Formation, isolation and composition of dense granules from mitochondria

256

BIOCHIMICA ET BIOPHYbICA AC'FA

BBA 25842

FORMATION, ISOLATION AND COMPOSITION OF DENSE GRANULES FROM MITOCHONDRIA EUGENE

C. W E I N B A C H

AND THI~;O1)t)R V()N B R A N D

Laboratory of Parasitic Diseases, Nal;onal lnstitt~te of Allergy a~d [nfecliotts l)isea.~es, Natio~ml lnslilutes of Heallh, U.S. l)elartment of Health, Edztcation and IVelfare, I3ethesda, Md. (t'.~;.A.I (Received March ioth, I907) (Revised m a n u s c r i p t received May 22nd, t()07)

SUMMARY

I. An investigation was undertaken to determine if the electron-dense granules which accumulate in mitochondria under conditions of massive Ca 2 -loading could be isolated and identified. 2. Four methods are described for the isolation of dense granules from rat liver mitochondria. Three of these methods involve chemical destruction of tile mitochondria and liberation of tile granules ; the fourth involves disruption by sonication. 3. The granules are anlorphous, and consist of both organic and inorganic constituents. 4. Incineration of the granules destroyed the organic nloietv and induced crystallization of the im/rganic constituents. 5. Chemical analysis disclosed that Ca 2+ and Pi are the major inorganic constituents. Mg2¢- and COa"- also are present in significant amounts. 6. X-ray diffraction analysis indicated that the patterns of the incinerated granules correspond to either hydroxyapatite, Calo(PO4)6(OH),, , whitlockite, Caa(PO4)2, or to a mixture of both. 7. The isolation of the granules enabled a study of some of the factors which influence this active ion translocation in a more direct way than was hitherto possible.

INTRODUCTION

One of tile energy-linked functions of isolated mitochondria is a capacity for ion translocation 1. Under appropriate conditions, massive amounts of divalent cations can be accumulated, and in the case of calcium, loading is accompanied by the uptake of orthophosphate and the formation of electron-dense granules within tile mitochondria ~ 4. Although this phenomenon has been studied extensively (for references, see CHANCES), the isolation and characterization of the granules have not heen described in detail. BRIERLY AND SLAUTTERBACK2 proposed that the granules were deposits of calcium phosphate, and RossI AND LEHNINGER6 found that the molar ratio of Ca: P taken up from the medium corresponded to that of hydroxyapatite. In a preliminary report 7, we described methods for the isolation of dense Biochi~2. Biophys..4eta, I4~ (1907) 256 26~

DENSE GRANULES FROM MITOCHONDRIA

257

granules from rat liver mitochondria after calcium loading. Analysis of the isolated material, after incineration to remove the organic components, showed that hydroxyapatite, Calo(PO4)e(OH)2, and whitlockite, Ca3(PO4) 2 were the major inorganic constituents. The present communication describes the effects of various experimental conditions on the yield and composition of the isolated granules, and presents the earlier findings in greater detail. METHODS

