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A subvital muscle preparation with contractile and oxidative-phosphorylative activity
BIOCHIMICA ET BIOPHYSICA ACTA
525
BBA 3715
A S U B V I T A L MUSCLE P R E P A R A T I O N W I T H C O N T R A C T I L E AND O X I D A T I V E - P H O S P H O R Y L A T I V E
ACTIVITY*
RICHARD J. GUILLORY**, W. F. H. M. MOMMAERTS AND KOKI UCHIDA***
Department of Medicine, The Los Angeles County Heart Association, Cardiovascular Research Laboratory, The University of California, Los Angeles, Calif. (U.S.A.) Received January 3oth, 1962)
SUMMARY
It has been shown that by a process of extraction with sucrose media, muscle preparations can be obtained which display contractility as well as oxidation and phosphorylation. The P:O ratio is below that of isolated mitochondria, presumably because of continuous splitting of the synthesized ATP by adjacent myofibrils. This synthesized ATP can be used for contraction, thus contraction can be elicited with small amounts of ADP together with oxygen and oxidizable substrate.
INTRODUCTION
The glycerol-extracted muscle preparation, developed by SZENT-GYrRGYI 1, represents, by its contractile response towards extraneously added ATP, a basic feature of the contractile mechanism of muscle. Dependent on the mode of preparation, it may or may not retain its relaxing-factor activity; otherwise, although it may hold some retained enzymes as accidental contaminants, it has no metabolism other than the decomposition of added ATP. Since it has no excitability, its activity depends on the addition of substances. The preparation described in this paper is one step higher in complexity, in that the biochemical integrity of its mitochondria has been preserved. Consequently, it can respire when oxidizable substrates are added, and it can contract n o t only with added ATP, but also with the ATP formed as the result of its own metabolism. Like the glycerol-extracted fiber, it still is inexcitable, and its activity is still contingent upon extraneously provided materials; but a greater variety of biochemical responses becomes possible. We have developed this preparation in order to study whether the relations between mechanical parameters and biochemical kinetics, such as the effect of length upon metabolic rate, are determined by factors dependent on the integrity of the cell; and also for making the study of contraction and relaxation phenomena * For preliminary publication see R. J. GUILLORY AND W. F. H. M. MOMMAERTS, Circulation
Research, 6 [1958) 73 o. ** Present address: University of Amsterdam, Department of Physiological Chemistry, Jonas Daniel Meijerplein 3, Amsterdam [C) (The Netherlands). *** Former address: Department of Physiology, Sapporo Medical College, Sapporo (Japan).
Biochim. Biophys. Acta, 62 (1962) 525-533
526
R . J . GUILLORY, W. F. H. M. MOMMAERTS, K. UCHIDA
less dependent on the limitations posed by diffusion. This publication describes the general features of the preparation, as a preliminary toward the quantitative investigations now in progress. EXPERIMENTAL
For the purpose of surveying both its biochemical and its contractile properties, our preparation was designed ill tWO forms : the one a suspension of small particles suitable for manometric experiments; the other in the familiar form of a fiber bundle of which the contractile responses can be measured. In later work, only briefly alluded to in this paper, both contraction and oxygen consumption were measured on the same fiber, but the dual methodology was found advantageous for the initial stage of the work. Muscles from various sources have been used; the extracted particles are best prepared from tissues that can be sliced, i.e. cardiac muscle; the fiber bundles ale best made from skeletal muscles, preferably from those with a significant mitochondrial count 2. We shall specifically describe the handling of the two objects on which most of the communicated experiments were performed.
Minced rat heart muscle Rat hearts were excised, rinsed with Ringer solution and blotted, and cut into sections of 2 mm thickness with a STADIE-RIGGSs tissue slicer. These were then placed on filter paper wetted with saline on the object holder of a MclLWAIN4 tissue chopper, and cut successively along two perpendicular directions, thus yielding prismatic particles of 0.2 × 0.2 × 2 mm. These were freed from filter-paper shreds and from larger tissue fragments by suspending and decanting, and were then extracted with 0.88 or 0.32 M sucrose lor varying lengths of time, usually 48 h at o °.
Pigeon breast muscle fiber bundle A preparation entirely analogous to the SZENT-GY6RGYI model was made by extraction with 0.88 M sucrose instead of aqueous glycerol, with or without other constituents added to the basic sucrose medium; often, the composition was 0.88 M sucrose, o.ooi M EDTA. This could be applied to all muscles studied: rat and rabbit psoas, heart muscle and pectoral muscle of the pigeon. For mechanical measurements, a fiber was mounted in a suitable cuvet and connected to one of the isotonic or isometric variable capacitance gages of this laboratory 5, the output being recorded on a Brush Mark II recorder or a Texas Instruments Corporation Recti-Riter. For the simultaneous recording of oxygen consumption, an oscillating platinum electrode was used, but this part of the methodology is still being improved, and will be described fully at a later occasion.
Oxygen consumption by minced tissue Suspensions of prismatic heart-muscle particles were measured into Warburg vessels, and their oxygen consumption was determined by the usual methods, at 27 °. The composition of the medium differed in individual experiments. In typical cases, it consisted of I.O ml of a solution of o.Io M KC1, 0.005 M MgC12 and 0.04 M glycylglycine (pH 7.o), with substrate and nucleotide additions; to which was added 0.20 ml of tissue suspension in 0.88 M sucrose. Biochim. Biophys. Acta, 62 (I962) 525-533
SUBVITAL MUSCLE PREPARATION
527
Such extracted particles showed little or no endogenous respiration, but responded markedly to the addition of oxidizable substrates. Table I illustrates this, and also shows that significant respiration is obtained without the addition of nucleotides, but that it is enhanced by ADP. Apparently, enough ADP was retained in these preparations6, 7 to act as a phosphate acceptor, and ATP was not dephosphorylated rapidly enough to make a significant difference. On the other hand, the addition of ADP led to a direct increase in the phosphate-acceptor concentration. This is only one of several possible results, and later work with skeletal-muscle bundles led to preparations which were dependent on added ADP. The effect of succinate rose sharply at low concentrations, and displayed a broad maximum above 0.02 M. Fig. i shows respiration experiments with different substrates. It is seen that =-ketoglutarate and ~-hydroxybutyrate give lower respiratory rates and faster inactivation than succinate. The differences in rates may be ascribed to different yields of phosphorylation, but the faster inactivation (which was not reversed by added DPN) is, in addition, indicative of the loss of required enzymes. TABLE I
RESPIRATION OF CARDIAC-MUSCLEPARTICLES 5 ° m g of tissue per W a r b u r g vessel, extracted 24 h in o.88 M sucrose; composition of m e d i u m : o. IO M KC1, 0.0o 5 M MgClz, 0.08 M succinate, 0.04 M glycylglycine at p H 7.0; nucleotides, w h e n added, 0.3" lO -8 M each; gas phase, oxygen; t e m p e r a t u r e , 27°; m e a s u r e m e n t over a period of 3 ° min.
