t-pa and plasminogen embedded in casein rule its degradation
TRANSCRIPT
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t-PA and plasminogen embedded in casein rule
its degradation
Nissim Silanikovea*, Fira Shapiroa , Gabriel Leitnerb
aa Biology of Laction Lab., Institute of Animal Science, Agricultural Research Organization, The Volcani Center, P.O.
Box 6, Bet Dagan 50250, Israel
b bNational Mastitis Reference Center, Kimron Veterinary Institute, Bet Dagan 50250, Israel
*Corresponding author: email: [email protected]
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Abstract
The aims of this study were to test the assumption that tissue-
plasminogen activator (t-PA) and plasminogen are closely associated to
the casein micelle and form a functional complex that rule casein
degradation. This assumption was essentially verified for bovine’s milk.
It was also shown that the second type of plasminogen activator presented
in milk, urokinase-PA (u-PA), was not involved in casein degradation. It
was found that t-PA and plasminogen are found in freshly secreted milk
(less than 10 min from its secretion), strongly suggesting that they are
secreted as complex by the mammary gland epithelial cells.
Keywords: t-PA, u-PA, plasminogen, plasmin, casein, milk, bovine
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Introduction
Milk secretion rate in various mammals is tightly associated with
the fluctuating offspring nutritional demands and mammary gland (MG)
development stages are closely related to the nurse’s reproduction cycles
[1-2]. The regulation of day-to-day variation in milk secretion [2-3] and
induction of MG involution [4] are ruled by milk-borne negative
feedback (MBNF) system. The plsminogen activator (PA)-plasminogen
(PG)- plasmin (PL) enzymatic system is ubiquitously expressed in the
milk of human [5], rodents [6], and ruminants [4] and was found to be
associated both with regulation of milk secretion in cows and goats [7-8]
and activation of involution in rodents [6], goats [9] and cows [10-12].
As in other body tissues, PL is presented in milk mainly by its
inactive zymogen form PG, whose conversion to PL is modulated by PAs
[12]. The two types of PAs that exist in mammals systemic fluids,
urokinase-type PA (u-PA) and tissue-type PA (t-PA) are also presented in
milk [6,12-14]. In milk, PL, PG, and t-PA are closely associated with the
casein micelles, whereas u-PA is associated with neutrophils in close
association to its specific receptor [12, 14].
The MBNF system associated with regulation of milk secretion
was shown to comprise the PA- PG- PL system that specifically forms a
β-casein (CN) fragment (f) (1–28) from β-CN, which in return, serves as
the negative control signal by closing potassium channels on the apical
membrane of the epithelial cells of the MG [7-8]. Down-regulation of
these channels induces undefined inwardly directed cellular signals that
inhibit milk secretion. Interestingly, a further activation of the PA-PG-PL
system, which was coupled to more extensive degradation of casein
induced involution of the MG in lactating goats and cows and forcefully
activated the innate immune system [9-11]. Based on these findings, a
casein hydrolyzate (CNH) preparation was developed to reduce the
suffering from MG engorgement associated with abrupt cessation of
milking (the conventional procedure to induce involution in modern dairy
cows) [15] and to treat and prevent common clinical and subclinical
infections of the udder in dairy cows [16-18]. It was also found that CN-
derived peptides induced by PL activity inhibits milk clotting [19-20],
which is important during mastitis and milk stasis in preventing
uncontrolled inflammation [21].
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There are convincing evidences that under physiological situations
t-PA is the main factor that involve in casein hydrolysis [6, 12, 22].
However, the picture remained unclear because: i. there is a lot of data
showing that u-PA is involved as a major factor in CN hydrolysis in
stored milk [23], and ii. That the increase of u-PA secretion by MG
epithelial cells under inflammation (12, 22] was claimed to be responsible
for CN degradation in goat’s milk under mastitis [24].
The association of t-PA with the CN micelles suggests that it
constitutes a pivotal component of the MBNF system; however, as noted
above, evidence for the involvement of u-PA confronts this assumption.
The ability of CNH to up regulate the PL system activity in a manner that
imitate in accelerated manner the events associated with MG involution
stage 1 [9-11] represent an opportunity to test the assumption regarding
the pivotal role of t-PA in CN degradation under relevant physiological
situation.
