- Email: [email protected]
Interaction of Ca2+ with endoplasmic reticulum of rat liver: A standardized procedure for the isolation of rat liver microsomes
ANALYTICAL
BIOCHEMISTRY
Interaction
48, 53-61 (1972)
of Ca2+
with Endoplasmic of Rat Liver:
Reticulum
A Standardized Procedure for the Isolation of Rat liver Microsomes S. A. KAMATHl llepl~i-tme~~t of
Food
AND
K. ANANTH
Science, University Illinois
of Illinois 61801
Received September
NARAYAN2 at
Champaign-Urbana,
1. 1971
Most commonly, the differential centrifugation technique of Hogeboom (l-3) or its modification (4) is used for the separation of rat liver subcellular fractions. According to the classical procedure (4), the nuclear and mitochondrial fractions in a crude-liver homogenate are removed stepwise by centrifugation at 7009 and lO,OOOg,respectively, for 10 min, while the microsomes present in the postmitochondrial supernatant are obtained by clarifying at 105,OOOgin an ult.racentrifuge for 60 min (5). In comparison to the other subcellular fractions, the isolation of microsomal fraction and its subsequent purification consume a lot of time and, in addition, it requires the use of an expensive ultracentrifuge. Recently we reported (6) that, under certain conditions using Ca2+ ions, a microsomal fraction from rat liver can be routinely obtained by low-speed centrifugation at 1,500g. In brief, the procedure (7), which involved enormous dilution of the postmitochondrial supernatant, was as follows: The crude-liver homogenate (20%) was prepared in a buffer containing sucrose (0.25 M), MgCl, (5 mu), KC1 (25 mM), CaClz (8 mM), and Tris-WC!, pH 7.5 (0.05 M) using a Dounce homogenizer. The postmitochondrial supernatant (lO,OOOg,10 min) diluted 5 times with 0.05 M Tris-HCl buffer, pH 7.5, or 0.0125 M sucrose, each containing 5 mM MgZ+ and 8 mM Ca?+,was centrifuged at 1,500g for 10 min to isolate the aggregated microsomal pellet. The studies on this 1,500g pellet were further extended (8) to compare and provide evidence that the micro’ Present address: Department of Pathology, Mount Sinai School of Medicine of the City University of New York, New York 10029. * Reprints may be requested from this author at the following address: Nutrition Division, Food Laboratory, U. S. Army Natick Laboratories, Natick, Massachusetts 01760. 53 @ 1972 by Academic Press. Inc.
54
KAMATH
AND
h-ARAYAK
somes prepared using Ca2+ were not intrinsically different from normal microsomes with respect to their chemical composition and biochemical properties. In this present communication, we report a standardized procedure using divalent, cations for the isolation of rat-liver microsomes that is simpler and more direct than the classical method as well as the previous method using Ca”+ ions (71. The enzymic activities and RNA and lipid compositions of normal microsomes, and of microsomes prepared according to the present procedure, as well as of smooth and rough microsomes prepared in the presence and absence of Ca”+ ions, have been compared. Metal ions other than Ca’+ were also investigated for their ability to precipitate the microsomes by low-speed ccntrifugation. MATERIALS
AND
METHODS
Extraction of lipids and their estimations, protein determination assays for 5’-nucleotidase, glucose-6-phosphat’ase,Rig”+-K+-Na+-activated adenosine triphosphatase, succinic dchydrogenase, and monoamine oxidase were done as described earlier (9). Enzymes involved in the electron transport system of microsomes were determined according to published methods (10-12). RNA was assayed according to the method of Schneider (13). Ca?+and Mg’+ were analyzed using a Beckman atomic absorption xpectrophotometer with an air-acetylene flame. For the isolation of rat-liver micr~osomes,rats weighing between 300 and 400 gm (Holtzman Co., Madison, Wisconsin~ were starved overnight in order to reduce the glycogen load. The animals were given Nembutal anesthesia and were bled from the abdominal aorta. All the wlx5cq11et1t steps were performed between 0” and 4°C. The livers were excised and kept in ice-cold isotonic sucrose (141, blotted free of blood clots, and homogenized using isotonic sucrose ( 2625% homogenate) in a Douncr homogenizer with about 8-12 strokes. The crutle homogenate was centrifuged at 10,OOOgfor 10 min twice, so that all particles approximately above 300-500 nm vvcre removed. The clear postmitochondrial supernatant was used for the preparation of either normal microsomcs or Ca2+ microsonies. For preparing the normal microsomes, the postmitochondrial supernatant was spun at 105,OOOgfor 60 min in a Beckman L2-65 ultracentrifuge and the pellet obtained was further washed twice, each time rehomogenizing in ieot’onic sucrose, and recentrifuging at 105,OOOgfor 60 min. For the preparation of Ca”+ microsomes, the postmitochondrial supernatant was made 8 mM with respect to calcium chloride (pH 7.5) and was allowed to stand at 4°C for about 3-5 min. The large dilution of the supernatant required for the previous method (7) was eliminated under the present modified conditions. At the end of this period, the entire so-
RAT
LIVER
(-‘:~“+-51l(:ROSO~lF.S
5.i
