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ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
Vol. 227, No. 1, November, pp. 154-163, 1983
Proteolysis in Mitochondrial Preparations and in Lysosomal Preparations Derived from Rat Liver
BRIAN DEAN
Department of Biochemistry, University of Shefield, Western Bank, She&&l 510 2TN, United Kingdom
Received April 7, 1983, and in revised form July 13, 1983
A method of preparing rat liver mitochondria with low residual contamination by lysosomal proteases is described. Preparations of mitochondria are divided into two equal portions, one of which is supplemented with a lysosomal fraction. The addition of the lysosomal fraction causes an increase in proteolysis of between 26- and 56-fold at pH 5.0 in four similar experiments. This increase matches the increase in the lysosomal marker @glucuronidase and indicates that all proteolysis at pH 5.0 is due to enzymes of the lysosomal fraction. Above pH 7.0, the addition of a lysosomal supplement increases proteolysis by 1.5- to 5-fold only, suggesting that in the absence of a lysosomal supplement very little of the observed proteolysis is due to enzymes of lysosomal origin. A method of calculating the contribution to total proteolysis of enzymes of the lysosomal fraction or of the mitochondrial fraction is described. The calculations show that at pH 7.0 and above, more than 93% of the observed proteolysis is due to enzymes originating in the mitochondrial fraction. The results support the view of other workers that rat liver mitochondria contain an endogenous neutral proteolytic system capable of degrading mitochondrial proteins to acid-soluble products.
The existence of neutral proteases en- dogenous to mitochondria has been pro- posed by a number of workers (l-6). These workers used mitochondrial preparations contaminated by lysosomal material which might have accounted for the observed neutral proteolysis.
More recently some studies have been published of proteolysis in mitochondrial preparations with very low levels of ly- sosomal contamination.
Subramanian et al. (7) detected proteo- lytic activity in high-speed supernatants from sonicated rat liver mitochondria which had been exposed to digitonin at suf- ficient concentration to lyse lysosomal membranes but not mitochondrial mem- branes, and then washed free of lysosomal contamination. The preparations were ac- tive between pH 4 and 8 with a pronounced optimum at pH 6.0.
Rubio and Grisolia (8) measured pro- teolytic activity in rat liver mitocondria
prepared without special precautions to reduce lysosomal contamination and in mitoplasts prepared after exposure to dig- itonin at concentrations sufficient to lyse lysosomes and also the mitochondrial outer membrane. They showed that proteolysis at pH 7.4 and also the loss of activity of carbamoyl phosphate synthetase were re- duced by digitonin treatment roughly in proportion to the reduction in the lyso- somal marker N-acetyl+glucosaminidase. They concluded that lysosomes are re- sponsible for the larger portion if not all protein degradation of mitochondria.
Duque-Magalhaes (9) prepared rat liver mitochondria with and without exposure to digitonin at concentrations sufficient to lyse lysosomes but not the mitochondrial outer membrane. The mitochondria were broken by osmotic shock and a high-speed supernatant was prepared. The proteolytic activity of the supernatant toward ‘251-la- beled casein and toward mitochondrial cy-
0003-9861/83 $3.00 Copyright Q 1983 by Academic Press, Inc. All rights of reproduction in any form reserved.
154
PROTEOLYSIS IN RAT LIVER MITOCHONDRIA AND LYSOSOMES 155
tochromes was determined over the pH range 4-9. Digitonin treatment abolished the acidic activity toward both substrates and reduced the neutral activity toward casein. The neutral activity toward mito- chondrial cytochromes was little affected and had an activity peak at pH 7.0. It was concluded that this residual neutral activ- ity represents a proteolytic system of true mitochondrial nature.
Manjunath et aZ. (10) prepared rat liver mitochondria by a method in which digi- tonin is used to lyse lysosomes without af- fecting mitochondria and also by a method in which no special precautions are taken to reduce lysosomal contamination. They measured rates of proteolysis over the pH range 5.5-8.5. They found that digitonin treatment removed 85% of the neutral protease activity. The residual activity in- creased as the pH increased over the range tested but without a clear activity peak. They concluded that the residual activity was of mitochondrial origin.