Oxygen (tat/l*) No succinate, A D P No succinate, ATP Succinate, no nucleotide Succinate, A D P Succinate, ATP Succinate, A D P + ATP
15 16 75 119 68 155
150 20[ ~ . HYDROXY-
/
SUCCtNATE
IO
o
0
,
1
100
,l
:::L
/
SO
E~O" a
- KETOGLUTARATE i
~0
40 2o I
I
I
20 TIME IN MINUTES
40
0 20
40
TIME IN MINUTES
Fig. i. Respiration of cardiac-muscle particles. Composition of the m e d i u m as in Table I, w i t h 0.6 m M A D P and ATP. Curves I, I I and I I I obtained with p r e p a r a t i o n s extracted for 24, 48 and 72 h respectively.
Biochim. Biopkys. Acta, 62 (1962) 525-533
528
R. J . G U I L L O R Y , W . F. H . M. M O M M A E R T S ,
K. U C H I D A
No additional cofactors, DPN, TPN, or cytochrome c, were required, and such additions, when made, had no marked effect. The respiration was inhibited by cyanide and, in phosphate media, by fluoride.
Oxidative phosphorylation Phosphorylation was determined by analysis of the vessel contents after a respiration measurement, after deproteinization with perchloric acid. It was measured in the presence of glucose and hexokinase, and expressed in terms of phosphate uptake or of the production of glucose 6-phosphate. The particles, during the washing process, were apparently freed of glycolytic enzymes to a sufficient extent, so that glucose 6-phosphate was not further converted to a significant degree. We have also used deoxyglucose with hexokinase as a trapping agent; if sufficient hexokinase was added, this procedure gave results identical to those with glucose. TABLE
II
OXIDATIVE PHOSPHORYLATION IN HEART PARTICLES WITH 0~-KETOGLUTARATE C o m p o s i t i o n of t h e m e d i u m (cf. SLATER AND HOLTONg), p H 7 ' 4 , 3 0 m M K - p h o s p h a t e , 5 m M MgClz, 5 m M a - k e t o g l u t a r a t e , 3 0 m M g l u c o s e , 0 . 6 m M A D P , 2 0 0 u n i t s h e x o k i n a s e , 5 ° m g o f h e a r t p a r t i c l e s , t o t a l v o l . 1.2 m l . l,moles Pi added to tissue O 20 3° 4° 5°
i~moles glucose 6-phosphate ]ormed
I~moles O, cons*4med 2.90 lo.30 9.O8 lO.42 lo.18
2.94 9.2o 9.O5 9.65 IO.IO
TABLE
P: 0 ratio
I.OI O.9O 1.0o 0.92 o.99
III
RESPIRATION AND PHOSPHORYLATION IN HEART PARTICLES WITH SUCCINATE C o m p o s i t i o n of t h e m e d i u m a s i n T a b l e I,moles succinate added O 5 25 5° 75
II, e x c e p t Izmol~s 03 consumed O 4.20 .10.28 9-54 6.58
f o r t h e r e p l a c e m e n t of k e t o g l u t a r a t e l~moles glucose 6-phosphate formed
P: 0 ratio
O O.612 O.831 0.757 °.494
-O.149 0.08O 0.079 °-°75
by succinate.
Some typical results are summarized in Tables II and III. In the first, it is shown that variation of the added phosphate quantity does not affect the P: 0 ratio, although with only the intrinsic phosphate present the respiratory rate is greatly diminished. In the second, it is shown that with succinate, the yield of phosphorylation is optimal at low substrate concentrations, below that required for maximal respiration. Generally, P : O ratios of O.l-O.3 were obtained for succinate, approx. I.O for **-ketoglutarate oxidation. B i o c h i m . B i o p h y s . A c t a , 62 (1962) 5 2 5 - 5 3 3
529
SUBVITAL MUSCLE PREPARATION
Integrity of the mitochondria These low values of the P : 0 ratios, compared to those of 2.0 and 4.0 for succinate and a-ketoglutarate that are said to be theoretically expected, or of I.O and 2.5 as are readily obtained with cardiac mitochondria s, require an explanation. One reason might be a functional destruction of the mitochondria b y some feature of the extraction technique. The following experiment was designed to test this possibility. R a t hearts were minced as described, and the material divided into three portions. From one, mitochondria were prepared directly, following the procedure of SLATERAND HOLTON9. TABLE IV DETERMINATION OF OXIDATIVE PHOSPHORYLATION IN MITOCHONDRIA AND EXTRACT]~D MINCED HEART-MUSCLE PARTICLES C o m p o s i t i o n of m e d i u m as in T a b l e I I . D u r a t i o n of e x p e r i m e n t , I h.
