Indian J. Dairy Sci., 30, 3, 1977, pp 229-242
PURIFICATION AND PROPERTIES OF ALKALINE PHOSPHATASE
ISOLATED FROM BUFFALO MILK·
R.S. SHARMAi' and N.C. GANGULI
National Dairy Research Institute, Kamal, Haryana
Roceived on 6 September, 1975
acrylamide was "Cyanogum 41", a product
of American Cyanamide Co. The rennet was
Hansen's products and trypsin was from
E-Merck. EngJa~d. Cysteine was from Calbiocbern, Los Angeles, USA.
INTRODUCTION
Several workers (Zittle and Della Monica.
1952; Morton, 1953; Lyster and Aschaifenburg,
1962) bave purified alkaline pbosphatase (EC
3.1.3.1) from the skim milk and creamery
phases of bovine milk.
Methods
Only limited attempts bave been made to
study this enzyme in buffalo milk (Ganguli,
1968; Laxminarayana and Dastur, 1968).
Recently, Sharma and Ganguli (\9710, 1971 b)
reported that the enzyme is nearly equally
distributed in the skim milk and cream phase of
buffalo milk. Utilizing such information, the
present investigation was undertaken to purify
and study the properties of the enzyme
from both cream and skim milk phase of
buffalo milk. To know the species differences,
if any, cow milk enzyme was also studied.
Alka line phospho ta se activity was assessed
by the method of Aschaffenburg (1963) using
p-nitrophenyl phosphate as substrate. While
using other substrates, the
released
inorganic phosphate was measured according to the method of Fiske and Subbarow
(1925).
Protein was estimated by the procedure of
Lowry of al. (1951). Sialic acid was estimated
by Warren's thiobarbituric acid procedure
(1959) as descrihcd by Gupta and Ganguli
( 1965).
One unit of t he enzyme was equivalent to a
preparation liberating 1·0 I'g of p-nitrophenoi
in 30 min at 37"C and specific aC1ivity was units
of enzyme activity,lmg protein.
Milk was skimmed in Alfa-Lava! cream
separator and whole milk samples were spun
at 73.000 g for 30 min in Beckman Model L
preparative ultracentrifuge.
The Km value for p-nitrophenyl phosphate
was determined by the method of Lineweaver
and Burk (1934) as modified by Dixon (1957).
For thermal inactivation, purified enzyme
preparation in Tris-HCI buffer of pH 6·8,
MATERIALS AND METHODS
Materials
Milk samples: Fresh compo,>ite samples of
milk from the Murrah buffaloes and Tharparkar
cows were collected from the herd ma intained
at this Institute.
Chemicals: p-Nitrophenyl phosphate was
prepared following the procedure of Bessey
and Love (1952). Sephadex G-200 used was a
product of Pharmacia , Upsala; agar used was
"Agar Noble" a Difeo product and poly-
·N.D.R.I. Publicalion No. 75-125.
tPrtstllt .""'OIIS: Department of Dairy Chemistry, Gujarat Agricultural University, Anand·388110, Gujaral.
I
2
Alkaline Phosphatase from Buffalo Milk
was taken in thin walled glass tubes and exposed
to different temperatures ranging from m' to
7S"C for 15 sec and then cooled immed iately
in ice-water. The residual activity of the
enzylDe was then determined.
The DNP-derivative was prepared following the method used by Sud (1963) for casein.
Urea and mercaptoet.hanol (2-MCE) treatments
were carried out at different concentrations
for different periods at 3TC. Dialysis
of 2-MCE and urea treated enzyme samples
was carried out at TC, first against distilled
water and then aganst Tris-HC I buffer solution
(pH 6'8).
The enzyme was preincubaled with cysteine
for 10 min at 37'C and then assayed for the
activity.
A 5·0 ml aliquot of enzyme at pH 7'0 was
incubated with 0·2 ml. of rennet solution
(100 mg!ml distilled water) at 37'C and at
every 30 min interval 1·0 ml aliquot of this
mixture was w t hdrawn for assay. The conlrol
samples contaioc(\ 0·2 ml distilled water in
place of renoet solution.