Incubation Mitochondria, isolated from rat livers, were incubated under conditions favorable for m a x i m u m accumulation of Ca 2+ and P I a s described by VASINGTON AND lVIVRPI~Y9. Each reaction vessel contained IO mM Tris-maleate buffer, p H 7.0; 4 mM PI; 8 mM NaC1; IO mM succinate; io mM MgCle; 3 mM ATP; and 2.5 mM CaC12 in a final volume of 15 ml. One ml of rat liver mitochondria, suspended in 0.25 M sucrose, and adjusted to contain 20 mg of protein, was added and the mixture gently shaken at 3 °° for 20 min. Isolation For each experiment, the combined contents of 30 vessels (unless specified otherwise) were chilled in an ice bath and centrifuged at IOOOO × g for IO min. The mitochondrial pellet (Fig. i) was washed once with 60 ml of chilled 0.25 M sucrose buffered at p H 7.0 with 5 mM Tris-maleate. Isolation of the granules was accomplished by one of the following procedures 7. (A) The washed mitochondrial pellet was suspended in 3.0 ml of ethylenediamine and heated at IOO° for 1. 5 h (ref. 13). During heating it was advantageous to redisperse the suspended material frequently. The suspension was centrifuged at 16000 × g for 5 rain, and the precipitate successively washed twice with water, once with absolute ethanol, and once with diethyl ether. The granules were air-dried overnight, then heated at IiO ° for i h prior to weighing. (B) The dense granules were liberated by suspending the mitochondrial pellet in 3 ml of IO % K O H and heating for 30 min at IOO°. The granules were collected by centrifugation, washed and dried as described above. (C) The mitochondrial pellet was made into a smooth paste with a minimum of water; 30 ml of 3 % sodium deoxycholate (pH 8.0) and 6 ml of I M NaOH were added, and the volume adjusted to 60 ml with water. To aid dissolution of the mitochondria, the suspension was kept at room temperature for 30 min during which time it was intermittently shaken. The released granules were removed by centrifugation, washed and dried as described above. (D) The mitochondrial pellet was suspended in 30 ml of 0.25 M sucrose and 15 ml portions were subjected to sonic oscillation in a 20 KC Branson sonifier, Model S-75, equipped with the o.5o-inch probe. The instrument was tuned to m a x i m u m intensity with a power output of approximately 6 amperes (power setting at 8). The temperature was maintained at 4-6 ° by immersing the sonication chamber* in a salt-ice bath. Examination of the suspension by phase-contrast microscopy revealed that adequate disruption of the mitochondria occurred after 3 min of sonication. The granules were collected by centrifugation, washed and dried as described above. * Rosett ref. 14).

Cooling Cell, Branson I n s t r u m e n t s , Incorporated, Danbury, Connecticut (see

Biochim. B ophys. Acta, 148 (1967) 256-266

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E. C, "~VEINB~CH, T. V(~N I¢I~AX[~

B Fig. I. E l e c t r o n m i c r o g r a p h s of m i t o c h o n d r i a i n c u b a ~ t h e absence and presence ()I" (]a ~ . A, C o n t r o l m i t o c h o n d r i a i n c u b a t e d u n d e r the c o n d i t i o n s described in the t e x t except t h a t ( a ( ] 2 was o m i t t e d . B, M i t o c h o n d r i a i n c u b a t e d in t h e complete m e d i u m containinR -'..5 m M ('a('l.,. Tim dense granules are i n d i c a t e d by. t h e arrow. The m i t o c h o n d r i a ] pellets were fixed in buffered ( ) s O l ( p H 7.4) as d e s c r i b e d b y ])ALADETM, d e h y d r a t e d .n a l c o h o l , e m b e d d e d in l'ipon H , ~ ' c t i o n e d a n d s t a i n e d w i t h l e a d 1~. . ,~oooo.

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DENSE GRANULES FROM MITOCHONDRIA

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In control experiments where CaC12 was omitted from the incubation medium no significant amounts of granules were obtained.

Analytical methods Carbonate was assayed manometrically, and N determined by a micro-Kjeldahl procedure. Trace elements were detected by emission spectroscopy*. X-ray diffraction patterns were obtained as described by SCOTT et al. 15. The granules were incinerated in a muffle furnace at 600 ° for i8 h, and portions dissolved in 3 M HC1 for chemical or radiochemical analyses. Ca 2÷ and Pl were determined by previously described methods 7. Radioactivity was determined in a liquid scintillation spectrometer.

Materials All of the reagents employed were of the highest purity commercially available. ATP, labeled in the gamma position with ~2p, prepared and assayed as described by GLYNN AND CHAPPELL 16, had a specific activity of 6. 9 tzC//,mole. Carbonate-free, glass-distilled water was used throughout. RESULTS

Electron microscopy The granules isolated with ethylenediamine appeared as finely dispersed, amorphous material shown in Fig. 2A, or when isolated with deoxycholate, as the larger amorphous aggregates shown in Fig. 2C. Incineration of the granules at 600 ° for I8 h induced the formation of the larger particles shown in Fig. 2B and D. Examination of these particles by X-ray diffraction revealed the crystallinity of the incinerated granules as will be shown below. TABLE I COMPOSITION OF THE ISOLATED GRANULES

Method of isolation

Total yield*

N (%)

Ethylenediamine KOH Deoxycholate Sonication

o.29 o.17 o.i8 o.25

5.4 o.4 1.2 7.8

COs2(%) 8.9 9.6 io.2 7.0

Wt. loss upon Inorganic incineration(%) yield* 42.2 16.2 i9.8 60.0

o.17 o.14 o. 15 o.15

* Expressed as mg per m g of mitochondrial protein.

