373

Biochimica et Biophysica Acta, 543 (1978) 373--382 © Elsevier/North-Holland Biomedical Press

BBA 28696

CHARACTERIZATION OF AN AGGLUTININ FROM HUMAN SERUM

INGER SCHOUSBOE

Department of Biochemistry C, University of Copenhagen, Panum Institute, Blegdamsvej 3, DK-2200 Copenhagen N (Denmark)

(Received March 10th, 1978)

Summary

After exposure to serum, an agglutination of mitochondria from yeast, liver, heart and kidney was observed. The degree of agglutination was dependent on the ratio between the amount of serum proteins and mitochondrial protein. The serum protein which induced agglutination was bound irreversibly to the mitochondria , was heat stable and partly resistant to acidification. Maximal agglutination was observed at an ionic strength equal to 40 mM Tris, at pH 6.0--7.5. Preincubation of mitochondria with calcium ions at slightly acidic pH prevented the agglutination. Neuraminidase t rea tment of either serum or mitochondria had no effect upon the agglutination.

In t roduct ion

In studies of the subcellular localization of intracellular lipase using serum- stabilized substrates [1,2] it was observed that under certain conditions the mitochondria agglutinated.

For the lipase studies this agglutination was circumvented by using a rela- tively high buffer concentrat ion.

The present study was under taken to elucidate further the cause and the exact conditions for the observed agglutination of mitochondria .

Materials and Methods

Materials. Baker's yeast was a gift from De Danske Spritfabrikker A/S Den- mark; pig orgmls and human blood were delivered from the University Hospital. Cytochrome c and Clostr id ium per fr ingens neuraminidase were purchased from Boehringer (Mannheim, G.F.R.). Sephadex G-200 was obtained from Pharmacia Fine Chemicals (Uppsala, Sweden). Rabbit anti-human whole serum protein was delivered from Dakopat t (Copenhagen, Denmark). All other chemicals

374

were of purest grade available from regular commercial sources. Preparation of mitochondria. Yeast mitochondria were isolated and purified

from baker's yeast as described previously [2] and finally suspended in 0.3 M mannitol /50 mM Tris-HC1, pH 7.4. Pig heart mi tochondr ia were isolated by differential centrifugation of homogenates prepared according to Crane et al. [3] and finally suspended in 0.3 M sucrose/10 mM potassium phosphate pH 7.4/0.2% (w/v) bovine serum albumin. Pig liver mitochondria were isolated and purified as described by Morr+ [4] and mitochondria from pig kidney were obtained using the technique of Shibko and Tappel [5].

Serum. Outdated acid-citrate-dextrose plasma was centrifuged (12 000 ,; g, 15 rain). The supernatant was dialyzed twice against 15 vols. 25 mM Tris-HC1, pH 7.5, and centrifuged (12 000 >Z g, 15 rain). Foetal calf serum was dialyzed twice against 15 vols. 25 mM Tris-HC1, pH 7.5. Both sera were stored at --20°C until use.

Agglutination test. In the standard assay, 6 vols. of an ice-cold isotonic mito- chondrial suspension were added to 4 vols. of dialyzed ice-cold serum. The mix- ture was incubated in an ice bath for 30 min and then centrifuged at low speed (630 × g, 5 min) with precooled test-tube adapters.

Samples of the supernatant and non-centrifuged test mixture were diluted with 4 vols. 0.1% Triton X-100 and stored at --20°C. The relative amount of mitochondria in suspension after centrifugation was calculated from cyto- chrome c-oxidase activity in these samples, as 100% minus the percentage of cy tochrome c-oxidase activity remaining in the supernatant after eentrifuga- tion. In all experiments controls were run using dialysis buffer instead of serum.

Gel filtration. Dialyzed serum was applied on a Sephadex G-200 column (2.5 × 80 cm) equilibrated with 0.05 M NaC1/0.1 M Tris-HC1, pH 8.0. The proteins were eluted with the same buffer. The fractions within each of the three peaks obtained were pooled, concentrated by ultrafiltration with an Amicon PM-10 membrane and dialyzed against 100 vols. of 25 mM Tris-HC1, pH 7.5.

