Pergamon Prese
Life Sciences Vol . 18, pp .215-222 Printed is the U .S .A .
AMINO ACID INCORPORATION BY NUCLEAR MEMBRANE FRACTION OF RAT LIVER r r* ** H . Ono , T . Ono and 0 . 1Vada `Department of Physiology, Faculty of Medicine, University of Tokyo ** Department of Hygiene and Preventive Medicine, Faculty of Medicine, University of Tokyo (Received is final form December 30, 1975) Summary Nuclear membrane fraction of rat liver is able to incorporate 14C_leucine into its proteins in vitro . The incorporation of 1 4 Cleucine into the nuclear membrane-motion was almost completely inhibited by chloramphenicol, but the inhibition by cycloheximide and puromycin was not so remarkable . RNase and DNase were not effective . The incorporation was also inhibited by several reagents known to interfere with energy metabolism . These characteristics of the incorporation of 14 C-leucine by the nuclear membrane fraction are quite similar to those of the incorporation by nuclei isolated from rat liver and mitochondrial fraction, but seem to be different from those of the ordinary protein synthetic system in microsomal fraction . 14 C-Leucine was preferentially incorporated into intrapolypeptide or C-terminal residues but not into N-terminal residues . Acrylamide gel electrophoresis showed that three protein species were mainly labelled . The incorporating activity of the nuclear membrane fraction obtained from regenerating liver 17 h after partial hepatectomy showed 220 $ of the control . The possibility that the contaminated mitochondrial fraction might be responsible for the incorporation of 14 C-leucine by the nuclear membrane fraction was ruled out . In the preceding paper, we have reported that rat liver nuclei can incorporate 14 C-leucine into nuclear proteins in vitro (1) . The incorporation of 1 4C-leucine into nuclear proteins was marc~ed~nhibited by chloramphenicol but not by puromycin, cycloheximide and RNase . It was found that 14 C-leucine was mainly incorporated into nuclear residual fraction remained after successive extraction of nuclei with 50 mM Tris-HC1 buffer (pH 7 .4) and 1 M NaCl . It has also been reported that the nuclear residual fraction is more highly labeled by radioactive amino acid in vitro (2-4) . The incorporation of 14C-leucine by nuclei isolated from regenerating liver was found to increase as compared with control and show a maximum activity 17 h after partial hepatectomy . Moreover, it was demonstrated that the increased incorporation by nuclei from regenerating liver was mainly due to increased incorporation into the nuclear residual fraction but not into other nuclear proteins (5) . In studying about the nuclear residual fraction, we found that nuclear membrane fraction from rat liver could also incorporate 14C-leucine into its proteins . In this paper we report some characteristics of the incorporation of 14 C-leucine by nuclear membrane fraction . 215
216
Amino Acid Incorporation by Nuclear Membrane
Vol. 18, No . 2
Methods Pr aration of Nuclear Membrane Fraction---Male Wistar rats weighing 150250 g were used . The proc ure or solating nuclei from rat liver was as described in the preceding paper (1) . Nuclear pellet was suspended in 0 .25 $ Triton X-100 in 0 .25 M sucrose-50 mM Tris-HC1 buffer (pH 7 .4)-3 mM MgC1 2 -20 mM KC1 (Buffer A) and centrifuged immediately . Nuclear membrane fraction was prepared by the method of Berezney, Funk and Crane (6) with a slight modification . The Triton treated nuclear pellet was washed twice with Buffer A and digested with DNase ~5 Pg/mg nuclear protein) and RNase (2 pg/mg nuclear protein) in Buffer A at 0 C overnight . The nuclease treated nuclear suspension was centrifuged at 20000 G for 15 min and pellet was suspenden in 1 M NaCl, followed by centrifugation at 20000 G for 15 min. This procedure was repeated twice more and the membranous pellet was washed with Buffer A and finally suspended in 0 .25 M sucrose-3toM MgC12-20 mM KC1 . The amount of protein of this nuclear membrane fraction was about 1 $ of the total nuclear proteins . Pre aration of Mitochondria---The liver homogenate was centrifuged at 1000 G for 10 n to precipitate nuclear fraction . Mitochondrial fraction was prepared by centrifuging the supernatant recovered from 1000 G run, at 20000 G for 20 min . Mitochondrial pellet was washed with Buffer A and 1 M NaCl successively according to the method for preparing the nuclear membrane fraction . Partial H atecto anesthesia as ascribe
---Rats were partially hepatectomized under ether by Higgins and Anderson (7) .
