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Basic protein induction of low amplitude energy-linked mitochondrial swelling
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
Basic Protein
131, 31&315
Induction
Energy-Linked C. L. JOHNSON,
(1969)
of Low Amplitude
Mitochondrial J. ORO,
Swelling
A. SCHWARTZ
_4ND
Department of Pharmacology, Baylor University College of Medicine, Houston, Texas ?+YOdb,and Department of Biophysical Sciences, University of Houston, Houston, Texas 77004 Received
January
4, 1969
Polycationic proteins cause an energy-dependent, low amplitude swelling of isolated mitochondria, supported either by substrate oxidation or by adenosine triphosphate. Inorganic phosphate (or arsenate) is required for maximal rate of swelling. Sodium or potassium significantly inhibits the basic protein-induced effect. Magnesium completely prevents the reaction regardless of the ionic constitution of the medium. An energy-dependent potassium ion efflux accompanies the basic protein-induced swelling. These effects are similar to those observed with parathyroid hormone.
Previous reports from this laboratory have demonstrated effects of histones and other polycationic proteins on various aspects of mitochondrial structure and function. In micromolar concentrations, e.g., histones stimulate oxygen consumption (I, 2), adenosine triphosphatase (3, 4), and potassium ion efflux in isolated liver and heart mitochondria (2, 5, 6). An earlier paper also reported an induction of mitochondrial swelling by histones, measured by turbidity changes (7). These experiments, however, were carried out under conditions usually employed in “high amplitude” swelling studies (i.e., in decimolar salt solutions and at low mitochondrial protein concentrations (8)) and the reaction appeared to be energy-independent. In the present communication, we report the “low amplitude,” energy-linked swelling of mitochondria induced by polycationic proteins. MATERIALS
AND METHODS
The histone fractions were isolated from ox thymus or rat liver and characterized as described in the literature (7, 9, 10). We gratefully acknowledge generous supplies of basic proteins donated by Dr. W. C. Starbuck. Some of the properties are shown in Table I. The basic proteins Peaks II and III isolated from the parathyroid gland and highly
purified and partially purified parathyroid hormone (PTH), were gifts from Dr. G. D. Aurbach. Poly-L-lysine (mol wt 230,000)) spermine, oligomytin, and rotenone were obtained from Sigma Chemical Company. Rat liver mitochondria were isolated by the method of Schneider (11) in a medium containing 0.25 M sucrose and 1 mu Tris-ethylene diamine tetraacetate (EDTA), pH 7.0. The mitochondria were suspended in this medium at a concentration of about 50 mg protein/ml. Protein was determined by the biuret method (12). Swelling was estimated by measuring decreases in optical density of a mitochondrial suspension at 520 rnN in a Gilford model 2000 recording spectrophotometer. The linear absorbance slide wire of this instrument from 0 to 3 OD units allows 180” light transmission measurements to be made at high protein concentrations (up to about 2 me/ml).
In most of the experiments
the assay medium
consisted
mM Tris-phosphate,
of 0.25
reported M
here,
sucrose,
and 3.1 mM succinate
9.4
(Tris
salt) at pH 7.4 in a final volume of 4.0 ml. The mitochondrial
protein
experiments
varied
(initial optical OD units). (about
low amplitude scribed 310
in these
density ranged between 2.0 and 2.3 at lower
1.0 mg/ml) for
used
1.75 and 1.95 mg/ml
Other experiments
were performed procedure
concentration between
with
measuring swelling
by Packer
(13).
not reported
protein identical the
results.
characteristics
are similar
here
concentrations
to those
The of de-
HISTONES
ON
MITOCHONDRIAL TABLE
NOMENCLATURE Histone fraction P Ps Ps WB
Classification Arginine-rich Arginine-rich Arginine-rich Lysine-rich
311
SWELLING I
OF HISTONES Mol wt
(AR) (AL) (ARs)
Purity
-
Crude Electrophoreticslly 90% pure Electrophoretically
12,000 30,000 20,000
RESULTS
f75,ug
BASIC
Energy Requirements for HistoneInduced Swelling The energy required for histone-induced swelling could be provided either by electron transport or by ATP (Fig. 2). ATP-supported histone-induced swelling is prevented by oligomycin. The latter does not block substrate-supported swelling. Glutamate oxidation supports the basic protein effect, although at a slightly reduced rate, compared to succinate oxidation.
pure
PROTEIN -A
Swelling Inducecl by Various Basic Proteins All of the polycationic proteins studied, except spermine, caused a decrease in the optical density of mitochondrial suspensions (Fig. 1). The lack of effect of spermine on mitochondria has been previously noted (7) and may be related to the low molecular weight of this polypeptide compared to the other proteins studied. In these experiments both lysine and arginine-rich proteins appeared to exert equal effects. Because of the still tentative molecular weight assignments of the various basic proteins, molar stoichiometry was not studied. A partially purified parathyroid hormone preparation reacted similarly to the histones, although 4-5 times as much PTH was required (data not shown). A constant basic protein to mitochondrial protein ratio of about 10 pg/mg, was maintained throughout all of the experiments. Concentrations in excess of 20-30 pg/mg, produce non-energy-linked optical density changes, due probably to mitochondrial aggregation. The threshold concentration for energy-dependent swelling is approximately 2-3 pg/mg, which is in the same range as that found for histone-induced I<+ efflux (5).
pure
‘I
FIG. 1. chondrial M sucrose, succinate, protein/ml; the final micrograms added as spermine; mone; C, F, p-histone; histone.