Three of these authors consider that their results support the existence of en- dogenous mitochondrial neutral proteases but there is no agreement on the pH op- timum of the activity. Also there is some small residual activity of the lysosomal marker after digitonin treatment which may account for the residual neutral pro- teolysis.
Several neutral proteinases which had been isolated, characterized, and ascribed to a mitochondrial origin have now been shown to originate in high-density gran- ules of mast cell origin which can be sed- imented through 1.7 M sucrose (11). These enzymes are now thought to be identical to the mast cell chymase which has a pH optimum of 8.5 (12,13). This throws further doubt on the existence of endogenous mi- tochondrial neutral proteases.
This paper reports the preparation of rat liver mitochondria by a method which employs digitonin to reduce lysosomal contamination to low levels and in which the mitochondria are sedimented onto a cushion of 1.4 M sucrose to allow nuclei and high-density granules of mast cell origin to be rejected. A lysosomal fraction was also prepared and added as a supplement
to half of the mitochondrial preparation. Proteolysis in identical aliquots of mito- chondria, one with a lysosomal supplement and one without, has been measured over the pH range 4.5-8.0. In the absence of a lysosomal supplement, proteolysis was measurable over the whole pH range but was greater at neutral pH than at acid pH. Upon addition of a lysosomal supplement proteolysis at acid pH increased in pro- portion to the increase in the lysosomal marker P-glucuronidase. Proteolysis at neutral pH also increased but to a much smaller extent. It can be calculated that all proteolysis at pH 5.0 is of lysosomal origin, while in the absence of a lysosomal supplement less than 7% of the proteolysis above pH 7.0 is of lysosomal origin. The results support the existence of an endog- enous neutral proteolytic system in rat liver mitochondria.
MATERIALS AND METHODS
Animals. Rats were of a Wistar strain obtained from the Sheffield University breeding colony. Males
weighing between 175 and 225 g were used. Chemicals. Digitonin was obtained from BDH
Chemicals Limited, Atherstone, U. K. Phenolphthalein glucuronide was obtained from Koch Light Labora-
tories Limited, Colnbrook, U. K.. All other chemicals were of analytical grade or the highest quality com-
mercially available. Preparation of mitochondria The technique of pre-
paring mitochondria is crucial in the study of pro-
teolysis because small changes in procedure may have large effects on contamination by proteases origi-
nating elsewhere than in hepatocyte mitochondria. As a prelude to the preparation described here, rats were killed by stunning and cervical dislocation. Livers
were removed without delay and cooled in ice-cold 0.25 M sucrose containing 5 mM Tris-chloride, pH 7.6.
When cold, the livers were passed through the stain- less-steel grid of a Fischer mincer. The Fischer mincer
has a screw-operated plunger in a barrel 2.5 cm in diameter which forces the tissue through a static grid with holes 1 mm square. The grid retains 7% of the
tissue which is the vascular tissue and associated connective tissue (14). Most of the nonparenehymal cells, mast cells, and leucocytes should also be re- moved. The resulting pulp is used as starting material for the mitochondrial preparation.
For every two rats used the following solutions were prepared: A, 300 ml of 0.25 M sucrose containing 5 mM Tris-chloride, pH 7.6: B, 100 ml of solution A containing also 0.2 mM Ca&: C, 100 ml of solution
156 BRIAN DEAN
A containing also 1 mM EGTA’: D, 20 ml of solution
A containing also 1.25 mg/ml of digitonin dissolved to give a clear solution either by heating or by son-
ication: E, 10 ml of 1.4 M sucrose (480 g/liter) con- taining 30 mM Tris-chloride, pH 7.6, and 0.2 mM CaCl,.
