Three enzyme reactions related to asparagine synthesis
were studied in rat tissues : formation of aspartylhydroxamate,
either from aspartate or by transfer from asparagine, and
actual synthesis of asparagine from aspartate. Actual asparagine
synthesis occurred at one-thousandth the rate of the other two
reactions.
Optimal conditions for quantitative assay of asparagine synthesis
were determined in fetal liver extract, which is a rich source
of the enzyme. Demonstrable activity in liver fell 6 days after birth to 20% of the fetal
value and decreased slowly thereafter to the low adult value. Adult pancreas was the most
active tissue found.
The asparagine synthetase of fetal liver extracts was significantly inhibited when combined
with adult liver or tumor extracts. The inhibitor fractionated with ammonium
sulfate in close association with the asparagine synthetase. Therefore, demonstrable activities
of asparagine synthetase in tissue extracts, measured in the presence of this inhibitor,
do not necessarily parallel the concentrations of the enzyme present.
The purification of glutamine synthetase (GS) from
rat liver demonstrates that a small portion of glutamine-hydroxylamine-
glutamyltransferase activity (GT) remains associated with
GS activity (GT(S)). As GS is purified from the water extract, the
ratio between GT(S) and GS remains constant. The major portion
of the GT activity (GT(T)) is left to be extracted by KC1 from the pellet and, on
further purification, appears to be independent of GS activity. Subtle differences in pH
optimum, substrate requirement and reaction rates on addition of cofactors and amino
acids in vitro and in responses to hormonal stimuli in vivo indicate that the glutamine
transfer reaction may be catalyzed by two distinguishable proteins; only the minor component
may be identical to GS.
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