lution was centrifuged at 10,OOOy for 10 min in a Sorvall centrifuge. The well-sedimented pellet was washed twice by rcsuspending it in isotonic sucrose solution and rcccntrifuging at 10,OOOg for 10 min. Both t’ypes of microsomal preparations were stored at -20°C until use hut, for enzymic assays, fresh preparations were used. Xcrosomal preparation derived from 250 mg liver was ntorc,d in I ml of sucrorc ::olution and isotonic sucrose was used for diluting the microsomes. The smooth and rough microsomes were l)rcl)ared from a 25% crudeliver homogenate in 0.25 111 sucrose solution essentially according to the procedure of Bergstrand and Dallner ( 151. The crude homogenate was clarified at 10,OOOy for 15 min and tbe “10,OOOg supernate” was made to 15 mM with C&l. In the case of Ca”- smooth and rough microsomcs, the “10,OOOg supernate” was made 8 ml11 and 15 mX with Ca’+ and C&l, respectively. An aliquot of 4.5 ml of this “lO,OOOg supernate” was layered over 2 ml of 1.3 M sucrose/l5 mill &Cl. The tubes were centrifuged at 102,OOOg for 120 min in a 40.3 rotor of the Beckman L2-65 ultracentrifuge. After 2 hr, t’hc smooth microsomes separated at 0.25 X,/l.3 M sucrose zone and rough microsomcs sedimcnted as pellets were collected, dispersed in 0.25 M sucrose. These were recentrifuged at 105,OOOg for 60 min before final dispersion and storage at - 20°C in 0.25 111sucrose until use. To study the binding of rat liver microsomes with various metal ions, the postmitochondrial supernatant prepared in isotonic sucrose was made 8 mM final concentration of different metal ions as listed in Table 3 and centrifuged at 10,OOOg for 10 min. No attempts were made to determine the marker enzyme activities in these experiments, but the protein contents were determined in the pellets. H ESULTS
The present studies were directed toward standardizing a useful and rapid method for the isolation of rat liver microsomes and their possible application to other tissues and species. The observations made by Sanui et al. (16,171 that a divalent cation like Ca2+binds to rat liver microsomes formed the basis of the method previously reported (71 and presently described. In the initial experiment, different volumes ranging from 0.01 ml to 0.32 ml of 1 M CaCl, (,pH 7.5) were added to a 15 ml portion of postmitochondrial supernatant (approximately 3.75 gm of tissue). As can be seen from Fig. 1, as the concentration of Ca*+ increased, the yield was also increased until a final Ca”+ concentration of 6 mM (0.08 ml of 1 M CaCl,) was reached, when all the microsomes were precipitated quantitatively. The specific activity (13.2) of the marker enzyme glucose-6phosphatase, RNA-to-protein ratio (0.19-0.22’1, total lipids (0.40-0.43
56
KAMATH
301
AND
0 02
004
NARAYAN
008
0 I6
032
ml OF 1M CaCI,
FIG. 1. Effect of amount of Ca’+ on yield, total lipids, RN.4, marker enzyme activity, and amount of calcium bound to microsomes OG addition of calcium to 15 ml postmitochondrial supernatant : (X) yield. (0) RNil, ( 8) t,otal lipids, (A) glucose6-phosphatasc, (A) calcium.
mg/mg protein), and Ca’+ i30-35 j/-g bound/mg protein) remained constant at different Ca’+ concentrations. In the subsequent experiments, a concentration of Ca”+ equivalent to 8 mM was used as standard for precipitating the microsomes. The characteristics of the above CaO+ microsomal preparation were next compared with that of normal high-speed microsomal preparation (Table 1). The hydrolytic enzymes assayed (glucose-6-phosphatase, ionosine diphosphatase, and adenosine mono- and triphosphatases) were found to be of similar specific activities in both kinds of preparations. The total lipids, phospholipid, cholesterol, and RNA were also noted to be similar with the exception of Ca’+, which was found to be as much as 4-fold more in the Ca2+ microsomes. From these results it, was concluded that there was no significant difference in the two preparations except that, according to the present, method, a preparation of acceptable purity could be prepared with much greater ease. As t’he addition of Ca2+ would be expected to have an effect on the electron transport system rather than on t,he hydrolytic enzymes or chemical composition, a study of the enzymes involved in the microsomal electron transport system was undertaken. These aspects included: the CO-binding hemoprotein, cytochrome P-450, and cytochrome 6, contents, type I (aniline) and type II (ethylmorphine) substrates, and three different cyto-
RAT
Enzyme
Activities,
Lipid
Assa! Yield, mg/gm liver (;lucose-6-phosphatase” Ionosine diphoxphatasecL 5’-Nucleotidase” Mg2+-Ii+-Na+-activated xdenosine triphoaphathse” Succinic dehydrogenase* Monoamine oxidasec Cytochrome P-450d Cytochrome bjd hniline hydroxylase< Ethylmorphine demethylltse’ C Y tochrome c reductaseg NAI)H-cytorhrome c reductase” NADPH-cytochrome c reduct,ase” Total lipids, mg/mg protein Phospholipids as 0;. of total lipids Cholestserol as (,-i of total lipids RNA, mg/mg protein Caz+, pgjmg protein w+, rg /mg proteilI
LIVER
57
Ca’+-MICROSOMES
TABLK Composition,
1 and RNA
Content,s
of Microsome?;
Ca2+ microsomes 17.9 1:3.4 7.9 7.4 6.0 0.23 0.8 0.73 0.53 0.81 ‘I 6 184.0 0 60 0.045 0 .4 92.4 5 8 0.21
f f f + + i I + * + +_ + + + * + + + :30.:! 6X.0
Normal miprosomes 1.1 0.6 0.4 0.4 0.1
17,:s 1s. 1 7.6 6 .i r,.!)