Perchloric acid treatment of serum. On an ice-bath dialyzed serum was mixed with perchloric acid and carefully stirred for 15 min. The mixture was neutralized with 1 M KOH and centrifuged (12 000 × g, 10 min). The supernatant was concentra ted by ultrafiltration with an Amicon PM-10 membrane.

Neuraminidase treatment of mitoehondria. Mitochondria were incubated with neuraminidase as described for neuraminidase t rea tment of liver plasma membranes [6]. The incubation mixture was kept approximately isotonic with mannitol, and 37 munits of C. perfringens neuraminidase was used per mg mitochondrial protein instead of 4 munits of Vibrio cholera neuraminidase per mg plasma membrane protein.

The mitochondria were incubated with neuraminidase at 25°C for 30 rain. The reaction was stopped by cooling on an ice-bath. The mitochondr ia were freed from neuraminidase by three centrifugations (10 000 ;~ g, 15 min) and resuspensions in 0.3 M mannitol /50 mM Tris-HC1, pH 7.5.

Neuraminidase treatment of serum. Dialyzed serum was treated with neur- aminidase as described for orosomucoid by Hudgin et al. [7]. 3 ml of 0.5 M acetate buffer, pH 5.0, was mixed with 7 ml of dialyzed serum (62 mg pro- tein/ml) and incubated with 500 munits. C. perfringens neuraminidase for 18 h

375

at 30~'C. Sialic acid was removed from the protein by three dialyses at 4°C against 10 vols. of 25 mM Tris-HC1, pH 7.5.

Cytochrome c-oxidase activity. The cytochrome c-oxidase activity was deter- mined as described by Smith [8] .

Immunoelectrophoresis. Crossed immunoelectrophoresis [9] was performed on 1.5-ram thick agarose gels coated on 100 ~ 100 mm glass plates. The gel consisted of 1% (w/v) agarose in 20 mM sodium barbital buffer, pH 8.6. The first dimension was run for 1 h at 10 V/cm, the second for 20 h at 3 V/cm. In general, 3 ~l diluted dialyzed serum (approx. 90 ~g protein) was applied in the application well, and the antibody content of the gel was 13 ~l/cm 2.

The amount of certain proteins in the serum was measured relative to the amount of prealbumin in the same serum electrophoresed on the same plate. The height of the peak was used as a measure of the protein amount.

Other methods. Protein concentration was determined by the method of Lowry et al. [10] with crystalline bovine serum albumin as standard. For elec- tron microscopy pellets of mitochondria preincubated at 0°C with and without serum were fixed in 3% (w/v) glutaraldehyde in 0.1 M caeodylate buffer, pH 6.8, and 1% (w/v) OsO4 in 0.1 M caeodylate buffer, pH 7. After dehydration and embedding, thin sections were stained with uranyl acetate and lead citrate and examined in a Philips 201 electron microscope.

Results

Agglutination of mitochondria was not observed in buffer solutions alone in the range of pH 6.0--9 .1 of a Tris concentration from 10 to 160 mM. Nor could the presence of 0 - 1 4 0 mM KC1 induce the mitochondria to agglutinate.

~" 100

_ ~ 80- ~ o S 60- T~40-

20 2 u 10 20 30 40 50 ~ I n c u b a t i o n t i m e , r a i n

13-

"S ol

"D .,c o ~ \ w o

01 "~ 55 60 65 70 '75 80 85 90

pH

100 ~6 c 8O

-~ 4O

Z 2o

0

• o

/ o %

20 40 60 80 100 120 140 160 C o n c e n t r a t i o n of Tris, mM

t 0 20 40 60 80 100 120 140

C o n c e n t r a t i o n of KC[,mM

Fig. 1. T i m e c o u r s e o f t h e r e l a t i v e a m o u n t o f m i t o c h o n d r i a a g g l u t i n a t e d a t 0°C (o :~) a n d a t 30°C (e e) . T h e c o n c e n t r a t i o n s o f m i t o c h o n d r i a a n d s e r u m w e r e 1 . 0 a n d 6.3 m g p r o t e i n p e r m l a s s a y m e -

d i u m , r e s p e c t i v e l y .