Composition of the granules The analytical data summarized in Table I show that the granules as isolated consisted of both organic and inorganic constituents. The total yield of the initially isolated product was variable, and was dependent on the method used for isolation. For example, the yields were consistently higher when ethylenediamine or sonication was used. Such granules also contained more organic material than those isolated * These analyses were conducted by Melpar, Inc., Arlington, Va.

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E . C . WEINBACH, T. V()N BRANI)

Fig. z. l•lectron m i c r o s c o p y of tile i s o l a t e d granules. A, G r a n u l e s i s o l a t e d w i t h e t h y l e n e d i a m i n c ; t3, G r a n u l e s i s o l a t e d w i t h e t h y l e n e d i a m i n e a n d h e a t e d a t ¢ooo: for t8 h; C, G r a n u l e s i s o l a t e d \~ith d e o x y c h o l a t e , D, G r a n u l e s i s o l a t e d w i t h d e o x y c h o l a t e a n d h e a t e d a t 6oo: for ~8 h. The s a m p l e s were p r e p a r e d for electron m i c r o s c o p y by t h e p r o c e d u r e c i t e d in Fig. t e x c e p t t h a t lead s t a i n i n v was f ound to be u n n e c e s s a r y owing to t h e i n t r i n s i c elect ron o p a c i t y of t he granuh's. Soooo.

Biochim. Igiophys. Acla, r.tX (I9~,7) -'50 e()(,

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DENSE GRANULES FROM MITOCHONDRIA

with KOH or deoxycholate, as reflected by a larger content of N, and by a greater weight loss upon incineration. It is unlikely that the substantial amount of carbonate found in the freshly isolated (unincinerated) granules is a preparative artifact associated with the alkalinity of the reagents used in the chemical isolation procedures. Granules isolated mechanically by sonication under an atmosphere of argon also contained comparable quantities of carbonate. It may be seen in Table I that the average inorganic yield obtained after incineration at 6o0 ° for 18 h was approximately the same with each of the methods of isolation.

Organic constituents The organic moiety of the granules as isolated contained N and gave a positive biuret reaction, indicating the presence of protein. No N was detected in the incinerated samples. Carbohydrate, shown to be present by spot tests x7,was identified by paper chromatography as ribose. No other sugar was detected. Only traces of P1 (less than 1% of the total PI found in the incinerated granules) were found in acid digests of the organic fraction.

Inorganic constituents These were determined in the incinerated granules. The analytical findings summarized in Table II show that Ca2+, P I and Mg2+ were the major inorganic constituents. The percentage of these constituents did not vary greatly and appeared to be unrelated to the method of isolation. Analysis by emission spectroscopy showed that in addition to these major components, trace amounts of other elements, including A1, Cd, Cu and Fe were present. Most of the carbonate present in the non-incinerated samples (Table I) was lost during incineration, suggesting that the carbonate was present initially as MgCQ, which in contrast to CaC03, is unstable at 6oo °. TABLE II INORGANIC CONSTITUENTS The inorganic constituents were determined after incineration and expressed as per cent of the total residual weight.

Method of isolation

Ca ~+

Pl

M g 2+

Ethylenediamine t(OH Deoxycholate Sonication

30. 4 34.3 3o.o 27.I

17.6 14.6 18.5 17.2

4.0 3.4 3.9 2.o

X-ray diffraction analysis In Fig. 3, representative X-ray diffraction patterns of the isolated granules are compared with authentic mineral samples. The granules as isolated by the ethylenediamine, deoxycholate or sonication procedures were amorphous. One of these patterns is shown in Fig. 3A. On the other hand, samples isolated with KOH showed definite lines of hydroxyapatite before incineration*. These lines became very pronounced * The ease by which K O H induces crystallization u n d e r relatively mild conditions (ioo ° for 3 ° rain) is in m a r k e d c o n t r a s t to the observation t h a t granules isolated b y either of the o t h e r procedures did not crystallize even w h e n heated to 300 ° for 18 h.