The aims of the present experiment were: i. to test the assumption
that t-PA and PG embedded within casein micelles form a functional
complex that rule casein hydrolysis by analyzing the distribution of the
PL system in different fraction of bovine milk in control and in response
to CNH treatment, ii. to test the assumption that t-PA, PG and CN micelle
are secreted by MG cells as functional unit by verifying the presence of t-
PA and PLG in CN micelle in freshly secreted milk, and iii. to exclude
role of u-PA in CN hydrolysis under the experimental conditions.
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Material and Methods
Materials. H-D-Norleucyl-hexahydrotyrosol-lysine-pnitroanilide
diacetate [Spectrozyme PL (SpecPL)], bovine PG, and cyanogens
bromide fibrinogen digest (FIBGN) were purchased from American
Diagnostica (Greenwich, CT, USA). N-methylsulphonyl-D-Phe-Gly-Arg-
4-nitroanilide acetate substrate was obtained from Boehringer Mannheim
(Chromozym t-PA; UK, East Sussex, UK). Polyclonal rabbit anti-human
t-PA IgG was obtained from Oxford Biomedical Research (Oxford, UK)
and Plasminogen activator inhibitor-1 (PAI-1), was from Calbiochem
(USA). Other mentioned chemicals were purchased from Sigma
(Rheovot, Israel).
Ethical considerations. All protocols were approved by the Institutional
Animal Care Committee of the Agricultural Research Organization,
which is the legitimate body for such authorizations in Israel.
Experiment 1. Six Israeli Holstein heifers with low leukocyte content, as
indicated by low somatic cell count (< 70,000 cells/ml) and no bacterial
finding according to preliminary analysis [17], yielding ~36 l/day-1
milk,
at their second to third were lactation used. Two MGs, one front and one
rear quarters, were infused with sterile saline solution while the other two
counter were infused with casein hydrolyzate (CNH).
The experiment was carried out during November under natural
lighting regimen, with typical noon temperatures of 24 C0
and night
temperatures of 12 C0, which is within the thermoneutral zone of cows
[25]. The cows were milked thrice daily (0530, 1230, and 2130) and milk
yield and exact milking times were individually recorded automatically
[18].
All the experimental procedures were carried out during the noon
milking. Milk samples (100 ml) were taken from every gland of each cow
at -24 h, 0 h, +18 h relative to treatment with saline solution and CNH,
where 0 h refer to day of infusion. Milk samples on day 0 were taken
prior to the infusion. At day 0, a dose of 10 mL of CNH [17], with a
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peptide concentration of 7 mg mL− 1
was infused into each treated
gland with a special applicator following careful sterile cleaning of the
teat. The control glands of the cows in the first group were infused with
10 ml of sterile saline. Milk yield was discarded for 3 days following the
infusion.
Analytical procedures: One set of samples (10 ml) was send to central
laboratory for the determination of total protein, fat, lactose and somatic
cell count [8]. A second set of milk samples (2 x 10 ml) were defatted
under cold conditions [26] and analyzed as follow according to
previously described procedures: concentration of lactose, protein, fat,
casein, whey protein, proteose peptones, lactoferrin, albumin, Na+ and K
+
and the activity of xanthine oxidase, lactoperoxidase, and the
concentration of nitrite (by the DAN reagent), nitrate (by the Griess
reaction), and uric acid [11]. A sub-set of skim milk was ultracentrifuged
and clear milk serum (whey) devoid of membranous particles and casein
micelle pellet were separated [26].
A third set of samples (70 ml) was used to isolate somatic cells from
milk. The samples were centrifuged at 2,000 × g for 30 min at 4°C;
then the fatty fraction and supernatant were removed. Cells from the
bottom layer were suspended in 500 μl of PBS (pH 7.4) containing
0.02% NaN3 and centrifuged twice (400 × g for 15 min at 4 C0) to
concentrate cells. A cell concentration of at least 1 × 107 cells/ml was
obtained, which was measured using Fossomatic 90 (Foss Electric,
Hillerød, Denmark). After separation the cells were lysed by at least 3
freeze-thaw cycles.