Another 5·0 ml aliquot of the enzyme solulion in Tris-HCI bulTer (pH g·O; O·05M) was
incubated at 37'C WIth 0·3 ml of a solution
prepared by dissolving 15·0 mg of trypsin/ml
of disti led water and )·0 ml aliquot of th is
mixture at 30 min interval was assayed for
alkaline phosphatase. To the control samples
0· 3 ml of distilled water was added in place
of trypsin solution.
The enzyme preparation obtained at the
2nd butanol treatment step (Figs. 1 and 2)
was fractionated on Sephadex 0-200 column
2·4 X 80 em). The preparation of gel and
other essential steps were as described by
Porath and Flodin (1959). Six ml aliquot of
eluting buffer was collected in each tube during
the fitration of the sample througb t he gel bed.
Agar gel electrophoresis was carried out
according to Wieme (1965) using LKB (Sweden)
electrophoresis appa,dtus a nd veronal buffer,
pH 8·4
a nd
O·05M.
Elcctrophorc ie
separation was allowed for 3 hr at 150 volts.
Stain used was Amido Black 108 and acetic
acid (7 % v /v) solution was used for washing.
For detection of enzyme bands, the gel plates
were incubated at 37'C with p-nitrophenyl
phosphate buffer substrate having 0'2 %
(w Iv) magnesium chloride. The characteristic yellow bands developed on the gel were
visually observed and the distance moved was
measured.
Polyacrylamide disc electrophoresis was
performed according to Tombs and Akroyd
(1967) in Shandon apparatus. A continuous
buffer system (Tris glycine, pH 6·8; 0·05 M)
was used. Electrophoretic run was carried
using 2·0 rnA current per gel rod (0'5 X 8'5 em)
for 2 hr. Staining, deslaining and enzyme
detection procedures were salDe as followed in
agar gel electrophoresis.
RES U LTS
A. Purification of the enzyme
(i) Purification from the skim milk phase .The procedure followed fo r the purification
of the enzyme from the skim milk phase is
outlined in F ig. 1. T he ,ummary of purification results for a single batch is given in Table 1.
The enzyme could be purified to about 490a nd 350-fold from buffalo and cow milk,
respectively, wi th a recove ry of about 14 %.
(ii) Purification from the cream phase: The
proced ure adopted to purify the enzyme
from the cream phase i, outlined in Fig. 2
and the results obtained are presented in Table 2.
The results reveal that the enzyme could be
purified to about 980- and lOOO-fold, respectively , in case of buffalo and cow.
B. Properties of the eDzyme
1.
General properties of the enzyme
(i) Effect of .w bstrate cOrlcentratioll:
The rate of enzymic hydrolysis of p-nitrophenyl
phosphate (Fig. 3) indicates a simple MichaelisMemen type kinetics. The Michaelis constant
(Km)
for p·nitrop~enyl
phospate
was
6·6 X IO- 4M fo r the skim milk and 2·6 x 10- 4
R.S. Sharma and N.C. Ganguli
FIz. 1.
3
Plow Doe! sllowiaa: .tep. fo1l<>wtd In ""if\""'ion of alkalin. pbo.ph,ta .. fr.,. !be oJd.. aallk ph...
(Opol"""Dt Jayer).
MILK
Fa.
Ultraeentrifugal
\
M~llar
Opalescent layer
<:aseba
SCCUm
diluted by
10 vol.,
n-butanol 40%
(vfv) 8 t 37"C for
15 mio centrifu ged
- - - ---- -
- - --
- ---
I
n~Butanol
Aqueous layer
layer
I. centrifuged
pH 6-8 •
-- --- - -1
SUpcfutant
Precipitate
pH 6'8.
ammonium sUlph ate 70 %
saturation,
1
,
i
centrifugation
-
- -- - - --- -
----
- --
I
Supernatant
Precipitate
!
djss olved in Tris·HCt buffer,
pH 6'8, dialysis
I
aa n im;~
water.
dialysis aaaiast buffe r
Dialysed solution
n-butanol 30% (vlv). at
37::> C fOT to min, centrifuged
I- -
--
Aqueous layer
I
Sephadex G·200 gel filtration
---- -Butanollayet
....