\H
Effect of various basic proteins on mitoswelling. The medium consisted of 0.25 9.4 mix Tris-phosphate, 3.1 mM TrispII 7.4, and 1.75 mg mitochondrial final volume 4.0 ml. In all of the figures concentrations are listed. Seventy-five of the following basic proteins were shown at the arrow: A, no addition or B, partially purified parathyroid horPeak II; D, Peak III; E, poly-L-lysine; G, Be-histone; H, &-histone; I, 01~s-
Anion Requirementsfor HistoneInduced X~oelling Either phosphate or arsenate is required for maximal swelling rate whether electron transport or ATP supplies the energy (Fig. 3). Oligomycin was included in experiment E of Fig. 3 to prevent the uncoupling effect of arsenate. Minimal swelling occurs in a medium containing chloride or sulfate as the principal anion. It would appear from these data, therefore, that a permeant anion or specifically phosphate, is required for optimal basic protein-induced swelling.
312
JOHNSON.
ORO,
AND
SCHWARTZ
E$ect of Monovalent Cations on HistoneInduced Swelling A
Replacing sucrose with KC1 or NaCl in a phosphate medium substantially decreases the extent of swelling although initial rates of swelling are increased (Fig. 4 and Table
I I MIN
FIG. 2. Energy requirements for histone-induced swelling. The medium consisted of 0.25 M sucrose, 9.4 mu Tris-phosphate, pH 7.4, and 1.85 mg mitochondrial protein/ml; final volume 4.0 ml. In addition: A contained 3.1 mu Tris-succinate, 1 pg antimycin A and 10 pg oligomycin; B contained 3.1 mu Tris-succinate and 1 rg antimycin A; C contained 3.1 mu Tris-glutamate; D contained 3.1 mM Tris-succinate. &,-Histone was added to each curve as shown; ATP was added to curves A and B only. The antimycin was added to block substrate-supported swelling. +-75j~g
PqHISTONE
L -----A
FIG. 4. Effect of monovalent cations on histone-induced swelling. The medium consisted of 9.4 nnw Tris-phosphate, 3.1 nnw Tris-succinate, pH 7.4, and 1.75 mg mitochondrial protein/ml; final volume 4.0 ml. In addition: A contained 0.25 M sucrose; B contained 0.125 M KCl; C contained 0.125 M NaCl. &Histone was added to each curve aa shown. TABLE EFFECT
iw
OF KC1
II
CONCENTRATION
HISTONEINDUCED SWELLING SUBSEQUENT CONTRACTIONS KC1
0 31.3 62.5 78.1 93.7 109.3 125.0
lMlN
FIG. 3. Effect of swelling. The medium 3.1 mu Tris-succinate, chondrial protein/ml; dition, the following final concentration of C, sulfate; D, acetate; gomycin; F, inorganic _. _ added to each curve
(mu)
anions on histone-induced consisted of 0.25 M sucrose, pH 7.4, and 1.75 mg mitofinal volume 4.0 ml. In adTris salts were added at a 9.4 mM: A, none; B, chloride; E, arsenate plus 10 pg oliphosphate. Ps-Histone was as shown.
Swelling rate (AOD/min)
0.165 0.400 0.500 0.490 0.440 0.410 0.380
Extent of swelling (AOD)
Contraction rate (AOD/min)
0.780 0.250 0.245 0.250 0.245 0.245 0.228
0.065 0.075 0.063 0.033 0.028
ON AND
Extent of contraction (AON
0 0 0.035 0.085 0.130 0.103 0.100
a The medium consisted of 9.4 mm Tris-phosphate, 3.1 mM Tris-succinate, pH 7.4, and 1.75 mg mitochondrial protein/ml. In addition, the medium contained KC1 at the concentrations shown and sufficient sucrose to bring the osmolarity to 0.25. After a 2-min equilibration period, 75 pg ,% histone was added and the rates and extent of swelling and subsequent contraction were determined. The final volume was 4.0 ml.