The activities of PL, PG, PA, t-PA and u-PA were determined in the
whey and in the re-dissolved casein micelle pellets and isolated somatic
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cells. Whey, casein micelle pellets (1 mg/ml), cell lysates and other
reagents were dissolved and diluted in 0.05 modified Tris buffer (MTB)
composed of 0.05 M Tris, 0.1 M NaCl, 0.01% twin 80, pH 7.6. The actual
amount of proteins in the reaction mixtures was determined by the
Bradford method.
Plasmin and PG activities of the isolated fractions were measured by
chromogenic assays [27] in a plate reader (Bio Kinetics Reader EL
340; Bio-Tek Instruments) in triplicate, with minor modifications in the
volumes and concentrations used. For PL determination, sample (25 µL)
was added to 225 µL MTB that contained 1.6 mM SpecPL and allowed to
react for 1 h at 37°C in water bath and then read at 405 nm. For PG
determination, sample (100 µL) was added to 100 µL MTB that
contained 3.2 mM SpecPL and 100 µL MTB that contained human u-PA
(280 IU/mL), and the reaction mixture was allowed to react for 1 h at
37°Cin a water bath and then read at 405 nm. Proper blank and control
mixtures were prepared.
Plasminogen activators and their subtypes were determined by
chromogenic reactions as follow: The t-PA activity was measured
according to published procedure [27] by incubating 25 µL samples
dissolved in MTB with 225 µL of 60 mM Tris-HCl, pH 8·5, 0·09%
Tween 20, with 0·375 mg/ml N-methylsulphonyl-D-Phe-Gly-Arg-4-
nitroanilide acetate substrate (Chromozym t-PA). The absorbance at 405
nm was measured for 60 min and the generation of nitroaniline was
determined by the rate of change in absorbance.
The types of PA present in the CN micelles were further
established as follow: (i) Plasminogen activator activity was determined
reaction system that was composed of 25 µL samples nixed with 225 µL
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MTB that contained bovine PG (32 µg/ml), 1.6 mM SpecPL, with and
without fibrinogen fractions (FIBGN; 16.2 µg/ml), with and without 4
mM of amiloride. Amiloride, was used to inhibit u-PA activity, and the
fibrinogen fractions, to stimulate t-PA activity, which enables to
differentiate between t-PA activity from u-PA [14]. Tissue-PA was
scored as the activity obtained in the presence of fibrinogen fragments
and amiloride, subtracting the activity obtained in the presence of
amiloride, but in the absence of fibrinogen fragments. Plasminogen
activator activity caused by u-PA was scored as the activity obtained in
the absence of fibrinogen fragments, but in the presence of anti-human t-
PA IgG, subtracting the activity obtained in the presence of amiloride.
(ii) Polyclonal rabbit anti-human t-PA IgG was included to a final
concentration of 0.1 mg/ml in the reaction mixture for direct
determination of t-PA activity, and (iii) The effects of PAI-1 at final
concentration of 500 ng/ml was determined in the CN micelles under the
condition of the direct t-PA analysis [28].
Experiment 2. Milk (∼50 ml per cow) was obtained from udders of six
Israeli-Holstein cows producing ~ 40l/day. The sample from each cow
was taken from a mixed yield of a bacterial-free (see experiment 1) single
udder. and was designated as mature milk (MM). After that milking, the
sampled glands in these cows were completely emptied by hand milking.
When no more milk could be obtained by hand milking, the cows were
injected intramuscularly with a dose of 20 international units of oxytocin
(Vetimex, Bladel, Holland). After 3–5 min the MG was hand milked
again, to ensure that any residual milk left in the alveolus was evacuated.
The cows were injected intramuscularly with a dose of 30 international
units of oxytocin and after 3–5 min 30–50 ml of milk were sampled from
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each of the previously sampled glands; this milk was designated as fresh
milk (FM). The MM and FM samples were stored in dry ice immediately
after sampling, and were transported to a nearby laboratory, where they
arrived at a temperature of 6–10 °C, and were analyzed within less than
20 min for the content of xanthine + hypoxanthine as described before
[29]. The milk defined as FM was fractionated to obtained whey, CN
micelle and somatic cells and these fractions were analyzed for the
content of t-PA, u-PA, PG and PL as described for experiment 1.