Incubation Conditions---The reaction mixture contained 20 mM sodü~ phosphate bu er (pH 6 .0), 0.1 M sucrose, 1 mM MgC12, 8 mM KC1, 0.1 pCi of 14C-leucine (312 mCi/mmole, The Radiochemical Centre, Amersham, England), and membrane fraction equivalent to 100-300 pg protein in a final volume of 0 .5 ml, was incubated at 37 C for 60 min. The reaction was terminated by addition of cold 5 $ trichloroacetic acid containing 0.25 $ Na2W04 . The acid-insoluble precipitates were collected on glass fiber discs by filtration and washed with 5 $ trichloroacetic acid, ethanol-ether mixture (3 :1, v/v) and ether, successively . Dried filter discs were put into toluene-PPO-POPOP scintillator and counted in a liquid scintillation counter . Determination of 14C-Leucine Inco orated in N-Terminals---The distribution o incorporate C-leucine among N-terminal or ntrapolypeptide residues of the nuclear membranous protein was determined by the dinitro phenylation method of Sangar (8) . The nuclear membrane fraction after incubating with 14 C-leucine, was dinitrophenylated with 1-fluoro-2,4-dinitrobenzene and hydrolyzed in 6 N HC1 at 105°C for 12 h . Then dinitrophenylated The (DNP)-amino acids derived from N-terminals were extracted with ether. radioactivity of the ether extract and the aqueous phase, which contained free amino acids derived from intrapolypeptide and C-terminal residues, and a few ether-unextractable DNP-amino acids was counted .
Ac lamide Gel Electro horesis---The nuclear membrane fraction, incubated in the presence o C-leucine was washed with cold Buffer A and then solubilized with 8 M urea~50 mM NaHC03-Na2C03 (pH 9 .5) . The gel contained 7 .5 $ acrylamide, 0 .2 $ N,N -methylenebisacrylamide, 0.15 M Tris-HC1 buffer (pH 9 .5) and 8 M deionized urea . The urea extract contained 300-500 Kg protein was put on the gel and electrophoresed in 0.05 M Tris-glycine buffer (pH 8 .6) at 3mA/ tube for 45 min. The gel was frozen on dry ice and cut into 1 mm thick slices . The gel slices xere then solubilized in 0 .2 ml of 30 $ H202 at 60 °C for 4 h and dryad . The residues were solubilized in 0.3 ml of hyamine solution and counted in a toluene-PPO-POPOP scintillation liquid .
Amino Acid Incorporation by Nuclear Membrane
Vol . 18, No . 2
21 7
Enz e Assa ---Monoamine oxidase activity was assayed by the procedure of McEwen, Jr . 9 . Protein was assayed by the method of Lowry et al . (10) . Results Characteristics of Amino Acid Inco ration b the Nuclear Membrane Fraction--- e nuclear membrane fraction incorporate C-leucine almost 1l n~ëarly during 2 h incubation . Optimal H of the incorporation was found between 6 .0 and 6 .5 . The rate at which 1 ~C-leucine was incorporated into protein was proportional to the added amount of the membrane fraction up to 400 pg protein . TABLE I Effect of Various Substances on the Incorporation of 14 C- Leucine by Nuclear Membrane Fraction in vitro Exptl . No .
Additions
Incorporation of 14 C- Leucine cpm/mg protein
$ activity
I
Control Puromycin, 100 pg/ml Cycloheximide, 100 pg/ml Chloramphenicol, 100 pg/ml Lipase, 100 pg/ml Creatine phosphate, 10 mM + Creatine kinase, 100 pg/ml
14730 10900 13130 490 2430 12650
100 74 89 3 17 86
II
Control ATP, 0 .5 mM GTP, 0 .5 mM
14100 7650 13400
100 54 95
12350 5620 2140 8100 2550 720
100 45 17 66 21 6
III
Control Amino acid mixture, 2 pM Trypsin, 5 pg/ml Sodium deoxycholate, 0 .05 $ Sodium dodecyl sulfate, 0 .05 $ - Preheating, 70 ° C for 10 min
Table I gives the characteristics of the incorporation of 14C-leucine into the nuclear membrane fraction . Non-specific adsorption of 14 C-leucine onto the nuclear membrane fraction was negligible (100 cpm/mg protein) . The incorporation of 14C-leucine was strongly inhibited by chloramphenicol at the concentration of 100 pg/ml, but only slightly by puromycin and cycloheximide at same concentration . Addition of ATP, ATP-generating system and GfP was not necessary for the incorporating system of the nuclear membrane fraction . (hn the contrary, presence of the first two substances inhibited the reaction . The incorporation of 14 C-leucine proceeded without supply of amino acid mixture, suggesting that amino acid pool was still present in the nuclear membrane fraction which might be sufficient to support protein synthesis during 2 h incubation period . To see if the incorporating activity could be enhanced by addition of exogenous amino acids, 2 EiM of each amino acid mixture minus leucine was added to the incubation medium . The incorporation of 14C-leucine was inhibited to 45 $ of control by addition of amino acid mixture . Whether this inhibition is caused by competition among 14 C-leucine and the different amino acids or unknown factors) is concerned, remains unexplained . Trypsin had a strong inhibitory effect on the incorporating system and this incorporating system was thereto-labile . The membrane fraction preheated at 70 ° C for