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Fig. 3. Diliraction p a t t e r n s of dense granules isolated from mitochondria. The arrows over the p a t t e r n point to some of the diagnostic lines for the c o m p o u n d indicated ill the 1hatching legend. A, A m o r p h o u s p a t t e r n of granules as isolated with deoxycholate. Identical a m o r p h o u s p a t t e r n s were seen in grannies isolated with ethylenediamine or by sonication. B, H y d r o x y a p a t i t e p a t t e r n obtained with granules isolated with bt()H and heated to ooo" for ~8 h. C, ( o m p a r i s o n s t a n d a r d of authentic h y d r o x y a p a t i t e , (~a10(I)O4)~(()H)2. I), H y d r o x y a p a t i t e and whitlockite p a t t e r n (left arrows) obtained with granules isolated with deoxycholate and heated to t)oo for 18 h. "l'h~ right arrow indicates Mg(). E, Comparison s t a n d a r d of all equimolar mixture of authentic h y d r o x y apatite, Ca10(PO4)~(OH)2, and whitlockite, Caa(PO4) 2. F, \Vhitlockite pattern obtained with granules isolated by' sonication and heated to 0oo ° for r8 h. A similar patter~ ~ a s seen with incinerated granules isolated with ethylenediamine.

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DENSE GRANULES FROM MITOCHONDRIA

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after heating the KOH samples at 6oo ° for 18 h (Fig. 3B) and correspond closely to the authentic hydroxyapatite pattern shown in Fig. 3C. Patterns of incinerated granules isolated with deoxycholate showed the presence of whitlockite in addition to that of hydroxyapatite (Fig. 3D and E). The patterns of heated granules isolated by either sonication or ethylenediamine correspond to that of authentic whitlockite only (Fig. 3F). In addition to hydroxyapatite and whitlockite, traces of MgO were evident in the incinerated granules isolated with deoxycholate (Fig. 3D).

Granule formation Conditions for accumulation of granules. The data collected in Table III represent the results of experiments in which the effects of various experimental conditions were tested. Utilizing the complete medium and under the conditions described in M E T H O D S the average yield of granules was 52 rag. Omission of the respiratory substrate, or Pt substantially reduced the yield, and in the absence of Ca 2+, no significant amount of granules could be isolated. Likewise, ATP was found to be an absolute requirement for granule formation, thus eliminating the possibility that the isolation procedures were merely concentrating non-specific calcium phosphate or other precipitates*. On the other hand, oligomycin was without significant effect. TABLE III CONDITIONS FOR ACCUMULATION OF GRANULF~SBy MITOCHONDRIA Fifteen flasks containing a total of a p p r o x i m a t e l y 300 mg of mitochondrial protein were used for each p a r a m e t e r tested. I n c u b a t e d for 20 min at 3 o°. The granules were isolated b y the deoxycholate method. The concentration of oligomycin was 2/zg per nag of mitochondrial protein.

Conditions

Yield (rag)

Complete m e d i u m (See METHODS.) Succinate omitted Pt o m i t t e d Ca 2+ o m i t t e d ATP omitted Oligomycin added

52 19 33 o o 49

A D P in place of ATP Succinate omitted Pl o m i t t e d Oligomycin added

34 o 14 3°

Of other nucleotides tested, only ADP could replace ATP to any extent. However, when ADP was the nucleotide supporting granule formation, quantitative differences in the other requirements became apparent. Other experiments showed that pentachlorophenol (5"10-5 M), or antimycin A (2 /~g per mg of mitochondrial protein) completely suppressed granule formation. These findings agree with the earlier data of VASINGTON AND I~URPHY9 based on Ca 2+ uptake studies with rat kidney mitochondria, of SARISTM and of RossI AND LEHNINGERn with rat liver mitochondria. Isotope experiments. Labeled Pt or ATP was included in the incubation medium and analyses of radioactivity and PI concentration were done on samples removed * The p a u c i t y of granules isolated in the absence of Ca 2+ or ATP also prevented a comparison of the composition of the intrinsic granules with t h a t of the granules produced w h e n mitochondria were incubated in the complete medium.