Statistical analysis. The results of experiment 1 were analyzed by using
repeated-measures analysis to model correlated residuals within cow as
described previously [10]. The analysis concentrated on the effects of
treatment, day, and treatment × day interactions. The effects of parity
and of days in milk were not significant (P > 0.25) and therefore were not
included in the analyses presented here.
Results
Treating MG with CNH induced dramatic changes in the secretion
and composition of the treated glands, whereas no significant changes
were recorded in the treated gland (Table 1). Milk yield fell 5.5 folds in
comparison to pre-treated or control levels, and lactose concentration
drooped 7.7 folds, so that lactose secretion was reduced by ~42 folds.
Na+ concentration increased ~ 4 folds whereas K
+ concentration
decreased ~ 4 folds, so that their level in milk of treated glands resembled
the expected level of Na+ and K
+ in blood plasma. Protein concentration
increased in the skim milk of treated glands by ~22%, which could be
related to increase of 78% in whey protein concentration. This was
particularly associated with dramatic increase in the concentration of
proteose peptones (CN degradation peptides), and soluble components of
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the acquired (IgG,), and innate (lactoferrin, albumin, lactoperoxidase,
xanthine oxidase) and related metabolites (uric acid, nitrite and nitrate).
CNH treatment was also associated by ~ 14 folds increase in the treated
glands in the count somatic cells, which are composed mainly of
leukocytes.
Most of PA activity in pre-treated and control glands was
associated with the CN micelle and only minority was found in milk
serum and somatic cells (Table 2). CNH treatment induced increase in PA
activity in CN micelle and somatic cells. However, the activity of PA in
CN micelle per ml milk was 4 folds higher than in somatic cells. No PG
activity could be detected in milk serum or in the somatic cells (data not
shown in a table).
t-PA activity in CN micelle in pre-treated, control and treated gland
accounted for the vast majority of PA activity (Table 3). CNH treatment
induced dramatic reduction in PG activity without change in total PG +
PL activity. Thus, the large increase in PL activity in the CNH treated
glands can be related to conversion of PG to PL and was associated with
dramatic reduction in PG to PL ratio (Table 3).
PA activity was measured with the following additions in CN
micelles and somatic cells (Table 4): Amiloride and anti-u-PA antibody
did not affect PA activity in CN micelles, but reduced it in somatic cells.
PAI-1 and anti t-PA antibody dramatically reduced PA activity in CN
micelles, but not in somatic cells. Addition of fibrin increased PA activity
in CN micelles, but not on somatic cells.
The concentration of xanthine + hypoxanthine in milk defined as
freshly secreted was 38.5 ± 5 µM. On the other hand, no xanthine +
hypoxanthine could be detected in milk defined as mature milk, or in
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freshly secreted milk stored at 37 0C for 30 min (data not shown in a
table). Based on that, we can conclude that freshly secreted milk
represent milk that was sampled within 10 min or less after being secreted
[29]. The content of t-PA, PG and PL in freshly secreted milk was found
to be similar to those found in the pre-treated and control glands (Table
4).
Discussion
It is well established that amiloride affect u-PA but not t-PA, that
PA-1 affect t-PA but not u-PA and that fibrin accelerate t-PA activity but
not u-PA activity [12-14 23, 28]. Based on that, it can be concluded that
under physiological conditions, t-PA in milk is vastly associated with the
CN micelle, whereas, no u-PA cannot be traced in the micelle. This
conclusion is further supported by the interaction with respective anti-
bodies to t-PA and u-PA. As mentioned, this conclusion is also consistent
with some previous reports [6,12, 22]. However, what’s unique to this
study is that the great increase in t-PA activity was induced by CNH. In
previous studies, we show that CNH induce accelerated MG involution,
which is associated with intense activation of the MG immune system [9-
11]. This conclusion is supported by the data presented in Table 1, which
demonstrate that the treatment with CNH precipitously reduced
mammary secretion, disrupts the tight junction (increase Na+ and
decrease in K+ concentrations), induce degradation of CNH, and
activates various elements of the innate and acquired immune system.
These aspects were considered in detail in previous publications [4, 9-11]
and therefore will not consider here in detail. However, our data is
consistent with the theory that the PL system plays a key role in inducing
MG involution by degradation of CN micelle and liberating or inducing
the formation active components that in turn affect MG epithelial cells to
commit involution. In this study, we have identified t-PA as the principal
PA, which is responsible for the conversion of PG to PL.
