HASHIMOTO, SHINYA, KITAO FIJUWARA, KEIICHIRO FUWA, AND

Limnol. Oceanogr., 30(3), 1985, 631-645
0 1985, by the American
Society of Limnology
and Oceanography,
Inc.
Distribution and characteristics of carboxypeptidase activity in
pond, river, and seawaters in the vicinity of Tokyo’
Shinya Hashimoto,
Kitao Fujiwara,2
and Keiichiro
Fuwa
Department of Chemistry, University of Tokyo, Hongo, Bunkyo-ku, Tokyo 113, Japan
Toshiro Saino
Ocean Research Institute, University of Tokyo, Minamidai, Nakano-ku, Tokyo 164
Abstract
Enzymatic hydrolysis of the peptide bond between glycine and tryptophan was measured in river
water, pond water, and seawater near Tokyo. Enzymatic activities were in the range between 0.1
and 3 mU liter-l, 1O-50% of the activity was due to cell-free (dissolved) enzymes (1 mU expresses
the rate of hydrolyzing substrate at 1 nM min-* at 25°C). According to gel permeation chromatography, the dissolved activity included several macromolecules with molecular weights between
30,000 and 500,000 daltons. The horizontal and vertical distributions of this activity were measured
in Tokyo Bay and the open sea, where an appreciable amount was detected even at 1,400-m depth.
Among various factors investigated (including pH, salinity, water temperature, metal ions, and
phosphorus concentrations), total organic carbon, particulate organic carbon, and especially chlorophyll u were positively correlated to the enzymatic activity in Tokyo Bay. The slope of regressions
of chlorophyll a on carboxypeptidase activity for individual sampling points showed that the activity
per unit of chlorophyll a increased when the sampling station was closer to the open sea. Although
whole protein molecules are not decomposed in the dissolved state, small peptides may be degraded
by the free dissolved enzymes.
Proteolytic activity by organisms is important in the distribution
and degradation
of proteins in natural waters (Handa and
Tominaga 1969; Seki 1973; Seki et al. 1974;
Andrews and Williams 197 1; Jannasch et
al. 197 1; Ogura 1972). For example, it is
necessary for solubilizing
particulate proteins, which make up as much as half of the
total particulate organic carbon in some
areas (Krogh and Lange 1932; Ogura 1975).
Enzymatic activities are carried out by organisms in natural waters and, as in the case
of alkaline phosphatase and nitrate reductase, free dissolved enzymes are also active
there (Berman 1969; Kobayashi et al. 1982;
Wetzel 198 1; Stewart and Wetzel 1982;
Kishibe et al. 1982). Among several reports
of the isolation and identification
of microorganisms possessing proteolytic
activity
(Litchfield and Prescott 1970a,b; Merkel et
al. 1964; Tanaka and Iuchi 197 l), Daatselaar and Harder (1974) isolated a marine
bacterium
which produces extracellular
proteolytic
enzymes. However, cell-free
1This work ws supported by Grant-in-Aid 58540349,
57030020, and 5803004 1 from the Ministry ofscience,
Culture and Education, Japan.
2 To whom correspondence should be addressed.
protease activity in natural water has not
been detected (Little et al. 1979) except in
specialized microenvironments
(Sizemore
and Stevenson 1974). Hollibaugh and Azam
(1983) pointed out that the energetic cost is
too high for extracellular enzymes to contribute to the degradation of protein in natural water because of diffusion.
Among the proteases, exo-peptidase,
which works on the release of terminal amino acids from polypeptides, is divided into
amino and carboxypeptidases.
Carboxypeptidase hydrolyzes the peptide bond in
the carboxy terminal of the polypeptide. For
the sake of sensitivity in the detection of the
enzymatic activity, we focus here on the carboxypeptidase activity (CPA) in the water.
Tryptophan can be sensitively detected by
fluorimetry,
and it is possible to measure
the rate at which tryptophan is cleaved from
dansylglycyl-L-tryptophan
in natural water
without any concentration. We have investigated the distributions of the hydrolysis of
the peptide bond between glycine and tryptophan in pond water, river water, and seawater and the relationship between CPA and
various chemical species in eutrophic Tokyo Bay and the less eutrophic open sea.
Also, an attempt was made to detect the
631
632
Hashimoto
distribution
of the dissolved cell-free enzyme (DCPA) in natural water, as well as
the total (cell-associated) carboxypeptidase
activity
(TCPA). The characteristics
of
DCPA from natural water were compared
with the commercial carboxypeptidase
in
terms of pH dependence, thermal and
chemical denaturation, and molecular distribution (gel permeation chromatography).