by the min dependent been incorporation asControl Mthe assaying sodium III of nuclear lost deoxycholate effect NaCl membrane nuclei shown sodium in structures No contaminated treated fraction 14C-leucine, treated acid the 2mM 20 The the 2of Treated fraction fluoride mM These mM of in membrane cyanide, membrane the was Monoamine Additions activity and specific Table with yeast Acid ract was nuclei were 0Contamination---In activity inhibited dehydroacetic facts of 14C-Leucine acid, mitochondria acid, mitochondrial 1showed expressed Incorporation Metabolic $the RNA II, on Mnecessary fraction sodium fraction, activity of 1Oxidase completely m1seem NaCl 2mM aand extent mM ght of the mM amarked the to monoamine calf azide less in be NaCl-treated bywas as Inhibitors in on of the incorporation, for acid suggest of Nuclear contaminated 100 thymus inhibitory pronounced fraction Subcellular h/m~ by which mitochondrial the 5and Lipase, the order same benzaldehyde/ $also Nuclear III oxidase 2,4-dinitrophenol II enzyme ~rotein incorporation that of Membrane manner The DNA the to showed on besodium mitochondrial Monoamine know enzyme effect incorporation the (native Membrane by responsible Practions inprotein suggesting asmitochondrial contamination strong energy Incorporation Fraction the preparing $dodecyl by activity onof result -and possibility respiratory the There yieldin of inhibitory heat was fraction that for activity of Rat sulfate incorporation was observed the of was the denatured) 1~C-leucine intact was fraction Liver shown the nuclear system nodeterminthat incorpoand and inhibitors effects, 18, Triton stimumemin that the No Monoand which isof on 2
218
Amino
10 sodium branous latory the
Vol . . . .
As such iodoacetic while 14C_leucine . present is .
.
TABLE
Effect of Exptl . I
II
.
Incorporation cpm/mg
.
l4C-Leucine $
KCN, NaN3, 2,4-Dinitrophenol,
11940 530 2130 70
100 5 18 1
Dehydroacetic Monoiodoacetic NaF,-
10550 3340 80 6440
100 32 1 - 61
Mitochondrial nuclear that ration ed Table . had membrane treated of Activity Fractions Homogenate Mitochondria 1 Nuclei Triton Membrane
-
.
The
.
.4
. TABLE Activity nmole 3 354 298 525 10 1 .7 27
Oxidase activity 67 .4 56 .8 100 1 .9 0 .4 5 .1
Vol . 18, No . 2
Amino Acid Incorporation by Nuclear Membrane
21 9
Addition of mitochondrial fraction to the incubation medium inhibited the incorporation of 14 C-leucine . The incorporation of 14 C-leucine by mitochondrial fraction itself under the same conditions for the nuclear membrane fraction was 3 $ as compared with the incorporating activity of the nuclear membrane fraction . These facts suggest that the incorporating activity of the nuclear membrane fraction is not ascribed to the contaminated mitochondria . Partial Characterization of the Labeled Product---In order to elucidate the distribution o incorporated -leucine among N-terminal and intrapolypeptide residues of the nuclear membranous protein, N-terminal analysis was carried out . As shown in Table IV, the incorporated 14C-leucine was not located in N-terminal residues and most of the radioactivity seemed to incorporate into intrapolypeptide or C-terminal residues of the membranous protein . The degree of dinitrophenylation as measured by a ratio of £-DNP-lysine to DNP-lysine plus free-lysine, was about 90 $. TABLE IV Distribution of Incorporated 14 C- Leucine between N-Terminals and Intrapolypeptides of the Nuclear Membrane Fraction 14 C_ Leucine Incorporation cpm
Fractions Ether extracts (N-terminal DNP-amino acids) Aqueous phase (free amino acids from intrapolypeptides or C-terminals, and ether unextractable DNP-amino acids)
18 865
+~ u m ao
1001
N N .U .a
m
100
NO
H
50
I
IY
I
r
I ~
1
~ I 1 (-)
I 1
I
III~~
I
,~~ 1 2 3 4 (+) distance from origin (cm) Fig . 1
a
d
U
~
50
I II ,
II ;
8 16 24 time after partial hepatectomy (h) Fig . 2
Fig. 1 Acrylamide gel electrophoresis of the nuclear membrane fraction . Anode and the direction of movement were to the right .
Fig . 2 Incorporation of 14 C-leucine by the nuclear membrane fraction from regenerating liver as a function of time after partial hepatectomy . Closed circles are average of two values for one experiment group and bars indicate average of two experiments . One group consists of S animals .