Fig. 2. T h e r e l a t i v e a m o u n t o f m i t o c h o n d r i a a g g l u t i n a t e d as a f u n c t i o n o f p H : 30 mM p o t a s s i u m c a c o d y - l a t e (c> ©), 30 mM Tris (e --). T h e c o n c e n t r a t i o n s o f m i t o c h o n d r i a a n d s e r u m are as in t h e

l e g e n d t o Fig. 1.

Fig. 3. T h e r e l a t i v e a m o u n t o f m i t o e h o n d r i a a g g l u t i n a t e d as a f u n c t i o n o f t h e i o n i c s t r e n g t h . W i t h a m a n -

n i t o l c o n c e n t r a t i o n o f 90 mM, t h e i o n i c s t r e n g t h in t h e i n c u b a t i o n m i x t u r e w a s v a r i e d b y KCI C; ©) o r b y T r i s (,~ ~). With a m a n n i t o l c o n c e n t r a t i o n o f 180 m M t h e i o n i c s t r e n g t h w a s v a r i e d b y KCI (¢ "-). T h e c o n c e n t r a t i o n s o f m i t o c h o n d r i a a n d s e r u m w e r e as i n l e g e n d t o Fig. 1.

376

Agglut inat ion of m i tochond r i a was, however , induced by the presence of d ia lyzed human serum. The agglut inat ion was maximal a f te r 10 min incuba t ion at 3 0 C and af ter 30 min incuba t ion at 0°C (Fig. 1). The pH d e p e n d e n c y is shown in Fig. 2 and ionic s t rength d e p e n d e n c y is shown in Fig. 3. It is seen that maximal agglut inat ion was ob ta ined at pH 6.5--7 .5 and at an ionic s trength equal to 40 mM Tris or 12 mM Tris plus 28 mM KC1. The ionic s t rength d e p e n d e n c y was the same in the presence of 180 mM manni to l as in the presence of 90 mM manni to l . In the presence of a cons t an t a m o u n t o f serum prote ins the ex t en t of agglut inat ion at varying concen t ra t ions of m i t o c h o n d r i a fo l lowed a bell-shaped func t ion (Fig. 4). At a cons t an t mi tochondr i a l pro te in concen t r a t i on of 1 rag/ ml, the e x t e n t of agglut inat ion was a sigmoid func t ion o f the serum pro te in concen t r a t i on (Fig. 5). Doubl ing or tripling of the incuba t ion vo lume was with- ou t e f fec t upon the relative a m o u n t of m i t o c h o n d r i a agglutinated.

The a m o u n t of serum prote ins necessary to induce 50% of a mi tochondr ia l suspension at 1 mg pro te in /ml to agglut inate varied f rom ba tch to batch, in tha t one batch of serum was ob ta ined f rom b lood f rom one person. As mea- sured by the peak height of the prec ip i ta te in a crossed immunoe lec t rophores i s [9] this a m o u n t varied with the a m o u n t of ~-1C globulin, as shown in Fig. 6. Here it is seen tha t in the batches with high concen t r a t i on o f ~-1C globulin there is a high c oncen t r a t i on of agglutinin.

F rac t iona t ion of the serum prote ins on Sephadex G-200 showed tha t the serum c o m p o n e n t which induces agglut inat ion was to be fo u n d among prote ins with a molecu la r weight of 30 000- -105 000 (Fig. 7) indicat ing tha t it is n o t an immunoglobu l in . The c o m p o u n d in ques t ion was re ta ined when f i l tered on an Amicon PM-10 membrane .

Extens ive washing of m i t o c h o n d r i a p re incuba ted with serum (Table I) was no t able to abolish the agglut inat ion of these, since the m i to ch o n d r i a aggluti- na ted to the same or to an even increased e x t e n t af ter washing.