0.01 0.04 0.10 0. OS 0.07 I.2 10.6 0.07 0.00s 0 04 6.:3 0.2 0.02
0.64 I.:! 0.72 0.54 0.72 8 3 136 0 0.56 0.049 0.37 03 :3 6.0 0.20
All values are mean + S.D. of 4 preparations except, iu the magnesium, which are the mean of two values. ‘* rmoles inorganic phosphate liberated per hr per mg protein. ‘) /*moles Iodonitrotetrazolium formnzan per hr per mg protein. ’ mrmoles benzylamine per mitt per mg protein. d mpmoles per mg protein. c mpmoles p-aminopheuol per min per mg protein. ( mpmoles HCHO formed per mirl per mg protein. u mpmoles cytochrome c reduced per min per mg protein. h rmoles NAl)H or NAI)PH oxidised per mill per mg proteitl.
case
rf: * f f f
1.5 (I.!1 0.9 0.5 0.:;
f 0.0.5 + 0.0s &- 0.16 + 0.10 + 0.06 + 0.9 & 11.6 i 0.06 & 0.007 * 0.02 f 2 :: * 0.3 + 0.01 8.1 -54.1
of calcium
and
chrome c reductases after initially checking the microsomal preparations for possible contamination in terms of succinic dehydrogenase and monoamine oxidare. The aniline hydroxylase and cthylmorphine demethylase activities were noted to be somewhat higher in Ca”+ microsomes than in normal mirro$omes, while the specific activities of various reductases were about equal in both preparat,ions (Table 1). The mitochondrial marker enzymes succinic dehydrogenase and monoamine oxidase were present in low concentrat,ions in both preparations. It was of considerable interest to determine whether the addition of Ca2+ would have some effect in modifying the properties of smooth and
58
IiAMATH
Studies Assay Yield, mg/gm liver Glucose-6-phosphat,ae* 5’-Nucleotidase* Mgz+-KC-Na+-activated adenosine triphosphat,aseh PhospholipS Cholesterol KNh Calcium< Magnesiumc
AND
on Smooth
TABLE 2 and Rough
Smoot.h 6.8 12.8 6.8 6.4
NARAYAA-
rt + If: k
Microsomes
Smootha 0.4 0.5 0.3 0.6
0.3L3 0.034 0.08 0.0104 0.080
6.1 12.1 7.2 6.1
k ?c f +
0.35 0.035 0.08 0.025 0.076
Rough 0.5 0.3 0.5 1.0
8.X IL1.8 5.1 6.1
f * * +
0.24 0.014 0.32 0.0049 0.025
Rougha 0.6 0.8 0.4 0.5
9.1 12.1 5.4 5.9
* + * +
1.1 1.6 0.7 0.3
0, 23 0.015 0.31 0.0185 0.030
a Prepared by adding calcium chloride. * pmoles P, liberated per mg protein per hr. c E:xpressed as mg per mg protein.
rough microsomes. The characteristics of smooth and rough microsomes, prepared with and without the addition of Ca”+, are compared in Table 2. The addition of Ca2+ did not seem to change the properties of the two fractions of microsomes studied. The results are in agreement with the reported work (18-211 in that the concentration of rough microsomes with attached RNP” particles being more in microsomal preparation than smooth microsomcs and that the levels of phospholipid and cholesterol were more in the latter preparation but the specific activities of the three enzymes assayed were found to be similar. Of interest was the fact that the smooth microsomea (with or without Ca”+l had more rapacity to bind the added Ca’+ than the corresponding rough microsomes. Within each class of microsomes the YIg” content did not vary with or without the addition of Ca”+ when compared on the basis of mg cation bound/mg protein. The effect of other metallic ions was next investigated for their ability to precipitate the microsomes. It was observed that the monovalent and t,rivalent ions iuvest’igatcd here did not, appreciably precipitate the microsomes while all the divalent ions did aggregate the microsomrs, enabling them to sediment in 10 min at 10,OOOg.
The method described here for the isolatiou of rat liver microsomee has some obvious advantages over the earlier published method (7‘1, although both avoid the critical ultracentrifugal step and, thus, have an edge over ’ RNP.
Kit)ont[cleo~rrotPin
the classical method. In comparison with the earlier method (7)) the present one avoids large dilution and permits substantial saving of time. Also, during the studies on microsomee prepared according to the previous procedure (71, we often noticed that careful precautions are to be cxercised whilr making uw of the Ca?+ microsomt~s. This was mainly due to the fact that the size of the Ca’+-aggregated microsomal particles was dependent on the manner in which the tissue was homogenized, the particular medium wed, md the experimental conditions employed. Thus, these factors determined the minimum centrifugal force and the time required to sediment them. It is very likely that larger aggregates are formed when the earlier procedure (7) was used becauac they invariably settled to the bottom of the tutw lrl~on standing for a short time. This difficulty was overcome in the present procedure by obt.aining comparatively smaller aggregates, which do not sediment appreciably at 1,500g but do sediment at 10,OOOg for 10 min. The present method is particularly useful when large amounts of tissues are to hc processed for microsomes in a single experiment and when there are limitations as to the type of rotors available in the laboratory for the Sorvall centrifuge. Our experience with the three preparations, namely, normal microsomcs, Ca’+ microsomes (7)) and Ca”+ microsomes prepared according to the present procedure using a Sorvall rotor 8934, indicate that, for a 20 gm liver tissue, the present method took only a third of time, while the previous method took half the time as compared to the time required for obtaining the normal preparation using an ultracentrifuge. However, if the preparations are to be washed further, the times for the present and previous methods ww reduced markedly to a fourth and a third, respectively, of that required for isolating normal microsomes. An estimation of the recovery of cytochrome P-450 and cytochrome b5 in CaZ+ microsomes and high-speed microsomes revealed similar high amounts in both preparations (Table 1). On the other hand, the hydroxylation and demethylation reactions with aniline and ethylmorphine as substrates showed a small increase in specific activities in Ca’+ microsomes. The cytothrome c reductase activities did not indicate any striking differences in both type of microsomes. Although it is certain that the addition of Ca”+ leads to aggregation of microsomes (22,231 resulting in their precipitation, the mode of action of cation binding to microsomes still remains to be solved. Earlier studies of Gross (23,241 indicate that. a small quantity of Ca”+, added to the supernatant after clarifying the liver homogenate to eliminate the uricase activity, precipitated the particles bearing glucose-6-phosphatase activity. No experimental conditions were provided but it was concluded that most likely a colloidal type of reaction occurred between Ca?+ and small micro-
60
KAMATH
AND
NARAYAN
somal particles. In our present, st’udies, it seems probable that the addition of Ca2+ to the postmitochondrial supernatant, resulted in the binding of both smooth and rough particles. The separation of smooth and rough microsomes with or without Ca’)+ led to the conclusion that the properties of these two microsomal fractions were not altered, and was consistent with our previous t1at.a (8 1. Various studies have demonstrated the aggregation of microsometi (l&25,26). hut the precipitation by diralent ions other than Cat”+ is very interesting because only Ca?+ has, apparently, t’he unique and useful feature of aggregating the microsomes to larger and smaller fragments, depending upon the experimental conditions, and enables them to sediment at 1,500~ and 10,OOOg in 10 min, respectively. For example, although a similar divalent cation like Mg2+ has the capacity, separately or t.ogether with Ca?+, to bind normal microsomes (16) and can sediment according to the present procedure, it was not, able to do so under the conditions of the earlier procedure (7). SUMMARY
A standardized method has been developed for the rapid isolation of rat liver microsomes using Ca”* and its advantages over other available methods have been outlined. In addition t.o hydroIytic enzymes and chemical composition, the important) enzymes in thr electron transport system were determined in CaZ+ microsomes and normal 105,OOOg microsomes and indicat)e only minor differences between the two preparations. Two classes of microsomes-smoot’h and rough particles prepared with or
Effect,
of Metal
Cation
TABLE 3 Ions on Aggregation
10,OOOg sediment (rnn protein/gm liver)
added
Fe”+ .413+ CW+ Mg*+ Fez+ Zll2+ HgZf Ba*+ K+ Na+ To 10 ml of postmitochondrialsupernatant (pH 7.5) and the precipitate obtained according to Lowry’s met,hod.