.
220
Amino Acid Incorporation by Nuclear Membrane
Vol . 18, No . 2
Next, polyacrylamide gel electrophoresis was employed to analyze the 14 C_leucine-incorporated membranous protein . It was obvious in Fig . 1 that 14 C_leucine was incorporated into three major protein species . Using SDS polyacrylamide gel electrophoresis, molecular weight of these protein species were determined . The molecular weights of them were about 50000, 70000 and 75000 . Berezney and Coffey have reported that the molecular weights of the nuclear protein matrix of rat liver are 62000, 66000 and 69000 (11) . 14C_Leucine Inco ratin Activit after Partial H atecto ---Fig . 2 shows t e results on the incorporat ono C-leucine nto proteins of the nuclear membrane fraction obtained from regenerating rat liver . Values for regenerating liver are expressed as percentages of those for normal liver . Incorporation of 14C-leucine 4 h after partial hepatectomy increased slightly and reached a maximum about 17 h after the operation . After 21 h, the incorporating activity showed a decrease and returned to the control level 24 h after the operation . Discussion The characteristics of the incorporation of 14 C-leucine by the nuclear membrane fraction of rat liver are quite similar to those of the incorporation of amino acids by nuclei isolated from rat liver (1,2) . In both cases the o timal pH of the incorporation is slightly acidic . The incorporation of l~C-leucine is strongly inhibited by chloramphenicol, while RNase, DNase, cycloheximide and puromycin show almost no remarkable effect on the incorporation . The change of the incorporating activity of the nuclear membrane fraction as a function of time after partial hepatectomy is also similar to that of isolated nuclei (5) . Therefore, it seems probable that the membrane fraction is an important place in nuclei, where amino acid incorporation is carried out . It has been reported that amino acid incorporation by mitochondria is inhibited by chloxamphenicol but insensitive to the addition of RNase and cycloheximide (12-16) . The possibility that the contaminated mitochondrial fraction might be responsible for the incorporation of 14C-leucine by the nuclear membrane fraction could be ruled out from the following reasons . 1) The nuclei were treated with Triton X-100 for the removal of the outer nuclear membrane and cytoplasmic constituents (17) . The nuclei thus treated with Triton could incorporate 14C-leucine in the same degree as untreated nuclei . It suggests that the outer envelope and the cytoplasmic constituents are not responsible for the incorporation of amino acid by nuclei . 2) Electronmicroscopic investigation denied the contamination of mitochondria in the nuclear membrane fraction . 3) The activity of monoamine oxidase in the nuclear membrane fraction was almost negligible . 4) Furthermore the addition of mitochondrial fraction inhibited the incorporation of 1 ~C-leucine by the nuclear membrane fraction . References 1. 2. 3. 4. 5. 6. 7. 8.
H . Ono and H . Terayama, Biochim . Bio h s . Acta 166 175-185 (1968) . R . Rendi, Expt~ . Cell Research 19 489-498 1960 T . Y . Wang, Biochim .-~B3ïPfi~.~ Ys Acta 68 52-61 (1963) . P . H . Helmsing Bioch ophys . Acta 232 733-735 (1971) . H . Ono and F . T aku, J . Biochim . 72 1567-1569 (1972) . R . Berezney, L . K . Funk, ~ .L F . Crane, Biochim . Biophys . Acta _203 531-546 (1970) . G . M . Higgins and R . M . Anderson, Arch . Pathol . 12 186-202 (1931) . F . Sanger, Biochim . J . 39 507-515 1945) .
Vol . 18, No . 2 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 .
Amino Acid Incorporation by Nuclear Membrane
221
C . M . McEwen, Jr ., Methods in Enz lo , vol . 17B, p . 692-698 Academic Press, New Yor 197 1 0 . H . Lowry, N . J . Rosebrough, A . L . Farr, and R . Randall, _J . Biol . Chem . 193 265-275 (1951) . .Berezney and D . S . Coffey, Biochem . Biophys . Res . Commun . 60 1410-1417 R (1974) . A . M . Kroon, C . Saccone, and M . J . Botman, Biochim . Biophys . Acta _142 552-554 (1967) . D . S . Beattie, R . E . Basford, and S . B . Korits, Biochemistry _6 3099-3106 (1967) . M . Ashwell and T . S . Work, Biochem . Biophys . Res . Commtm . 3 2 1006-1011 (1968) . L . I . Malkin, Proc . Natl . Acad . Sci . US . 6 7 1695-1702 (1970) . D . Halder, Biochem . whys . ResCommon . . 40 129-134 (1970) . G . Blobel and. Potter, S~ence 154 1662-1665 (1966) .