We thank A. Hattori for his help and suggestions during cruise KT83-9 of the Tansei-maru, and X. Maruyama and M. Sakamoto for their support of this study. We
also thank T. Yoshida and personnel of the
Misaki Marine Biological Station for their
help in the collection and analyses of seawater.
Experimental
Chemicals-- Carboxypeptidase
A from
bovine pancreas (Sigma Chem. Co.) was
used as the reference enzyme. DansylglycylL-tryptophan (Sigma) was used as the substrate and L-tryptophan as the fluorescence
standard. All chemicals were of analytical
grade and were dissolved in Tris-HCl buffer
(pH 7.5) prepared in water from a subboiling distiller. Molecular weights in the gel
chromatography
were calibrated against
standard proteins (Boehringer
Manheim
Co.): ferritin (indicator protein, mol wt =
450,000); catalase (from beef liver, mol
wt = 240,000); aldolase (from rabbit muscle, mol wt = 158,000); albumin (from bovine serum, mol wt = 68,000); albumin
(from hen egg, mol wt = 45,000); and cytochrome c (from horse heart, mol wt =
12,500). Alkaline phosphatase (from Escherichia coli, mol wt = 89,000) and bovine
carbonic anhydrase (mol wt = 3 1,000) were
also used. The void volume (Vo) was determined with blue dextran 2,000 (Pharmacia Fine Chem.).
Instruments-Fluorescence
of tryptophan was measured with a Hitachi type 65060 or 204 fluorometer with a lo- X lo-mm
quartz cell. All enzymatic reactions were determined at 25°C in a thermostatic bath
(Hirayama Manufacturing
Co., model TR900). Sephadex G-200 gel (Pharmacia) and
TSK-GEL HW55S (Toyo Soda Chem. Co.)
were the Chromatographic resins used for
the determination of molecular weights for
et al.
dissolved
carboxypeptidases
in natural
water. A K-16/70 column (1.6-cm diameter, 70 cm long) with an A- 16 adaptor (Pharmacia) and a GF 44 x 60 column (4.4-cm
diameter, 60 cm long) with an AA44 adjuster (Amicon) were used for gel permeation chromatography.
The UV absorption
of the Chromatographic effluent was monitored with a single path monitor UV-l
(Pharmacia) and a recorder (type EPR-200A,
Tao Instr. Co.). A mini-pump (type TMP15E, Toyo Kagaku Sangyo Co.) and drop
time unit (type DCT- 1,OOOK) were used as
the driver and collector of the effluent.
A membrane filter with a pore size of 0.2
pm (mixed cellulose ester type, Toyo Kagaku Sangyo, TM-4; diameter 47 mm and
293 mm) was used for excluding particulate
matter from the sample with filter holders
(type xx 15-046-00, Millipore,
and type
KSP-293-3, Toyo Kagaku Sangyo). This filter excludes Pseudomonas diminuta and
Rickettsiaprowazekii.
Also, this filter is biochemically neutral, and protein adsorption
to it is negligible. A few pg of 1251-labeled
bovine serum albumin were added to suspensions of several concentrations (O-20%)
of fetal calf serum, the 10% solution containing 4.4 mg ml-l of protein. When 50
ml of these solutions (suspension) were filtered at 120 mm of Hg, O-2.4% of the protein was adsorbed on the filter.
Macromolecular
compounds in the water
sampled were preconcentrated by reverse
osmosis with a water purifier (type RO- 1OS,
Shibata Instr. Co.) and by ultrafiltration with
a cell (type 202, Amicon), ultrafilters, and
a sample reservoir (type RG-5, Amicon).
Ultrafiltration
was performed at 4°C in a
thermostatic bath circulator (model TRL11 lSP, Thomas Sci. Co.), in which a filter of
UK 10 (Toyo Kagaku Sangyo) was used for
concentrating the macromolecules of molecular weight over 10,000 daltons.
Sampling and pretreatment
of natural
water- Water samples were taken from Tokyo Bay, off the coast of Misaki (Sagami
Bay), the Shinobazu-no-ike
Pond (Tokyo),
the open sea, and the Tama River (Fig. 1).
Tokyo Bay and open seawater were collected
on cruises of the Hiyodori-maru
(Tokyo
Univ. Fish.), 12-14 October 1982, and of
cruise KT83-9 of the Tansei-maru (Ocean
Res. Inst., Univ. Tokyo), 13-18 June 1983,
Carboxypeptidase
Sumir’-
D;t,nr
) Arakawa
a
I
36’ N
eiver
.
