Printed in Sweden Copyright 0 I975 by Academic Press, Inc. A/I rights of reproduction in my form rcsrrcrd
Experimental
MEMBRANES X. Adenosine
Cell Research 90 (1975) 253-260
OF MAMMARY
Triphosphate
by Golgi Apparatus
dependent
Rich Fractions
C. R. BAUMRUCKER Department
of Animal
Sciences,
Purdue
from
GLAND Calcium
Accumulation
Bovine Mammary
Gland
and T. W. KEENAN University,
West Lafayette,
Ind. 47907,
USA
SUMMARY Golgi apparatus rich fractions from lactating bovine mammary gland had an Mg*+-dependent, Ca2+-stimulated adenosine triphosphatase. These Golgi apparatus fractions also accumulated Ca*+ in vitro. Accumulation of Ca2+ required ATP and could be abolished by treatment either with low concentrations of deoxycholate followed by ulttasound, or by heating at 100°C for IO min. The adenosine triphosphatase activity of Golgi apparatus was strongly stimulated by low concentrations of Ca2+ and moderately stimulated by high concentrations of K+. This activity was unaffected by Na+ and was not inhibited by ouabain. The pH optimum for the Mg*+dependent hydrolysis of ATP was 7.5, the K,,, was 5 x lO-5 M and the activation energy was 6 000 calories/mole. This Mg2 +-dependent adenosine triphosphatase activity was also found in rough endoplasmic reticulum, smooth mictosomes and milk fat globule membrane, the latter membrane being derived directly from the apical plasma membrane. All of these membrane fractions had the ability to specifically accumulate Ca %+. Specific accumulation was highest with smooth microsomes and lowest with milk fat globule membrane with Golgi apparatus and rough endoplasmic reticulum being intermediate. These observations provide one plausible explanation for intracellular Ca2+ accumulation and secretion into milk. Further, these results help explain the ultrastructural observations of casein micelle formation in secretory vesicles elaborated by Golgi apparatus.
Research during the last decade has revealed the relaxing factor of contracted muscle to be a membrane-bound, Mg2+-dependent adenosine triphosphatase (ATPase) (ATP phosphohydrolase, E.C. 3.6.1.3) which acts as a calcium pump. This ATPase activity has been found in muscle sarcoplasmic reticulum [l] and, more recently, in plasma membranes from muscle cells [2]. The efficacy of this pump mechanism for Ca2+ is such that Ca2+ can reach concentrations in sarcoplasmic reticulum vesiclesthat are some20 times higher than in the extravesicular fluid [3]. Another tissue in which Ca2+ is concentrated is in the lactating mammary gland. Although the mechanismsare unknown, Ca2+ 17-
741804
is accumulated from the blood, where the concentration is 2 to 3 mM, and secretedwith milk, where it is present in a concentration of about 30 mM [4, 51. Thus, Ca2+ must passa concentration gradient, indicating the existence of an energy-requiring process [6]. One can envisage the existence of two mechanisms involved in translocation of Ca2+ from blood to milk. One mechanism would function in accumulation of blood Ca2+by secretory cells and the other would account for the discharge of this ion into the gland lumen. In contrast to Ca2+, the mechanismsof Na+, K+ and Clsecretion into milk have been extensively studied [7-l 11. The mechanism involved in intracellular Exptl
Ceil Res 90 (1975)
254
Baumrucker
and Keenan
accumulation and secretion of Ca2+ may be located in Golgi apparatus. Caseins, the major proteins of milk, are known to be packaged into secretory vesicles elaborated by Golgi apparatus [12-151. After formation, these vesicles migrate to the apical region of the cell where they fuse with the plasma membrane and discharge their contents:into the lumen [12, 16-191. In addition to caseins, there is evidence that other milk proteins 120, 211, lactose [17, 20, 211 and certain of the minerals of milk [22] are contained in these secretory vesicles. The caseins, which are phosphoproteins, exist in milk in the form of micelles [23]. Calcium is necessary for formation of these micelles, forming salt bonds with the covalently bonded phosphates of neighboring casein molecules [23]. In fact, the majority of the calcium of milk is associated with casein micelles [24, 251. Within the cell casein apoproteins are synthesized on ribosomes of the rough endoplasmic reticulum [13, 18, 26, 271. Phosphorus is added after completion of the apoprotein chain by a protein kinase which is concentrated in Golgi apparatus [28]. There is abundant morphological evidence indicating that casein micelles form within secretory vesicles [12-151. This implies that Ca2+ is also contained in the vesicles and suggests a role for Golgi apparatus in accumulation of Ca2+ from cytosol. To explore this possibility we have characterized an ATPase in bovine mammary Golgi apparatus. Results obtained have demonstrated the presence of a Mg2+-dependent, Ca2+-stimulated ATPase in Golgi apparatus. In addition, the ability of Golgi apparatus fractions to accumulate Ca2+ in vitro is demonstrated. METHODS
AND MATERIALS
Isolation procedures Mammary tissue was obtained from lactating Holstein
cows(BOSiypicuspritnigenius) at slaughter and cooled Exptl
Cell Res 90 (1975)
on ice during transportation to the laboratory. Golgi apparatus fractions were isolated as described previously [21,29]. Rough endoplasmic reticulum, smooth microsomes and total microsomes were also isolated [IS, 291. Milk fat globule membranes were prepated from fresh milk by churning as described previously [IO, 301. All membrane fractions were washed and resuspended in the buffers used for enzyme assay or determination of Ca2+ uptake.
Enzyme assays ATPase was assayed in a reaction mixture containing 10 mM histidine buffer. oH 7.5. 100 mM KCI. 5 mM MgCI,, 5 mM ATP and from 6.05 to 0.2 mg of fraction protein in a final volume of 1 ml. Incubation was at 30°C for various times in order to determine hydrolytic rates by regression analysis. Reactions were terminated by addition of 1 ml of 10 % trichloroacetic acid. Reactions were initiated by addition of enzyme, and blanks consisting either of heat-inactivated enzyme or no enzyme were always run to correct for nonspecific hydrolysis of ATP. Released phosphate was measured calorimetrically. Succinic dehydrogenase activity was assayed as succinate-2-(p-indophenyl)-3-(p-nitrophenyl)-j-phenyltetrazolium (INT) reductase accordinn to Pennington [31]. Galactosyl transferase activiiy of Golgi apparatus fractions was determined as in previous studies with N-acetylglucosamine as the galactose acceptor 121, 221. Caz+ accumulation was measured according to Meissner & Fleischer [33, 341. Reaction mixtures contained 10 mM histidine buffer, pH 7.5, 10 mM MgCl,, 100 mM KCI, 5 mM ATP, 0.5 mM WaCl,, I mM potassium oxalate and about 0.1 mg membrane protein in a final volume of 2 ml. Incubation was at 30°C for time intervals up to 6 min. Reactions were terminated by filtration of the reaction mixture through a 0.45 pm pore diameter Millipore filter under vacuum. After washing. the radioactivity retained on the filter was measured in Bray’s [35] scintillation fluid containing 60 g naphthalene, 4 g 2,5-diphenyloxazole, 200 mg 1,4-his-(2-(5-phenylosazolyl))-benzene. 100 ml methanol. 20 ml ethvlene glycol and sufficient p-dioxane to yield I I. Control samples without membrane. with membrane fractions treated with 0.1 % sodium deoxycholate and sonicated for 10 to 15 set (Branson Model S125, setting 3), or fractions heated for 10 min in boiling water were assayed to determine nonspecific binding of Vaz+. Protein was determined according to Lowry et al. [36] with crystalline bovine serum albumin as the standard. Inorganic phosphate was measured according to Fiske & SubbaRow [37].