of the Microsomes
2 4 2 h 16.8 16.2 13 .!I 15.6 16.1 16.X 0.8 1.2
after
were added 0.08 ml 1 M metal salt solutions 10,OOOg for 10 min was estimated for prot,ein
without the addition of Ca”--were compared for their chemical and biochemical properties and indicated little differences within each microsomal fraction. The ability of other divalent cations like Mg2+, Fe’+, Ba”+, Zn?+, and Hg”+ to aggregate the microeomes was observed while the monovalent and kivalent cations tested did not appreciably sediment the microsomcs under the present expcrimcntal conditions. .~CKSOWI,EDGMESTS This work was supported in part by Research Grant CA-61932 from the National Cancer Institute, USPHS. and Research Carter Development Award 5K3-CA-31,063 from the National Cancer Institute, USPHS. to one of us (K.A.N.). We are grateful to Dr. J. M. Bergen for calcium and magnesium dctc-rminations. I~EI’EIII1:NCES 1. HOGEBOOM, G. H., QCHSEIUER, W. C., AZD PALADE. G. E., J. Biol. Chem. 172, 619 (1948). 2. SCHNEIDER, IV. C., J. BioL Che~na. 176, 259 (1948). 3. SCHNEIDER, W. C., .~ND HOGEBOOM. G. H., J. Viol. Cilenl. 183, 123 (1950). 4. HOGEBOOM, G. H., in “Methods of Enzymology” (S. P. Colo\virk and N. 0. Kaplan, eds.), Vol. 2, p. 16. ilcademic Press, New York. 1955. 5. SIE~EVITZ, P., in “Methods in Enzymology” (8. P. Colowick and N. 0. Kaplan. eds.), Vol. 5, p. 61. Academic Press, New York, 1962. 6. K.~MATH, S. A., KUMMEROW, F. A., ~SD .~N.UTFI N.~R.~Y.~N> I~., Fed. Proc. 30, 582, (1971). 7. KAMATH, S. A., KUMMEROW, F. A., AND ANANTH NARAY.~N, Ei., FEBS Lett. 17, 90 (1971). 8. KAMATH, S. A., AND ANANTH NARAYAN, K., submitted for publication. 3. CHAX~DRASEKNARA, 3.. AND ANANTH NARATAN. K., Cro~cer 8e.s. 30, 2876 (1976). 10. OMURA, T., AND SATO, R., J. Biol. Chem. 239, 2370 (1964). 11. ki.4~0, R., AND GILLETTE, J. R., J. Phnrmrrcol. Exp. B’hernp. 150, 279 (1965). 12. DALLNER, G., SICKEVITZ, P., AND PALADE, G. E., J. Cell Biol. 30, 97 (1966). 13. SCHNEIDER, W. C., in “Methods of Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. 3, p. 680. Academic Press, New York, 1957. 14. POFFER, V. R., AND RECHNAGEL, R. O., in “Phosphorus Metabolism” (W. D. McElroy and B. Glass, eds.), p. 377. Johns Hopkins Press, Baltimore, 1951. 15. BERGSTRAND, A., AND DALLNER, G., Anal. Biochem. 29, 351 (1969). 16. SANUI. H., CARVALHO, A. P., AND PACE. N., J. Cell Ph2/sioZ. 59, 261 (1959). 17. SANUI, H., AND PACE, N., J. Cell Physiol. 69, 11 (1967). 18. GLAUMANN, M., AND DALLXER, G., J. Lipid Res. 9, 720 (1968). 19. MANGANIELLO, V. C., .~ND PHILLIPS, A. M.. J. Bioi. Chem. 240, 3941 (1965). 20. SCHNEIDER, W. C.. J. Bid. Chem. 238, 3572 (1963). 21. DALLNER, G., Acta Pathol. Microbial. Scud. Suppl. 166. 1 (1963). 22. SCHNEIDER, W. C., J. Biol. Chem. 166, 595 (1946). 23. GROSS, P. R., Trans. N. 1.. Acad. Sci. 20, 154 (1957). 24. GROSS, P. R., J. Cell Physiol. 47, 429 (1956). 25. GLAUMANN, H., AND DALLNER, G., J. Cell Biol. 47, 34 (1970). 26. REINERT, J. C., .~ND DAVIS, J. I,.. Biochem. Bioph?~s. Actn 241, 921 (1971).
