KINETIC
STUDIES ON ALKALINE
PHOSPHATASE
FROM ECHINOPLUTEI?
Sidney C. Hsiao
Department
of Zoology,
University
of Hawaii,
Honolulu
ABSTRACT
Using mass culture of the sea urchin Tripneustm gratilln ( L. ) eggs, a method was worked
out for extracting fairly large quantities of the phosphomonoesterase
alkaline phosphatase.
The extracted enzyme showed a single ultraviolet
absorbance band after elution from a
Dowex 2 column, and the material that absorbed most strongly at this wave length contained alkaline phosphatase.
It showed two characteristic
bands in starch-gel electrophoresis.
When 6.25 rn~~ p-nitrophenyl
phosphate was hydrolyzed
in glycine buffer at 38C, the
optimum pH was 10.5. The hydrolysis conformed to a first order reaction, and the reaction
for enzymatic action was 25-3OC,
constant was 0.00864 min-l. The optimum temperature
coinciding
with the range of fluctuation
of the animal’s ambient temperature.
The temperature-activity
curve showed a close conformity
to the Arrhenius equation, and the activation energy was 11,250 Cal/mole.
The value of K, (Michaelis
constant)
obtained at pH
10.5 (at 2%) is 2.197 x 10-O M of p-NPl?/liter.
INTRODUCTION
Although
a number of fine chemical
studies on alkaline phosphatase ( a phosphomonoes terase) from vertebrate and bacterial sources have been made, no isolation, purification,
and chemical characterization of this enzyme has been made
from sea urchins-a
marine counterpart of
laboratory rats and chicks. The literature
on alkaline phosphatase in echinoids as examined by Hsiao and Fujii (1963) is
limited to, histochemical localization
and
simple bio-chemical analysis. There have
been no physico-chemical studies done that
are comparable, for instance, to the work of
Garen and Levinthal ( 1960), Rothman and
Byrne ( 1963), or Heppel, Harknes, and
Hilmoe ( 1962), on Escherichia
coli, of
Anagnostopoulos
and Matsudaira
( 1958)
on human placenta, of Grossberg, Harris,
and Schlamowitz (1961) on human adult
tissues, of Motzok (1959) on the chick, or
of Motzok and Branion ( 1959) on the birds
and mammals. We have been interested in
the ontogenesis of this enzyme in the Hawaiian echinoids and in its isolation, chemical
characterization, and physiologic functions,
By developing a method for mass culture of
l Contribution
No. 232 from the Hawaiian
Marine Laboratory,
University
of Hawaii.
This work
was partially
supported by National
Institute
of
Health Grant AM-2519
sea urchin eggs, it has been possible to
isolate usable quantities of extracts from
echinoplutei with phosphomonoesterase activity and to examine the enzyme chemically. This paper will report on some of the
physico-chemical
characteristics of echinopluteus alkaline phosphatase.
The author is grateful to Mr. W. K. Fujii
for technical assistance and to Dr. S. M.
Trefz for reading the typescript.
MATERIAL
AND
METHODS
Preparation of alkaline phosphatase
from echinoplutei
The sperm and eggs were collected from
male and female specimens of the Hawaiian sea urchin Tripnewtes
gratillix (L. )
either by spontaneous spawning or by injecting a small volume of 0.5 N KC1 solution into an animal’s body cavity. The
sperm were kept undiluted while the eggs
were strained through a few layers of
cheesecloth to remove extraneous material
(for example, broken spines, fecal pellets,
and so on) and washed several times with
freshly filtered seawater by gentle agitation; this was followed by sedimentation
and decantation of the supernatant fluid.
