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The enzymatic composition of rat liver microsomes during liver regeneration
Experimental
Cell Research 19, 591-604 (1960)
591
THE ENZ~ATIC COMPOSITION OF RAT LIVER~ICROSO~~S DURING LIVER REGENERATION ALEXANDRA The Wenner-Gren
VON
DER
Institute for ExperimentaE
DECKEN Biology,
and T.HULTIN University
of Stockholm, Sweden
Received June 18, 1959
the course of the liver regeneration after partial hepatectomy the properties of rat liver microsomes become modified in a characteristic way [6j. When compared in cell-free incorporation systems, microsomes from regenerating livers incorporate labeled amino acids into protein more rapidly than normal microsomes. The activity increase begins about 12-14 hours after the operation and culminates some 15-20 hours later. At the same time the RNA1 content of the microsomal preparations becomes higher. As is well known, the microsomal RNA is concentrated in small particles scattered over the surfaces of the microsomal membranes [31,32]. Since the ribonucleoprotein particles are primarily involved in the incorporation of amino acids into protein [16, 23, 301, these observations suggest that the membranes become more tightly covered with particles as a response to the growth induction (cf. [l]). On the basis of equal protein contents in the microsomal preparations the proportion of the membrane material should be expected to slightly decrease during regeneration, as the nucleoprotein concentration becomes higher. It has in fact been shown [6] that there is a slight decrease of the glucose-6phosphatase (G-&Pase) activity in the microsomal fractions simultaneously with the relative increase of the microsomal nucleoprotein. G-6-Pase is one of the enzymes, which are localized in the membrane part of the microsomes [177. In connection with these observations it became of interest to know, whether the components of the membranes respond to the growth stimulation uniformly and in the same way as G-6-Pase, or vary independently of each other. The experiments described in this paper show that the response may be entirely different in different enzyme groups. IN
1 The following abbreviations are used: RNA, ribonucleic acid; DPNH, reduced diphosphopyridine nucleotide; TPN, TPNH, triphosphopyridine nucleotide, oxidized and reduced; G-6-P, glucose-6-phosphate; G-6-Pase, glucose-6-phosphatase; TCA, trichloroacetic acid; Tris, tris(hydroxymethyl)aminomethane; [WJAF, 2-amino-Q[W]-fluorene; [W)AN, 2-amino-8[%]-naphthalene; MAB, pmonomethylaminoazobenzene; [WIMAB, MAB labeled with 14C in the prime ring; AB, p-aminoazobenzene; N-microsomes, microsomes from livers of normal control rats; R-microsomes, microsomes from livers of hepatectomized rats. Experimentai
Cell Research 19
592
Alexandra von der Decken and T. H&tin
An account of the investigation Biochemical Society, Stockholm,
was presented at the meeting of the Swedish March 8, 1958 [20].
EXPERIMENTAL
Male albino rats weighing approximately 250 g were used. Partial hepatectomy was performed by a modification of the method of Higgins and Anderson [13, 211. The rats were kept without food for 15-20 hours [17] before being killed by decapitation. The livers were perfused with cold 0.15 M KCl, removed and chilled in an icecold medium containing 0.25 M sucrose, 0.035 M Tris pH 7.8, 0.025 M KC1 and 0.01 M MgCl,. They were rapidly weighed, minced and homogenized in a glass homogenizer with 2 volumes of the same medium. After centrifugation for 7 minutes at 12,000 x g (0%) the middle portion of the supernatant was transferred to the tubes of a Spinco preparative ultracentrifuge. The lipid layers were carefully avoided. The microsomal fraction was separated by centrifugation for 55 minutes at 105,000 x g. Each experiment included 2 control rats and 2-3 hepatectomized rats. Determination of proteins.-Protein contents were determined by the method of Lowry et al. [24] cristallized bovine plasma albumin (Armour Lab.) being used as a standard. Determination of G-b-Pase activity.-The determination of G-6-Pase was carried out by the method of de Duve et al. IS]. Measurement
of DPNH-
and
TPNH
cytochrome
c reductase activities.-DPNH-
and TPNH cytochrome c reductases were determined by the method of Hogeboom and Schneider [14, 151 by the use of a Beckman DK-2 spectrophotometer. kfeasurement of DPNH- and TPhTH diaphorase activities.-The activities of DPNHand TPNH diaphorases were determined in the same way as the cytochrome c reductases but dichlorophenolindophenol in a final concentration of 3 x IO-5 M was used instead of oxidized cytochrome c as electron acceptor [17]. Determination of cytochrome b,.-The content of cytochrome b, in the deoxycholate extract of the microsomal fractions was calculated from the difference in optical density at 557 rnp before and after reduction with dithionite [33]. Measurement of oxidative demethylation.-The activity of the microsomal suspensions of oxidatively demethylating MAB was measured by the chromatographic method of Mueller and Miller [2x]. The incubation mixtures contained in a final volume of 2.2 ml: 120 pmoles of nicotinamide, 15 pmoles of G-6-P, 0.1 pmole of TPN, 0.5 ,umoles of MAB in 0.1 ml of 90 per cent ethanol and 0.5 ml of a soluble fraction from normal rat livers. Since the experiments were carried out immediately after the preparation of the liver fractions it was not possible to determine the protein content of the microsomal fractions before the start of the experiments. The microsomes therefore were added to the incubation tubes in a series of increasing concentrations with protein contents in the range of 0.4 to 2.5 mg per tube. After 15 minutes of incubation at 35°C the reaction was stopped by the addition of 3 ml of acetone. The mixtures were shaken twice with acetone-benzene (3: 4) and the AB formed by the reaction was separated from MAB by chromatography on Al,O, columns. The AB values obtained for the microsomes of the normal and regenerating livers were Experimentul Cell Research 19
Microsomes from regenerafing liver
593
compared on the basis of equal protein contents (Fig. 1, curve b). The remaining amounts of non-metabolized MAB were compared in the same way (Fig. 1, curve a). Binding to protein of labelled metabolites of carcinogenic amines.-The same system was used as for the determination of demethylase activity but with the difference that the amount of the soluble fraction was increased to 1.0 ml. The microsomal fractions were added at increasing concentrations varying between I to 6 mg of c ounlslminlcm2
hB 06
40
D6
30
04
2a
02
1c
mg of microsomal
0
0.4
0.6 Fig. 1.
1.2
d
2
1
protem
3
Fig. 2.