HISTONES
ON
MITOCHONDRIAL
II). In addition, in an ionic medium, a spontaneous reversal of the hi&one-induced swelling is observed after about 1 min (Fig. 4). The nature of the reversal process is not clear at present although the extent of reversal appears to be dependent on the
SWELLING
monovalent ion concentration (Table II). In an ionic medium, in contrast to the sucrose medium, the nature of the permeant anion appears to have little effect (Fig. 5). It is possible, considering the high concentration of chloride ion used in these experiments (0.125 M), that Cl- may act as the permeant anion. E$ect of Mg2+ on Histone-Induced
C
I MIN
0
313
Xwelhg
Magnesium prevents the hi&one-induced swelling whether the basic protein effect is supported by electron transport or by ATP and regardless of the anion or monovalent cation composition of the medium (Fig. 6). Similar effects of Mg2+ were noted with histone-stimulated oxygen consumption and K+ efflux (2, 5). DISCUSSION
-w
FIG. 5. Effect of anions in presence or absence of monovalent cations on histone-induced swelling. The medium consisted of 3.1 mM Tris-succinate, pH 7.4, and 1.75 mg mitochondrial protein/ml; final volume 4.0 ml. In addition: A contained 0.25 x sucrose and 9.4 mM Tris-acetate; B contained 0.25 M sucrose and 9.4 mM Tris-phosphate; C contained 0.125 M KC1 and 9.4 mM Tris-phosphate; D contained 0.125 M KC1 and 9.4 rnM Tris-acetate. Ps-Histone was added to each curve as shown. f75Ag
a-.-<, \ \
B-
&-HISTONE
oTo5 0.0. YNITS
I MIN -
\
\ FIG. 6. Effect of Mg*+ on histone-induced swelling. The medium consisted of 0.25 M sucrose, 9.4 rn>r Tris-phosphate, 3.1 mnr Tris-succinate, pH 7.4, and 1.75 mg mitochondrial protein/ml; final volume 4.0 ml. In addition, B contained 5 rnM ?rlgCls. Bs-Histone was added to each curve as shown.
It is clear from the present results that hi&one-like basic proteins cause an energydependent decrease in optical density of mitochondrial suspensions. Similar observations have been reported by Utsumi and Yamamoto (14). Such decreases in turbidity are usually interpreted in terms of a swelling of the mitochondria. However, as pointed out by numerous investigators, relationships between turbidity, actual volume, and membrane conformational changes are not yet clear. It is obvious, however, that the effects induced by the basic proteins cannot be interpreted on t.he basis of nonspecific, irreversible aggregation of the mitochondria or passive membrane permeability effects. It has been postulated that the intermediates involved in both ion transport and the swelling-contraction cycle lie off the main pathway of oxidative phosphorylation (15, 16). *$ccording to this, both ion transport and swelling are supported by either electron transport or ATP. The results presented in this and the previous communication on K+ efflux (5) are consistent with this supposition. According to this chemical theory of oxidative-phosphorylation, the basic proteinstimulated smelling, K+ efflux, and oxygen consumption would not occur during State 3 respiration because of a competition between ATP synthesis and the side pathway for common high-energy intermediates. Although there is perfect correlation be-
314
JOHNSON,
ORO, AND SCHWARTZ
tween h&tone-induced swelling and Kz+ efflux in terms of energy and anion requirements and divalent cation effects, the stoichiometric or kinetic relationship appears to be complex. The rate of K+ efflux, induced by histone usually drops rapidly after about 1 min (data not shown) whereas the rate of swelling remains high for 4-5 min. Furthermore, in the ionic medium where a spontaneous reversal of swelling (after about 1 min) ,occurs, no apparent change in oxygen consumption is observed. It is possible that some of the histone effects are secondary (but not independent) and that another process (possibly anion transport, basic protein binding or accumulation, or divalent cation transport) may be the major site of action of these basic proteins. For example, the inhibition of histone effects by Mg2+ and the recent report of Harris et al. (17) demonstrating that histone causes an efflux of imply a role for divalent cation transport in the observed effects. Furthermore, requirement for a permeant anion suggests the importance of anion uptake with respect to histone action, The efflux of K+ might occur as a result of an exchange of the polycationic protein for intramitochondrial monovalent cation. This last suggestion is of interest in light of the exchange of K+ for substituted amines rereported by Chappell and Crofts (18). The spontaneous reversal of the histoneinduced swelling which occurs in the presence of monovalent cations appears to be analogous to the oscillatory states of mitochondria induced by EDTA (19), in that both require an energy source (either electron transport or ATP) and a monovalent cation salt of a permeant anion. Packer suggests that EDTA removes mitochondrial RIg2+; Mg2+ causes an inhibition of oscillations. Since histone as well as EDTA cause the release of mitochondrial Mg2+ (17), it is possible that the oscillations reported in the present study, are due to Rlg2+ loss. It is of interest that essentially identical results to those reported in this paper have been found in the case of parathyroid hormone (PTH)-induced swelling (20). For example, Mg2+ inhibits PTH-induced swelling. In addition, Rasmussenand Ogata (21) have recently shown that PTH stimulates