TABL E 1
Purification of alkal..., pbosplmt.." from buJJalo ad cowailk.
Total units
Specific activity
(units/mg protein)
Total protein
--- -..
Purification steps
B'
C·
B
Foldputilkation
Recovery ( %)
~.
C
B
C
B
C
B
C
~
-~
S'
<II
~
1.
Milk
2.
Opalescent
la~t
3. First butanol
<:I .
47,02,j
193,730
20,315
17,325
2-3
11 '2
1'0
1'0
100'0
100-0
~
11 ,712
46,800
244
630
47'7
74'3
26-7
6'7
24-9
23-9
I:l
10,796
42,930
212
575
50-6
83-3
21-9
7'S
22'9
21 '9
~
i:i
....,
'1>
~
treatment
<:I
4,
Casein free serunl
8,78 1
34,500
119
165
73-9
209'1
32-()
18-8
18'7
17'6
3
5.
Ammonium sulpha te
fraclion
7,380
30,430
80
87
91 '6
384' 5
39'6
31-3
Is-?
IS'S
§I:l
6_
Second butanol
treatment
7,240
29,990
75
76
9'-S
394-5
41-S
35'6
IS'S
15'2
7_ Sephadex G,200
6,800
21,020
6
6
1133'j
4012-8
490-6
359'8
14'3
1H
fraction
OR Bnd C stand for buffalo milk and cow milk, respectively.
tx:J
<:I
~
-,
~
R.S. Sharma and N.C. Ganguli
Fie. 1. Fl ... _ I ,bowing .Ieps (oll ...e<! ill ,1Ir1llcn Uon or .lk. Ii•• phosphatase Ir.", tbe "'''-IT
,ba...
Ml LK
Sk imming
I
I
Skimrnl",
Cream
<tiluted by equal volume of distilled
water~ n·butanol 40% (v/v), eo! 37-tl C
for 15 min. centrifuged
. -T
r
Aqueous l.yer
···_···- 1
n·Butano! layer
Fat- butanol-aqueous gel
I
diluted by distilled water,
n·butanol, 30% (vlv), a l
3T'C for 10 min, centrifuged
- _._-_ .-
I
nMButanoJ laye!."
diluted. by distilled water,
.-butanol, 30% (vlv), at
37°C for to mjo~ centrifuged
--·r
...._--/- --_._-- .
n-Butanol layer
melted at 40' C,
centrifuged
I
pH 4·6,
.centrifueed
I
Precip itate
-
-
n-Butanolillyer
- - - _.. -
• .• - • .• .. ,
I
Supernatllnt
pH 6-7, ammon.ium :>; uJphatc
10 ); saturation, C'.!ntrifuged
I
I
Freci pitate
SUptrnil ta nt
dissolved jn Tris-HCI buffer pH 6'8,
dialyse d against di:;tilled water ,
agaimt Tris. buffer n-buttlDO\ 30% (v t v) ,
at 3rC for 10 min, cen! rifugt:d
-::-;=:,:;I
~::::-=-.-
n·ButaDol layer
... -..... - - _... ....__._ -.._
..
=="'--"--Aq ueous layer
I
Sephadax G-2()O s et filt ration
~
TABLE 2
Purlficalloa or a",ali.., pl!o.phawe fro .. cream.
Total unit.
Pu~ation
Total protein (mg)
Sllecific activity
uoits/mg
Fold purifica lion
Recovery (%)
stePs
~
B'
C'
B
B
C
C
0
C
B
C
--""~
s::.
:::t
1.