BIOCHEMISTRY
Interaction
48, 53-61 (1972)
of Ca2+
with Endoplasmic of Rat Liver:
Reticulum
A Standardized Procedure for the Isolation of Rat liver Microsomes S. A. KAMATHl llepl~i-tme~~t of
Food
AND
K. ANANTH
Science, University Illinois
of Illinois 61801
Received September
NARAYAN2 at
Champaign-Urbana,
1. 1971
Most commonly, the differential centrifugation technique of Hogeboom (l-3) or its modification (4) is used for the separation of rat liver subcellular fractions. According to the classical procedure (4), the nuclear and mitochondrial fractions in a crude-liver homogenate are removed stepwise by centrifugation at 7009 and lO,OOOg,respectively, for 10 min, while the microsomes present in the postmitochondrial supernatant are obtained by clarifying at 105,OOOgin an ult.racentrifuge for 60 min (5). In comparison to the other subcellular fractions, the isolation of microsomal fraction and its subsequent purification consume a lot of time and, in addition, it requires the use of an expensive ultracentrifuge. Recently we reported (6) that, under certain conditions using Ca2+ ions, a microsomal fraction from rat liver can be routinely obtained by low-speed centrifugation at 1,500g. In brief, the procedure (7), which involved enormous dilution of the postmitochondrial supernatant, was as follows: The crude-liver homogenate (20%) was prepared in a buffer containing sucrose (0.25 M), MgCl, (5 mu), KC1 (25 mM), CaClz (8 mM), and Tris-WC!, pH 7.5 (0.05 M) using a Dounce homogenizer. The postmitochondrial supernatant (lO,OOOg,10 min) diluted 5 times with 0.05 M Tris-HCl buffer, pH 7.5, or 0.0125 M sucrose, each containing 5 mM MgZ+ and 8 mM Ca?+,was centrifuged at 1,500g for 10 min to isolate the aggregated microsomal pellet. The studies on this 1,500g pellet were further extended (8) to compare and provide evidence that the micro’ Present address: Department of Pathology, Mount Sinai School of Medicine of the City University of New York, New York 10029. * Reprints may be requested from this author at the following address: Nutrition Division, Food Laboratory, U. S. Army Natick Laboratories, Natick, Massachusetts 01760. 53 @ 1972 by Academic Press. Inc.
54
KAMATH
AND
h-ARAYAK
somes prepared using Ca2+ were not intrinsically different from normal microsomes with respect to their chemical composition and biochemical properties. In this present communication, we report a standardized procedure using divalent, cations for the isolation of rat-liver microsomes that is simpler and more direct than the classical method as well as the previous method using Ca”+ ions (71. The enzymic activities and RNA and lipid compositions of normal microsomes, and of microsomes prepared according to the present procedure, as well as of smooth and rough microsomes prepared in the presence and absence of Ca”+ ions, have been compared. Metal ions other than Ca’+ were also investigated for their ability to precipitate the microsomes by low-speed ccntrifugation. MATERIALS
AND
METHODS
Extraction of lipids and their estimations, protein determination assays for 5’-nucleotidase, glucose-6-phosphat’ase,Rig”+-K+-Na+-activated adenosine triphosphatase, succinic dchydrogenase, and monoamine oxidase were done as described earlier (9). Enzymes involved in the electron transport system of microsomes were determined according to published methods (10-12). RNA was assayed according to the method of Schneider (13). Ca?+and Mg’+ were analyzed using a Beckman atomic absorption xpectrophotometer with an air-acetylene flame. For the isolation of rat-liver micr~osomes,rats weighing between 300 and 400 gm (Holtzman Co., Madison, Wisconsin~ were starved overnight in order to reduce the glycogen load. The animals were given Nembutal anesthesia and were bled from the abdominal aorta. All the wlx5cq11et1t steps were performed between 0” and 4°C. The livers were excised and kept in ice-cold isotonic sucrose (141, blotted free of blood clots, and homogenized using isotonic sucrose ( 2625% homogenate) in a Douncr homogenizer with about 8-12 strokes. The crutle homogenate was centrifuged at 10,OOOgfor 10 min twice, so that all particles approximately above 300-500 nm vvcre removed. The clear postmitochondrial supernatant was used for the preparation of either normal microsomcs or Ca2+ microsonies. For preparing the normal microsomes, the postmitochondrial supernatant was spun at 105,OOOgfor 60 min in a Beckman L2-65 ultracentrifuge and the pellet obtained was further washed twice, each time rehomogenizing in ieot’onic sucrose, and recentrifuging at 105,OOOgfor 60 min. For the preparation of Ca”+ microsomes, the postmitochondrial supernatant was made 8 mM with respect to calcium chloride (pH 7.5) and was allowed to stand at 4°C for about 3-5 min. The large dilution of the supernatant required for the previous method (7) was eliminated under the present modified conditions. At the end of this period, the entire so-
RAT
LIVER
(-‘:~“+-51l(:ROSO~lF.S
5.i