I. F r e s h m i t o c h o n d r i a I I . 24-h e x t r a c t e d p a r t i c l e s I I I . M i t o c h o n d r i a p r e p a r e d from I I
I~moles 03 used
l, moles glucose 6~phosphate formed
P: 0 ratio
3.29 4.53 5.16
7.46 3.44 8.74
2.27 o.76 1.69
The others were extracted with sucrose as usual; of these, one portion was used for determining the P : O ratio directly, while mitochondria were now prepared from the other, and their P : O ratio measured. The results (Table IV) show that directly prepared mitochondria display a P : 0 ratio similar to that commonly reported and that, again, in the extracted particles it is much less. While an effort was made to match the amount of mitochondria to that of the quantity of tissue in Expt. 2, this was only approximate, and the absolute amounts of oxygen consumed were not meant to be compared, Mitochondria prepared afterwards from the minced particles (Expt. 3) gave again the higher P : O ratio. This, it is true, was below that in Expt. I, but we believe (without especially designed experiment in that direction) that this decrease is less than would be expected for isolated mitochondria during the same period of time. I t is concluded, therefore, that the mitochondria in our preparation are fully preserved, and that the low phosphorylation yield is due to another cause, probably the hydrolysis of ATP by the myofibrillar ATPase which represents such a large part of the preparation.
Contractile phenomena in minced-tissue particles While the prismatic tissue particles evidently do not lend themselves to mechanical measurements, their contraction can be qualitatively detected by judging the sediment volume after centrifugation of the suspension. This has, among others, been applied to the contents of Warburg vessels after a manometric run. As in the "gel volume" changes studied b y MARSI-ITMin the discovery of the relaxation factor, contraction leads to a reduction of particle volume. Fig. 2 shows an example of such measurement, illustrating that with an oxidizable substrate, contraction is independent of externally added ATP; this experiment parallels that of Table I in suggesting that some ADP was retained, and capable of participating in metabolism B i o c h i m . B i o p h y s . A c t a , 62 (1962) 525-533
530
R.J. GUILLORY, W. F. H. M. MOMMAERTS, K. UCHIDA
a n d contraction, which will not be universally true. While this m e t h o d is useful for such observations, it is of course less informative t h a n the explicit m e a s u r e m e n t of contraction in sucrose-extracted fiber bundles.
Contractile responses and oxygen consumption of the sucrose-extracted fiber bundle A selection of various types of experiments will be described to characterize the behavior of the preparation. Those presented in Figs. 3 a n d 4 were done with pigeon breast muscle. This did not contract with A D P alone, b u t did respond moderately, after a brief lag period, when a-ketoglutarate was added. S u b s e q u e n t addition of A T P caused relaxation, p r o m p t l y reversed b y the addition of calcium, which b r o u g h t out the full contractile response. Thus, the preparation displays the m a j o r features f o u n d in the glycerol-extracted preparation with relaxation-factor activity. The dependence of these responses u p o n respiration has been shown in two ways. On the one hand, the described responses do not occur in the presence of cyanide
WI~
CONTRACTION WITH SUCCINATE
o
20
o
o
~ -o
~1
o
I0
•
•
CONTRACTION
I
0. 0(~
I
0
I I0
I
I 20
I
I
310
concn. (f'/ x 10 "s)
ATP
Fig. 2. Contraction of minced heart-muscle particles, as judged by sediment volume. Description in the text. Composition of medium as in Table I . F-
/io
I
/
,
~o
•
/,'?" //
zW
7
0 :Z
~
°
~" o
,
T
'
4ram
._//m®
0 . S m M Co
~------'D-- - - o - - - c . o
T: 4 mM ADP i 0
T ATP
~ a-KG
8mht i i
I 2 TIME
4 m h f ATP I 3 IN
T 0.Smhf I 4
Co I 5
r 6
I 7
MINUTES
Fig. 3. Contractile responses of a 2-day extracted pigeon breast-muscle preparation in the presence of oxygen, as such ( Q - - O ) and with io mM cyanide (O---O). Medium: io mM K-phosphate {pH 7.3), 5 mM MgSO4, 3o mM KF and 5 mM AMP (the latter two substances serving to reduce myokinase activity), Additions as indicated by arrows. Shortening expressed as percentage of the original length, load o.I g.
Biochim. Biophys. Acta, 62 (1962) 525-533
531
SUBVITAL MUSCLE PREPARATION
(Fig. 3), although the cyanide-poisoned muscle contracts fully with ATP and Ca. On the other hand (Fig. 4), when ADP and oxidizable substrate are given anaerobically, no response ensues until after the admission of oxygen. As an example of the simultaneous measurement of contraction and oxygen consumption, we refer to an experiment on rabbit heart muscle (Fig. 5). This had a small and declining intrinsic respiration, not raised by ADP, but greatly enhanced by ~-ketoglutarate, which also initiated contraction after a brief delay. At this occasion, we restrict ourselves, to the descriptive presentation of these survey experiments, quantitative work now being pursued.
2¢
)=. o
/
ANAEROBIC
w
/
°.2
÷
T
8 rn/~fa-KG
4rnh/ ADP
I
TIME IN MINUTES
Fig. 4- Contractile r e s p o n s e s of a 4 - d a y e x t r a c t e d pigeon b r e a s t - m u s c l e p r e p a r a t i o n in t h e absence a n d presence of oxygen. Details as in Fig. 3. 4C 490
1.12 mp Oxygen/ml/rnln
3¢
420 0~ 6.3 rnp Oxygen/rnl//rnJn
::L
I,-
z
o
,~
¢
9
z bJ
/ I
0
•
l,
i~
i •
IO
TiME
o~
15
I
20
215
310
iN M I N U T E S
Fig. 5. O x y g e n c o n s u m p t i o n ( c o n t i n u o u s c u r v e a n d r i g h t - h a n d ordinate) a n d c o n t r a c t i o n (curve w i t h m e a s u r i n g p o i n t s a n d l e f t - h a n d ordinate) of r a b b i t h e a r t - m u s c l e strip (length 15 m m , a v e r a g e cross section 2 m m 2) e x t r a c t e d for 6 d a y s in o.88 M sucrose m e d i u m followed b y i d a y in o.32 M sucrose w i t h o u t E D T A . M e d i u m : 3o m M glycylglycine (pH 7.4), IO m M p h o s p h a t e (pH 7.4), 5 m M MgSO4, 25 m M K F , 2.6 m M A M P ; 8o m M t o t a l K a n d N a ions. L o a d 0.2 g, t e m p . 25 °.