Reagents Bovine serum albumin, the 5’-nucleotide di- and triphosphates, IV-acetylglucosamine and UDP-galactose were from Sigma Chemical Company, St Louis, MO. UDP-(WJ-galactose (298 mCi/mmole) was obtained from New England Nuclear, Boston, Mass. *YIaCl, (1.63 x 10m3 mCi/mmole) was from International
Calcium accumulation by Golgi apparatus Chemical and Nuclear, Cleveland, Ohio. Millipore filters were from the Millipore Corporation, Bedford, Mass. Statistical analyses were performed with the aid of a PDP 11 computer.
RESULTS Subcellular fractions used in the present study have previously been characterized with respect to homogeneity and composition [17, 18, 291. Milk fat globule membrane is known to be derived from the apical plasma membrane of mammary secretory cells (for reviews seerefs [8, 18, 381).This membrane fraction is virtually free of contamination by mitochondria, endoplasmic reticulum and Golgi apparatus [17, 18, 381. Rough endoplasmic reticulum fractions as prepared are nearly completely free of contamination by other subcellular fractions [17, 18,291. In particular, the rough endoplasmic reticulum as well as the smooth microsomal fractions contain only negligible quantities of mitochondria. Golgi apparatus fractions, while being nearly 90 % pure with respect to membranous contaminants, contain low but variable amounts of mitochondria [17, 18, 291. Since the presence of a Ca2+ binding protein and ATPase activity in mitochondria are well known [39], care was taken to exclude mitochondrial contaminants from Golgi apparatus fractions used in this study. That this was achieved was determined by assay for succinate INT reductase, a known mitochondrial marker enzyme. Golgi apparatus fractions had a specific activity in this enzyme of 0.05 pmole INT reduced/h/mg protein. The ratio of the activity in Golgi apparatus to that in total homogenates was 0.025. This compares with the g-fold enrichment of this activity in mammary mitochondria relative to total homogenates [18]. From these data it can be calculated that, on a protein basis, mitochondria accounted for less than 1% of the material in Golgi apparatus fractions. Simi-
255
Table 1. Hydrolysis ojnucleotide triphosphates and diphosphatesby Golgi apparatus fractions from bovine mammary gland Substrate ATP GTP UTP CTP ADP GDP UDP CDP
Specific activity’ 2.50 2.40 2.55 2.61
0.50 0.76 0.75
0.41
R2 0.92 0.94 0.97 0.95 0.85 0.95 0.98 0.80
a Specific activity, a rate determined by computerassisted regression analysis, is pmoles of substrate hydrolysed/mg protein/h. Complete incubation mixtures contained 10 mM histidine, pH 7.5,5 mM MgCI,, 100 mM KCI, 5 mM nucleotide tri- or diphosphate and 0.1 mg Golgi apparatus protein in a final volume of 1 ml. Incubation was at 3o’C for various intervals from 1 to 20 min. R2 values are correlation coefficients.
larly, galactosyl transferase, an enzyme specifically localized in Golgi apparatus [18, 21, 29, 321, was enriched 20 times in Golgi apparatus fractions. This figure compares favorably with the 16- to 18-fold enrichment of this enzyme previously measured in morphologically homogeneous Golgi apparatus fractions [18, 21, 291. Multiple Golgi apparatus fractions from bovine mammary gland were obtained and all were found to hydrolyse ATP. The ratio of specific activity in Golgi apparatus to that in total homogenates was, on the average, 5.8. Hydrolysis of ATP was linear up to about 0.15 mg of Golgi apparatus protein/ml. The rate of ATP hydrolysis at 30°C was linear for all times tested. The average specific activity of Golgi apparatus fractions was about 2.5 pmoles of inorganic phosphate liberated from ATP/h/mg protein. In addition to ATP other nucleotide triphosphates were hydrolysed by Golgi apparatus fractions (table 1). The fraction was most active with CTP. The Exptl
Cell Res 90 (1975)
256
Baumrucker
I
Oo
I
.7
and Keenan
I
I
50
I 100
150
I
I
I
I
I I
I 2
I 3
I 4
200
250
C
1
.F
Similarly, the rates of hydrolysis of GDP, CDP and UDP ranged from about I5 to 30 % of the rate of nucleotide triphosphate hydrolysis (table 1). Thus further hydrolysis of the nucleotide triphosphates would not contribute substantially to the results obtained. ATPase activity of Golgi apparatus fractions was absolutely dependent on the presence of Mg2+ in the reaction mixture (fig. 1). Maximum activation was attained at a Mg2+ concentration of 10 mM; higher concentrations of Mg2+ were without additional effect. Addition of K+ to the Mg2+-stimulated enzyme resulted in further enhancement of activity. Maximal activation of the enzyme by Kf was attained at concentrations of about 100 mM; however, higher concentrations of K+ were inhibitory (fig. 1). Addition of Na+ to reaction mixtures containing Mg2+ and K+ gave no measurable enhancement of activity. In concentrations of up to 200 mM Na+ had no effect on hydrolysis of ATP. Na+ was also without effect at half maximal Kf concentrations. Addition of Ca2+ to the Mg2+,
.e
.d1
.?,i 0
mM of Mg2+; (B) K+, (C) Ca*+; phosphate/mg protein/l0 min. Effect of ions on ATP hydrolysis by Golgi apparatus fractions from bovine mammary gland. Incubation mixtures contained 10 mM histidine, pH 7.5, 5 mM ATP, 0.15 mg Golgi apparatus protein and ions (A) Mg2+; (B) 5 mM Mg2+ plus K+; (C) 5 mM Mg2+, 100 mM K+ plus Ca2+. The final volume was 1 ml and incubation was at 30°C for various times to determine a rate (each point) by regression analysis. Fig. 1. Abscissa: (A) ordinate: pmoles of
other nucleotide triphosphates were hydrolysed in the order UTP, ATP and GTP. In contrast, the rate of ADP hydrolysis by this fraction was only about 20% of the rate of hydrolysis of the nucleotide triphosphates. Exptl
Cell Res 90 (1975)
3--
I
I -40
l
-20
I 0
20
I 40
I 60
1 80
I
I
100
120
Fig. 2. Abscissa: reciprocal of substrate concentration expressed as mM; ordinate: reciprocal of velocity expressed as pmoles of phosphorus/mg protein/l0 min. Lineweaver and Burke plot of the rate of phosphate release from ATP by bovine mammary Golgi apparatus fractions. Reaction mixtures contained 10 mM histidine, pH 7.5, 10 mM MgCl,, 0.5 mM CaCl,, 100 mM KCI. the indicated amounts of ATP and 0.1 mg of Golgi apparatus protein in a final volume of 1 ml. Incubation was at 30°C for various times to determine a rate by regression analysis with the aid of a computer.
