Localization of enzymes by means of proteases

Biochimica et Biophysica Aeta, 297 (1973) 203-212

Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA 27023

L O C A L I Z A T I O N OF E N Z Y M E S BY M E A N S OF P R O T E A S E S

D. BRDICZKA and W. KREBS With technical assistance of P. KLOOCK Faehbereieh Biologic und Faehbereieh Physik der Universitiit Konstanz, Postfaeh 733, BRD-775, Konstanz (Germany)

(Received September 4th, 1972)

SUMMARY A method is described which makes it possible to determine whether a given enzyme is located on the outer surface of a mitochondrial membrane, or whether it is localized within a mitochondrial compartment. The method combines the use of proteases with digitonin. Depending on the concentration of digitonin, enzymes behind the microsomal membranes and the outer mitochondrial membrane may be successively exposed to the action of proteases. Thus, enzymes located within microsomes contaminating the mitochondrial fraction may be easily distinguished from true intramitochondrial enzymes. Applying this technique to the mitochondrial fraction of rat liver, it is shown that lactate dehydrogenase (L-lactate: N A D + oxidoreductase, EC 1.1.1.27) is located on the outer surface of the mitochondria and within microsomal contaminants.

INTRODUCTION During the last few years two methods have been applied to the separation of external and internal mitochondrial membranes and to the study of submitochondrial enzyme distribution. Both methods, the digitonin method 1,a as well as the swellingshrinking method a, 4, have provided evidence that there exist two main mitochondrial compartments in which soluble enzymes are located. For example, adenylate kinase (ATP: A M P phosphotransferase, EC 2.7.4.3) has been localized within the intermembrane space (external compartment) of liver mitochondria, whereas the soluble enzymes of the citric acid cycle and 3-hydroxyacyl-CoA dehydrogenase (L-3-hydroxyacyl-CoA: N A D + oxidoreductase, EC 1.1.1.35) have been localized within the matrix space (internal compartment) a,4,2. Although these methods allow separate extraction of the two compartments and separation of the surrounding membranes, they cannot be used to unambiguously localize enzymes absorbed to one or other membrane system. For this purpose a method is needed which makes it possible to distinguish between binding or adsorption to the outer or inner face of a membrane. This is especially important for distinguishing true intramitochondrial location (within the external membrane) from adsorption of an enzyme to the outer face of

204

D. BRD[CZKA, W. KREBS

the external membrane and inclusion within contaminating vesicles. The use of proteases to inactivate enzymes in intact and lysed mitochondria provides a method making this distinction possible. This technique has been previously used to localize mitochondrial rotenone-insensitive N A D H : cytochrome c reductase s in rat liver, creatine kinase (EC 2.7.3.2) in heart muscle mitochondria 6, long chain fatty acid activating enzyme in skeletal muscle 7 and rat liver mitochondria 8 and, recently, mitochondrial hexokinase (EC 2.7.1.1 ) in intestinal mucosa and guinea pig brain 9. The present study combines this protease technique with the digitonin method for the examination of mitochondrial enzyme localization. In order to prove the efficiency of the technique, the experiments reported are concerned with the localization of lactate dehydrogenase (L-lactate: NAD + oxidoreductase, EC 1.1.1.27)adenylate kinase, 3-hydroxyacyl-CoA dehydrogenase and isocitrate dehydrogenase (NADP +) (threo-Ds-isocitrate: NADP + oxidoreductase decarboxylating, EC 1.1.1.42) in mitochondrial fractions of liver (i.e. enzymes of relatively well-known intracellular distribution of activity). MATERIAL AND METHODS

Reagents Subtilisin (proteinase VII) (EC 3.4.4.16), trypsin (EC 3.4.4.4.) and trypsin inhibitor were purchased from Sigma Chemical Co., St. Louis, Mo. U.S.A.; proteinase K was a gift from E. Merck, Darmstadt, Germany; Lubrol PX was a gift from ICI Deutschland GmbH, Frankfurt, Germany. Digitonin (Calbiochem. Los Angeles, Calif. U.S.A.) was twice recrystallized from ethanol. All other reagents were supplied by Boehringer Mannheim and by E. Merck, Darmstadt, Germany.

