EFFECT OF PREGNANCY GLYCOSYLTRANSFERASE CALGARY
S. PARK and T. W. KEENAN
ALFRED A. BUSHWAY.CHUWG Laboratory
of Mammary
AND LACTATION ON ACTIVITIES OF RAT GLAND
Biology Department of Animal Science. West Lafayette. IN 47907. U.S.A. (Received
12 July
Purdue
University.
1978)
EfTect of stage of pregnancy and lactation on levels of protein, DNA, lactose synthetase. CMP-N-acetylneuraminic acid: GM, sialyltransferase. CMP-N-acetylneuraminic acid synthctase. CMP-N-acetylneuraminic acid: GM, sialyltransferase. and UDP-galactose:glucosyl ceramide galactosyltransferase in rat mammary gland were examined. 2. In all cases protein content and enzyme activities began to increase one to 2 weeks before parturition and demonstrated sharp increases one to three days after parturition. 3. Maximum enzyme activity. expressed per unit protein, occurred around day thirteen of lactation for all enzymes examined except for CMP-N-acetylneuraminic acid: GM, si~iyltransferase, 4. There was a strong positive correlation between the levels of lactose synthetase. CMP-N-acetylneuraminic acid synthetase. CMP-N-acetyineuraminic acid:GM, sialyltransferase and UDP-galactose:glucosyl ceramide galactosyl-transferase on any given day. 5. Analysis of variance demonstrated that enzyme levels showed linear, quadratic and cubic properties over the course of the experimental period. Abstract-l.
intermediate involved in the formation of new plasma membrane. To our knowledge no one has examined the effect of stage of differentiation on activity of glycosyltransferases involved in giycolipid and glycoprotein biosynthesis in m~mary gtand. The membrane flow theory would predict that levels of nucleotide-sugar synthetases and glycosyitransfer~es involved in glycolipid and glycoprotein biosynthesis would be high during the same period that milk synthesis was the greatest. As a further test of this hypothesis, we studied the effect of pregnancy and lactation on levels of protein, DNA, lactose synthetase, CMP-N-acetylneuraminic acid synthetase, CMP-N-acetylneuraminic acid: GM, sialyltransferase, CMP-N-acetylneuraminic acid: GM, sialyltransferase, and UDPgalactose: glucosylceramide galactosyltransferase in rat mammary gland.
INTRODL'CTION The theory of membrane flow (Franke et al., 1971; Morrt? & Mollenhauer 1974; MO& et al., 1974) states that formation of new plasma membrane is initiated in the rough endoplasmic reticulum and that discrete membrane domains progress through the Golgi apparatus to secretory vesicles which ultimately fuse with the plasma membrane. The initial experiments were preformed with rat liver (Morre et al., 1974), but the hypothesis has now been extended to include the lactating 1972; Keenan
mammary gland et al., 1974).
(Keenan
&
Huang,
During lactation, mammary gland epithelial cell apical plasma membrane is constantly being lost as milk fat globule membrane which envelops milk lipid droplets as they are discharged from the cell into the gland lumen (Patton & Fowkes, 1967; Bargmann & Welsch, 1969: Keenan er al.. 1970; Linzell & Peaker, 1971). As apical plasma membrane is lost, new plasma membrane must be synthesized to maintain the integrit? of the surface membrane. Keenan & Huang (1972) have shown the protein and lipid composition of bovine mammary gland Golgi apparatus to be intermediate between that of endoplasmic reticulum and plasma membrane. Bovine mammary gland Golgi apparatus has also been shown to contain glycosyltransferases involved in the synthesis of monosialo- and disialogangliosides (Keenan, 1974b). These studies suggested that Golgi apparatus may be the
MATERIALS AND
METHODS
Female rats of the Sprague-Dawley strain. weighing 15&175 g, were obtained from Murphy Breeding Laboratories, Plainfield, IN U.S.A. Male rats of the Long-Evans strain, weighing 250-300 g. were obtained from the-Biology rat colony, Purdue University, West Lafayette, IN. Glucosylceramide was isolated from bovine milk fat globule membrane preparations (Kayser & Patton. 1970). Other glycosphingolipids were isolated as in earlier studies (Keenan, 1974a,b). Unlabeled CMP-N-acetylneuraminic acid was prepared according to Kean (1970). The ion exchange resin AGl-X8 (chloride form) was from Bio-Rad. Richmond, CA. Detergents. z-IactaIbumin, U DP-galactose, N-acetylneuraminic acid and hyamine hydroxide were from Sigma, St. Louis, MO. [U-‘“Cl UDP-gaiactose (230 Ciimole). CMP-N-[4,5.6.7,8,9,~~C]-acetylneuraminic acid (214 Ci/mole and N-[4.5.6,7.8.9.“C]-acetyineuraminic
Abbrrciations: DNA. deoxyribonucleic acid; CMP, cytidine~5’-monophosphat~; Glucosyl ceramide. glucose-ceramide: Lactosyl ceramide. galactose-glucose-ceramide; GM3. sialic acid-galactose-glucose-ceramide; GM ,, galactose-N-acetylgalactosamine-(sialic aczd)-galactose-glucoseceramidc. 147
148
ALI-HI.II
A.
BMHWAY.
CHUM;
S. PARK
and T. W.
