272
JOHN RUNNSTRÖM
able material for these studies. After a certain latency period there was
a decrease of the negative potential of the egg surface, often with an overshoot to the positive side. After about 1 min the potential returned to its
resting value. During the same time there was a decrease in the membrane resistance that indicates an increased permeability. This may
allow the entrance of certain cations, such as Ca
2 +
, the role of which will
be discussed below (Sections III,C,D and IV,B).
D. The Role of Enzymes in the Cortical Changes
Örström (1942) found an increase in the concentration of free amino
acids upon fertilization. This result was later confirmed and extended
(Kavanau, 1953, 1954; Ricotta, 1956). Lundblad (1954; for further
references see Runnström et al., 1959) characterized some different
proteolytic enzymes found in extracts or homogenates of
Paracentrotus
eggs. The activity was tested in several ways but usually by measuring
the changes in viscosity of high-polymer gelatin after exposure to the
enzyme-containing extract or homogenate. A cathepsin belonging to
type Β had a pH optimum around pH 4.5. Three proteolytic enzymes,
Ε 1-E 3, had optima in a region pH 6.7-7.8; Ε 2 is inhibited, Ε 1 and Ε 3
are activated by cysteine or glutathione. In the fertilization, the enzymes
Ε 1-E 3 are activated, probably in the order Ε 2, Ε 1, Ε 3 (Lundblad,
1954, Fig. 3). Their activity subsides soon, however, and about 15 min
after insemination it has almost disappeared. A gelating enzyme is activated in whole unfertilized eggs of Paracentrotus
and
Psammechinus
exposed to low doses of trypsin. This process requires Ca
2+
(Runnström,
1961a). Moreover it is removed by treatment of the eggs with reduced
glutathione (Runnström and Kriszat, 1962). In extracts or homogenates,
the enzyme Ε 2 is also dependent for its activity on the presence of Ca
2 +
,
but it is, as mentioned, inhibited by cysteine or by reduced glutathione. It
was inferred that the gelating enzyme is identical with Ε 2, which may
only split a few linkages in proteins, in this way giving rise to new
interactions leading to gel formation.
Enzymes Ε 1 and Ε 3 seem to be more pronouncedly hydrolytic than
Ε 2 (Lundblad and Runnström, 1962). Maggio (1957) demonstrated
(with casein substrate) in egg homogenates of Paracentrotus
lividus
the presence of a protease with the optimum activity in the range pH
5.4r-5.2. In the mitochondria-free cytoplasm this activity undergoes
an increase at fertilization.
No pronounced difference in activity of cathepsin Β was found when
testing extracts or homogenates of unfertilized and fertilized eggs.
Within the egg, however, an activation may occur due to acid formation
which may bring down the pH value in a cortical region so that it ap-
JOHN RUNNSTRÖM
able material for these studies. After a certain latency period there was
a decrease of the negative potential of the egg surface, often with an overshoot to the positive side. After about 1 min the potential returned to its
resting value. During the same time there was a decrease in the membrane resistance that indicates an increased permeability. This may
allow the entrance of certain cations, such as Ca
2 +
, the role of which will
be discussed below (Sections III,C,D and IV,B).
D. The Role of Enzymes in the Cortical Changes
Örström (1942) found an increase in the concentration of free amino
acids upon fertilization. This result was later confirmed and extended
(Kavanau, 1953, 1954; Ricotta, 1956). Lundblad (1954; for further
references see Runnström et al., 1959) characterized some different
proteolytic enzymes found in extracts or homogenates of
Paracentrotus
eggs. The activity was tested in several ways but usually by measuring
the changes in viscosity of high-polymer gelatin after exposure to the
enzyme-containing extract or homogenate. A cathepsin belonging to
type Β had a pH optimum around pH 4.5. Three proteolytic enzymes,
Ε 1-E 3, had optima in a region pH 6.7-7.8; Ε 2 is inhibited, Ε 1 and Ε 3
are activated by cysteine or glutathione. In the fertilization, the enzymes
Ε 1-E 3 are activated, probably in the order Ε 2, Ε 1, Ε 3 (Lundblad,
1954, Fig. 3). Their activity subsides soon, however, and about 15 min
after insemination it has almost disappeared. A gelating enzyme is activated in whole unfertilized eggs of Paracentrotus
and
Psammechinus
exposed to low doses of trypsin. This process requires Ca
2+
(Runnström,
1961a). Moreover it is removed by treatment of the eggs with reduced
glutathione (Runnström and Kriszat, 1962). In extracts or homogenates,
the enzyme Ε 2 is also dependent for its activity on the presence of Ca
2 +
,
but it is, as mentioned, inhibited by cysteine or by reduced glutathione. It
was inferred that the gelating enzyme is identical with Ε 2, which may
only split a few linkages in proteins, in this way giving rise to new
interactions leading to gel formation.
Enzymes Ε 1 and Ε 3 seem to be more pronouncedly hydrolytic than
Ε 2 (Lundblad and Runnström, 1962). Maggio (1957) demonstrated
(with casein substrate) in egg homogenates of Paracentrotus
lividus
the presence of a protease with the optimum activity in the range pH
5.4r-5.2. In the mitochondria-free cytoplasm this activity undergoes
an increase at fertilization.
No pronounced difference in activity of cathepsin Β was found when
testing extracts or homogenates of unfertilized and fertilized eggs.
Within the egg, however, an activation may occur due to acid formation
which may bring down the pH value in a cortical region so that it ap-