Biochim. Biophys. Acta, 62 (1962) 525-533
532
R. J. GUILLORY, W. F. H. M. MOMMAERTS, K. UCHIDA
DISCUSSION We believe that the type of preparation described in this paper is of interest, as an example of levels of organization intermediate between those representing the integrity of the whole cell, and those constituting lower degrees of biochemical complexity. Evidently, there is a broad spectrum of such "subvital" preparations, not sharply delineated on either side of the range. As the simplest case we might regard the glycerol-extracted muscle preparation with an added enzyme such as creatine kinase 11, and also systems with retained or added relaxation factor. On the opposite end of the scale, there would be surviving muscle with altered membrane properties, e.g. potassium-depolarized muscle with or without calcium 1~. A systematic conceptualization and experimental investigation of the systems of intermediate complexity m a y be highly rewarding, in establishing which biochemical regulations depend on higher or lower levels of biochemical architecture. Such investigations do not need to be restricted to muscle. Our work took its origin in an investigation by BIRO AND SZENT-GY6RGY113who measured the succinic oxidase activity of washed ground muscle. This was found to display a sensitivity toward K and other ions reminiscent of the precipitation and ATPase activity of actomyosin, and was enhanced b y ATP. These connections remained unspecified, but prompted our work, a preliminary communication of which appeared in 1958 (see ref. 14). Recently, WATANABE AND PACKER15 described a system in which a glycerol-extracted muscle preparation is bathed in a suspension of respiring mitochondria. This allows an elegant measurement of the ATP consumed by the fiber, but the ATP still has to reach the fiber b y diffusion from the outside. The results presented so far are not free of discrepancies and ambiguities. Thus, the cardiac-muscle preparation with added substrate showed respiration and contraction without added nucleotide, although additional ADP enhanced the oxygen uptake. The pigeon b r e a s t - a n d other skeletal-muscle preparations were entirely dependent on added ADP for their contraction and largely for their oxidation. This m a y well reflect differences between tissues with respect to nucleotide retention during the extraction, on which we have no explicit information. The question arises, however, to what extent this bound ADP participates in metabolism and contractility. In our skeletal-muscle preparation, it would be either absent or inaccessible; the latter would agree with the tight binding of ADP to fibrous actin 16 in which condition it is not accessible to enzymic action~7, is. In cardiac muscle, on the other hand, we would have to conclude that the actin-bound nucleotide is more accessible, or that nucleotide is retained in other forms. It has been shown that, while mitochondria isolated subsequently from our preparation display a normal P : O ratio, the particles themselves give only low yields of measurable phosphorylation. Undoubtedly, this is due to splitting of the formed ATP by the myofibrils (cf. the work of NIEMEYER A N D JALIL on mitochondrial respiration in the presence of added ATPase19). The competition for ATP by the myofibrils with the hexokinase-glucose acceptor system seems very effective; presumably, this is completely attributable to the close proximity of the fibrils to the mitochondria, and must not be looked upon as indicative, b y itself, of direct connections between the mitochondria and the myofibrils. The highest respiratory rates observed were of the order of magnitude of 3 t~moles Biochim. Biophys. Acta, 62 (I962) 525-533
SUBVITAL MUSCLE PREPARATION
533
OJg/min at the experimental temperature, or about IO/*moles/g/min at 37 °. EVANS2° has found an oxygen consumption in canine hearts in moderate activity corresponding to 2.5/,moles of 0Jg/min. Even when considering the presumably higher metabolism of the rat heart in full activity, it can nevertheless be concluded that the total possible oxidation rate in cardiac tissue is reasonably well accounted for in our preparation. ACKNOWLEDGEMENT
This investigation was supported by research grant No. H-3o67 from the National Heart Institute of the National Institutes of Health. REFERENCES 1 A. SZENT-GY6RGYI, Biol. Bull., 96 (1949) 14o. 2 M. H. PAUL AND E. SPERLING, Proc. Soc. Exptl. Biol. Med., 79 (19521 352. 3 W. C. STADIE AND B. C. RIGGS, J. Biol. Chem., 154 (1944) 687. 4 H. MclLWAIN AND H. L. BUDDLE, Biochem. J., 53 ~I953) 412. 5 M. O. SCHILLING, Rev. of Sci. Instr., 31 (I96O) i i . s S. V. PERRY, Bioehem. J., 51 (1952) 495. 7 K. SERAYDARIAN, W. 1V~OMMAERTSAND A. WALLNER, Biochim. Biophys. Acta, in the press. 8 E. C. SLATER, in G. BOURNE, The Structure and Function of Muscle, Vol. 2, Academic Press, Inc., New York, I96O, p. lO 5. 9 E. C. SLATER AND F. A. HOLTON, Biochem. J., 56 (1954) 28. 10 B. B. MARSH, Bioehim. Biophys. Acta, 9 (1952) 247. n E. BOZLER, J. Gen. Physiol., 37 (1953) 63. 13 L. KAYE AND V~. MOMMAERTS, J. Gen. Physiol., 44 (196o) 2. 13 N. A. BIRO AND A. G. SZENT-G¥6RGYI, Hungarian Physiol. Acta, I (1946) 9. 14 R. J. GUILLORY AND W. MOMMAERTS, Circulation Research, 6 (1958) 5. 15 S. WATANABE AND L. PACKER, J. Biol. Chem., 236 (1961) 12Ol. le W. F. H. M. MOMMAERTS, J. Biol. Chem., I98 (1952) i. 1T R. C. STROHMAN, Biochim. Biophys. Acta, 32 ~I939) 436. 18 A. MARTONOSI, H. GOUVEA AND J. GERGELY, J. Biol. Chem., 235 (196o) 17oo. 13 H. NIEMEYER AND J. JALIL, Biochim. Biophys. Acta, 12 (1953) 492. 2o C. LOVATT EVANS, in Lovatt Evans' Recent Advances in Physiology, 5 (1936) 33.