KLENAN
Giycosyltransferase acid (259 Ci/mole) were from Am~rsham-Searie~ Arlington Heights, IL. Folin reagent was from Fisher, Fair Lawn. NJ and Ready-Solv HP scintillation fluid was from Beckman Instruments, Irvine. CA. Treatments
qf animals
Rats were housed under conditions of controlled temperature and lighting. A diet of Wayne Lab Block was fed freely. Each afternoon one male was placed in a cage with 2-4 females. The following morning males were removed and vaginal smears taken from each female. Those females which were sperm positive were placed in individual cages and the date of breeding recorded. At birth the number of pups per mother was fixed at from 6 to 10. Four rats were used for each day pre- and post-partum. Rats were sacrificed by cervical dislocation and the inguinal mammary glands were removed and weighed. All subsequent procedures were performed at 0-4’C. Tissue was trimmed, finely minced and placed in 30 ml of 0.32 M sucrose: 14 mm 2-mercaptoethanol. Tissue was homogenized with a Polytron Homogenizer (Kinematica. Lucerne Switzerland). Portions of the homogenate were retained for protein (Lowry et al., 1951) and DNA (Burton. 1956) assays. The remaining homogenate was filtered and centrifuged at 8009 for 1Omin to remove debris. Supernatants were centrifuged at 176,OOOg for 1 hr and the particulates were resuspended in sucrose-metcaptoethanol and used as the enzyme source.
For lactose synthetase assay complete reaction mixtures contained. in a final volume of 0.1 ml, the following: Tris -HCI, pH 7.4. 20mM: MnC12, 20 mM; D-glUCOSe, 20mM; (12.5 x lo6 CPM,ipmole). UDP-galactose 0.25mM: a-lactalbumin, 2Opg; Triton X-100, 0.5”; and 100 pg of total particulate protein. Incubations were at 37’C for 4min and reactions were stopped by addition of EDTA to 200mM. Contents of the reaction tube were placed onto a 0.5 x 8 cm column of Bio-Rad AGI-X8 resin packed in a Pasteur pipette. Columns were eluted with 2 x 0.5ml of water and radioactivity in the eluate was measured in a Beckman liquid scintillation spectrometer (Fitzgerald et al., 1970a.b). For IMP-N-a~tvlneuraminic acid synthetast assay complete reaction mixtures contained, in a final volume of O2ml. the following: Tris acetate buffer. pH 8.5. 100mM: CTP. 2SmM; MgCI,. 2SmM; t-mercaptoethanal, 1.25 mM; N-acetylneuraminic acid (22 x 106 CPM/ Imole). OSmM; Triton X-100. OS?;, and 1OOpg of total particulate protein. Incubation was for 10 min at 37 C and reactions were stopped by the addition of 50~1 EDTA (200 mM). Reaction mixtures were quantitatively streaked onto 5 x 40 cm strips of Whatman 3 MM chromatography paper IOcm from the origin. Papers were developed. descending. for 20hr in ethanol: I M ammontum acetate (5::. by vol) at 4-C. Papers were dried. areas eorresponding to CMP-N-acetylneuraminic acid cut out and radioactivity was determined in a Beckman scintillation spectrometer (Kean. 1970: Warren & Blacklow, 1962). For each set of reactions a parallel strip streaked with [14C]-CMPN-acetylneuraminic acid was developed to act as a standard. For incorporation of galactose into giucosylceramide, complete reaction mixlures contained in a final volume of 0.1 ml: Glucosylceramide. 0.5 mM; Triton CF-54, 400 pg; cacodylate-HCl. pH 7.3, 150 mM; MnCI,. 300 mM; .2-mercaptoethanol, 1OOmM: UDP-galactose (12.5 x lo6 CPM/pmole), 0.5 mM; and 0.5 mg particulate protein. For sialyltransferase assays complete reaction mixtures contained (in Almoles) in a final iolume of 0.1 ml: Glycolipid acceptor, 0.5 mM; Triton CF-54, 4OOpg; cacodylate-HCl, pH 6.35; 150 mM; MgC!,, 300 mM; CMP-N-
activities
rn rat mammary
gland
149
acetylneuraminic acid (2.0 x t0’ CPM/~moie). 0.5 mM and 0.5 mg particulate protein. Incubations were for 1 hr at 37C at which time reactions were stopped by the addition of 1.2 ml of chloroform-methanol (2: 1, by voi). The reaction mixtures were dried under a stream of Nz and the residue taken up in 0.57ml of chloroform-methanal-H,Q (60:30:4.5). This solution was applied to a column of 0.6 g Sephadex G-25 fine which had been conditioned in chloroform-methanol-water (60: 30:4.5. by vol) (3 rinses over a 2 hr period and then allowed to stand in solvent overnight). The column was eluted with 1.2ml of the above solvent followed by 1.2 ml of chloroform methanol (2:l. by vol). The eluate was evaporated under nitrogen in a water bath at 37 C and the residue solubllized in 1ml hyamme hydroxide. Sclntiilation fluid (5g PPO and 0.1 g POPOP:‘titer of toluene) was added and radicactivity was determmed in a Beckman scintillation spectrometer (Carter & Kanfer, 1973). Specific activity values were calculated from the amount of radioactive sugar transferred to acceptor per unit total particulate protein. In all cases blanks without added acceptor were assayed in parallel to determine the amount of sugar incorporated into endogenous acceptors; this value was subtracted to determine incorporation into exogenous acceptor. All values were obtained under conditions of linear kinetics with respect to incubation time and enzyme concentration.