Batches of washed eggs were suspended in
large dishes containing about 500 ml of
freshly filtered seawater. Each batch was
fertilized with either a drop of undiluted
R129
11130
SIDNEY
sperm or a few ml of sperm freshly suspended in seawater. After agitating the
eggs for a few minutes, they were allowed
to settle, the cloudy supernatant was decanted, and the fertilized eggs remaining
on the bottom were transferred to large jars
containing about 2 liters of filtered seawater. The jars (usually 20-30 were used
in each run) were covered and left in
place where the ambient temperature fluctuated only slightly (2sZSC ) . When the
eggs developed (in about 50 hr) into echinoplutci with arms about one-half the body
length, the upper layer of the medium containing actively swimming echinoplutci was
gently siphoned off through large bore tubing leaving the scttlcd material undisturbed
at the bottom of the jar. The larvae in the
medium collected by siphoning were filtered through a sintcrcd glass funnel, They
were transferred to graduated tubes, and
the volumes of animals were recorded. The
cchinoplutei were then quickly frozen and
stored until used for enzyme extraction.
About 100 ml of echinoplutei (weighing
a little over 100 g wet wt) were mixed with
an equal volume of distilled water and
homogenized in a Virtis blcndcr with the
blending chamber kept in an ice-brine bath.
The homogenate had about 1-2 units of
alkaline phosphatase/ml, one unit being the
amount of enzyme capable of liberating
1
pmole of p-nitrophenol/hr
from p-nitrophcnyl phosphate (p-NPP).
The homogenate was brought to p’II 9 by the addition of.
2 N NaOH
while monitored by a Beckman
Zeromatic pH meter. To each 10 ml of
homogenate, 1 ml of 2% trypsin and 0.5 ml
of toluene were added, and the mixture was
incubated at 37C for 20-24 hr. The digest
was centrifuged at 1O4x g KCF for 10 min
to recover the supernatant, and the residue
was washed with 30 ml of distilled water,
centrifuged, and decanted. This was repeated three times and the decanted washings were combined with the supernatant
liquid
and the solids discarded.
Solid
( NH4) 2 SOLi was added to the combined
liquid in the amount of 60 g/100 ml, and
the mixture was allowed to stand in the
cold over night, The precipitate was col-
C.
IISIAO
lected over Hyflo Super-ccl (infusorial
earth) with the help of suction, The filtrate had about 0.1 units of enzyme/ml.
The precipitate was redissolved in 100 ml
of Tris (hydroxyme thyl) aminomethane bu Ffcr, pH 8.6, and diluted to 300 ml by adding redistilled
water. This solution was
trcatcd with one-third its volume of nbutanol. The aqueous fraction recovered
from this treatment was dialyzed against
Tris buffer, “pH 8.7, for 48 hr in the cold,
and the enzyme was reprecipitated in the
cold by adding 50 g of ( NH,) 2 SO4 to each
100 ml of solution, The precipitate was
collected by filtration through Hyflo Supcrccl, and the filtrate, which contained about
0.7 units/ml, was discarded. The prccipitate was redissolved in redistilled water, dialyzed successively against tap and distilled
water ( 24 hr against each). The fluid in
the dialyzing
bag was transferred to a
Virtis apparatus and freeze-dried. From 100
g of wet echinoplutei, 1.08 g of white dry
powder was recovered.
Estimation of alkaline phosphatase activity
As substrate, 4 mg of p-nitrophenyl phosphate (p-NPP, supplied by Sigma Chcmical Co.) was freshly dissolved in each ml of
triple distilled water. Into each tube 0.5 ml
of 0.1 M glycine buffer, pH 10.5, was added
together with an equal volume of substrate
and 0.1 ml of enzyme solution. The mixture
was incubated at a selected temperature for
a specific length of time. At the end of incubation, 2 ml of 0.02 N NaOH was added
to stop the reaction, and the absorbance
of the mixture was read on a Beckman DB
spectrophotometer at 410 rnp against H20.
The correction for reagent blank was obtaincd by acidifying with 2 drops of coned
HCl and the absorbance again read at 410
mp. This is essentially the method of
Besscy, Lowry, and Brock ( 1946), with
slight modifications
for using the p-NPP
substrate prepared by Sigma Chemical Co.