Fig. l.-Metabolic degradation of MAB by microsomes from normal ( l ) and regenerating (0) livers, 39 hours after partial hepatectomy. Amounts of (a) MAB and (5) AB after the incubation. The system contained in a final volume of 2.2 ml: 120 pmoles of nicotinamide, 15 pmoles of G-6-P, 0.1 pmole of TPN, 0.5 ymoles of MAB, 0.5 ml of a soluble fraction and increasing amounts of N-microsomes or R-microsomes. The tubes were incubated for 15 minutes at 35°C. Fig. 2.-Release of reactive metabolites of [W]AN from microsomes of normal ( l ) or regenerating (0) livers, and their binding to the proteins of a soluble fraction from normal liver. The system contained in a final volume of 2.2 ml: 120 pmoles of nicotinamide, 15 pmoles of G-6-P, 0.1 ymole of TPN, 0.25 pmoles of ‘%-AN, 1.0 ml of the soluble fraction and varied amounts of the microsomal suspensions. The tubes were incubated for 20 minutes at 35°C. The experiment was performed 39 hours after partial hepatectomy.
proteins. In preliminary experiments 0.7 ml of 30 per cent mitochondria-free homogenates were used instead of the isolated fractions. In some experiments the TPNH generating system, G-6-P, TPN and the soluble fraction was replaced by 1 pmole of TPNH. The following amines were used: [l*C]AN (2.43 me/m-mole), [14C]AF (0.35 me/m-mole) or [l*C]MAB (0.5 me/m-mole). To each tube 0.25 pmoles of amines were added, dissolved in 0.05 ml 90 per cent ethanol. The tubes were incubated for 20 minutes and the microsomal and the soluble fractions were then usually separated again by recentrifugation at 105,000 x g for 55 minutes. Both fractions were precipitated by the addition of 3 ml of acetone or 2 ml of ice-cold 10 per cent TCA. The proteins were washed twice with cold ethanol, twice with varm ethanol and then extracted in a Sohxlet extractor with methanol for 48 hours and with ether for 4 hours. Experimental
Cell Research 19
Alexandra uon tier Decken and T. Hr.&in The proteins were plated on planchets with an area of 1 cmz. The radioactivity was measured with a thin window Geiger-angler tube and scaler. The specific activities were calculated for infinite sample thickness on the basis of an empirical activity saturation curve. As was mentioned before, it was not possible for technical reasons to determine the protein contents of the microsomal fractions before starting the experiments. Therefore the microsomal fractions were added at different concentrations to the incubation mixtures. The soluble proteins served as acceptor system for such reactive metabolites which were released from the microsomal membranes. With increasing microsomal concentrations the radioactivity of the acceptor protein increased gradually (Fig. 2). No significant binding was observed, when the microsomal fraction was omitted from the incubation mixture. The isotope content of the samples of acceptor protein incubated with microsomes from normal and regenerating livers in the same experiment were compared at equal microsomal concentrations. The method is illustrated by the experiment shown in Fig. 2. &r&strafes.-The carcinogen [r*C]AF was synthesized from [l*C]BaCO,, essentially by the method of Ray and Gieser (291. [l*C]MAB was synthesized from (l*CJanilin (0.5 mc~m-mole), and purified on alumina columns [26]. The corresponding unlabeled MAB was synthesized in the same way. [l*C]AN, [l*C]anilin and [lPC]BaCO, were obtained from the Radiochemical Centre, Amersham. G-6-P, DPNH, TPNH, TPN and cytochrome c were obtained from the Sigma Chemical Co.
RESULTS As has been shown previously [6] there is a slight but significant decrease during rat liver regeneration of the G-6-Pase activity of the liver microsomes measured on the basis of the protein content. Also in the present series of experiments G-6-Pase determinations were carried out as a standard method for the characterization of microsomal suspensions, and some of these results are summarized in Table I. As in the previous material the activity of the enzyme began to decrease about 15 hours after the hepatectomy. The decrease was not very pronounced. At the most active stages of the regeneration cycle the activity reached down to about 85 per cent of that of the normal mierosomes. in a number of the microsomal suspensions the activities of the cytochrome c reductases were determined in parallel with the G-6-Pase activity. As is shown by Table I, the activity of DPNH cytochrome c reductase behaved in about the same way as the G-6-Pase activity, and also the TPNH cytochrome c reductase showed a similar picture. About 18 hours after the hepatectomy the microsomal activities of these enzymes had become approximately 10 per cent lower than in the normal microsomes, and this was the case also 40 hours after the operation. The activity of the DPNH- and TPNH diaphorases were determined in
~~croso~es from regenerating liver
595
several of the microsomal suspensions. At 26 and 40 hours of regeneration a decrease by 15-20 per cent was observed in the activities of these enzymes (Table II). TABLE
I. Activities of G-6-Puse and DPNH cytochrome c reductase in rat liver microsomes at different periods after partial hepatectom y.
The activity values are calculated as per cent of the corresponding values for N-microsomes. Average activities for N-microsomes: G-6-Pase 1.9 pmoles of phosphate released per 10 minutes per mg protein; DPNH cytochrome c reductase 4.6 units (25) per mg protein.
Enzyme
0
G-6-Pase DPNH cytochrome c reductase
TABLE
II.
TPNH
7
11
100 101 106 100 97 86
Hours after hepatectomy 15 18 20 26
40
44
90
98 95
87 82
87 -
93 86
84 -
92 97
c~~ochrome c reductase and diaphorase somes from hepa tectomized livers.
92 93
activities
in micro-
The data are given as per cent of the corresponding values for N-microsomes. Average activities for normal microsomes: TPNH cytochrome c reductase 0.29 units (25) per mg protein; DPNH diaphorase 0.12 pmoles of dichlorophenolindophenol reduced per minute per mg protein; TPNH diaphorase 0.026 pmoles of dichlorophenolindophenol reduced per minute per mg protein.
Enzyme TPNH cytochrome c reductase DPNH diaphorase TPNH diaphorase
0
18
100 100 100
90 98 98
Hours after operation 22 23
40
84 91
89 78 81
93 -
It may be concluded from these experiments that the growth induction following partial hepatectomy had only limited effects on the activities per mg protein of G-6-Pase, DPNH- and TPNH cytochrome c reductases or DPNHand TPNH diaphorases. There may occur certain minor individual differences in the activities of these enzymes, particularly at the later stages of regeneration but on the whole the slight general decrease during the regeneration cycle may be due mainly to a changed proportion between the membranous material and the nucleoproteins in the microsomal preparations [6]. In comparison with these results another group. of enzymes in the microsomal membranes gave a more conspicuous response to the growth induction. When carcinogenic amines or a variety of other compounds are incubated
A~exu~dru UORder decked and T. ~u~~~~
596
with rat liver microsomes in the presence of TPNH they become oxidatively metabolized 12, 3, 18, 281. In the present investigation the oxidative demethylation of MAB was studied as an example of these oxidative reactions. As is illustrated by Fig. 3 the activity of the microsomal suspensions to demethylate MAB decreased quite markedly in the regenerating livers. The decrease became apparent after a lag period of about 12-14 hours. After 25-30 hours the activity was only 50-60 per cent of that of the normal microsomes and this low activity persisted until 72 hours or longer. Per cent
of normalactivity .
r, 0
20
40
Hours after hepotectomy 60 80
Fig. 3.--The activity of mierosomes for oxidative demethylation of MAB at different periods after partial hepatectomy. The data obtained for the R-microsomes were calculated as per cent of thb values of the controls by a procedure, illustrated by Fig. 1. The mean value for the control N-. microsomes was 0.06 pmoles of AB per mg protein per 15 minutes.
The reductive cleavage of MAB which takes place under the same conditions as the oxidative demethylation [3, 18, 271, was measured in a few experiments. As is shown by Table III the capacity of the microsomes fox reductive cleavage was significantly diminished in the regenerating livers. In connection with the oxidative metabolism of aromatic compounds by liver microsomes a binding to protein of these compounds or some of their metabolic derivatives may take place [ 11, 12, 18, 191. The binding reaction was studied in the present investigation by the use of different kinds of 14Clabeled aromatic amines, ( [14C]AN, [14C]AF and [W]MAB) all of which, under suitable conditions, have revealed carcinogenic properties. Some preliminary experiments were made on mitochondria-free homogenates from normal and regenerating livers. As is shown in Table IV, the Experimental
Cell Researcfi 19
~~~rosornes f~orn rege~er~~i~g liver
597
binding of isotope from [%]AF and [W]MAB to proteins of such systems was markedly reduced in the regenerating livers. The values were compared with each other on the basis of equal liver weight and a decrease by about 20 per cent was observed for both [14C]AF and [14C]MAB at 20-25 hours after the operation. TABLE
III.