Calcium accumulation by Golgi apparatus
257
(A) pH; (B) temperature (“C); ordinate: plmoles of phosphorus/mg protein/IO min. Effect of pH and incubation temperature on the rate of ATP hydrolysis by Golgi apparatus fractions from bovine mammary gland. Reaction conditions are identical to those in fig. 2 except that the ATP concentt ation was 5 mM and the pH or temperature was varied as indicated. Fig. 3. Abscissa:
K+-stimulated enzyme yielded a pronounced stimulation in ATP hydrolysis (fig. 1). A very sharp maximum in Ca2+ stimulation was attained at a concentration of 0.5 mM. At concentrations above 1.0 mM Ca2+ slightly inhibited the enzyme activity. This Ca2+ effect is very similar to that displayed by the ATPase of skeletal sarcoplasmic reticulum [l]. Straight line relationships were obtained when the reciprocal of the velocity of Golgi apparatus-mediated ATP hydrolysis was plotted against reciprocal substrate concentration (fig. 2) [40]. The K,,, calculated for ATP was 5 x 10-j M and the corresponding maximal velocity was 3.0 mmoles of phosphate liberated/h/mg protein. Phosphate liberation was maximal at pH 7.5 (fig. 3). The enzyme was inactive at pH 6 and below. ATP was hydrolysed over a rather broad pH range; the rate of hydrolysis at pH 9 was about 50 % of the rate at pH 7.5. Maximum rates of phosphate liberation occurred on incubation at 45°C for 10 min and the rate of hydrolysis decreased at higher temperatures (fig. 3). Rates of ATP hydrolysis increased over the temperature range of 25 to 45°C. The activation energy, calculated from Arrhenius plots, was 6 027 calories/mole. Ouabain, a cardiac glycoside which is a potent inhibitor of Naf, K+-ATPases involved in ion translocation through membranes [41], was without effect on the Golgi
apparatus ATPase at concentrations up to 0.4 mM (fig. 4). At higher concentrations an inhibitory effect was noted. At 0.8 mM ouabain had decreased the reaction velocity by about 50 %. In initial assays, we noted a rather pronounced variation in specific activity of the Golgi apparatus ATPase. This appeared to be a function of the time during which the tissue or fraction was held before assay. Accordingly a study of the storage stability was made. Tissue was held on ice or at 4°C for various intervals before the Golgi apparatus fraction was isolated and the ATPase assayed. Results obtained (fig. 5) revealed a rather linear decrease in ATPase activity between 8 and 48 h. The minimum time required for tissue procurement, transportation, isolation and assay was 8 h. Similar decreases in specific activity were noted when isolated fractions were held in the cold before assay. Thus, for comparative studies it is necessary to control the time interval between death of the animal and enzyme assay. In addition to Golgi apparatus, K+, Mg2+ATPase activity was detected in rough endoplasmic reticulum, smooth microsome and milk fat globule membrane fractions. Highest specific activities were observed with smooth microsomesfollowed by Golgi apparatus and the lowest levels of activity were encountered Exptl
Cell Res 90 (1975)
258
.4
0
Baumrucker
I .2
and Keenan
I .4
I .6
I .8
I .o
Fig. 4. Abscissa: mM ouabain; ordinate: pmoles of phosphorus/mg protein/IO min. Effect of ouabain on the rate of hydrolysis of ATP by Golgi apparatus fractions from bovine mammary gland. Reaction conditions are those given in fig. 2 except that the ATP concentration was 5 mM and reaction mixtures contained the indicated amount of ouabain.
in milk fat globule membrane. The distribution pattern and characteristics of this activity in these other endomembrane fractions will be detailed in a subsequentpublication. Golgi apparatus fractions displayed Ca2+ accumulation patterns typical of those found with skeletal sarcoplasmic reticulum (fig. 6). The accumulation of Ca2+ was rapid and nearly linear throughout 6 min of incubation. When the fractions were heated prior to ATP addition there was no measurable uptake of Ca2+ over the time course (fig. 6). An R2 value of 0.28 obtained for the heated controls is consistent with little, if any, specific uptake per unit time. Golgi apparatus fractions treated with deoxycholate and ultrasound showed only very low levels of Ca2+ uptake with time; the R2 value for the slope of this control was 0.24. In contrast, untreated Golgi apparatus fractions accumulated Ca2+ at a specific rate of 1.81 nmoles Ca2+/min/mg protein, with a linear regression value of 0.98. Ca2+ uptake was ATP-dependent, as evidenced by very low levels of Ca2+accumulation by Golgi apparatus fractions in the absence of ATP (R2=0.38) (fig. 6). This Exptl
Cell Res 90 (1975)
indicates that the uptake is an energy dependent process. Similarly, of the multiple Golgi apparatus fractions assayedduring the course of this study, two were found to have ATPase activities barely above the detection limit. These two fractions also had only a very slight ability to accumulate Ca2L. The ability of endomembrane components [42] from mammary gland to accumulate Ca2+ is compared in fig. 7. Smooth microsomes had the highest Ca2+ accumulation rates. Specific accumulation by Golgi apparatus fractions was slightly lower than the rate with smooth microsomes. Total microsomes had a specific uptake rate which was about 21% of the rate with smooth microsomes.Milk fat globule membranesdisplayed very little ability to accumulate or bind Ca2+. Rough endoplasmic reticulum (not shown in fig. 7) accumulated Ca2+ at about 29 “/bof the rate obtained with smooth microsomes. DISCUSSION The results presented herein show that Golgi apparatus fractions from lactating bovine mammary gland contain an ATPase activity
.8
-
.4
-
.2-
8
16
24
32
40
48
time (hours); ordinate: jcmoles of phosphorus/mg protein/i0 min. Time-deoendent loss in ability of bovine mammary Golgi apparatus fractions to hydrolyse ATP. Tissue was held on ice or at 4°C for intervals before fractions were isolated and assayed. The time points plotted are the hours after procurement that the fraction was assayed. Reaction conditions as in fig. 4 except that ouabain was not used. Fin.
5. Abscissa:
Calcium accumulation by Golgi apparatus which is Mg 2+-dependent, which is markedly stimulated by Ca2+ and, to a much lesser extent, by Kf. The lack of stimulation by Na+ and the ineffectiveness of ouabain as an inhibitor distinguish this ATPase from the classical Na+, K’ pump ATPase found in a wide variety of tissues [41]. These observations confirm and extend previous histochemical [I 11, physiological [7] and biochemical [9, lo] results which suggested that Na+ is discharged passively from mammary secretory cells. In addition to Golgi apparatus, other endomembrane components of the mammary gland also contain a Mg2 +-dependent ATPase. In terms of specific activity per unit protein, this activity is greatest in smooth microsomes, intermediate in Golgi apparatus and lowest in the (apical plasma membrane derived) milk fat globule membrane. The patterns of Mg2+-dependency and Ca2+-stimulation of the Golgi apparatus ATPase are quite similar to those reported for skeletal muscle Ca2’-pump ATPase [l].