Zone electrophoresis in starch-gels
Smithies’ (1955) method with some modification was used for zone electrophoresis
of the freeze-dried extracts from echino-
KINICTICS
OF
IXHINOPLUTEI
plutei. The hydrolyzed starch, supplied by
Fisher Scientific Company, was prepared
with 0.08, M Tris-citrate
buffer, PI-I 8.7.
The Pt electrodes were bathed in 0.03 M,
~$1 8.2, borate buffer which was connected
to the starch-gel with 2-mm thick sheets of
spongy rubber. The cnzymc sample was introduced either on filter paper or in a simple
slot. The potential gradient from a variablevoltage IIcathkit
Power Supply (O-400 v
d-c, O-100 ma) was 6 v/cm and applied for
2-3 hr or until the front band, visible
through the covering Saran Wrap (a plastic
film used to prevent evaporation from the
starch-gel), had traveled 5-6 cm (in about
2 hr when a 100 v 20 ma current was used).
A tray of ordinary ice cubes was placed
above the starch-gel and served effectively
in keeping the temperature at about 5C during electrophoresis. To detect alkaline phosphatase, the starch-gel was split along its
length in a horizontal plane, and each half
was placed for 1 hr in 200 ml of an incubation-staining medium of freshly prepared
Tris (Poulik)
buffer containing
alphanaphthyl acid phosphate, 0.2 g; fast blue
RR salt, 0.2 g; polyvinyl pyrolidone, 1 g;
NaCl, 4 g; and trace amounts of MnCl2
and MgC12. The starch-gel was destained
and washed in several changes of a solution
made of methanol, acetic acid, and water
(5 : 1 : 5) until no color came off. These
white starch-gel sheets with dark bands
were packed in Saran Wrap, labeled, and
stored in a covered glass container in the
cold.
Column chromatography
An anion exchange resin, Dowex 2 8X
(200-400 mesh) was “sieved” by mixing the
rcquircd volume of the resin with 2 parts of
water in a volumetric cylinder, scdimenting
for a few minutes, and decanting into a
Elask leaving the larger particles in the
bottom of the cylinder, The resin in the
flask was washed with 1 N I-ICI under
vacuum from a water pump. About 35 ml
of the resin was transferred to a 40 x 20-cm
Pyrex glass Chromatographic tube with fritted disc sealed into the lower member of
the standard-taper joint. The column was
washed with one-half of dead volume of
ALKALTNI’:
*I
R131
PEIOSl?IIATASE
2
3
4
5
of alkaline
phosFIG. 1. Elcc trophorogram
phatasc cxtractcd from adult tissues and echinoplutci of the sea urchin Tfipnmstes
grutilln
(L. ).
Electrophorogram
No. l-cchinoplutei,
No. 2test of adult sea urchin, No. 3-mixture
of Nos. 1,
2, 4, and 5, No. 4-adult
intestine, and No. 5adult body fluid.
0.2 M Tris buffer followed by 6-8 dead
volumes of the starting buffer. The freezedried enzyme material was dissolved in
water and dialyzed against 0.01 M Tris
buffer, pH 7.2, and absorbed on the resin
column. Elution was carried out with the
gradient of Tris buffer increasing by steps
from 0.01 to 1.0 M at the selected pH level,
and the eluate was collected in 3-ml fractions in separate tubes on a fraction collector. An aliquot from each tube of eluate
was examined at 280 mp for absorbance
with a spectrophotometer and assayed for
alkaline phosphatase activity
using the
method described above.
RIZSULTS
Homogeneity of the enzyme extract
The enzyme material extracted from the
early echi~oplutei
echinoplutei was, after freeze-drying,
a light white powder. This crude extract,
when examined by zone clectrophorcsis
on starch-gel and column chromatography
with Dowex 2, showed consistent uniformity among different batches. Under the
conditions used in this study,_, the enzyme
extract showed two constant bands on its
clectrophorogram:
a fast o,nc at 3.5 cm, and
K132
SIDNEY
0.02M
0.04M
0.8
0.06M
0.16M
0.25M
0.5M
3
1
111
a. 0.7
E
g 0.6
N
; 0.5
22
2 0.4
6P 03
a 0.2
.-._.-.