Reductive
cleavage of MAB
by normal
and regenerating
livers.
Each pair of values for normal and regenerating livers were obtained from experiments run in parallel. Incubation system as in Fig. 1. Hours after hepateetomy ymoles of reduced ~B~rng protein~l5 min. Microsomes from normal livers Mierosomes from regenerating livers
TABLE
21
22
39
0.080 0.070
0.080 0.045
0.10 0.075
IV. Binding
of labeled carcinogenic amines to proteins by mifochondriafree homogenates from normal and regenerating liuers.
Incubation system as in Fig. 2, but the soluble and microsomal fractions replaced by 0.7 ml of a 30 per cent mitochondria-free homogenate.
Labeled compound [“C]MAB [‘“CIAN [W]AF TABLE
normal
Hours after operation 21 26 26
Counts per minute Regenerating Normal liver liver 130 202 461
112 169 363
V. Binding
of isotope from [14CfMAB to microsomal proteins from and regenerating liuers at different periods after partial hepafectomy.
The values were compared at a microsomal concentration of about 6 mg of protein. Incubation system as in Fig. 2. Hours after operation 14 24 27 48 48 39 - 603703
Counts per minute N-microsomes R-microsomes 298 215 229 298 330
Per cent R/N
306 146 133 170 100
102 69 60 57 30 Experi~enfaf
Cell Research 19
Alexandra uon der lecher and T. Hu~iin
598
For a more detailed study of the binding reaction isolated microsomes from normal and regenerating livers were incubated with TPN and G-6-P in the presence of standard amounts of a soluble fraction from normal liver. As isotopic components were added [14C]MAB or [14C]AF. The soluble cent of normal
0
activity
20
40
60
80
Fig. 4.-The binding in vitro of radioactive metabolites of [l’C]MAB ( A ) and [‘*C]AF (A) to the proteins of liver microsomes at different periods after partial hepatectomy. The data obtained for the R-microsomes were compared with the values of the controls on a percentual basis. Incubation system as in Fig. 2.
protein fraction had the double function of generating TPNH and of serving as an acceptor for the reactive, isotopic metabolites effusing from the microsomes [lS]. After the incubation the microsomes and the soluble fractions were separated again. The specific activities of the purified proteins were determined both in the microsomes and in the soluble acceptor proteins. Some data on the isotope contents of microsomal proteins from incubation systems of this kind are shown in Table V. Results from a larger number of experiments are summarized in Fig. 4. It is observed that the binding to microsomal proteins of isotope from [14C]MAB was still unchanged about 14 hours after the hepatectomy. At about 20 hours, however, a significant decrease had occurred and at about 40 hours after the operation the binding was reduced by one half or more. A similar effect was obtained when [14C] MAB was replaced by [14C]AF. The specific activities of the solubie proteins were determined with the aim of getting a relative measure of the effusion of reactive metabolites from the microsomes under the same experimental conditions. As is shown by Experimental
Cell Research 19
Microsomes from regenerating liver
599
Fig. 5, the binding of reactive metabolites to the soluble proteins decreased after 12-15 hours. The decrease, which in our material culminated already 20-25 hours after the operation, never became as pronounced as the decrease observed in the microsomal proteins (Fig. 4). The experiments suggest that Pur cent 100
80.
~ 60,
. . .
40,
20
.
l
0
0
I1
Hours after
L 0
20
hepalectomy
60
40
80
Fig. B.-The binding to soluble proteins of radioactive metabolites of [W]MAB (0) and [‘*C]AN ( l ) released from liver microsomes at different periods after partial hepatectomy. The values obtained from the system with R-microsomes are shown as per cent of the corresponding control values. Incubation system as in Fig. 2. Per cent
of normal activity
loo-
80. l<
AA
60.
“1
. 0
.
20
Hoyrs after 40
hopatecfomy
60
80
Fig. 6.-The binding of radioactive metabolites of [W]MAB ( A ) or [W]AF (A) to the proteins of liver microsomes isolated at different periods after partial hepatectomy. The data obtained from R-microsomes were expressed as per cent of the values of the corresponding control N-microsomes. Incubation system as in Fig. 4, but with TPNH replacing the TPNH-generating system. Experimental
Cell Research 19
600
Alexundra von der Decken and T. Hultin
smaller amounts of reactive metabolites were produced in the microsomal membranes of regenerating livers but that these metabolites had a higher probability of effusing into the surrounding medium. This may be due to a lower capacity of the proteins of these membranes to react with the labeled metabolites (cf. [ 181).
. . 20.
Hours after 0
20
CO
60
hepatectomy
60
Fig. 7.-Cytochrome b, content of liver microsomes, determined at different periods after partial hepatectomy. The data obtained for the R-microsomes are shown as per cent of the values of the control N-microsomes.
In a number of experiments the TPNH-generating system (G-6-P, TPN and soluble enzymes) was replaced by TPNH. The specific activities of the microsomal proteins were determined in the same way as in the previous experiments. As is illustrated by Fig. 6, the binding reaction was markedly decreased in the microsomes from regenerating livers also when measured by this more direct method. The content of cytochrome b, was determined in many of the microsomal preparations. In the normal microsomes the average concentration of cytochrome b, was 5 X 1O-4 pmoles per mg protein. This value is lower than that of Klingenberg [22]. The difference may mainly be due to the fact that the homogenization medium used in our experiments contained 0.01 M MgCl,. In the course of the regeneration cycle there was a pronounced decrease of the cytochrome b, content (Fig. 7). The time course of this decrease was approximately parallel with that of the oxidative demethylation shown in Fig. 3.