All solutions were cooled in ice. Liver pulp from two rats was homogenized in 4 vol
of solution B. A motor-driven glass-Teflon homoge- nizer was used and four passes of the pestle were
made. The homogenate was strained through surgical gauze and centrifuged at 3200g for 15 min. The su-
pernatant was used as starting material for the prep-
aration of a lysosomal fraction. The pellet, which con- tained mitochondria, nuclei, and 20-40% of the /3-
glucuronidase activity of the whole homogenate, was homogenized again in 1.5 vol of solution B and trans-
ferred to a single 50-ml polycarbonate centrifuge tube. A 20-ml syringe fitted with a length of silicone-rubber
tubing in place of a needle was used to put 10 ml of solution E below the mitochondrial suspension. The
tube was centrifuged at 3200~ for 15 min to sediment nuclei, dense lysosomes, and high-density granules
of mast cell origin through the lower sucrose layer, leaving the mitochondria at the interface. The su-
pernatant was discarded and the mitochondrial ma- terial was drawn into the syringe used earlier to place
the lower layer into the tube. The mitochondria were dispersed in solution A to a volume of 1 ml for each
gram of liver pulp. This suspension was stirred mag- netically in an ice bath. An equal volume of solution D was added and stirring was continued for 20 min
to lyse the remaining lysosomes. The suspension was then diluted fourfold with solution A and centrifuged
at 14,OOOg for 5 min. The supernatant was discarded and the fluffy layer was transferred to a separate tube. Both the mitochondrial pellet and the fluffy layer
were suspended in solution C to a volume of 40 ml
and centrifuged at 14,000g for 5 min. This washing removes residual calcium ions and recovers mito- chondria from the fluffy layer. The supernatant and
fluffy layer were discarded. The mitochondrial pellets were pooled and washed again in solution A to remove
residual EGTA. All volumes are relative to liver pulp weight. The temperature throughout was 0-5°C. Typ-
ical yields from two rats were 15 g liver pulp, 1 g mitochondrial wet wt, 280 mg mitochondrial protein.
Preparation of a lyso.so?nal fraction. The first su- pernatant from the mitochondrial preparation was
used as starting material. This was centrifuged at 14,000g for 30 min. The supernatant was discarded. The pellet was resuspended in 4 vol of solution C and centrifuged at 14,OOOg for 10 min. The supernatant
was discarded. The pellet was resuspended in 1.5 vol of solution A and centrifuged at 24,000g for 10 min.
’ Abbreviation, EGTA: Ethylene glyeol his@-ami- noethyl ether)-N,N’-tetraacetic acid.
The supernatant was discarded. Volumes are rela-
tive to liver pulp weight. The temperature throughout was 0-5°C.
Assay of &gluouronidase. The assay used was based
on the method of de Duve et al. (15) in which the liberation of phenolphthalein from phenolphthalein
glucuronide is measured. In the original method the
reaction is stopped by adding a glycine buffer, pH 10.7, which converts the phenolphthalein liberated in
the incubation to its red-colored ionic form. When large samples were used to assay the very low activity
found in mitochondria prepared by the above method, protein was inadequately removed. The method was
modified by inclusion of 0.1 M ZnSOl in the glycine buffer to improve the removal of protein by centrif-
ugation and avoid falsely high extinction readings. The activity is expressed relative to the mitochondrial
protein content of the suspensions. Assay of protein. Protein was assayed by the mi-
crobiuret method of Burgi et al (16). The mean of four samples was found.
Assay of proteolysis. The mitochondria were sus-
pended in a buffered saline of the following compo- sition: KCL, 0.193 M; MgClz, 0.0186 M; phosphate, 0.0146
M, pH 7.6 (potassium salt); and chloramphenicol, 0.02 g/liter. The suspension was divided into two equal
portions and the lysosomal fraction was suspended in one of these. A volume of saline equal to the wet
weight of the lysosomal fraction was added to the other portion of the mitochondrial suspension to en- sure that the two suspensions were identical in mi-
tochondrial content while differing in their lysosomal content. For each suspension, 20 tubes were prepared, each containing 0.5 ml of the buffered saline. To these
tubes microliter quantities of 2 M HCl or 2 M KOH were added to give a range of pH values from 3 to 9.
Each tube received 0.5 ml of the mitochondrial sus-
pension. Tubes were incubated without shaking at 37°C for 19 h. Samples of 0.2 ml were withdrawn after 7 and 19 h and deproteinized with 0.6 ml of 0.5 M
HCIOI. Acid-soluble ninhydrin-reactive substances were measured in the samples as described by Alberti
and Bartley (17). Assays were referred to aglutamate standard. The pH of each tube was measured with a combined electrode several times during the course
of the incubation. Over a period of 19 h the pH changed by less than 0.1 pH unit in the pH range 4.5-8.0. Outside these limits pH variation was increasingly observed.
The concentration of acid-soluble ninhydrin-reac-
tive substances in the mitochondria at zero time was determined as the mean of four samples.