\ ,Edo
w
a23
633
in natural water
River
Shinobazu-no-ike
I
pond
3,Okm
0
24
0
Fig. 1. Sampling locations. Numbers in the lower figure show the sampling stations on Tank-maru
KT83-9 (13-18 June 1983).
cruise
634
Hashimoto
applied
pressure
(atm 1
Fig. 2. DCPA found in the filtrate with various
applied pressures. The sample was collected at Shinobazu-no-ike Pond on 14 January 1983 and was filtered with pressure filtration (filter pore size, 0.2 pm).
Arrow shows the present experimental condition.
with Niskin bottles. River water and pond
water were collected with Teflon samplers.
All samples were stored in iceboxes to
avoid denaturation of enzymatic activity after collection. A portion of the water was
filtered as soon as possible through double
filters, a glass-fiber filter (pore size 1.O pm,
type GA-100, Toyo Kagaku Sangyo) superimposed on a membrane filter (pore size 0.2
pm) to eliminate microorganisms. There are
two advantages to using double filters. The
pressure applied can be reduced, and the
speed of filtration is expedited.
The enzyme activity in the filtrate is defined as “dissolved.” The filtration was carried out under low pressure (1.1 atm) to the
filter so as not to rupture the cells (pressure
< 1.25 atm is recommended by Florence and
Batley 1980). Figure 2 shows the dependence of DCPA in the filtrate of eutrophic
pond water on filtration pressure. Almost
the same result was obtained for the oligotrophic seawater, showing that any artificial increase in DCPA was low under the
conditions used.
A few drops of chloroform were added to
the unfiltered sample (2% vol/vol) to sterilize and cause cell disintegration (Berman
1970). The carboxypeptidase activity of this
solution is defined as “total.”
DCPA and TCPA were determined with-
et al.
in 2-4 h after sample collection; the loss of
activity before the measurement was ~-5%.
When the enzymatic activity in the sample was too low for chromatography,
we
concentrated the filtered sample by ultrafiltration; Preconcentration
by reverse osmosis was also used when a large amount
of sample had to be concentrated. All the
procedures for ultrafiltration
were done at
4°C. To minimize the adsorption of dissolved species into the ultrafilter, the concentration procedure was ended when the
solution was concentrated 5-fold-l O-fold.
Since some adsorption of protein is inevitable in this procedure, the concentrated
samples were used only for chromatography. About 80-90% of the enzymatic activity was preserved during this process, calculated by comparing the initial enzymatic
activity in the prefiltered sample to that after ultrafiltration.
Also, each peak of enzymatic activity in the chromatogram of the
concentrated sample corresponded to one
in the chromatogram of the initial sample
without ultrafiltration.
All glassware was
sterilized by soaking with 7 M nitric acid or
with 40% ethanol or by dry-heating at 110°C
for 2 h. The enzymatic activity detected was
not disturbed by this sterilization:
chromatograms of various samples showed no
peaks at void volume. This means that there
was no contamination
by microorganisms
through this procedure.
Dependences of DCPA on pH under the
existence of enzyme inhibitorsFor characterizing DCPA, we measured enzymatic
activity with variation of pH. Buffer solutions used were 0.05 M acetic acid-sodium
acetate with 1 M NaCl (pH 2.9-5.2), 0.05
M potassium dihydrophosphate-potassium
hydrophosphate
with 1 M NaCl (pH 4.86.8), and 0.05 M Tris-HCl with 1 M NaCl.
As inhibitors
for carboxypeptidases
from
various sources, we added EDTA-sodium
salt, and phenylmethylsulfonyl
fluoride
(PMSF) to the buffer solutions at concentrations of 0.91 and 0.88 mM.
of carboxyFluorometric
determination
peptidase activity-The
detection of carboxypeptidase activity was based on the hydrolysis of the peptide bond between glycine
and tryptophan and the production rate of
Carboxypeptidase
free tryptophan. We applied this method,
developed for detecting carboxypeptidase
in mammalian tissue (Latt et al. 1972a), to
measurement of the enzyme activity of natural water. The substrate was dansyl-glycylL-tryptophan,
0.4 mM of which was dissolved into 1 M NaCl bufler solution (0.05
M Tris-HCl at pH 7.5). One milliliter
of
sample was added to 2 ml of substrate solution and incubated at 25°C for 2 h. Before
mixing sample and substrate, the solutions
were preincubated for 20 min at 25°C. The
enzymatic activity was measured by the increasing rate of fluorescence of tryptophan
(Af min- l) at 36 1 nm under excitation at
28 1 nm with a xenon lamp. For calibrating
the fluorescence intensity, we added a known
amount of tryptoph, n to the above mixture
after measuring the activity. This process
also eliminates the interference of coexistent substances to the fluorescence of tryptophan. The enzymatic activity (A) was calculated from the following equation:
A = (Af min-l)
x b x r
where r is the coefficient of correction obtained from the recovery of fluorescence
when tryptophan at a known concentration
was added to the reaction mixture. The factor b shows the dilution of sample (b = 3 in
this experiment). The activity found in distilled water (the value of Af min- l for distilled water), which is due to the natural
hydrolysis of substrate, was taken as a blank
and subtracted from the values of sample
activity. All measurements were repeated at
least three times for each sample. The enzymatic activity was indicated as U (unit)
in which 1 U shows the rate of hydrolyzing
substrate at 1 PM min- 1 at 25°C.
