310
J . A . L U C Y
contain both an increased total quantity of bound protease and more
protease per unit wet weight as compared with control cultures at the
end of the experiment. This observation indicates that the vitamin
increases the synthesis of the protease in addition to stimulating release
of the enzyme in an active form (Fell and Dingle, 1963).
It is of interest that Carrel and Baker (1926) in experiments on the
constituents of embryo juice responsible for cell growth showed that
when the juice was incubated at a p H between 7*6 and 9-0 no increase
in amino nitrogen was apparent. At p H 6*2, and especially at p H 5,
however, the quantity of amino nitrogen increased rapidly, indicating
the presence of a proteolytic enzyme with a p H optimum in the acid
region. They suggested that the p H at the surface of cells in culture
might be sufficiently low to enable enzymes present in the embryo
juice to hydrolyse proteins into proteoses that could be absorbed and
utilized by the tissues.
Little is known about the mechanism of resorption of collagen
in vitro. Harkness (1961) has pointed out that resorption must be capable of close control and localization, and that this may be brought
about by the local secretion of enzymes at the surface of the cell
involved. The factors controlling the resorption of bone collagen can
be conveniently studied in tissue culture, especially in view of the fact
that excess vitamin A has been shown to cause rapid resorption of bone
in vitro (Fell and Mellanby, 1952). In preliminary experiments, it has
been found that treatment of mouse bone in culture with an excess
of vitamin A causes a decrease in the hydroxyproline content of the
bone while control cultures gain in hydroxyproline during the same
period (Fell and Lucy, unpublished observations).
Hayashi, Tokuda and Udaka (1960) have used tissue culture
techniques to study the problem of the activation of proteolytic
enzyme-systems in allergic reactions. Cultures of freshly isolated
monocytes from the peritoneal fluid of sensitized rabbits were used,
and it was found that introduction of antigen into the culture medium
caused a release of protease within 10 min: there was no further increase
in activity with time. The released protease was inhibited by />-chloromercuribenzoate, and this inhibition was overcome by cysteine. The
enzyme had optimum activity at p H 7*4. Observations made with
a phase-contrast microscope showed that retraction of cytoplasmic
processes, swelling ofthe cytoplasm and formation of a fibrous reticulum
in the ground substance accompanied the release of the protease. It
was concluded therefore that the site of protease activation was the
"cytoplasmic ground substance". About 1 h after the release of enzyme,
filamentous mitochondria were disintegrated into granules. In related
work, it was found that the protease activity of the culture decreased
J . A . L U C Y
contain both an increased total quantity of bound protease and more
protease per unit wet weight as compared with control cultures at the
end of the experiment. This observation indicates that the vitamin
increases the synthesis of the protease in addition to stimulating release
of the enzyme in an active form (Fell and Dingle, 1963).
It is of interest that Carrel and Baker (1926) in experiments on the
constituents of embryo juice responsible for cell growth showed that
when the juice was incubated at a p H between 7*6 and 9-0 no increase
in amino nitrogen was apparent. At p H 6*2, and especially at p H 5,
however, the quantity of amino nitrogen increased rapidly, indicating
the presence of a proteolytic enzyme with a p H optimum in the acid
region. They suggested that the p H at the surface of cells in culture
might be sufficiently low to enable enzymes present in the embryo
juice to hydrolyse proteins into proteoses that could be absorbed and
utilized by the tissues.
Little is known about the mechanism of resorption of collagen
in vitro. Harkness (1961) has pointed out that resorption must be capable of close control and localization, and that this may be brought
about by the local secretion of enzymes at the surface of the cell
involved. The factors controlling the resorption of bone collagen can
be conveniently studied in tissue culture, especially in view of the fact
that excess vitamin A has been shown to cause rapid resorption of bone
in vitro (Fell and Mellanby, 1952). In preliminary experiments, it has
been found that treatment of mouse bone in culture with an excess
of vitamin A causes a decrease in the hydroxyproline content of the
bone while control cultures gain in hydroxyproline during the same
period (Fell and Lucy, unpublished observations).
Hayashi, Tokuda and Udaka (1960) have used tissue culture
techniques to study the problem of the activation of proteolytic
enzyme-systems in allergic reactions. Cultures of freshly isolated
monocytes from the peritoneal fluid of sensitized rabbits were used,
and it was found that introduction of antigen into the culture medium
caused a release of protease within 10 min: there was no further increase
in activity with time. The released protease was inhibited by />-chloromercuribenzoate, and this inhibition was overcome by cysteine. The
enzyme had optimum activity at p H 7*4. Observations made with
a phase-contrast microscope showed that retraction of cytoplasmic
processes, swelling ofthe cytoplasm and formation of a fibrous reticulum
in the ground substance accompanied the release of the protease. It
was concluded therefore that the site of protease activation was the
"cytoplasmic ground substance". About 1 h after the release of enzyme,
filamentous mitochondria were disintegrated into granules. In related
work, it was found that the protease activity of the culture decreased