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The respiratory activity of isolated mammalian nuclei. II. The NADH oxidase of rat liver nucleoli
Lüe Sciences Vol. S, pp . 1459-1464, 1964. Pergamon Press, Inc . Printed in the United ßta.tes.
~S RESPIRATaRY ACTMTY OF ISCB.A~D MAM
(Received 25 August 1964; in final form 2 October 1964) We have found that liver nuclei isolated in dense sucrose catalyze an aerobic phosphor~latSonl~ 2 which is stimulated by e~oogenous cytochrome c . 3 In seeking to understand this process, we have found that such nuclei exhibit NADH-neotetrazolium reduotaae, and NAÛi oxidaee in the presence of exogeaous oytoohrome c~; but they are incapable of oxidising aucoinate .
mere is ample
enzymiol ~ 2 and electron microsoopio4 evidence that mitochondria play no part in these activities .
Such reau0.ts verity and extend previous reporta 5 ~ 6 under
unequivocal oonditiona .
Since nuclei isolated in dense sucrose provide such
an ideal syatern from a purity standpoint, we have used them to extend our knowledge of nuclear electron transport .
In such studies xe have found that
isolated muoleoli contain much or all of the original nuclear NADH oxidaee, and that nuclei, and nucleoli, contain only very law levels of heawprotein enzymes which could be involved in the observed respiratory activity. Nuclei were isolated tram livers of mature . male 3prague_Da~ley rats by described prooednrea . 2
Nucleoli were isolated by the procedure of Husah,
NADH et a1 . 7 oxidaae was measured with a Clark electrode (Yellox Springs Inatruaent Co ., Sallow 9pringa, Ohio), employing a f~ o~graph for a constant electrode potential and recording ayataa .~ _NAtB and oytoohraee o were obtained from Sigma fhenical Co ., St. Lovia, lio . ; the oytochrame o exhibited no detectable antoxidation xith excess aaoorbate .
Nucleic acids were
dstexminsd by the procedure of 1leanev sad Markov, 8 and protein by biurst. 2 " 9
1459
NADH OICZDASE OF RAT LIVER NUCLEOLI
1460
Vol . S, No . 12
Cytochrames in the nuclei or nucleoli were determined by ultrasensitiv loa: temperature
spectroscopy
using a Cart' model lI coupled with an on..line oar.
puter.l0 Thus far, fifteen rniolei preparations have s~hrnm NAD[i oaidase responsive to
cytochrome c; from six of these preparations nucleoli hav been isolated and
found similarly active .
Table I ahowa the data of four experiments deemed TA~E I
Conditions:
1 ml . of nuclear, or micleolar, suapeasion ; 450 yanolea sucrose ; uncle cytochrome c; SOOYPES* (in the presence of nucleoli) ; and 1.1 Poole ~ -PADS in final wlume of 5 .2 ml . Incubation temperature, 30oC. *Polyethyleneaulfonate (av. mol. wt. 12g00), kindly pa~ovided by the Upjohn Co., Kalamasoo, Mich. 180 ~amolea Trls, pä 6 .9 ; 36 pmolea Hgß2 ; 0 .2
~ayme
Addition
1
-
_
n ~-
-
~atams e0/hour/mg . protein Nuclei
403
418
9'15
554
Nucleoli
73~+
569
878
644
Nucleoli
0 .2 mM NaCN
0
Nucleoli
0 .4
mM Chlorpromasine
Nucleoli
0 .6
mM Atebrin
0 685
mg,
protein/al.
Nuclei
9 .7
11.8
6 .0
8 .7
Nucleoli
2 .$
4.9
3 .0
2 .8
satisfactory in that no mic7.eolar plumping or oontaminatdon was observed siicrosoopically.
In each case nuclei and nucleoli hav demonstrable NADß osidase ;
snah prsparatioaa ahoR no aucdnats oxidation ± oytochrane c. nucleoli
there
is
considerable loss
always a loss of~original nuclear activity of DNA
(58-88;6)
and protein
(Sn-71~)t
In isolatAng (~634~)
and a
but, with one eocoeption,
isolated nnaleoli hav higher specific activities than the nuclei from ~enoe
Vol. 3, No. 12 they came .
NADH OXIDASE OF RAT LIVER NUCLEOLI
1461
Hoes et a1 . 6 have found that, semen nuclei are fraotioaated by so na-
tion ~td centrifugation, electron transport activity resides in oertai.n lipidrich aubnuclear fractions thought to be originally associated With the nucleoli .
