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“Microsomes” in heart-muscle homogenates
B1OCHIMICA ET BIOPHYS1CA ACTA
"MICROSOMES" IN HEART-MUSCLE
I
HOMOGENATES*
H. A. M. H U L S M A N S
Laboratory o] Physiological Chemistry, University o/ Amsterdam (The Netherlands) (Received April 28th, 1961)
SUMMARY
I. Microsomes have never been clearly identified in homogenates of heart muscle, although electron micrographs of tissue sections have revealed a sarcoplasmic reticulum resembling the endoplasmic reticulum of other tissue. 2. By means of differential centrifugation four fractions were obtained from rat heart muscle and liver. A fraction sedimented at 105000 × g from heart muscle was found to contain the highest concentration of esterase, RNA and DPN + nucleosidase. Since previous work has shown t h a t these cell constituents are concentrated in the microsome fraction separated from liver homogenates, it is concluded t h a t heart-muscle homogenates also contain microsomes. 3-Based on the distribution of DNA (characteristic for nuclei), cytochrome oxidase (characteristic for mitochondria) and of esterase in the various fractions, the proportions of the cell components, on a protein basis, were calculated to be as follows (first value for heart, second for liver): nuclei + myofibrils + erythrocytes + unbroken cells + cell debris, 49, 15 ; mitochondria, 22, 25 ; microsomes, 15, 31 ; "soluble protein", I3, 32. 4. The cytochrome oxidase activity of heart mitochondria is 3.5 times that of liver mitochondria. The RNA concentration in liver microsomes is 3.1 times that of heart microsomes, and the esterase activity 12.4 times. Heart-muscle microsomes contain no measurable glucose-6-phosphatase activity. 5. No DPN + pyrophosphorylase could be detected in any of the heart-muscle fractions (or in brain nuclei). 6. Acid phosphatase and ribonuclease were concentrated in a "mitochondria + microsome" fraction of heart muscle, suggesting that this tissue contains lysosomes. However, this could not be stated with certainty since no increase in the activity of these enzymes was obtained after hypotonic or sonic treatment.
INTRODUCTION
The study of the fractions obtained by differential centrifugation of liver homogenates has greatly increased our knowledge of the properties of various microscopically identifiable cell components, and especially of the localization of intracellular enzymes within them. Comparable studies with muscle homogenates have mostly been restricted * T h i s work is p a r t of t h e M. D. thesis 1 of t h e a u t h o r , w h i c h w a s p u b l i s h e d (in D u t c h ) in M a y 196o.
Biochim. Biophys. Acta, 54 (1961) 1-14
2
I~. A. M. HULSMANS
to the identification of the respiratory granules in this tissue (reviewed in ref. 2). I t is now firmly established that intracellular respiration in muscle is localized in the sarcosomes of RETZIUS 3, which correspond to the mitochondria of other tissues. Other components of the heart-muscle cell have been much less studied b y this method. In particular, "microsomes" have never been definitely identified in homogenates of heart muscle, although electron micrographs 4 have clearly revealed that between the myofibrils a fine network exists which resembles the endoplasmic reticulum found in other tissues 5. I t has been shown 6 that the "microsome" fraction isolated from liver homogenates 7 is derived by fragmentation of the endoplasmic reticulum, which consists of a system of membranes forming a network of channels, with granules attached to the membranes. The reticulum of heart muscle differs from that of other tissues in that these granules are absent s. I t has been suggested that the sarcoplasmic reticulum is involved in the conduction of the nervous impulse from the cell membrane to the myofibril9. For these reasons it appeared to be desirable to a t t e m p t to prepare a microsome fraction from heart muscle and to determine its enzyme content. By means of differential centrifugation a homogenate of rat-heart muscle was separated into four fractions : (i) a mixture of nuclei, myofibrils and cell debris; (ii) a sarcosome fraction ; (iii) a small fraction whose properties will be further described; (iv) a supernatant fraction containing the "soluble" components of the cell. The distribution of nuclei, sarcosomes and "microsomes" in the three particulate fractions was determined b y the procedure introduced by DE DUVE1°, using DNA to characterize the nuclei, cytochrome oxidase for the sarcosomes, and esterase, DPN+ nucleosidase and RNA for the microsomes. MATERIALS AND METHODS
Chemicals Unless otherwise stated, all reagents were of analytical grade and dissolved in glass-distilled water, p-Phenylenediamine, E D T A and nicotinamide were obtained from British Drug Houses Ltd. Glucose 6-phosphate (barium salt), NMN, DPN + and ATP were from Sigma Chemical Co. D P N H was prepared from DPN + with alcohol dehydrogenase obtained from Boehringer & S6hne. Antimycin was kindly supplied b y the K y o w a Fermentation Co. Ltd. Cytochrome c was prepared from horse heart as described b y MARGOLIASH11.
Preparation of tissue fractions Heart muscle. 5 Wistar rats each weighing about 200-300 g were killed b y a blow on the head and as quickly as possible the throat was cut so as to remove the blood from the body. The thorax was then opened and the heart removed. The aorta and auricles were cut away, the heart opened and the blood soaked up with filter paper. The five hearts were then placed in IO ml of ice-cold 0.25 M sucrose and cut into small pieces with scissors. The solution was poured off and the finely cut heart tissue was washed out twice with ice-cold sucrose. The washed tissue was then "homogenized" in an amount of 0.25 M sucrose sufficient to give about a IO °/o "homogenate". The homogenizer was of the Potter-Elvehjem type with a Teflon plunger. Separate portions of the heart tissue were "homogenized" for 1-1.5 min, while the homogenizer tube Biochim. Biophys. Acta, 54 (I96I) i 14
"MICROSOMES" 1N HEART-MUSCLE HOMOGENATES
3
was kept in an ice bath. Care was taken to avoid any appreciable increase in temperature during the homogenization. The combined "homogenate" was centrifuged in the "multi-speed a t t a c h m e n t " of a M.S.E. Major refrigerated centrifuge for 3 min at 800 x g (all values of g refer to the middle of the tube). The supernatant was carefully poured off and the sediment further "homogenized" in a small volume of 0.25 M sucrose. The "homogenate" was centrifuged for 3 rain at 500 × g, the lowest speed sufficient for almost complete sedimentation of the erythrocytes, nuclei and myofibrils. The supernatant was carefully poured off and combined with the first supernatant to give what was called the "cytoplasmic (E) fraction". The sediment was "homogenized" in about 20 ml 0.25 M sucrose to give what was named the "nuclear (N) fraction", although it consisted for the most part of myofibrils and contained a considerable amount of sarcosomes and also some erythrocytes and cell debris as well as the nuclei. Balance studies were made only with the E fraction, because it was difficult to take representative samples from the so-called "homogenate" which in fact contained large pieces of myofibrils and connective tissues. After removal of a sample, the cytoplasmic fraction was fractionated in the Spinco preparative ultracentrifuge, model L, with rotor No. 4 o. After IO min at 12500 x g, the supernatant was removed, and the sediment homogenized in a small volume of 0.25 M sucrose and again centrifuged for IO min at 12500 × g. The supernatant was removed and combined with the previous, while the sediment was homogenized with about IO ml 0.25 M sucrose to give the "sarcosome (M) fraction". The combined supernatant was centrifuged for 60 min at lO5 400 × g to give a small amount of a red transparent pellet. After pouring off the supernatant the surface of the sediment was washed with a little 0.25 M sucrose, and the sediments were taken up in about 5 ml 0.25 M sucrose and homogenized to yield the " P fraction". The final supernatant (about 50 ml) was called the "S fraction". The symbols used for the different fractions follow DE DUVE et al. 1°. The entire fractionation required about 2.5 h. The temperature of the tissue material was kept at about o ° throughout the fractionation. Liver. R a t liver was homogenized in the same way as the heart muscle. The fractionation was carried out similarly, except for a slight difference in the preparation of the N fraction. The homogenate was first centrifuged in the "multi-speed attachm e n t " of the M.S.E. centrifuge for 5 min at 125 ° × g. The supernatant was carefully poured off and the sediment suspended and homogenized in a small volume of 0.25 M sucrose. This homogenate was then centrifuged for 5 min at 800 × g and the carefully decanted supernatant added to the previous. The combined supernatants formed the E fraction which was further fractionated in exactly the same way as for heart. The sediment was suspended in 0.25 M sucrose to give the N fraction.