lution was centrifuged at 10,OOOy for 10 min in a Sorvall centrifuge. The well-sedimented pellet was washed twice by rcsuspending it in isotonic sucrose solution and rcccntrifuging at 10,OOOg for 10 min. Both t’ypes of microsomal preparations were stored at -20°C until use hut, for enzymic assays, fresh preparations were used. Xcrosomal preparation derived from 250 mg liver was ntorc,d in I ml of sucrorc ::olution and isotonic sucrose was used for diluting the microsomes. The smooth and rough microsomes were l)rcl)ared from a 25% crudeliver homogenate in 0.25 111 sucrose solution essentially according to the procedure of Bergstrand and Dallner ( 151. The crude homogenate was clarified at 10,OOOy for 15 min and tbe “10,OOOg supernate” was made to 15 mM with C&l. In the case of Ca”- smooth and rough microsomcs, the “10,OOOg supernate” was made 8 ml11 and 15 mX with Ca’+ and C&l, respectively. An aliquot of 4.5 ml of this “lO,OOOg supernate” was layered over 2 ml of 1.3 M sucrose/l5 mill &Cl. The tubes were centrifuged at 102,OOOg for 120 min in a 40.3 rotor of the Beckman L2-65 ultracentrifuge. After 2 hr, t’hc smooth microsomes separated at 0.25 X,/l.3 M sucrose zone and rough microsomcs sedimcnted as pellets were collected, dispersed in 0.25 M sucrose. These were recentrifuged at 105,OOOg for 60 min before final dispersion and storage at - 20°C in 0.25 111sucrose until use. To study the binding of rat liver microsomes with various metal ions, the postmitochondrial supernatant prepared in isotonic sucrose was made 8 mM final concentration of different metal ions as listed in Table 3 and centrifuged at 10,OOOg for 10 min. No attempts were made to determine the marker enzyme activities in these experiments, but the protein contents were determined in the pellets. H ESULTS
The present studies were directed toward standardizing a useful and rapid method for the isolation of rat liver microsomes and their possible application to other tissues and species. The observations made by Sanui et al. (16,171 that a divalent cation like Ca2+binds to rat liver microsomes formed the basis of the method previously reported (71 and presently described. In the initial experiment, different volumes ranging from 0.01 ml to 0.32 ml of 1 M CaCl, (,pH 7.5) were added to a 15 ml portion of postmitochondrial supernatant (approximately 3.75 gm of tissue). As can be seen from Fig. 1, as the concentration of Ca*+ increased, the yield was also increased until a final Ca”+ concentration of 6 mM (0.08 ml of 1 M CaCl,) was reached, when all the microsomes were precipitated quantitatively. The specific activity (13.2) of the marker enzyme glucose-6phosphatase, RNA-to-protein ratio (0.19-0.22’1, total lipids (0.40-0.43
56
KAMATH
301
AND
0 02
004
NARAYAN
008
0 I6
032
ml OF 1M CaCI,
FIG. 1. Effect of amount of Ca’+ on yield, total lipids, RN.4, marker enzyme activity, and amount of calcium bound to microsomes OG addition of calcium to 15 ml postmitochondrial supernatant : (X) yield. (0) RNil, ( 8) t,otal lipids, (A) glucose6-phosphatasc, (A) calcium.
mg/mg protein), and Ca’+ i30-35 j/-g bound/mg protein) remained constant at different Ca’+ concentrations. In the subsequent experiments, a concentration of Ca”+ equivalent to 8 mM was used as standard for precipitating the microsomes. The characteristics of the above CaO+ microsomal preparation were next compared with that of normal high-speed microsomal preparation (Table 1). The hydrolytic enzymes assayed (glucose-6-phosphatase, ionosine diphosphatase, and adenosine mono- and triphosphatases) were found to be of similar specific activities in both kinds of preparations. The total lipids, phospholipid, cholesterol, and RNA were also noted to be similar with the exception of Ca’+, which was found to be as much as 4-fold more in the Ca2+ microsomes. From these results it, was concluded that there was no significant difference in the two preparations except that, according to the present, method, a preparation of acceptable purity could be prepared with much greater ease. As t’he addition of Ca2+ would be expected to have an effect on the electron transport system rather than on t,he hydrolytic enzymes or chemical composition, a study of the enzymes involved in the microsomal electron transport system was undertaken. These aspects included: the CO-binding hemoprotein, cytochrome P-450, and cytochrome 6, contents, type I (aniline) and type II (ethylmorphine) substrates, and three different cyto-
RAT
Enzyme
Activities,
Lipid
Assa! Yield, mg/gm liver (;lucose-6-phosphatase” Ionosine diphoxphatasecL 5’-Nucleotidase” Mg2+-Ii+-Na+-activated xdenosine triphoaphathse” Succinic dehydrogenase* Monoamine oxidasec Cytochrome P-450d Cytochrome bjd hniline hydroxylase< Ethylmorphine demethylltse’ C Y tochrome c reductaseg NAI)H-cytorhrome c reductase” NADPH-cytochrome c reduct,ase” Total lipids, mg/mg protein Phospholipids as 0;. of total lipids Cholestserol as (,-i of total lipids RNA, mg/mg protein Caz+, pgjmg protein w+, rg /mg proteilI
LIVER
57
Ca’+-MICROSOMES
TABLK Composition,
1 and RNA
Content,s
of Microsome?;
Ca2+ microsomes 17.9 1:3.4 7.9 7.4 6.0 0.23 0.8 0.73 0.53 0.81 ‘I 6 184.0 0 60 0.045 0 .4 92.4 5 8 0.21
f f f + + i I + * + +_ + + + * + + + :30.:! 6X.0
Normal miprosomes 1.1 0.6 0.4 0.4 0.1
17,:s 1s. 1 7.6 6 .i r,.!)
0.01 0.04 0.10 0. OS 0.07 I.2 10.6 0.07 0.00s 0 04 6.:3 0.2 0.02
0.64 I.:! 0.72 0.54 0.72 8 3 136 0 0.56 0.049 0.37 03 :3 6.0 0.20
All values are mean + S.D. of 4 preparations except, iu the magnesium, which are the mean of two values. ‘* rmoles inorganic phosphate liberated per hr per mg protein. ‘) /*moles Iodonitrotetrazolium formnzan per hr per mg protein. ’ mrmoles benzylamine per mitt per mg protein. d mpmoles per mg protein. c mpmoles p-aminopheuol per min per mg protein. ( mpmoles HCHO formed per mirl per mg protein. u mpmoles cytochrome c reduced per min per mg protein. h rmoles NAl)H or NAI)PH oxidised per mill per mg proteitl.