We have tried, Without success, to fractionate nuclei bY this procedure . 6
As xith nuclei, 4 nuoleolar NADS o~ddase is quite sensitive to NaCN and ahlorpra~aasine but it is unaffected by Antimycin A .
Nnoleoli e~ibit no NADFi osir
dace in the absence of e~oogenous aßrtochrome c ; they are stimulated to a degree by polyethylene sulfonate (PES) prior to DN9aae treatment .
àfter DNAase,
nucleoli e~ibit no activity without added PES, presumably to overcame inhibition by released histonea .
Despite these evidences that DNAase affects the
nucleoli, nucleolar-associated ahramatin remains in our preparations . RNA/DNA ratios of the starting nuclei (0 .10 -
0 .20)
are raised to only
bus,
0 .53 -
0 .86 in the final nucleoli preparations . In extending these findings, we have used law temperature spectroscopy to verifp purity and to assess the presence of arty hemoproteins which could be involved in electron transport ; to date, six nnalei preparations active in phosphory~ation and/or NADH oxidase have been ao examined .
Such experiments
shoo consistently that liver nuclei contain small amounts of cytochromea e and occasional]y band traces of
but no a + a3 .
Fig . 1 presents a representa-
and
oc l
peaks of cytochraae c can be seen at
mp as yell as shoulders at
554
and
tive speotr~na in Which the of
545-549
cl,
2
cytochromea of and b, roapectively. a + a3 have been found.
562 mu
denoting small amounts of
No distinct peaks for oytochromes b 5 or
In only one preparation has a Weak shoulder for a + a3
been observed in the reduced spectran in the 440 my~ region .
Preliminary eapsrS.-
nents With nucleoli indicate that their isolation removes the bulk of the original nuclear oytoohranea ; however, xith
75
a persistent but i++~pfi+r~ ble absorption in the
scaua it is possible to observe
550
causes for this absorption are under investigation .
mK and Soret regions .
~e
Recent data of Couover and
$iebert~ ro ~everal points of this report with both aqueous and nonaqueoua liver nuclei ; the presence in nuclei of oyto~xrome c and the absence of cyto-
NADH OXiDASE OF RAT LIVER NUCLEOLI
1462
475
48S
SOS
495
SI5
S2S
M~
S3S
S4S
Vol . 3, No. 12
55S
S65
575
Hedn:ced llbsorption Spectra of a Nuclei Preparation . ~e spectra were recorded from ice plates of the rnxolsi suspended in M/4 aucroaet5 mM triethaTiolamine, PH 7 .0s3 mM MgC12, With dithionite added: 4.7 mg. protein/nl. ~e plates Were 2 mss . thick and supported in an alumimms spacer itmaerssd in liquid nitrogen at 77o K . ~e C~ry Model 11 apectrop~hotometer ~+aa operated at dynode 1 With 0 .04 mm, alit Width at 550 mu to record the tracing ahotm in the insert. ~e main tracing Was made by repeating the scan txenty five times and su4mdng the scans in an on-line computer. Bath spectra are apparent absolute spectra ; and the absorbante scale is sho~m as measured . lthae not been corrected for inareaaed light path due to multiple reYletnon . ohramea a + a3; and a measurable capacity for NAÛi oxidation. It seems anomalous that uualei should oo~ntain oytochratne a and yet respond to emgenons aytochrome c in NADEi oxidation.
However, nuclei do bind cytochrome
off , and it is possible that sates or all of the endogenous cytochraae t of nuclei is bound in nonflmcnonal state.
Zhe fart that nucleoli contain little or no
dswnatrabls cytoohrate c, and a tomplste depe~anos on it for NAÛi ozS.dation, is consistent With this.
Althon~t our stork to date ahaxa no apparent relation-
ship betttesn nuclear pbosphorylat.ionl^'3 and nuclear,4 or nucleolar, NADS osidass, the annulanon of both activities by cyytochrome c is most interesting .
Vol . 3, No. 12
NADH OXIDASE OF RAT LIVER NUCLEOLI
1483
We feel the clues to an utderatanding of this pussle lie in solving the relationships between the observations of these studies and the capability for interaction displayed by mitodzandria and trudei or nudeoli . l~15 Thin Work Was supported in part by grants from the National Soisnce Foundation (a23g57) and the National Institutes of Health (H$-06088 ; R~-062Jî1 ; 1-GS-95) .
W.D .C . Eras supported in part by $II-(~Ja04, and W .H .$ . is tire recip
ient of a Research Career Program Award
(5-g~C$4 15514) .
The authors wish to
thank Drs . T. E . Conover and (3r. Siebart for snaking their paper available prior to publicat3.on ; and Dr . (~ . F . Doebbler of the Linda Co . for a generous supply of liquid nitrogen .