Determination of enzyme activities Cytochrome oxidase. This enzyme was determined manometrically at 26 ° with p-phenylenediamine as reducing agent. The reaction mixture contained 50 m M potassium phosphate buffer (pH 7-4), 0.3 m M EDTA (pH 7.4), 55/~M cytochrome c, 15 m M p-phenylenediamine (pH 7.4) and an aliquot of the tissue fraction in a total volume of 3 ml. The reaction was begun, after temperature equilibration, b y tipping the p-phenylenediamine solution from the side-arm into the main compartment of Biochirn. Biophys. Acta, 54 (I96I)
1
14
4
H. &. M. HULSMANS
the manometric vessel. Readings commenced 3 min later and the initial rate of 0 2 uptake was measured. Since, above I O O / , 1 0 , / h , the rate of 0 3 uptake was not proportional to the concentration of tissue fraction, the concentration of the enzyme was calculated from a calibration curve. The 0 3 uptake never exceeded 200/,l/h. The conditions were so chosen as to promote swelling of the mitochondria so that the full cytochrome oxidase activity would be determined. Glucose-6-phosphatase. The method used was based on that described b y DE DUVE et al. 1°. The reaction mixture contained 7 m M histidine buffer (pH 6.5), I m M E D T A (pH 6.5), 40 m M glucose 6-phosphate (pH 6.5) and an aliquot of the tissue fraction in a final volume of I ml. The reaction was begun by addition of the glucose 6phosphate and was stopped after IO min at 320 by the addition of 2 ml IO % (w/v) trichloroacetic acid. The precipitated protein was removed b y centrifugation and the phosphate content of the supernatant determined by the method of SUMNER12. A zero-time control (to correct for phosphate in the tissue preparation) and a no-tissue control (to correct for any non-enzymic hydrolysis) were subtracted from the values found. The amount of inorganic phosphate set free was linear with time and proportional to the concentration of tissue fraction, Esterase. The esterase activity was measured manometrically in NaHCOs-CO 2 buffer with p-nitrophenyl acetate as substrate. The difficultly soluble substrate was ground in a mortar with an equal amount of gum arabic and ten times this amount of water. The reaction mixture contained 25 m M NaHCO3, 0.02 ml of the substrate suspension and an aliquot of the tissue fraction, in a total volume of i . i ml. The p H was maintained at p H 7.4 by having 5 % C02-95 % N2 as the gas phase. The reaction was begun by tipping in the substrate from the side-arm. The COs evolution was followed for 20 rain at 37 °, and the initial rate of evolution determined. The gas evolution was constant for 6-1o rain, at a rate which was proportional to the concentration of tissue fraction. DPNH-cytochrome c reductase. This was measured spectrophotometrically at 550 m/z and at room temperature by the method described by HOGEBOOM13. The reaction mixture contained 75 m M potassium phosphate buffer (pH 7.2), I m M EDTA, o.i m M D P N H , 38/zM cytochrome c, I m M KCN, 1. 5 ~g/ml antimycin (about 50 ~g/mg protein) and an aliquot of the tissue fraction in a total volume of 3 ml. The solution in the reference cell had the same composition, except that it contained no D P N H or cytochrome c. The cyanide was added immediately before the addition of the tissue fraction which began the reaction. The increase of absorbancy was linear with time for 1.5-2 min. The rate of the reaction was proportional to the concentration of the tissue fraction. DPN+ nucleosidase. This enzyme activity was determined as described by KAPLAN 14.
DPN+ pyrophosphorylase. This enzyme activity was determined as described by KORNBERG 15.
Analytical methods Nucleic acids. The concentration of the nucleic acids was determined b y MoOL~lvs modification of the method of SCHMIDT AND THAN'NHAUSER17. The low concentration of RNA in heart muscle made it necessary to correct for interference caused b y the presence of polypeptides in the solution. This was done by the method of MclNDoE Biochim. Biophys. Acta, 54 (1961) 1-14
"MICROSOMES"
IN HEART-MUSCLE HOMOGENATES
5
AND DAVlDSONls in which the difference between the absorbancies at 260 m ~ and 290 m ~ was used. The amount of nucleic acid was calculated from the extinction coefficients at 260 m/~ of the pure nucleic acids, neglecting absorption of the latter at 29 ° m~. The extinction coefficient of heart-muscle RNA was calculated from the nucleotide composition, determined b y paper-chromatographic separation of the nucleotides present in the hydrolysed RNA (cf. MAGANASIKel al. 19) and measurement of the ultraviolet absorption spectra. From these spectra and the data of ELSON et al. 2°, the composition expressed as moles/Ioo moles nucleotides was found to be: adenylic acid, 20.9; guanidylic acid, 21.5; cytidylic acid, 37.o and uridylic acid, 20.6, from which the molar extinction coefficient (per P atom) was calculated to be IO 78o. The same value was used for calculating the amount of liver RNA. For DNA, it was assumed that the molar extinction coefficient (per P atom) was the same as for t h y m u s nucleic acid 21, namely 6400. Protein. Protein was determined by the biuret method of GORNALL et al. 22, as applied to particulate material b y CLELAND AND SLATER23. RESULTS
Protein. In Table I the distributions of the protein in the different fractions isolated from homogenates of rat heart and rat liver are compared. A striking difference between the two tissues is the amount of material sedimented at low speed, i.e. found in fraction N. Most of the protein of heart-muscle homogenate is found in this fraction, which in this tissue consists largely of myofibrils. The percentage of the total protein found in the M fraction is similar in the two tissues. The P fraction, on the other hand, makes up a much smaller part of the total protein in heart muscle than in liver, even when expressed as percentage of the E fraction. The S fractions represent about the same proportion of the E fraction in the two tissues. Cytochrome oxidase. Table I gives also the distribution of the cytochrome oxidase in the different fractions. In agreement with previous findings 24, 23, z, the cytochrome oxidase is most active in the mitochondrial (sarcosomal) fractions. The greater TABLE
I
DISTRIBUTION OF PROTEIN AND CYTOCHROME OXII)ASE ACTIVITY IN FRACTIONS SEPARATED BY DIFFERENTIAL CENTRIFUGATION OF HOMOGENATES OF RAT HEART MUSCLE .&ND LIVER T h e v a l u e s g i v e n f o r h e a r t m u s c l e a r e t h e m e a n s of 3 e x p e r i m e n t s , t h o s e f o r l i v e r of 2 e x p e r i m e n t s .