Experimental
Cell Research 19
Microsomes from regenerating liver
601
DISCUSSION
It has recently been shown that certain enzymes in the membranes of rat liver microsomes may increase in amount several times, when the animals are treated in vivo with suitable kinds of inducing agents [4, 5, 91. The response is remarkably specific, and it becomes manifest within 15-20 hours. In experiments on the effects of amino acid analogues on rat liver slices it has previously been observed that the activity of membrane-bound microsomal enzymes may decrease quite rapidly as soon as the protein metabolism of the slices becomes impaired [19]. Taken together, these data indicate that the enzyme pattern of the microsomal membranes is of a remarkably dynamic nature and susceptible to alterations of the metabolic situation of the cell. In view of this metabolic instability of many of the microsomal enzymes it is not surprising that a considerable scattering was met with in the course of the investigation of the microsomal activities. In certain cases rats of different strains were tried. We had the impression that they differed not only with respect to their enzyme levels but also with respect to the time course and intensity of their response to partial hepatectomy. There were also indications of season variations, possibly due to minor changes in the diet. In spite of this general variability the experiments clearly show that different groups of membrane-bound enzymes give different responses to the growth stimulation after partial hepatectomy. In contrast to the nucleoprotein components of the microsomes, which gave a positive response under these conditions [6]. the effect on the investigated membrane-bound enzymes always was negative. In the case of the G-6-Pase the reduction was not very marked. The microsomal constituents throughout were determined on the basis of the total protein concentrations of the microsomal suspensions. The reduction of the G-6-Pase/protein ratio (Table I) is therefore mainly regarded as a consequence of the higher proportion of nucleoprotein material in the microsomes during regeneration. The situation is probably the same in the case of the diaphorases and cytochrome c reductases (Tables I, II). The microsomal membranes of liver have been shown to contain a wide group of enzymes, related to metabolic detoxication [2, 3, 18, 271. These enzymes are characterized by their simultaneous demand for oxygen and reduced pyridine nucleotides, primarily TPNH. They are represented in this study by the oxidative enzyme, which catalyzes the demethylation of MAB to AB. In the case of this enzyme the decrease in activity during the period Experimental
Cell Research 19
of rapid growth was much more pronounced than in the group of enzymes mentioned in the previous section (Fig. 3). In the course of several of the oxidative microsomal reactions a formation of reactive oxidation products has been observed [ 11, 12, 18, 191. These reactive metabolites may mainly be intermediates or side-products in hydroxylation reactions. They can be demonstrated by means of their capacity of becoming bound by covalent bonds to cell constituents, particularly proteins. aromatic amines, all of them In the present experiments some l*C-labeled with carcinogenic properties, were used as substrates for the microsomal enzymes in order to make the extent of the binding-reaction measurable. The experiments showed that the total amounts of protein-bound isotope became markedly reduced in systems containing microsomes from regenerating livers (Figs. 4, 5, 6). Both the oxidative demethylase and the enzymes responsible for the formation of reactive metabolites from the carcinogenic amines apparently are incapable of maintaining their normal concentration levels in the microsomal membranes in the period of active growth, in which an increased competition may influence the synthesis of individual proteins [34]. The situation becomes different, however, if a compound with inductive capacity [4] is injected into the hepatectomized animal. In hepatectomized rats, treated with methylcholanthrene, the activity of oxidative demethylation in the liver microsomes rapidly increases again [7]. Another component of the microsomal membranes, which becomes strikingly reduced during liver regeneration, is cytochrome b, (Fig. 7). There is no direct evidence that this compound is involved in the electron transfer reactions which are coupled with the oxidative microsomaf functions. However, also the cytochrome b, content is significantly increased in the microsomes of regenerating livers under the influence of methylcholanthrene [7].
SUMMARY
Microsomal suspensions were prepared in parallel from normal rat livers and from livers after partial hepatectomy. The activities of different enzymes were compared in the course of the r~eneration cycle on the basis of equal protein content. The growth induction had only limited effects on the activities of glucose-ephosphatase, DPNH- and TPNH cytochrome c reductases and DPNH- and TPNH diaphorases. Experimental Cell Research 19
Microsomes from regenerating liver
603
The activity of oxidative demethylation of MAB on the other hand rapidly decreased after a lag period of about 12-14 hours. In the presence of liver microsomes carcinogenic amines become bound to proteins. The same conditions as for metabolic oxidation are required. The binding to proteins of isotope from labeled carcinogenic amines was studied in different systems: (a) mitochondria-free homogenates fortified with a TPNH-generating system, (b) isolated microsomes combined with a TPNHgenerating system, (c) isolated microsomes in the presence of added TPNH. In all of these systems the activity of the microsomes to effect a binding of isotope to proteins showed a marked decrease during the liver regeneration. In some experiments the effusion from the microsomes of reactive metabolites of the labeled amines was measured separately by means of added, soluble acceptor proteins which were isolated again after the incubation. The binding of isotope to these proteins also decreased during regeneration, but not quite as much as the binding to the microsomal proteins. The reasons for this are discussed. The content of cytochrome b, was determined in a number of the microsomal preparations. In the microsomes from regenerating livers a pronounced decrease was observed. The work was supported by a grant from the Swedish Cancer Society. The authors wish to thank Miss Margareta Gerlijw for valuable technical assistance. REFERENCES 1. BERNHARD, W. and ROUILLER, C., J. Biophys. Biochem. Cytol. Suppl. 2, 73 (1956). 2. BRODIE, B. B., AXELROD, J., COOPER, J. R., GAUDETTE, L., LA Du, B. N., MITOMA, C. and UDENFRIEND, S., Science 121, 603 (1955). 3. CONNEY, A. H., BROWN, R. R., MILLER, J. A. and MILLER, E. C., Cancer Research 17, 628 (1957). 4. CONNEY, A. H., MILLER, E. C. and MILLER, J. A., ibid. 16, 450 (1956). J. Biol. Chem. 228, 753 (1957). 5. 6. v. D. DECKEN, A. and HULTIN, T., Exptl. Cell Research 14, 88 (1958). Actu Chem. Stand. 13, 2129 (1959). Ii: DUVE, C. DE, PRESSMAN, B. C., GIARUTTO, R., WATTIAUX, R. and APPELMAUS, F., Biochem. J. 60, 604 (1955). 9. FREEDLAND, R. A. and HARPER, A. E., J. Biol. Chem. 233,1 (1958). 10. GARFINKEL, D., Arch. Biochem. Biophys. 71, 111 (1957). 11. HECKER, E., Federation Proc. 16, 194 (1957). 12. HECKER, E. and MUELLER, G. C., J. Biol. Chem. 233,991 (1958). 13. HIOOINS, G. M. and ANDERSON, R. M., Arch. Pathol. 12, 186 (1931). 14. HOQEBOOM, G. H.. J. Biol. Chem. 177, 847 (1949). 15. HO~EBOOM, G. H. and SCHNEIDER, W. C., J. Biol. Chem. 186,417 (1950). 16. HULTIN, T., Bxptl. Cell Research, Suppl. 3, 210 (1955). ibid. 12, 290 (1957). 17. ibid. 13, 47 (1957). 18. ibid. 18, 112 (1959). 19. Experimental
Cell Research 19
604 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34.
Alexandra von der Decken and T. Hultin
HULTIN, T. and v. D. DECKEN, A., Acta Chem. Stand. 12, 596 (1958). EXDK Cell Research 13, 83 (1957). KLING&BERG, M., Arch. B&hem. Bidphys. 71, 111 (1957). LITTLEFIELD, J. W.. KELLER. E. B., GROSS, J. and ZAMECNIK, P. C.. J. Biol. Chem.217.111 (1955): ’ LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L. and RASDALL, R. J., J. Biol. Chem. 193, 265 (1951). MAHLER, H. K., SARKAR,N. K., VERNON, L. P. ~~~ALBERTY, R. A.,J. Biol. Chem. 199,585 (1952). MILLER, J. A. and BAUMAXX, C. A., Cuncer Research 5, 157 (1945). MUELLER, G. C. and MILLER, J. A., J. Biol. Chem. 180, 1125 (1949). ~ ibid. 202, 579 (1953). RAY, F. E. and GIE~ER, k. E., Cancer Research 10, 616 (1950). RENDI, R. and HULTIN, T., Expptl. Cell Research 19, 253 (1960). PALADE, G. E. and SIEKEVITZ, P., .7. Biophys. Biochem. Cytol. 2, 171 (1956). SLAUTTERBACK, D. B., Eqd. Cell Research 5, 173 (1953). STRITTMATTER, C. F. and BALL, E. G., J. Cellular Comp. Physiol. 43, 57 (1954). SWANN, M. M., Cancer Research 18. 1118 (1958).
