanine
binding activity, the plasma membrane marker enzyme 5'-nucleotidase, and
the mitochondrial marker succinate cytochrome c reductase, but not the
microsomal marker NADH cytochrome c redu.ctase. Binding of L-[OH]alanine
was measured using a Millipore filter method in which correction for
non-specific binding was also determined. Time, temperature, and pH for
measuring binding activity were established. At the optimal pH of 7.8, the
KD for L-alanine is 4.8 X 10(-6) M. The first order dissociation rate
constant at 6 degrees is 3.8 X 10(-4) s-1 and at 24 degrees it is 12.1 X
10(-4) s-1. The second order rate constant for association is between 10(2)
and 10(3) M-1 S-1. Reversibility of the binding interaction was also
demonstrates by the rapid displacement of bound L-[3H]alanine by a large
excess of unlabeled L-alanine. That the binding does not represent
incorporation into protein was confirmed by the lack of effect of
puromycin. The amounts bound of several other chemostimulatory amino acids
werealso determined.
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