nIg2+,
K+ efflux from mitochondria although energy requirements were not discussed. These authors also observed an inhibition of K+ efllux by 51g2+ ions. It is clear, therefore, that at least some of the effects of PTH on mitochondria can be duplicated by basic proteins. Rasmussenet al. (22), on the other hand, have reported that parathyroid hormone has specific effects on the decarboxylation of succinate by mitochondria. The hormone-stimulated decarboxylation was not observed if chloride was the only anion present in a Rlg2+-containing medium; the hormone did have an effect, however, in a chloride medium in the absence of RIg2+ while polylysine, histone, and a basic, nonhormonal polypeptide from the parathyroid gland stimulated decarboxylation in a chloride medium in the presence or absence of i\lg2+ ions. The inhibition of PTH-stimulated succinate decarboxylation by ;\lg2+ is difficult to interpret in light of an earlier report by Rasmussen and Ogata (21) that Jlg2+ increased PTH-stimulated respiration in a chloride medium. Although it would appear that succinate decarboxylation is directly related to oxygen consumption measurements, a direct correlation between succinate decarboxylation and respiration has not to our knowledge been reported. Preliminary experiments in this laboratory with highly purified PTH have clearly demonstrated RIg2+inhibition of both oxygen consumption (assayed polarographically) and K+ efflux induced by the hormone (unpublished observations) . At present, it is attractive to speculate that the histone might enter the mitochondrion via an energy-dependent uptake mechanism probably in combination with a permeant anion like phosphate. Phosphorylation of histone has in fact been recently described (23). Once inside the mitochondrion, dissociation of the basic proteinanion complex would lead to loss of K+ (and possibly Mg2+) in exchange for the polycationic protein. Swelling might either be due to increased osmotic pressure or interaction of the positively charged protein with negative receptor sites within or on the membrane leading to conformational changes. Another possible mechanism involves an energy-requiring binding of histone to spe-
HISTONES
Ori
MITOCHO~D1LIAL
cific negative sites which produces permeability changes resulting in the observed phenomena. A recent interesting report, describing an energy-dependent TI;+-efflux induced by certain heavy metals particularly Zdf (24) may be related t#othe present data. Utochondria bind zinc by energ\,-dependent and independent processes (2.5). Further investigations into t’he eBect,s of basic proteins on mitochondria are of importance in terms of the possible physiological role of histones, and perhaps of basic polypcptide hormones. Histones arc among the very few naturally occurring compounds which are present in suficient concentration within the cell of most tissues and which cn11 in very low concentration exert specific eft’ccbs 011 the mitochondrion. ACKNOWLEI)GMEh’TS This study was slyported by the following grants: HE-07906-07 and IfE-5435-09, P.8, from the USPIIS, National IIesrt 1nst)itute; by GB;6895 from the National Science Foundation; and by grants from the Texas Heart Assoriation and the Houston Heart, Association. Dr. Schwartz is a recipient of a Career Research Development Award from the USPIIS, National Heart Institute (K, HE 11, 875-05). Mr. Johnson acknowledges support by Nl>Eh Title 11. Fellowship tici-8251.
7. SCHWARTZ, UUCK, W. 8. HUNTER, F. in Bnzymol. 9. BTXCH, I-I., in Cancer 10. BVSCH, II., C., T.\nort, 11. 12.
13. l-1. 15, 1G. 17. 18.
19. 20.
REFEREXCES 1. SCHW.~RTZ, A., J. Biol. them. 240, 939 (1965). 2. JOHNSOS. C. L., SAFER, H., AND SCHWARTZ, A., J. Biol. Chem. 241, 4513 (196G). :3. SCH~vlKTZ, A., J. Viol. Chem. 240, 9id (1965). 4. L.w;TE:R, -4-1. I-I., JOI~NX, C. I~., .lND S:(~HV.\RTZ, -4., l’exas Rep. Biol. 3fed. 24, 605 (1966). 5. JOHNMN, C. L., MAURITZCN, C.IzI., STARIWCIG W. C., .\A-D SCHWARTZ, A., Biochemistry 6, 1121 (1967). 6. SAFI~;R, B., AND SCHW.\RTZ, A., Circulation Res. 21, 25 (1967).
SWELLIPL’G
21.
22. 23.
24. 25.