Milk
1,525,000
2,275,00'
234,000
208,000
6-S
10'9
1'0
1-0
100-0
100'0
~
Q
2. Cream
690,000
1,050,000
10,500
8,640
65-7
121'S
10'1
11.1
45,2
46-0
3. First butanol
treatment
615,000
960,000
5,625
5,340
109-3
179'3
16-7
16-4
4{j'3
42-2
4_
Caseio-free serum
<40,000
789,000
675
570
800'0
1384-2
122'9
126-6
35'4
34'7
5_
Ammonium sulphate
fractionalion
480,000
719,000
485
508
969-7
1415-4
152'0
129-5
31"
31-7
475,000
680,000
375
436
1266-7
1559-6
194'S
142'7
31'1
29'8
6_ S<eond butanol
Sepb.dcx G-200
Peak I
Pe.I< II
to
and
c
Q
Ir.,
:l
""
':;-.
Q
3
b:l
~
s::.
--,a::
<;:)
treatment
7.
~
;;::-.
~
212,500
225,000
120,000
406,000
.tand for bulfaJo and cow milk, respectiVl)ly,
76
35
S5
1796-1
35
6429-5
1181'S
11600-0
429'5
987'6
200-1
1061')
13'9
14'7
5'3
11'8
R.S. Sharma and N.C. Gangult
for cream enzyme (peaks I and JI). The Km
calculated was ;ndent;cal for both buffalo a nd
. cow enz~me .
7
studied at various temperatures for a holding
period of 15 sec (Fig. 4) revealed that enzyme
from the milk of both the species was inactivated at the same rate and at 70·C, the enzyme
activity was lost completely with IS sec
holding time.
(ii) Effect ofpH; The influence of pH on
the activity of the enzyme (Fig. 3) suggests of
pH optimum at 9·5 a nd was similar for
buffalo and cow enzymes.
2. Substrate speclflcity
(iii) Th ermal inactivation ;
The kinetics
of thermal inactivatio n of a lkaline phosphatase
0) Action 0/1 organic phosphares .. The
enzyme from the milk of both the species
FitI·3
.-.-e,
Effect of """.Iule ..,a.,._1ioJI (ouler tune) lUld pH (o..e, btock) 0_ oIlI.allDo
Butralo
0-0-0 CO".
30
30
>- 20
~
:;;
;:::
o
«
II>
~
~
~ 10
>N
10
...
Z
2
!>-NITROPHcNYL
PHOSPHAn
(10-3 M)
phoopa'''' octirily.
Alkaline Phosphatase from Buffalo Milk
8
FIg. 4
Rate of loactlvatioo of all",l1... ph••
holding temperature.
"""!.,,,
TABLE 3
";I~
Actt.., or mlIk .Ikidl.. pkospbltue on dlff.reJ>t
\lIlaopbote est...
Inore.nic pho'pbate (ug)
100
Substrate·
P-nitropbcnyl phosphate
Glucose-6-phos phate
Gluco;e-I-pho,phate
Galactosc-6.phosphate
Fruclo;e· 6·phosphale
Manoose-6-ph05phate
Ribose-6.phosphale
PyridoJtai phosp-hate
Sodium pyrophospha te
80
....,
60
~
&
z
~
...
~
40
Cow
12'80
]4'40
S'12
3'84
3'20
7'04
4'30
5-80
5'80
4'48
3'36
1'92
40
5'91
5·92
4'80
2'40
1'92
"Values represent the average of duplicate experiment
with a single cnz.yme preparation in each caSe.
0-
~
,;
20
!fOLDING
T~M9.
hydrolysed different phosphoric ester corn·
pounds at varying rates (Table 3). 1" general,
most of the sugar 6-phosphate compounds were
hydrolysed at a comparable rate, but slower
than the reference substrate, i.e. p-llitrophenyl
phosphate. The enzyme hydrolysed glucose6-phosphate faster than glucose-l-phospltatc.
3.
Buffalo
Characteristics of the enzyme
<i) lIzfiuence of metal ions melal chclatiilgagents
Thlol compounds and Ihiol blocking compoW/ds: Addition of MgH to the enzyme assay
system stimulated the activity whereas the
addition of EDTA at various concentrations inhibited the enzyme. Such inhibition
could be restored by the addition of
magensiulll "hlo ride
solution
to
the
EDTA-treated enzyme. Addition of cysteine
up to 0-05 mM showed an activating effect and
above this concentration it acted as an inhibitor.