Animals Male rats of the FP 59 strain Thomae Biberach/Riss, Germany, weighing 250300 g, were used. The animals had free access to food and water and were killed by cervical fracture.

Mitochondrial preparation Mitochondria from rat liver were isolated in 0.3 M sucrose (buffered with 10 mM triethanolamine, 2 mM EDTA, pH 7.4) as described recently 2. The mitochondrial preparations were suspended to a final concentration of approximately 20 mg mitochondrial protein per ml in 0.3 M sucrose (buffered with 10 mM Tris (pH 8.0), hereafter referred to as sucrose/Tris).

Incubation with proteases When proteases are to be used in a localization experiment, it is necessary to determine which kind of protease is required to inactivate a given enzyme under the conditions of the experiment. This was determined by digestion experiments: The tissue extracts were prepared by thorough disintegration in sucrose/Tris with a Polytron homogenizer (Kinematica Luzern, Switzerland). After centrifugation for 30 min at 183 000 ×g, an aliquot of the supernatant was incubated for 30 min with 1 mg of the protease per mg protein of the extract (Table I).

LOCALIZATION OF ENZYMES

205

TABLE I EFFECT OF PROTEASES ON D I F F E R E N T ENZYMES IN EXTRACTS OF THE RAT LIVER Activities are given as units per ml of the respective tissue extract. The concentration of each protease was 1 mg per mg protein of the extract.

Subtilisin

Trypsin

Proteinase K

Control .4later (units/ml) 30 min incubation (units/ rnl)

Control After (units/ml) 30 min incubation (units / ml)

Control After (units/ml) 30 rain incubation (units/ ml)

Triosephosphate dehydrogenase (EC 1.2.1.12) 13.6 Triosephosphate isomerase (EC 5.3.1.1) 121 Fructosediphosphate aldolase (EC 4.1.2.13) 1.1 Pyruvate kinase (EC 2.7.1.40) 2.4 Enolase (EC 4.2.1.11 ) 2.4 Hexokinase liver (EC 2.7.1.1 ) 0.024 Hexokinase heart . . Lactate dehydrogenase (EC 1.1.1.27) 24 Adenylate kinase (EC 2.7.4.3) 1.3 3-Hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) 3.8 Isocitrate dehydrogenase (NADP ÷) (EC 1.1.1.42) 0.08 fumarase (EC 4.2.1.2) 0.6 Malate dehydrogenase (EC 1.1.1.37) 4.3 Glutamate dehydrogenase (EC 1.4.1.2) 3.6 Lipoamide dehydrogenase (EC 1.6.4.3.) 1.45

15.1

13.6

91

13.6

121

13.6

109

121

13.6

103

0.07

1.1

0.06

1.1

0.07

0.3

2.4

0.6

2.4

0.24

1.0

2.4

0.0

2.4

1.3

0.024

0.00

0.024 0.017

0.00 0.003

0.00 .

.

0.0

24

25

24

0.0

1.3

0.0

0.33

2.3

0.27

--

0.1 --

1.0

0.00

.

.

.

.

0.07

.

.

.

.

0.048

5.9

3.8

1.8

--

--

4.2

1.6

0.35

--

--

1.7

.

.

.

.

After the determination of the effective protease, 0.2 ml suspension of mitochondria or of subfractions from the liver homogenate was incubated for 30 min at 2 °C with 1 mg per mg protein of the respective protease (subtilisin, trypsin, proteinase K) in a total volume of 0.5 ml sucrose/Tris. Digitonin was added to some samples in a concentration of 0.2 or 2 mg per 10 mg protein. Enzyme activities still present in the particle fractions after incubation with proteases were released by the addition

206

D. B R D I C Z K A , W. KREBS

to the test mixture of 1 mg Lubrol PX per 7 mg protein. Lubrol did not affect the free activity of the investigated enzymes. To avoid rapid inactivation of the enzymes by the protease after the addition of Lubrol PX, the respective enzyme substrates were added to the assay mixture prior to the addition of the detergent.