Life Sciences Vol . 18, pp .215-222 Printed is the U .S .A .
AMINO ACID INCORPORATION BY NUCLEAR MEMBRANE FRACTION OF RAT LIVER r r* ** H . Ono , T . Ono and 0 . 1Vada `Department of Physiology, Faculty of Medicine, University of Tokyo ** Department of Hygiene and Preventive Medicine, Faculty of Medicine, University of Tokyo (Received is final form December 30, 1975) Summary Nuclear membrane fraction of rat liver is able to incorporate 14C_leucine into its proteins in vitro . The incorporation of 1 4 Cleucine into the nuclear membrane-motion was almost completely inhibited by chloramphenicol, but the inhibition by cycloheximide and puromycin was not so remarkable . RNase and DNase were not effective . The incorporation was also inhibited by several reagents known to interfere with energy metabolism . These characteristics of the incorporation of 14 C-leucine by the nuclear membrane fraction are quite similar to those of the incorporation by nuclei isolated from rat liver and mitochondrial fraction, but seem to be different from those of the ordinary protein synthetic system in microsomal fraction . 14 C-Leucine was preferentially incorporated into intrapolypeptide or C-terminal residues but not into N-terminal residues . Acrylamide gel electrophoresis showed that three protein species were mainly labelled . The incorporating activity of the nuclear membrane fraction obtained from regenerating liver 17 h after partial hepatectomy showed 220 $ of the control . The possibility that the contaminated mitochondrial fraction might be responsible for the incorporation of 14 C-leucine by the nuclear membrane fraction was ruled out . In the preceding paper, we have reported that rat liver nuclei can incorporate 14 C-leucine into nuclear proteins in vitro (1) . The incorporation of 1 4C-leucine into nuclear proteins was marc~ed~nhibited by chloramphenicol but not by puromycin, cycloheximide and RNase . It was found that 14 C-leucine was mainly incorporated into nuclear residual fraction remained after successive extraction of nuclei with 50 mM Tris-HC1 buffer (pH 7 .4) and 1 M NaCl . It has also been reported that the nuclear residual fraction is more highly labeled by radioactive amino acid in vitro (2-4) . The incorporation of 14C-leucine by nuclei isolated from regenerating liver was found to increase as compared with control and show a maximum activity 17 h after partial hepatectomy . Moreover, it was demonstrated that the increased incorporation by nuclei from regenerating liver was mainly due to increased incorporation into the nuclear residual fraction but not into other nuclear proteins (5) . In studying about the nuclear residual fraction, we found that nuclear membrane fraction from rat liver could also incorporate 14C-leucine into its proteins . In this paper we report some characteristics of the incorporation of 14 C-leucine by nuclear membrane fraction . 215
216
Amino Acid Incorporation by Nuclear Membrane
Vol. 18, No . 2
Methods Pr aration of Nuclear Membrane Fraction---Male Wistar rats weighing 150250 g were used . The proc ure or solating nuclei from rat liver was as described in the preceding paper (1) . Nuclear pellet was suspended in 0 .25 $ Triton X-100 in 0 .25 M sucrose-50 mM Tris-HC1 buffer (pH 7 .4)-3 mM MgC1 2 -20 mM KC1 (Buffer A) and centrifuged immediately . Nuclear membrane fraction was prepared by the method of Berezney, Funk and Crane (6) with a slight modification . The Triton treated nuclear pellet was washed twice with Buffer A and digested with DNase ~5 Pg/mg nuclear protein) and RNase (2 pg/mg nuclear protein) in Buffer A at 0 C overnight . The nuclease treated nuclear suspension was centrifuged at 20000 G for 15 min and pellet was suspenden in 1 M NaCl, followed by centrifugation at 20000 G for 15 min. This procedure was repeated twice more and the membranous pellet was washed with Buffer A and finally suspended in 0 .25 M sucrose-3toM MgC12-20 mM KC1 . The amount of protein of this nuclear membrane fraction was about 1 $ of the total nuclear proteins . Pre aration of Mitochondria---The liver homogenate was centrifuged at 1000 G for 10 n to precipitate nuclear fraction . Mitochondrial fraction was prepared by centrifuging the supernatant recovered from 1000 G run, at 20000 G for 20 min . Mitochondrial pellet was washed with Buffer A and 1 M NaCl successively according to the method for preparing the nuclear membrane fraction . Partial H atecto anesthesia as ascribe
---Rats were partially hepatectomized under ether by Higgins and Anderson (7) .