Bioehim. Biophys. Acta, 62 (1962) 525-533
525
BBA 3715
A S U B V I T A L MUSCLE P R E P A R A T I O N W I T H C O N T R A C T I L E AND O X I D A T I V E - P H O S P H O R Y L A T I V E
ACTIVITY*
RICHARD J. GUILLORY**, W. F. H. M. MOMMAERTS AND KOKI UCHIDA***
Department of Medicine, The Los Angeles County Heart Association, Cardiovascular Research Laboratory, The University of California, Los Angeles, Calif. (U.S.A.) Received January 3oth, 1962)
SUMMARY
It has been shown that by a process of extraction with sucrose media, muscle preparations can be obtained which display contractility as well as oxidation and phosphorylation. The P:O ratio is below that of isolated mitochondria, presumably because of continuous splitting of the synthesized ATP by adjacent myofibrils. This synthesized ATP can be used for contraction, thus contraction can be elicited with small amounts of ADP together with oxygen and oxidizable substrate.
INTRODUCTION
The glycerol-extracted muscle preparation, developed by SZENT-GYrRGYI 1, represents, by its contractile response towards extraneously added ATP, a basic feature of the contractile mechanism of muscle. Dependent on the mode of preparation, it may or may not retain its relaxing-factor activity; otherwise, although it may hold some retained enzymes as accidental contaminants, it has no metabolism other than the decomposition of added ATP. Since it has no excitability, its activity depends on the addition of substances. The preparation described in this paper is one step higher in complexity, in that the biochemical integrity of its mitochondria has been preserved. Consequently, it can respire when oxidizable substrates are added, and it can contract n o t only with added ATP, but also with the ATP formed as the result of its own metabolism. Like the glycerol-extracted fiber, it still is inexcitable, and its activity is still contingent upon extraneously provided materials; but a greater variety of biochemical responses becomes possible. We have developed this preparation in order to study whether the relations between mechanical parameters and biochemical kinetics, such as the effect of length upon metabolic rate, are determined by factors dependent on the integrity of the cell; and also for making the study of contraction and relaxation phenomena * For preliminary publication see R. J. GUILLORY AND W. F. H. M. MOMMAERTS, Circulation
Research, 6 [1958) 73 o. ** Present address: University of Amsterdam, Department of Physiological Chemistry, Jonas Daniel Meijerplein 3, Amsterdam [C) (The Netherlands). *** Former address: Department of Physiology, Sapporo Medical College, Sapporo (Japan).
Biochim. Biophys. Acta, 62 (1962) 525-533
526
R . J . GUILLORY, W. F. H. M. MOMMAERTS, K. UCHIDA
less dependent on the limitations posed by diffusion. This publication describes the general features of the preparation, as a preliminary toward the quantitative investigations now in progress. EXPERIMENTAL
For the purpose of surveying both its biochemical and its contractile properties, our preparation was designed ill tWO forms : the one a suspension of small particles suitable for manometric experiments; the other in the familiar form of a fiber bundle of which the contractile responses can be measured. In later work, only briefly alluded to in this paper, both contraction and oxygen consumption were measured on the same fiber, but the dual methodology was found advantageous for the initial stage of the work. Muscles from various sources have been used; the extracted particles are best prepared from tissues that can be sliced, i.e. cardiac muscle; the fiber bundles ale best made from skeletal muscles, preferably from those with a significant mitochondrial count 2. We shall specifically describe the handling of the two objects on which most of the communicated experiments were performed.
Minced rat heart muscle Rat hearts were excised, rinsed with Ringer solution and blotted, and cut into sections of 2 mm thickness with a STADIE-RIGGSs tissue slicer. These were then placed on filter paper wetted with saline on the object holder of a MclLWAIN4 tissue chopper, and cut successively along two perpendicular directions, thus yielding prismatic particles of 0.2 × 0.2 × 2 mm. These were freed from filter-paper shreds and from larger tissue fragments by suspending and decanting, and were then extracted with 0.88 or 0.32 M sucrose lor varying lengths of time, usually 48 h at o °.
Pigeon breast muscle fiber bundle A preparation entirely analogous to the SZENT-GY6RGYI model was made by extraction with 0.88 M sucrose instead of aqueous glycerol, with or without other constituents added to the basic sucrose medium; often, the composition was 0.88 M sucrose, o.ooi M EDTA. This could be applied to all muscles studied: rat and rabbit psoas, heart muscle and pectoral muscle of the pigeon. For mechanical measurements, a fiber was mounted in a suitable cuvet and connected to one of the isotonic or isometric variable capacitance gages of this laboratory 5, the output being recorded on a Brush Mark II recorder or a Texas Instruments Corporation Recti-Riter. For the simultaneous recording of oxygen consumption, an oscillating platinum electrode was used, but this part of the methodology is still being improved, and will be described fully at a later occasion.
Oxygen consumption by minced tissue Suspensions of prismatic heart-muscle particles were measured into Warburg vessels, and their oxygen consumption was determined by the usual methods, at 27 °. The composition of the medium differed in individual experiments. In typical cases, it consisted of I.O ml of a solution of o.Io M KC1, 0.005 M MgC12 and 0.04 M glycylglycine (pH 7.o), with substrate and nucleotide additions; to which was added 0.20 ml of tissue suspension in 0.88 M sucrose. Biochim. Biophys. Acta, 62 (I962) 525-533
SUBVITAL MUSCLE PREPARATION
527
Such extracted particles showed little or no endogenous respiration, but responded markedly to the addition of oxidizable substrates. Table I illustrates this, and also shows that significant respiration is obtained without the addition of nucleotides, but that it is enhanced by ADP. Apparently, enough ADP was retained in these preparations6, 7 to act as a phosphate acceptor, and ATP was not dephosphorylated rapidly enough to make a significant difference. On the other hand, the addition of ADP led to a direct increase in the phosphate-acceptor concentration. This is only one of several possible results, and later work with skeletal-muscle bundles led to preparations which were dependent on added ADP. The effect of succinate rose sharply at low concentrations, and displayed a broad maximum above 0.02 M. Fig. i shows respiration experiments with different substrates. It is seen that =-ketoglutarate and ~-hydroxybutyrate give lower respiratory rates and faster inactivation than succinate. The differences in rates may be ascribed to different yields of phosphorylation, but the faster inactivation (which was not reversed by added DPN) is, in addition, indicative of the loss of required enzymes. TABLE I
RESPIRATION OF CARDIAC-MUSCLEPARTICLES 5 ° m g of tissue per W a r b u r g vessel, extracted 24 h in o.88 M sucrose; composition of m e d i u m : o. IO M KC1, 0.0o 5 M MgClz, 0.08 M succinate, 0.04 M glycylglycine at p H 7.0; nucleotides, w h e n added, 0.3" lO -8 M each; gas phase, oxygen; t e m p e r a t u r e , 27°; m e a s u r e m e n t over a period of 3 ° min.