Enzyme assays Adenylate kinase, 3-hydroxyacyl-CoA dehydrogenase and NADP*-specific isocitrate dehydrogenase were assayed as described previously-'. Lactate dehydrogenase was determined according to the method of Delbrfick et al.1 o. All test systems contained 5 #M rotenone.

Electron microscopy All specimens were fixed as a suspension in 0.3 M sucrose/Tris containing 0.5 °/o glutaraldehyde. After 10-15 min at 4 °C, the suspension was filtered through a 0.2pm filter. The filter was postfixed with 1 ~o OsO, in 100 mM phosphate buffer (pH 7.2) for 15 rain. After dehydration with ethanol, epon was used for embedding. Silver to grey sections were stained with uranyl acetate and lead citrate. RESULTS A N D DISCUSSION

Distribution of lactate dehydrogenase in subcellular fractions A subfractionation of a liver homogenate by differential centrifugation is shown in Table II. The mitochondrial enzymes (such as adenylate kinase and 3-hyT A B L E I1 DISTRIBUTION OF M I T O C H O N D R I A L AND E X T R A M I T O C H O N D R I A L ENZYMES IN SUBFRACTIONS OF A RAT LIVER " P O T T E R " H O M O G E N A T E 5 g of minced rat liver were homogenized in 45 ml 0.3 M sucrose buffered with 10 m M Tris, p H 8.0 (sucrose/Tris) with a Teflon homogenizer. The homogenate was centrifuged for 10 rain at 500,~g in a Sorvall RC-2B centrifuge with rotor SS-34. The sediment was resuspended to a volume of 50 ml with 0.3 M sucrose/Tris to give Fraction P1. The supernatant was spun for 10 rain at 8000~:# in the same rotor. The sediment was resuspended in sucrose/Tris to a volume of 10 ml (Fraction P2). The next supernatant was centrifuged for 10 rain at 27 000 • g and the resulting sediment resuspended to 10 ml volume with sucrose/Tris (Fraction P3). 30 ml of the remaining supernatant were spun for 30 min at 300 000 : g in a Spinco centrifuge with rotor 60 Ti. The pellet was resuspended to a volume of 6 ml with sucrose/Tris (Fraction P4). The final supernatant was designated $5. Values are presented in percent of total activity in all fractions. Relative specific activity (i.e. spec. act. of a given enzyme in each fraction relative to the spec. act. in the homogenate) is given in parentheses.

Enzyme activity, % (rel. units~my protein) Adenylate kinase

Pyruvate kinase

Lactate dehydrogenase

3-HydroxyacylCoA dehydrogenase

Intact cells, nuclei Mitochondria Mitochondria, microsomes Microsomes Soluble supernatant

PI P2 P3 P4 $5

61 (2.0) 28 (2.6) 2 (0.26) 1.5 (0.26) 7.5 (0.16)

10 0 6 3 81

(0.32) (0.00) (0.85) (0.56) (1.80)

8 3 5 2 82

(0.28) (0.29) (0.79) (0.45) (1.75)

62 30 2 I 5

(2.1) (4.2) (0.3) (0.1) (0.1)