Statistical procedures used were analysis of variance for main treatment effects (Snedccor & Cochran. 1967). Duncan’s test (Steel & Torrie. 1960) for comparmg treatment means. and regression analysis (Snedecor & Cochran, 1967). RESULTS AND DISCUSSION
The means and standard errors for each of the parameters for each day they were measured are given in Table 1. The maximum activity for lactose synthetase, CMP-N-acetylneuraminic acid synthetase, and UDP-galactose:glucosylceramide galactosyltransferase occurred at about day I3 of lactation. while maximum activities in CMP-N-acetylneuraminic acid: GM, and GM, sialyltransferase were attained during the latter stages of pregnancy or the first days of lactation. The coefficients of variation (CV) for the enzymes measured in this study indicated that the amounts of variation in measurements were very small. CV values for the enzymes ranged from 92, for CMP-N-acetylneuraminic acid synthetase to l9yd) for CMP-N-acetylneuraminic acid: GM, sialyltransferase (Table 1). From the r’ values one can deduce that there was a strong relationship between day preor post-partum and the levels of protein, lactose synthetase, CMP-N-acetylneuraminic acid synthetase and CMP-N-acetykeuraminic acid : GM 1 sialyltransferase in rat mammary gland (Table 1). The effect of day pre- and post-partum on the level of CMP-Nacetylneuraminic acid: GM, sialyltransferase and UDP-galactose: glucosyi-ceramide gaIactosyltransferase is not as pronounced (Table 1). Other factors must affect the expression of these two enzymes. In Table 2 is presented an analysis of variance on the activity of three enzymes (CMP-N-acetylneuraminic acid synthetase. CMP-N-acetylneuraminic acid: GM, sialyltransferase and UDP-galactose: glucosylceramide galactosyltransferase). The effect of day preand post-partum on enzyme levels showed linear, quadratic and cubic properties over the course of the
ALFRED A. BUSHWAY. CHUNG S. PARK and T. W. KEENAN
150 Table
2. Error and mean squares associated with enzyme activities of rat mammary tisstles
source
d.f:’
Total Day
59 14
Linear Quadratic
Cubic Residual Error
0.64** 1.42** 5.34** 0.24** 0.17** 0.01
45
.’ d.f. = Degrcrs h Abhrcviations ** P i 0.01.
of freedom. as in Table
0.51** 0.12** 6.06** 0.10** 0.07** 0.01
0.36** 0.03 2.7X** 0.40** 0.17** 0.01
I.
IO
6
14
18
0
8
4
period.
Figures
IL8
show
the
12
16
20
24
28
DAY (pre and post pa&m)
Fig.
1. Effect
of day
pre-
and
post-partum calculated
,O
6
Fig. 2. Effect of day
14
18
0
in weight of rat mammary by least squares procedure.
4
8
12
16
20
tissues.
24
Best fit curve
was
28
DAY (pre ond post porturn) pre-
was calculated
and
post-partum
by least squares
best
fit
curves for the parameters measured. Included with the best fit curves for the enzyme data are plots of the actual data obtained from the assays. From these plots of the original data it can be seen why enzyme activities demonstrated linear, quadratic and cubic properties over the course of the experimentation period (Figs 3-7). For all enzymes studied activity began to increase l-2 weeks before parturition and increased sharply 1-3 days after parturition. Maximum enzyme activity occurred around day 13 of lactation for all enzymes examined except for CMP-Nacetylneuraminic acid: GM, and GM, sialyltransferases (Figs 3-7). For lactose synthetase and CMP-N-acetylneuraminic acid synthetase maximum activity occurred in
Mean squares GMI” Gal transh
CMPh
I I I II
experimental
on protein procedure.
levels in rat mammary gland. Best fit curve Assay conditions are given in text.
Glycosyltransferase
activities
in rat mammary
gland
Y
F = -I
9435,
t 2 41932x
- 31409x2
*
01067X’
x IO
6
I8
14
0 DAY (pre
and post porturn)
Fig. 3. Effect of day pre- and post-partum on lactose synthetase levels in rat mammary particulate fractions. (0). Best fit curve ucas calculated by least squares procedure. (2). Assay conditions are given in the text.
IO z
c
14
18
0 DAY
total data.
x
4
(pre
gland Actual
8
and
12
16
20
24
28
post porturn)
Fig. 4. Effect of day pre- and post-partum on CMP-N-acetylneuraminic acid synthetase levels In rat mammary gland total particulate fractions. (0). Best fit curve was calculated by least squares proccdurc. (0). Actual data. Assay conditions arc given in the text.
DAY
(pre
and
post parlum)
Fig. 5. Effect of day pre- and post-partum on CMP-~-acetylneuraminic acid: GM3 siaiyltransferase levels in rat mammary gland total particulate fractions. (0). Best fit curve was calculated by least squares procedures. (O), Actual data. Assay conditions are given in the text.
ALFREV A. BUSHWAY. CHUWG S. PARK
152
and T. W.
KEENAN
1
2400 -
La
14
6
I8
0 DAY
4
8
I2
16
20
24
28
’
(pre and post porturn)
Fig. 6. Effect of day pre- and post-partum on CMP-N-acetylneuraminic acid: GM, sialyltransferase levels in rat mammary gland total particulate fractions. (0). Best fit curve was calculated by least squares procedure. (Cl), Actual data. Assay conditions are given in the test.
6
IO
14
18
0 __., ,
4
8
1;
UYT rpre ano. posr. porturn)
Fig. 7. Effect of day pre- and post-partum on UDP-galactose: Glucosylceramid~ ga~actosyltransferase levels in rat mammary gland total particulate fractions. (Of, Best fit curve was calculated by least squares procedure. (0). Actual data. Assay conditions are given in the text.