Fraction
Heart
N E M P S M + P + S
Cylochrome c oxidase
Protein (% of total protein in #action*) Liver
70 3° 12.o 3.7 13. 5
27 73 14. 5 29.0 32.5
29.2
76.o
Heart Q02
43 ° 132o 26oo I29O 20
Liver
% total activity in fraction*
43.1 56.9 44.9 6.7 O. 4
QO.
34 ° 202 820 47 IO
% total activity in fraction*
38.5 t)1.5 5° 5.7 1.3
52.o
57.o
* E x p r e s s e d as p e r c e n t a g e of t h a t f o u n d in N f r a c t i o n + t h a t f o u n d in E f r a c t i o n .
Biochim. Biophys..qcta,
54 (1961) t I4
H. A. M. HULSMANS
specific activity of the heart sarcosomes compared with the liver mitochondria reflects the greater respiratory activity of this tissue ~. In both cases, a substantial amount of the cytochrome oxidase is found in the N fraction, presumably due to mitochondria either not extracted from the tissue or sedimented at low speed. Activity is also found in the P fraction; when expressed as specific activity this is much more marked in the case of heart (cf. ref. 23). ,
,
i
~
,
J
,
,
J
20
o
....
~
o
,~,
,~o,-~--o
5
1o
P/'otein (rng)
:Fig. I, Glucose-6-phosphatase activity of h o m o g e n a t e s of r a t liver ( × - - × ) and h e a r t muscle ( O - - O ) , The protein c o n t e n t was determined b y the Kjeldahl m e t h o d (N × 6.25).
Glucose-6-phosphatase. HERS et al. 2~ showed that this enzyme was exclusively localized in liver microsomes. The great difference between the ability of liver and heart homogenates to hydrolyse glucose 6-phosphate is shown in Fig. z. The almost insignificant activity in heart homogenates (cf. AvI-DOR et al3 e) could be due to nonspecific phosphomonoesterase activity. From this result, it must be concluded either that heart-muscle homogenates do not contain microsomes or, if they do, that these microsomes lack glucose-6-phosphatase. Esterase. UNDERHAY et al3 7 have shown that certain esterases are distributed in the different fractions of a liver homogenate in the same way as glucose-6-phosphatase, which is good evidence that these enzymes are also localized in the microsomes. In Table II are compared the esterase activities, measured with p-nitrophenyl acetate, of the fractions obtained from both rat liver and rat heart. With this substrate TABLE II DISTRIBUTION BY
DIFFERENTIAL
OF
ESTERASE
CENTRIFUGATION
OF
ACTIVITY HOMOGENATES
IN
FRACTIONS OF
RAT
SEPARATED
HEART
MUSCLE
AND
LIVI~R
The values are for the same fractionations as in Table I. Heart Fraction QC02
N E M P S M+P+S
186 392 164 678 472
Liver
% of total activity in fraction*
51.5 48.5 7.8 9.1 25.2
QC02
1221 5599 1398 13 745 891
% of total activity in fraction*
7.6 92.4 4.7 9o.5 6.3
42.1
I O I "5
* E x p r e s s e d as percentage of t h a t found in N fraction + E fraction.
Biochim. Biophys. Acta, 54 (1961) 1-14
"MICROSOMES"
IN
HEART-MUSCLE
HOMOGENATES
7
essentially only the ali-esterase and the aromatic esterase are measured ~. It is clear from the results shown in Table I I that the esterase activity is much higher (about 2u-fold) in liver than in heart muscle. In both tissues, the highest specific activity was found in the P fraction. A much greater fraction of the activity was found in the supernatant fraction of heart muscle than of liver, although the specific activity was no greater. No difference between the esterases in the P and S fractions of heart muscle could be found by use of the inhibitors neostygmine (6-8 % inhibition by o.oi mg/ml) or diisopropylfluorophosphate (47-50 % inhibition by 0.5 mM). It is possible that serum esterase is responsible for the activity in the supernatant. DPNH-cytochrome c reductase. Liver homogenates appear to contain three systems catalysing the reduction of added ferricytochrome c by added D P N H , viz. (I) the antimycin- and Amytal-sensitive system present in the mitochondria; (2) the antimycin- and Amytal-insensitive reaction catalysed by the microsomes; and (3) an obscure pathway, also insensitive to antimycin and Amytal, which appears to be present on the exterior of the mitochondria 29. Table I I I shows that the highest activity of the antimycin-insensitive D P N H cytochrome c reductase of heart-muscle homogenates is found in the P fraction. TABLE III DISTRIBUTION OF ANTIMYCIN-INSENSITIVE DPNH--CYTOCHROME C REDUCTASE ACTIVITY IN F R A C T I O N S S E P A R A T E D BY D I F F E R E N T I A L C E N T R I F U G A T I O N OF H O M O G E N A T E S OF R A T - H E A R T M U S C L E
The values are for the same fractionations with heart muscle as in Table I. Fraction
A A 550 m#lmin/mg protein
°/o of total activity in fraction*
N E M
o.o63 o.264 0.224
21.8
P
o.816
29. 5
S M+P+S
0.055
35-7 64.3
6.0 57.3
* Expressed as percentage of that found in N fraction + E fraction. D P N + pyrophosphorylase. In agreement with HOGEBOOM AND SCHNEIDERs°, DPN + pyrophosphorylase was found to be concentrated in the nuclear fraction isolated from rat-liver homogenates. However, no activity of this enzyme could be found in any of the fractions separated from the heart-muscle homogenate. (Furthermore, no activity could be found in brain nuclei.) D P N + nucleosidase. This enzyme is localized in the microsome fraction of rat-liver homogenatesZl, 3~. Table IV shows that the P fraction of heart-muscle homogenates also contains the greatest activity. Nucleic acids. It is long known from histochemical studies that DNA is exclusively located in the cell nucleus (see ref. 33) and this was confirmed for liver homogenates by HOGEBOOMAND SCHNEIDER34. Table V shows a comparison between the distribution of DNA in the fractions isolated from rat heart and rat liver in our studies. The liver contained rather more DNA than the heart. In both tissues the DNA is largely confined to the N fraction, but an appreciable amount was found in the heart M Biochim. Biophys. Acta, 54 (196I) 1-14
8
H.A.M.
HULSMANS
TABLE IV DISTRIBUTION
OF
BY DIFFERENTIAL
DPN +
NUCLEOSIDASE
CENTRIFUGATION
ACTIVITY IN
OF H O M O G E N A T E S
FRACTIONS OF
SEPARATED
RAT-HEART
MUSCLE
T h e v a l u e s g i v e n are t h e m e a n s of 3 f r a e t i o n a t i o n s n o t i d e n t i c a l w i t h t h o s e s h o w n in T a b l e I, Protein Fraction
D P N + nucleosidase i~mole D P N + split/rag protein/h
% of total protein in fraction*
N E M P S M+P+S
66 34
o.525 o.546 o.496 1.341 0.238
19.4 3.2 lO. 7
% of total activity in fraction*
65 35 18.o 8.1 4.9
33.3
31
* E x p r e s s e d as p e r c e n t a g e of t h a t f o u n d in N f r a c t i o n + E fraction. TABLE V DISTRIBUTION
OF DNA
IN
FRACTIONS
OF HOMOGENATES
SEPARATED
OF RAT
HEART
BY DIFFERENTIAL MUSCLE
CENTRIFUGATION
AND LIVER
T h e v a l u e s g i v e n for h e a r t m u s c l e are t h e m e a n s of 3 e x p e r i m e n t s (the s a m e f r a c t i o n a t i o n s as i n T a b l e I), t h o s e for l i v e r are for one e x p e r i m e n t (one of t h o s e s h o w n in T a b l e I).