The thiol group binding reagents namely pCMB
and NEM inhibited the enzyme from milk of
both hulTalo and cow with increasing concentration (0 to 10 mM).
(ii) influence. q( N-Ierminal amino group
blocking reagents: The N-terminal group of the
enzyme was blocked by treatment with f1uorodinitrobenzene_ It was observed that the
addition of sodium bicarbonate to the enzyme
re,,,lted in a loss of ahout 45 /~ of the enzyme
activity.
Effect of urea and 2-merraptoethanol
The effects of various concentrations of urea on the enzyme for various periods
of incubation at 37' C arc presented in Fig. 5.
Los> in enzyme ac ti vity was observed with the
increase in conccntration of urea and with incubation period as well. A loss of about 50
and 100% in enzyme activity was also apparent
in presence of 3 M and 6M urea concentration.
(iii)
(2~,}lCE):
R.S. Sharma and NC. Ganguli
Fig. 5
Elrod or""", (loCI) and 2-MCE (rigbt) alkali•• pbospbatase
100
100
80
90
10
8
70-
,
50
z
z
0
..,~
4()
~
l;! 30
i
II:
40
'0
10
15
5
0
(WITH ..1£1,)
respectively, and the enzyme preparations from
both the species showed similar trend.
The effect of incubation of buffalo and cow
milk alkaline phosphatase with various concentrations of 2-MCE is depicted in Fig. 5.
At a concentration of 0·4 % the enzyme was inactivated completely.
About 75 to 88 %
activity of the enzyme could be revived in case
of 2-MCE treated samples after dialysis, but no
such appearance of activity was noticed in urea
treated and dialysed enzyme from both the
species.
Effect of prot.olytic enzymes
Trypsin treated enzymes showed an increase
in the activity of the enzyme and such effect
appeared to be more pronounced (3()~'-;;)
in case of cow enzyme than buffalo (16 ~-~).
Treatment with rennet had no effect on the
activity of the enzyme.
~ 40
u
•
10
.,
:(,,0
0
20
S-
1
;::
to
MINUTES
4.
SO
0
0
BulJalo. Q-O-o Co..,
...".60
-.;..
"'-
l60
.,
e_._.
9
~ 20
0-'
Qo4
0-' 02-M[RCAPTOHHAHOL (-/0)
•
UREA
5.
4
5
..
7
("')
Isozymic studies
(i) From the Sephadex G-200 gel filtration pattern of the enzyme preparations (Fig. 6)
it is apparent that the cream phase enzyme
contained 2 isozymes of alkaline phosphatase, namely peak 1 and peak II, whereas
the skim milk enzyme preparation contained
only one enzyme corresponding to the
peak II enzyme of cream phase. The 2 isozymes of cream were distinctly different in
their molecular sizes, peak I enzyme being
heavier than the peak IT enzyme. The results
further suggested that the 2 enzymes in cream
phase of both the species were almost identical
in their molecular sizes with a distinct difference in the ratio of peak I and peak II. Such
ratio was about I ; I (45 and 55 %) in case of
buffalo milk cream whereas I ; 4 (20 and 80 %)
in case of cow milk cream, respectively. However, repeated experiments in different samples
of milk did not show any consistency towards
10
Alkaline Phosphatase from Buffalo Milk
Fill- 6
SepiladeJ: G-lOO eel 1lltratloo ,ott..". for albllue pllo.pbata •• from bulf.1<! milk (loft) .ad
eo" milk (rigbt).
~oo
500
••
400
.. ZOO
5.
, I
40<1
~EI"
.00
.'~;N
:
ZOQ
zz
I
>8
TUBE
this ratio. Tllis could be due to individual
variation in the milk.