315
A., JOHNSON, C. I,., AND STARC.> J. Biol. Chom. 241,4505 (1966). B., JR., AND SMITH, I<. E., Methods 10, 689 (1967). .mu X~UAITZICN, C. 31., Methods Res. 3, 391 (1967). R~AURITZEN, C. AI., STARHUC~, W. C. W., .YND G~tooax, D. E., Gann Monograph 4, 113-148 (1968). S~IIXKIDIX, W. C., J. Biol. Ch,em. 176, 259 (1948). J.\com, E. I<., J\co~, AI., S.\X.tDI, 11. k, AND UR.\DLI~;Y, I,. B., J. Biol. Chen. 223, 147 (1956). P.\C!I~~~R, L., Jlefhods in Enzymol. 10, 685 (1907). UTSUMI, K., AND Y~~MAMOTO, G., Biochim. Biophys. Acta 100, 600 (1965). MOORE, C., AND PRESSM.\N, B. C., Biochem. Riophys. Res. Commun. 15, 562 (1964). LUDY, II. A., CON.VILLI., J. L., .WD JOHNSON, Il., Biochemislry 3, 1961 (1964). HARRIS, E. J., C.\,rLIN, G., AND PILESSMAN, B. C., Biochemistry 6, 1360 (1967). CHAPPELL, J. B., AND CROFTS, A Ii., in “Regulation of Xetabolic Processes in Mitochondria,” (J. RI. Tager, S. Papa, E. Quagliariello, and F. C. Slater, Eds.), BBA Library 1’01. 7, p. 293. Elsevier, Amsterdam (1966). P.\crcls~l, L., UTSU.\II, K., AND Mus~r.wA, M. G., Arch. Biochem. Biophys. 117, 381 (1966). TJwrr>xr, K., S.IIAIS, J. D., AND D~:mc,\, II. F., J. Bid. Chem., 241, 1128 (1966). k.\sh~usseN, IT., .\SD OG.YIYL, E., Riochemistry 5, 733 (1966). I:.ZYMUSSL;N, H., SHIRASU, H., Oo.Yr.\, E., AND II.\wIG:R, C., J. Biol. Chem. 242,4G69 (1967). GUTIEHI~Z, It. AI., AND HNILICA, L. S., Abstr., 7th 4nnual Meeting American Society for Cell Biology 11967) p. 5lA; Science 167, 1324 (196i). BRIERIAY, (;. I'., AND SETTLEMIRE, C. T., J. Biol. Chem., 242, 4322 (1967). BRIERLIX,G. P., AND KNIGHT, V.A.,Biochemistry 6, 3892 (1967).
OF
BIOCHEMISTRY
AND
BIOPHYSICS
Basic Protein
131, 31&315
Induction
Energy-Linked C. L. JOHNSON,
(1969)
of Low Amplitude
Mitochondrial J. ORO,
Swelling
A. SCHWARTZ
_4ND
Department of Pharmacology, Baylor University College of Medicine, Houston, Texas ?+YOdb,and Department of Biophysical Sciences, University of Houston, Houston, Texas 77004 Received
January
4, 1969
Polycationic proteins cause an energy-dependent, low amplitude swelling of isolated mitochondria, supported either by substrate oxidation or by adenosine triphosphate. Inorganic phosphate (or arsenate) is required for maximal rate of swelling. Sodium or potassium significantly inhibits the basic protein-induced effect. Magnesium completely prevents the reaction regardless of the ionic constitution of the medium. An energy-dependent potassium ion efflux accompanies the basic protein-induced swelling. These effects are similar to those observed with parathyroid hormone.
Previous reports from this laboratory have demonstrated effects of histones and other polycationic proteins on various aspects of mitochondrial structure and function. In micromolar concentrations, e.g., histones stimulate oxygen consumption (I, 2), adenosine triphosphatase (3, 4), and potassium ion efflux in isolated liver and heart mitochondria (2, 5, 6). An earlier paper also reported an induction of mitochondrial swelling by histones, measured by turbidity changes (7). These experiments, however, were carried out under conditions usually employed in “high amplitude” swelling studies (i.e., in decimolar salt solutions and at low mitochondrial protein concentrations (8)) and the reaction appeared to be energy-independent. In the present communication, we report the “low amplitude,” energy-linked swelling of mitochondria induced by polycationic proteins. MATERIALS
AND METHODS
The histone fractions were isolated from ox thymus or rat liver and characterized as described in the literature (7, 9, 10). We gratefully acknowledge generous supplies of basic proteins donated by Dr. W. C. Starbuck. Some of the properties are shown in Table I. The basic proteins Peaks II and III isolated from the parathyroid gland and highly
purified and partially purified parathyroid hormone (PTH), were gifts from Dr. G. D. Aurbach. Poly-L-lysine (mol wt 230,000)) spermine, oligomytin, and rotenone were obtained from Sigma Chemical Company. Rat liver mitochondria were isolated by the method of Schneider (11) in a medium containing 0.25 M sucrose and 1 mu Tris-ethylene diamine tetraacetate (EDTA), pH 7.0. The mitochondria were suspended in this medium at a concentration of about 50 mg protein/ml. Protein was determined by the biuret method (12). Swelling was estimated by measuring decreases in optical density of a mitochondrial suspension at 520 rnN in a Gilford model 2000 recording spectrophotometer. The linear absorbance slide wire of this instrument from 0 to 3 OD units allows 180” light transmission measurements to be made at high protein concentrations (up to about 2 me/ml).