Fraction
Heart
N E M P S M + P + S
DNA
Protein (% of total protein in fraction*) Liver
7° 3° 12 3.7 13. 5
25 75 13 27 34
29.2
Heart pmoles Pig protein
29-4 5.5 9-3 4.5 1.6
74
Liver
% of total amount in fraction*
92.7 7.3 5 .1 0.8 0.9 6.8
#mole Pig protein
12.7 8 4 IO 4
% of total amount in fraction*
83.2 16.8 1-4 6.9 3.5 11.8
* E x p r e s s e d as p e r c e n t a g e of t h a t found in N f r a c t i o n + E fraction.
fraction and in the liver P fraction, indicating contamination of these fractions b y small amounts of nuclei or nuclear fragments. This was confirmed microscopically (Feulgen staining) for the heart M fraction. CLAUDE7 and SCHNEIDER84, 35 have shown that the highest concentration of RNA is found in the microsome fraction of liver homogenates, although this is not the only cell component containing RNA. Table VI shows that liver homogenates contain very much more RNA than heart homogenates. In both cases, b y far the highest concentration was found in the P fraction. DISCUSSION
Clearly the fractions as separated by differential centrifugation are very impure, especially those obtained from the heart-muscle homogenate. The relative proportions of the various fractions reflect rather poorly the proportions of the various cell Biochim. Biophys. Acta, 54 (1961) 1-14
"MICROSOMES" IN HEART-MUSCLE HOMOGENATES TABLE
9
VI
DISTRIBUTION OF R N A IN FRACTIONS SEPARATED BY DIFFERENTIAL CENTRIFUGATION OF HOMOGENATES OF RAT HEART MUSCLE AND LIVER
T h e v a l u e s g i v e n are for t h e s a m e f r a e t i o n a t i o n s as s h o w n in Table V. RNA Heart
Fraction
Liver
I~mole Pig
% of total amount
t~moleP/g
% of total amount
protein
in fraction*
protein
in fraction*
N
25
70
69
15.8
E
24
3°
122
84.2
M
23
12.1
26
3.1
P
51
7.6
254
62.5
S
io
5.2
41
13.o
M + P + S
78.6
24.9
* E x p r e s s e d as p e r c e n t a g e of t h a t f o u n d in N fraction + E fraction.
TABLE
VII
PROPORTION OF CELL COMPONENTS IN HOMOGENATES OF RAT HEART MUSCLE AND LIVER
Calculated f r o m t h e d a t a in Tables I and II, on the basis of a s s u m p t i o n s given in t h e text. Celt component
% of total protein Heart
Nuclei, etc. * Mitochondria Microsomes "Soluble" p r o t e i n
Liver
49
15
22
25
15
3i
13
32
99
lO3
* I n c l u d i n g u n b r o k e n cells, cell debris, e r y t h r o c y t e s and, in t h e case of heart, myofibrils.
components present in the homogenate. However, by making certain assumptions, it is possible to obtain a much more accurate, although still approximate, picture. The assumptions are: (I) cytochrome oxidase is present only in the mitochondria; (2) the M and P fractions consist only of mitochondria and microsomes; (3) the S fraction is not contaminated by any appreciable amount of microsomes; (4) "soluble" protein is present only in the S fraction; (5) there are both a microsomal-bound and a soluble esterase. On the basis of these assumptions Table VII can be derived from the data given in Tables I and II (see APPENDIX). About one half of the protein of the heart-muscle homogenate consists of the so-called nuclear fraction, which in fact is largely made up of myofibrils. This fraction constitutes only 15 % of the liver homogenate. In both tissues, mitochondria make up about one quarter of the protein. The higher respiratory activity of heart is due to the greater concentration of the cytochrome system in heart mitochondria ~. When microsomal contamination is allowed for, the heart sarcosomes have 3.5 times the Biochim. Biophys. A c t a , 54 (1961) 1-14-
I0
H.A.M.
HULSMANS
cytochrome oxidase activity of fiver mitochondria. More than 6o % of the total protein of a liver homogenate is made up of the microsomes (31%) and "soluble" protein (3z %). These components form a considerably smaller proportion of a heartmuscle homogenate. In Table VIII are shown the compositions of the various fractions isolated by differential centrifugation. The N fraction is heavily contaminated with mitochondria (especially in liver) and with microsomes. The M fraction contains appreciable TABLE
VIII
COMPOSITION OF FRACTIONS SEPARATED BY DIFFERENTIAL CENTRIFUGATION" OF HOMOGENATES OF RAT HEART MUSCLE AND LIVER Calculated from the data in Tables I and II, on the basis of assumptions
given in the text.
Composition of reactions Cell component
N
M
heart
liver
P
heart
liver
Nuclei, etc. *
7o
54
.
14
38
86
91
42
5
Microsomes
I6
8
14
9
58
95
"Soluble" protein
. IOO
* Including unbroken
. IOO
.
S liver
Mitochondria
.
.
heart
. IOO
.
TABLE
.
IOO
liver
.
.
IOO
cells, cell d e b r i s , e r y t h r o c y t e s
.
heart
IOO
I
I
--
--
99
99
IOO
IOO
a n d , i n t h e c a s e of h e a r t , u l y o f i b r i l s .
IX
DISTRIBUTION OF MITOCHONDRIA AND MICROSOMES IN VARIOUS FRACTIONS C a l c u l a t e d f r o m t h e d a t a i n p r e v i o u s T a b l e s . T h e v a l u e s g i v e n r e p r e s e n t t h e p e r c e n t a g e s o f t h e cell constituent or component found in the various fractious.