Experimental
Cell Research 19
Cell Research 19, 591-604 (1960)
591
THE ENZ~ATIC COMPOSITION OF RAT LIVER~ICROSO~~S DURING LIVER REGENERATION ALEXANDRA The Wenner-Gren
VON
DER
Institute for ExperimentaE
DECKEN Biology,
and T.HULTIN University
of Stockholm, Sweden
Received June 18, 1959
the course of the liver regeneration after partial hepatectomy the properties of rat liver microsomes become modified in a characteristic way [6j. When compared in cell-free incorporation systems, microsomes from regenerating livers incorporate labeled amino acids into protein more rapidly than normal microsomes. The activity increase begins about 12-14 hours after the operation and culminates some 15-20 hours later. At the same time the RNA1 content of the microsomal preparations becomes higher. As is well known, the microsomal RNA is concentrated in small particles scattered over the surfaces of the microsomal membranes [31,32]. Since the ribonucleoprotein particles are primarily involved in the incorporation of amino acids into protein [16, 23, 301, these observations suggest that the membranes become more tightly covered with particles as a response to the growth induction (cf. [l]). On the basis of equal protein contents in the microsomal preparations the proportion of the membrane material should be expected to slightly decrease during regeneration, as the nucleoprotein concentration becomes higher. It has in fact been shown [6] that there is a slight decrease of the glucose-6phosphatase (G-&Pase) activity in the microsomal fractions simultaneously with the relative increase of the microsomal nucleoprotein. G-6-Pase is one of the enzymes, which are localized in the membrane part of the microsomes [177. In connection with these observations it became of interest to know, whether the components of the membranes respond to the growth stimulation uniformly and in the same way as G-6-Pase, or vary independently of each other. The experiments described in this paper show that the response may be entirely different in different enzyme groups. IN
1 The following abbreviations are used: RNA, ribonucleic acid; DPNH, reduced diphosphopyridine nucleotide; TPN, TPNH, triphosphopyridine nucleotide, oxidized and reduced; G-6-P, glucose-6-phosphate; G-6-Pase, glucose-6-phosphatase; TCA, trichloroacetic acid; Tris, tris(hydroxymethyl)aminomethane; [WJAF, 2-amino-Q[W]-fluorene; [W)AN, 2-amino-8[%]-naphthalene; MAB, pmonomethylaminoazobenzene; [WIMAB, MAB labeled with 14C in the prime ring; AB, p-aminoazobenzene; N-microsomes, microsomes from livers of normal control rats; R-microsomes, microsomes from livers of hepatectomized rats. Experimentai
Cell Research 19
592
Alexandra von der Decken and T. H&tin
An account of the investigation Biochemical Society, Stockholm,
was presented at the meeting of the Swedish March 8, 1958 [20].
EXPERIMENTAL
Male albino rats weighing approximately 250 g were used. Partial hepatectomy was performed by a modification of the method of Higgins and Anderson [13, 211. The rats were kept without food for 15-20 hours [17] before being killed by decapitation. The livers were perfused with cold 0.15 M KCl, removed and chilled in an icecold medium containing 0.25 M sucrose, 0.035 M Tris pH 7.8, 0.025 M KC1 and 0.01 M MgCl,. They were rapidly weighed, minced and homogenized in a glass homogenizer with 2 volumes of the same medium. After centrifugation for 7 minutes at 12,000 x g (0%) the middle portion of the supernatant was transferred to the tubes of a Spinco preparative ultracentrifuge. The lipid layers were carefully avoided. The microsomal fraction was separated by centrifugation for 55 minutes at 105,000 x g. Each experiment included 2 control rats and 2-3 hepatectomized rats. Determination of proteins.-Protein contents were determined by the method of Lowry et al. [24] cristallized bovine plasma albumin (Armour Lab.) being used as a standard. Determination of G-b-Pase activity.-The determination of G-6-Pase was carried out by the method of de Duve et al. IS]. Measurement
of DPNH-
and
TPNH
cytochrome
c reductase activities.-DPNH-
and TPNH cytochrome c reductases were determined by the method of Hogeboom and Schneider [14, 151 by the use of a Beckman DK-2 spectrophotometer. kfeasurement of DPNH- and TPhTH diaphorase activities.-The activities of DPNHand TPNH diaphorases were determined in the same way as the cytochrome c reductases but dichlorophenolindophenol in a final concentration of 3 x IO-5 M was used instead of oxidized cytochrome c as electron acceptor [17]. Determination of cytochrome b,.-The content of cytochrome b, in the deoxycholate extract of the microsomal fractions was calculated from the difference in optical density at 557 rnp before and after reduction with dithionite [33]. Measurement of oxidative demethylation.-The activity of the microsomal suspensions of oxidatively demethylating MAB was measured by the chromatographic method of Mueller and Miller [2x]. The incubation mixtures contained in a final volume of 2.2 ml: 120 pmoles of nicotinamide, 15 pmoles of G-6-P, 0.1 pmole of TPN, 0.5 ,umoles of MAB in 0.1 ml of 90 per cent ethanol and 0.5 ml of a soluble fraction from normal rat livers. Since the experiments were carried out immediately after the preparation of the liver fractions it was not possible to determine the protein content of the microsomal fractions before the start of the experiments. The microsomes therefore were added to the incubation tubes in a series of increasing concentrations with protein contents in the range of 0.4 to 2.5 mg per tube. After 15 minutes of incubation at 35°C the reaction was stopped by the addition of 3 ml of acetone. The mixtures were shaken twice with acetone-benzene (3: 4) and the AB formed by the reaction was separated from MAB by chromatography on Al,O, columns. The AB values obtained for the microsomes of the normal and regenerating livers were Experimentul Cell Research 19
Microsomes from regenerafing liver
593
compared on the basis of equal protein contents (Fig. 1, curve b). The remaining amounts of non-metabolized MAB were compared in the same way (Fig. 1, curve a). Binding to protein of labelled metabolites of carcinogenic amines.-The same system was used as for the determination of demethylase activity but with the difference that the amount of the soluble fraction was increased to 1.0 ml. The microsomal fractions were added at increasing concentrations varying between I to 6 mg of c ounlslminlcm2
hB 06
40
D6
30
04
2a
02
1c
mg of microsomal
0
0.4
0.6 Fig. 1.
1.2
d
2
1
protem
3
Fig. 2.