The detection limit of tryptophan in this
fluorometric system was 0.3 nM. When the
sample enzyme is added to the substrate,
the fluorescence due to the product increases linearly for 7 h, so that the lowest
limit of detecting carboxypeptidase activity
is 2.1 pM min-l (2.1 PU liter-l) for a 7-h
incubation.
This detection limit is about
5,000 times that of the conventional
spectrophotometric
method using hippuryl-Lphenylalanine.
Under the above experimental conditions,
in natural
water
635
carboxypeptidase
activity can be determined
The
relative standard deviation of activity was
about 2% at the level of 2 mU liter- ‘. The
blank; the hydrolysis of this substrate in distilled water, was so low that the substrate
solution could be stored at 4°C for 1 month.
It has been confirmed that the concentration
of substrate is sufficiently higher than the
Michaelis constant found in all the samples.
When total carboxypeptidase
activities
were measured in unfiltered samples, the
addition of 2% chloroform did not interfere
with the fluorescence of tryptophan and the
carboxypeptidase activity.
of other chemical facMeasurements
tors-Chlorophyll
a was extracted with acetone from the filtration residue (filter: magnesium carbonate
coated GFC-2) and
determined by spectrophotometry
(UNESCO/SCOR method) or fluorometry.
Total
organic carbon (TOC) and dissolved organic carbon (DOC) were analyzed according
to Menzel and Vaccaro (1964). Particulate
organic carbon (POC) was defined by difference of DOC from TOC. Total organic
phosphorus (TOP), dissolved organic phosphorus (DOP), and orthophosphate (Pi) were
determined by the procedure of Menzel and
Corwin ( 196 5). Particulate organic phosphorus (POP) was defined by difference of
DOP from TOP. Analyses of metal ions will
be described elsewhere (Akagi et al. in prep.).
POC and particulate organic nitrogen (PON)
(see Fig. 8) were measured according to the
method of analyzing combustion gas of filtration residue on GFC-2 by quadrapole
mass spectrometry (Saino in prep.).
‘in the range of 5 PI-J-1 0 U liter-‘.
Results and discussion
Distribution
of carboxypeptidase activity
in Tokyo Bay and the open sea-Figure
3A
and B show the distributions
of total and
dissolved carboxypeptidase activities at the
surface of Tokyo Bay and the open sea, and
those in the estuaries of major rivers emptying into Tokyo Bay are listed in Table 1.
Dissolved carboxypeptidase activities in the
surface water of Tokyo Bay ranged from 0.1
to 0.5 mU liter- 1in fall 1982 (Fig. 3A). The
activity was high in the area from the estuary of the Tama River to the mouth of
636
Hashimoto
l
et al.
: 1 mU/ I iter
Fig. 3. Horizontal distributions of DCPA, TCPA, Chl a, and particulate phosphorus in surface water of
Tokyo Bay. Area of circle represents the magnitude of enzymatic activity or concentration of each species found
at each sampling point, and magnitude is the linear relation to area of the circle. In panel B, open area in the
circle indicates DCPA. A. DCPA (samples collected during Hiyodori-maru cruise, 12-14 October 1982). B.
DCPA and TCPA (samples collected during Tansei-maru cruise KT83-9, 13-l 8 June 1983). C. Chlorophyll a
(samples collected during Tans&-maru cruise KT83-9). D. Particulate phosphorus (samples collected during
Tansei-maru cruise KT83-9).
Tokyo Bay and decreased toward the open
sea and the shore of Chiba prefecture (east
shore of Tokyo Bay) (Fig. 3A).
DCPA values found in summer 19 8 3 (Fig.
3B) were in the range of 0. l-l .2 mU liter-‘.
The trend in DCPA distribution
in Fig. 3B
is quite different from that in Fig. 3A. Activities of carboxypeptidase were rather high
even in the open sea, where the magnitudes
of TCPA (or DCPA) were almost equal to
those in Tokyo Bay. The ratio of DCPA:
TCPA is almost constant (about 30%) (Fig.
3B).