Oxygen (tat/l*) No succinate, A D P No succinate, ATP Succinate, no nucleotide Succinate, A D P Succinate, ATP Succinate, A D P + ATP
15 16 75 119 68 155
150 20[ ~ . HYDROXY-
/
SUCCtNATE
IO
o
0
,
1
100
,l
:::L
/
SO
E~O" a
- KETOGLUTARATE i
~0
40 2o I
I
I
20 TIME IN MINUTES
40
0 20
40
TIME IN MINUTES
Fig. i. Respiration of cardiac-muscle particles. Composition of the m e d i u m as in Table I, w i t h 0.6 m M A D P and ATP. Curves I, I I and I I I obtained with p r e p a r a t i o n s extracted for 24, 48 and 72 h respectively.
Biochim. Biopkys. Acta, 62 (1962) 525-533
528
R. J . G U I L L O R Y , W . F. H . M. M O M M A E R T S ,
K. U C H I D A
No additional cofactors, DPN, TPN, or cytochrome c, were required, and such additions, when made, had no marked effect. The respiration was inhibited by cyanide and, in phosphate media, by fluoride.
Oxidative phosphorylation Phosphorylation was determined by analysis of the vessel contents after a respiration measurement, after deproteinization with perchloric acid. It was measured in the presence of glucose and hexokinase, and expressed in terms of phosphate uptake or of the production of glucose 6-phosphate. The particles, during the washing process, were apparently freed of glycolytic enzymes to a sufficient extent, so that glucose 6-phosphate was not further converted to a significant degree. We have also used deoxyglucose with hexokinase as a trapping agent; if sufficient hexokinase was added, this procedure gave results identical to those with glucose. TABLE
II
OXIDATIVE PHOSPHORYLATION IN HEART PARTICLES WITH 0~-KETOGLUTARATE C o m p o s i t i o n of t h e m e d i u m (cf. SLATER AND HOLTONg), p H 7 ' 4 , 3 0 m M K - p h o s p h a t e , 5 m M MgClz, 5 m M a - k e t o g l u t a r a t e , 3 0 m M g l u c o s e , 0 . 6 m M A D P , 2 0 0 u n i t s h e x o k i n a s e , 5 ° m g o f h e a r t p a r t i c l e s , t o t a l v o l . 1.2 m l . l,moles Pi added to tissue O 20 3° 4° 5°
i~moles glucose 6-phosphate ]ormed
I~moles O, cons*4med 2.90 lo.30 9.O8 lO.42 lo.18
2.94 9.2o 9.O5 9.65 IO.IO
TABLE
P: 0 ratio
I.OI O.9O 1.0o 0.92 o.99
III
RESPIRATION AND PHOSPHORYLATION IN HEART PARTICLES WITH SUCCINATE C o m p o s i t i o n of t h e m e d i u m a s i n T a b l e I,moles succinate added O 5 25 5° 75
II, e x c e p t Izmol~s 03 consumed O 4.20 .10.28 9-54 6.58
f o r t h e r e p l a c e m e n t of k e t o g l u t a r a t e l~moles glucose 6-phosphate formed
P: 0 ratio
O O.612 O.831 0.757 °.494
-O.149 0.08O 0.079 °-°75
by succinate.
Some typical results are summarized in Tables II and III. In the first, it is shown that variation of the added phosphate quantity does not affect the P: 0 ratio, although with only the intrinsic phosphate present the respiratory rate is greatly diminished. In the second, it is shown that with succinate, the yield of phosphorylation is optimal at low substrate concentrations, below that required for maximal respiration. Generally, P : O ratios of O.l-O.3 were obtained for succinate, approx. I.O for **-ketoglutarate oxidation. B i o c h i m . B i o p h y s . A c t a , 62 (1962) 5 2 5 - 5 3 3
529
SUBVITAL MUSCLE PREPARATION
Integrity of the mitochondria These low values of the P : 0 ratios, compared to those of 2.0 and 4.0 for succinate and a-ketoglutarate that are said to be theoretically expected, or of I.O and 2.5 as are readily obtained with cardiac mitochondria s, require an explanation. One reason might be a functional destruction of the mitochondria b y some feature of the extraction technique. The following experiment was designed to test this possibility. R a t hearts were minced as described, and the material divided into three portions. From one, mitochondria were prepared directly, following the procedure of SLATERAND HOLTON9. TABLE IV DETERMINATION OF OXIDATIVE PHOSPHORYLATION IN MITOCHONDRIA AND EXTRACT]~D MINCED HEART-MUSCLE PARTICLES C o m p o s i t i o n of m e d i u m as in T a b l e I I . D u r a t i o n of e x p e r i m e n t , I h.