Fig. l.-Metabolic degradation of MAB by microsomes from normal ( l ) and regenerating (0) livers, 39 hours after partial hepatectomy. Amounts of (a) MAB and (5) AB after the incubation. The system contained in a final volume of 2.2 ml: 120 pmoles of nicotinamide, 15 pmoles of G-6-P, 0.1 pmole of TPN, 0.5 ymoles of MAB, 0.5 ml of a soluble fraction and increasing amounts of N-microsomes or R-microsomes. The tubes were incubated for 15 minutes at 35°C. Fig. 2.-Release of reactive metabolites of [W]AN from microsomes of normal ( l ) or regenerating (0) livers, and their binding to the proteins of a soluble fraction from normal liver. The system contained in a final volume of 2.2 ml: 120 pmoles of nicotinamide, 15 pmoles of G-6-P, 0.1 ymole of TPN, 0.25 pmoles of ‘%-AN, 1.0 ml of the soluble fraction and varied amounts of the microsomal suspensions. The tubes were incubated for 20 minutes at 35°C. The experiment was performed 39 hours after partial hepatectomy.
proteins. In preliminary experiments 0.7 ml of 30 per cent mitochondria-free homogenates were used instead of the isolated fractions. In some experiments the TPNH generating system, G-6-P, TPN and the soluble fraction was replaced by 1 pmole of TPNH. The following amines were used: [l*C]AN (2.43 me/m-mole), [14C]AF (0.35 me/m-mole) or [l*C]MAB (0.5 me/m-mole). To each tube 0.25 pmoles of amines were added, dissolved in 0.05 ml 90 per cent ethanol. The tubes were incubated for 20 minutes and the microsomal and the soluble fractions were then usually separated again by recentrifugation at 105,000 x g for 55 minutes. Both fractions were precipitated by the addition of 3 ml of acetone or 2 ml of ice-cold 10 per cent TCA. The proteins were washed twice with cold ethanol, twice with varm ethanol and then extracted in a Sohxlet extractor with methanol for 48 hours and with ether for 4 hours. Experimental
Cell Research 19
Alexandra uon tier Decken and T. Hr.&in The proteins were plated on planchets with an area of 1 cmz. The radioactivity was measured with a thin window Geiger-angler tube and scaler. The specific activities were calculated for infinite sample thickness on the basis of an empirical activity saturation curve. As was mentioned before, it was not possible for technical reasons to determine the protein contents of the microsomal fractions before starting the experiments. Therefore the microsomal fractions were added at different concentrations to the incubation mixtures. The soluble proteins served as acceptor system for such reactive metabolites which were released from the microsomal membranes. With increasing microsomal concentrations the radioactivity of the acceptor protein increased gradually (Fig. 2). No significant binding was observed, when the microsomal fraction was omitted from the incubation mixture. The isotope content of the samples of acceptor protein incubated with microsomes from normal and regenerating livers in the same experiment were compared at equal microsomal concentrations. The method is illustrated by the experiment shown in Fig. 2. &r&strafes.-The carcinogen [r*C]AF was synthesized from [l*C]BaCO,, essentially by the method of Ray and Gieser (291. [l*C]MAB was synthesized from (l*CJanilin (0.5 mc~m-mole), and purified on alumina columns [26]. The corresponding unlabeled MAB was synthesized in the same way. [l*C]AN, [l*C]anilin and [lPC]BaCO, were obtained from the Radiochemical Centre, Amersham. G-6-P, DPNH, TPNH, TPN and cytochrome c were obtained from the Sigma Chemical Co.
RESULTS As has been shown previously [6] there is a slight but significant decrease during rat liver regeneration of the G-6-Pase activity of the liver microsomes measured on the basis of the protein content. Also in the present series of experiments G-6-Pase determinations were carried out as a standard method for the characterization of microsomal suspensions, and some of these results are summarized in Table I. As in the previous material the activity of the enzyme began to decrease about 15 hours after the hepatectomy. The decrease was not very pronounced. At the most active stages of the regeneration cycle the activity reached down to about 85 per cent of that of the normal mierosomes. in a number of the microsomal suspensions the activities of the cytochrome c reductases were determined in parallel with the G-6-Pase activity. As is shown by Table I, the activity of DPNH cytochrome c reductase behaved in about the same way as the G-6-Pase activity, and also the TPNH cytochrome c reductase showed a similar picture. About 18 hours after the hepatectomy the microsomal activities of these enzymes had become approximately 10 per cent lower than in the normal microsomes, and this was the case also 40 hours after the operation. The activity of the DPNH- and TPNH diaphorases were determined in
~~croso~es from regenerating liver
595
several of the microsomal suspensions. At 26 and 40 hours of regeneration a decrease by 15-20 per cent was observed in the activities of these enzymes (Table II). TABLE
I. Activities of G-6-Puse and DPNH cytochrome c reductase in rat liver microsomes at different periods after partial hepatectom y.
The activity values are calculated as per cent of the corresponding values for N-microsomes. Average activities for N-microsomes: G-6-Pase 1.9 pmoles of phosphate released per 10 minutes per mg protein; DPNH cytochrome c reductase 4.6 units (25) per mg protein.
Enzyme
0
G-6-Pase DPNH cytochrome c reductase
TABLE
II.
TPNH
7
11
100 101 106 100 97 86
Hours after hepatectomy 15 18 20 26
40
44
90
98 95
87 82
87 -
93 86
84 -
92 97
c~~ochrome c reductase and diaphorase somes from hepa tectomized livers.
92 93
activities
in micro-
The data are given as per cent of the corresponding values for N-microsomes. Average activities for normal microsomes: TPNH cytochrome c reductase 0.29 units (25) per mg protein; DPNH diaphorase 0.12 pmoles of dichlorophenolindophenol reduced per minute per mg protein; TPNH diaphorase 0.026 pmoles of dichlorophenolindophenol reduced per minute per mg protein.
Enzyme TPNH cytochrome c reductase DPNH diaphorase TPNH diaphorase
0
18
100 100 100
90 98 98
Hours after operation 22 23
40
84 91
89 78 81
93 -
It may be concluded from these experiments that the growth induction following partial hepatectomy had only limited effects on the activities per mg protein of G-6-Pase, DPNH- and TPNH cytochrome c reductases or DPNHand TPNH diaphorases. There may occur certain minor individual differences in the activities of these enzymes, particularly at the later stages of regeneration but on the whole the slight general decrease during the regeneration cycle may be due mainly to a changed proportion between the membranous material and the nucleoproteins in the microsomal preparations [6]. In comparison with these results another group. of enzymes in the microsomal membranes gave a more conspicuous response to the growth induction. When carcinogenic amines or a variety of other compounds are incubated
A~exu~dru UORder decked and T. ~u~~~~
596
with rat liver microsomes in the presence of TPNH they become oxidatively metabolized 12, 3, 18, 281. In the present investigation the oxidative demethylation of MAB was studied as an example of these oxidative reactions. As is illustrated by Fig. 3 the activity of the microsomal suspensions to demethylate MAB decreased quite markedly in the regenerating livers. The decrease became apparent after a lag period of about 12-14 hours. After 25-30 hours the activity was only 50-60 per cent of that of the normal microsomes and this low activity persisted until 72 hours or longer. Per cent
of normalactivity .
r, 0
20
40
Hours after hepotectomy 60 80
Fig. 3.--The activity of mierosomes for oxidative demethylation of MAB at different periods after partial hepatectomy. The data obtained for the R-microsomes were calculated as per cent of thb values of the controls by a procedure, illustrated by Fig. 1. The mean value for the control N-. microsomes was 0.06 pmoles of AB per mg protein per 15 minutes.
The reductive cleavage of MAB which takes place under the same conditions as the oxidative demethylation [3, 18, 271, was measured in a few experiments. As is shown by Table III the capacity of the microsomes fox reductive cleavage was significantly diminished in the regenerating livers. In connection with the oxidative metabolism of aromatic compounds by liver microsomes a binding to protein of these compounds or some of their metabolic derivatives may take place [ 11, 12, 18, 191. The binding reaction was studied in the present investigation by the use of different kinds of 14Clabeled aromatic amines, ( [14C]AN, [14C]AF and [W]MAB) all of which, under suitable conditions, have revealed carcinogenic properties. Some preliminary experiments were made on mitochondria-free homogenates from normal and regenerating livers. As is shown in Table IV, the Experimental
Cell Researcfi 19
~~~rosornes f~orn rege~er~~i~g liver
597
binding of isotope from [%]AF and [W]MAB to proteins of such systems was markedly reduced in the regenerating livers. The values were compared with each other on the basis of equal liver weight and a decrease by about 20 per cent was observed for both [14C]AF and [14C]MAB at 20-25 hours after the operation. TABLE
III.
