378
J .
T . D I N G L E A N D M .
W E B B
glutamine (Leloir and Cardini, 1953; Pogell, 1956; Lowther and
Rogers, 1956; Pogell and Koenig, 1959). The relative importance of
these pathways in different tissues has yet to be determined. The enzymic synthesis of glucosamine as its 6 - P 0 4
3_ from hexosephosphate and
glutamine was demonstrated clearly by Leloir and Cardini (1953),
Pogell and Gryder (1957) and Pogell and Koenig (1959). More recently,
Ghosh, Blumenthal, Davidson and Roseman (1960) have shown that
fructose-6-phosphate, derived from glucose-1-phosphate by the action
of phosphoglucoisomerase, is the active acceptor in this synthesis.
The incorporation of ammonia nitrogen into glucosamine also
occurs via fructose-6-phosphate and requires the presence of glucosamine-6-phosphate deaminase. Normally, equilibrium lies far towards
fructose-6-phosphate, but it can be shifted by coupling with glucosamine-6-phosphate-acetylase and acetyl CoA. The presence of this
enzyme system, which occurs in rat and pig liver (Leloir and Cardini,
1956) and E. coli (Comb and Roseman, 1956) has been detected in
tissue cultures of fibroblasts (Gaines, 1960).
Recent work of Markovitz, Cifonelli and Dorfman (1958, 1959)
and Markovitz and Dorfman (1962a, b) has shown that: (a) in the
synthesis of mucopolysaccharides the monosaccharide precursors are
utilized as their U D P derivatives; and (b) glucosamine is acetylated
before incorporation, but provides no information of the mechanism
of formation ofthe polysaccharide chain. Schoenberg and Moore (1958)
have produced evidence for the existence for a tri- or tetra-saccharide
precursor of hyaluronic acid and/or chondroitin sulphate in both the
cytoplasm of fibroblasts and the extracellular material of umbilical
cord. The mechanism of the synthesis of the complete polysaccharide
chain, however, remains to be established for both the sulphated and
non-sulphated mucopolysaccharides.
A number of recent observations (see Bostrdm, 1959) have shown
that inorganic sulphate is incorporated into mucopolysaccharides and
other conjugated sulphates of mammalian tissues in vivo and in vitro,
but not to any significant extent into sulphur compounds (e.g. methionine, cysteine and taurine). Thus, in mammalian tissues at least, the
uptake of
3 5
S 0 4
2 _ leads to essentially selective labelling of conjugated
sulphates. It is generally assumed that autoradiography, coupled with
histological examination of tissue sections from animals injected with
3 5
S 0 4
2 _ gives a reliable indication of the presence of mucopolysaccharide
sulphates in areas in which radioactivity and metachromatic staining
coincide. Sulphate labelled with
3 5
S has been used frequently to study
mucopolysaccharide synthesis in culture. Mancini, Lustig, Nunez and
Roffo (1956), for example, used a stripping-film technique to study the
incorporation of
3 5
S 0 4
2 ~ by embryonic connective tissue in culture, and
J .
T . D I N G L E A N D M .
W E B B
glutamine (Leloir and Cardini, 1953; Pogell, 1956; Lowther and
Rogers, 1956; Pogell and Koenig, 1959). The relative importance of
these pathways in different tissues has yet to be determined. The enzymic synthesis of glucosamine as its 6 - P 0 4
3_ from hexosephosphate and
glutamine was demonstrated clearly by Leloir and Cardini (1953),
Pogell and Gryder (1957) and Pogell and Koenig (1959). More recently,
Ghosh, Blumenthal, Davidson and Roseman (1960) have shown that
fructose-6-phosphate, derived from glucose-1-phosphate by the action
of phosphoglucoisomerase, is the active acceptor in this synthesis.
The incorporation of ammonia nitrogen into glucosamine also
occurs via fructose-6-phosphate and requires the presence of glucosamine-6-phosphate deaminase. Normally, equilibrium lies far towards
fructose-6-phosphate, but it can be shifted by coupling with glucosamine-6-phosphate-acetylase and acetyl CoA. The presence of this
enzyme system, which occurs in rat and pig liver (Leloir and Cardini,
1956) and E. coli (Comb and Roseman, 1956) has been detected in
tissue cultures of fibroblasts (Gaines, 1960).
Recent work of Markovitz, Cifonelli and Dorfman (1958, 1959)
and Markovitz and Dorfman (1962a, b) has shown that: (a) in the
synthesis of mucopolysaccharides the monosaccharide precursors are
utilized as their U D P derivatives; and (b) glucosamine is acetylated
before incorporation, but provides no information of the mechanism
of formation ofthe polysaccharide chain. Schoenberg and Moore (1958)
have produced evidence for the existence for a tri- or tetra-saccharide
precursor of hyaluronic acid and/or chondroitin sulphate in both the
cytoplasm of fibroblasts and the extracellular material of umbilical
cord. The mechanism of the synthesis of the complete polysaccharide
chain, however, remains to be established for both the sulphated and
non-sulphated mucopolysaccharides.
A number of recent observations (see Bostrdm, 1959) have shown
that inorganic sulphate is incorporated into mucopolysaccharides and
other conjugated sulphates of mammalian tissues in vivo and in vitro,
but not to any significant extent into sulphur compounds (e.g. methionine, cysteine and taurine). Thus, in mammalian tissues at least, the
uptake of
3 5
S 0 4
2 _ leads to essentially selective labelling of conjugated
sulphates. It is generally assumed that autoradiography, coupled with
histological examination of tissue sections from animals injected with
3 5
S 0 4
2 _ gives a reliable indication of the presence of mucopolysaccharide
sulphates in areas in which radioactivity and metachromatic staining
coincide. Sulphate labelled with
3 5
S has been used frequently to study
mucopolysaccharide synthesis in culture. Mancini, Lustig, Nunez and
Roffo (1956), for example, used a stripping-film technique to study the
incorporation of
3 5
S 0 4
2 ~ by embryonic connective tissue in culture, and