Incubation Conditions---The reaction mixture contained 20 mM sodü~ phosphate bu er (pH 6 .0), 0.1 M sucrose, 1 mM MgC12, 8 mM KC1, 0.1 pCi of 14C-leucine (312 mCi/mmole, The Radiochemical Centre, Amersham, England), and membrane fraction equivalent to 100-300 pg protein in a final volume of 0 .5 ml, was incubated at 37 C for 60 min. The reaction was terminated by addition of cold 5 $ trichloroacetic acid containing 0.25 $ Na2W04 . The acid-insoluble precipitates were collected on glass fiber discs by filtration and washed with 5 $ trichloroacetic acid, ethanol-ether mixture (3 :1, v/v) and ether, successively . Dried filter discs were put into toluene-PPO-POPOP scintillator and counted in a liquid scintillation counter . Determination of 14C-Leucine Inco orated in N-Terminals---The distribution o incorporate C-leucine among N-terminal or ntrapolypeptide residues of the nuclear membranous protein was determined by the dinitro phenylation method of Sangar (8) . The nuclear membrane fraction after incubating with 14 C-leucine, was dinitrophenylated with 1-fluoro-2,4-dinitrobenzene and hydrolyzed in 6 N HC1 at 105°C for 12 h . Then dinitrophenylated The (DNP)-amino acids derived from N-terminals were extracted with ether. radioactivity of the ether extract and the aqueous phase, which contained free amino acids derived from intrapolypeptide and C-terminal residues, and a few ether-unextractable DNP-amino acids was counted .
Ac lamide Gel Electro horesis---The nuclear membrane fraction, incubated in the presence o C-leucine was washed with cold Buffer A and then solubilized with 8 M urea~50 mM NaHC03-Na2C03 (pH 9 .5) . The gel contained 7 .5 $ acrylamide, 0 .2 $ N,N -methylenebisacrylamide, 0.15 M Tris-HC1 buffer (pH 9 .5) and 8 M deionized urea . The urea extract contained 300-500 Kg protein was put on the gel and electrophoresed in 0.05 M Tris-glycine buffer (pH 8 .6) at 3mA/ tube for 45 min. The gel was frozen on dry ice and cut into 1 mm thick slices . The gel slices xere then solubilized in 0 .2 ml of 30 $ H202 at 60 °C for 4 h and dryad . The residues were solubilized in 0.3 ml of hyamine solution and counted in a toluene-PPO-POPOP scintillation liquid .
Amino Acid Incorporation by Nuclear Membrane
Vol . 18, No . 2
21 7
Enz e Assa ---Monoamine oxidase activity was assayed by the procedure of McEwen, Jr . 9 . Protein was assayed by the method of Lowry et al . (10) . Results Characteristics of Amino Acid Inco ration b the Nuclear Membrane Fraction--- e nuclear membrane fraction incorporate C-leucine almost 1l n~ëarly during 2 h incubation . Optimal H of the incorporation was found between 6 .0 and 6 .5 . The rate at which 1 ~C-leucine was incorporated into protein was proportional to the added amount of the membrane fraction up to 400 pg protein . TABLE I Effect of Various Substances on the Incorporation of 14 C- Leucine by Nuclear Membrane Fraction in vitro Exptl . No .
Additions
Incorporation of 14 C- Leucine cpm/mg protein
$ activity
I
Control Puromycin, 100 pg/ml Cycloheximide, 100 pg/ml Chloramphenicol, 100 pg/ml Lipase, 100 pg/ml Creatine phosphate, 10 mM + Creatine kinase, 100 pg/ml
14730 10900 13130 490 2430 12650
100 74 89 3 17 86
II
Control ATP, 0 .5 mM GTP, 0 .5 mM
14100 7650 13400
100 54 95
12350 5620 2140 8100 2550 720
100 45 17 66 21 6
III
Control Amino acid mixture, 2 pM Trypsin, 5 pg/ml Sodium deoxycholate, 0 .05 $ Sodium dodecyl sulfate, 0 .05 $ - Preheating, 70 ° C for 10 min
Table I gives the characteristics of the incorporation of 14C-leucine into the nuclear membrane fraction . Non-specific adsorption of 14 C-leucine onto the nuclear membrane fraction was negligible (100 cpm/mg protein) . The incorporation of 14C-leucine was strongly inhibited by chloramphenicol at the concentration of 100 pg/ml, but only slightly by puromycin and cycloheximide at same concentration . Addition of ATP, ATP-generating system and GfP was not necessary for the incorporating system of the nuclear membrane fraction . (hn the contrary, presence of the first two substances inhibited the reaction . The incorporation of 14 C-leucine proceeded without supply of amino acid mixture, suggesting that amino acid pool was still present in the nuclear membrane fraction which might be sufficient to support protein synthesis during 2 h incubation period . To see if the incorporating activity could be enhanced by addition of exogenous amino acids, 2 EiM of each amino acid mixture minus leucine was added to the incubation medium . The incorporation of 14C-leucine was inhibited to 45 $ of control by addition of amino acid mixture . Whether this inhibition is caused by competition among 14 C-leucine and the different amino acids or unknown factors) is concerned, remains unexplained . Trypsin had a strong inhibitory effect on the incorporating system and this incorporating system was thereto-labile . The membrane fraction preheated at 70 ° C for