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t';. C. x,VEINBA('H, T. VON BRANI)

d u r i n g various stages of tile experiments. Tile d a t a of Table IV summarize the morv significant findings. TABLE IV FORMATION

OF GRANULES

I N P R I , 2 S E N C E O F ;1~1)1 O R

[a2P ATI'

tgifteen flasks containing a total of approximately 300 mg of mitochondrial protein were used t(>teach experiment. In Expt. ~, the medium contained 9oo t~moles of Pt labeled with 2.5 //(" !,f carrier-free a2p and 675/m~oles of ATP, in addition to the other components described in M),;THOOS. In Expt. 2, the medimn contained 9oo [m~oles of t)t and 675 /,moles of ATP labeled with -'.5 , t ' of ~a2PATP, in addition to the other components described in METHODS. ()ther conditions as described in the legend t<) Table III. Expl.

Condition, s

~\'o.

I 2

,1Pl iJl medium (¢ot2oles)

Pt labeled with a2p ATP labeled with a2PTATP

Pl i~z gramdes (/~moles)

,S'pecific aclivi/y
b~ t/u' gramdcs (% ,~f i~iliat)

23

19o

r,7

29

2I 5

1()

I n the first e x p e r i m e n t there was relatively little net change in the a m o u n t (>f Pi in the i n c u b a t i o n m e d i u m although there was a large a c c u m u l a t i o n of Pi in the granules*. The fact t h a t the specific a c t i v i t y of the Pi in the isolated material was lower t h a n t h a t initially present in tile m e d i u m indicates t h a t a t)ortion of tile Pi was derived from ATP. This could occur b y at least three routes : (a) D i l u t i o n of the radioa c t i v i t y of the a2pi pool b y the P i - A T P exchange reaction 19, (b) direct incorporation of PI from A T P into the granules v i a an energized i n t e r i n e d i a t e a or (c) hydr()lysis ()f ATP. I n d e p e n d e n t experiments showed t h a t the P i - A T P exchange did not occur u n d e r these conditions. If Pi was incorporated i n t o the granules directly fr()m ATP a n d no hydrolysis occurred, then the specific a c t i v i t y of the m e d i u m should renlain constant. However, it was found t h a t the specific a c t i v i t y of tile m e d i u n l after inc u b a t i o n had declined t() a p p r o x i m a t e l y 6o '1o of the initial wtlue, indicative ~)f subs t a n t i a l A T P hydrolysis 6. Tile d a t a of the second experiment support this interpretation. I n this c o m p l e m e n t a r y experiment, A T P labeled in the g a m m a position with a2p was used, a n d again there was little net disappearance of Pi fronl the m e d i u m although the granules contained 2I 5 /,moles of Pi. Analysis of the m e d i u m b y a modified MARTIN aXl) DOTY')° procedure to determine the d i s t r i b u t i o n of the isot~)pe disclosed t h a t a p p r o x i n l a t e l y 4o % of tile r a d i o a c t i v i t y was lost from the ATP fraction after incubation, This was s u b s t a n t i a t e d by direct analysis for A T P with luciferase 21. However, most of tile Pi found in the granules a p p a r e n t l y had its ()rigin in the Pi initially present in the m e d i u m , as only 16 % of the initial specific a c t i v i t y of the labeled A T P was f(mnd in tile granules. DISCUSSION

The d a t a presented in this paper d e m o n s t r a t e tile feasibility of isolating the dense granules which a c c u m u l a t e in rat liver m i t o c h o n d r i a d u r i n g calcium loading. * It must be recognized that because of unavoidable mechanical losses during the isolation of the dense granules a quantitative recovery was not possible. Thus, the total amouut of Pi found in the isolated granules represents an underestinaation of the true amount and should be regarded as an approximation. Biochim. Biophys. ~lcga, 14,~;(~9()7) 25o zoo>