Reductive
cleavage of MAB
by normal
and regenerating
livers.
Each pair of values for normal and regenerating livers were obtained from experiments run in parallel. Incubation system as in Fig. 1. Hours after hepateetomy ymoles of reduced ~B~rng protein~l5 min. Microsomes from normal livers Mierosomes from regenerating livers
TABLE
21
22
39
0.080 0.070
0.080 0.045
0.10 0.075
IV. Binding
of labeled carcinogenic amines to proteins by mifochondriafree homogenates from normal and regenerating liuers.
Incubation system as in Fig. 2, but the soluble and microsomal fractions replaced by 0.7 ml of a 30 per cent mitochondria-free homogenate.
Labeled compound [“C]MAB [‘“CIAN [W]AF TABLE
normal
Hours after operation 21 26 26
Counts per minute Regenerating Normal liver liver 130 202 461
112 169 363
V. Binding
of isotope from [14CfMAB to microsomal proteins from and regenerating liuers at different periods after partial hepafectomy.
The values were compared at a microsomal concentration of about 6 mg of protein. Incubation system as in Fig. 2. Hours after operation 14 24 27 48 48 39 - 603703
Counts per minute N-microsomes R-microsomes 298 215 229 298 330
Per cent R/N
306 146 133 170 100
102 69 60 57 30 Experi~enfaf
Cell Research 19
Alexandra uon der lecher and T. Hu~iin
598
For a more detailed study of the binding reaction isolated microsomes from normal and regenerating livers were incubated with TPN and G-6-P in the presence of standard amounts of a soluble fraction from normal liver. As isotopic components were added [14C]MAB or [14C]AF. The soluble cent of normal
0
activity
20
40
60
80
Fig. 4.-The binding in vitro of radioactive metabolites of [l’C]MAB ( A ) and [‘*C]AF (A) to the proteins of liver microsomes at different periods after partial hepatectomy. The data obtained for the R-microsomes were compared with the values of the controls on a percentual basis. Incubation system as in Fig. 2.
protein fraction had the double function of generating TPNH and of serving as an acceptor for the reactive, isotopic metabolites effusing from the microsomes [lS]. After the incubation the microsomes and the soluble fractions were separated again. The specific activities of the purified proteins were determined both in the microsomes and in the soluble acceptor proteins. Some data on the isotope contents of microsomal proteins from incubation systems of this kind are shown in Table V. Results from a larger number of experiments are summarized in Fig. 4. It is observed that the binding to microsomal proteins of isotope from [14C]MAB was still unchanged about 14 hours after the hepatectomy. At about 20 hours, however, a significant decrease had occurred and at about 40 hours after the operation the binding was reduced by one half or more. A similar effect was obtained when [14C] MAB was replaced by [14C]AF. The specific activities of the solubie proteins were determined with the aim of getting a relative measure of the effusion of reactive metabolites from the microsomes under the same experimental conditions. As is shown by Experimental
Cell Research 19
Microsomes from regenerating liver
599
Fig. 5, the binding of reactive metabolites to the soluble proteins decreased after 12-15 hours. The decrease, which in our material culminated already 20-25 hours after the operation, never became as pronounced as the decrease observed in the microsomal proteins (Fig. 4). The experiments suggest that Pur cent 100
80.
~ 60,
. . .
40,
20
.
l
0
0
I1
Hours after
L 0
20
hepalectomy
60
40
80
Fig. B.-The binding to soluble proteins of radioactive metabolites of [W]MAB (0) and [‘*C]AN ( l ) released from liver microsomes at different periods after partial hepatectomy. The values obtained from the system with R-microsomes are shown as per cent of the corresponding control values. Incubation system as in Fig. 2. Per cent
of normal activity
loo-
80. l<
AA
60.
“1
. 0
.
20
Hoyrs after 40
hopatecfomy
60
80
Fig. 6.-The binding of radioactive metabolites of [W]MAB ( A ) or [W]AF (A) to the proteins of liver microsomes isolated at different periods after partial hepatectomy. The data obtained from R-microsomes were expressed as per cent of the values of the corresponding control N-microsomes. Incubation system as in Fig. 4, but with TPNH replacing the TPNH-generating system. Experimental
Cell Research 19
600
Alexundra von der Decken and T. Hultin
smaller amounts of reactive metabolites were produced in the microsomal membranes of regenerating livers but that these metabolites had a higher probability of effusing into the surrounding medium. This may be due to a lower capacity of the proteins of these membranes to react with the labeled metabolites (cf. [ 181).
. . 20.
Hours after 0
20
CO
60
hepatectomy
60
Fig. 7.-Cytochrome b, content of liver microsomes, determined at different periods after partial hepatectomy. The data obtained for the R-microsomes are shown as per cent of the values of the control N-microsomes.
In a number of experiments the TPNH-generating system (G-6-P, TPN and soluble enzymes) was replaced by TPNH. The specific activities of the microsomal proteins were determined in the same way as in the previous experiments. As is illustrated by Fig. 6, the binding reaction was markedly decreased in the microsomes from regenerating livers also when measured by this more direct method. The content of cytochrome b, was determined in many of the microsomal preparations. In the normal microsomes the average concentration of cytochrome b, was 5 X 1O-4 pmoles per mg protein. This value is lower than that of Klingenberg [22]. The difference may mainly be due to the fact that the homogenization medium used in our experiments contained 0.01 M MgCl,. In the course of the regeneration cycle there was a pronounced decrease of the cytochrome b, content (Fig. 7). The time course of this decrease was approximately parallel with that of the oxidative demethylation shown in Fig. 3.