Our results are also consistent with previous ones, which show that
PG is closely associated with the casein micelle [12, 24]. However,
perhaps the most novel finding in this study, namely, the presences of t-
PA and PG in the CN micelles of freshly secreted milk provide a new
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insight into the setting of PL-casein interaction. It was already
demonstrated that t-PA is produced and secreted by MG cells [12, 30].
Thus, the close presence of t-PA and CN micelles should not be
surprising as they both share the same excretory pathway through
secretory vesicles released from the Golgi apparatus and because t-PA
has high affinity to the CN micelles [16, 23]. Following early suggestion
[16], PG is generally considered to leak to milk from blood plasma.
Though, to the best of our knowledge; there is no evidence that support
this notion. The presence of PG in fresh milk strongly suggest that it is
secreted into milk through the secretory vesicles route embedded within
the CN micelles along with its activator, t-PA and based on the results
Sorrel et al [28] , most likely along with PA-1 because it secreted by MG
cells [30].
The close association between PL, t-PA, PA-1 and CN suggests
that these components serve as functional complex that regulate the
liberation of active components from the CN micelle. Such an
arrangement allows effective fine tuning of the CN micelles degradation
process: i. it allow the complex to function as time machine, milk stasis
will result in longest exposure and thus higher degradation, and ii. It
allows fast responsive reaction to relevant systemic hormonal effects,
which either attenuate or stimulate casein hydrolysis [4].
The localization of u-PA with somatic cells and lack of u-PA
activity in milk serum is consistent with previous reports [12, 14, 22]. In
more detailed studies, it was demonstrated that milk u-PA is bound
mostly to u-PA receptors on polymorphonuclear cells. Recently, it was
shown that MG cells respond to lipopolysacharide challenge (pro-
inflammatory stress) by increasing the expression of u-PA [31]. The
physiological role of u-PA was attributed to its ability to induce basal
membrane degradation and thus help to induce inflow of
polymorphonuclear to injure of infected tissue [31].
According to present findings, the reported correlation between
increased u-PA activity and CN hydrolysis during mastitis [24], merely
reflect the fact that they response similarly to the inflammatory stress.
Our results in consistent with those of others [12-14, 22], clearly
indicated that there is no direct relation between u-PA activity and CN
hydrolysis in raw milk during inflammation. The substantial evidence for
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major role of u-PA in casein degradation in stored milk may be explained
by the dissociation of u-PA from its receptor and the tendency of u-PA to
form interaction with CN. There are many reports that show that heat
treatment, such as that applied during pasteurization, inactivate PAI-1 and
increase the association between u-PA and CN [23].
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Table 1. Effect of treatment with CNH on milk yield (on a single gland level) and skim milk
composition of protein, casein degradation products (proteose peptpnes), Na, K and
components of the immune system in pre-treated, control and treated glands (mean ± SD)
Measures Pre-treated* Control* Treated*
Milk yield, l/day 8.5 ± 1.5a 8.2 ± 1.4 a 1.5 ± 0.9 b
Lactose, mM 148 ± 5.5 a 142± 6.2 a 19 2. ± 3 b.5
Na+, mM 26.2 ± 4.4 a 29.1 ± 4.6 a 110.0 ± 8.5 b
K+, mM 37.2 ± 5.1 a 33.1 ± 4.8 a 8.2 ± 1.9 b
Total protein, mg/ml 32.4 ± 2.5 a 32.8 ± 2.6 a 39.8 ± 3.1 b
Casein, mg/ml 24.6 ±2.7 a 24.9 ± 2.8 a 25.8 ± 3.4 b
Whey protein, mg/ml 7.8 ± 1.9 a 7. 9 ± 2.0 a 14.0± 2.2 b
Proteose peptones, µg/ml 465 ± 29.5 a 472± 31.2 a 1037 ± 37.5 b
IgG, µg/ml 200.5 ± 19.9 a 307.9 ±25.2 a 998.5 ± 31.3 b
Lactoferrin, µg/ml 175.2 ± 19.1 a 200.1 ± 23.8 a 1425 ± 41.3 b
albumin, µg/ml 127.0 ± 17.9 a 255.6± 18.5 a 600.5 ± 24.9 b
Uric acid, µM 34.7 ± 5.9 a 35.2 ± 6.6 a 75.2 ± 8.7 b
nitrite, µM 0.6 ± 0.2 a 0.8 ± 0.3 a 8.1± 1.1 b
nitrate, µM 25.1 ± 4.7 a 27.1 ± 5.1 a 145.6 ± 9.9 b
Lactoperoxidase, unit/ml 2.5 ± 0.5 a 3.1 ± 1.1 a 14.7± 1.3 b
Xanthine oxidase, unit/ml 11.6 ± 2.9 a 12.6 ± 3.3 a 70.9 ± 5.7 b
Somatic cell count. Number/ml
75000 ± 8200 a 82000 ± 9300 a 1050000 ± 12100 b
*Results from experiment 1.