RESULTS
The values for proteolysis obtained from four similar experiments are shown in Fig. 1. Values given are for glutamic acid
PROTEOLYSIS IN RAT LIVER MITOCHONDRIA AND LYSOSOMES 157
7 hr 5
.‘; 19 hr
3 4 5 6 7 8 9 pH 3 4 5 6 7 8 9
FIG. 1. Proteolysis observed in four experiments conducted as described under Materials and Methods. 0, A, 0, V, mitochondria incubated as prepared, Experiment 1 to 4.0, A, n , V, mitochondria
incubated with a lysosomal supplement, Experiment 1 to 4.
equivalents formed in excess of the zero- time value. The values have been corrected for total protein content to take account of the variation between the four mito- chondrial preparations and to allow for the small amount of extra protein added with the lysosomal supplement. In each case the addition of a lysosomal supplement greatly increases the proteolysis at acid pH values and produces a much smaller increase at neutral pH values. The amount of lyso- somal material added as a supplement varies and determines the extent of pro- teolysis in the four supplemented mito- chondrial incubations.
The increases in protein concentration, P-glucuronidase (a lysosomal marker), and
proteolysis upon addition of a lysosomal supplement are quantified in Table I. Here comparable values have been taken from the curves in Fig. 1 at set pH values over the range 4.5-8.0.
It can be seen from the ratios of lyso- some-supplemented to nonsupplemented values that the increase in protein on ad- dition of lysosomal supplement was max- imally 15% in experiment 2 and minimally 6% in experiment 4. The increase in p-gluc- uronidase on addition of a lysosomal sup- plement was maximally 56-fold in exper- iment 3 and minimally 26-fold in experi- ment 1. In every case the increase in proteolysis at pH 4.5 and 5.0 is very similar to the increase in the lysosomal marker /3-
TABL
E I
EFFE
CT
OF A
LYS
OSOM
AL
SUPP
LEM
ENT
ON A
MIN
O AC
ID
FORM
ATIO
N
Expe
rimen
t In
cuba
tion
Lyso
som
al
time
(h)
supp
lem
ent
Prot
ein
&Giu
curo
nida
se
Amin
o ac
id
form
atio
n (p
mol
/g
tota
l pr
otein
) at
th
e in
dica
ted
pH
(mg/
(p
mol
/g
mito
-
ml)
chon
dria
l pr
otein
) 4.
5 5.
0 5.
5 6.
0 6.
5 7.
0 7.
5 8.
0
7 19 7 19 7 19 7 19
+ - ra
tio
+/-
+ 9.
47
226
- 8.
30
8.65
ra
tio
+/-
1.14
26
.1
+ - ra
tio
+/-
+ -
ratio
+/
-
+ -
ratio
+/
-
+ - ra
tio
+/-
+ -
ratio
+/
-
+ -
ratio
+/
-
9.47
22
6
8.30
8.
65
1.14
26
.1
26.5
35
8 23
.0
7.85
1.15
45
.6
26.5
35
8
23.0
7.
85
1.15
45
.6
13.6
36
9
12.5
6.
5 1.
08
56.7
13.6
36
9
12.5
6.
5 1.
08
56.7
16.1
15.1
1.
06
16.1
15
.1
1.06
184 4.
8 38
.3
184 4.
8 38
.3
1870
19
50
1860
11
20
71.4
74
.7
70.5
69
.3
26.2
26
.1
26.4
16
.2
2880
32
10
2500
16
00
111
121
103
142
25.9
26
.5
24.3
11
.3
2680
25
10
2090
11
50
59.2
58
.0
53.8
53
.5
45.3
43
.3
38.8
21
.5
4030
48
60
4530
31
40
92.1
11
7 14
7 19
0 43
.8
41.5
30
.8
16.5
2330
24
70
2360
12
40
38.6
44
.7
43.5
37
.5
60.4
55
.3
54.3
33
.1
3160
34
60
3600
18
40
59.8
65
.9
74.9
11
1 52
.8
52.5
48
.1
16.6
668
744
684
301
17.2
19
.1
21.7
21
.1
38.8
38
.9
31.5
14
.3
1380
16
60
1450
79
1 35
.6
44.2
48
.8
79.9
38
.8
37.6
29
.7
9.90
189
201
199
164
87.9
10
3 11
7 10
5 2.