Vertical distributions of DCPA and TCPA
at several sampling points were also determined (Fig. 4). In Tokyo Bay, the activity
was generally higher in the surface water and
decreased toward the bottom (Fig. 4A-C).
At the open sea station (sta. 24: Fig. 4E),
the maximum activity was at the surface,
but another distinctive peak was found at
about 30-m depth. Below 30 m the activity
gradually decreased with depth, but there
was appreciable activity even at 1,400 m
(TCPA: 0.08 mU liter-l, DCPA: 0.02 mU
liter- l). At the coastal station (sta. 2 1: Fig.
Carboxypeptidase
in natural
637
water
0: QWliter
.
0
0
.
Fig. 3. Continued.
4D), DCPA and TCPA were also maximal
at the surface, become minimal at 50 m,
and increased again just above bottom.
The vertical distribution pattern of DCPA
was parallel to that of TCPA;
constantly about 30% of TCPA
ure 5 shows the correlation (r2
tween TCPA and DCPA from
DCPA was
(Fig. 4). Fig= 0.968) beall samples
Table 1. Carboxypeptidase activity in natural water. TCPA and DCPA in mU liter-‘. The measurements
were repeated three times (standard deviations given). The sampling points for river water were in the estuaries
at Tokyo Bay.
Sampling
point
Off Misaki
Shinobazu-no-ike
Sumida River
Edo River
Arakawa River
Tama River
Sampling
date (1982)
5 Aug
16 Sep
15 Dee
8 Jun
20 Jul
23 Jul
9 Nov
4 Nov
4 Nov
4 Nov
18 Nov
TCPA
1.9_+0.1
2.2kO.2
0.51f0.09
0.78 kO.08
2.220.1
2.lkO.l
2.3kO.2
l.OkO.2
3.OkO.3
1.5kO.2
1.6-tO.l
DCPA
0.64kO.03
l.l-tO.1
0.12-1-0.03
0.28kO.05
1.1kO.l
0.92t-0.04
1.2&O. 1
0.4940.14
0.80+0.04
0.45kO.22
0.34*0.10
DCPA:TCPA
0.34
0.50
0.24
0.36
0.50
0.44
0.52
0.40
0.27
0.30
0.21
638
Hashimoto
activity
A)
(mU/
et al.
liter)
activity
(mu/liter)
1.0
activity
B)
(mu/liter)
activity
C)
(mu/
D)
activity
rl
2.0
(mu/
1.0
aI
0
liter)
1 .o
0
liter)
2.0
1000
z
z
u&
1500
50
2343
IOCI-
ww
11E i-
on the Tansei-maru KT83-9 cruise. The correlation indicates that a constant proportion
of the enzymes produced by the organisms
is dissolved into the seawater. This is different from the relation in river water (Fig.
6).
Distributions of TCPA and DCPA in the
Tama River-The
Tama River is 138 km
long and flows into Tokyo Bay. Figure 6
shows the distributions
of activities found
in the Tama River on two dates. The salinities measured on 26 August (Fig. 6A) were
20, 3.3, and 2.47~ at the sampling stations
going upstream. Both DCPA and TCPA increased downstream, however the increase
was greater for TCPA than for DCPA; there
was a lo-fold difference in TCPA between
the upper river and its estuary, but less than
a 2-fold difference in DCPA. This trend was
Fig. 4. Vertical distributions of DCPA (0) and
TCPA (0) in Tokyo Bay and the open sea. A. Station
3 (Tokyo Bay). B. Station 6 (Tokyo Bay). C. Station 8
(Tokyo Bay). D. Station 2 1 (Tateyama Bay). E. Station
24 (open sea). Location of sampling stations shown in
Fig. 1.
common to both sampling seasons; DCPA
represented 60% of the TCPA in the upper
stream, but only 20% in the estuary.
Correlation of carboxypeptidase activity
with other conditions-Table
2 shows the
correlation coefficients between DCPA and
TOC, POC, DOC, Chl a, TOP, POP, DOP,
water temperature, pH, salinity, dissolved
oxygen (DO), and turbidity for the combined 11 sampling points in the vicinity of
the estuary of the Tama River (Tokyo Bay,
sampling date: October 1982) and the correlations between DCPA and water temperature, pH, salinity, DO, and several metal
ions (Zn, Fe, Al, Cu, Ni, Co, Ti, and Y) for
the whole area of Tokyo Bay (sampling
points: 25; sampling date: October 1982).
Correlation coefficients of DCPA were high
with TOC, POC, and, in particular, Chl a.
Carboxypeptidase
I
I
2
1
DCPA(mW/liter)
Fig. 5. Correlation between TCPA and DCPA. All
data obtained on the Tank-maru KT83-9 cruise are
given.