0. I
7
,“,
x
.-
4
.-.-.
8
,
---.-
ELUATE
, .-*-.,*,.
12
16
_.- .-.-.
-._.-.
FRACTIONS ( 3ml
20
/
24
- JY
L
‘5
28
each)
FIG. 2.
Chromatography
of alkaline phosphatase
on Dowex 2. Numbers along abscissa arc number
of tubes each containing 3 ml of cluate. Each dot
represents absorbance reading made on each tube
with a Beckman DB spcctrophotomcter.
The fractions were assays for alkaline phosphatase activity
with p-NPP (crosses and dotted curve).
Each vertical arrow indicates when the buffer of a specific
molar strength was introduced
above the column.
a slo,w one at 2.7 cm from the origin. The
fast-moving band is much darker than the
slower one. These bands were different
from alkaline phosphatase extracted from
adult tissues ( Fig. 1). This figure shows
the results when five different
samples
were applied by the filter paper method
simultaneously to a single piece of starchgel. The electrophorogram
of alkaline
phosphatase from early echinoplutci shows
the same constant bands 3.5 and 2.7 cm
from the origin. The single fast but different band shown by the enzyme extracted
from the adult test is also a- constant one
I
0.02M
1
1
0.04M
1
Oa08M
O.IM
1
1
0.32M
1
0.64M
J
FIG. 3. Chromatogranhy
of echinopluteus
alkaline phosphatase on DoGei 2. Experimental
conditions similar to those of Fig. 2 except the eluting
solution was buffered
at PE-I 9.0.
C.
HSIAO
obtained under the specific conditions of
this study. The electrophorogram
of the
enzyme extracted from the intestinal tissue,
No. 4, is very different from the first two,
while the extract from adult body fluid was
so low in enzyme content that the elcctrophorogram contained very faint bands
that do not show in this photograph. In the
case of electrophorogram No. 3, produced
by combining equal quantities of four different enzyme materials and introducing
the mixture into the starch-gel by filter
paper of the same size as the others so that
only one-fourth as much of each type of
enzyme was used in the electrophoresis,
there is not only a dilution effect as shown
by the lighter color of its bands, but the
separation is not clear either, indicating a
possible interference effect.
The enzyme adsorbed on Dowex 2 was
eluted by increasing concentrations of pH
7.2 Tris buffer and collected in successive
3-ml fractions. The absorbance at 280 rnp
by each fraction is shown in Fig. 2. It will
be seen that 0.02 M Tris buffer eluted nearly
all the ultraviolet
absorbing protein that
was collected by the first six tubes, and
little or no protein was eluted by more
concentrated buffer solutions. The ultraviolet absorbance graphs of the eluted fractions of all the batches of enzyme extracts
from echinoplutei showed the same pattern.
An aliquot from each fraction was used
for estimating alkaline phosphatase activity
against p-NPP as substrate and glycineNaOII as buffer. The results (Fig. 2, dotted
line) show that the peaks of both curves
are in the same fraction. No enzyme activity was detected after the eighth tube.
Similar results were obtained when “pH 9.0
Tris buffer was used (Fig. 3). The maximum ultraviolet absorbance occurs in tube
No, 4, and the alkaline phosphatase activity
curve parallels that of the cluted fractionultraviolet
absorbance curve. The use of
the several batches of enzyme as a single
material for comparative experiments in
this study appears to be justified because
their homogeneity is strongly indicated by
the fact that the bands in the zone electrophorograms
of enzyme material from
KlNETlCS
OF IXIIlNOPLUT~I
ALKALINE
R133
PIIOSl?IIATASE
3
A:
Expl.
