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The effects of histones on swelling and contraction of mitochondria
Life Sciences Vol. 4, pp . 1555-1560, 1985 . Pergamon Press Ltd. Great Britain.
Printed in
THE EFFECTS OF HISTONES ON SWELLING AND CONTRACTION OF MITOCHONDRIA Arnold Schwartz and Carl L . Johnson Department oß Pharmacology, Baylor University College oß Medicine Houston, Texas (Received 11 May 1965 ; in final form 9 June 1965) The results of experiments conducted recently in this laboratory suggest that histones and certain other basic compounds aßfect various membrane-bound systems .
For example, very small amounts
oß arginine-rich or lysine-rich histones obtained ßrom a variety oß sources including heart muscle, inhibit super-precipitation and ATPase activity oß actomyosin or myoßibrillar suspensíons l , markedly stimulate mitochondrial ATPase activity and oxygen consumption 2 and inhibit a ouabain-sensitive ATPase in heart3 .
The
present report is concerned with histone-mitochondrial membrane interactions, and is relevant to recent observations concerning 4 the possible presence oß a contractile system in mitochondria . Methods The preparation oß mitochondria from rat liver as well as the isolation and properties oß the histones ßrom rat liver, mor, heart muscle or calf thymus have been described2 ' S .
tu-
Com-
mercially-prepared histones (arginine and lysine-rich histones isolated from calf thymus) and spermine were purchased ßrom Worthington Biochemical Company, Freehold, New Jersey . Swelling (spontaneous or calcium-induced) and contraction oß mitochondria were studied employing the procedure oß Lehninger with some modißication oß the media .
Three types were used :
Medium A - 0 .25 M SC1, 0 .04 M tris (pH 7 .4) i Medium B = 0 .125 M 1555
6
1558
SWELLING & CONTRACTION OF MITOCHONDRIA
Vol. 4, No. 16
BC1, 0 .02 Y tris (pH 7 .4) ; Medium C - oxidative-phosphorylation cocktail" previously described7 . mAdium were qualitatively similar .
The results obtained with each Acetylation oß the histones
was carried out by the method oß Allßrey, Faulàner and Yiraky 8 . The experiments were usually carried out at a temperature oß 37°C . Results and Discussion The curves shown in Figure 1 demonstrate that very low concentrations oß histones prepared either ßrom rat liver or obtained commercially induced a marked swelling oß rat liver mitochondria .
Histones prepared ßrom other sources such as
.soo
I
0
7
q
Ib YO 26 TIYE lY1NUTE9)
SO
36
FIG. 1 Histone-Induced Swelling oß Rat Liver Mitochondria . Swelling was studied as described in the text, aßter the addition oß the ßollowing to a total volume oß 3 ml : 1 - control (0 .1 ml . H20) ; 2 - spermine, 0 .01, 0 .05, 0 .1 and 0 .2 mg . (separate experiments) ; 3 - lysine-rich histone (worthington), y00 4~g . ; 4 F'2a (rat liver), 4 fig " ; 5 - F2a (rat liver), 10 4~g " ; 6 Arginine-rich histone (worthington), 100 4~g " ; 7 - F2a (rat liver), 20 4+8 " Medium "A" was eaployed . llitochondrial protein = 1 .25 mg .
Vol. 4, No. 16
SWELLING & CONTRACTION OF MITOCHONDRIA
1557
llalker tumor, rat heart, rat skeletal muscle, erythrocytes, or guinea pig brain, showed similar effects although the F2a fraction Brom rat liver, was the most active preparation with respect to swelling .
This !rac
oon
was also most active in its eßßects
on mitochondria! oxidation, ATPase activity and exchangereactions2 .
In agreement with Tabor9spermine, a "stabilizing"
laait protein, even in relatively high concentrations did not Although not shown, small
swell the mitochondria (Figure 1) .
amounts oß poly Irlysine produced swelling but not as marked as It would appear ßrom
comparable concentrations oß the histones .
Figure 1 that the "arginine-rich" histone oß ~llorthington was a more active swelling agent than the "lysine-rich" histone ßrom the same source .
.soo .500 .400 a E O .300 N
i .200 O Ó
. 100 1 0
1
8
1
1
1
1
10 15 20 26 TIIAE f YIINUTES)
1
30
FIG . 2 BPßect of Acetylatioa on Histone-Induced Swelling oß Mitochondria . The conditions were the same as in Figure 1 except that !tedium "C" was used . The code is as ßollows : 1 - control ; 2 - acetylated F2a (rat liver), equivalent oß 20 4~g histone ; 3 e acetylated control F a (the histone carried through the process of acetylatiop with ~he omission oß acetic anhydride), 20 Fig ; 4 - F2a, untreated, 20 118 " Mitochondria! protein ~ 1 .34 mg .