case
rf: * f f f
1.5 (I.!1 0.9 0.5 0.:;
f 0.0.5 + 0.0s &- 0.16 + 0.10 + 0.06 + 0.9 & 11.6 i 0.06 & 0.007 * 0.02 f 2 :: * 0.3 + 0.01 8.1 -54.1
of calcium
and
chrome c reductases after initially checking the microsomal preparations for possible contamination in terms of succinic dehydrogenase and monoamine oxidare. The aniline hydroxylase and cthylmorphine demethylase activities were noted to be somewhat higher in Ca”+ microsomes than in normal mirro$omes, while the specific activities of various reductases were about equal in both preparat,ions (Table 1). The mitochondrial marker enzymes succinic dehydrogenase and monoamine oxidase were present in low concentrat,ions in both preparations. It was of considerable interest to determine whether the addition of Ca2+ would have some effect in modifying the properties of smooth and
58
IiAMATH
Studies Assay Yield, mg/gm liver Glucose-6-phosphat,ae* 5’-Nucleotidase* Mgz+-KC-Na+-activated adenosine triphosphat,aseh PhospholipS Cholesterol KNh Calcium< Magnesiumc
AND
on Smooth
TABLE 2 and Rough
Smoot.h 6.8 12.8 6.8 6.4
NARAYAA-
rt + If: k
Microsomes
Smootha 0.4 0.5 0.3 0.6
0.3L3 0.034 0.08 0.0104 0.080
6.1 12.1 7.2 6.1
k ?c f +
0.35 0.035 0.08 0.025 0.076
Rough 0.5 0.3 0.5 1.0
8.X IL1.8 5.1 6.1
f * * +
0.24 0.014 0.32 0.0049 0.025
Rougha 0.6 0.8 0.4 0.5
9.1 12.1 5.4 5.9
* + * +
1.1 1.6 0.7 0.3
0, 23 0.015 0.31 0.0185 0.030
a Prepared by adding calcium chloride. * pmoles P, liberated per mg protein per hr. c E:xpressed as mg per mg protein.
rough microsomes. The characteristics of smooth and rough microsomes, prepared with and without the addition of Ca”+, are compared in Table 2. The addition of Ca2+ did not seem to change the properties of the two fractions of microsomes studied. The results are in agreement with the reported work (18-211 in that the concentration of rough microsomes with attached RNP” particles being more in microsomal preparation than smooth microsomcs and that the levels of phospholipid and cholesterol were more in the latter preparation but the specific activities of the three enzymes assayed were found to be similar. Of interest was the fact that the smooth microsomea (with or without Ca”+l had more rapacity to bind the added Ca’+ than the corresponding rough microsomes. Within each class of microsomes the YIg” content did not vary with or without the addition of Ca”+ when compared on the basis of mg cation bound/mg protein. The effect of other metallic ions was next investigated for their ability to precipitate the microsomes. It was observed that the monovalent and t,rivalent ions iuvest’igatcd here did not, appreciably precipitate the microsomes while all the divalent ions did aggregate the microsomrs, enabling them to sediment in 10 min at 10,OOOg.
The method described here for the isolatiou of rat liver microsomee has some obvious advantages over the earlier published method (7‘1, although both avoid the critical ultracentrifugal step and, thus, have an edge over ’ RNP.
Kit)ont[cleo~rrotPin
the classical method. In comparison with the earlier method (7)) the present one avoids large dilution and permits substantial saving of time. Also, during the studies on microsomee prepared according to the previous procedure (71, we often noticed that careful precautions are to be cxercised whilr making uw of the Ca?+ microsomt~s. This was mainly due to the fact that the size of the Ca’+-aggregated microsomal particles was dependent on the manner in which the tissue was homogenized, the particular medium wed, md the experimental conditions employed. Thus, these factors determined the minimum centrifugal force and the time required to sediment them. It is very likely that larger aggregates are formed when the earlier procedure (7) was used becauac they invariably settled to the bottom of the tutw lrl~on standing for a short time. This difficulty was overcome in the present procedure by obt.aining comparatively smaller aggregates, which do not sediment appreciably at 1,500g but do sediment at 10,OOOg for 10 min. The present method is particularly useful when large amounts of tissues are to hc processed for microsomes in a single experiment and when there are limitations as to the type of rotors available in the laboratory for the Sorvall centrifuge. Our experience with the three preparations, namely, normal microsomcs, Ca’+ microsomes (7)) and Ca”+ microsomes prepared according to the present procedure using a Sorvall rotor 8934, indicate that, for a 20 gm liver tissue, the present method took only a third of time, while the previous method took half the time as compared to the time required for obtaining the normal preparation using an ultracentrifuge. However, if the preparations are to be washed further, the times for the present and previous methods ww reduced markedly to a fourth and a third, respectively, of that required for isolating normal microsomes. An estimation of the recovery of cytochrome P-450 and cytochrome b5 in CaZ+ microsomes and high-speed microsomes revealed similar high amounts in both preparations (Table 1). On the other hand, the hydroxylation and demethylation reactions with aniline and ethylmorphine as substrates showed a small increase in specific activities in Ca’+ microsomes. The cytothrome c reductase activities did not indicate any striking differences in both type of microsomes. Although it is certain that the addition of Ca”+ leads to aggregation of microsomes (22,231 resulting in their precipitation, the mode of action of cation binding to microsomes still remains to be solved. Earlier studies of Gross (23,241 indicate that. a small quantity of Ca”+, added to the supernatant after clarifying the liver homogenate to eliminate the uricase activity, precipitated the particles bearing glucose-6-phosphatase activity. No experimental conditions were provided but it was concluded that most likely a colloidal type of reaction occurred between Ca?+ and small micro-
60
KAMATH
AND
NARAYAN
somal particles. In our present, st’udies, it seems probable that the addition of Ca2+ to the postmitochondrial supernatant, resulted in the binding of both smooth and rough particles. The separation of smooth and rough microsomes with or without Ca’)+ led to the conclusion that the properties of these two microsomal fractions were not altered, and was consistent with our previous t1at.a (8 1. Various studies have demonstrated the aggregation of microsometi (l&25,26). hut the precipitation by diralent ions other than Cat”+ is very interesting because only Ca?+ has, apparently, t’he unique and useful feature of aggregating the microsomes to larger and smaller fragments, depending upon the experimental conditions, and enables them to sediment at 1,500~ and 10,OOOg in 10 min, respectively. For example, although a similar divalent cation like Mg2+ has the capacity, separately or t.ogether with Ca?+, to bind normal microsomes (16) and can sediment according to the present procedure, it was not, able to do so under the conditions of the earlier procedure (7). SUMMARY
A standardized method has been developed for the rapid isolation of rat liver microsomes using Ca”* and its advantages over other available methods have been outlined. In addition t.o hydroIytic enzymes and chemical composition, the important) enzymes in thr electron transport system were determined in CaZ+ microsomes and normal 105,OOOg microsomes and indicat)e only minor differences between the two preparations. Two classes of microsomes-smoot’h and rough particles prepared with or
Effect,
of Metal
Cation
TABLE 3 Ions on Aggregation
10,OOOg sediment (rnn protein/gm liver)
added
Fe”+ .413+ CW+ Mg*+ Fez+ Zll2+ HgZf Ba*+ K+ Na+ To 10 ml of postmitochondrialsupernatant (pH 7.5) and the precipitate obtained according to Lowry’s met,hod.