mid to late lactation (Figs 3 and 4). This observation supports results of McKenzie et al. (1971) for lactose synthetase in rats. These workers found that there was very little lactose synthetase activity prior to parturition. This was followed by a sharp increase with maximum activity occurring between the 13th and 16th day of Iactation and then by a rapid drop during involution. Kuhn (1968) has shown, in the rat, that lactose synthetase activity in the absence of a-lactalbumin is essentially zero until 24 hr prior to parturition, but in the presence of Aactalbumin there is a low but definite activity prior to parturition. To our knowledge, this is the first time that the effect of day pre- and post-partum on glycolipid glycosyltransferase activity in mammary gland has been studied. If one examines data for CMP-N-acetylneuraminic acid: GM, sialyltransferase (GM,), it can be seen that there is no significant difference in enzyme activity in early pregnancy and late lactation (Fig. 6). Highest enzyme activity occurred at parturition
and between the 13th and 16th day of lactation (Fig. 6). Similar results were obtained for UDP-galactose: glucosylceramide galactosyltransferase although the enzyme demonstrated significant pre-partum activity (Fig. 7). The enzyme CMP-N-acetylneuraminic acid: GM, sialyltransferase demonstrated a slightly different pattern of activity. Enzyme activity increased significantiy during pregnancy and again at parturition (Fig. 5). Enzyme activity was high between day 13 and 16 of lactation (Fig. 5). From Figs 5 and 7 it can be seen that CMP-N-acetyfneuraminic acid: GM, sialyltransferase and UDPgalactose: glucosylceramide galactosyltransferase increased during mid pregnancy. Since these enzymes are involved in early steps of ganglioside biosynthesis (Basu et al., 1965, 1973; Kaufman et al., 1966, 1968; Roseman, 1970). this initial increase may be important in new plasma membrane synthesis as cell numbers and cell size increase in mammary gland. Table 3 demonstrates the correlation among
Glycosyltransferase
activittes
in rat mammary
153
gland
9I IO
6
I
I 14
I,,
I 0
I8
DAY
I (pre
I 4
I
and
I 8
I
I 12
I
II I6
I 20
I
II 24
I 28
I* x
posf parturn)
Fig. 8. Effect of day pre- and post-partum was calculated
by least squares
Table Tissue Tissue Protein LX CMP GM3 GMI Gal trans DNA
1.00 0.44 0.74** 0.78** 0.0 1 0.3 I 0.48 0.92**
)’ Abbreviations * P < 0.05. ** P -galactosc: glycolipid galactosyltransferase. J. hid. Chert~. 240, 41 I5 41 17. BASU S., KAUFMAN B. & ROSEMANS. (1973) Enzymatic synthesis of glucocerebroside by a glucosyltransferase from embryonic chicken brain. J. hiol. Chem. 248, 1388- 1394. BURTON K. (1956) A study of the conditions and mechanism of the diphenylamine reaction for the calorimetric estimation of deoxyribonucleic acid. Biochem. J. 62, 315-322.
CARTER T. P. & KANFER J. (1973) Methodology for separation of gangliosides from potential water-soluble precursors. Lipids 8, 537-548. FITZGERALD D. K., BRODBECK U.. KIYOSAWA I.. MAWAL R., COLVIN B. & EBNER K. E. (1970a) n-Lactalbumin and the lactose synthetase reaction. J. hiol. Chew. 245, 2103-2108.
I54
ALFK~.I>A. BUSHWAY. CHUNC; S. PARK and T. W. KEENAN
FITZGERALI) D. K.. COLVIU B.. MAWAL R. & ERNEK K. E. (t970b) Enrymic assay for galactosyl transferase activity of lactose synthetase and a-lactalbumin in purified and crude systems. il&yr. Biodwm. 06, 43-61. FRANKL- W. W., M~KR~ D. J.. &rlMLING B.. CHFFTHAM R. D.. KART~NB~TK J.. JARASCI-I E. D. & ZENTGRAE H. (1971) Synthesis and turnover of membrane proteins m rat liver: An examination of the membrane flow hypot bests Z. Nnturfo~.sc~l~. 26B, 103 I 1039. KAIJI’MAN B., BA.& S. & ROSEMAN S. ( 1966) In Inborn Disor&s of Spltinqolipid Mcttrho/i.w~ (Edited by Aronson & Volk). pp. 193-213. Pergamon Press. New York. KAI:I-MAN B., BASU S. 8: ROSI.MANS. (IP6SIEnzymatic synthesis of disi~lo~an~iiosides from monosilogangliosides by sialyitr~~nsfer~scs from embryonic chick&n brain. J. hicii. t&m. 243, 5804.S807. KAVSER S. G. & PATTON S. (1970) The function of very long chain fatty actds in membrane structure: Evidence from milk cerebrosides. Riochcn~. hioph\,s. Res. Commun. 41, 1572-1578. KI;AN E. L. (1970) Nuclear cytldine S’-monophosphosialic acid synthetase. J. hiol. C/IU?I. 245, 2301-2308. KE~NAN T. W. (1974a) Composition and synthesis of gangllosides in mammary gland and milk of the bovine. BioC~hirn. hiopllyc. Acrrr 337, 75% 270. KE~N.~N T. W. (1974b) Membranes of mammary gland. IX. Concentration of glycosphingolipid galactosyl and sialyltransferases in Golgi apparatus from bovine mnmmary gland. J. Dolry Sci. 57. I87 192. Kttxhx T. W. & Hu,~s(; C. M. (1972) Membranes of rn~~rnrn~ry gland. VI. Lipid and protein compositit~n of Golg apparatus and rough endopiasmic reticulum from bovine mammary gland. J. Dair!. %i. $5. I%&- 1596. KAFKA*; T. W.. M~>RR$.D. J. & H~AN(; C. M. (1974) Membranes of the mammary gland In Lal,r[~ion (Edited by LARSON & SZUTHI. Vol. II. pp. 191 23.1, Academic PreTs. Ncv, York. KEENAN T. W., MORRF( D. J.. OLSON D. E.. YCJNGHANS W. N & P.~TTON S. ( 1970) Biochemical and morphologl-
cal comparison of plasma membrane and milk fat globule membrane from bovine mammary gland. .i. 3n enqme from Nciswrin rtwtlimqitidis. J. hrol. Chi’m 237. 35’7.-3637.