1558
SWELLING & CONTRACTION OF MITOCHONDRIA
Vol. 4, No. 16
Acetylatioa of the histone (F~a) removed its property oß swelling mitochondria (Figure 2) .
It is of interest that an ace-
tylated or trypsin-treated histone also did not affect mito chondrial oxidation or ATPase activity2 .
These observations
suggest that both the peptide bonding and perhaps subtle structural configurations oß the molecule are necessary for histone-membrane interactions .
Similar conclusions were reached
by Allfrey, Faulkner and Husky$ concerning the inhibitory effects of histones on DNA-dependent RNA synthesis . In preliminary experiments whole gangliosides but not Nacetylneuraminic acid, and, also DNA blocked the swelling efßect of the histones . The results shown in Figure 3 indicate that histones may reverse or inhibit "contraction" oP mitochondria after swelling was induced by Ca .
These observations appear to be analagous to
those recently reported by Baltscheffsky and Baltscheffsky l0 concerning the "relaxing" properties of a soluble substance or factor on mitochondrial contraction, and are .also related to recent findings that histones may act as "relaxing substances" on actomyosin or myofibrillar suspensions ßrom heart or skeletal muscle l .
The histone appears to inhibit contraction oß mito-
chondria whether it is added to the system at the same time the "contracting mixture" (ATp, Hg, BSA) is added (Figure 3) or if it is added before or after contraction has commenced . The similarity of effects oP thehistoaes and certain other basic ca~mpounds on the "contractile-relaxation" cycle of liver mitochondria, as compared to muscle actomyosin systems l , supports the contention that the mitochondrion possesses a contractile . system 4 ' 10 Chile the site of binding of thehistoae to the mitochondrial
Vol. 4, No . 16
SWELLING & CONTRACTION OF NIITOCHONDRIA
1559
membrane (iß such a reaction in Pact does occur) is unknown, a number oß possible acidic compounds present in mitochondria are being considered, including neuraminic acid derivatives, phosphoproteins, phaspholipids and nucleotides .
Ia view oß recent
reports concerning the presence oß DNA in mitochondrial and 11,12 erythrocyte membranes the possibilit y oß a DNA-histone binding in mitochondria is attractive .
FIG . 3 Inhibition oß Contraction oß Mitochondria by Histone . The conditions were the same as in Figure 1 except that Medium "B" was used, and 1 mM CaCl2 (Final concentration) added at zero time, was the "swélling agent" . At the arrow, 1 - 3 mM MgCl2, 3 mM ATP and 2 mg/ml oß bovine serum albumin weré added, or 2 -éame as 1 plus 20 hg oß F2a (rat liver) . Mitochondrial protein - 1 .24 mg . Acknowledgement : This study was supported by grants ßrom the D9PHS (H8 0790803 and HS 05435-05) and Brom the Texas Heart Association (64G4N) . Dr . Schwartz is a recipient oß a Career Research Development Award (iJ3IyH9, HS 11,875-01),
Appreciation is extended to
1580
SWELLING & CONTRACTION OF MITOCHONDRIA
Vol. 4, No. 18
Dr . A . C . 3tarbuck and to Yr . C . A . Taylor Por their generous help in isolation oß the histones . References 1.
A . BCHwARTZ and A . H . LASSPER, Li ße Sciences 4, 145 (1965) .
2.
A . BCH~ARTZ, J . Biol . Chem ., 240, 939 ;944 (1965) .
3.
A . SCH~ARTZ, Biochim . Biophys . Acta , 100, 202 (1985) .
4.
T . OHNISHI and T . OHNISHI, J . Biochem . (Tok) 52, 230 (1962) .
5.
L . S . HNILICA and H . BUSCH, J . Biol . Chea ., 238, 918 (1963) .
6.
A . L. LEHNINGER, J . Biol . Chem ., 234, 2465 (1959) .
7.
A . SCHIIARTZ and K . S . LEE, Circulat ion Res ., 10, 321 (1962) .
B.
V . G . ALLFREY, R. FAULKNER and A . E . MIRSRY, Froc . Nat'1 Acad . Sci ., 51, 786 (1964) .
9.
C. lf . TABOR, Biochem . Biophys . Res . Coma ., 2, 117 (1960) .
10 .
H. BALTSCHEFFSKX and Y . BALTSCHEFFSKY, Biochem . Biophys . Res . Comm a, 17, 220 (1964) .
11 .
G. F . KALF, Biochemistry , 3,
12 .