15
1.95
1.
70
1.56
590
467
435
393
194
224
229
206
3.04
2.
08
1.90
1.
91
386
228
186
57.5
73
.5
77.8
6.
71
3.10
2.
39
203 69
.9
1260
62
8 58
9 22
5 31
7 31
0
5.60
1.
98
1.90
530
279
279
39.6
54
.4
64.5
13
.4
5.13
4.
33
2.90
B
533
282
z
1.89
z
227
5 61
.0
3.72
916
588
570
530
167
216
240
250
5.49
2.
72
2.38
2.
12
165
148
145
123
38.1
53
.0
42.9
36
.7
4.33
2.
79
3.38
3.
35
379
310
315
276
114
129
142
115
3.32
2.
40
2.22
2.
40
Note
. Co
mpa
rable
va
lues
of
am
ino
acid
fo
rmat
ion
at
the
indi
cate
d pH
va
lues
ha
ve
been
ta
ken
from
th
e cu
rves
sh
own
in
Fig.
1.
The
in
crem
ents
in
pr
otei
n co
ncen
tratio
n,
P-gl
ucur
onid
ase
activ
ity,
and
amin
o ac
id
form
atio
n up
on
addi
tion
of
a lys
osom
al
supp
lem
ent
are
given
as
th
e ra
tio
of
supp
lem
ente
d to
no
nsup
plem
ente
d va
lues
.
PROTEOLYSIS IN RAT LIVER MITOCHONDRIA AND LYSOSOMES 159
glucuronidase and is exactly so in exper- iment 1 at pH 5 and at 7 h incubation time. This suggests that the proteolytic enzymes active at these pH values have the same origin as the P-glucuronidase, i.e., a lyso- somal origin. Between pH 5.5 and 6.5 the increase in proteolysis on addition of ly- sosomal supplement falls, suggesting that enzymes of lysosomal origin make a pro- gressively smaller contribution to total proteolysis and that enzymes of mitochon- drial origin are increasingly contributing to total proteolysis. Above pH 7.0 the ly- sosomal contributions increase proteolysis by 1.56- to 5.13-fold, suggesting that en- zymes of lysosomal origin contribute 36 to 80% of total proteolysis in the supple- mented incubations.
It is of interest to try to calculate the contribution of enzymes of lysosomal or- igin to the proteolysis observed in the ab- sence of a lysosomal supplement to deter- mine the extent to which the observed pro- teolysis is due to residual lysosomal contamination. This can be done if two as- sumptions are made. The first assumption is that at any one pH value, proteolytic enzymes originating in the mitochondrial preparation would have the same activity, irrespective of the level of lysosomal con- tamination. If this was so, a plot of pro- teolysis (ordinate) against lysosomal con- tent (abscissa) would give a horizontal line if all proteolytic activity was of mitochon- drial origin.
The second assumption is that enzymes originating in the lysosomal fraction would have constant relative activities at any one pH value. If this was so, the percentage increase in the activity of the lysosomal marker P-glucuronidase when a lysosomal supplement was added to a mitochondrial preparation would be matched by an iden- tical percentage increase in the proteolytic activity at that pH value, if that activity was all of lysosomal origin. In this case a plot of proteolysis (ordinate) against p- glucuronidase (abscissa) would indicate a slope passing through the origin.
Specimen plots of this kind are shown in Fig. 2 for proteolysis at pH 5.0 and at pH 7.5. The plot at pH 5.0 (Fig. 2A) passes through the origin and so indicates that
all proteolysis is due to enzymes of lyso- somal origin. The plot at pH 7.5 (Fig. 2B) indicates that the proteolysis is partly of lysosomal origin and partly of mitochon- drial origin.
A method of calculating the contribution of lysosomal enzymes to total proteolysis in the absence of a lysosomal supplement (P in Fig. 2B) and in the presence of a lysosomal supplement (S in Fig. 2B) is given in Fig. 2. Here the difference between P-glucuronidase values for the two prep- arations has been used, but the difference between proteolysis at pH 5.0 could equally well be used as this meets the requirements for a lysosomal marker in the second as- sumption inherent in this method.