(ii) Associatioll of sialic acid.' The analytical data on the sialic acid content ofgkim milk
alkaline phosphatru>e (Table 4) revealed tllat
buffalo milk enzyme contained higher sialic
acid per unit weight ofl'rotein and also per unit
activity of the enzyme. Similar data on the
peak I and II enzyme of cream phase are presented in Table 5. It will be seell from
Table 5 that peak I enzyme from cream of both
Ihe species contained higher sialic acid per unit
protein as well · as per unit enzyme activity than
the peak II enzyme. The results al,o sbow that
the cream phase enzyme of buffalo milk contained lower sialic acid than the corresponding
cow milk enzyme.
(iii) Agar gel eleclrophoresis: Tbe agar
gel electrophoresis of the enzyme preparations
(Fig. 7) reveal that cream alkaline phosphatase
0
••
.,.'
Z2
T"IIE
~6
30
-..
~.
'"
resolved into 3 enzyme bands namely A, Band
C. The C band migrated towards tbe negative
electrode. The skim milk phase enzyme resolved into only ODe spot corresponding to
B band of the cream phase. The pattern of
isozymes was almost identical in case of botb
buffalo and cow.
0") Polyacrylamide disc electrophoresis:
The alkaline phosphatase positive peak I and 11
whcn subjected to polyacrylamide disc electrophoresi, (Fig. 7) revealed that peak I enzyme
of cream phase resolved into 2 alkaline phosphatase bands, namely Al and A2, and also
contained some non-migrating enzyme protein.
The peak II enzyme of cream phase also resol¥ed to give a similar pattern except that tbe
uon-migrating enzyme band at the point of
application was absent. Tbe skim milk
enzyme peak also sbowed the bands corresponding to AI and A2.
R.S. Sharma and N.c. Ganguli
A_lyti ..1 data
Spedes··
Butralo
Cow
Jl
TBBLE 4
tIIo .klm milk . _•.
OB
Activity
Protein
Sialic ae-id
(units/ml)
(lls/m1)
<).Ill/lUI)
protein
Sialic acidl
activity
0'0024
0'0015
0'013
0'004
5'71
31'~
0'076
2993
75'16
0'121
Sialic add!
·Combined eluanu of tube No. 11 to 16 when fractionated On Sephade,= G·200 column of 2'5 X 30 em.
··Values represent analytical data or a single expedment io each case.
TABLE 5
Alkaline phosphata.. attivit" protein .Ia sialic acid eo.te.t of peak I .Dd p••k II • ..,.... of
CrfJIm pbase (combl.ed .Iua.t. of tobe No. 15 to 13 for P I and tube No. 35 ror P 11).
Specie!-
En;r.yme
Act!vjty
peak
(unit'/ml)
Protein
(J.lBlml)
76
80
19
184
110
52
40
29
Buff.lo
I' I
Cow
PII
I' I
I'll
Sjalic acid/
Sialic acid/
Sialic acid!
rol
protein
a~tivity
1'66
0'80
1'43
0'015
0'015
0'36
0'021
Oill0
0'075
0'77
0'026
0 -041
.Values represent the analy tical data of a single experiment in each case.
2'4xSO em.
DISCUSSION
Puri fication of al kaline phosphatase from
cream and the skim milk phase of buffalo milk
has been attempted for the fi rst r;me. The
enzyme- from these 2 fractions of buffalo milk
<:ould be purified to abou t 950- and 450-fold,
respectively (Table I). Similar results have been
obtained from the cow milk enzyme also (Table
2). However, this enzyme from bovine skim
m ilk and cream has been purified to 1000- and
5000-fold by Zittle and Della Monica (1952)
and Morton (1953). respectively. Thelinal
preparation of alkaline phosplk'ltase from
buffalo milk had lower specific activi ty as
compared to that of cow milk enzyme.
The pH optimum for alkaline phosphatase
from the milk of both the species appear to be
at about 9·5 (Fig. 3). A wide variation of
pH optimum has been observed by various
The column size in this case was
workers for different substrates in different
buffer systems. Aschaffenburg (1963) found
an optimum pH of 10·0 for bovine milk enzyme
using p-nitrophcnyl phosphate in carbonate-bicarbonate buffer system. Mohamed and
EI-Rafey (1956) reported an optimum pH at
9·5 for bulTalo and cow milk enzyme using
disodium phenyl phosphate as substrate.