L. PHILIDBON and 0 . ZSPTERQVIST, Biochim . Biophys . Acta , 91 171 (1964) . -
1702 (1964) .
Printed in
THE EFFECTS OF HISTONES ON SWELLING AND CONTRACTION OF MITOCHONDRIA Arnold Schwartz and Carl L . Johnson Department oß Pharmacology, Baylor University College oß Medicine Houston, Texas (Received 11 May 1965 ; in final form 9 June 1965) The results of experiments conducted recently in this laboratory suggest that histones and certain other basic compounds aßfect various membrane-bound systems .
For example, very small amounts
oß arginine-rich or lysine-rich histones obtained ßrom a variety oß sources including heart muscle, inhibit super-precipitation and ATPase activity oß actomyosin or myoßibrillar suspensíons l , markedly stimulate mitochondrial ATPase activity and oxygen consumption 2 and inhibit a ouabain-sensitive ATPase in heart3 .
The
present report is concerned with histone-mitochondrial membrane interactions, and is relevant to recent observations concerning 4 the possible presence oß a contractile system in mitochondria . Methods The preparation oß mitochondria from rat liver as well as the isolation and properties oß the histones ßrom rat liver, mor, heart muscle or calf thymus have been described2 ' S .
tu-
Com-
mercially-prepared histones (arginine and lysine-rich histones isolated from calf thymus) and spermine were purchased ßrom Worthington Biochemical Company, Freehold, New Jersey . Swelling (spontaneous or calcium-induced) and contraction oß mitochondria were studied employing the procedure oß Lehninger with some modißication oß the media .
Three types were used :
Medium A - 0 .25 M SC1, 0 .04 M tris (pH 7 .4) i Medium B = 0 .125 M 1555
6
1558
SWELLING & CONTRACTION OF MITOCHONDRIA
Vol. 4, No. 16
BC1, 0 .02 Y tris (pH 7 .4) ; Medium C - oxidative-phosphorylation cocktail" previously described7 . mAdium were qualitatively similar .
The results obtained with each Acetylation oß the histones
was carried out by the method oß Allßrey, Faulàner and Yiraky 8 . The experiments were usually carried out at a temperature oß 37°C . Results and Discussion The curves shown in Figure 1 demonstrate that very low concentrations oß histones prepared either ßrom rat liver or obtained commercially induced a marked swelling oß rat liver mitochondria .
Histones prepared ßrom other sources such as
.soo
I
0
7
q
Ib YO 26 TIYE lY1NUTE9)
SO
36
FIG. 1 Histone-Induced Swelling oß Rat Liver Mitochondria . Swelling was studied as described in the text, aßter the addition oß the ßollowing to a total volume oß 3 ml : 1 - control (0 .1 ml . H20) ; 2 - spermine, 0 .01, 0 .05, 0 .1 and 0 .2 mg . (separate experiments) ; 3 - lysine-rich histone (worthington), y00 4~g . ; 4 F'2a (rat liver), 4 fig " ; 5 - F2a (rat liver), 10 4~g " ; 6 Arginine-rich histone (worthington), 100 4~g " ; 7 - F2a (rat liver), 20 4+8 " Medium "A" was eaployed . llitochondrial protein = 1 .25 mg .
Vol. 4, No. 16
SWELLING & CONTRACTION OF MITOCHONDRIA
1557
llalker tumor, rat heart, rat skeletal muscle, erythrocytes, or guinea pig brain, showed similar effects although the F2a fraction Brom rat liver, was the most active preparation with respect to swelling .
This !rac
oon
was also most active in its eßßects
on mitochondria! oxidation, ATPase activity and exchangereactions2 .
In agreement with Tabor9spermine, a "stabilizing"
laait protein, even in relatively high concentrations did not Although not shown, small
swell the mitochondria (Figure 1) .
amounts oß poly Irlysine produced swelling but not as marked as It would appear ßrom
comparable concentrations oß the histones .
Figure 1 that the "arginine-rich" histone oß ~llorthington was a more active swelling agent than the "lysine-rich" histone ßrom the same source .
.soo .500 .400 a E O .300 N
i .200 O Ó
. 100 1 0
1
8
1
1
1
1
10 15 20 26 TIIAE f YIINUTES)
1
30
FIG . 2 BPßect of Acetylatioa on Histone-Induced Swelling oß Mitochondria . The conditions were the same as in Figure 1 except that !tedium "C" was used . The code is as ßollows : 1 - control ; 2 - acetylated F2a (rat liver), equivalent oß 20 4~g histone ; 3 e acetylated control F a (the histone carried through the process of acetylatiop with ~he omission oß acetic anhydride), 20 Fig ; 4 - F2a, untreated, 20 118 " Mitochondria! protein ~ 1 .34 mg .