S. PARK and T. W. KEENAN
ALFRED A. BUSHWAY.CHUWG Laboratory
of Mammary
AND LACTATION ON ACTIVITIES OF RAT GLAND
Biology Department of Animal Science. West Lafayette. IN 47907. U.S.A. (Received
12 July
Purdue
University.
1978)
EfTect of stage of pregnancy and lactation on levels of protein, DNA, lactose synthetase. CMP-N-acetylneuraminic acid: GM, sialyltransferase. CMP-N-acetylneuraminic acid synthctase. CMP-N-acetylneuraminic acid: GM, sialyltransferase. and UDP-galactose:glucosyl ceramide galactosyltransferase in rat mammary gland were examined. 2. In all cases protein content and enzyme activities began to increase one to 2 weeks before parturition and demonstrated sharp increases one to three days after parturition. 3. Maximum enzyme activity. expressed per unit protein, occurred around day thirteen of lactation for all enzymes examined except for CMP-N-acetylneuraminic acid: GM, si~iyltransferase, 4. There was a strong positive correlation between the levels of lactose synthetase. CMP-N-acetylneuraminic acid synthetase. CMP-N-acetyineuraminic acid:GM, sialyltransferase and UDP-galactose:glucosyl ceramide galactosyl-transferase on any given day. 5. Analysis of variance demonstrated that enzyme levels showed linear, quadratic and cubic properties over the course of the experimental period. Abstract-l.
intermediate involved in the formation of new plasma membrane. To our knowledge no one has examined the effect of stage of differentiation on activity of glycosyltransferases involved in giycolipid and glycoprotein biosynthesis in m~mary gtand. The membrane flow theory would predict that levels of nucleotide-sugar synthetases and glycosyitransfer~es involved in glycolipid and glycoprotein biosynthesis would be high during the same period that milk synthesis was the greatest. As a further test of this hypothesis, we studied the effect of pregnancy and lactation on levels of protein, DNA, lactose synthetase, CMP-N-acetylneuraminic acid synthetase, CMP-N-acetylneuraminic acid: GM, sialyltransferase, CMP-N-acetylneuraminic acid: GM, sialyltransferase, and UDPgalactose: glucosylceramide galactosyltransferase in rat mammary gland.
INTRODL'CTION The theory of membrane flow (Franke et al., 1971; Morrt? & Mollenhauer 1974; MO& et al., 1974) states that formation of new plasma membrane is initiated in the rough endoplasmic reticulum and that discrete membrane domains progress through the Golgi apparatus to secretory vesicles which ultimately fuse with the plasma membrane. The initial experiments were preformed with rat liver (Morre et al., 1974), but the hypothesis has now been extended to include the lactating 1972; Keenan
mammary gland et al., 1974).
(Keenan
&
Huang,
During lactation, mammary gland epithelial cell apical plasma membrane is constantly being lost as milk fat globule membrane which envelops milk lipid droplets as they are discharged from the cell into the gland lumen (Patton & Fowkes, 1967; Bargmann & Welsch, 1969: Keenan er al.. 1970; Linzell & Peaker, 1971). As apical plasma membrane is lost, new plasma membrane must be synthesized to maintain the integrit? of the surface membrane. Keenan & Huang (1972) have shown the protein and lipid composition of bovine mammary gland Golgi apparatus to be intermediate between that of endoplasmic reticulum and plasma membrane. Bovine mammary gland Golgi apparatus has also been shown to contain glycosyltransferases involved in the synthesis of monosialo- and disialogangliosides (Keenan, 1974b). These studies suggested that Golgi apparatus may be the
MATERIALS AND
METHODS
Female rats of the Sprague-Dawley strain. weighing 15&175 g, were obtained from Murphy Breeding Laboratories, Plainfield, IN U.S.A. Male rats of the Long-Evans strain, weighing 250-300 g. were obtained from the-Biology rat colony, Purdue University, West Lafayette, IN. Glucosylceramide was isolated from bovine milk fat globule membrane preparations (Kayser & Patton. 1970). Other glycosphingolipids were isolated as in earlier studies (Keenan, 1974a,b). Unlabeled CMP-N-acetylneuraminic acid was prepared according to Kean (1970). The ion exchange resin AGl-X8 (chloride form) was from Bio-Rad. Richmond, CA. Detergents. z-IactaIbumin, U DP-galactose, N-acetylneuraminic acid and hyamine hydroxide were from Sigma, St. Louis, MO. [U-‘“Cl UDP-gaiactose (230 Ciimole). CMP-N-[4,5.6.7,8,9,~~C]-acetylneuraminic acid (214 Ci/mole and N-[4.5.6,7.8.9.“C]-acetyineuraminic
Abbrrciations: DNA. deoxyribonucleic acid; CMP, cytidine~5’-monophosphat~; Glucosyl ceramide. glucose-ceramide: Lactosyl ceramide. galactose-glucose-ceramide; GM3. sialic acid-galactose-glucose-ceramide; GM ,, galactose-N-acetylgalactosamine-(sialic aczd)-galactose-glucoseceramidc. 147
148
ALI-HI.II
A.
BMHWAY.
CHUM;
S. PARK
and T. W.