26 Awg
in natural
water
For the whole of Tokyo Bay and the open
sea, however, the correlation beween Chl a
and CPA (DCPA and TCPA) was rather
poor. Figure 3C and D show the surface
distributions of Chl a and particulate phosphorus for Tokyo Bay and the open sea
measured at the same time as the data of
Fig. 3B. Although both Chl a and particulate phosphorus decreased toward the open
sea, TCPA did not decrease (Fig. 3B), i.e.
the oligotrophic open sea does not always
have lower peptidase activity than the inside of eutrophic Tokyo Bay. Consequently,
the correlation coefficient between TCPA
and Chl a was only 0.2 for the total sampling
points in Table 3.
While the correlation coefficients between
TCPA and Chl a were calculated for each
sampling station, Table 3 shows correlation
coefficients based on the water sampled at
various depths at each station. These correlation coefficients were high, i.e. TCPA is
1982
50 km
639
4
Fig. 6. Distributions of TCPA and DCPA in the Tama River. Shaded areas indicate DCPA.
640
Hashimoto
et al.
Table 2. Correlation coefficients between DCPA and various factors.
A. Vicinity of Tama River estuary (sampling date: 12-14 October 1982; number of sampling points: 11)
Chl a:
0.75
TOC:
0.53
POC:
0.51
DO:
0.53
Water temp:
0.22
DOC:
0.17
Turbidity:
0.12
Salinity:
0.07
DOP:
0.06
POP:
0.01
TOP:
9.8 x 1O-3
pH:
2.3 x lo-’
B. Whole Tokyo Bay (sampling date: 12-14 October 1982; number of sampling points: 25)
Water Temp:
0.03
Fe:
0.08
Y:
Zn:
0.17
Ti:
0.07
DO:
co:
3.7 x 10-S
pH:
0.01
Cu:
Salinity:
0.08
Al:
0.08
Ni:
positively correlated with Chl a in the vertical samples at the same stations, as is
DCPA. This implies that both intracellular
and dissolved CPA are dominated by factors closely correlated to the amount of Chl
a, i.e. that CPA came either from phytoplankton or from something else correlated
with phytoplankton
distributions.
To explain the low correlation coefficients
obtained from the total data in Table 3, we
should note the slope of the regression curves
of CPA and Chl a at each station, also shown
in Table 3. The inclination of the regression
curve (enzymatic activity per Chl a amount)
increased when the station was close to the
open sea, i.e. the values at stations 24 and
2 1 were about 15-20 times those in Tokyo
Bay. As an example, we have plotted TCPA
vs. Chl a at stations 24 (open sea) and 8
(Tokyo Bay) (Fig. 7).
Correlation between Chl a and POC (Fig.
8A), and Chl a and PON (Fig. 8B), obtained
at stations 4 and 6 (Fig. 1) are based on
samples collected at the same time as those
for CPA. There is a linear relationship between POC (or PON) and Chl a. Since Chl
a represents phytoplankton,
Fig. 8 shows
0.02
1.7x10-4
0.02
7.7 x 10-S
that the major organic materials were autochthonous. Therefore, CPA may appear
in association with primary production in
Tokyo Bay. Also, in Tokyo Bay, dissolved
organic carbon and dissolved protein decreased linearly with an increase of salinity
(Ogura 1978). Although the amount of substrate was not measured in this study, the
correlations can be interpreted as an increase of the activity of carboxypeptidase in
organisms toward the open sea where the
substrate was poor. Bacteria or zooplankton
may also contribute to CPA in the open sea.
Although
the magnitude
of TCPA (or
DCPA) is related to the Chl a in a limited
(or vertical) area, the relations of CPA to
biomass varied for each sampling station so
that correlation coefficients were low for the
whole area of Tokyo Bay and the open sea.
Deactivation of commercial carboxypeptidase was rapid (the half-life was 2.5 h) in
intact sea and pond waters, although this
enzyme was stable in the filtered or autoclaved sea or pond water (the half-life was
about 12-l 7 h) (Table 4). This suggests that
the residence time of active DCPA in natural water is extremely short, which in turn
Table 3. Correlation coefficients and slopes of regression curve between TCPA and Chl a or DCPA and Chl
a (r-correlation coefficient; a-slope of regression curve).