Fr~t~n
Mitochondrm
Mwrosomcs
DPNHcytochromec reduct~e
DPN+ n~leosidase
RNA
Heart N
46
75
36
65
7o
M
47
II
22
18
I2
P
7
I4
3o
8
8
S
0. 4
--
6
5
5
IOO
94
96
95
16
lOO. 4 Liver N
4°
7
--
--
M
52
4
--
--
P
6
88
--
---
62
S
1.6
--
---
--
13
99.6
IOO
3
94
B i o c h i m . B i o p h y s . d c t a , 54 (1961) 1 - 1 4
"MICROSOMES" 1N HEART-MUSCLE HOMOGENATES
II
amounts of microsomes, especially that obtained from heart. The P fraction isolated from liver consists of 95 % microsomes, while that isolated from heart is heavily (42 %1 contaminated with mitochondria. The reason for this is t h a t the amount of microsomes in the P fraction in heart is only 7.7 % of the corresponding value in liver, so that a similar contamination with the mitochondria (the amounts of mitochondria in the P fractions from the two tissues are in fact identical) has relatively a much greater effect in the case of the heart. The distribution of the mitochondria and microsomes in the various fractions is given in Table IX, which also shows for comparison with the microsomes the data on the distribution of the antimycin-resistant D P N H - c y t o c h r o m e c reductase, the DPN+ nucleosidase and the RNA, taken from Tables III, IV and VI, respectively. With both heart and liver, only about one-half of the mitochondria are obtained in the M fraction, most of the remainder being sedimented with the nuclei and myofibrils in the N fraction. The distribution of the microsomes presents a very different picture in the two tissues. Most (88 °/o) of the liver microsomes were obtained in the P fraction, but only 14 °/o of the heart microsomes were found in this fraction. Most (75 %) were sedimented with the myofibrils and nuclei which strongly suggests that they were not fragmented and set free in the original homogenization. If they are in close association with the myofibrilss, they will be protected from fragmentation during homogenization. I t is clear that a different technique should be used to obtain a good yield of reasonably pure microsomes from a heart-muscle homogenate. The value (75 %) given for the percentage of the heart microsomes present in the N fraction is based on the particulate esterase activity, on the assumption that this is confined to the microsomes. This assumption is supported by the distributions of the DPN+ nucleosidase and RNA, which are quite similar to that of the particulate esterase. Thus, it appears reasonable to conclude that heart-muscle homogenates contain microsomes characterized like liver microsomes b y the localization therein of all the particulate esterase, the RNA and the DPN+ nucleosidase. Any one of these three components can be used to follow the distribution of the microsomes. The antimycin-resistant D P N H - c y t o c h r o m e c reductase is much less suitable for this purpose since, although the highest specific activity of this enzyme was also present in the P fraction, much less of the enzyme was found in the N fraction, and more in the M and P fractions than would be expected from the other distributions. I t appears that the little-understood antimycin-resistant D P N H - c y t o c h r o m e c reductase in mitochondria (the so-called "external pathway") is confusing the picture (cf. ref. 26). The possible presence in heart muscle of DE DUVE'S lysosomes 1° has been ignored in the above treatment. In fact, small activities of acid phosphatase and RNAase, typical lysosomal enzymes in liver, were found in heart-muscle homogenates. The highest specific activity was obtained in a combined (M + P) fraction. The activities of the two enzymes were distributed over the N, (M + P) and S fractions in a very similar manner. From these results, it seems rather likely that some lysosomes are present in heart muscle. However, this cannot be stated with certainty since no increase in the activity of these enzymes was obtained after hypotonic or sonic treatment. This is one of the most important characteristics of lysosomal enzymes in liver. I t is possible that the lysosomes are disrupted during homogenization of the heart muscle. This could also be the reason why 16-2o °/o of the enzyme activities were found in the S fraction. Biochim. Biophys. Acta, 54 (1961) l-14
12
H.A.M.
HULSMANS
In most tissues, the endoplasmic reticulum, from which the microsome fraction is derived, consists of lipoprotein membranes and RNA-containing granules. In heart mucle, however, these granules are lacking s. Nevertheless, the RNA appears to be associated with the microsome fraction in this tissue also. If the concentrations of RNA in the P fractions (Table VI) are corrected for the mitochondria in these fractions (Table VIII), and assuming that the mitochondria contain no RNA, it can be calculated that heart microsomes contain 87 and liver microsomes 267 ~moles RNA-P/g protein, i.e. a difference of a factor of 3. ACKNOWLEDGEMENTS
This work was supported in part by a grant from the Foundation "De Drie Lichten". I wish to thank Professor E. C. SLATERfor his interest and advice, and Dr. P. BORST and Dr. C. VEEGER for their help given in many time-consuming experiments. The help given by Mr. F. AMONS and the skilful technical assistance of Miss M. VAN U F F E L E N , Miss A. SEARLEand Mr. A. KOEKEBAKKERare also gratefully acknowledged.
APPENDIX
Calculation of the proportion of the various cell components in homogenates of rat heart muscle and liver The following assumptions are made: (I) Cytochrome oxidase is present only in the mitochondria. (2) The M and P fractions contain only mitochondria and microsomes. (3) The S fraction does not contain any microsomes. (4) Soluble protein is present only in the S fraction. (5) Esterase is present both in the microsomes and the "soluble" fraction. Suppose that the M fraction consists of x % mitochondria and ( I O O - x ) % microsomes and that the P fraction consists of y ~o mitochondria and (IOO - - y ) % microsomes. Then the Qo2 (cytochrome oxidase) of pure mitocbondria equals (IOO/X) ((202 of M fraction) = (IOO/y) (Qo2 of P fraction) x
i.e .
.
Y
Qo~ of M fraction
. . Qo~ of p fraction
/1
(I)
Similarly, Qco2 (esterase) of pure microsomes equals ElOO/(ioo- x)]Qcoz of M fraction = [ioo/(ioo--Y)] Qco2 of P fraction IOO
-
-
i.e. i o o - - ~
x
Qco~ of M fraction Qco~ of p fraction
= [2
(2)
The solution of equations (I) and (2) gives y
IOO --
IOO [2
[t--t2
;x =]Iy Biochim. Biophys. Acta, 54 (1961) I - I 4
13
"M1CROSOMES" 1N HEART-MUSCLE HOMOGENATES
The values in Tables I and I I give ]1 [2 11 - - [2 I 0 0 - - I 0 0 [2
Y x Qo2
( c y t o c h r o m e oxidase) pure mitochondria Qco2 (esterase) pure microsomes
Heart
Liver
2.02 0.24 1.7 8 75.8 42.5 86
17. 5 0,I I7. 4 89.8 5.2 90.5
3020
906
118o
14 500
The distribution of mitochondria in the various fractions can be calculated from the data of Table I Heart Fraction
protein N M P S
Liver
%
7° 12 3.7 13. 5
o;
OO~ 43 ° 26oo 129o 20
99.2
(0
(2)
(3)
protein
Q02
(t)
(2)
(3)
o.142 o.86 0.425 0.007
IO.O lO. 3 1.57 0.09
45.6 47.o 7.2 0. 4
27 14. 5 29 32.5
34 ° 820 47 io
0.376 0.905 o.o52 O.Oli
lO.2 13.i 1.5 0. 4
40.5 52.o 5.9 1.6
~'(2) = 21.96
lOO.2
lO3.O
~(2) = 25.2
IOO.O
( I ) = p r o p o r t i o n of p r o t e i n in f r a c t i o n c o n s i s t i n g of m i t o c h o n d r i a = Qoz/(Qo2 of p u r e m i t o chondria). (2) = % of t o t a l p r o t e i n in t h i s f r a c t i o n c o n s i s t i n g of m i t o c h o n d r i a = (i) × (% p r o t e i n ) . I00
(3) = % of t o t a l m i t o c h o n d r i a p r e s e n t in f r a c t i o n -- -
-
X (2)
21(2)
Similarly, the distribution of the microsomes in the various fractions can be calculated from the data of Table I I Heart Fraction
N M P
%
Liver
%
protein
QCO~
(~)
(2)
(3)
protein
7° 12.o 3.7
186 164 678
o.158 o.139 0.575
11.o 7 1.67 2.13
74.6 11.2 14. 4
27.0
~7(2) = 14.87
lOO.2
14. 5 29.0
QCO2 1221 1398 13 745
(#
(2)
(,1)
0.084 0.097 0.948
2. 3 1.4 27-5
7.4 4-5 88.4
~'(2) = 31.2
lOO.3
The composition of the N fraction is then % mitochondria % microsomes
% nuclei, etc. % of t o t a l p r o t e i n found in N f r a c t i o n % of t o t a l p r o t e i n c o n s i s t i n g of nuclei, etc.
Heart
Liver
I4.2 15.8
37-6 8.4
30.0
46.o
7 °.0
54.o
7°
27
49
14.6
Biochim. Biophys. dcta, 54 (1961) t - I 4
14
H. A. M. HULSMANS
The composition of the S fraction is % mitochondria ~o " s o l u b l e " % of total protein found in S fraction % of total protein consisting of "soluble" protein
Heaet
Liver
0. 7 99.3
I.I 98.9
13. 5
32.5
13. 4
32.2
The results of these calculations are summarized in Tables V I I - I X .