I-Hr
.o, I
-0,2
\
I
’
’
n
TIME
L
>
’
’
’
’
’
IN MINUTES
-0.2
c
a
PH
4. Effect of pH on the activity of cchinoplutcus
alkaline
phosphatase
against p-NPP.
The solutions were buffcrcd
with 0.1 M glycincNaOH, the substrate, 6.25 mM p-NPP, and the
activities
mcasurcd at 38C, 30 min incubation.
The points are averages of three observations,
and
the short horizontal
lines above and below each
point indicate the range of activity
at each pH
value. The circles rcprcscnt single observations.
FIG.
w
>
F
-0.4
a
= -0.6
a
I
0
120
TIME
cchinoplutei
were constant, and they differed consistently from bands produced by
extracts from adult tissues. Moreover,
homogeneity is supported by the appearance of the same narrow band in all the
ultraviolet
absorbance curves of elutcd
fractions from the Dowex 2 column.
Effect of pII
on alkaline phosp’h&ase
activity
The influence of pH on the echinoplutcus
alkaline phosphatase activity at the level of
substrate concentration used (the variation
in enzyme activity with the pH of the reaction mixture expressed as PM of p-NPP
hydrolyzed per hr) is shown in Fig. 4. This
pII-cnzymc activity curve shows that when
6.25 mna of p-NPP was used under the conditions of this study, the optimum pH was
10.5.
Time course of enzyme action
Three series o!f ,expcriments with &ifferent periods of incubation were carried
out. The enzyme, substrate, and buffer
solutions were separately equilibrated
at
38C with the water bath and mixed at the
I
I
I
60
I80
6
I
240
300
IN MINUTES
FIG. 5. Rate of hydrolysis
of p-NPP by alkaline
phosphatase
from echinoplutei.
Initial
substrate
was 9.122 ,UM, 0.1 mg enzyme powder per sample,
glycine buffer ??H 10.5. Incubated for periods as
indicated in the graph. Curve A, 1-hr cxpcrimcnt
duplicates.
Curve B, 2-hr cxpcrimcnt
duplicates.
Curve C, 5-hr cxpcriment duplicates.
beginning of each time period. At the end
of the selected time period, the reaction
was arrested by the addition of NaOH, and
the amount of p-NPP hydrolyzed was estimated spcctrophotometrically.
The first series of experiments was run
for about 1 hr, with duplicate samples incubated for periods of 2, 4, 8, 16, 32, and 64
min. The second, or 2-hr experiment, had
a series of duplicates incubated for 5, 10,
20, 40, 60, 80, 100, and 120 min. The last,
or 5-hr experiment, used incubation periods
of %, %, 1, 2, 3, 4, and 5 hr.
When the logarithm of relative activity
is plotted against time, the data fall along
a straight line (Fig. 5). The points are
based on duplicate determinations, and the
straight
lines were calculated
by the
method of least squares. It is obvious that
all three reactions are of the first order.
R134
SIDNEY
C. IISIAO
IF. . 5
./C.-*
~ ISII5
F
0
QO.! s-’
\
.
8
-I
CI- \
6.
of alkaline
l+G.
5
20
25
30
TEMPERATURE
in
“C
IO
15
Infhlcnce of temperature
phosphatase.
35
40
On0032
45
On0033
0,0034
OaOO35
0.0036
‘IT
on the activity
Statistical analysis of the slopes of thcsc
curves by the method of analysis of variance indicates that there is no significant
difference between them, The 50% hydrolysis .timc or ts, the Eirst order reaction rate,
K’, and the slope are summarized in Table
1. There is a decrease in the reaction rate,
with concomitant change in slope and tx,
as the time of the experiment increases, but
the difference is not statistically significant
by “t” test or Bartlett’s ( 1937) test.