by the min dependent been incorporation asControl Mthe assaying sodium III of nuclear lost deoxycholate effect NaCl membrane nuclei shown sodium in structures No contaminated treated fraction 14C-leucine, treated acid the 2mM 20 The the 2of Treated fraction fluoride mM These mM of in membrane cyanide, membrane the was Monoamine Additions activity and specific Table with yeast Acid ract was nuclei were 0Contamination---In activity inhibited dehydroacetic facts of 14C-Leucine acid, mitochondria acid, mitochondrial 1showed expressed Incorporation Metabolic $the RNA II, on Mnecessary fraction sodium fraction, activity of 1Oxidase completely m1seem NaCl 2mM aand extent mM ght of the mM amarked the to monoamine calf azide less in be NaCl-treated bywas as Inhibitors in on of the incorporation, for acid suggest of Nuclear contaminated 100 thymus inhibitory pronounced fraction Subcellular h/m~ by which mitochondrial the 5and Lipase, the order same benzaldehyde/ $also Nuclear III oxidase 2,4-dinitrophenol II enzyme ~rotein incorporation that of Membrane manner The DNA the to showed on besodium mitochondrial Monoamine know enzyme effect incorporation the (native Membrane by responsible Practions inprotein suggesting asmitochondrial contamination strong energy Incorporation Fraction the preparing $dodecyl by activity onof result -and possibility respiratory the There yieldin of inhibitory heat was fraction that for activity of Rat sulfate incorporation was observed the of was the denatured) 1~C-leucine intact was fraction Liver shown the nuclear system nodeterminthat incorpoand and inhibitors effects, 18, Triton stimumemin that the No Monoand which isof on 2
218
Amino
10 sodium branous latory the
Vol . . . .
As such iodoacetic while 14C_leucine . present is .
.
TABLE
Effect of Exptl . I
II
.
Incorporation cpm/mg
.
l4C-Leucine $
KCN, NaN3, 2,4-Dinitrophenol,
11940 530 2130 70
100 5 18 1
Dehydroacetic Monoiodoacetic NaF,-
10550 3340 80 6440
100 32 1 - 61
Mitochondrial nuclear that ration ed Table . had membrane treated of Activity Fractions Homogenate Mitochondria 1 Nuclei Triton Membrane
-
.
The
.
.4
. TABLE Activity nmole 3 354 298 525 10 1 .7 27
Oxidase activity 67 .4 56 .8 100 1 .9 0 .4 5 .1
Vol . 18, No . 2
Amino Acid Incorporation by Nuclear Membrane
21 9
Addition of mitochondrial fraction to the incubation medium inhibited the incorporation of 14 C-leucine . The incorporation of 14 C-leucine by mitochondrial fraction itself under the same conditions for the nuclear membrane fraction was 3 $ as compared with the incorporating activity of the nuclear membrane fraction . These facts suggest that the incorporating activity of the nuclear membrane fraction is not ascribed to the contaminated mitochondria . Partial Characterization of the Labeled Product---In order to elucidate the distribution o incorporated -leucine among N-terminal and intrapolypeptide residues of the nuclear membranous protein, N-terminal analysis was carried out . As shown in Table IV, the incorporated 14C-leucine was not located in N-terminal residues and most of the radioactivity seemed to incorporate into intrapolypeptide or C-terminal residues of the membranous protein . The degree of dinitrophenylation as measured by a ratio of £-DNP-lysine to DNP-lysine plus free-lysine, was about 90 $. TABLE IV Distribution of Incorporated 14 C- Leucine between N-Terminals and Intrapolypeptides of the Nuclear Membrane Fraction 14 C_ Leucine Incorporation cpm
Fractions Ether extracts (N-terminal DNP-amino acids) Aqueous phase (free amino acids from intrapolypeptides or C-terminals, and ether unextractable DNP-amino acids)
18 865
+~ u m ao
1001
N N .U .a
m
100
NO
H
50
I
IY
I
r
I ~
1
~ I 1 (-)
I 1
I
III~~
I
,~~ 1 2 3 4 (+) distance from origin (cm) Fig . 1
a
d
U
~
50
I II ,
II ;
8 16 24 time after partial hepatectomy (h) Fig . 2
Fig. 1 Acrylamide gel electrophoresis of the nuclear membrane fraction . Anode and the direction of movement were to the right .
Fig . 2 Incorporation of 14 C-leucine by the nuclear membrane fraction from regenerating liver as a function of time after partial hepatectomy . Closed circles are average of two values for one experiment group and bars indicate average of two experiments . One group consists of S animals .