Fig. 6. Abscissa: time (set); ordinsre: nmoles of calcium accumulated/mg protein. Adenosine triphosphate dependent calcium accumulation by bovine mammary Golgi apparatus fractions in vitro. Assay methods are described in the text. No significant accumulation of Cat+ occurred in the boiled control or the deoxycholate and ultrasound control. The regression lines plotted were determined with the aid of a computer. A-A, Golgi apparatus + ATP; v-7, Golgi apparatus without ATP; O-O, Golgi apparatus +ATP and treated with deoxycholate and ultrasound; H-E, boiled Golgi apparatus ~- ATP.
259
Fig. 7. Absrissa: time (min); ordinate: nmoles of calcium accumulated/mg protein. Calcium accumulation by endomzmbrane components from bovine mammary gland. Assay conditions are described in the text. Data are plotted as the computer determined rsgression lines. W-E, smooth microsome:; O-O, Golgi apparatus; 8-0, total microsomes; A-A, milk fat globule membrane.
However, the specific rates of Ca2+accumulation are lower than the range of values reported for skeletal muscles [l]. Treatment with deoxycholate and ultrasound or with heat abolished specific accumulation of Ca2+ by Golgi apparatus; similar effects have been found with skeletal sarcoplasmic reticulum [43]. In the latter, loss of Ca2t uptake has been correlated with loss of ATPase activity caused by treatment with detergents and ultrasound [43]. The necessity of ATP for Ca2t accumulation by Golgi apparatus confirms that this is an energy-requiring process. The results obtained in this study do not bear on the mechanism of accumulation of blood Ca2+by mammary secretory cells. However, the fact that Golgi apparatus rich fractions have both an Mg2+-dependent, Ca2-kstimulated ATPase and the ability to accumulate Ca2+ in vitro suggeststhat this endomembrane component is involved in accumulation of intracellular Ca2+ for secretion. Thus calcium may be secreted as are milk proteins by incorporation into secretory vesicles. This pathway would explain the Exptl
Cell Res 90 (1975)
260
Baumrucker
and Keenan
ultrastructural observations of casein micelle formation in secretory vesicles. In this manner a supply of Ca2+ would be available in the cisternal lumina and secretory vesicles of Golgi apparatus and this would permit micelle formation after phosphorylation of the casein apoproteins at the level of the Golgi apparatus. Once it entered the lumina of the Golgi apparatus cisterna, Ca2+ would be effectively trapped through interaction with phosphate groups of caseins. This would provide for a unidirecticnal flow of the ion. This work was supported by grant GB 25110 from the NSF and by grant GM 18760 from the National Institute of General Medical Science. T. W. K. is supported by USPHS Research Career Development Award GM 70596 from the National Institute of General Medical Science. We thank the nersonnel of Project MIRACLE, Purdue University, for assisting with the computer analyses and Professor W. W. Franke, Deutsches Krebsforschungszentrum, for aid in preparation of the manuscript. Purdue University AES Journal Paper No. 5422.
14. 15. 16. 17. 18.
19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.
REFERENCES I. Martonsi, A, Biomembranes (ed L Manson) vol. 1, p. 191. Plenum Press, New York (1971). 2. Sulakhe, P V, Drummond, G I & Ng, D C, J biol them 248 (1972) 4150. 3. Ebashi, S & Lipmann, F, J cell biol 14 (1962) 389. 4. Jenness, R & Patton, S, Principles of dairy chemistry, p. 158. Wiley and Sons, New York (1956). 5. Corbin. E A & Whittier. E 0. Fundamentals of dairy chemistry (ed B H’Webb & A D Johnson) p. 1. Avi Publishing _ Comoanv. Conn. __, Westport. . (1965). 6. Swanson, E W, Monroe, R A, Zilversmit, D B, Visek, W J PCComar, C L, J dairy sci 39 (1956) 1594. 7. Linzell, J L & Peaker, M, J physiol 216 (1971) 683. 8. - Physiol rev 51 (1971) 564. 9. Patton, S & Trams, E G, FEBS letters I4 (1971) 230. IO. Huang, C M & Keenan, T W, Comp biochem physiol 43B (1972) 277. 1 I. Kinura, T, J Japan obstet gynecol sot 21 (1969) 301. 12. Wellings, S R, Deome, K B & Pitelka, D R, J natl cancer inst 25 (1960) 393. 13. Saacke, R G & Heald, W, Lactation: A compre-
Exptl
Cell
Res 90 (1975)
30. 31. 32. 33. 34.
35. 36. 37. 38. 39. 40. 41. 42. 43.
hensive treatise (ed B L Larson & V R Smith) vol. 2, p. 137. Academic Press, New York (1974). Berry, K E, Hood, L F & Patton, S, J dairy sci 54 (1971) 911. Farrell, H M & Thompson, M P, J dairy sci 54 (1971) 1219. Patton, S & Fowkes, F M, J theoret biol 15 (1967) 274. Keenan, T W & Huang, C M, J dairy sci 55 (1972) 1586. Keenan, T W, Morrt, D J & Huang, C M, Lactation: A comprehensive treatise (ed B L Larson & V R Smith) vol. 2, p. 191. Academic Press, New York (1974). Bargmann, W & Welsch, U, Lactogenesis (ed M Reynolds & S J Folley) p. 43. University of Pennsylvania Press, Philadelphia, Pa (1969). Brew, K, Nature 222 (1969) 67. Keenan, T W, Morn?, D J & Cheetham, R D, Nature 228 (1970) 1105. Silcock, W R & Patton, S, J cell physiol 79 (1972) 151. Farrell, H M Jr, J dairy sci 56 (1973) 1195. Davis, D T & White, J C, J dairy res 27 (1960) 171. Demott, B J, J dairy sci 51 (1968) 1008. Turkinnton, R W & Topuer. Y J. Biochim biophys asa 127 (1966) 366:. Verley, J M & Hollmann, K M, Z Zellforsch mikrosk Anat 75 (1966) 605. Bingham, E W, Farrell, H M & Basch, J J, J biol them 247 (1972) 8193. Keenan, T W, Huang, C M & Morre, D J, J dairy sci 55 (1972) 1577. Huang, C M & Keenan, T W, Biochim biophys acta 274 (1972) 246. Penninaton. R S. Biochem i 80 (1961) 649. Morn&-D J, Merlin, L M h Keenan, T W, Biothem biophys res commun 37 (1969) 813. Meissner, G & Fleischer, S, Biochim biophys acta 241 (1971) 356. Meissner, G, Conner, G E & Fleischer, S, Biochim biophys acta 298 (1973) 246. Bray, G A, Anal biochem 1 (1960) 279. Lowry, 0 H, Rosebrough, N J, Farr, A L & Randall, R J, J biol them 193 (1951) 265. Fiske, C H & SubbaRow, Y, J biol them 66 (1925) 375. Keenan, T W, Morre, D J, Olson, D E, Yunghans, W N & Patton, S, J cell biol 44 (1970) 80. Lehninger, A L, Carafoli, E & Rossi, C S, Adv enzymol 29 (1967) 259. Lineweaver, H & Burk, D, J Am them sot 56 (1934) 658. Skou, J C, Physiol rev 45 (1965) 596. Morn&, D J & Mollenhauer, H H, Dynamics of plant ultrastructure (ed A W Robards) p. 84. McGraw-Hill, London (1974). Selinger, Z, Klein, M & Amsterdam, A, Biochim biophys acta 183 (1969) 19.