I. F r e s h m i t o c h o n d r i a I I . 24-h e x t r a c t e d p a r t i c l e s I I I . M i t o c h o n d r i a p r e p a r e d from I I
I~moles 03 used
l, moles glucose 6~phosphate formed
P: 0 ratio
3.29 4.53 5.16
7.46 3.44 8.74
2.27 o.76 1.69
The others were extracted with sucrose as usual; of these, one portion was used for determining the P : O ratio directly, while mitochondria were now prepared from the other, and their P : O ratio measured. The results (Table IV) show that directly prepared mitochondria display a P : 0 ratio similar to that commonly reported and that, again, in the extracted particles it is much less. While an effort was made to match the amount of mitochondria to that of the quantity of tissue in Expt. 2, this was only approximate, and the absolute amounts of oxygen consumed were not meant to be compared, Mitochondria prepared afterwards from the minced particles (Expt. 3) gave again the higher P : O ratio. This, it is true, was below that in Expt. I, but we believe (without especially designed experiment in that direction) that this decrease is less than would be expected for isolated mitochondria during the same period of time. I t is concluded, therefore, that the mitochondria in our preparation are fully preserved, and that the low phosphorylation yield is due to another cause, probably the hydrolysis of ATP by the myofibrillar ATPase which represents such a large part of the preparation.
Contractile phenomena in minced-tissue particles While the prismatic tissue particles evidently do not lend themselves to mechanical measurements, their contraction can be qualitatively detected by judging the sediment volume after centrifugation of the suspension. This has, among others, been applied to the contents of Warburg vessels after a manometric run. As in the "gel volume" changes studied b y MARSI-ITMin the discovery of the relaxation factor, contraction leads to a reduction of particle volume. Fig. 2 shows an example of such measurement, illustrating that with an oxidizable substrate, contraction is independent of externally added ATP; this experiment parallels that of Table I in suggesting that some ADP was retained, and capable of participating in metabolism B i o c h i m . B i o p h y s . A c t a , 62 (1962) 525-533
530
R.J. GUILLORY, W. F. H. M. MOMMAERTS, K. UCHIDA
a n d contraction, which will not be universally true. While this m e t h o d is useful for such observations, it is of course less informative t h a n the explicit m e a s u r e m e n t of contraction in sucrose-extracted fiber bundles.
Contractile responses and oxygen consumption of the sucrose-extracted fiber bundle A selection of various types of experiments will be described to characterize the behavior of the preparation. Those presented in Figs. 3 a n d 4 were done with pigeon breast muscle. This did not contract with A D P alone, b u t did respond moderately, after a brief lag period, when a-ketoglutarate was added. S u b s e q u e n t addition of A T P caused relaxation, p r o m p t l y reversed b y the addition of calcium, which b r o u g h t out the full contractile response. Thus, the preparation displays the m a j o r features f o u n d in the glycerol-extracted preparation with relaxation-factor activity. The dependence of these responses u p o n respiration has been shown in two ways. On the one hand, the described responses do not occur in the presence of cyanide
WI~
CONTRACTION WITH SUCCINATE
o
20
o
o
~ -o
~1
o
I0
•
•
CONTRACTION
I
0. 0(~
I
0
I I0
I
I 20
I
I
310
concn. (f'/ x 10 "s)
ATP
Fig. 2. Contraction of minced heart-muscle particles, as judged by sediment volume. Description in the text. Composition of medium as in Table I . F-
/io
I
/
,
~o
•
/,'?" //
zW
7
0 :Z
~
°
~" o
,
T
'
4ram
._//m®
0 . S m M Co
~------'D-- - - o - - - c . o
T: 4 mM ADP i 0
T ATP
~ a-KG
8mht i i
I 2 TIME
4 m h f ATP I 3 IN
T 0.Smhf I 4
Co I 5
r 6
I 7
MINUTES
Fig. 3. Contractile responses of a 2-day extracted pigeon breast-muscle preparation in the presence of oxygen, as such ( Q - - O ) and with io mM cyanide (O---O). Medium: io mM K-phosphate {pH 7.3), 5 mM MgSO4, 3o mM KF and 5 mM AMP (the latter two substances serving to reduce myokinase activity), Additions as indicated by arrows. Shortening expressed as percentage of the original length, load o.I g.
Biochim. Biophys. Acta, 62 (1962) 525-533
531
SUBVITAL MUSCLE PREPARATION
(Fig. 3), although the cyanide-poisoned muscle contracts fully with ATP and Ca. On the other hand (Fig. 4), when ADP and oxidizable substrate are given anaerobically, no response ensues until after the admission of oxygen. As an example of the simultaneous measurement of contraction and oxygen consumption, we refer to an experiment on rabbit heart muscle (Fig. 5). This had a small and declining intrinsic respiration, not raised by ADP, but greatly enhanced by ~-ketoglutarate, which also initiated contraction after a brief delay. At this occasion, we restrict ourselves, to the descriptive presentation of these survey experiments, quantitative work now being pursued.
2¢
)=. o
/
ANAEROBIC
w
/
°.2
÷
T
8 rn/~fa-KG
4rnh/ ADP
I
TIME IN MINUTES
Fig. 4- Contractile r e s p o n s e s of a 4 - d a y e x t r a c t e d pigeon b r e a s t - m u s c l e p r e p a r a t i o n in t h e absence a n d presence of oxygen. Details as in Fig. 3. 4C 490
1.12 mp Oxygen/ml/rnln
3¢
420 0~ 6.3 rnp Oxygen/rnl//rnJn
::L
I,-
z
o
,~
¢
9
z bJ
/ I
0
•
l,
i~
i •
IO
TiME
o~
15
I
20
215
310
iN M I N U T E S
Fig. 5. O x y g e n c o n s u m p t i o n ( c o n t i n u o u s c u r v e a n d r i g h t - h a n d ordinate) a n d c o n t r a c t i o n (curve w i t h m e a s u r i n g p o i n t s a n d l e f t - h a n d ordinate) of r a b b i t h e a r t - m u s c l e strip (length 15 m m , a v e r a g e cross section 2 m m 2) e x t r a c t e d for 6 d a y s in o.88 M sucrose m e d i u m followed b y i d a y in o.32 M sucrose w i t h o u t E D T A . M e d i u m : 3o m M glycylglycine (pH 7.4), IO m M p h o s p h a t e (pH 7.4), 5 m M MgSO4, 25 m M K F , 2.6 m M A M P ; 8o m M t o t a l K a n d N a ions. L o a d 0.2 g, t e m p . 25 °.