of the Microsomes
2 4 2 h 16.8 16.2 13 .!I 15.6 16.1 16.X 0.8 1.2
after
were added 0.08 ml 1 M metal salt solutions 10,OOOg for 10 min was estimated for prot,ein
without the addition of Ca”--were compared for their chemical and biochemical properties and indicated little differences within each microsomal fraction. The ability of other divalent cations like Mg2+, Fe’+, Ba”+, Zn?+, and Hg”+ to aggregate the microeomes was observed while the monovalent and kivalent cations tested did not appreciably sediment the microsomcs under the present expcrimcntal conditions. .~CKSOWI,EDGMESTS This work was supported in part by Research Grant CA-61932 from the National Cancer Institute, USPHS. and Research Carter Development Award 5K3-CA-31,063 from the National Cancer Institute, USPHS. to one of us (K.A.N.). We are grateful to Dr. J. M. Bergen for calcium and magnesium dctc-rminations. I~EI’EIII1:NCES 1. HOGEBOOM, G. H., QCHSEIUER, W. C., AZD PALADE. G. E., J. Biol. Chem. 172, 619 (1948). 2. SCHNEIDER, IV. C., J. BioL Che~na. 176, 259 (1948). 3. SCHNEIDER, W. C., .~ND HOGEBOOM. G. H., J. Viol. Cilenl. 183, 123 (1950). 4. HOGEBOOM, G. H., in “Methods of Enzymology” (S. P. Colo\virk and N. 0. Kaplan, eds.), Vol. 2, p. 16. ilcademic Press, New York. 1955. 5. SIE~EVITZ, P., in “Methods in Enzymology” (8. P. Colowick and N. 0. Kaplan. eds.), Vol. 5, p. 61. Academic Press, New York, 1962. 6. K.~MATH, S. A., KUMMEROW, F. A., ~SD .~N.UTFI N.~R.~Y.~N> I~., Fed. Proc. 30, 582, (1971). 7. KAMATH, S. A., KUMMEROW, F. A., AND ANANTH NARAY.~N, Ei., FEBS Lett. 17, 90 (1971). 8. KAMATH, S. A., AND ANANTH NARAYAN, K., submitted for publication. 3. CHAX~DRASEKNARA, 3.. AND ANANTH NARATAN. K., Cro~cer 8e.s. 30, 2876 (1976). 10. OMURA, T., AND SATO, R., J. Biol. Chem. 239, 2370 (1964). 11. ki.4~0, R., AND GILLETTE, J. R., J. Phnrmrrcol. Exp. B’hernp. 150, 279 (1965). 12. DALLNER, G., SICKEVITZ, P., AND PALADE, G. E., J. Cell Biol. 30, 97 (1966). 13. SCHNEIDER, W. C., in “Methods of Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. 3, p. 680. Academic Press, New York, 1957. 14. POFFER, V. R., AND RECHNAGEL, R. O., in “Phosphorus Metabolism” (W. D. McElroy and B. Glass, eds.), p. 377. Johns Hopkins Press, Baltimore, 1951. 15. BERGSTRAND, A., AND DALLNER, G., Anal. Biochem. 29, 351 (1969). 16. SANUI. H., CARVALHO, A. P., AND PACE. N., J. Cell Ph2/sioZ. 59, 261 (1959). 17. SANUI, H., AND PACE, N., J. Cell Physiol. 69, 11 (1967). 18. GLAUMANN, M., AND DALLXER, G., J. Lipid Res. 9, 720 (1968). 19. MANGANIELLO, V. C., .~ND PHILLIPS, A. M.. J. Bioi. Chem. 240, 3941 (1965). 20. SCHNEIDER, W. C.. J. Bid. Chem. 238, 3572 (1963). 21. DALLNER, G., Acta Pathol. Microbial. Scud. Suppl. 166. 1 (1963). 22. SCHNEIDER, W. C., J. Biol. Chem. 166, 595 (1946). 23. GROSS, P. R., Trans. N. 1.. Acad. Sci. 20, 154 (1957). 24. GROSS, P. R., J. Cell Physiol. 47, 429 (1956). 25. GLAUMANN, H., AND DALLNER, G., J. Cell Biol. 47, 34 (1970). 26. REINERT, J. C., .~ND DAVIS, J. I,.. Biochem. Bioph?~s. Actn 241, 921 (1971).