KLENAN
Giycosyltransferase acid (259 Ci/mole) were from Am~rsham-Searie~ Arlington Heights, IL. Folin reagent was from Fisher, Fair Lawn. NJ and Ready-Solv HP scintillation fluid was from Beckman Instruments, Irvine. CA. Treatments
qf animals
Rats were housed under conditions of controlled temperature and lighting. A diet of Wayne Lab Block was fed freely. Each afternoon one male was placed in a cage with 2-4 females. The following morning males were removed and vaginal smears taken from each female. Those females which were sperm positive were placed in individual cages and the date of breeding recorded. At birth the number of pups per mother was fixed at from 6 to 10. Four rats were used for each day pre- and post-partum. Rats were sacrificed by cervical dislocation and the inguinal mammary glands were removed and weighed. All subsequent procedures were performed at 0-4’C. Tissue was trimmed, finely minced and placed in 30 ml of 0.32 M sucrose: 14 mm 2-mercaptoethanol. Tissue was homogenized with a Polytron Homogenizer (Kinematica. Lucerne Switzerland). Portions of the homogenate were retained for protein (Lowry et al., 1951) and DNA (Burton. 1956) assays. The remaining homogenate was filtered and centrifuged at 8009 for 1Omin to remove debris. Supernatants were centrifuged at 176,OOOg for 1 hr and the particulates were resuspended in sucrose-metcaptoethanol and used as the enzyme source.
For lactose synthetase assay complete reaction mixtures contained. in a final volume of 0.1 ml, the following: Tris -HCI, pH 7.4. 20mM: MnC12, 20 mM; D-glUCOSe, 20mM; (12.5 x lo6 CPM,ipmole). UDP-galactose 0.25mM: a-lactalbumin, 2Opg; Triton X-100, 0.5”; and 100 pg of total particulate protein. Incubations were at 37’C for 4min and reactions were stopped by addition of EDTA to 200mM. Contents of the reaction tube were placed onto a 0.5 x 8 cm column of Bio-Rad AGI-X8 resin packed in a Pasteur pipette. Columns were eluted with 2 x 0.5ml of water and radioactivity in the eluate was measured in a Beckman liquid scintillation spectrometer (Fitzgerald et al., 1970a.b). For IMP-N-a~tvlneuraminic acid synthetast assay complete reaction mixtures contained, in a final volume of O2ml. the following: Tris acetate buffer. pH 8.5. 100mM: CTP. 2SmM; MgCI,. 2SmM; t-mercaptoethanal, 1.25 mM; N-acetylneuraminic acid (22 x 106 CPM/ Imole). OSmM; Triton X-100. OS?;, and 1OOpg of total particulate protein. Incubation was for 10 min at 37 C and reactions were stopped by the addition of 50~1 EDTA (200 mM). Reaction mixtures were quantitatively streaked onto 5 x 40 cm strips of Whatman 3 MM chromatography paper IOcm from the origin. Papers were developed. descending. for 20hr in ethanol: I M ammontum acetate (5::. by vol) at 4-C. Papers were dried. areas eorresponding to CMP-N-acetylneuraminic acid cut out and radioactivity was determined in a Beckman scintillation spectrometer (Kean. 1970: Warren & Blacklow, 1962). For each set of reactions a parallel strip streaked with [14C]-CMPN-acetylneuraminic acid was developed to act as a standard. For incorporation of galactose into giucosylceramide, complete reaction mixlures contained in a final volume of 0.1 ml: Glucosylceramide. 0.5 mM; Triton CF-54, 400 pg; cacodylate-HCl. pH 7.3, 150 mM; MnCI,. 300 mM; .2-mercaptoethanol, 1OOmM: UDP-galactose (12.5 x lo6 CPM/pmole), 0.5 mM; and 0.5 mg particulate protein. For sialyltransferase assays complete reaction mixtures contained (in Almoles) in a final iolume of 0.1 ml: Glycolipid acceptor, 0.5 mM; Triton CF-54, 4OOpg; cacodylate-HCl, pH 6.35; 150 mM; MgC!,, 300 mM; CMP-N-
activities
rn rat mammary
gland
149
acetylneuraminic acid (2.0 x t0’ CPM/~moie). 0.5 mM and 0.5 mg particulate protein. Incubations were for 1 hr at 37C at which time reactions were stopped by the addition of 1.2 ml of chloroform-methanol (2: 1, by voi). The reaction mixtures were dried under a stream of Nz and the residue taken up in 0.57ml of chloroform-methanal-H,Q (60:30:4.5). This solution was applied to a column of 0.6 g Sephadex G-25 fine which had been conditioned in chloroform-methanol-water (60: 30:4.5. by vol) (3 rinses over a 2 hr period and then allowed to stand in solvent overnight). The column was eluted with 1.2ml of the above solvent followed by 1.2 ml of chloroform methanol (2:l. by vol). The eluate was evaporated under nitrogen in a water bath at 37 C and the residue solubllized in 1ml hyamme hydroxide. Sclntiilation fluid (5g PPO and 0.1 g POPOP:‘titer of toluene) was added and radicactivity was determmed in a Beckman scintillation spectrometer (Carter & Kanfer, 1973). Specific activity values were calculated from the amount of radioactive sugar transferred to acceptor per unit total particulate protein. In all cases blanks without added acceptor were assayed in parallel to determine the amount of sugar incorporated into endogenous acceptors; this value was subtracted to determine incorporation into exogenous acceptor. All values were obtained under conditions of linear kinetics with respect to incubation time and enzyme concentration.