The observed thermal inactivation of the
purified enzyme suggests that almost complete
loss of enzyme activity occurs at 70'C with a
holding period of 15 sec and such thermal
inact ivation is similar for both huffalo and cow
milk el1Zyme, (Fig. 4). Mohamed aud ElRafey (J 956) reported that the enzyme in milk
system gets inactivated at 70·C.
The enzyme from both the species hydrolysed various sugar phosphates at comparable
rates, but slower than the reference substrate
p-nitrophenyl phosphate (Table 3).
Alkaline Phosphatase from Buffalo Milk
12
Fig. 7
Electropber<>gram, of alkali.e phosphot.,. on agar gel (left) aod polyacrylamide gel
.,
c
:
I
B
r
•
•
A
B
(rig~t).
AI
A2
f3
•
0<:
,
~
________ --L--______
~
2
The purified enzyme from both buffalo and
cow milk was activated by Mg++. EDTA
iuhibited the enzyme and
addition of
Mgt+ ions could reverse the inhibition. The
observations of Anderson (1961) lend snpport
to the present findings.
Inhibition of the enzyme by the addition of
pCMB snggested tbe involvement of SH group
of the enzyme in its catalytic action_ A similar
conclusion is drawn from the inhibition of the
enzyme by N-ethylmaleimide. The par\lclpation of free-SH groups of alkaline phosphatase from other sources has been well esta!i,hed (Fishman and Ghosh, 1967). On the basis
of the present experimental resnlts, metal.
thiol and amino groups can be implicated in the
mechanism of catalysis of hydrolysis of monophosphoric acid esters by mi Ik alkaline pho,phatase.
The irreversible inhibition of alkaline phosphatase with urea suggests an irreversible
2
3
4
denaturation of the enzyme (Fig. 5). Such
stndies have been reported on liver, kidney
and intestinal enzymes (Bntterworth and Moss,
1967). The results of present stndy relate the
milk and intestinal alkaline phosphatase as
regards the irreversible denatnration of the
enzyme. The inactivation effect of 2-MCE
was found to be reversible after dialysis (Fig. 5).
This inhibition may be because of inhibitory
effect of the reagent or it may be due to the
cleavage of the -S-S- linkage of the enzyme
molecule.
The enzyme purified from the milk of both
bnffalo and cow contained bound sialic acid
(Tahles 4 ,wd 5) and it was found to be high in
the enzyme prenaration from Cream phase
(Table 5). The findings of Peereboom (1968)
that neuraminidase releases sialic acid from
alkaline phosphatase of milk leads the clne to
their
occnrrence
in
our preparations.
Sharma and Ganguli (969) have also reported
13
R.S. Sharma and N.C. Ganguli
the association of sialic acid with buffalo and
cow skim milk enzyme. An alteration in
the electrophoretic mobilities of alkaline
phosphatase due to neuraminidase action
was also ohserved by Peereboom (1968).
Association of sialic acid with alkaline
phosphatase of other sources like kidney,
human placenta and sheep brain was also
reported by Moss et al. (1966), Ghosh et al.
(1967) and Saraswathi and Bachhawat (1968),
respectively.
The molecular sieving of the whole cream
extract (2nd butanol step, Fig. 2) on Sephadex
0·200 column resolved int.o 2 enzyme peaks
(Fig. 6), namely peak I (high molecular
weight) and peak II (low molecular weight).
The enzyme prepacatiou from the skim
milk phase resolved to give only one peak
corresponding to the P II of the cream phase
enzyme (Fig. 6). These observatiom suggested
that the cream pbase contained at least 2 multi"
pIe forms of the enzyme and the P I (high
molecular weight) enzyme appeared to be
missing in the skim milk phase. Peereboom
(1968) found that the Cream phase enzyme
resolved to give 2 isozyme spots on Sephadex
gel by permeation chromatography.