1558
SWELLING & CONTRACTION OF MITOCHONDRIA
Vol. 4, No. 16
Acetylatioa of the histone (F~a) removed its property oß swelling mitochondria (Figure 2) .
It is of interest that an ace-
tylated or trypsin-treated histone also did not affect mito chondrial oxidation or ATPase activity2 .
These observations
suggest that both the peptide bonding and perhaps subtle structural configurations oß the molecule are necessary for histone-membrane interactions .
Similar conclusions were reached
by Allfrey, Faulkner and Husky$ concerning the inhibitory effects of histones on DNA-dependent RNA synthesis . In preliminary experiments whole gangliosides but not Nacetylneuraminic acid, and, also DNA blocked the swelling efßect of the histones . The results shown in Figure 3 indicate that histones may reverse or inhibit "contraction" oP mitochondria after swelling was induced by Ca .
These observations appear to be analagous to
those recently reported by Baltscheffsky and Baltscheffsky l0 concerning the "relaxing" properties of a soluble substance or factor on mitochondrial contraction, and are .also related to recent findings that histones may act as "relaxing substances" on actomyosin or myofibrillar suspensions ßrom heart or skeletal muscle l .
The histone appears to inhibit contraction oß mito-
chondria whether it is added to the system at the same time the "contracting mixture" (ATp, Hg, BSA) is added (Figure 3) or if it is added before or after contraction has commenced . The similarity of effects oP thehistoaes and certain other basic ca~mpounds on the "contractile-relaxation" cycle of liver mitochondria, as compared to muscle actomyosin systems l , supports the contention that the mitochondrion possesses a contractile . system 4 ' 10 Chile the site of binding of thehistoae to the mitochondrial
Vol. 4, No . 16
SWELLING & CONTRACTION OF NIITOCHONDRIA
1559
membrane (iß such a reaction in Pact does occur) is unknown, a number oß possible acidic compounds present in mitochondria are being considered, including neuraminic acid derivatives, phosphoproteins, phaspholipids and nucleotides .
Ia view oß recent
reports concerning the presence oß DNA in mitochondrial and 11,12 erythrocyte membranes the possibilit y oß a DNA-histone binding in mitochondria is attractive .
FIG . 3 Inhibition oß Contraction oß Mitochondria by Histone . The conditions were the same as in Figure 1 except that Medium "B" was used, and 1 mM CaCl2 (Final concentration) added at zero time, was the "swélling agent" . At the arrow, 1 - 3 mM MgCl2, 3 mM ATP and 2 mg/ml oß bovine serum albumin weré added, or 2 -éame as 1 plus 20 hg oß F2a (rat liver) . Mitochondrial protein - 1 .24 mg . Acknowledgement : This study was supported by grants ßrom the D9PHS (H8 0790803 and HS 05435-05) and Brom the Texas Heart Association (64G4N) . Dr . Schwartz is a recipient oß a Career Research Development Award (iJ3IyH9, HS 11,875-01),
Appreciation is extended to
1580
SWELLING & CONTRACTION OF MITOCHONDRIA
Vol. 4, No. 18
Dr . A . C . 3tarbuck and to Yr . C . A . Taylor Por their generous help in isolation oß the histones . References 1.
A . BCHwARTZ and A . H . LASSPER, Li ße Sciences 4, 145 (1965) .
2.
A . BCH~ARTZ, J . Biol . Chem ., 240, 939 ;944 (1965) .
3.
A . SCH~ARTZ, Biochim . Biophys . Acta , 100, 202 (1985) .
4.
T . OHNISHI and T . OHNISHI, J . Biochem . (Tok) 52, 230 (1962) .
5.
L . S . HNILICA and H . BUSCH, J . Biol . Chea ., 238, 918 (1963) .
6.
A . L. LEHNINGER, J . Biol . Chem ., 234, 2465 (1959) .
7.
A . SCHIIARTZ and K . S . LEE, Circulat ion Res ., 10, 321 (1962) .
B.
V . G . ALLFREY, R. FAULKNER and A . E . MIRSRY, Froc . Nat'1 Acad . Sci ., 51, 786 (1964) .
9.
C. lf . TABOR, Biochem . Biophys . Res . Coma ., 2, 117 (1960) .
10 .
H. BALTSCHEFFSKX and Y . BALTSCHEFFSKY, Biochem . Biophys . Res . Comm a, 17, 220 (1964) .
11 .
G. F . KALF, Biochemistry , 3,
12 .
L. PHILIDBON and 0 . ZSPTERQVIST, Biochim . Biophys . Acta , 91 171 (1964) . -
1702 (1964) .