This Work will be .sukmitted by W.D .C . in partial fu1fi11-
ment of requirements for the Ph .D . degree of this institution . References
Z,
1.
Pent>i .oll, R ., Liu, S . H. and 3annders, J . P ., Bioahia . Hiophya . Acta, 170 (1963) .
2.
Perutia]l, R., Saundera, J . P . atd Liu, S . M . ; Panzdai? , R . atd 3auadera, J . P . , Bioohanietry, ia put~sas .
3.
Pension, R., anbmittsd to Noturs .
4.
Pentdall, R., Cnrrie, w . D., MaComnell, N . R . aad Bibb, ü . R., aalaitted to Biochem . Biophya . Ree . Catom .
5.
Rees, K. R. and Rowl.atd, Q . F ., Hiochem. J., ~, 89 (1961) .
6.
R~ees, B . R ., Rutund, a. F . atd Varcoe, J . 9 ., Biochem . J ., 86, l30 (1963) .
7"
Husch . H ., Fotostau, M., ~d~ea, H ., 3tesls, W . J ., Liau, M. C . and 9oetana, H ., E~ . Cell Res ., 9ufPl ., Q, l50 (1963) "
8.
Taanev, R. atd Harkov, ß. C~., Hloohim . Biophya . Aota, 4~2, ~ (1960) .
9.
Oornall, A . Q ., Bsrdawill, C . S . and David, M. M ., J . Biol . Chea ., ]~, 75l (1949) .
lo .
E111ott, W . B ., Hayford, R . B, and Tonaki, W., Biochim . Biopitya . Acta, in press .
11 .
Conover, T . E . and 9iebert, (3 ., autanitted to Hiocinin . BLophys . Acts, and unpublished data .
12 .
Heinsrt, H ., J . Hiol . Cbem ., ~0, 287 (1951) "
13 .
Potter, V. R ., Lyle, G. a. atd Sahneider, w. C., J . Biol . Gàoa ., ~0, 293 (1951) .
1484
NADH OXIDASE OF RAT LIVER NUCLEOLI
Vol . 3, No. 12
lei .
Johnson, R . H. and Ackermann, W. W., J . ß1o1 . mem., 200 . 263 (1953) "
15.
Moses, M. J., Cytolo® and Os11 Physiology ((3. H. Hourne, Fd.) pp" x-559, Aaademio Press, N. Y. (1964) "
~S RESPIRATaRY ACTMTY OF ISCB.A~D MAM
(Received 25 August 1964; in final form 2 October 1964) We have found that liver nuclei isolated in dense sucrose catalyze an aerobic phosphor~latSonl~ 2 which is stimulated by e~oogenous cytochrome c . 3 In seeking to understand this process, we have found that such nuclei exhibit NADH-neotetrazolium reduotaae, and NAÛi oxidaee in the presence of exogeaous oytoohrome c~; but they are incapable of oxidising aucoinate .
mere is ample
enzymiol ~ 2 and electron microsoopio4 evidence that mitochondria play no part in these activities .
Such reau0.ts verity and extend previous reporta 5 ~ 6 under
unequivocal oonditiona .
Since nuclei isolated in dense sucrose provide such
an ideal syatern from a purity standpoint, we have used them to extend our knowledge of nuclear electron transport .
In such studies xe have found that
isolated muoleoli contain much or all of the original nuclear NADH oxidaee, and that nuclei, and nucleoli, contain only very law levels of heawprotein enzymes which could be involved in the observed respiratory activity. Nuclei were isolated tram livers of mature . male 3prague_Da~ley rats by described prooednrea . 2
Nucleoli were isolated by the procedure of Husah,
NADH et a1 . 7 oxidaae was measured with a Clark electrode (Yellox Springs Inatruaent Co ., Sallow 9pringa, Ohio), employing a f~ o~graph for a constant electrode potential and recording ayataa .~ _NAtB and oytoohraee o were obtained from Sigma fhenical Co ., St. Lovia, lio . ; the oytochrame o exhibited no detectable antoxidation xith excess aaoorbate .
Nucleic acids were
dstexminsd by the procedure of 1leanev sad Markov, 8 and protein by biurst. 2 " 9
1459
NADH OICZDASE OF RAT LIVER NUCLEOLI
1460
Vol . S, No . 12
Cytochrames in the nuclei or nucleoli were determined by ultrasensitiv loa: temperature
spectroscopy
using a Cart' model lI coupled with an on..line oar.
puter.l0 Thus far, fifteen rniolei preparations have s~hrnm NAD[i oaidase responsive to
cytochrome c; from six of these preparations nucleoli hav been isolated and
found similarly active .
