9. P R O T E I N M E T A B O L I S M IN C U L T U R E
299
and before degenerative changes occur in organized structures. For
example, it has been shown that embryonic chick tibiotarsi increase
in length, and in wet and dry weights on initial cultivation in chemically defined media (see V o l . 2, Chapter 4 ) . Nutritional requirements
can be demonstrated during the first few days in vitro before the onset of
a failure to maintain the normal structure of the cartilaginous matrix,
which occurs after about the sixth day of cultivation (Biggers, Webb,
Parker and Healy, 1957; Biggers, 1960; Biggers and Lucy, 1960).
B. " N O N - E S S E N T I A L " A M I N O A C I D S
Morgan and Morton (1957), using nutritionally depleted explants
of chick embryonic heart cultured in defined medium M 150, found
that glycine, serine, glutamine and isoleucine were not essential for
maximum survival. Omission of alanine, proline, hydroxyproline,
aspartic acid and glutamic acid was beneficial; despite this, however,
omission of alanine, proline and glutamic acid simultaneously from the
medium did not increase the survival time of the explants. It was
suggested that the inhibitory action of glutamic acid, aspartic acid and
alanine was associated with the accumulation of these compounds in
the culture medium. Gothoskar, Raina, Tate and Ramakrishnan (1960)
have reported that explants of chick heart contain glutamine synthetase
and therefore do not need glutamine, unlike explants of rat heart and
human hypertrophic cervical tissue which require this amino acid.
Experiments with isotopically-labelled metabolites can be used to
provide data on the metabolic characteristics of tissues, which are
complementary to those obtained by nutritional techniques. Thus, it
was shown that aspartic acid, glutamic acid, alanine, serine, glycine
and proline in the protein of chick heart-fibroblasts became labelled
during cultivation of the tissue in a natural medium containing
radioactive glucose (Fischer, Fischer, Landschutz, Ehrensvard, Rafelson
and Stjernholm, 1952-3). Similar observations have been made on
freshly isolated cells from the pectoral muscle of the embryonic chick
(Lucy and Rinaldini, 1959), while Markert (1956) demonstrated that
explants of skin, liver, spleen, kidney, heart and the pigmented layer
of the retina from the embryonic chick synthesized aspartic acid,
glutamic acid, serine, alanine, proline and cysteine, from labelled
glucose.
With regard to organ cultures, glycine, serine, alanine, proline,
hydroxyproline, aspartic acid, glutamic acid, cystine and cysteine were
found to be non-essential for the embryonic chick tibia cultivated
in a defined medium, (Biggers, Webb, Parker and Healy, 1957).
Furthermore, Webb and Biggers (1961) showed that glycine, serine,
299
and before degenerative changes occur in organized structures. For
example, it has been shown that embryonic chick tibiotarsi increase
in length, and in wet and dry weights on initial cultivation in chemically defined media (see V o l . 2, Chapter 4 ) . Nutritional requirements
can be demonstrated during the first few days in vitro before the onset of
a failure to maintain the normal structure of the cartilaginous matrix,
which occurs after about the sixth day of cultivation (Biggers, Webb,
Parker and Healy, 1957; Biggers, 1960; Biggers and Lucy, 1960).
B. " N O N - E S S E N T I A L " A M I N O A C I D S
Morgan and Morton (1957), using nutritionally depleted explants
of chick embryonic heart cultured in defined medium M 150, found
that glycine, serine, glutamine and isoleucine were not essential for
maximum survival. Omission of alanine, proline, hydroxyproline,
aspartic acid and glutamic acid was beneficial; despite this, however,
omission of alanine, proline and glutamic acid simultaneously from the
medium did not increase the survival time of the explants. It was
suggested that the inhibitory action of glutamic acid, aspartic acid and
alanine was associated with the accumulation of these compounds in
the culture medium. Gothoskar, Raina, Tate and Ramakrishnan (1960)
have reported that explants of chick heart contain glutamine synthetase
and therefore do not need glutamine, unlike explants of rat heart and
human hypertrophic cervical tissue which require this amino acid.
Experiments with isotopically-labelled metabolites can be used to
provide data on the metabolic characteristics of tissues, which are
complementary to those obtained by nutritional techniques. Thus, it
was shown that aspartic acid, glutamic acid, alanine, serine, glycine
and proline in the protein of chick heart-fibroblasts became labelled
during cultivation of the tissue in a natural medium containing
radioactive glucose (Fischer, Fischer, Landschutz, Ehrensvard, Rafelson
and Stjernholm, 1952-3). Similar observations have been made on
freshly isolated cells from the pectoral muscle of the embryonic chick
(Lucy and Rinaldini, 1959), while Markert (1956) demonstrated that
explants of skin, liver, spleen, kidney, heart and the pigmented layer
of the retina from the embryonic chick synthesized aspartic acid,
glutamic acid, serine, alanine, proline and cysteine, from labelled
glucose.
With regard to organ cultures, glycine, serine, alanine, proline,
hydroxyproline, aspartic acid, glutamic acid, cystine and cysteine were
found to be non-essential for the embryonic chick tibia cultivated
in a defined medium, (Biggers, Webb, Parker and Healy, 1957).
Furthermore, Webb and Biggers (1961) showed that glycine, serine,