T ha t the persis tence of the agglut inat ion af ter extensive washing (Table I) was no t an a r te fac t caused by damage was indicated no t only by the absence of agglut inat ion of cont ro ls t rea ted in the same way bu t also by the observa- t ion tha t e lec t ron micrographs of m i t o c h o n d r i a incuba ted with and w i th o u t

I00 I _ 8o! 5 ~ 60 u 40

~ 2o

~ o

Mi tochondr laL prote~n

c o n c e n t r a t i o n , m g / m [

]D

~6 c ~5

o o

o o o

100

8 0 ~

60

4O

2O

°i 2 4 6 8 10

Serum protein concen t ra t i on ,mg/m l

Fig. 4. The re la t ive a m o u n t o f m i t o c h o n d r i a a g g l u t i n a t e d as a f u n c t i o n of the m i t o c h o n d r i a l p r o t e i n con - c e n t r a t i o n a t t w o d i f f e r e n t s e r u m p r o t e i n c o n c e n t r a t i o n s , 21 .8 m g p r o t e i n pe r ml ( ) - - 0 a n d 5.5 m g pe r ml (o-- o) .

Fig. 5. The re la t ive a m o u n t o f m i t o c h o n d r i a a g g l u t i n a t e d as a f u n c t i o n o f s e r u m p r o t e i n c o n c e n t r a t i o n . The m i t o c h o n d r i a l p r o t e i n c o n c e n t r a t i o n was 1 rng /ml .

377

tm E 80

c 70

e 60 o

c I o u E 40~

30

030 040 050 060

Retative amount of I']-1C to preolbumm F i g . 6 . T h e r e l a t i o n b e t w e e n t h e a m o u n t o f s e r u m ( o r d i n a t e ) n e c e s s a r y t o i n d u c e a g g l u t i n a t i o n o f 5 0 % o f a m i t o c h o n d r i a l s u s p e n s i o n a t 1 m g / m l u n d e r s t a n d a r d c o n d i t i o n s a n d t h e a m o u n t o f f i - l C g l o b u l i n r e l a t i v e t o p r e a l b u m i n as d e t e r m i n e d b y t h e p e a k h e i g h t o f t h e p r e c i p i t a t e s i n a c r o s s e d i m m u n o c l c c t r o - p h o r e s i s a g a i n s t r a b b i t a n t i - h u m a n w h o l e s e r u m p r o t e i n .

serum showed no morphological changes and by the observation that serum simultaneously lost its ability to induce agglutination of a new portion of mitochondria (Fig. 8). It is seen that maximally 40% of the mitoehondria could be agglutinated by the preineubated serum, compared to 95% in the control experiment.

The protein that induces mitochondria to agglutinate is also present in foetal serum. Agglutination induced by foetal calf serum could not be abolished by 60 min preheating at 56°C of the serum but after 60 min preheating at 80°C no agglutination could be induced. Besides being relative heat stable the agglu- tinin also showed some resistance to acid treatment. Independent of the per- chloric acid concentration and serum protein concentration used about 40% of the serum proteins were soluble after acidification-neutralization treatment. From Table II is seen that the ability of serum to induce agglutination of mira-

B

m

1 O0 I

050 i 3 0 ~ ~

40 45 50 55 60 65 70 75

Froctron no F i g . 7 . S e p h a d e x G - 2 0 0 c o l u m n c h r o m a t o g r a p h y o f s e r u m p r o t e i n s . D i a l y z e d s e r u m w a s a p p l i e d a n d e l u t e d w i t h 0 . 1 M T r i s - H C 1 / 0 . 5 M N a C l , p H 8 . 0 . F l o w r a t e , 1 2 m l / h . 4 m l p e r f r a c t i o n .

378

T A B L E I

A G G L U T I N A T I O N O F M I T O C H O N D R 1 A A F T E R P R E I N C U B A T I O N

S e r u m a n d m i t o c h o n d r i a o r b u f f e r a n d m i t o c h o n d r i a we re i n c u b a t e d a n d a l i q u o t s t a k e n b e f o r e a n d a f t e r

l o w s p e e d c e n t r i f u g a t i o n fo r d e t e r m i n a t i o n o f t he a g g l u t i n a t i o n . A f t e r l o w speed c e n t r i f u g a t i o n t h e t u b e s

were c e n t r i f u g e d 12 0 0 0 X Z fo r 10 ra in . T h e s u p c r n a t a n t was d i s c a r d e d a n d t he p e l l e t r e s u s p e n d e d in