The agar gel electrophoresis of the whole
cream extract (2nd butanol step. Fig. 2)
showed the presence of 3 alkaline phosphatase
zones, namely 'A' (slow moving) 'B' (fast
moving) and 'C' migrating towards the negative
electrode (Fig. 7). The skim milk phase enzyme
preparation isolated from the opalescent layer
had only 'B' enzyme zone (Fig. 7). This ob·
servation suggested a differential isozyme
pattern of alkaline phosphatase in the 2 phases
of milk of both the species.
The polyacrylamide disc electrophoresis of
the whole cream extract showed the presence
of at least 3 alkaline phosphatase bands, namely
'A2' (fast moving) 'AI' (slow moving) and
another 'B' band which did not migrate much
from the point of application of the gel
rod (Fig. 7). The peak I enzyme of cream also
showed similar pattern (Fig. 7). The peak II
(low molecular weight) resolved only into 2
bands (AI and A,) as in the peak I enzyme.
although it showed high concentration of Al
and A2 enzymes. From the foregoing discussion
it may be concluded that the isozyme pattern
of buffalo milk alkaline phosphatase closely
resembles the bovine milk alkaline phos.
phatase.
On the basis of electrophoretic mobilities
and the molecular size, if a faster moving
(Band B) lower molecular weight enzyme
(Peak Il) is named as alkaline phosphatase
conventionally the slower moving (Band A)
higher molecular weight enzyme (Peak I) becomes the ~ and the enzyme migrating towards
the negative electrode may be termed as
"I·isozyme of alkaline phospbatase (Fig. 7).
Peereboom (1966, 1968) also observed presence
of at least 3 enzyme zones in cream phase.
The skim milk phase enzyme may, therefore,
represent the a-alkaline phosphatase since it
showed the similar mobility and molecular size
as the a-isozyme in cream phase (Fig. 7).
The results thus showed that in cream fraction
more number of isozymes are present than the
skim milk.
.
SUMMARY
Buffalo milk alkaline phospbatase (EC
3.1.3.1) was purified to about 98()· and 49()·
fold from the cream and skim milk phase, respectively. The optimum pH was 9'5 and the
Km value for p.nitropbenyl phosphate was 6·6
x 1O--4M and 2'6 x 1O-4M for the skim milk
and cream enzyme, respectively. The purified
enzyme also hydrolyses monophosphoric
esters of various sugars. The enzyme was
activated by Mg++ and inhibited by EDTA.
pCMB and NEM. Cysteine at low concentration (0·05 mM) stimulated the enzyme whereas
at concentration higher than 0·05 mM, inhibited.
Treatment with DNP caused about 30% loss in
enzyme activity. Rennet treatment did not
14
Alkaline Phosphatase from Buffalo Milk
show any effect but trypsin appeared to sti- Fiske CH. and Subbarow, Y. (1925) f . BIo[. Cl>em.,
66, 375.
mulate. The enzyme was inactivated irreversia.Dguli
, N.C. (1968) Ind[an f. V.t , Sci, and A.H"
bly by 6 M urea whereas reversibly by 1 %
38,
I.
(v/v) 2-mercaptoethanol. Enzyme activity
Gho,h,
N.K.,
Goldman, g,S, and Fiwman, W.H.
was lost at 70"C with 15 sec holding period
(1967) Enzy mologia, 31, 113.
at pH 6·8.
Resolution of the cream phase enzyme on
Sephadex G -200 showed 2 enzyme peaks (P I
and P II) of which P I being of larger molecular
size. The skim milk enzyme resolved to give
only peak II. Agar gel electrophoresis of the
cream phase enzyme showed prese nce of (l-,
p- and r-isozyme. The polyacrylamide disc
electrophoresis also showed the presence of at
least 3 isozymes, but skim milk 'showed only
2 isozymes. The peak J enzyme contained higher sialic acid than peak II enzyme. Buffalo
cream enzyme contained lower sialic acid than
the cow cream enzyme. Other properties of
buffalo milk enzyme were identical to tbat
of cow milk enzyme.
ACKNOWLEDGEMENT
Mr. R.S. Sharma is indebted to Indian
Council of AgriCUltural Research, New Delhi
for the award of a senior fellowship d uring
the course of investigation.
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