In most of the experiments
the assay medium
consisted
mM Tris-phosphate,
of 0.25
reported M
here,
sucrose,
and 3.1 mM succinate
9.4
(Tris
salt) at pH 7.4 in a final volume of 4.0 ml. The mitochondrial
protein
experiments
varied
(initial optical OD units). (about
low amplitude scribed 310
in these
density ranged between 2.0 and 2.3 at lower
1.0 mg/ml) for
used
1.75 and 1.95 mg/ml
Other experiments
were performed procedure
concentration between
with
measuring swelling
by Packer
(13).
not reported
protein identical the
results.
characteristics
are similar
here
concentrations
to those
The of de-
HISTONES
ON
MITOCHONDRIAL TABLE
NOMENCLATURE Histone fraction P Ps Ps WB
Classification Arginine-rich Arginine-rich Arginine-rich Lysine-rich
311
SWELLING I
OF HISTONES Mol wt
(AR) (AL) (ARs)
Purity
-
Crude Electrophoreticslly 90% pure Electrophoretically
12,000 30,000 20,000
RESULTS
f75,ug
BASIC
Energy Requirements for HistoneInduced Swelling The energy required for histone-induced swelling could be provided either by electron transport or by ATP (Fig. 2). ATP-supported histone-induced swelling is prevented by oligomycin. The latter does not block substrate-supported swelling. Glutamate oxidation supports the basic protein effect, although at a slightly reduced rate, compared to succinate oxidation.
pure
PROTEIN -A
Swelling Inducecl by Various Basic Proteins All of the polycationic proteins studied, except spermine, caused a decrease in the optical density of mitochondrial suspensions (Fig. 1). The lack of effect of spermine on mitochondria has been previously noted (7) and may be related to the low molecular weight of this polypeptide compared to the other proteins studied. In these experiments both lysine and arginine-rich proteins appeared to exert equal effects. Because of the still tentative molecular weight assignments of the various basic proteins, molar stoichiometry was not studied. A partially purified parathyroid hormone preparation reacted similarly to the histones, although 4-5 times as much PTH was required (data not shown). A constant basic protein to mitochondrial protein ratio of about 10 pg/mg, was maintained throughout all of the experiments. Concentrations in excess of 20-30 pg/mg, produce non-energy-linked optical density changes, due probably to mitochondrial aggregation. The threshold concentration for energy-dependent swelling is approximately 2-3 pg/mg, which is in the same range as that found for histone-induced I<+ efflux (5).
pure
‘I
FIG. 1. chondrial M sucrose, succinate, protein/ml; the final micrograms added as spermine; mone; C, F, p-histone; histone.
\H
Effect of various basic proteins on mitoswelling. The medium consisted of 0.25 9.4 mix Tris-phosphate, 3.1 mM TrispII 7.4, and 1.75 mg mitochondrial final volume 4.0 ml. In all of the figures concentrations are listed. Seventy-five of the following basic proteins were shown at the arrow: A, no addition or B, partially purified parathyroid horPeak II; D, Peak III; E, poly-L-lysine; G, Be-histone; H, &-histone; I, 01~s-
Anion Requirementsfor HistoneInduced X~oelling Either phosphate or arsenate is required for maximal swelling rate whether electron transport or ATP supplies the energy (Fig. 3). Oligomycin was included in experiment E of Fig. 3 to prevent the uncoupling effect of arsenate. Minimal swelling occurs in a medium containing chloride or sulfate as the principal anion. It would appear from these data, therefore, that a permeant anion or specifically phosphate, is required for optimal basic protein-induced swelling.
312
JOHNSON.
ORO,
AND
SCHWARTZ
E$ect of Monovalent Cations on HistoneInduced Swelling A
Replacing sucrose with KC1 or NaCl in a phosphate medium substantially decreases the extent of swelling although initial rates of swelling are increased (Fig. 4 and Table
I I MIN
FIG. 2. Energy requirements for histone-induced swelling. The medium consisted of 0.25 M sucrose, 9.4 mu Tris-phosphate, pH 7.4, and 1.85 mg mitochondrial protein/ml; final volume 4.0 ml. In addition: A contained 3.1 mu Tris-succinate, 1 pg antimycin A and 10 pg oligomycin; B contained 3.1 mu Tris-succinate and 1 rg antimycin A; C contained 3.1 mu Tris-glutamate; D contained 3.1 mM Tris-succinate. &,-Histone was added to each curve as shown; ATP was added to curves A and B only. The antimycin was added to block substrate-supported swelling. +-75j~g
PqHISTONE
L -----A
FIG. 4. Effect of monovalent cations on histone-induced swelling. The medium consisted of 9.4 nnw Tris-phosphate, 3.1 nnw Tris-succinate, pH 7.4, and 1.75 mg mitochondrial protein/ml; final volume 4.0 ml. In addition: A contained 0.25 M sucrose; B contained 0.125 M KCl; C contained 0.125 M NaCl. &Histone was added to each curve aa shown. TABLE EFFECT
iw
OF KC1
II
CONCENTRATION
HISTONEINDUCED SWELLING SUBSEQUENT CONTRACTIONS KC1
0 31.3 62.5 78.1 93.7 109.3 125.0
lMlN
FIG. 3. Effect of swelling. The medium 3.1 mu Tris-succinate, chondrial protein/ml; dition, the following final concentration of C, sulfate; D, acetate; gomycin; F, inorganic _. _ added to each curve
(mu)
anions on histone-induced consisted of 0.25 M sucrose, pH 7.4, and 1.75 mg mitofinal volume 4.0 ml. In adTris salts were added at a 9.4 mM: A, none; B, chloride; E, arsenate plus 10 pg oliphosphate. Ps-Histone was as shown.