0 .3 M m a n n i t o l / 1 0 m M Tris-HC1, p t I 7 .5 , b y use o f a P o t t e r E l v e h j e m h o m o g e n i z e r . A f t e r t w o f u r t h e r

w a s h i n g s t he m i t o c h o n d r i a were r e s u s p e n d e d b y h o m o g e n i z a t i o n in t he c o m p l e t e a g g l u t i n a t i o n t e s t

m e d i u m ( 5 0 r a M Tr i s -HCl , p H 7 . 5 : 0 . 3 M m a n n i t o l / 1 0 m M Tr i s -HCl , p t t 7 .5 (4 : 6, v / v ) ) t o a f i n a l c o n c e n -

t r a t i o n o f 1 . 0 r a g p r o t e i n / m l a n d i n c u b a t e d a t 0 ° C f o r 3 0 r a in a n d s a m p l e s were t a k e n fo r c y t o c h r o m c

c - o x i d a s e d e t e r m i n a t i o n as d e s c r i b e d a b o v e .

E x p e r i m e n t C o n d i t i o n s o f P e r c e n t M i t o e h o n d r i a

No . p r e i n c u b a t i o n a g g l u t i n a t e d

B e f o r e A f t e r

w a s h i n g w a s h i n g

+ S e r u m p r o t e i n s 88 81

- - S e r u m p r o t e i n s 0

+ S e p h a d e x G - 2 0 0 f r a c t i o n 9 5 92

- - S e p h a d e x G - 2 0 0 f r a c t i o n 0 0

+ S e r u m p r o t e i n s 54 95

- - S e r u m p r o t e i n s 0 0

+ S e r u m p r o t e i n s 65 88

- - S e r u m p r o t e i n s 7 7

chondria is only partly lost after acidification and neutralization. Most activity is left after treatment with 0.2 N perchloric acid.

Neuraminidase treatment of human serum had no effect upon the ability to induce agglutination of mitochondria.

I00"

80 5

60 cJ

o 4 0 nD c 20 o

\

i i 5 L Mitochondria[ protein concentrat ion, rng/rot

Fig . 8. The e f f e c t o f p r e i n e u b a t i o n o f s e r u m w i t h m i t o c h o n d r i a o n the a b i l i t y t o a g g l u t i n a t e a new" po r -

t i o n o f m i t o c h o n d r i a . T h e r e l a t i v e a m o u n t o f m i t o c h o n d r i a a g g l u t i n a t e d as a f u n c t i o n o f t h e m i t o c h o n * d r i a l p r o t e i n c o n c e n t r a t i o n in t he p r e s e n c e o f h u m a n s e r u m p r e i n c u b a t e d w i t h m i t o c h o n d r i a ( c , - - )

o r w i t h o u t m i t o c h o n d r i a (o e ) . In a t o t a l v o l u m e o f 1 0 5 m l 5 7 2 m g o f s e r u m p r o t e i n was pre-

i n c u b a t e d w i t h m i t o c h o n d r i a ( 4 1 5 m g p r o t e i n ) o r w i t h s u s p e n s i o n m e d i u m fo r 3 0 r a in a t 0°C. A f t e r cen- t r i f u g a t i o n ( 1 2 0 0 0 × g fo r 15 r a i n ) t he s u p e r n a t a n t was c o n c e n t r a t e d by u l t r a f i l t r a t i o n w i t h an A m i c o n P M - 1 0 m e m b r a n e a n d e x t e n s i v e l y d i a l y z e d . T h e a b i l i t y o f t he c o n c e n t r a t e d a n d d i a l y z e d s u p e r n a t a n t to

i n d u c e a g g l u t i n a t i o n was t e s t e d o n a n e w P o r t i o n o f m i t o c h o n d r i a .

3 7 9

T A B L E II

T H E E F F E C T O F A C I D I F I C A T I O N O F S E R U M ON T H E A B I L I T Y TO I N D U C E A G G L U T I N A T I O N

O F M I T O C H O N D R I A

C o n c e n t r a t i o n o f C o n c e n t r a t i o n o f C o n c e n t r a t i o n o f P e r c e n t A g g l u t i n a t i o n

p e r c h l o r i e ac id s e r u m p r o t e i n s s e r u m p r o t e i n s in o f m i t o c h o n d r i a

(N) d u r i n g a c i d i f i c a t i o n t h e assay

( m g / m l ) ( m g / m l )