a, bValues mark by different superscript letter are significantly different, P <0.01 or
lower.
21
Table 2. Effect of CNH treatments on the distribution of PA activity (unit/ml*) in milk
fractions (mean ± SD).
Glands Pre-treated** Control** Treated**
Plasminogen activator (PA)
Casein micelle 115.1 ± 5.8 d 118.0 ±6.6 d 463.2 ± 11.7 e
Milk serum 5.5 ± 2.2a 4.5± 2.5 a 11.2 ± 3.7 b
Milk somatic cells b.d 35.5 ± 5. c 101.1 ± 9.9 d
ml* - Using dilution and protein content activity of PA was calculated to be based on
ml of reconstituted raw milk
** results from experiment 1
a,b,c,d,e Values marked by different superscript letter are significantly differebt, P <
0.01 or lower.
21
Table 3. Effect of CNH treatments on the activities of the PA-PLG-PL (unit/ml*) in isolated
casein micelles in pre-treated, control, freshly secreted milk and treated gland (mean ± SD).
Pre-treated** Freshly secreted***
Control** Treated**
PA unit/ml 115.1 ± 5.8 a 114.7 ± 5.9 a 118.0 ±6.6 a 463.2 ± 11.7 b
t-PA 109.5 ± 5.5 a 110.0 ± 5.7 a 110.5 ± 6.3 a 461. 2 ± 10.7 b
plasminogen 37.5 ± 3.8 a 38.1 ± 4.9 a 40.4 ± 4.3 a 6.1± 3.8 b
Plasmin 5.1 ± 1.9 a 5.2 ± 2.8 a 5.3 ± 3.8 a 42.4 ± 5.2 b
Plasminogen +plasmin
42.6 ± 6.1 a 43.1 ± 6.8 a 42.7 ± 5.8 a 48.5 ± 7.3 a
Plasminogen/plasmin ratio
8.4 ± 1.1 a 7.3 ± 1.5 a 8.6 ± 2.1 a 1.1 ± 0.5 b
ml* - Using dilution and protein content activities of PA, PLG and PL were calculated
to be based on ml of reconstituted raw milk
** results from experiment 1
*** results from experiment 2
a,b,c,d,e Values marked by different superscript letter are significantly differebt, P <
0.01 or lower.
22
Table 4. Effect of amiloride, anti-U-PA, PA-I and fibrin on PA activity (unit/ml*) in isolated
casein micelles and isolated somatic cells (mean ± SD).
treatments Casein micelle somatic cells
Pre-treated** 120.2 ± 5.8 a 10.5 ± 1.1 a
Amiloride 118 ± 6.1 a. 1.9 ± 0.9b
Anti u-PA 115 ± 7.1 a 0.9± 1.1b
Anti t-PA 15.7 ± 4.9b 10.4 ± 1.3 a
Fibrin 185 ± 7.9b 10.5 ± 1.5 a
PA-I 25.0 ± 5.5b 10.3 ± 1.4 a
ml* - Using dilution and protein content activity of PA was calculated to be based on
ml of reconstituted raw milk
Milk from pre-treated gland (6 cows) was used to isolate casen micelle and somatic
cells.
a,b Values mark by different superscript letters are significantly different from the
pre-treated values by pair-t-test analysis ; P <0.01 or lower.