Swelling rate (AOD/min)
0.165 0.400 0.500 0.490 0.440 0.410 0.380
Extent of swelling (AOD)
Contraction rate (AOD/min)
0.780 0.250 0.245 0.250 0.245 0.245 0.228
0.065 0.075 0.063 0.033 0.028
ON AND
Extent of contraction (AON
0 0 0.035 0.085 0.130 0.103 0.100
a The medium consisted of 9.4 mm Tris-phosphate, 3.1 mM Tris-succinate, pH 7.4, and 1.75 mg mitochondrial protein/ml. In addition, the medium contained KC1 at the concentrations shown and sufficient sucrose to bring the osmolarity to 0.25. After a 2-min equilibration period, 75 pg ,% histone was added and the rates and extent of swelling and subsequent contraction were determined. The final volume was 4.0 ml.
HISTONES
ON
MITOCHONDRIAL
II). In addition, in an ionic medium, a spontaneous reversal of the hi&one-induced swelling is observed after about 1 min (Fig. 4). The nature of the reversal process is not clear at present although the extent of reversal appears to be dependent on the
SWELLING
monovalent ion concentration (Table II). In an ionic medium, in contrast to the sucrose medium, the nature of the permeant anion appears to have little effect (Fig. 5). It is possible, considering the high concentration of chloride ion used in these experiments (0.125 M), that Cl- may act as the permeant anion. E$ect of Mg2+ on Histone-Induced
C
I MIN
0
313
Xwelhg
Magnesium prevents the hi&one-induced swelling whether the basic protein effect is supported by electron transport or by ATP and regardless of the anion or monovalent cation composition of the medium (Fig. 6). Similar effects of Mg2+ were noted with histone-stimulated oxygen consumption and K+ efflux (2, 5). DISCUSSION
-w
FIG. 5. Effect of anions in presence or absence of monovalent cations on histone-induced swelling. The medium consisted of 3.1 mM Tris-succinate, pH 7.4, and 1.75 mg mitochondrial protein/ml; final volume 4.0 ml. In addition: A contained 0.25 x sucrose and 9.4 mM Tris-acetate; B contained 0.25 M sucrose and 9.4 mM Tris-phosphate; C contained 0.125 M KC1 and 9.4 mM Tris-phosphate; D contained 0.125 M KC1 and 9.4 rnM Tris-acetate. Ps-Histone was added to each curve as shown. f75Ag
a-.-<, \ \
B-
&-HISTONE
oTo5 0.0. YNITS
I MIN -
\
\ FIG. 6. Effect of Mg*+ on histone-induced swelling. The medium consisted of 0.25 M sucrose, 9.4 rn>r Tris-phosphate, 3.1 mnr Tris-succinate, pH 7.4, and 1.75 mg mitochondrial protein/ml; final volume 4.0 ml. In addition, B contained 5 rnM ?rlgCls. Bs-Histone was added to each curve as shown.
It is clear from the present results that hi&one-like basic proteins cause an energydependent decrease in optical density of mitochondrial suspensions. Similar observations have been reported by Utsumi and Yamamoto (14). Such decreases in turbidity are usually interpreted in terms of a swelling of the mitochondria. However, as pointed out by numerous investigators, relationships between turbidity, actual volume, and membrane conformational changes are not yet clear. It is obvious, however, that the effects induced by the basic proteins cannot be interpreted on t.he basis of nonspecific, irreversible aggregation of the mitochondria or passive membrane permeability effects. It has been postulated that the intermediates involved in both ion transport and the swelling-contraction cycle lie off the main pathway of oxidative phosphorylation (15, 16). *$ccording to this, both ion transport and swelling are supported by either electron transport or ATP. The results presented in this and the previous communication on K+ efflux (5) are consistent with this supposition. According to this chemical theory of oxidative-phosphorylation, the basic proteinstimulated smelling, K+ efflux, and oxygen consumption would not occur during State 3 respiration because of a competition between ATP synthesis and the side pathway for common high-energy intermediates. Although there is perfect correlation be-
314
JOHNSON,
ORO, AND SCHWARTZ
tween h&tone-induced swelling and Kz+ efflux in terms of energy and anion requirements and divalent cation effects, the stoichiometric or kinetic relationship appears to be complex. The rate of K+ efflux, induced by histone usually drops rapidly after about 1 min (data not shown) whereas the rate of swelling remains high for 4-5 min. Furthermore, in the ionic medium where a spontaneous reversal of swelling (after about 1 min) ,occurs, no apparent change in oxygen consumption is observed. It is possible that some of the histone effects are secondary (but not independent) and that another process (possibly anion transport, basic protein binding or accumulation, or divalent cation transport) may be the major site of action of these basic proteins. For example, the inhibition of histone effects by Mg2+ and the recent report of Harris et al. (17) demonstrating that histone causes an efflux of imply a role for divalent cation transport in the observed effects. Furthermore, requirement