Statistical procedures used were analysis of variance for main treatment effects (Snedccor & Cochran. 1967). Duncan’s test (Steel & Torrie. 1960) for comparmg treatment means. and regression analysis (Snedecor & Cochran, 1967). RESULTS AND DISCUSSION
The means and standard errors for each of the parameters for each day they were measured are given in Table 1. The maximum activity for lactose synthetase, CMP-N-acetylneuraminic acid synthetase, and UDP-galactose:glucosylceramide galactosyltransferase occurred at about day I3 of lactation. while maximum activities in CMP-N-acetylneuraminic acid: GM, and GM, sialyltransferase were attained during the latter stages of pregnancy or the first days of lactation. The coefficients of variation (CV) for the enzymes measured in this study indicated that the amounts of variation in measurements were very small. CV values for the enzymes ranged from 92, for CMP-N-acetylneuraminic acid synthetase to l9yd) for CMP-N-acetylneuraminic acid: GM, sialyltransferase (Table 1). From the r’ values one can deduce that there was a strong relationship between day preor post-partum and the levels of protein, lactose synthetase, CMP-N-acetylneuraminic acid synthetase and CMP-N-acetykeuraminic acid : GM 1 sialyltransferase in rat mammary gland (Table 1). The effect of day pre- and post-partum on the level of CMP-Nacetylneuraminic acid: GM, sialyltransferase and UDP-galactose: glucosyi-ceramide gaIactosyltransferase is not as pronounced (Table 1). Other factors must affect the expression of these two enzymes. In Table 2 is presented an analysis of variance on the activity of three enzymes (CMP-N-acetylneuraminic acid synthetase. CMP-N-acetylneuraminic acid: GM, sialyltransferase and UDP-galactose: glucosylceramide galactosyltransferase). The effect of day preand post-partum on enzyme levels showed linear, quadratic and cubic properties over the course of the
ALFRED A. BUSHWAY. CHUNG S. PARK and T. W. KEENAN
150 Table
2. Error and mean squares associated with enzyme activities of rat mammary tisstles
source
d.f:’
Total Day
59 14
Linear Quadratic
Cubic Residual Error
0.64** 1.42** 5.34** 0.24** 0.17** 0.01
45
.’ d.f. = Degrcrs h Abhrcviations ** P i 0.01.
of freedom. as in Table
0.51** 0.12** 6.06** 0.10** 0.07** 0.01
0.36** 0.03 2.7X** 0.40** 0.17** 0.01
I.
IO
6
14
18
0
8
4
period.
Figures
IL8
show
the
12
16
20
24
28
DAY (pre and post pa&m)
Fig.
1. Effect
of day
pre-
and
post-partum calculated
,O
6
Fig. 2. Effect of day
14
18
0
in weight of rat mammary by least squares procedure.
4
8
12
16
20
tissues.
24
Best fit curve
was
28
DAY (pre ond post porturn) pre-
was calculated
and
post-partum
by least squares
best
fit
curves for the parameters measured. Included with the best fit curves for the enzyme data are plots of the actual data obtained from the assays. From these plots of the original data it can be seen why enzyme activities demonstrated linear, quadratic and cubic properties over the course of the experimentation period (Figs 3-7). For all enzymes studied activity began to increase l-2 weeks before parturition and increased sharply 1-3 days after parturition. Maximum enzyme activity occurred around day 13 of lactation for all enzymes examined except for CMP-Nacetylneuraminic acid: GM, and GM, sialyltransferases (Figs 3-7). For lactose synthetase and CMP-N-acetylneuraminic acid synthetase maximum activity occurred in
Mean squares GMI” Gal transh
CMPh
I I I II
experimental
on protein procedure.
levels in rat mammary gland. Best fit curve Assay conditions are given in text.
Glycosyltransferase
activities
in rat mammary
gland
Y
F = -I
9435,
t 2 41932x
- 31409x2
*
01067X’
x IO
6
I8
14
0 DAY (pre
and post porturn)
Fig. 3. Effect of day pre- and post-partum on lactose synthetase levels in rat mammary particulate fractions. (0). Best fit curve ucas calculated by least squares procedure. (2). Assay conditions are given in the text.
IO z
c
14
18
0 DAY
total data.
x
4
(pre
gland Actual
8
and
12
16
20
24
28
post porturn)
Fig. 4. Effect of day pre- and post-partum on CMP-N-acetylneuraminic acid synthetase levels In rat mammary gland total particulate fractions. (0). Best fit curve was calculated by least squares proccdurc. (0). Actual data. Assay conditions arc given in the text.
DAY
(pre
and
post parlum)
Fig. 5. Effect of day pre- and post-partum on CMP-~-acetylneuraminic acid: GM3 siaiyltransferase levels in rat mammary gland total particulate fractions. (0). Best fit curve was calculated by least squares procedures. (O), Actual data. Assay conditions are given in the text.
ALFREV A. BUSHWAY. CHUWG S. PARK
152
and T. W.
KEENAN
1
2400 -
La
14
6
I8
0 DAY
4
8
I2
16
20
24
28
’
(pre and post porturn)
Fig. 6. Effect of day pre- and post-partum on CMP-N-acetylneuraminic acid: GM, sialyltransferase levels in rat mammary gland total particulate fractions. (0). Best fit curve was calculated by least squares procedure. (Cl), Actual data. Assay conditions are given in the test.
6
IO
14
18
0 __., ,
4
8
1;
UYT rpre ano. posr. porturn)
Fig. 7. Effect of day pre- and post-partum on UDP-galactose: Glucosylceramid~ ga~actosyltransferase levels in rat mammary gland total particulate fractions. (Of, Best fit curve was calculated by least squares procedure. (0). Actual data. Assay conditions are given in the text.