.
220
Amino Acid Incorporation by Nuclear Membrane
Vol . 18, No . 2
Next, polyacrylamide gel electrophoresis was employed to analyze the 14 C_leucine-incorporated membranous protein . It was obvious in Fig . 1 that 14 C_leucine was incorporated into three major protein species . Using SDS polyacrylamide gel electrophoresis, molecular weight of these protein species were determined . The molecular weights of them were about 50000, 70000 and 75000 . Berezney and Coffey have reported that the molecular weights of the nuclear protein matrix of rat liver are 62000, 66000 and 69000 (11) . 14C_Leucine Inco ratin Activit after Partial H atecto ---Fig . 2 shows t e results on the incorporat ono C-leucine nto proteins of the nuclear membrane fraction obtained from regenerating rat liver . Values for regenerating liver are expressed as percentages of those for normal liver . Incorporation of 14C-leucine 4 h after partial hepatectomy increased slightly and reached a maximum about 17 h after the operation . After 21 h, the incorporating activity showed a decrease and returned to the control level 24 h after the operation . Discussion The characteristics of the incorporation of 14 C-leucine by the nuclear membrane fraction of rat liver are quite similar to those of the incorporation of amino acids by nuclei isolated from rat liver (1,2) . In both cases the o timal pH of the incorporation is slightly acidic . The incorporation of l~C-leucine is strongly inhibited by chloramphenicol, while RNase, DNase, cycloheximide and puromycin show almost no remarkable effect on the incorporation . The change of the incorporating activity of the nuclear membrane fraction as a function of time after partial hepatectomy is also similar to that of isolated nuclei (5) . Therefore, it seems probable that the membrane fraction is an important place in nuclei, where amino acid incorporation is carried out . It has been reported that amino acid incorporation by mitochondria is inhibited by chloxamphenicol but insensitive to the addition of RNase and cycloheximide (12-16) . The possibility that the contaminated mitochondrial fraction might be responsible for the incorporation of 14C-leucine by the nuclear membrane fraction could be ruled out from the following reasons . 1) The nuclei were treated with Triton X-100 for the removal of the outer nuclear membrane and cytoplasmic constituents (17) . The nuclei thus treated with Triton could incorporate 14C-leucine in the same degree as untreated nuclei . It suggests that the outer envelope and the cytoplasmic constituents are not responsible for the incorporation of amino acid by nuclei . 2) Electronmicroscopic investigation denied the contamination of mitochondria in the nuclear membrane fraction . 3) The activity of monoamine oxidase in the nuclear membrane fraction was almost negligible . 4) Furthermore the addition of mitochondrial fraction inhibited the incorporation of 1 ~C-leucine by the nuclear membrane fraction . References 1. 2. 3. 4. 5. 6. 7. 8.
H . Ono and H . Terayama, Biochim . Bio h s . Acta 166 175-185 (1968) . R . Rendi, Expt~ . Cell Research 19 489-498 1960 T . Y . Wang, Biochim .-~B3ïPfi~.~ Ys Acta 68 52-61 (1963) . P . H . Helmsing Bioch ophys . Acta 232 733-735 (1971) . H . Ono and F . T aku, J . Biochim . 72 1567-1569 (1972) . R . Berezney, L . K . Funk, ~ .L F . Crane, Biochim . Biophys . Acta _203 531-546 (1970) . G . M . Higgins and R . M . Anderson, Arch . Pathol . 12 186-202 (1931) . F . Sanger, Biochim . J . 39 507-515 1945) .
Vol . 18, No . 2 9. 10 . 11 . 12 . 13 . 14 . 15 . 16 . 17 .
Amino Acid Incorporation by Nuclear Membrane
221
C . M . McEwen, Jr ., Methods in Enz lo , vol . 17B, p . 692-698 Academic Press, New Yor 197 1 0 . H . Lowry, N . J . Rosebrough, A . L . Farr, and R . Randall, _J . Biol . Chem . 193 265-275 (1951) . .Berezney and D . S . Coffey, Biochem . Biophys . Res . Commun . 60 1410-1417 R (1974) . A . M . Kroon, C . Saccone, and M . J . Botman, Biochim . Biophys . Acta _142 552-554 (1967) . D . S . Beattie, R . E . Basford, and S . B . Korits, Biochemistry _6 3099-3106 (1967) . M . Ashwell and T . S . Work, Biochem . Biophys . Res . Commtm . 3 2 1006-1011 (1968) . L . I . Malkin, Proc . Natl . Acad . Sci . US . 6 7 1695-1702 (1970) . D . Halder, Biochem . whys . ResCommon . . 40 129-134 (1970) . G . Blobel and. Potter, S~ence 154 1662-1665 (1966) .