Experimental
Cell Research 19
Microsomes from regenerating liver
601
DISCUSSION
It has recently been shown that certain enzymes in the membranes of rat liver microsomes may increase in amount several times, when the animals are treated in vivo with suitable kinds of inducing agents [4, 5, 91. The response is remarkably specific, and it becomes manifest within 15-20 hours. In experiments on the effects of amino acid analogues on rat liver slices it has previously been observed that the activity of membrane-bound microsomal enzymes may decrease quite rapidly as soon as the protein metabolism of the slices becomes impaired [19]. Taken together, these data indicate that the enzyme pattern of the microsomal membranes is of a remarkably dynamic nature and susceptible to alterations of the metabolic situation of the cell. In view of this metabolic instability of many of the microsomal enzymes it is not surprising that a considerable scattering was met with in the course of the investigation of the microsomal activities. In certain cases rats of different strains were tried. We had the impression that they differed not only with respect to their enzyme levels but also with respect to the time course and intensity of their response to partial hepatectomy. There were also indications of season variations, possibly due to minor changes in the diet. In spite of this general variability the experiments clearly show that different groups of membrane-bound enzymes give different responses to the growth stimulation after partial hepatectomy. In contrast to the nucleoprotein components of the microsomes, which gave a positive response under these conditions [6]. the effect on the investigated membrane-bound enzymes always was negative. In the case of the G-6-Pase the reduction was not very marked. The microsomal constituents throughout were determined on the basis of the total protein concentrations of the microsomal suspensions. The reduction of the G-6-Pase/protein ratio (Table I) is therefore mainly regarded as a consequence of the higher proportion of nucleoprotein material in the microsomes during regeneration. The situation is probably the same in the case of the diaphorases and cytochrome c reductases (Tables I, II). The microsomal membranes of liver have been shown to contain a wide group of enzymes, related to metabolic detoxication [2, 3, 18, 271. These enzymes are characterized by their simultaneous demand for oxygen and reduced pyridine nucleotides, primarily TPNH. They are represented in this study by the oxidative enzyme, which catalyzes the demethylation of MAB to AB. In the case of this enzyme the decrease in activity during the period Experimental
Cell Research 19
of rapid growth was much more pronounced than in the group of enzymes mentioned in the previous section (Fig. 3). In the course of several of the oxidative microsomal reactions a formation of reactive oxidation products has been observed [ 11, 12, 18, 191. These reactive metabolites may mainly be intermediates or side-products in hydroxylation reactions. They can be demonstrated by means of their capacity of becoming bound by covalent bonds to cell constituents, particularly proteins. aromatic amines, all of them In the present experiments some l*C-labeled with carcinogenic properties, were used as substrates for the microsomal enzymes in order to make the extent of the binding-reaction measurable. The experiments showed that the total amounts of protein-bound isotope became markedly reduced in systems containing microsomes from regenerating livers (Figs. 4, 5, 6). Both the oxidative demethylase and the enzymes responsible for the formation of reactive metabolites from the carcinogenic amines apparently are incapable of maintaining their normal concentration levels in the microsomal membranes in the period of active growth, in which an increased competition may influence the synthesis of individual proteins [34]. The situation becomes different, however, if a compound with inductive capacity [4] is injected into the hepatectomized animal. In hepatectomized rats, treated with methylcholanthrene, the activity of oxidative demethylation in the liver microsomes rapidly increases again [7]. Another component of the microsomal membranes, which becomes strikingly reduced during liver regeneration, is cytochrome b, (Fig. 7). There is no direct evidence that this compound is involved in the electron transfer reactions which are coupled with the oxidative microsomaf functions. However, also the cytochrome b, content is significantly increased in the microsomes of regenerating livers under the influence of methylcholanthrene [7].
SUMMARY
Microsomal suspensions were prepared in parallel from normal rat livers and from livers after partial hepatectomy. The activities of different enzymes were compared in the course of the r~eneration cycle on the basis of equal protein content. The growth induction had only limited effects on the activities of glucose-ephosphatase, DPNH- and TPNH cytochrome c reductases and DPNH- and TPNH diaphorases. Experimental Cell Research 19
Microsomes from regenerating liver
603
The activity of oxidative demethylation of MAB on the other hand rapidly decreased after a lag period of about 12-14 hours. In the presence of liver microsomes carcinogenic amines become bound to proteins. The same conditions as for metabolic oxidation are required. The binding to proteins of isotope from labeled carcinogenic amines was studied in different systems: (a) mitochondria-free homogenates fortified with a TPNH-generating system, (b) isolated microsomes combined with a TPNHgenerating system, (c) isolated microsomes in the presence of added TPNH. In all of these systems the activity of the microsomes to effect a binding of isotope to proteins showed a marked decrease during the liver regeneration. In some experiments the effusion from the microsomes of reactive metabolites of the labeled amines was measured separately by means of added, soluble acceptor proteins which were isolated again after the incubation. The binding of isotope to these proteins also decreased during regeneration, but not quite as much as the binding to the microsomal proteins. The reasons for this are discussed. The content of cytochrome b, was determined in a number of the microsomal preparations. In the microsomes from regenerating livers a pronounced decrease was observed. The work was supported by a grant from the Swedish Cancer Society. The authors wish to thank Miss Margareta Gerlijw for valuable technical assistance. REFERENCES 1. BERNHARD, W. and ROUILLER, C., J. Biophys. Biochem. Cytol. Suppl. 2, 73 (1956). 2. BRODIE, B. B., AXELROD, J., COOPER, J. R., GAUDETTE, L., LA Du, B. N., MITOMA, C. and UDENFRIEND, S., Science 121, 603 (1955). 3. CONNEY, A. H., BROWN, R. R., MILLER, J. A. and MILLER, E. C., Cancer Research 17, 628 (1957). 4. CONNEY, A. H., MILLER, E. C. and MILLER, J. A., ibid. 16, 450 (1956). J. Biol. Chem. 228, 753 (1957). 5. 6. v. D. DECKEN, A. and HULTIN, T., Exptl. Cell Research 14, 88 (1958). Actu Chem. Stand. 13, 2129 (1959). Ii: DUVE, C. DE, PRESSMAN, B. C., GIARUTTO, R., WATTIAUX, R. and APPELMAUS, F., Biochem. J. 60, 604 (1955). 9. FREEDLAND, R. A. and HARPER, A. E., J. Biol. Chem. 233,1 (1958). 10. GARFINKEL, D., Arch. Biochem. Biophys. 71, 111 (1957). 11. HECKER, E., Federation Proc. 16, 194 (1957). 12. HECKER, E. and MUELLER, G. C., J. Biol. Chem. 233,991 (1958). 13. HIOOINS, G. M. and ANDERSON, R. M., Arch. Pathol. 12, 186 (1931). 14. HOQEBOOM, G. H.. J. Biol. Chem. 177, 847 (1949). 15. HO~EBOOM, G. H. and SCHNEIDER, W. C., J. Biol. Chem. 186,417 (1950). 16. HULTIN, T., Bxptl. Cell Research, Suppl. 3, 210 (1955). ibid. 12, 290 (1957). 17. ibid. 13, 47 (1957). 18. ibid. 18, 112 (1959). 19. Experimental
Cell Research 19
604 20. 21. 22. 23. 24. 25. 26. 27. 28. 29. 30. 31. 32. 33. 34.
Alexandra von der Decken and T. Hultin
HULTIN, T. and v. D. DECKEN, A., Acta Chem. Stand. 12, 596 (1958). EXDK Cell Research 13, 83 (1957). KLING&BERG, M., Arch. B&hem. Bidphys. 71, 111 (1957). LITTLEFIELD, J. W.. KELLER. E. B., GROSS, J. and ZAMECNIK, P. C.. J. Biol. Chem.217.111 (1955): ’ LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L. and RASDALL, R. J., J. Biol. Chem. 193, 265 (1951). MAHLER, H. K., SARKAR,N. K., VERNON, L. P. ~~~ALBERTY, R. A.,J. Biol. Chem. 199,585 (1952). MILLER, J. A. and BAUMAXX, C. A., Cuncer Research 5, 157 (1945). MUELLER, G. C. and MILLER, J. A., J. Biol. Chem. 180, 1125 (1949). ~ ibid. 202, 579 (1953). RAY, F. E. and GIE~ER, k. E., Cancer Research 10, 616 (1950). RENDI, R. and HULTIN, T., Expptl. Cell Research 19, 253 (1960). PALADE, G. E. and SIEKEVITZ, P., .7. Biophys. Biochem. Cytol. 2, 171 (1956). SLAUTTERBACK, D. B., Eqd. Cell Research 5, 173 (1953). STRITTMATTER, C. F. and BALL, E. G., J. Cellular Comp. Physiol. 43, 57 (1954). SWANN, M. M., Cancer Research 18. 1118 (1958).
Experimental
Cell Research 19