290
H A R R Y
E A G L E
A N D L E O N
L E V I N T O W
utilization may in fact involve its prior incorporation into glutamic
acid.
Individual cell lines have been found with a nutritional requirement
for normally "non-essential" amino acids (Table I V ) . In most of these
cases, the cell in fact synthesizes enough of the amino acid for survival
and growth, and its dependence results from a loss of the product to
the medium in amounts which exceed the biosynthetic capacity of the
cell. In consequence, the requirement is population-dependent, and
disappears at cell densities sufficiently high to "condition" the medium
(i.e. to build up the extracellular and intracellular concentrations to
levels consistent with their normal metabolism) before the cells die
of the specific amino acid deficiency (cf. Table II).
B . A M I N O A C I D T R A N S P O R T , A M I N O A C I D
P O O L S
A N D T H E S Y N T H E S I S O F C E L L U L A R
P R O T E I N
Cultured cells contain a large pool of free amino acids in dynamic
equilibrium with the surrounding fluid. When the amino acids are
provided, they are concentrated within the cell to reach levels three
to fifty times that in the medium, the degree of concentration varying
with the specific compound, its absolute concentration in the medium,
and the cell-type. With some amino acids (e.g. valine), the degree of
concentration is, within wide limits, independent of the absolute
external level; with others, however (e.g. threonine), the degree of
concentration is much greater at low external levels than at high, as if
a transport mechanism were being saturated at the higher levels
(Fig. 1). When the concentration in the environment is abruptly
increased or decreased, the new equilibrium is largely established within
15 min.
The amino acids synthesized by the cell are retained concentratively,
so that the intracellular levels may be as much as 20-50 times those
in the surrounding fluid. However, since the cells constitute so small
a proportion ofthe total volume ofthe culture (approximately 1/2500th
at 100,000 cells/ml) it follows that even if the cell: medium ratio is
50 : 1, in absolute amounts, 50 times as much of the newly synthesized
amino acid has been lost to the medium as is retained by the cell.
Because small numbers of cells can be grown in relatively large
volumes of fluid, the environmental amino acid levels can be kept constant even at concentrations so low as to be growth limiting. This
has made it possible to determine the minimum intracellular concentration of amino acid required for protein synthesis. (Eagle, Piez and Levy,
1961). There proves to be a critical threshold level (0-01-0-05 IIIM)
below which there is no appreciable protein synthesis, and no demon-
H A R R Y
E A G L E
A N D L E O N
L E V I N T O W
utilization may in fact involve its prior incorporation into glutamic
acid.
Individual cell lines have been found with a nutritional requirement
for normally "non-essential" amino acids (Table I V ) . In most of these
cases, the cell in fact synthesizes enough of the amino acid for survival
and growth, and its dependence results from a loss of the product to
the medium in amounts which exceed the biosynthetic capacity of the
cell. In consequence, the requirement is population-dependent, and
disappears at cell densities sufficiently high to "condition" the medium
(i.e. to build up the extracellular and intracellular concentrations to
levels consistent with their normal metabolism) before the cells die
of the specific amino acid deficiency (cf. Table II).
B . A M I N O A C I D T R A N S P O R T , A M I N O A C I D
P O O L S
A N D T H E S Y N T H E S I S O F C E L L U L A R
P R O T E I N
Cultured cells contain a large pool of free amino acids in dynamic
equilibrium with the surrounding fluid. When the amino acids are
provided, they are concentrated within the cell to reach levels three
to fifty times that in the medium, the degree of concentration varying
with the specific compound, its absolute concentration in the medium,
and the cell-type. With some amino acids (e.g. valine), the degree of
concentration is, within wide limits, independent of the absolute
external level; with others, however (e.g. threonine), the degree of
concentration is much greater at low external levels than at high, as if
a transport mechanism were being saturated at the higher levels
(Fig. 1). When the concentration in the environment is abruptly
increased or decreased, the new equilibrium is largely established within
15 min.
The amino acids synthesized by the cell are retained concentratively,
so that the intracellular levels may be as much as 20-50 times those
in the surrounding fluid. However, since the cells constitute so small
a proportion ofthe total volume ofthe culture (approximately 1/2500th
at 100,000 cells/ml) it follows that even if the cell: medium ratio is
50 : 1, in absolute amounts, 50 times as much of the newly synthesized
amino acid has been lost to the medium as is retained by the cell.
Because small numbers of cells can be grown in relatively large
volumes of fluid, the environmental amino acid levels can be kept constant even at concentrations so low as to be growth limiting. This
has made it possible to determine the minimum intracellular concentration of amino acid required for protein synthesis. (Eagle, Piez and Levy,
1961). There proves to be a critical threshold level (0-01-0-05 IIIM)
below which there is no appreciable protein synthesis, and no demon-