lnf luence 0f temperature
In studying the effect of temperature upon
alkaline phosphatase activity, solutions of
6.25 mM p-NPP buffered by 0.1 M glycineNaOH at pH 10.5 were brought to the
selected temperature
of the bath and
hydrolyzed
by a standard quantity
of
cnzymc equal to 0.1 mg of freeze-dried
powder that was brought to the same
temperature immediately before each experiment. The temperature ranged from 5
to 40C in steps of 5C. At the end of 30-min
incubation, the released nitrophenol
was
measured by spectrophotometry
at 410 rnp
(Fig. 6). The optimum temperature is in
the range of 25-3OC, and activity decreases
very rapidly beyond 35C. As the mean surface temperature of Hawaiian waters fluctuates, according to Sverdrup, Johnson, and
Fleming ( 1942), from 23C in February to
26C in August, this optimum temperature
for enzymatic activity coincides with the
range of the ambient temperature.
For a
1OC increase in temperature at the steep
7. Hydrolysis
of p-NPP by echinopluteus
alkaline phosphatase.
Each of the first three points
on the right represents the average of four obscrvations while the other points are based on duplicates. Initial p-NPP substrate for all cxpcrimcnts
6.25 mM, glycine-NaOH
buffer 0.1 M, 1111 10.5;
enzyme used, 0.1 mg; incubated
at the specific
temperature
for 30 min.
FIG.
part of the curve, from lO--2OC, the activity
increases by a factor of 2 or more while
astride the optimum temperature, the corresponding factor is M-1.3.
In Fig. 7, values of ln in activity ( ,UM of
phosphate hydrolyzed 1 hr) of the enzyme
are plotted against the reciprocals of temperature on the K&in scale. A straight line
is fitted by the method of least squares.
The equation is y = 21.605 - 5661.7 x. As
the points fall closely along a straight line,
it is concluded that over this range of temperature, the enzymatic process, as a whole,
shows a close conformity to the Arrhcnius
equation. Using this equation in the form
In 7c= -E/ET + const, the activation energy
is calculated as 11,250 Cal/mole.
TABLE
--
1.
1-hr
2-hr
5-h]
-
of p-NPP by echinopluteus
phosphatase
_________
____
--__~---
Expcrinwnt
--
Hydrolysis
alkaline
--
-
Slope of
log rclativc
activity--time
cnrve
----
0.00376
0.00307
0.00293
- -
--
$AlK)
-
- -. -- -79.8
87.7
102.4
- - --
--
.-
First order
reaction rate
constant, KL,
(min-1)
50~0.J~yclrol-
.
- - .-
0.00864
0.00706
0.00674
Substrate concentration
The effect of different concentrations of
p-NPP on th e activity of alkaline phosphatase at pH 10.5 buffcrcd by 0.1 M gly-
KINETICS
OJ? ECFILNOPLUTFI
RI,KRLlXE
R135
PIIOSIWATASE
tine was studied by 30-min incubation
using a substrate ranging from 1.4 X 10-l to
1.8 x 10 pmolcs. The results are plotted in
the double reciprocal manner suggested by
Lincweaver and Burk ( 1934) and a straight
Zinc fitted to the data by the method of
least squares (Fig. 8). All the observations
fall very near a straight lint, indicating
close conformity to the Michaclis-Mcnten
(1913) equation. The slope of the best fit
curve is 0.54 and the intcrccpt 0.264; the
Michaelis constant K,, is 2.197 X 10dGM by
using the equation l/V = l/V,,l,, + K,/
V,,,,, x l/S; and a reaction velocity of 4.07
pM/hr when saturated with substrate is dcduced. When the curve is extrapolated
across the l/V axis, as suggested by Dixon
( 1953)) it cuts the l/S axis at -0.455. This
gives the same value of K,,, graphically as
the reciprocal of l/S.
II
n
VS
8. Relationship
bctwecn reaction velocity
and substrate concentration
plotted in the double
reciprocal
manner according
to Lincwcavcr
and
Rurk ( 1934.).
FIG.
