1 1 . M U C O P O L Y S A C C H A R I D E S IN T I S S U E C U L T U R E
381
merits and late foetal mouse bones. These included a uniform loss of
metachromasia similar to that found in explants grown in the presence of
excess vitamin A, although the changes in the cartilage cells that occur
in the presence of vitamin A (see p. 383) were not observed in the
papain-treated explants. Moreover, the enzyme did not cause the
rapid resorption of bone that occurred in the presence of the vitamin.
These findings in vitro are similar to those of Thomas, McCluskey,
Potter and Weissmann (1960) in vivo.
Decrease in metachromasia in the bone rudiments was associated
with the rapid loss of hexosamine (Dingle et al., 1961). Recently, Fell
and Dingle (1963) have shown that a protease preparation from rat
liver lysosomes can degrade the matrix of cartilaginous rudiments with
the liberation of chondroitin sulphate and loss of metachromasia. These
enzymes probably act by the hydrolysis of a small number of linkages
in the protein component of the mucoprotein.
The ability of the cells to change the extracellular material can be
inferred from the experiments of Lucy, Dingle and Fell (1961) in which
hypotonic treatment of rudiments caused the loss of both chondroitin
sulphate and metachromasia. This effect was shown to be pH- and
temperature-dependent, and was accompanied by the release of an acid
protease from the ruptured cells. Incubation of such hypotonically
treated rudiments greatly reduced the metachromatic staining of the
matrix and caused the loss of hexosamine, mostly as polysaccharide of
high molecular weight; at the same time protein components were
liberated into the buffer. Amino acid analysis of the protein material
associated with the liberated mucopolysaccharides indicated that it was
probably derived from non-collagenous protein. It differed significantly
from that of the non-collagenous protein of the whole chick limb-bone
rudiment, however, in having considerably less glutamic acid, aspartic
acid and leucine, and more serine and proline relative to glycine.
From these and other observations the authors suggested that the
degradation of non-collagenous protein possibly results from enzymic
action on the linkage between glycine and glutamic acid, and concluded that the cells of embryonic chick cartilage contain an enzyme
that is able to degrade the extracellular material. This, or a similar
enzyme, has recently been shown to be present in bound form, in
mammalian and avian embryonic cartilage and is probably present
in most tissues (Fell and Dingle, 1963). Recent work in this laboratory
(discussed in Chapter 17 of this volume) has shown that this bound
enzyme (or enzymes) can be released by treatment with vitamin A. It is
probably, however, that the release of these enzymes can be modified by
a number of compounds of physiological importance, e.g. hydrocortisone (Weissmann and Dingle, 1961). The relationship of these released
381
merits and late foetal mouse bones. These included a uniform loss of
metachromasia similar to that found in explants grown in the presence of
excess vitamin A, although the changes in the cartilage cells that occur
in the presence of vitamin A (see p. 383) were not observed in the
papain-treated explants. Moreover, the enzyme did not cause the
rapid resorption of bone that occurred in the presence of the vitamin.
These findings in vitro are similar to those of Thomas, McCluskey,
Potter and Weissmann (1960) in vivo.
Decrease in metachromasia in the bone rudiments was associated
with the rapid loss of hexosamine (Dingle et al., 1961). Recently, Fell
and Dingle (1963) have shown that a protease preparation from rat
liver lysosomes can degrade the matrix of cartilaginous rudiments with
the liberation of chondroitin sulphate and loss of metachromasia. These
enzymes probably act by the hydrolysis of a small number of linkages
in the protein component of the mucoprotein.
The ability of the cells to change the extracellular material can be
inferred from the experiments of Lucy, Dingle and Fell (1961) in which
hypotonic treatment of rudiments caused the loss of both chondroitin
sulphate and metachromasia. This effect was shown to be pH- and
temperature-dependent, and was accompanied by the release of an acid
protease from the ruptured cells. Incubation of such hypotonically
treated rudiments greatly reduced the metachromatic staining of the
matrix and caused the loss of hexosamine, mostly as polysaccharide of
high molecular weight; at the same time protein components were
liberated into the buffer. Amino acid analysis of the protein material
associated with the liberated mucopolysaccharides indicated that it was
probably derived from non-collagenous protein. It differed significantly
from that of the non-collagenous protein of the whole chick limb-bone
rudiment, however, in having considerably less glutamic acid, aspartic
acid and leucine, and more serine and proline relative to glycine.
From these and other observations the authors suggested that the
degradation of non-collagenous protein possibly results from enzymic
action on the linkage between glycine and glutamic acid, and concluded that the cells of embryonic chick cartilage contain an enzyme
that is able to degrade the extracellular material. This, or a similar
enzyme, has recently been shown to be present in bound form, in
mammalian and avian embryonic cartilage and is probably present
in most tissues (Fell and Dingle, 1963). Recent work in this laboratory
(discussed in Chapter 17 of this volume) has shown that this bound
enzyme (or enzymes) can be released by treatment with vitamin A. It is
probably, however, that the release of these enzymes can be modified by
a number of compounds of physiological importance, e.g. hydrocortisone (Weissmann and Dingle, 1961). The relationship of these released