REFERENCES 1 H. A. M. HULSMANS, Verdeling van enzymactiviteiten by [ractionering van hartspierwee]sel, A m s t e r d a m , P o o r t p e r s N.V., 196o. 2 E. C. SLATER, in G. H. BOURNE, The Structure and Function o] Muscle, Vol. 2, Academic Press, Inc., N e w York, 196o, p. lO5. 3 G. RETZlUS, Biol. Untersuch. (I~.F.), I (189o) 1. 4 H. S. BENNETT AND K. R. PORTER, Am. J. Anat., 93 (1953) 6I. 5 G. E. PALADE, J. Biophys. Biochem. Cytol., Suppl. No. 2 (1956) 85. G. E. PALADE, J. Biophys. Biochem. Cytol., i (1955) 59. 7 A. CLAUDE, J. Exptl. Med., 80 (1944) 19. 8 K. R. PORTER AND G. E. PALADE, J. Biophys. Biochem. Cytol., 3 (1957) 269. 9 A. F. HUXLEY AND R. E. TAYLOR, J. Physiol., 13o (1955) 4910 C. DE DUVE, B. C. PRESSMAN, R. GIANETTO, R. WATTIAUX AND F. APPELMANS, Biochem. J., 6o (1955) 604. ll E. MARGOLIASH, Biochem. J., 56 (1954) 529. 12 j . B. SUMNER, Science, ioo (1944) 413 . 13 G. H. HOGEBOOM, J. Biol. Chem., 177 (1949) 847. 14 N. O. KAPLAN, in Methods in Enzymology, Vol. 2, Academic Press, Inc., New York, 1955, p. 660. 15 A. KORNBERG, J. Biol. Chem., 182 (195o) 779. is y . MOULA, Arch. sci. physiol., 7 (1953) 161, 241. 17 G. SCHMIDT AND S. J. THANNHAUSER, J. Biol. Chem., 161 (1945) 83. as W. M. MclNDOE AND J. N. DAVIDSON, Brit. J. Cancer, 6 (1952) 200. 19 n . MAGASANIK, E. VISCHER, R. DONIGER, D. ELSON AND E. CHARGAFF,J. Biol. Chem., I86 (195o) 37. 20 D. ELSON, T. GUSTAFSON AND E. CHARGAFF, J. Biol. Chem., 209 (1954) 285. 21 E. CHARGAFF, E. VISCHER, R. DONIGER, C. GREEN AND F. MISANI, J. Biol. Chem., 177 (1949) 405 • 22 A. G. GORNALL, C. J. BARDAWILL AND M. M. DAVID, J. Biol. Chem., 177 (1949) 751. 23 K. W. CLELAND AND E. C. SLATER, Biochem. J., 53 (1953) 547. 24 O. H. HOGEBOOM, A. CLAUDE AND R. D. HOTCHKISS, J. Biol. Chem., 165 (1946) 615. 25 H. G. HERS, J. BERTHET, L. BERTHET AND C. DE DUVE, Bull. soc. chim. biol., 33 (1951) 21. 2n y . AvI-DoR, A. TRAUB AND J. MAGER, Biochim. Biophys. Acta, 3 ° ~I958) 164. 27 E. UNDERHAY, S. J. HOLT, H. BEAUFAY AND C. DE I)UVE, J. Biophys. Biochem. Cytol., 2 (1956) 635. 28 D. I~. MYERS, Studies on selective esterase inhibitors, Ph.D. thesis, The Hague, Excelsior, 1954. 22 L. ERNSTER, Exptl. Cell Research, IO (1956) 721. 30 G. H. HOGEBOOM AND W. C. SCHNEIDER, J. Biol. Chem., 186 (195 o) 417 . 31 S. C. SUNG AND J. N. WILLIAMS, J. Biol. Chem., 197 (1952) 175. 32 K. B. JACOESON AND N. O. KAPLAN, J. Biophys. Biochem. Cytol., 3 (1957) 31. 33 j . BRACHET, Biochemical Cytology, Academic Press, Inc., New York, 1957. 34 W. C. SCHNEIDER, J. Biol. Chem., 165 (1946) 585 • 35 W. C. SCHNEIDER, J. Biol. Chem., 176 (1948) 259. 3e F. A. HOLTON, W. C. Hi3LSMANN, D. t{. MYERS AND E. C. SLATER, Biochem. J., 67 ~I957) 579.
Biochim. Biophys..4cta, 54 (1961) 1-14
"MICROSOMES" IN HEART-MUSCLE
I
HOMOGENATES*
H. A. M. H U L S M A N S
Laboratory o] Physiological Chemistry, University o/ Amsterdam (The Netherlands) (Received April 28th, 1961)
SUMMARY
I. Microsomes have never been clearly identified in homogenates of heart muscle, although electron micrographs of tissue sections have revealed a sarcoplasmic reticulum resembling the endoplasmic reticulum of other tissue. 2. By means of differential centrifugation four fractions were obtained from rat heart muscle and liver. A fraction sedimented at 105000 × g from heart muscle was found to contain the highest concentration of esterase, RNA and DPN + nucleosidase. Since previous work has shown t h a t these cell constituents are concentrated in the microsome fraction separated from liver homogenates, it is concluded t h a t heart-muscle homogenates also contain microsomes. 3-Based on the distribution of DNA (characteristic for nuclei), cytochrome oxidase (characteristic for mitochondria) and of esterase in the various fractions, the proportions of the cell components, on a protein basis, were calculated to be as follows (first value for heart, second for liver): nuclei + myofibrils + erythrocytes + unbroken cells + cell debris, 49, 15 ; mitochondria, 22, 25 ; microsomes, 15, 31 ; "soluble protein", I3, 32. 4. The cytochrome oxidase activity of heart mitochondria is 3.5 times that of liver mitochondria. The RNA concentration in liver microsomes is 3.1 times that of heart microsomes, and the esterase activity 12.4 times. Heart-muscle microsomes contain no measurable glucose-6-phosphatase activity. 5. No DPN + pyrophosphorylase could be detected in any of the heart-muscle fractions (or in brain nuclei). 6. Acid phosphatase and ribonuclease were concentrated in a "mitochondria + microsome" fraction of heart muscle, suggesting that this tissue contains lysosomes. However, this could not be stated with certainty since no increase in the activity of these enzymes was obtained after hypotonic or sonic treatment.
INTRODUCTION
The study of the fractions obtained by differential centrifugation of liver homogenates has greatly increased our knowledge of the properties of various microscopically identifiable cell components, and especially of the localization of intracellular enzymes within them. Comparable studies with muscle homogenates have mostly been restricted * T h i s work is p a r t of t h e M. D. thesis 1 of t h e a u t h o r , w h i c h w a s p u b l i s h e d (in D u t c h ) in M a y 196o.