for a permeant anion suggests the importance of anion uptake with respect to histone action, The efflux of K+ might occur as a result of an exchange of the polycationic protein for intramitochondrial monovalent cation. This last suggestion is of interest in light of the exchange of K+ for substituted amines rereported by Chappell and Crofts (18). The spontaneous reversal of the histoneinduced swelling which occurs in the presence of monovalent cations appears to be analogous to the oscillatory states of mitochondria induced by EDTA (19), in that both require an energy source (either electron transport or ATP) and a monovalent cation salt of a permeant anion. Packer suggests that EDTA removes mitochondrial RIg2+; Mg2+ causes an inhibition of oscillations. Since histone as well as EDTA cause the release of mitochondrial Mg2+ (17), it is possible that the oscillations reported in the present study, are due to Rlg2+ loss. It is of interest that essentially identical results to those reported in this paper have been found in the case of parathyroid hormone (PTH)-induced swelling (20). For example, Mg2+ inhibits PTH-induced swelling. In addition, Rasmussenand Ogata (21) have recently shown that PTH stimulates
nIg2+,
K+ efflux from mitochondria although energy requirements were not discussed. These authors also observed an inhibition of K+ efllux by 51g2+ ions. It is clear, therefore, that at least some of the effects of PTH on mitochondria can be duplicated by basic proteins. Rasmussenet al. (22), on the other hand, have reported that parathyroid hormone has specific effects on the decarboxylation of succinate by mitochondria. The hormone-stimulated decarboxylation was not observed if chloride was the only anion present in a Rlg2+-containing medium; the hormone did have an effect, however, in a chloride medium in the absence of RIg2+ while polylysine, histone, and a basic, nonhormonal polypeptide from the parathyroid gland stimulated decarboxylation in a chloride medium in the presence or absence of i\lg2+ ions. The inhibition of PTH-stimulated succinate decarboxylation by ;\lg2+ is difficult to interpret in light of an earlier report by Rasmussen and Ogata (21) that Jlg2+ increased PTH-stimulated respiration in a chloride medium. Although it would appear that succinate decarboxylation is directly related to oxygen consumption measurements, a direct correlation between succinate decarboxylation and respiration has not to our knowledge been reported. Preliminary experiments in this laboratory with highly purified PTH have clearly demonstrated RIg2+inhibition of both oxygen consumption (assayed polarographically) and K+ efflux induced by the hormone (unpublished observations) . At present, it is attractive to speculate that the histone might enter the mitochondrion via an energy-dependent uptake mechanism probably in combination with a permeant anion like phosphate. Phosphorylation of histone has in fact been recently described (23). Once inside the mitochondrion, dissociation of the basic proteinanion complex would lead to loss of K+ (and possibly Mg2+) in exchange for the polycationic protein. Swelling might either be due to increased osmotic pressure or interaction of the positively charged protein with negative receptor sites within or on the membrane leading to conformational changes. Another possible mechanism involves an energy-requiring binding of histone to spe-
HISTONES
Ori
MITOCHO~D1LIAL
cific negative sites which produces permeability changes resulting in the observed phenomena. A recent interesting report, describing an energy-dependent TI;+-efflux induced by certain heavy metals particularly Zdf (24) may be related t#othe present data. Utochondria bind zinc by energ\,-dependent and independent processes (2.5). Further investigations into t’he eBect,s of basic proteins on mitochondria are of importance in terms of the possible physiological role of histones, and perhaps of basic polypcptide hormones. Histones arc among the very few naturally occurring compounds which are present in suficient concentration within the cell of most tissues and which cn11 in very low concentration exert specific eft’ccbs 011 the mitochondrion. ACKNOWLEI)GMEh’TS This study was slyported by the following grants: HE-07906-07 and IfE-5435-09, P.8, from the USPIIS, National IIesrt 1nst)itute; by GB;6895 from the National Science Foundation; and by grants from the Texas Heart Assoriation and the Houston Heart, Association. Dr. Schwartz is a recipient of a Career Research Development Award from the USPIIS, National Heart Institute (K, HE 11, 875-05). Mr. Johnson acknowledges support by Nl>Eh Title 11. Fellowship tici-8251.
7. SCHWARTZ, UUCK, W. 8. HUNTER, F. in Bnzymol. 9. BTXCH, I-I., in Cancer 10. BVSCH, II., C., T.\nort, 11. 12.
13. l-1. 15, 1G. 17. 18.
19. 20.
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