DENSE GRANULES FROM MITOCHONDRIA

265

It is difficult to decide which one of the four procedures yields granules which correspond most closely to those existing in situ. Undoubtedly, each of the methods produces artifacts in the final isolated product. This is seen in the variable amount of organic material contained in the granules as isolated (Table I). Presumably, treatment with hot K O H destroyed a large part of the organic component. On the other hand, granules isolated b y mechanical means contained such a substantial amount of organic constituents that it is likely that extraneous material was included in these samples. Granules isolated with either ethylenediamine or with deoxycholate contained organic material in amounts intermediate between these extremes. Because of the uncertainty regarding the authenticity of the organic constituents, this fraction was not studied in detail. Regardless of the unknown nature of the organic moiety of the isolated granules, definite conclusions concerning composition and mineralogical structure can be made on the basis of our findings. The intra-mitochondrial granules as isolated are amorphous*, and consist of both organic and inorganic components. After incineration which readily induced crystallization, two types of calcium phosphate salts were found: hydroxyapatite, Calo(POa)e(OH ) 2 and whitlockite, Caz(P04)2. The appearance of two different calcium salts m a y be related to differences in the alkalinity of the reagents used in the isolation of the granules. In addition to the above minerals, MgO was detected in the crystallized granules; a component presumably derived from MgCO 3 initially present. The significant amount of carbonate present appears to be authentic. This is of special interest as m a n y of the apatite-eontaining structures in nature also contain carbonate 22-24. The amount of carbonate found in the mitochondrial granules is substantially smaller than that found in tl~e calcareous corpuscles of cestodes 15. An examination of some of the factors which influence the formation of the intra-mitochondrial granules (Table III) fully substantiates the previous findings of others, based on uptake studies, that massive accumulation of Ca 2+ is an energydependent process, requiring exogenous ATP (for references, see CHANCE1, 5). Results of the isotopic experiments (Table IV) indicate that the nucleotide undergoes hydrolysis coincident with the uptake of Ca ~+. Moreover, the data are consistent with the explanation that the terminal P I of ATP undergoes transfer to the pool of PI in the reaction medium prior to its incorporation into the dense granules. It appears unlikely that ATP participates directly to any large extent in granule formation. This is concordant with the suggestion of ERNSTER AND LEE ~5 that the role of ATP in the accumulation of Ca 2+ is to counteract the uncoupling effect of this cation. However, we found previously by the use of [14C~ATP that 4 % of the added nucleotide counts were recovered in the mitochondria, and 0. 5 % was incorporated in the granules. CARAFOLI, RossI AND L E H N I N G E R 26 also have shown that adenine nueleotides are taken up during the massive accumulation of Ca 2+ and Pt by respiring rat liver mitochondria. It is recognized that the massive uptake of Ca 2+ under the conditions described in this report represents a physiological extreme. The possible physiological significance of Ca 2+ accumulation by mitochondria has been reviewed by CHANCE5. On the other hand, the composition of the intramitochondrial granules formed under these * T h e a p p e a r a n c e of h y d r o x y a p a t i t e in t h e n o n - i n c i n e r a t e d g r a n u l e s isolated w i t h K O H u n d o u b t e d l y is i n d u c e d b y t h e action of t h e h o t alkali a n d is n o t characteristic of t h e g r a n u l e s in situ. A similar o b s e r v a t i o n w a s m a d e w i t h calcareous corpuscles of cestodes 13.

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i n vitro c o n d i t i o n s is s i m i l a r i n n l a n y r e s p e c t s t o t h a t of t h e c a l c a r e o u s c o r p u s c l e s f o r m e d i n vivo by" c e s t o d e s " ; , a n d t o t h e i n t r a c e l l u l a r d e t ) o s i t i o n o f h y d r ~ ) x y a p a t i t e in t h e c i l i a t e S p i r o s t o m u m a m b i g u u m 2s. W h e t h e r t h e s i m i l a r i t i e s in c o m p o s i t i ~ n a n d mineralogical granules

structure

of v e r t e b r a t e

ing ion deposition

of t h e c a l c a r e o u s mitochondria

is a s u b j e c t

i n c l u s i o n s of i n v e r t e b r a t e s

reflect a basic physiologieal

for f u r t h e r

comparative

and the matrix

mechanism

reffulat-

micrographs,

and to

studies.