Biochim. Biophys. Acta, 54 (1961) 1-14
2
I~. A. M. HULSMANS
to the identification of the respiratory granules in this tissue (reviewed in ref. 2). I t is now firmly established that intracellular respiration in muscle is localized in the sarcosomes of RETZIUS 3, which correspond to the mitochondria of other tissues. Other components of the heart-muscle cell have been much less studied b y this method. In particular, "microsomes" have never been definitely identified in homogenates of heart muscle, although electron micrographs 4 have clearly revealed that between the myofibrils a fine network exists which resembles the endoplasmic reticulum found in other tissues 5. I t has been shown 6 that the "microsome" fraction isolated from liver homogenates 7 is derived by fragmentation of the endoplasmic reticulum, which consists of a system of membranes forming a network of channels, with granules attached to the membranes. The reticulum of heart muscle differs from that of other tissues in that these granules are absent s. I t has been suggested that the sarcoplasmic reticulum is involved in the conduction of the nervous impulse from the cell membrane to the myofibril9. For these reasons it appeared to be desirable to a t t e m p t to prepare a microsome fraction from heart muscle and to determine its enzyme content. By means of differential centrifugation a homogenate of rat-heart muscle was separated into four fractions : (i) a mixture of nuclei, myofibrils and cell debris; (ii) a sarcosome fraction ; (iii) a small fraction whose properties will be further described; (iv) a supernatant fraction containing the "soluble" components of the cell. The distribution of nuclei, sarcosomes and "microsomes" in the three particulate fractions was determined b y the procedure introduced by DE DUVE1°, using DNA to characterize the nuclei, cytochrome oxidase for the sarcosomes, and esterase, DPN+ nucleosidase and RNA for the microsomes. MATERIALS AND METHODS
Chemicals Unless otherwise stated, all reagents were of analytical grade and dissolved in glass-distilled water, p-Phenylenediamine, E D T A and nicotinamide were obtained from British Drug Houses Ltd. Glucose 6-phosphate (barium salt), NMN, DPN + and ATP were from Sigma Chemical Co. D P N H was prepared from DPN + with alcohol dehydrogenase obtained from Boehringer & S6hne. Antimycin was kindly supplied b y the K y o w a Fermentation Co. Ltd. Cytochrome c was prepared from horse heart as described b y MARGOLIASH11.
Preparation of tissue fractions Heart muscle. 5 Wistar rats each weighing about 200-300 g were killed b y a blow on the head and as quickly as possible the throat was cut so as to remove the blood from the body. The thorax was then opened and the heart removed. The aorta and auricles were cut away, the heart opened and the blood soaked up with filter paper. The five hearts were then placed in IO ml of ice-cold 0.25 M sucrose and cut into small pieces with scissors. The solution was poured off and the finely cut heart tissue was washed out twice with ice-cold sucrose. The washed tissue was then "homogenized" in an amount of 0.25 M sucrose sufficient to give about a IO °/o "homogenate". The homogenizer was of the Potter-Elvehjem type with a Teflon plunger. Separate portions of the heart tissue were "homogenized" for 1-1.5 min, while the homogenizer tube Biochim. Biophys. Acta, 54 (I96I) i 14
"MICROSOMES" 1N HEART-MUSCLE HOMOGENATES
3
was kept in an ice bath. Care was taken to avoid any appreciable increase in temperature during the homogenization. The combined "homogenate" was centrifuged in the "multi-speed a t t a c h m e n t " of a M.S.E. Major refrigerated centrifuge for 3 min at 800 x g (all values of g refer to the middle of the tube). The supernatant was carefully poured off and the sediment further "homogenized" in a small volume of 0.25 M sucrose. The "homogenate" was centrifuged for 3 rain at 500 × g, the lowest speed sufficient for almost complete sedimentation of the erythrocytes, nuclei and myofibrils. The supernatant was carefully poured off and combined with the first supernatant to give what was called the "cytoplasmic (E) fraction". The sediment was "homogenized" in about 20 ml 0.25 M sucrose to give what was named the "nuclear (N) fraction", although it consisted for the most part of myofibrils and contained a considerable amount of sarcosomes and also some erythrocytes and cell debris as well as the nuclei. Balance studies were made only with the E fraction, because it was difficult to take representative samples from the so-called "homogenate" which in fact contained large pieces of myofibrils and connective tissues. After removal of a sample, the cytoplasmic fraction was fractionated in the Spinco preparative ultracentrifuge, model L, with rotor No. 4 o. After IO min at 12500 x g, the supernatant was removed, and the sediment homogenized in a small volume of 0.25 M sucrose and again centrifuged for IO min at 12500 × g. The supernatant was removed and combined with the previous, while the sediment was homogenized with about IO ml 0.25 M sucrose to give the "sarcosome (M) fraction". The combined supernatant was centrifuged for 60 min at lO5 400 × g to give a small amount of a red transparent pellet. After pouring off the supernatant the surface of the sediment was washed with a little 0.25 M sucrose, and the sediments were taken up in about 5 ml 0.25 M sucrose and homogenized to yield the " P fraction". The final supernatant (about 50 ml) was called the "S fraction". The symbols used for the different fractions follow DE DUVE et al. 1°. The entire fractionation required about 2.5 h. The temperature of the tissue material was kept at about o ° throughout the fractionation. Liver. R a t liver was homogenized in the same way as the heart muscle. The fractionation was carried out similarly, except for a slight difference in the preparation of the N fraction. The homogenate was first centrifuged in the "multi-speed attachm e n t " of the M.S.E. centrifuge for 5 min at 125 ° × g. The supernatant was carefully poured off and the sediment suspended and homogenized in a small volume of 0.25 M sucrose. This homogenate was then centrifuged for 5 min at 800 × g and the carefully decanted supernatant added to the previous. The combined supernatants formed the E fraction which was further fractionated in exactly the same way as for heart. The sediment was suspended in 0.25 M sucrose to give the N fraction.
Determination of enzyme activities Cytochrome oxidase. This enzyme was determined manometrically at 26 ° with p-phenylenediamine as reducing agent. The reaction mixture contained 50 m M potassium phosphate buffer (pH 7-4), 0.3 m M EDTA (pH 7.4), 55/~M cytochrome c, 15 m M p-phenylenediamine (pH 7.4) and an aliquot of the tissue fraction in a total volume of 3 ml. The reaction was begun, after temperature equilibration, b y tipping the p-phenylenediamine solution from the side-arm into the main compartment of Biochirn. Biophys. Acta, 54 (I96I)
1
14
4
H. &. M. HULSMANS
the manometric vessel. Readings commenced 3 min later and the initial rate of 0 2 uptake was measured. Since, above I O O / , 1 0 , / h , the rate of 0 3 uptake was not proportional to the concentration of tissue fraction, the concentration of the enzyme was calculated from a calibration curve. The 0 3 uptake never exceeded 200/,l/h. The conditions were so chosen as to promote swelling of the mitochondria so that the full cytochrome oxidase activity would be determined. Glucose-6-phosphatase. The method used was based on that described b y DE DUVE et al. 1°. The reaction mixture contained 7 m M histidine buffer (pH 6.5), I m M E D T A (pH 6.5), 40 m M glucose 6-phosphate (pH 6.5) and an aliquot of the tissue fraction in a final volume of I ml. The reaction was begun by addition of the glucose 6phosphate and was stopped after IO min at 320 by the addition of 2 ml IO % (w/v) trichloroacetic acid. The precipitated protein was removed b y centrifugation and the phosphate content of the supernatant determined by the method of SUMNER12. A zero-time control (to correct for phosphate in the tissue preparation) and a no-tissue control (to correct for any non-enzymic hydrolysis) were subtracted from the values found. The amount of inorganic phosphate set free was linear with time and proportional to the concentration of tissue fraction, Esterase. The esterase activity was measured manometrically in NaHCOs-CO 2 buffer with p-nitrophenyl acetate as substrate. The difficultly soluble substrate was ground in a mortar with an equal amount of gum arabic and ten times this amount of water. The reaction mixture contained 25 m M NaHCO3, 0.02 ml of the substrate suspension and an aliquot of the tissue fraction, in a total volume of i . i ml. The p H was maintained at p H 7.4 by having 5 % C02-95 % N2 as the gas phase. The reaction was begun by tipping in the substrate from the side-arm. The COs evolution was followed for 20 rain at 37 °, and the initial rate of evolution determined. The gas evolution was constant for 6-1o rain, at a rate which was proportional to the concentration of tissue fraction. DPNH-cytochrome c reductase. This was measured spectrophotometrically at 550 m/z and at room temperature by the method described by HOGEBOOM13. The reaction mixture contained 75 m M potassium phosphate buffer (pH 7.2), I m M EDTA, o.i m M D P N H , 38/zM cytochrome c, I m M KCN, 1. 5 ~g/ml antimycin (about 50 ~g/mg protein) and an aliquot of the tissue fraction in a total volume of 3 ml. The solution in the reference cell had the same composition, except that it contained no D P N H or cytochrome c. The cyanide was added immediately before the addition of the tissue fraction which began the reaction. The increase of absorbancy was linear with time for 1.5-2 min. The rate of the reaction was proportional to the concentration of the tissue fraction. DPN+ nucleosidase. This enzyme activity was determined as described by KAPLAN 14.