0 .6 26 21 .1 2

5 .2 4 .7 37

0 .2 26 1 9 . 0 68

5 .2 4 .2 78

0 .0 - - 22 .9 9 6

- - 4 . 6 58

9 .1 9 5

In contrast to that neuraminidase pre t rea tment of mi tochondr ia in the pres- ence of relatively high concentra t ion (10 mM) of Ca 2+ as recommended by Pricer and Ashwell [6] abolished the agglutination. However, as shown in Table III, preincubation of mi tochondr ia under the same conditions as above, but wi thout neuraminidase, abolished the agglutination, while neuraminidase treat- ment in the absence of Ca :+ [11] had no effect upon the agglutination. An exchange of Mg 2÷ for Ca 2÷ or preincubation with Ca 2. at slightly basic pH could not prevent agglutination.

Agglutination o f mitochondria from mammalian tissue. In Table IV, the

T A B L E II I

A G G L U T I N A T I O N O F M I T O C H O N D R I A A F T E R P R E I N C U B A T I O N W I T H N E U R A M I N I D A S E A N D

D I V A L E N T C A T I O N S

In a f i na l v o l u m e o f 3 5 m l 1 6 . 1 0 m g m i t o c h o n d r i a l p r o t e i n was i n c u b a t e d a t 2 5 ° C fo r 3 0 r a in in t he

p r e s e n c e o f 1 0 m M b u f f e r / 0 . 2 7 m M m a n n i t o l / 3 . 5 m g b o v i n e s e r u m a l b u m i n / 1 0 m M d i v a l e n t c a t i o n

c h l o r i d e in t he p r e s e n c e o r a b s e n c e o f n e u r a m i n i d a s e (cf . M e t h o d ) , t r a n s f e r r e d t o an ice b a t h a n d cen t r i -

f u g e d a t 4 8 0 0 0 X g fo r 15 r a in . T h e p e l l e t was r e s u s p e n d e d i n 0 .3 M m a n n i t o l / 5 0 m M Tr i s -HCl , p H 7.5.

A f t e r o n e m o r e c e n t r i f u g a t i o n a n d r e s u s p e n s i o n t h e m i t o c h o n d r i a (1 .0 m g p r o t e i n ) were m i x e d w i t h

s e r u m ( 2 1 . 8 m g p r o t e i n ) in a f i n a l v o l u m e o f 1 . 0 0 m l a n d t h e a g g l u t i n a t i o n t e s t e d .

B u f f e r p H N e u r a m i n i - D i v a l e n t

s y s t e m dase c a t i o n

p r e s e n t

M i t o c h o n d r i a a g g l u t i n a t e d (%) *

E x p t . 1 E x p t . 2 E x p t . 3

M a l ea t e 6 . 5 + Ca 2+ +

- - - - 54 - - Ca 2+ 10

- - Mg 2+ 58

C a c o d y l a t e 6 .5 - - - - 54 - - Ca 2+ 6

- - Mg 2+ 56

T r i s -HCl 7 .5 - - - - 51 - - Ca 2+ 4 6

- - Mg 2+ 67

58

76

* T h e t h r e e e x p e r i m e n t s are p e r f o r m e d w i t h d i f f e r e n t b a t c h e s o f s e r u m a n d d i f f e r e n t p r e p a r a t i o n o f

m i t o c h o n d r i a .

380

T A B L E IV

S E R U M I N D U C E D A G G L U T I N A T I O N O F M I T O C H O N D R I A I S O L A T E D F R O M PIG H E A R T , KID- N E Y A N D L I V E R

T h e m i t o e h o n d r i a l p r o t e i n c o n c e n t r a t i o n in t he assay was 1 . 0 m g / m l a n d t he i o n i c s t r e n g t h e q u a l to 4 0 m M Tr i s a t p H 7 .5 .

S o u r c e o f C o n c e n t r a t i o n o f M i t o c h o n d r i a

m i t o c h o n d r i a h u m a n s e r u m p r o t e i n a g g l u t i n a t e d (%)

( m g / m l )

t t e a r t 0 5

12 .0 9 4

K i d n e y 0 5

12.7 70

L i v e r 0 30

11 .1 9 0

results obtained using mitochondria isolated from different mammalian tissues are shown. It is seen that serum is able to induce agglutination of mitochondria from kidney, liver and heart.