Received March 20, 1974 Revised version received August 19, 1974
Experimental
MEMBRANES X. Adenosine
Cell Research 90 (1975) 253-260
OF MAMMARY
Triphosphate
by Golgi Apparatus
dependent
Rich Fractions
C. R. BAUMRUCKER Department
of Animal
Sciences,
Purdue
from
GLAND Calcium
Accumulation
Bovine Mammary
Gland
and T. W. KEENAN University,
West Lafayette,
Ind. 47907,
USA
SUMMARY Golgi apparatus rich fractions from lactating bovine mammary gland had an Mg*+-dependent, Ca2+-stimulated adenosine triphosphatase. These Golgi apparatus fractions also accumulated Ca*+ in vitro. Accumulation of Ca2+ required ATP and could be abolished by treatment either with low concentrations of deoxycholate followed by ulttasound, or by heating at 100°C for IO min. The adenosine triphosphatase activity of Golgi apparatus was strongly stimulated by low concentrations of Ca2+ and moderately stimulated by high concentrations of K+. This activity was unaffected by Na+ and was not inhibited by ouabain. The pH optimum for the Mg*+dependent hydrolysis of ATP was 7.5, the K,,, was 5 x lO-5 M and the activation energy was 6 000 calories/mole. This Mg2 +-dependent adenosine triphosphatase activity was also found in rough endoplasmic reticulum, smooth mictosomes and milk fat globule membrane, the latter membrane being derived directly from the apical plasma membrane. All of these membrane fractions had the ability to specifically accumulate Ca %+. Specific accumulation was highest with smooth microsomes and lowest with milk fat globule membrane with Golgi apparatus and rough endoplasmic reticulum being intermediate. These observations provide one plausible explanation for intracellular Ca2+ accumulation and secretion into milk. Further, these results help explain the ultrastructural observations of casein micelle formation in secretory vesicles elaborated by Golgi apparatus.
Research during the last decade has revealed the relaxing factor of contracted muscle to be a membrane-bound, Mg2+-dependent adenosine triphosphatase (ATPase) (ATP phosphohydrolase, E.C. 3.6.1.3) which acts as a calcium pump. This ATPase activity has been found in muscle sarcoplasmic reticulum [l] and, more recently, in plasma membranes from muscle cells [2]. The efficacy of this pump mechanism for Ca2+ is such that Ca2+ can reach concentrations in sarcoplasmic reticulum vesiclesthat are some20 times higher than in the extravesicular fluid [3]. Another tissue in which Ca2+ is concentrated is in the lactating mammary gland. Although the mechanismsare unknown, Ca2+ 17-
741804
is accumulated from the blood, where the concentration is 2 to 3 mM, and secretedwith milk, where it is present in a concentration of about 30 mM [4, 51. Thus, Ca2+ must passa concentration gradient, indicating the existence of an energy-requiring process [6]. One can envisage the existence of two mechanisms involved in translocation of Ca2+ from blood to milk. One mechanism would function in accumulation of blood Ca2+by secretory cells and the other would account for the discharge of this ion into the gland lumen. In contrast to Ca2+, the mechanismsof Na+, K+ and Clsecretion into milk have been extensively studied [7-l 11. The mechanism involved in intracellular Exptl
Ceil Res 90 (1975)
254
Baumrucker
and Keenan
accumulation and secretion of Ca2+ may be located in Golgi apparatus. Caseins, the major proteins of milk, are known to be packaged into secretory vesicles elaborated by Golgi apparatus [12-151. After formation, these vesicles migrate to the apical region of the cell where they fuse with the plasma membrane and discharge their contents:into the lumen [12, 16-191. In addition to caseins, there is evidence that other milk proteins 120, 211, lactose [17, 20, 211 and certain of the minerals of milk [22] are contained in these secretory vesicles. The caseins, which are phosphoproteins, exist in milk in the form of micelles [23]. Calcium is necessary for formation of these micelles, forming salt bonds with the covalently bonded phosphates of neighboring casein molecules [23]. In fact, the majority of the calcium of milk is associated with casein micelles [24, 251. Within the cell casein apoproteins are synthesized on ribosomes of the rough endoplasmic reticulum [13, 18, 26, 271. Phosphorus is added after completion of the apoprotein chain by a protein kinase which is concentrated in Golgi apparatus [28]. There is abundant morphological evidence indicating that casein micelles form within secretory vesicles [12-151. This implies that Ca2+ is also contained in the vesicles and suggests a role for Golgi apparatus in accumulation of Ca2+ from cytosol. To explore this possibility we have characterized an ATPase in bovine mammary Golgi apparatus. Results obtained have demonstrated the presence of a Mg2+-dependent, Ca2+-stimulated ATPase in Golgi apparatus. In addition, the ability of Golgi apparatus fractions to accumulate Ca2+ in vitro is demonstrated. METHODS
AND MATERIALS
Isolation procedures Mammary tissue was obtained from lactating Holstein
cows(BOSiypicuspritnigenius) at slaughter and cooled Exptl
Cell Res 90 (1975)
on ice during transportation to the laboratory. Golgi apparatus fractions were isolated as described previously [21,29]. Rough endoplasmic reticulum, smooth microsomes and total microsomes were also isolated [IS, 291. Milk fat globule membranes were prepated from fresh milk by churning as described previously [IO, 301. All membrane fractions were washed and resuspended in the buffers used for enzyme assay or determination of Ca2+ uptake.
Enzyme assays ATPase was assayed in a reaction mixture containing 10 mM histidine buffer. oH 7.5. 100 mM KCI. 5 mM MgCI,, 5 mM ATP and from 6.05 to 0.2 mg of fraction protein in a final volume of 1 ml. Incubation was at 30°C for various times in order to determine hydrolytic rates by regression analysis. Reactions were terminated by addition of 1 ml of 10 % trichloroacetic acid. Reactions were initiated by addition of enzyme, and blanks consisting either of heat-inactivated enzyme or no enzyme were always run to correct for nonspecific hydrolysis of ATP. Released phosphate was measured calorimetrically. Succinic dehydrogenase activity was assayed as succinate-2-(p-indophenyl)-3-(p-nitrophenyl)-j-phenyltetrazolium (INT) reductase accordinn to Pennington [31]. Galactosyl transferase activiiy of Golgi apparatus fractions was determined as in previous studies with N-acetylglucosamine as the galactose acceptor 121, 221. Caz+ accumulation was measured according to Meissner & Fleischer [33, 341. Reaction mixtures contained 10 mM histidine buffer, pH 7.5, 10 mM MgCl,, 100 mM KCI, 5 mM ATP, 0.5 mM WaCl,, I mM potassium oxalate and about 0.1 mg membrane protein in a final volume of 2 ml. Incubation was at 30°C for time intervals up to 6 min. Reactions were terminated by filtration of the reaction mixture through a 0.45 pm pore diameter Millipore filter under vacuum. After washing. the radioactivity retained on the filter was measured in Bray’s [35] scintillation fluid containing 60 g naphthalene, 4 g 2,5-diphenyloxazole, 200 mg 1,4-his-(2-(5-phenylosazolyl))-benzene. 100 ml methanol. 20 ml ethvlene glycol and sufficient p-dioxane to yield I I. Control samples without membrane. with membrane fractions treated with 0.1 % sodium deoxycholate and sonicated for 10 to 15 set (Branson Model S125, setting 3), or fractions heated for 10 min in boiling water were assayed to determine nonspecific binding of Vaz+. Protein was determined according to Lowry et al. [36] with crystalline bovine serum albumin as the standard. Inorganic phosphate was measured according to Fiske & SubbaRow [37].