Table I ahowa the data of four experiments deemed TA~E I
Conditions:
1 ml . of nuclear, or micleolar, suapeasion ; 450 yanolea sucrose ; uncle cytochrome c; SOOYPES* (in the presence of nucleoli) ; and 1.1 Poole ~ -PADS in final wlume of 5 .2 ml . Incubation temperature, 30oC. *Polyethyleneaulfonate (av. mol. wt. 12g00), kindly pa~ovided by the Upjohn Co., Kalamasoo, Mich. 180 ~amolea Trls, pä 6 .9 ; 36 pmolea Hgß2 ; 0 .2
~ayme
Addition
1
-
_
n ~-
-
~atams e0/hour/mg . protein Nuclei
403
418
9'15
554
Nucleoli
73~+
569
878
644
Nucleoli
0 .2 mM NaCN
0
Nucleoli
0 .4
mM Chlorpromasine
Nucleoli
0 .6
mM Atebrin
0 685
mg,
protein/al.
Nuclei
9 .7
11.8
6 .0
8 .7
Nucleoli
2 .$
4.9
3 .0
2 .8
satisfactory in that no mic7.eolar plumping or oontaminatdon was observed siicrosoopically.
In each case nuclei and nucleoli hav demonstrable NADß osidase ;
snah prsparatioaa ahoR no aucdnats oxidation ± oytochrane c. nucleoli
there
is
considerable loss
always a loss of~original nuclear activity of DNA
(58-88;6)
and protein
(Sn-71~)t
In isolatAng (~634~)
and a
but, with one eocoeption,
isolated nnaleoli hav higher specific activities than the nuclei from ~enoe
Vol. 3, No. 12 they came .
NADH OXIDASE OF RAT LIVER NUCLEOLI
1461
Hoes et a1 . 6 have found that, semen nuclei are fraotioaated by so na-
tion ~td centrifugation, electron transport activity resides in oertai.n lipidrich aubnuclear fractions thought to be originally associated With the nucleoli .
We have tried, Without success, to fractionate nuclei bY this procedure . 6
As xith nuclei, 4 nuoleolar NADS o~ddase is quite sensitive to NaCN and ahlorpra~aasine but it is unaffected by Antimycin A .
Nnoleoli e~ibit no NADFi osir
dace in the absence of e~oogenous aßrtochrome c ; they are stimulated to a degree by polyethylene sulfonate (PES) prior to DN9aae treatment .
àfter DNAase,
nucleoli e~ibit no activity without added PES, presumably to overcame inhibition by released histonea .
Despite these evidences that DNAase affects the
nucleoli, nucleolar-associated ahramatin remains in our preparations . RNA/DNA ratios of the starting nuclei (0 .10 -
0 .20)
are raised to only
bus,
0 .53 -
0 .86 in the final nucleoli preparations . In extending these findings, we have used law temperature spectroscopy to verifp purity and to assess the presence of arty hemoproteins which could be involved in electron transport ; to date, six nnalei preparations active in phosphory~ation and/or NADH oxidase have been ao examined .
Such experiments
shoo consistently that liver nuclei contain small amounts of cytochromea e and occasional]y band traces of
but no a + a3 .
Fig . 1 presents a representa-
and
oc l
peaks of cytochraae c can be seen at
mp as yell as shoulders at
554
and
tive speotr~na in Which the of
545-549
cl,
2
cytochromea of and b, roapectively. a + a3 have been found.
562 mu
denoting small amounts of
No distinct peaks for oytochromes b 5 or
In only one preparation has a Weak shoulder for a + a3
been observed in the reduced spectran in the 440 my~ region .
Preliminary eapsrS.-
nents With nucleoli indicate that their isolation removes the bulk of the original nuclear oytoohranea ; however, xith
75
a persistent but i++~pfi+r~ ble absorption in the
scaua it is possible to observe
550
causes for this absorption are under investigation .
mK and Soret regions .