LOCALIZATION OF ENZYMES

207

d r o x y a c y l - C o A dehydrogenase) are concentrated in Fraction PI and especially in Fraction P21~,~2, whereas "soluble" extramitochondrial enzymes [pyruvate kinase (EC 2.7.1.40), lactate dehydrogenase] are concentrated in the particle-free supernatant $5. If the relative specific activity (i.e. specific activity of a given enzyme in each fraction relative to the specific activity in the homogenate) is determined, m a x i m u m activity o f the mitochondrial enzymes is observed in Fraction P2, while that o f the "soluble" extramitochondrial enzymes is maximal in Fraction $5. The activity o f lactate dehydrogenase in Fraction P2 is relatively high as compared to the activity o f pyruvate kinase. A l t h o u g h the lactate dehydrogenase activity in the mitochondrial Fraction P2 amounts to only 3 % o f the total cellular activity, the absolute value (0.64 unit per mg mitochondrial protein) is high when compared to other enzymes which show the bulk o f their activity in Fraction P2, such as citrate synthase (EC 4.1.3.7) (0.173 unit per mg mitochondrial protein) or 3-hydroxyacyl-CoA dehydrogenase (2.0 unit per mg mitochondrial protein). Thus if lactate dehydrogenase activity can be shown to have a true mitochondrial localization, the absolute a m o u n t becomes a question o f importance. This assumption would agree with the hypothesis that lactate dehydrogenase may be involved in hydrogen transfer across the inner mitochondrial membrane 13. Latency o f lactate dehydrogenase in subcellular fractions As noted by several authors 11,~2, 14, dehydrogenases in the inner mitochondrial c o m p a r t m e n t are not freely accessible to their substrates. If m e m b r a n e balriers are overcome by the use of osmotic pressure, mechanical forces or detergents, the socalled "latent" activity o f these enzymes is revealed. In contrast to enzymes situated in the inner mitochondrial compartment, enzymes located in the outer c o m p a r t m e n t show no latency. The same holds for enzymes which are adsorbed to the outer face

TABLE IIl LATENCY OF SEVERAL ENZYMES IN THE MITOCHONDRIAL AND MICROSOMAL FRACTION Activities of the respective enzymes were determined in the intact mitochondria or microsomes and in mitochondria or microsomes lysed by addition of Lubrol PX. It was proved that Lubrol PX did not affect the activity of the free enzymes. Enzyme activity (units~rag protein) Adenylate kinase

Intact mitochondria Lysed mitochondria (addition of Lubrol) Latent activity (% total activity) Intact microsomes Lysed microsomes (addition of Lubrol) Latent activity (% total activity)

3-Hydroxyacyl-CoA dehydrogenase

lsoeitrate dehydrogenase (NADP +)

Lactate dehydrogenase

1. I

0.29

0.002

0.032

1.08

3.64

0.03

0.102

2

--

92 --

94 --

-

-

-

-

- -

-

-

59 0.54 1.5 6 4

208

D. B R D I C Z K A , W. K R E B S

i ¸¸

! :~!i

Fig. 1 (a-d). Ultrathin section o f rat liver mitochondria. (a) After incubation with subtilisin (1 mg/mg mitochondrial protein). (b) After incubation with trypsin (1 mg/mg protein). (c) After incubation

LOCALIZATION OF ENZYMES

209

of the mitochondria. As demonstrated in Table III, adenylate kinase, which is situated in the outer compartment, is freely accessible to substrates, whereas 3-hydroxyacyl-CoA dehydrogenase and isocitrate dehydrogenase ( N A D P +) are not. Approximately 60 ~ of the total activity of lactate dehydrogenase in the mitochondrial fraction also exhibits latency. In the microsomal fraction, this enzyme is also not freely accessible to its substrates. Since it has been reported that endoplasmic reticulum is not penetrated by charged substances 15, the latency of lactate dehydrogenase in the mitochondrial fraction does not necessarily indicate location of the enzyme within the inner mitochondrial compartment. Its presence could be due to microsomal contamination, i.e. from the lactate dehydrogenase activity within the vesicles. Localization of enzymes by means of proteases The fact that some lactate dehydrogenase activity is not freely accessible to its substrates suggests that the enzyme is located behind a membrane barrier. Thus, 30 ~o of the activity of lactate dehydrogenase in the mitochondrial fraction is protected against degradation by subtilisin. However, as shown in Tables IV and V, almost no decrease in the activities of adenylate kinase, 3-hydroxyacyl-CoA dehydrogenase and isocitrate dehydrogenase ( N A D P ÷) is found when intact mitochondria are