The contribution of lysosomal enzymes to total proteolysis in the absence of a ly- sosomal supplement has been calculated on the basis of both ,&glucuronidase and pH 5.0 proteolysis. Similar results are ob- tained. The results are shown in Table II. At pH 7.0 and above, proteases of mito- chondrial origin account for more than 93% of observed proteolysis after 7 h incubation and for more than 95% of observed pro- teolysis after 19 h incubation. A specimen plot of the calculated contributions to total proteolysis of enzymes originating in the lysosomal fraction and in the mitochon- drial fraction is presented in Fig. 3. The curve of lysosomal activity has an acid pH optimum at 5.0 and a plateau in the neutral region with an activity about 4% of that at the acid peak. The curve of mitochon- drial activity is rising sharply at pH 6.0 and has an optimum at pH 7.5. Figure 1 indicates that both lysosomal and mito- chondrial neutral activity has declined to about half at pH 9.0.
DISCUSSION
Mitochondria prepared as described here showed measurable protease activity over the pH range 3.5-9.0. Maximum activity was found in the neutral region around pH 7.5. The activity at pH 7.5 varied from 6.1 to 7.5 pmol glutamate equivalent formed/ h/g total protein in a 7-h incubation and from 7.5 to 16.3 in a 19-h incubation. Com- parable values from the work of other au-
160 BRIAN DEAN
2000 - 200
1600 - t
160 n aao s
1200.
:,,::i+Z-:‘:: -
Q T
40 - I 0 40 80 120 160 200 240 0 40 80 120 160 200 240
@LUCURONIDASE ymol /g mitochondrial protein/h
FIG. 2. Calculation of the lysosomal contribution to the observed proteolysis. The activity of /3-
glucuronidase is plotted against amino acid formation for identical samples of mitochondria incubated with (M + L) or without (M) a lysosomal supplement in experiment 1 for ‘7 h. (Table I). When all
the amino acid formation is due to enzymes of the lysosomal supplement the two values should indicate a slope passing through the graph axis, as is seen at pH 5.0 (A). At pH 7.5 (B) the indicated
slope intercepts the ordinate at a value equal to the contribution of enzymes of the mitochondrial fraction, i.e., where the lysosomal activity is extrapolated to zero. The contribution of enzymes of
the lysosomal fraction to total amino acid formation at any value of &glucuronidase can be calculated by finding the value of amino acid formation/unit @-glucuronidase as
Aaa(M + L) - (M)
AG(M + L) - (M) .
This value multiplied by the measured fl-glucuronidase activity gives amino acid formation due to enzymes of the lysosomal fraction (P and S in (B)). Amino acid formation due to enzymes of the mitochondrial fraction can be found by subtracting the lysosomal contribution from the total (Q
and T in (B)). As proteolysis at pH 5.0 is entirely due to enzymes of the lysosomal fraction (A) the increase in amino acid formation (Aaa) at pH 5.0 on addition of a lysosomal supplement could
be used to replace AG as a basis for calculation at neutral pH values.
thors are 8 pmol/h/g protein in a 2-h in- showed a broad peak centred at about pH cubation at pH 7.5 (7) and 21.5 pmol/h/g 7.5 and extending down into the acid re- protein in a 7-h incubation at pH 7.4 (8). gion. This profile was partly due to residual The endogenous amino acid content of mi- contamination by lysosomal proteases. tochondria measured at zero time for the When the truly mitochondrial contribution incubations varied from 41 to 65 pmol/g was calculated and plotted (Fig. 3) the peak protein so that long incubation times were did not extend into the acid region. This necessary to give sufficient increase to al- profile was distinct from the sharp peak low accurate measurement. centred at pH 6.0 which is described by
The choice of incubation medium can Subramanian et al. (7). By contrast, Man- greatly influence the rates measured (17), junath et al. (10) found a pH profile similar and the use of several different buffer sys- to the one in Fig. 1 but continuing to rise terns to cover a pH range gives discontin- above pH 7.5 through to 8.5. This may be uous pH curves (9). A saline medium with due to the presence in their mitochondria low buffering capacity was used in this of mast cell chymase which has a pH op- work to minimize incubation medium ef- timum of 8.5 (12,13). No attempt was made fects. The addition of chloramphenicol here to verify the absence of mast cell chy- prevented bacterial growth and also in- mase but the decline of activity at pH 8.0 hibited mitochondrial protein synthesis and above suggests that chymase did not and the recycling of proteolysis products contribute significantly to the proteolysis into protein. measured here. Tests for the presence of
The pH profile for proteolysis in chymase by the use of specific substrates nonsupplemented mitochondria (Fig. 1) or inhibitors would not give significant
TABL
E II
THEC
ON
TRIB
UTI
ON
OFL
YSO
SOM
ALEN
ZYM
ESTO
TOTA
LAM
INO
ACID
FOR
MAT
ION
INTH
EABS
ENC
EOFA
LYSO
SOM
ALSU
PPLE
MEN
T.