Total
TCPA-Chl a
r
a
DCPA-Chl a
r
a
points
4
6
I
8
21
24
0.2
0.17
0.76
0.37
0.86
0.97
0.76
0.09
0.99
0.27
0.63
15.8
0.72
21.7
0.09
0.23
0.63
0.096
0.90
0.25
0.74
0.05
0.96
0.10
0.69
5.25
0.74
6.38
Carboxypeptidase
in natural
641
water
Table 4. Half-lives of commercial carboxypeptidase in various media. a-Filtered (0.2-pm pore size)
and autoclaved seawater; b - filtered seawater; c-autoclaved seawater; d-filtered and autoclaved pond
water; e-filtered pond water; f-seawater without
treatment. Autoclave was performed at 110°C for 30
min. Pond water was sampled at Shinobazu-no-ike on
7 August 1982 and seawater was sampled off Misaki
on 7 August 1982.
Half-life(h)
0
I
*
1
2
4
3
5
CHLOROPHYLL
6
7
17.1
14.9 14.3 11.9 12.6 2.51
6
a(pg/jiter)
Fig. 7. Relationships between TCPA and Chl a at
stations 24 (0: open sea) and 8 (0: Tokyo Bay).
explains the positive correlation between
DCPA and TCPA (Fig. 5).
Michaelis constant-The
Michaelis constant of bovine pancreas carboxypeptidase
reported in the literature is 110 PM (Latt et
al. 19723). The Michaelis constant obtained
by means of a Lineweaver-Burk
plot for
commercial carboxypeptidase was 130 PM;
that for pond water DCPA (Shinobazu-noike Pond, 19 July 1982) was 10 PM, very
low compared to the commercial enzyme.
pH dependence and response to inhibitors-Figure
9A shows the pH dependence
of DCPA in pond water, including effects
of the inhibitors EDTA and PMSF. The different response curves of DCPA to these
chemicals show the existence of two kinds
of enzymes: serine protease whose activity
2.
.
.
.
I’
0
O”
.
0
00
.
.
0’
qm
l
50
100
POC (pg C/liter)
150
(
10
PON W
26
N/liter)
Fig. 8. Correlations between POC and Chl a and between PON and Chl a inside Tokyo Bay. Samples
collected during cruise KT83-9, Tunsei-maw. Station 4-O; station 6-U.
642
Hashimoto
A)
Bj
_
b;ffer(pH)
a
buffer(pH)
pH dependence of DCPA in the presence of
enzyme inhibitors. A. Pond water (sampled at Shinobazu-no-ike Pond, 19 July 1982). B. Seawater (sampled
off Misaki, 5 August 1982). None added-w; EDTA
added-@; PMSF added-A.
is suppressed by PMSF, and chelator-sensitive metalloprotease which is deactivated
by chelators such as EDTA (Fahrney and
Gold 1963; Matsubara and Feder 197 1). In
Fig. 9A, the activity displayed in curve a is
almost equal to the sum of curves b and c,
showing that the DCPA values in pond water
consist of serine and metalloproteases. The
metalloprotease is about 30% of the total
DCPA. Figure 9B shows the pH dependence
of DCPA in seawater. Acidophil peptidase
was characteristic of seawater, but could not
be found in the pond water. As can be seen
in Fig. 9B, most of the acidophil protease
in seawater is the PMSF-sensitive
serine
protease. The activity of EDTA-sensitive
metalloprotease, which is optimal at pH 78, can be recovered by the addition of an
excess amount of Zn (1.3 mM) after treat-
et al.
ment with EDTA in both seawater and pond
water.
Little et al. (1979) pointed out that peptidase activity in natural waters might be
inhibited
by coexistent
chelating
compounds or poisonous metals such as Cu and
Pb (Matsubara and Feder 197 1). However,
at least for Tokyo Bay, this is quite unlikely
because of the low correlation
between
DCPA and heavy metals including Zn, Co,
and Cu (Table 2).
Determination
of molecular weight of
DCPA by gel chromatography-After
ultrafiltration of seawater, river water, and pond
water through a 0.2~pm membrane filter,
>95% of the CPA was found in the residue
(macromolecular
fraction
of mol wt
> 10,000). Figure 10A and B shows the
chromatograms of DCPA in seawater. Since
the activities in the seawater were not found
at void volume, the DCPA measured is due
to the enzyme molecules and not to contamination
by microorganisms.
The seawater sampled in May (Fig. 10A) shows a
distinct peak at about mol wt 450,000, while
in September (Fig. 10B) there were many
peaks in the range between 45,000-12,500.
The molecular weights of carboxypeptidases range from about 30,000 to 600,000, so
that these activity peaks correspond to the
carboxypeptidase enzymes generally found
in organisms. The chromatograms of DCPA
in the river (Fig. 1OC) and pond (Fig. 10D)
waters show most of the carboxypeptidase
activity or molecular weights in the range
of 500,000-30,000.