TABLE IV EFFECT OF TRYPSIN, DIGITONIN AND LUBROL PX ON SEVERAL ENZYMES IN THE MITOCHONDRIAL FRACTION FROM RAT LIVER Mitochondria were incubated for 30 min at 2 °C: (a) with trypsin (1 my/my protein); (b) with trypsin (1 mg/mg protein), and digitonin (2 mg/10 mg protein); (c) with trypsin (1 my/rag protein) and Lubrol PX (1 my/7 mg protein). After incubation, trypsin inhibitor (soja bean; 2 my/my trypsin was added). Enzyme activities in all samples were determined in presence of Lubrol PX. Enzyme activity (units~my)

Control mitochondria Mitochondria treated with trypsin Mitochondria treated with trypsin in the presence of digitonin Mitochondria treated with trypsin in the presence of Lubrol PX

Adenylate kinase

3-Hydroxyacyl-CoA dehydroyenase

lsocitrate dehydroyenase (NADP +)

1.72 2.10

3.84 3.54

0.028 0.031

0.00

4.15

0.0315

0.00

0.61

0.00

incubated with trypsin or subtilisin. It may be concluded that virtually all the activity of these enzymes is located behind a membrane barrier. As demonstrated by electron microscopy (Fig. 1), the mitochondrial and microsomal membranes remain intact during incubation with trypsin or subtilisin. In the presence of digitonin (2 mg per 10 mg mitochondrial protein), which ruptures the outer mitochondrial membrane, adenylate kinase and the inaccessible part o f the lactate dehydrogenase activity are with subtilisin (1 mg/mg protein) and digitonin (2mg/10mg protein), x60 000. (d) Ultrathin section of rat liver microsomes after incubation with trypsin (1 mg/mg mitochondrial protein). × 60 000.

210

D. B R D I C Z K A , W, KREBS

TABLE V E F F E C T O F SUBTIL[SIN, D I G I T O N I N A N D L U B R O L PX ON SEVERAL ENZYMES IN T H E M I T O C H O N D R I A L F R A C T I O N F R O M R A T LIVER Mitochondria were incubated for 30 min at 2 °C: (a) with subtilisin 1 mg/mg protein; (b) with subtilisin (I m g / m g protein) and digitonin (2 mg/10 mg protein); (c) with subtilisin (1 mg/mg protein) and Lubrol PX (1 mg/7 m g protein). After incubation, enzyme activities were determined in the presence of Lubrol. Lubrol was added after the test reactions had been started by addition of the respective substrates.

Enzyme activity (units/mg)

Control mitochondria Mitochondria treated with subtilisin Mitochondria treated with subtilisin in the presence of digitonin Mitochondria treated with subtilisin in the presence of Lubrol

Adenylate kinase

3-HydroxyaeylCoA dehydrogenase

Lactate dehydrogenase

Isocitrate dehydrogenase (NA DP +)

1.4

3.85

0.255

0.035

1.67

4.4

0.072

0.027

0.00

2.6

0.00

0.030

0.00

0.047

0.00

0.00

exposed to protease action and are then rapidly inactivated. The activity of 3-hydroxyacyl-CoA dehydrogenase and isocitrate dehydrogenase (NADP +) remains unaffected during this treatment. These enzymes are not inactivated by proteases until mitochondrial membranes are totally lysed by detergents or mechanical disruption. Electron microscopy in Fig. Ic shows that, after rupture of the outer membrane in the presence of digitonin, the inner mitochondrial membrane is not degraded by the action of proteases. That part of the lactate dehydrogenase activity in the mitochondrial fraction which is inaccessible to protease shows a different behaviour to that of adenylate kinase. Full lactate dehydrogenase activity is not shown unless the structure containing the enzyme is destroyed. However, enzymes located in the outer mitochondrial compartment would not show latency. It is also not likely that lactate dehydrogenase, while showing latency, is located in the inner mitochondrial compartment because it becomes accessible to proteases after digitonin incubation. The observed latency of lactate dehydrogenase might also be due to microsomal membrane barriers (el Table III). It may be suggested that digitonin should then render microsomal and outer mitochondrial membranes permeable to protease (el Table V). This is supported by the evidence given in Tables II and VI. In these experiments, subcellular fractions were isolated from a liver homogehate by differential centrifugation. Percentage distribution of total activities is given in Table II. Table VI shows the recoveries of these activities after the fractions were incubated with subtilisin in the presence of 0.2 mg digitonin per 10 mg protein. It was found that in the mitochondrial fraction especially, but also in the other particulate fractions, 3-hydroxyacyl-CoA dehydrogenase was stable and only 30--40 of the adenylate kinase was inactivated. By contrast, most of the lactate dehydroge-