Reagents Bovine serum albumin, the 5’-nucleotide di- and triphosphates, IV-acetylglucosamine and UDP-galactose were from Sigma Chemical Company, St Louis, MO. UDP-(WJ-galactose (298 mCi/mmole) was obtained from New England Nuclear, Boston, Mass. *YIaCl, (1.63 x 10m3 mCi/mmole) was from International
Calcium accumulation by Golgi apparatus Chemical and Nuclear, Cleveland, Ohio. Millipore filters were from the Millipore Corporation, Bedford, Mass. Statistical analyses were performed with the aid of a PDP 11 computer.
RESULTS Subcellular fractions used in the present study have previously been characterized with respect to homogeneity and composition [17, 18, 291. Milk fat globule membrane is known to be derived from the apical plasma membrane of mammary secretory cells (for reviews seerefs [8, 18, 381).This membrane fraction is virtually free of contamination by mitochondria, endoplasmic reticulum and Golgi apparatus [17, 18, 381. Rough endoplasmic reticulum fractions as prepared are nearly completely free of contamination by other subcellular fractions [17, 18,291. In particular, the rough endoplasmic reticulum as well as the smooth microsomal fractions contain only negligible quantities of mitochondria. Golgi apparatus fractions, while being nearly 90 % pure with respect to membranous contaminants, contain low but variable amounts of mitochondria [17, 18, 291. Since the presence of a Ca2+ binding protein and ATPase activity in mitochondria are well known [39], care was taken to exclude mitochondrial contaminants from Golgi apparatus fractions used in this study. That this was achieved was determined by assay for succinate INT reductase, a known mitochondrial marker enzyme. Golgi apparatus fractions had a specific activity in this enzyme of 0.05 pmole INT reduced/h/mg protein. The ratio of the activity in Golgi apparatus to that in total homogenates was 0.025. This compares with the g-fold enrichment of this activity in mammary mitochondria relative to total homogenates [18]. From these data it can be calculated that, on a protein basis, mitochondria accounted for less than 1% of the material in Golgi apparatus fractions. Simi-
255
Table 1. Hydrolysis ojnucleotide triphosphates and diphosphatesby Golgi apparatus fractions from bovine mammary gland Substrate ATP GTP UTP CTP ADP GDP UDP CDP
Specific activity’ 2.50 2.40 2.55 2.61
0.50 0.76 0.75
0.41
R2 0.92 0.94 0.97 0.95 0.85 0.95 0.98 0.80
a Specific activity, a rate determined by computerassisted regression analysis, is pmoles of substrate hydrolysed/mg protein/h. Complete incubation mixtures contained 10 mM histidine, pH 7.5,5 mM MgCI,, 100 mM KCI, 5 mM nucleotide tri- or diphosphate and 0.1 mg Golgi apparatus protein in a final volume of 1 ml. Incubation was at 3o’C for various intervals from 1 to 20 min. R2 values are correlation coefficients.
larly, galactosyl transferase, an enzyme specifically localized in Golgi apparatus [18, 21, 29, 321, was enriched 20 times in Golgi apparatus fractions. This figure compares favorably with the 16- to 18-fold enrichment of this enzyme previously measured in morphologically homogeneous Golgi apparatus fractions [18, 21, 291. Multiple Golgi apparatus fractions from bovine mammary gland were obtained and all were found to hydrolyse ATP. The ratio of specific activity in Golgi apparatus to that in total homogenates was, on the average, 5.8. Hydrolysis of ATP was linear up to about 0.15 mg of Golgi apparatus protein/ml. The rate of ATP hydrolysis at 30°C was linear for all times tested. The average specific activity of Golgi apparatus fractions was about 2.5 pmoles of inorganic phosphate liberated from ATP/h/mg protein. In addition to ATP other nucleotide triphosphates were hydrolysed by Golgi apparatus fractions (table 1). The fraction was most active with CTP. The Exptl
Cell Res 90 (1975)
256
Baumrucker
I
Oo
I
.7
and Keenan
I
I
50
I 100
150
I
I
I
I
I I
I 2
I 3
I 4
200
250
C
1
.F
Similarly, the rates of hydrolysis of GDP, CDP and UDP ranged from about I5 to 30 % of the rate of nucleotide triphosphate hydrolysis (table 1). Thus further hydrolysis of the nucleotide triphosphates would not contribute substantially to the results obtained. ATPase activity of Golgi apparatus fractions was absolutely dependent on the presence of Mg2+ in the reaction mixture (fig. 1). Maximum activation was attained at a Mg2+ concentration of 10 mM; higher concentrations of Mg2+ were without additional effect. Addition of K+ to the Mg2+-stimulated enzyme resulted in further enhancement of activity. Maximal activation of the enzyme by Kf was attained at concentrations of about 100 mM; however, higher concentrations of K+ were inhibitory (fig. 1). Addition of Na+ to reaction mixtures containing Mg2+ and K+ gave no measurable enhancement of activity. In concentrations of up to 200 mM Na+ had no effect on hydrolysis of ATP. Na+ was also without effect at half maximal Kf concentrations. Addition of Ca2+ to the Mg2+,
.e
.d1
.?,i 0
mM of Mg2+; (B) K+, (C) Ca*+; phosphate/mg protein/l0 min. Effect of ions on ATP hydrolysis by Golgi apparatus fractions from bovine mammary gland. Incubation mixtures contained 10 mM histidine, pH 7.5, 5 mM ATP, 0.15 mg Golgi apparatus protein and ions (A) Mg2+; (B) 5 mM Mg2+ plus K+; (C) 5 mM Mg2+, 100 mM K+ plus Ca2+. The final volume was 1 ml and incubation was at 30°C for various times to determine a rate (each point) by regression analysis. Fig. 1. Abscissa: (A) ordinate: pmoles of
other nucleotide triphosphates were hydrolysed in the order UTP, ATP and GTP. In contrast, the rate of ADP hydrolysis by this fraction was only about 20% of the rate of hydrolysis of the nucleotide triphosphates. Exptl
Cell Res 90 (1975)
3--
I
I -40
l
-20
I 0
20
I 40
I 60
1 80
I
I
100
120
Fig. 2. Abscissa: reciprocal of substrate concentration expressed as mM; ordinate: reciprocal of velocity expressed as pmoles of phosphorus/mg protein/l0 min. Lineweaver and Burke plot of the rate of phosphate release from ATP by bovine mammary Golgi apparatus fractions. Reaction mixtures contained 10 mM histidine, pH 7.5, 10 mM MgCl,, 0.5 mM CaCl,, 100 mM KCI. the indicated amounts of ATP and 0.1 mg of Golgi apparatus protein in a final volume of 1 ml. Incubation was at 30°C for various times to determine a rate by regression analysis with the aid of a computer.
