Selective Solubilization of Marker Enzymes of Buffalo
Milk Fat Globule Membrane by Detergents
ABSTRACT
Activities of alkaline phosphomono-
esterase, xanthine oxidase, 5'-nucleoti-
dase, and adenosine triphosphatase asso-
ciated with buffalo milk fat globule mem-
brane were assessed in the presence of de-
tergents Triton X-100, Tween-20, and de-
oxycholate. All detergents enhanced the
enzyme activities, except adenosine tri-
phosphatase, which was inhibited. After
treatment of the membrane material by
any of the detergents and subsequent
centrifugation at 100,000 × g for 1 h,
the recovery of alkaline phosphomono-
esterase, xanthine oxidase, and 5'-nucleo-
tidase activities in the supernatant varied.
Deoxycholate treatment of the mem-
brane material resulted in maximum
recovery of these enzymes in the super-
natant, but sodium dodecyl sulphate
at 2 mM inhibited all the enzymes.
INTRODUCTION
Milk fat globule membrane (MFGM) is a
lipoprotein complex that is solubilized sparing-
ly in water. But solubilization is prerequisite to
purification and characterization of proteins.
We have shown that alkaline phosphomono-
esterase, xanthine oxidase, and 5'-nucleotidase
are the major enzymes associated with buffalo
MFGM (1,2). In this study we solubilized these
enzymes with detergents to gain knowledge of
the organization of these enzymes on the mem-
brane and to determine whether the enzymes
can be purified with these detergents.
M A T E R I A L S A N D METHODS
Materials
Disodium paranitrophenyl phosphate was
purchased from Biochemical Unit, Patel Chest
Received July 12, 1977.
N.D.R.I. Publication No. 77-103.
1978 J Dairy Sci 61:697--700
M. K. B H A V A D A S A N and N. C. G A N G U L I
National Dairy Research Institute
Karnal 132001
India
Institute, New Delhi; triphenyl tetrazolium
chloride from British Drug House; sodium de-
oxycholate, sodium dodecyl sulphate (SDS),
Triton X-100, Tween-20, AMP, and Tris-ATP
from Sigma Chemical Company.
Isolation of Milk Fat Globule Membrane
Fresh composite buffalo milk was collected
from the Murrah buffaloes of the Institute
herd. The separated cream was washed by three
suspensions in .25 M sucrose at room tempera-
ture and separated with a cream separator. The
thrice-washed cream was diluted to 30% fat
with the sucrose solution, and MFGM was iso-
lated according to the freeze and thaw pro-
cedure of Kobylka and Carraway (9).
Assay of Enzymes
Alkaline phosphomonoesterase (E.C.3.1.3.
1.) was assayed according to Ostrowski and
Tsugita (11). Activities of xanthine oxidase
(E.C.1.2.3.2.), 5'-nucleotidase (E.C.3.1.3.5.),
and (Na + - K + - Mg ++) activated ATPase
(E.C.3.6.1.3.) were estimated by the proced-
ures of Zittle et al. (14), Huang and Keenan
(7), Dowben et al. (6), respectively. Protein
was estimated according to Lowry et al. (10).
Activity of Membrane Enzymes
in the Presence of Detergents
Samples of membrane suspensions (5 mg
protein) were incubated at 30 C for 30 min
with increasing amounts of detergents in a final
volume of 5.0 ml. After 15 min aliquots of the
sample were assayed for the enzymes and com-
pared with control comprising the membrane
suspension and no detergent.
Solubilization of Enzymes by Detergents
The MFGM was treated with increasing con-
centrations of Triton X-100 (.5 to 2.5 mM),
Tween-20 (.25 to 1.00%), or deoxycholate (.5
to 10.0 mM). The membrane material was sus-
pended in buffer for the enzyme assay along
with different concentrations of detergents.
697
698 BHAVADASAN AND GANGULI