AMIN
OAC
IDFO
RM
ATIO
N(~
~~~/
~TO
TALP
RO
TEIN
)ATT
HEI
ND
ICAT
ED~H
4.5
5.0
5.5
6.0
6.5
7.0
7.5
8.0
Incu
batio
n tim
e (h
) Ex
perim
ent
Obs
erve
d am
ino
acid
fo
rmat
ion
71.4
74
.7
70.5
69
.3
87.9
10
3 11
7 10
5
Lyso
som
al
cont
ribut
ion
G 71
.7
74.7
71
.3
41.9
4.
03
3.90
3.
27
2.35
Ly
soso
mal
co
ntrib
utio
n P
71.7
74
.7
71.3
41
.9
4.03
3.
39
3.27
2.
35
Obs
erve
d am
ino
acid
fo
rmat
ion
111
121
103
142
194
224
229
206
Lyso
som
al
cont
ribut
ion
G 11
0 12
3 95
.5
58.1
15
.8
9.68
8.
21
7.45
Ly
soso
mal
co
ntrib
utio
n P
108
121
93.9
57
.1
15.5
9.
52
8.07
7.
32
Obs
erve
d am
ino
acid
fo
rmat
ion
59.2
58
.0
53.8
53
.5
57.5
73
.5
77.8
69
.9
Lyso
som
al
cont
ribut
ion
G 58
.7
54.9
45
.6
24.6
7.
36
3.46
2.
42
2.98
Ly
soso
mal
co
ntrib
utio
n P
62.0
58
.0
48.2
25
.9
7.77
3.
65
2.56
3.
15
Obs
erve
d am
ino
acid
fo
rmat
ion
92.1
11
7 14
7 19
1 22
5 31
7 31
0 28
2
Lyso
som
al
cont
ribut
ion
G 88
.3
106
98.2
66
.1
23.2
6.
97
6.25
5.
63
Lyso
som
al
cont
ribut
ion
P 97
.1
117
108
72.8
25
.5
7.67
6.
88
6.19
Obs
erve
d am
ino
acid
fo
rmat
ion
38.6
44
.7
43.5
37
.5
39.6
58
.6
64.5
61
.0
Lyso
som
al
cont
ribut
ion
G 41
.1
43.5
41
.6
21.6
8.
80
3.96
3.
85
2.98
Ly
soso
mal
co
ntrib
utio
n P
42.2
44
.7
42.7
22
.2
9.04
4.
14
3.95
3.
06
Obs
erve
d am
ino
acid
fo
rmat
ion
59.8
65
.9
74.9
11
1 16
7 21
6 24
0 25
0 Ly
soso
mal
co
ntrib
utio
n G
55.6
60
.9
63.3
31
.0
13.4
6.
68
5.92
5.
03
Lyso
som
al
cont
ribut
ion
P 60
.2
65.9
68
.4
33.6
14
.5
7.22
6.
41
5.44
Obs
erve
d am
ino
acid
fo
rmat
ion
17.2
19
.1
21.7
21
.1
38.1
53
.0
42.9
36
.7
Lyso
som
al
cont
ribut
ion
G 17
.4
19.4
17
.7
7.50
3.
40
2.54
2.
74
2.31
Ly
soso
mal
co
ntrib
utio
n P
17.1
19
.1
17.5
7.
38
3.34
2.
50
2.69
2.
27
Obs
erve
d am
ino
acid
fo
rmat
ion
35.6
44
.2
48.8
79
.9
114
129
142
115
Lyso
som
al
cont
ribut
ion
G 36
.0
43.3
37
.5
19.0
7.