mid to late lactation (Figs 3 and 4). This observation supports results of McKenzie et al. (1971) for lactose synthetase in rats. These workers found that there was very little lactose synthetase activity prior to parturition. This was followed by a sharp increase with maximum activity occurring between the 13th and 16th day of Iactation and then by a rapid drop during involution. Kuhn (1968) has shown, in the rat, that lactose synthetase activity in the absence of a-lactalbumin is essentially zero until 24 hr prior to parturition, but in the presence of Aactalbumin there is a low but definite activity prior to parturition. To our knowledge, this is the first time that the effect of day pre- and post-partum on glycolipid glycosyltransferase activity in mammary gland has been studied. If one examines data for CMP-N-acetylneuraminic acid: GM, sialyltransferase (GM,), it can be seen that there is no significant difference in enzyme activity in early pregnancy and late lactation (Fig. 6). Highest enzyme activity occurred at parturition
and between the 13th and 16th day of lactation (Fig. 6). Similar results were obtained for UDP-galactose: glucosylceramide galactosyltransferase although the enzyme demonstrated significant pre-partum activity (Fig. 7). The enzyme CMP-N-acetylneuraminic acid: GM, sialyltransferase demonstrated a slightly different pattern of activity. Enzyme activity increased significantiy during pregnancy and again at parturition (Fig. 5). Enzyme activity was high between day 13 and 16 of lactation (Fig. 5). From Figs 5 and 7 it can be seen that CMP-N-acetyfneuraminic acid: GM, sialyltransferase and UDPgalactose: glucosylceramide galactosyltransferase increased during mid pregnancy. Since these enzymes are involved in early steps of ganglioside biosynthesis (Basu et al., 1965, 1973; Kaufman et al., 1966, 1968; Roseman, 1970). this initial increase may be important in new plasma membrane synthesis as cell numbers and cell size increase in mammary gland. Table 3 demonstrates the correlation among
Glycosyltransferase
activittes
in rat mammary
153
gland
9I IO
6
I
I 14
I,,
I 0
I8
DAY
I (pre
I 4
I
and
I 8
I
I 12
I
II I6
I 20
I
II 24
I 28
I* x
posf parturn)
Fig. 8. Effect of day pre- and post-partum was calculated
by least squares
Table Tissue Tissue Protein LX CMP GM3 GMI Gal trans DNA
1.00 0.44 0.74** 0.78** 0.0 1 0.3 I 0.48 0.92**
)’ Abbreviations * P < 0.05. ** P -galactosc: glycolipid galactosyltransferase. J. hid. Chert~. 240, 41 I5 41 17. BASU S., KAUFMAN B. & ROSEMANS. (1973) Enzymatic synthesis of glucocerebroside by a glucosyltransferase from embryonic chicken brain. J. hiol. Chem. 248, 1388- 1394. BURTON K. (1956) A study of the conditions and mechanism of the diphenylamine reaction for the calorimetric estimation of deoxyribonucleic acid. Biochem. J. 62, 315-322.
CARTER T. P. & KANFER J. (1973) Methodology for separation of gangliosides from potential water-soluble precursors. Lipids 8, 537-548. FITZGERALD D. K., BRODBECK U.. KIYOSAWA I.. MAWAL R., COLVIN B. & EBNER K. E. (1970a) n-Lactalbumin and the lactose synthetase reaction. J. hiol. Chew. 245, 2103-2108.
I54
ALFK~.I>A. BUSHWAY. CHUNC; S. PARK and T. W. KEENAN
FITZGERALI) D. K.. COLVIU B.. MAWAL R. & ERNEK K. E. (t970b) Enrymic assay for galactosyl transferase activity of lactose synthetase and a-lactalbumin in purified and crude systems. il&yr. Biodwm. 06, 43-61. FRANKL- W. W., M~KR~ D. J.. &rlMLING B.. CHFFTHAM R. D.. KART~NB~TK J.. JARASCI-I E. D. & ZENTGRAE H. (1971) Synthesis and turnover of membrane proteins m rat liver: An examination of the membrane flow hypot bests Z. Nnturfo~.sc~l~. 26B, 103 I 1039. KAIJI’MAN B., BA.& S. & ROSEMAN S. ( 1966) In Inborn Disor&s of Spltinqolipid Mcttrho/i.w~ (Edited by Aronson & Volk). pp. 193-213. Pergamon Press. New York. KAI:I-MAN B., BASU S. 8: ROSI.MANS. (IP6SIEnzymatic synthesis of disi~lo~an~iiosides from monosilogangliosides by sialyitr~~nsfer~scs from embryonic chick&n brain. J. hicii. t&m. 243, 5804.S807. KAVSER S. G. & PATTON S. (1970) The function of very long chain fatty actds in membrane structure: Evidence from milk cerebrosides. Riochcn~. hioph\,s. Res. Commun. 41, 1572-1578. KI;AN E. L. (1970) Nuclear cytldine S’-monophosphosialic acid synthetase. J. hiol. C/IU?I. 245, 2301-2308. KE~NAN T. W. (1974a) Composition and synthesis of gangllosides in mammary gland and milk of the bovine. BioC~hirn. hiopllyc. Acrrr 337, 75% 270. KE~N.~N T. W. (1974b) Membranes of mammary gland. IX. Concentration of glycosphingolipid galactosyl and sialyltransferases in Golgi apparatus from bovine mnmmary gland. J. Dolry Sci. 57. I87 192. Kttxhx T. W. & Hu,~s(; C. M. (1972) Membranes of rn~~rnrn~ry gland. VI. Lipid and protein compositit~n of Golg apparatus and rough endopiasmic reticulum from bovine mammary gland. J. Dair!. %i. $5. I%&- 1596. KAFKA*; T. W.. M~>RR$.D. J. & H~AN(; C. M. (1974) Membranes of the mammary gland In Lal,r[~ion (Edited by LARSON & SZUTHI. Vol. II. pp. 191 23.1, Academic PreTs. Ncv, York. KEENAN T. W., MORRF( D. J.. OLSON D. E.. YCJNGHANS W. N & P.~TTON S. ( 1970) Biochemical and morphologl-
cal comparison of plasma membrane and milk fat globule membrane from bovine mammary gland. .i. 3n enqme from Nciswrin rtwtlimqitidis. J. hrol. Chi’m 237. 35’7.-3637.