~e
Recent data of Couover and
$iebert~ ro ~everal points of this report with both aqueous and nonaqueoua liver nuclei ; the presence in nuclei of oyto~xrome c and the absence of cyto-
NADH OXiDASE OF RAT LIVER NUCLEOLI
1462
475
48S
SOS
495
SI5
S2S
M~
S3S
S4S
Vol . 3, No. 12
55S
S65
575
Hedn:ced llbsorption Spectra of a Nuclei Preparation . ~e spectra were recorded from ice plates of the rnxolsi suspended in M/4 aucroaet5 mM triethaTiolamine, PH 7 .0s3 mM MgC12, With dithionite added: 4.7 mg. protein/nl. ~e plates Were 2 mss . thick and supported in an alumimms spacer itmaerssd in liquid nitrogen at 77o K . ~e C~ry Model 11 apectrop~hotometer ~+aa operated at dynode 1 With 0 .04 mm, alit Width at 550 mu to record the tracing ahotm in the insert. ~e main tracing Was made by repeating the scan txenty five times and su4mdng the scans in an on-line computer. Bath spectra are apparent absolute spectra ; and the absorbante scale is sho~m as measured . lthae not been corrected for inareaaed light path due to multiple reYletnon . ohramea a + a3; and a measurable capacity for NAÛi oxidation. It seems anomalous that uualei should oo~ntain oytochratne a and yet respond to emgenons aytochrome c in NADEi oxidation.
However, nuclei do bind cytochrome
off , and it is possible that sates or all of the endogenous cytochraae t of nuclei is bound in nonflmcnonal state.
Zhe fart that nucleoli contain little or no
dswnatrabls cytoohrate c, and a tomplste depe~anos on it for NAÛi ozS.dation, is consistent With this.
Althon~t our stork to date ahaxa no apparent relation-
ship betttesn nuclear pbosphorylat.ionl^'3 and nuclear,4 or nucleolar, NADS osidass, the annulanon of both activities by cyytochrome c is most interesting .
Vol . 3, No. 12
NADH OXIDASE OF RAT LIVER NUCLEOLI
1483
We feel the clues to an utderatanding of this pussle lie in solving the relationships between the observations of these studies and the capability for interaction displayed by mitodzandria and trudei or nudeoli . l~15 Thin Work Was supported in part by grants from the National Soisnce Foundation (a23g57) and the National Institutes of Health (H$-06088 ; R~-062Jî1 ; 1-GS-95) .
W.D .C . Eras supported in part by $II-(~Ja04, and W .H .$ . is tire recip
ient of a Research Career Program Award
(5-g~C$4 15514) .
The authors wish to
thank Drs . T. E . Conover and (3r. Siebart for snaking their paper available prior to publicat3.on ; and Dr . (~ . F . Doebbler of the Linda Co . for a generous supply of liquid nitrogen .
This Work will be .sukmitted by W.D .C . in partial fu1fi11-
ment of requirements for the Ph .D . degree of this institution . References
Z,
1.
Pent>i .oll, R ., Liu, S . H. and 3annders, J . P ., Bioahia . Hiophya . Acta, 170 (1963) .
2.
Perutia]l, R., Saundera, J . P . atd Liu, S . M . ; Panzdai? , R . atd 3auadera, J . P . , Bioohanietry, ia put~sas .
3.
Pension, R., anbmittsd to Noturs .
4.
Pentdall, R., Cnrrie, w . D., MaComnell, N . R . aad Bibb, ü . R., aalaitted to Biochem . Biophya . Ree . Catom .
5.
Rees, K. R. and Rowl.atd, Q . F ., Hiochem. J., ~, 89 (1961) .
6.
R~ees, B . R ., Rutund, a. F . atd Varcoe, J . 9 ., Biochem . J ., 86, l30 (1963) .
7"
Husch . H ., Fotostau, M., ~d~ea, H ., 3tesls, W . J ., Liau, M. C . and 9oetana, H ., E~ . Cell Res ., 9ufPl ., Q, l50 (1963) "
8.
Taanev, R. atd Harkov, ß. C~., Hloohim . Biophya . Aota, 4~2, ~ (1960) .
9.
Oornall, A . Q ., Bsrdawill, C . S . and David, M. M ., J . Biol . Chea ., ]~, 75l (1949) .
lo .
E111ott, W . B ., Hayford, R . B, and Tonaki, W., Biochim . Biopitya . Acta, in press .
11 .
Conover, T . E . and 9iebert, (3 ., autanitted to Hiocinin . BLophys . Acts, and unpublished data .
12 .
Heinsrt, H ., J . Hiol . Cbem ., ~0, 287 (1951) "
13 .
Potter, V. R ., Lyle, G. a. atd Sahneider, w. C., J . Biol . Gàoa ., ~0, 293 (1951) .
1484
NADH OXIDASE OF RAT LIVER NUCLEOLI
Vol . 3, No. 12
lei .
Johnson, R . H. and Ackermann, W. W., J . ß1o1 . mem., 200 . 263 (1953) "
15.
Moses, M. J., Cytolo® and Os11 Physiology ((3. H. Hourne, Fd.) pp" x-559, Aaademio Press, N. Y. (1964) "