Protease-added detergents which contain
heat and chemical-resistant
enzymes such
as thermolysin have recently become commercially available. Enzyme-added detergents in this chromatography showed a peak
at about mol wt 100,000 daltons. However,
the activity peak attributable to the detergent could not be distinguished in the chromatographs of DCPA in the Tama River or
seawater (Fig. 10). The heat-denaturation
curves for DCPA in natural waters were
similar to that of the bovine pancreas carboxypeptidase; heating at 80°C for 20 min
completely destroyed the activity in the sea,
pond, and river waters. This indicates that
the carboxypeptidase activity was ascribable to enzymes and also suggests that the
Carboxypeptidase
1”
Fig. 10. Molecular distributions of DCPA. Vis the
volume of effluent and Vo is the void volume. A. Seawater (sampled off Misaki, 10 May 1982). Column:
Sephadex G-200 (16-mm i.d. x 65 cm long); carrier:
0.05 M Tris-HCl, 0.1 M NaCl, 0.5 mM 2-mercaptoethanol (pH 7.5); flow rate: 8.9 ml h-l. B. Seawater
(sampled off Misaki, 16 September 1982). Column:
Toyopearl HW 55s (44-mm i.d. x 54 cm long); carrier:
0.05 M Tris-HCl, 0.3 M NaCl (pH 8.0); flow rate: 156
ml h-l. C. River water (sampled at the estuary of the
Tama River, 19 November 1982). Experimental conditions as in panel B. D. Pond water (sampled at Shinobazu-no-ike, 9 November 1982). Experimental conditions as in panel B. Molecular standards: 1-ferritin;
2-catalase; 3 -aldolase; 4 -alkaline phosphatase; 5albumin (from bovine serum); 6-albumin (from hen
egg); 7-bovine serum carbonic anhydrase; 8 -cytochrome c.
activity due to the detergent was negligible
in the natural water; the peptidase activity
in the commercial detergents is not inactivated by this process.
As concerns bacterial proteolysis, Little
et al. (1979) did not detect any proteolytic
activity in lake water which was sterilized
643
in natural water
I
1.5
-V
I
2
by microfiltration
at 0.45-pm pore size.
Hollibaugh and Azam (1983) concluded that
particle-bound
activity in either free-living
or aggregated bacteria plays a dominant role
in the degradation of protein, mainly attacking proteins of molecular weight between 70,000 and 700. They suggested that
the amino acids produced in this process
were utilized preferentially
by the microorganisms. We may consider some reasons
for the difference between these studies and
ours. First, the substrates used differed. The
dansylglycyl+tryptophan
used in our experiment has a molecular weight of 494.6,
much smaller than the protein. It is well
known that proteases (and peptidase) are
enzymes which have low substrate specificity, and this specificity can decrease further
with simple substrates. The molecular distributions of DCPA (Fig. 10) suggest that
several kinds of enzymes are concerned with
the hydrolysis of dansylglycyl+tryptophan
644
Hashimoto
in natural water. Second, the experimental
conditions are different in terms of temperature, pH, salinity, and so on. Hollibaugh
and Azam ( 198 3) incubated seawater for 8
h at temperatures (17”-2 l°C) adjusted to the
seawater sampled, while we measured the
mixture of seawater and buffer (pH: 7.58.0, salinity: 4-5%0) at 25°C for 2 h. Considering the characteristics of enzymatic reactions, these do not seem to be decisive
factors to account for the different results.
However, even if the dissolved free enzyme
does not contribute to the degradation of
whole protein, it may still release amino
acids from small peptides in the dissolved
phase of natural water.
One of the objects of the work of Little
et al. (1979) and Hollibaugh
and Azam
(1983) was the estimation of the protein
degradation rate in natural water; therefore,
the experimental conditions were intended
to be as close to natural conditions as possible. On the other hand, one of our objects
in the present work was to provide evidence
of a latent potential of carboxypeptidase activity in natural water, so that experimental
conditions were kept uniform for all samples, including
measurement
of TCPA
(where chloroform was added to the unfiltered sample). Also, since we were interested in the distribution
of enzymatic activity
in natural waters, our techniques were designed to measure the activities in a large
number of samples in a limited time. Our
experimental
conditions
therefore rather
deviated from the in situ state. However, it
is impossible to perform the perfect in situ
experiment; at the very least, artificial substrate is added and the sample is incubated
in isolated vessels in the laboratory.
We conclude from our results that the
enzymatic activity necessary to hydrolyze
the peptide bond between glycine and tryptophan exists widely in natural waters, even
at a depth of 1,400 m in the open sea, at a
magnitude in the range between 0.1 and 3
mU liter-l at 25°C. From 10 to 50% of the
observed activity in natural water can be
attributed to dissolved enzymes.
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-,
Submitted: 24 February I984
Accepted: 26 November 1984