Discussion

The results obtained indicate strongly that a non-dialysable componen t in serum is able to induce sedimentation of mi tochondr ia at a much lower g value than under normal conditions. This phenomenon is probably caused by an agglutination of the mitochondria. This is supported by the electron micro- scopic demonstrat ion that no morphological change has been induced in the agglutinated mitochondria.

Hogeboom and Schneider [12] ment ion that nuclei isolated from livers which have not previously been perfused with 0.25 M sucrose containing CaC12 in the mM range, are contaminated with as much as 10% of the total amount of mitochondria and that the isolated nuclei are aggregated. However, when isolating the nuclei from livers perfused with and homogenized in a sucrose solution containing low concentrat ions of Ca 2÷ [13] the isolated nuclei were not aggregated and the nucleus fraction was contaminated with less than 0.5% of the mitochondria in the original homogenate.

Thus, aggregation of the nuclei and contaminat ion of this fraction with mitochondr ia apparently disappeared after removal of blood from the tissue before homogenisation and/or inclusion of Ca 2÷ in the medium.

In the present work preincubation of the mitochondria at slightly acidic pH in the presence of Ca 2+ prevented serum from inducing agglutination of these mitochondria . It may be that Ca 2÷ at slightly acid pH activates phospholipase C present either in contaminating subcellular organelles or in the mitochondrial membranes, resulting in alteration in the mitochondrial membrane which pre- vents agglutination. Alternatively, Ca > might block electronegative surface charges on the mitochondria through irreversible binding of Ca 2÷.

The results obtained with divalent cations at acidic pH and prevention of

3 8 1

agglutination at high ionic strength suggest that the binding of serum agglutinin to mi tochondr ia may be electrostatic.

Since dialyzed as well as partially purified protein fractions and ultrafil trated serum proteins were able to induce agglutination and since heating of the serum abolished this it is suggested that the agglutination is induced by a protein in the serum, called an agglutinin.

After incubation of serum with mitochondr ia and subsequent centrifugation the serum lost its ability to induce agglutination of mitochondria. The agglutination could not be reversed after extensive washing in the incubation medium, which indicates that the serum protein is tightly bound to the mito- chondria. This is also consistent with the findings that doubling or tripling of the incubation volume was wi thout effect on the relative amount of mito- chondria agglutinated.

The agglutination of mitochondria induced by serum proteins resemble the lectin induced cell agglutination and the format ion of an antogen • ant ibody complex. Serum-induced agglutination of mi tochondr ia was abolished in excess of mitochondria as the antigen • ant ibody precipitate disappears in excess of antigen. The agglutination showed an S-shaped dependency of the serum pro- tein concentrat ion indicating a positive cooperat ivi ty which has also been recognized for lectin binding to cells [14].

In view of the role of membrane-bound sialic acid for the binding of asialo- glycoproteins from serum to plasma membrane [6] it was of interest to exam- ine the effect of neuraminidase t rea tment of the mitochondria and of the serum. The results from these experiments indicate that sialic acid is not involved in the irreversible binding between serum proteins and mitochondria.

As indicated above, the agglutination of mitochondria resembles lectin- induced agglutination of cells, but none of the simple sugars that normally inhibits the lectin-induced agglutination [15,16] inhibited the serum-induced agglutination of mitochondria (results not shown).

The agglutination with regard to both mitochondr ia and serum seems to be non-specific. Foetal serum induced agglutination of yeast mi tochondr ia and human serum protein was able to induce agglutination of mitochondria from heart, kidney and liver.

In a number of diseases such as different forms of cirrhosis, rheumatoid diseases and syphilis, serum-induced agglutination is used as a diagnostic test [17]. This paper has dealt with an agglutinin which may be implicated in these diagnostic test systems. Purification of the agglutinin for subsequent ant ibody product ion is now in progress.

Acknowledgement

The expert technical assistance by Miss Ida Henningsen and Mrs. Ellen Philip- son is gratefully acknowledged. Dr. Birgit Bro is cordially thanked for perform- ing the electron microscopic investigations.

References

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