ACKNOWLEDGEMENTS \ V e a r e g r a t e f u I t o D r . H . G. SHF~FFIELI) f o r t h e e l e c t r o n D r . M. U . N V L ~ X f o r t h e X - r a y

diffraction

analyses. The

technical

assistance

(ff C

ELWOOD CLAGGETr is a c k n o w l e d g e d .

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B. CHANCE, Energy-l.inked Functions oj 34itochondria, \ c a d e m i c Press, New York, 19t, 3. G. P. BRIERLEY AND D. B. SLAUTTERBACK, Biochim. Biophys. Acta, 82 (191~4) ~83. L. D. PEACHEY, J . Cell Biol., 2o (1964) 95. J. \V. GREENAWALT, C. S. ROSSI AND A. L. LEHNIN(;ER, .]. (?ell Biol., 23 (r9(;4) 21. B. CHANCE, J. Biol. Chem., 24o (I965) 2729. C. S. R o s s I AND A. L. LEHNINGER, Biochenz. Z., 338 (1903) 098 . E. C. VVEINBACH AND T. VON BRAND, Bioehem. Biophys. Res. Commun., 19 (19(,5/ 133. E. C. WEINBACH, Anal. Biocheng., -, ([96I) 335F. D. VASlNOTON A,XD J. V. MURPHY, J . Biol. Chenz., 237 (I9(,2) 207o. G. E. PAL.aDZ, J. Exptl. Med., 95 (1952) 285}I. B. SPORN, T. WANKO AND W. I)INGMAN, ./r. Cell l~iol., 15 (1962) IO9. M. J. KARNOVSKY, J. Biophys. Biochem. Qvtol., II (1961) 729 . "[. VON BRAND, r . ]. MERCADO, M. U. NVI.FN AND .]). B. SCOTT, Expll. Parasihd., 9 (19t~o) 2o 5. T. ROSETT,, dplDl. ~,Vlicrobiol., 13 (~965) 254D. B. SCOTT, M. [:. NYLEN, T. VON BRAND AND N]. 11. PUOH, l:.xDtl. Parasilol., 12 (19o2)445. [. M. (;LYNN AND J. B. ('HAPPELL, Biochem..[., 9o (1904) 147. I;. FEIGL, Spot Tests i~z Organic Analysis, 5th. Engl. ed., Elsevier, New York, I95~, p. 389 • N. E. S.~_RIS, SOC. Sci. Fe~niea Comm, entationes Phy-Math., 28 (~963) i. P. D. BOYFR, A. B. FALCONE .aND \V. H. HARRISON, Nature, 174 (1954) 4 ol. J. B. MARTIN AND D. *I. DOTY, Anal. Chem., 2i (1949) 905. B. L. STREHLER AND W. 1). MCELROY, in S. P. COLOWICK AND N. (). I(APL\N, 3lethods i~z En~ymology, Vol. 3, A c a d e m i c Press, New York, 1957, p. 871. J. C. ELLIOTT, in J. L. HARDWlCK, J. P. DUSTIN AND tl. IC HELD, .4dvanccs iJl t:ht,,'i~zc Research and Dental Caries Preyed#ion, Macmillan, New York, 1963, p. 277. H. NEWESELY, Experientia, 19 (1963) 62o. D. R. SIMPSON, Scie~ce, 147 (1965) 5ol. L. ERNSTER AND C. P. LEE, Ann. Re'L. Biochem., 33 (1964) 729 • ]~. CARAFOL1, C. S. 1)tOSSI AND A. L. LEHNINGER, J. Biol. Chem., 240 (x965) 225. t. T. VON BRAND, Biochemistry qf Parasites, A c a d e m i c Press, New York, I96g), t). (~F. (;. E. PAUTARD, Biochim. Biophys. dcta, 35 (I959) 33-

I~iochim. l¢iophys. A cta, 148 (iq(,7) 256--200