10
4.85
4.
63
4.31
Ly
soso
mal
co
ntrib
utio
n P
36.8
44
.2
38.3
19
.5
7.25
4.
95
4.73
4.
40
7 19 7 19 7 19 7 19
Note
. Th
e lys
osom
al
cont
ribut
ion
has
been
ca
lcula
ted
as
set
out
in
Fig.
2,
us
ing
eith
er
the
incr
ease
in
@
-glu
curo
nida
se
(lyso
som
al
cont
ribut
ion
G)
or
the
incr
ease
in
am
ino
acid
fo
rmat
ion
at
pH
5.0
(lyso
som
al
cont
ribut
ion
P)
on
addi
tion
of
a lys
osom
al
supp
lem
ent
as
a ba
sis
for
calcu
latio
n.
162 BRIAN -- ’ - - U&AN
PH
FIG. 3. The contribution of lysosomal and mito-
chondrial enzymes to total amino acid formation. Data for experiment 1 with 7 h incubation are shown (Table
II). 0, amino acid formation due to lysosomal enzymes; A, amino acid formation due to mitochondrial en- zymes; 0, total amino acid formation.
positives because of the possible presence of unknown and uncharacterized enzymes which might have similar specificities.
The first assumption involved in the cal- culation of the lysosomal contribution is that the activity of mitochondrial enzymes will be unaffected by the addition of a ly- sosomal supplement. This might not be valid if the mitochondrial proteases only attacked a few minor proteins which were also attacked by lysosomal proteases. The addition of a lysosomal supplement might then lead to the exhaustion of available substrate during the course of the incu- bation so that the activity of the mito- chondrial proteases would appear to be less in the presence than in the absence of a lysosomal supplement. Rat liver mito- chondria contain several hundred proteins (18). It is unlikely that mitochondrial pro- teases would attack only a few of these. The proportion of the protein which is hy- drolyzed can be calculated on the assump- tion that the average molecular weight of amino acids when incorporated into protein
is 110. In these experiments the maximum amount of glutamate equivalent formed in the absence of a lysosomal supplement is 317 pmol/g protein in experiment 2 at pH 7.0 after 19 h. This is equivalent to 35 mg or 3.5% of the protein degraded. Further- more, because of the very low value of the lysosomal marker in the absence of a ly- sosomal supplement, a large error in the assay of proteolysis in the presence of a lysosomal supplement will make only a small difference to the estimate of the mi- tochondrial contribution (Fig. 2B).
The second assumption involved in the calculation of the lysosomal contribution is that at any one pH value, proteolysis due to enzymes of the lysosomal fraction would be proportional to the activity of p- glucuronidase. In these experiments the maximum amount of glutamate equivalent formed in the presence of a lysosomal sup- plement is 4860 pmol/g protein in exper- iment 2 at pH 5.0 after 19 h. This is equiv- alent to 535 mg or 53.5% of the protein degraded. Even in this case the ratio of proteolysis increase on addition of a ly- sosomal supplement, 41.5, is only 4% lower than the ratio of @glucuronidase increase on addition of a lysosomal supplement, 45.6 (Table I).
These considerations support the valid- ity of the method presented here for dis- tinguishing between proteases originating in the mitochondrial and in the lysosomal preparations.
The results presented in this paper pro- vide persuasive evidence for the existence of endogenous mitochondrial neutral pro- teases, but not absolute proof. The finding that supposed mitochondrial proteases were of mast cell origin (11) demonstrates that very low levels of contamination in protein terms can lead to substantial en- zyme activities. No mitochondrial prepa- ration can be absolutely pure. The origin of the neutral protease originating in the lysosomal fraction is also in doubt as the lysosomal preparation is not pure. It is un- likely that this neutral activity is due to the lysosomal acid proteases because these would not be expected to maintain activity through the neutral region to pH 9.0. The method of calculating the contribution to
PROTEOLYSIS IN RAT LIVER MITOCHONDRIA AND LYSOSOMES 163
total proteolysis, of enzymes originating in the mitochondrial, and in the lysosomal fraction provides a means of examining separately the response of these two pro- tease groups to inhibitors and to model substrates. This would establish similar- ities between the two groups and with other known cellular proteases. The neu- tral proteases may be novel enzymes not previously described.
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