Calcium accumulation by Golgi apparatus
257
(A) pH; (B) temperature (“C); ordinate: plmoles of phosphorus/mg protein/IO min. Effect of pH and incubation temperature on the rate of ATP hydrolysis by Golgi apparatus fractions from bovine mammary gland. Reaction conditions are identical to those in fig. 2 except that the ATP concentt ation was 5 mM and the pH or temperature was varied as indicated. Fig. 3. Abscissa:
K+-stimulated enzyme yielded a pronounced stimulation in ATP hydrolysis (fig. 1). A very sharp maximum in Ca2+ stimulation was attained at a concentration of 0.5 mM. At concentrations above 1.0 mM Ca2+ slightly inhibited the enzyme activity. This Ca2+ effect is very similar to that displayed by the ATPase of skeletal sarcoplasmic reticulum [l]. Straight line relationships were obtained when the reciprocal of the velocity of Golgi apparatus-mediated ATP hydrolysis was plotted against reciprocal substrate concentration (fig. 2) [40]. The K,,, calculated for ATP was 5 x 10-j M and the corresponding maximal velocity was 3.0 mmoles of phosphate liberated/h/mg protein. Phosphate liberation was maximal at pH 7.5 (fig. 3). The enzyme was inactive at pH 6 and below. ATP was hydrolysed over a rather broad pH range; the rate of hydrolysis at pH 9 was about 50 % of the rate at pH 7.5. Maximum rates of phosphate liberation occurred on incubation at 45°C for 10 min and the rate of hydrolysis decreased at higher temperatures (fig. 3). Rates of ATP hydrolysis increased over the temperature range of 25 to 45°C. The activation energy, calculated from Arrhenius plots, was 6 027 calories/mole. Ouabain, a cardiac glycoside which is a potent inhibitor of Naf, K+-ATPases involved in ion translocation through membranes [41], was without effect on the Golgi
apparatus ATPase at concentrations up to 0.4 mM (fig. 4). At higher concentrations an inhibitory effect was noted. At 0.8 mM ouabain had decreased the reaction velocity by about 50 %. In initial assays, we noted a rather pronounced variation in specific activity of the Golgi apparatus ATPase. This appeared to be a function of the time during which the tissue or fraction was held before assay. Accordingly a study of the storage stability was made. Tissue was held on ice or at 4°C for various intervals before the Golgi apparatus fraction was isolated and the ATPase assayed. Results obtained (fig. 5) revealed a rather linear decrease in ATPase activity between 8 and 48 h. The minimum time required for tissue procurement, transportation, isolation and assay was 8 h. Similar decreases in specific activity were noted when isolated fractions were held in the cold before assay. Thus, for comparative studies it is necessary to control the time interval between death of the animal and enzyme assay. In addition to Golgi apparatus, K+, Mg2+ATPase activity was detected in rough endoplasmic reticulum, smooth microsome and milk fat globule membrane fractions. Highest specific activities were observed with smooth microsomesfollowed by Golgi apparatus and the lowest levels of activity were encountered Exptl
Cell Res 90 (1975)
258
.4
0
Baumrucker
I .2
and Keenan
I .4
I .6
I .8
I .o
Fig. 4. Abscissa: mM ouabain; ordinate: pmoles of phosphorus/mg protein/IO min. Effect of ouabain on the rate of hydrolysis of ATP by Golgi apparatus fractions from bovine mammary gland. Reaction conditions are those given in fig. 2 except that the ATP concentration was 5 mM and reaction mixtures contained the indicated amount of ouabain.
in milk fat globule membrane. The distribution pattern and characteristics of this activity in these other endomembrane fractions will be detailed in a subsequentpublication. Golgi apparatus fractions displayed Ca2+ accumulation patterns typical of those found with skeletal sarcoplasmic reticulum (fig. 6). The accumulation of Ca2+ was rapid and nearly linear throughout 6 min of incubation. When the fractions were heated prior to ATP addition there was no measurable uptake of Ca2+ over the time course (fig. 6). An R2 value of 0.28 obtained for the heated controls is consistent with little, if any, specific uptake per unit time. Golgi apparatus fractions treated with deoxycholate and ultrasound showed only very low levels of Ca2+ uptake with time; the R2 value for the slope of this control was 0.24. In contrast, untreated Golgi apparatus fractions accumulated Ca2+ at a specific rate of 1.81 nmoles Ca2+/min/mg protein, with a linear regression value of 0.98. Ca2+ uptake was ATP-dependent, as evidenced by very low levels of Ca2+accumulation by Golgi apparatus fractions in the absence of ATP (R2=0.38) (fig. 6). This Exptl
Cell Res 90 (1975)
indicates that the uptake is an energy dependent process. Similarly, of the multiple Golgi apparatus fractions assayedduring the course of this study, two were found to have ATPase activities barely above the detection limit. These two fractions also had only a very slight ability to accumulate Ca2L. The ability of endomembrane components [42] from mammary gland to accumulate Ca2+ is compared in fig. 7. Smooth microsomes had the highest Ca2+ accumulation rates. Specific accumulation by Golgi apparatus fractions was slightly lower than the rate with smooth microsomes. Total microsomes had a specific uptake rate which was about 21% of the rate with smooth microsomes.Milk fat globule membranesdisplayed very little ability to accumulate or bind Ca2+. Rough endoplasmic reticulum (not shown in fig. 7) accumulated Ca2+ at about 29 “/bof the rate obtained with smooth microsomes. DISCUSSION The results presented herein show that Golgi apparatus fractions from lactating bovine mammary gland contain an ATPase activity
.8
-
.4
-
.2-
8
16
24
32
40
48
time (hours); ordinate: jcmoles of phosphorus/mg protein/i0 min. Time-deoendent loss in ability of bovine mammary Golgi apparatus fractions to hydrolyse ATP. Tissue was held on ice or at 4°C for intervals before fractions were isolated and assayed. The time points plotted are the hours after procurement that the fraction was assayed. Reaction conditions as in fig. 4 except that ouabain was not used. Fin.
5. Abscissa:
Calcium accumulation by Golgi apparatus which is Mg 2+-dependent, which is markedly stimulated by Ca2+ and, to a much lesser extent, by Kf. The lack of stimulation by Na+ and the ineffectiveness of ouabain as an inhibitor distinguish this ATPase from the classical Na+, K’ pump ATPase found in a wide variety of tissues [41]. These observations confirm and extend previous histochemical [I 11, physiological [7] and biochemical [9, lo] results which suggested that Na+ is discharged passively from mammary secretory cells. In addition to Golgi apparatus, other endomembrane components of the mammary gland also contain a Mg2 +-dependent ATPase. In terms of specific activity per unit protein, this activity is greatest in smooth microsomes, intermediate in Golgi apparatus and lowest in the (apical plasma membrane derived) milk fat globule membrane. The patterns of Mg2+-dependency and Ca2+-stimulation of the Golgi apparatus ATPase are quite similar to those reported for skeletal muscle Ca2’-pump ATPase [l].
Fig. 6. Abscissa: time (set); ordinsre: nmoles of calcium accumulated/mg protein. Adenosine triphosphate dependent calcium accumulation by bovine mammary Golgi apparatus fractions in vitro. Assay methods are described in the text. No significant accumulation of Cat+ occurred in the boiled control or the deoxycholate and ultrasound control. The regression lines plotted were determined with the aid of a computer. A-A, Golgi apparatus + ATP; v-7, Golgi apparatus without ATP; O-O, Golgi apparatus +ATP and treated with deoxycholate and ultrasound; H-E, boiled Golgi apparatus ~- ATP.