DISCUSSION
In their histochcmical study of the early
ontogenesis of echinopluteus phosphatase,
Hsiao and Fujii ( 1963) found this enzyme
in the skelctogenic mesenchyme and the invaginatcd archcnteron wall. The gut wall
and spicule-producing
cells rcmaincd rich
in alkaline phosphatase throughout the cchinoid’s early development to late cchinopluteus stage. But tissues such as the hydroenterocoels did not give positive reactions
for this enzyme after they separated from
the enzyme-rich gut wall. There is apparently a loss of alkaline phosphntasc synthesis in certain differentiating
tissues as
well as the appearance of new sites of its
synthesis in other tissues as development
proceeds. It would be interesting to asccrtain whether the embryonic phosphatase
enzyme is the same as that of the adult
tissues. Comparative studies, such as by
zone electrophoresis, on enzyme materials
prepared by identical procedures from adult
test, intestine, and body fluid have indicated that they arc different,
Motzok’s (1959) data on chick alkalino
phosphatase and the findings of Motxok
and Wynne ( 1950), Ross, Ely, and Archer
and Morton (1957) indicate that
uw,
the pH-activity
curves arc not similar in
shape for all concentrations of substrates.
This, of course, affects the accuracy of K,,
estimation if the range of substrate conccntrntion is large, Although the concentration
of substrate was in tha 10 to 10-l pmole
range, the K, estimation made in this study
must be considered a preliminary
figure.
Work is in progress on a systematic survey
of the relation between pII and enzyme
activity for a wide range of substrate concentrations.
Since no estimate of the molecular weight
of the cchinoplutcus
alkaline phosphatase
has been made, it is not possible to calculate the turnover number. For the same
reason, other thermodynamic
parameters
are not discussed.
SUMMARY
The phosphomonocsterase, alkaline phosphatase, has been isolated from the echinoplutci of Tripneustm g?~~ti&~ (L. ) in usable
quantity.
After preliminary
purification,
the enzyme extract was examined for some
of its chemical characteristics.
Its starchgel clcctrophorogram
shows two constant
R136
SIDNEY
bands, a fast moving band 3.5 cm from the
origin and a slower band 2.7 cm from the
starting line under the conditions used in
this study. The fast band is wider and
darker than the slower one. This electrophorogram is consistently different from
those of similar extracts from adult tissues
such as test, intestine, and body fluid. Enzyme preparation adsorbed on anion exchange resin, Dowex 2, is eluted by 0.02 M
Tris buffer whose pH does not affect the
elution. The optimum temperature of the
enzyme is near the ambient temperature of
the animal’s natural habitat which has a
mean temperature range of 23-26C. The
optimum pH is 10.5 when 6.25 mM of pnitrophenyl phosphate is hydrolyzed by this
enzyme at optimum temperature and with
glycine buffer. The overall hydrolysis of
phosphate ester by the echinopluteus phosphatase conforms to a first order reaction.
The calculated energy of activation
is
11,250 cal/molc and the Michaelis constant,
2.197
x 1o-6
M.
REFERERJCXS
C., AND H. MAT~UIIATRA.
1958.
Purification
and kinetic studies of the alkaline
phosphatase of human placenta. Proc. Intern.
Symp. Enzyme Chem., Tokyo Kyoto, 1957.
Ser. 2: 166-172.
Properties of sufficiency
BARTLFTIT,
M. S. 1937.
and statistical tests. Proc. Roy. Sot. (London),
Ser. A, 160: 268-282.
BESSEY, 0. A,, 0. H. LOWRY, AND M. J. BROCK.
1946. A method for rapid cletermination
of
alkaline
phosphatase
with five cubic millimctcrs of strum. J. Biol. Chcm., 164: 321329.
Determination
of enzymc1953.
DIXON,
M.
inhibitor
constants.
Biochem.
J., 55: 170171.
GAREN, A., AND C. LEVTNTEIAL.
1960. A finestructure genetics and chemical study of the
enzyme alkaline phosphatase
of E. cdi.
I.
ANAGNOSTOPOULOS,
C. EISIAO
Purification
and characterization
of alkaline
phosphatase.
Biochim.
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