The mixture was incubated at 30 C for 30 min ATPt~t

and was centrifuged at 100,000 × g for 1 h in ALKALINE PHOSPHOMONOEST ERASE

iP---~- --t1 5'- NUCLEOTIOASE
a Beckman Preparative Ultracentrifuge, Model o~---~---.~ XANTHINE OXIDASE
L. The material which did not sediment was de-
120 130
fined as soluble. Supernatant was removed with 1201
a syringe; the pellet was suspended in distilled HO

water and made to a definite volume. Aliquots ~OO

~_ I00
of suspended pellet and supernatant were
BO
assayed. ~ 9o
60
RESULTS A N D DISCUSSION -- 70
~ 6o 40
Activity of Enzymes in the 5(
Presence of Detergents 20
I0
\ 20

The activities of major enzymes of buffalo

,b sb ~b 4b '~ ?o ,!5 ~b
MFGM in the presence of detergents were esti- DEOXYCHOLATE CONCN. ('~M) SD$ CONCN, (~M)
mated to investigate the effect of detergents
upon enzyme activity• FIG. 2. Effect of concentration of deoxycholate
Mild detergent. Relative activities of the and SDS on enzyme activity.
major enzymes of buffalo MFGM in the pres-
ence of increasing concentrations of Triton X-
100 and Tween-20 are in Fig. 1. In the presence detergents may be due to alteration of inter-
of 1.5 mM Triton X-100 and 1% Tween-20, res- actions within the membrane or exposure of
pectively, 40% and 22% of ATPase activity buried enzyme molecules to the surface (3).
were lost. Tween-20 affected ATPase activity Strong detergent. Enzyme activities in the
less than Triton X-100, but one cannot tell presence of deoxycholate and SDS are in Fig.
whether the effect is due to solubilization or 2. The ATPase activity of buffalo MFGM was
denaturation. Activities of alkaline phospho- destroyed by .5 mM deoxycholate. Deoxycho-
monoesterase, xanthine oxidase, and 5'-nucleo- late treatment caused loss of ATPase activity in
tidase were enhanced by Triton X-100 and cell membrane (4). Deoxycholate was more
Tween-20. The activation of these enzymes by inhibitory to ATPase than Triton X-100 and
Tween-20. This agrees with the report of the
ATP~
effect of nonionic detergents on membrane-
e = ALKALINE PHOSPHOMONO- bound enzymes (5). But activities of alkaline
EST£RASE
~ - - ~ ' - ~ XANTHINE OXIDASE
phosphomonesterase, xanthine oxidase, and
e - - * - - 4 5LNUCLEOTIOASE 5'-nucleotidase were enhanced by deoxycho-
late. The apparent activation by deoxycholate
12C

IIC _ t/./"
~ - -
lOC I00 *tKACm~ ~HOSeHOMONOESrESASE
. . - . . 5'--NVCL~OV*D~SE
*.- o..-o ×~NTHmEOX]OAgE
~ 9c

~ 8c BC-
i so sol ,~

~ 7( 4 ° ~ 41 "*/'1~°
30 ~"~" . ......... °~i ,,w 60
6( GO
2o r;"/" ~ r/ / ' 4e /

5( 5 ,o .,7 ~o :ii
I
0 1.0 210 2 I. 5
TRITON X-IO0 CONCN.('~M) TWEEN-20 CONCN.( * / , )
o s ,o '25 ~o ~s ,~o zb ,~o s'o~o
TRtTON X--IO0 CONCfl.C~M) WEEN 20 ONCN ( / ) DEOXYCHOLATECONCN(~M)

FIG. 1. Effect of concentration of Triton X-IO0

and Tween-20 on enzyme activity. FIG. 3. Solubilization of enzymes by detergents.