Biochim. Biophys. Acta, 62 (1962) 525-533
532
R. J. GUILLORY, W. F. H. M. MOMMAERTS, K. UCHIDA
DISCUSSION We believe that the type of preparation described in this paper is of interest, as an example of levels of organization intermediate between those representing the integrity of the whole cell, and those constituting lower degrees of biochemical complexity. Evidently, there is a broad spectrum of such "subvital" preparations, not sharply delineated on either side of the range. As the simplest case we might regard the glycerol-extracted muscle preparation with an added enzyme such as creatine kinase 11, and also systems with retained or added relaxation factor. On the opposite end of the scale, there would be surviving muscle with altered membrane properties, e.g. potassium-depolarized muscle with or without calcium 1~. A systematic conceptualization and experimental investigation of the systems of intermediate complexity m a y be highly rewarding, in establishing which biochemical regulations depend on higher or lower levels of biochemical architecture. Such investigations do not need to be restricted to muscle. Our work took its origin in an investigation by BIRO AND SZENT-GY6RGY113who measured the succinic oxidase activity of washed ground muscle. This was found to display a sensitivity toward K and other ions reminiscent of the precipitation and ATPase activity of actomyosin, and was enhanced b y ATP. These connections remained unspecified, but prompted our work, a preliminary communication of which appeared in 1958 (see ref. 14). Recently, WATANABE AND PACKER15 described a system in which a glycerol-extracted muscle preparation is bathed in a suspension of respiring mitochondria. This allows an elegant measurement of the ATP consumed by the fiber, but the ATP still has to reach the fiber b y diffusion from the outside. The results presented so far are not free of discrepancies and ambiguities. Thus, the cardiac-muscle preparation with added substrate showed respiration and contraction without added nucleotide, although additional ADP enhanced the oxygen uptake. The pigeon b r e a s t - a n d other skeletal-muscle preparations were entirely dependent on added ADP for their contraction and largely for their oxidation. This m a y well reflect differences between tissues with respect to nucleotide retention during the extraction, on which we have no explicit information. The question arises, however, to what extent this bound ADP participates in metabolism and contractility. In our skeletal-muscle preparation, it would be either absent or inaccessible; the latter would agree with the tight binding of ADP to fibrous actin 16 in which condition it is not accessible to enzymic action~7, is. In cardiac muscle, on the other hand, we would have to conclude that the actin-bound nucleotide is more accessible, or that nucleotide is retained in other forms. It has been shown that, while mitochondria isolated subsequently from our preparation display a normal P : O ratio, the particles themselves give only low yields of measurable phosphorylation. Undoubtedly, this is due to splitting of the formed ATP by the myofibrils (cf. the work of NIEMEYER A N D JALIL on mitochondrial respiration in the presence of added ATPase19). The competition for ATP by the myofibrils with the hexokinase-glucose acceptor system seems very effective; presumably, this is completely attributable to the close proximity of the fibrils to the mitochondria, and must not be looked upon as indicative, b y itself, of direct connections between the mitochondria and the myofibrils. The highest respiratory rates observed were of the order of magnitude of 3 t~moles Biochim. Biophys. Acta, 62 (I962) 525-533
SUBVITAL MUSCLE PREPARATION
533
OJg/min at the experimental temperature, or about IO/*moles/g/min at 37 °. EVANS2° has found an oxygen consumption in canine hearts in moderate activity corresponding to 2.5/,moles of 0Jg/min. Even when considering the presumably higher metabolism of the rat heart in full activity, it can nevertheless be concluded that the total possible oxidation rate in cardiac tissue is reasonably well accounted for in our preparation. ACKNOWLEDGEMENT
This investigation was supported by research grant No. H-3o67 from the National Heart Institute of the National Institutes of Health. REFERENCES 1 A. SZENT-GY6RGYI, Biol. Bull., 96 (1949) 14o. 2 M. H. PAUL AND E. SPERLING, Proc. Soc. Exptl. Biol. Med., 79 (19521 352. 3 W. C. STADIE AND B. C. RIGGS, J. Biol. Chem., 154 (1944) 687. 4 H. MclLWAIN AND H. L. BUDDLE, Biochem. J., 53 ~I953) 412. 5 M. O. SCHILLING, Rev. of Sci. Instr., 31 (I96O) i i . s S. V. PERRY, Bioehem. J., 51 (1952) 495. 7 K. SERAYDARIAN, W. 1V~OMMAERTSAND A. WALLNER, Biochim. Biophys. Acta, in the press. 8 E. C. SLATER, in G. BOURNE, The Structure and Function of Muscle, Vol. 2, Academic Press, Inc., New York, I96O, p. lO 5. 9 E. C. SLATER AND F. A. HOLTON, Biochem. J., 56 (1954) 28. 10 B. B. MARSH, Bioehim. Biophys. Acta, 9 (1952) 247. n E. BOZLER, J. Gen. Physiol., 37 (1953) 63. 13 L. KAYE AND V~. MOMMAERTS, J. Gen. Physiol., 44 (196o) 2. 13 N. A. BIRO AND A. G. SZENT-G¥6RGYI, Hungarian Physiol. Acta, I (1946) 9. 14 R. J. GUILLORY AND W. MOMMAERTS, Circulation Research, 6 (1958) 5. 15 S. WATANABE AND L. PACKER, J. Biol. Chem., 236 (1961) 12Ol. le W. F. H. M. MOMMAERTS, J. Biol. Chem., I98 (1952) i. 1T R. C. STROHMAN, Biochim. Biophys. Acta, 32 ~I939) 436. 18 A. MARTONOSI, H. GOUVEA AND J. GERGELY, J. Biol. Chem., 235 (196o) 17oo. 13 H. NIEMEYER AND J. JALIL, Biochim. Biophys. Acta, 12 (1953) 492. 2o C. LOVATT EVANS, in Lovatt Evans' Recent Advances in Physiology, 5 (1936) 33.
Bioehim. Biophys. Acta, 62 (1962) 525-533