259
Fig. 7. Absrissa: time (min); ordinate: nmoles of calcium accumulated/mg protein. Calcium accumulation by endomzmbrane components from bovine mammary gland. Assay conditions are described in the text. Data are plotted as the computer determined rsgression lines. W-E, smooth microsome:; O-O, Golgi apparatus; 8-0, total microsomes; A-A, milk fat globule membrane.
However, the specific rates of Ca2+accumulation are lower than the range of values reported for skeletal muscles [l]. Treatment with deoxycholate and ultrasound or with heat abolished specific accumulation of Ca2+ by Golgi apparatus; similar effects have been found with skeletal sarcoplasmic reticulum [43]. In the latter, loss of Ca2t uptake has been correlated with loss of ATPase activity caused by treatment with detergents and ultrasound [43]. The necessity of ATP for Ca2t accumulation by Golgi apparatus confirms that this is an energy-requiring process. The results obtained in this study do not bear on the mechanism of accumulation of blood Ca2+by mammary secretory cells. However, the fact that Golgi apparatus rich fractions have both an Mg2+-dependent, Ca2-kstimulated ATPase and the ability to accumulate Ca2+ in vitro suggeststhat this endomembrane component is involved in accumulation of intracellular Ca2+ for secretion. Thus calcium may be secreted as are milk proteins by incorporation into secretory vesicles. This pathway would explain the Exptl
Cell Res 90 (1975)
260
Baumrucker
and Keenan
ultrastructural observations of casein micelle formation in secretory vesicles. In this manner a supply of Ca2+ would be available in the cisternal lumina and secretory vesicles of Golgi apparatus and this would permit micelle formation after phosphorylation of the casein apoproteins at the level of the Golgi apparatus. Once it entered the lumina of the Golgi apparatus cisterna, Ca2+ would be effectively trapped through interaction with phosphate groups of caseins. This would provide for a unidirecticnal flow of the ion. This work was supported by grant GB 25110 from the NSF and by grant GM 18760 from the National Institute of General Medical Science. T. W. K. is supported by USPHS Research Career Development Award GM 70596 from the National Institute of General Medical Science. We thank the nersonnel of Project MIRACLE, Purdue University, for assisting with the computer analyses and Professor W. W. Franke, Deutsches Krebsforschungszentrum, for aid in preparation of the manuscript. Purdue University AES Journal Paper No. 5422.
14. 15. 16. 17. 18.
19. 20. 21. 22. 23. 24. 25. 26. 27. 28. 29.
REFERENCES I. Martonsi, A, Biomembranes (ed L Manson) vol. 1, p. 191. Plenum Press, New York (1971). 2. Sulakhe, P V, Drummond, G I & Ng, D C, J biol them 248 (1972) 4150. 3. Ebashi, S & Lipmann, F, J cell biol 14 (1962) 389. 4. Jenness, R & Patton, S, Principles of dairy chemistry, p. 158. Wiley and Sons, New York (1956). 5. Corbin. E A & Whittier. E 0. Fundamentals of dairy chemistry (ed B H’Webb & A D Johnson) p. 1. Avi Publishing _ Comoanv. Conn. __, Westport. . (1965). 6. Swanson, E W, Monroe, R A, Zilversmit, D B, Visek, W J PCComar, C L, J dairy sci 39 (1956) 1594. 7. Linzell, J L & Peaker, M, J physiol 216 (1971) 683. 8. - Physiol rev 51 (1971) 564. 9. Patton, S & Trams, E G, FEBS letters I4 (1971) 230. IO. Huang, C M & Keenan, T W, Comp biochem physiol 43B (1972) 277. 1 I. Kinura, T, J Japan obstet gynecol sot 21 (1969) 301. 12. Wellings, S R, Deome, K B & Pitelka, D R, J natl cancer inst 25 (1960) 393. 13. Saacke, R G & Heald, W, Lactation: A compre-
Exptl
Cell
Res 90 (1975)
30. 31. 32. 33. 34.
35. 36. 37. 38. 39. 40. 41. 42. 43.
hensive treatise (ed B L Larson & V R Smith) vol. 2, p. 137. Academic Press, New York (1974). Berry, K E, Hood, L F & Patton, S, J dairy sci 54 (1971) 911. Farrell, H M & Thompson, M P, J dairy sci 54 (1971) 1219. Patton, S & Fowkes, F M, J theoret biol 15 (1967) 274. Keenan, T W & Huang, C M, J dairy sci 55 (1972) 1586. Keenan, T W, Morrt, D J & Huang, C M, Lactation: A comprehensive treatise (ed B L Larson & V R Smith) vol. 2, p. 191. Academic Press, New York (1974). Bargmann, W & Welsch, U, Lactogenesis (ed M Reynolds & S J Folley) p. 43. University of Pennsylvania Press, Philadelphia, Pa (1969). Brew, K, Nature 222 (1969) 67. Keenan, T W, Morn?, D J & Cheetham, R D, Nature 228 (1970) 1105. Silcock, W R & Patton, S, J cell physiol 79 (1972) 151. Farrell, H M Jr, J dairy sci 56 (1973) 1195. Davis, D T & White, J C, J dairy res 27 (1960) 171. Demott, B J, J dairy sci 51 (1968) 1008. Turkinnton, R W & Topuer. Y J. Biochim biophys asa 127 (1966) 366:. Verley, J M & Hollmann, K M, Z Zellforsch mikrosk Anat 75 (1966) 605. Bingham, E W, Farrell, H M & Basch, J J, J biol them 247 (1972) 8193. Keenan, T W, Huang, C M & Morre, D J, J dairy sci 55 (1972) 1577. Huang, C M & Keenan, T W, Biochim biophys acta 274 (1972) 246. Penninaton. R S. Biochem i 80 (1961) 649. Morn&-D J, Merlin, L M h Keenan, T W, Biothem biophys res commun 37 (1969) 813. Meissner, G & Fleischer, S, Biochim biophys acta 241 (1971) 356. Meissner, G, Conner, G E & Fleischer, S, Biochim biophys acta 298 (1973) 246. Bray, G A, Anal biochem 1 (1960) 279. Lowry, 0 H, Rosebrough, N J, Farr, A L & Randall, R J, J biol them 193 (1951) 265. Fiske, C H & SubbaRow, Y, J biol them 66 (1925) 375. Keenan, T W, Morre, D J, Olson, D E, Yunghans, W N & Patton, S, J cell biol 44 (1970) 80. Lehninger, A L, Carafoli, E & Rossi, C S, Adv enzymol 29 (1967) 259. Lineweaver, H & Burk, D, J Am them sot 56 (1934) 658. Skou, J C, Physiol rev 45 (1965) 596. Morn&, D J & Mollenhauer, H H, Dynamics of plant ultrastructure (ed A W Robards) p. 84. McGraw-Hill, London (1974). Selinger, Z, Klein, M & Amsterdam, A, Biochim biophys acta 183 (1969) 19.
Received March 20, 1974 Revised version received August 19, 1974