LOCALIZATION OF ENZYMES

211

TABLE VI RECOVERY OF ACTIVITY OF MITOCHONDRIAL AND EXTRAMITOCHONDRIAL ENZYMES IN SUBFRACTIONS OF RAT LIVER HOMOGENATE, AFTER INCUBATION WITH SUBTILIS1N (1 mg/mg PROTEIN) IN THE PRESENCE OF DIGITONIN (0.2 mg/ 10 mg PROTEIN The subfractions are the same as in Table II, their preparation is described in the legend of Table II. The values are presented as percentage of activity relative to the activity present in each fraction before incubation and as relative specific activities (i.e. specific activity of a given enzyme in each fraction relative to the specific activity of the respective enzyme in the homogenate). Enzyme activity, %o (rel. units~my protein)

Mitochondria P2 Mitochondria, microsomes P3 Microsomes P4 Soluble supernatant $5

Adenylate kinase

Lactate dehydrogenase

3-Hydroxyacyl-CoA dehydrogenase

58 59 46 6

7 4 11 0

100 88 57 12

(1.5) (0.16) (0.12) (0.001)

(0.02) (0.032) (0.05) (0.00)

(4.16) (0.29) (0.057) (0.012)

nase activity was d e g r a d e d in all fractions. T h u s it is evident t h a t d i g i t o n i n concentrations 10 times lower t h a n those n o r m a l l y used to r u p t u r e the o u t e r m i t o c h o n d r i a l m e m b r a n e were sufficient to expose the m e m b r a n e - p r o t e c t e d lactate d e h y d r o g e n a s e in the m i c r o s o m a l as well as in the m i t o c h o n d r i a l f r a c t i o n to the a c t i o n o f the protease. The results discussed so far have n o t t a k e n into a c c o u n t t h a t the m i t o c h o n d r i a l f r a c t i o n m a y also be c o n t a m i n a t e d with lysosomes a n d m i c r o b o d i e s . M e m b r a n e s o f these organelles are certainly also affected by the low d i g i t o n i n c o n c e n t r a t i o n s used in this study. Lowenstein et al. 16 have shown t h a t latency o f acid p h o s p h a t a s e is a b o lished by d i g i t o n i n c o n c e n t r a t i o n s which d o n o t release a d e n y l a t e kinase f r o m the o u t e r m i t o c h o n d r i a l c o m p a r t m e n t . As observed by electron m i c r o s c o p y , the m e m b r a n e s o f m i c r o b o d i e s are also lysed by low d i g i t o n i n c o n c e n t r a t i o n s (Krebs, W. a n d Brdiczka, D., u n p u b l i s h e d ) . Obviously, the o b s e r v e d difference in d i g i t o n i n sensitivity o f the external m i t o c h o n d r i a l m e m b r a n e a n d the m e m b r a n e o f microsomes, lysosomes a n d m i c r o b o d i e s reflects differences in m e m b r a n e structure. I n the case o f the m i c r o s o m a l m e m b r a n e a n d external m i t o c h o n d r i a l membrane,~the s a m e conclusion m a y be d r a w n f r o m the observed differences in p e r m e a b i l i t y ~5. ACKNOWLEDGEMENTS This study was s u p p o r t e d by grants f r o m the Deutsche F o r s c h u n g s g e m e i n schaft given to the F o r s c h e r g r u p p e "Membranfl~ichen u n d Spezifit~it".

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