Journal of Dairy Science Vol. 61, No. 6, 1978

 ENZVMESOFFATGLOBULE MEMBRANE
of some membrane-bound enzymes has been
ascribed to enzyme solubilization or dissocia-
tion of an inhibitor (12, 13).
The SDS at low concentration inhibited
ATPase activity. Alkaline phosphomonoester-
ase, xanthine oxidase, and 5'-nucleotidase also
were inhibited by 2 mM SDS. This concentra-
tion of SDS causes conformation changes in the
proteins of erythrocytc membrane (8), so the
inhibition of MFGM-bound enzymes may be
due to changes in conformation of these en-
zymes in the presence of SDS.
Solubilization of
Membrane Enzymes by Detergent
The percentages of the activities of MFGM
enzymes in the soluble fraction after treatment
with increasing concentrations of Triton X-100
and Tween-20 are in Fig. 3. The percentages
are based on the sum of the activity in the
supernatant and sedimented material. None of
the enzymes were inhibited by the concentra-
tions of detergents. The percentage of 5'-
nucleotidase in the supernatant was lower than
that of xanthine oxidase. Both Triton X-100
and Tween-20 solubilized more alkaline phos-
phomonoesterase and xanthine oxidase than
t
5-nucleotldase. The different enzyme solubil-
ities may reflect different binding to the mem-
brane. 5'-Nucleotidase was the most firmly
bound to MFGM and may be more characteris-
tic of MFGM proteins. Since Tween-20 could
solubilize all enzymes more than Triton X-100,
these enzymes can be purified better in Tween-
20 than Triton X-IO0.
Figure 3 also depicts the solubilization of
alkaline phosphomonoesterase, xanthine oxi-
dase, and 5'-nucleotidase from MFGM by de-
oxyeholate. At lower concentrations of de-
oxycholate, alkaline phosphomonoesterase was
TABLE 1. Optimum concentration of detergent to solubilize MFGM enzymes.
Concentration
Detergent of detergent
Triton X-100 5 mM
Tween-20 1%
Deoxycholate 80 mM
699
solubilized more easily than xanthine oxidase
and 5-nucleondase.
' At 20 mM deoxycholate,
75% alkaline phosphomonoesterase, 50% xan-
thine oxidase, and 30% 5'-nucleotidase were in
the supernatant. But at higher deoxycholate
concentrations, more than 80% of all these en-
zymes were solubilized. Although the ATPase
of MFGM is lost in deoxycholate, this detergent
can be used successfully to sotubilize and purify
the other three enzymes. This concurs with the
report of Huang and Keenan (7) on the effec-
tiveness of deoxycholate in releasing 5'-nucleo-
tidase from cow MFGM.
The concentrations of detergents for maxi-
mum enzyme recovery in the supernatant is in
Table 1. The activities of alkaline phospho-
monoesterase, xanthine oxidase, or 5'-nucleo-
tidase were not inhibited at these concentra-
tions. Since all the above detergents inhibited
the ATPase activity, it was not possible to as-
certain the percentage of this enzyme solubil-
ized. The SDS at high concentration inhibited
all these enzymes, so the extent of solubiliza-
tion of the enzyme by this detergent was not
assessed. Deoxycholate was the most effective
detergent for solubilizing alkaline phospho-
monoesterase, xanthine oxidase, and 5'-nucleo-
tidase.
This study indicates that all the enzymes
associated with MFGM are not bound similarly,
so organization of these enzymes on the mem-
brane may not be the same. This supports our
earlier observation (1, 2) that the enzyme activ-
ities associated with MFGM depended on the
procedure used for membrane isolation because
some enzymes are more strongly bound than
others.
ACKNOWLEDGMENTS
The authors are grateful to D. Sundaresan,
Enzyme activity solubilized (%)
Alkaline
phosphomono- Xanthine 5'-nucleo-
esterase oxidase tidase
40 35 32
58 45 40
91 82 80
Journal of Dairy Science Vol. 61, No. 6, 1978
700 BHAVADASAN AND GANGULI
Director, f o r his k i n d i n t e r e s t in this s t u d y . One
o f us (MKB) also is i n d e b t e d t o t h e Indian
Council o f Agricultural Research, N e w Dehli,
for financial assistance.
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