DPN+ pyrophosphorylase. This enzyme activity was determined as described by KORNBERG 15.
Analytical methods Nucleic acids. The concentration of the nucleic acids was determined b y MoOL~lvs modification of the method of SCHMIDT AND THAN'NHAUSER17. The low concentration of RNA in heart muscle made it necessary to correct for interference caused b y the presence of polypeptides in the solution. This was done by the method of MclNDoE Biochim. Biophys. Acta, 54 (1961) 1-14
"MICROSOMES"
IN HEART-MUSCLE HOMOGENATES
5
AND DAVlDSONls in which the difference between the absorbancies at 260 m ~ and 290 m ~ was used. The amount of nucleic acid was calculated from the extinction coefficients at 260 m/~ of the pure nucleic acids, neglecting absorption of the latter at 29 ° m~. The extinction coefficient of heart-muscle RNA was calculated from the nucleotide composition, determined b y paper-chromatographic separation of the nucleotides present in the hydrolysed RNA (cf. MAGANASIKel al. 19) and measurement of the ultraviolet absorption spectra. From these spectra and the data of ELSON et al. 2°, the composition expressed as moles/Ioo moles nucleotides was found to be: adenylic acid, 20.9; guanidylic acid, 21.5; cytidylic acid, 37.o and uridylic acid, 20.6, from which the molar extinction coefficient (per P atom) was calculated to be IO 78o. The same value was used for calculating the amount of liver RNA. For DNA, it was assumed that the molar extinction coefficient (per P atom) was the same as for t h y m u s nucleic acid 21, namely 6400. Protein. Protein was determined by the biuret method of GORNALL et al. 22, as applied to particulate material b y CLELAND AND SLATER23. RESULTS
Protein. In Table I the distributions of the protein in the different fractions isolated from homogenates of rat heart and rat liver are compared. A striking difference between the two tissues is the amount of material sedimented at low speed, i.e. found in fraction N. Most of the protein of heart-muscle homogenate is found in this fraction, which in this tissue consists largely of myofibrils. The percentage of the total protein found in the M fraction is similar in the two tissues. The P fraction, on the other hand, makes up a much smaller part of the total protein in heart muscle than in liver, even when expressed as percentage of the E fraction. The S fractions represent about the same proportion of the E fraction in the two tissues. Cytochrome oxidase. Table I gives also the distribution of the cytochrome oxidase in the different fractions. In agreement with previous findings 24, 23, z, the cytochrome oxidase is most active in the mitochondrial (sarcosomal) fractions. The greater TABLE
I
DISTRIBUTION OF PROTEIN AND CYTOCHROME OXII)ASE ACTIVITY IN FRACTIONS SEPARATED BY DIFFERENTIAL CENTRIFUGATION OF HOMOGENATES OF RAT HEART MUSCLE .&ND LIVER T h e v a l u e s g i v e n f o r h e a r t m u s c l e a r e t h e m e a n s of 3 e x p e r i m e n t s , t h o s e f o r l i v e r of 2 e x p e r i m e n t s .
Fraction
Heart
N E M P S M + P + S
Cylochrome c oxidase
Protein (% of total protein in #action*) Liver
70 3° 12.o 3.7 13. 5
27 73 14. 5 29.0 32.5
29.2
76.o
Heart Q02
43 ° 132o 26oo I29O 20
Liver
% total activity in fraction*
43.1 56.9 44.9 6.7 O. 4
QO.
34 ° 202 820 47 IO
% total activity in fraction*
38.5 t)1.5 5° 5.7 1.3
52.o
57.o
* E x p r e s s e d as p e r c e n t a g e of t h a t f o u n d in N f r a c t i o n + t h a t f o u n d in E f r a c t i o n .
Biochim. Biophys..qcta,
54 (1961) t I4
H. A. M. HULSMANS
specific activity of the heart sarcosomes compared with the liver mitochondria reflects the greater respiratory activity of this tissue ~. In both cases, a substantial amount of the cytochrome oxidase is found in the N fraction, presumably due to mitochondria either not extracted from the tissue or sedimented at low speed. Activity is also found in the P fraction; when expressed as specific activity this is much more marked in the case of heart (cf. ref. 23). ,
,
i
~
,
J
,
,
J
20
o
....
~
o
,~,
,~o,-~--o
5
1o
P/'otein (rng)
:Fig. I, Glucose-6-phosphatase activity of h o m o g e n a t e s of r a t liver ( × - - × ) and h e a r t muscle ( O - - O ) , The protein c o n t e n t was determined b y the Kjeldahl m e t h o d (N × 6.25).
Glucose-6-phosphatase. HERS et al. 2~ showed that this enzyme was exclusively localized in liver microsomes. The great difference between the ability of liver and heart homogenates to hydrolyse glucose 6-phosphate is shown in Fig. z. The almost insignificant activity in heart homogenates (cf. AvI-DOR et al3 e) could be due to nonspecific phosphomonoesterase activity. From this result, it must be concluded either that heart-muscle homogenates do not contain microsomes or, if they do, that these microsomes lack glucose-6-phosphatase. Esterase. UNDERHAY et al3 7 have shown that certain esterases are distributed in the different fractions of a liver homogenate in the same way as glucose-6-phosphatase, which is good evidence that these enzymes are also localized in the microsomes. In Table II are compared the esterase activities, measured with p-nitrophenyl acetate, of the fractions obtained from both rat liver and rat heart. With this substrate TABLE II DISTRIBUTION BY
DIFFERENTIAL
OF
ESTERASE
CENTRIFUGATION
OF
ACTIVITY HOMOGENATES
IN
FRACTIONS OF
RAT
SEPARATED
HEART
MUSCLE
AND
LIVI~R
The values are for the same fractionations as in Table I. Heart Fraction QC02
N E M P S M+P+S
186 392 164 678 472
Liver
% of total activity in fraction*
51.5 48.5 7.8 9.1 25.2
QC02
1221 5599 1398 13 745 891
% of total activity in fraction*
7.6 92.4 4.7 9o.5 6.3
42.1
I O I "5
* E x p r e s s e d as percentage of t h a t found in N fraction + E fraction.
Biochim. Biophys. Acta, 54 (1961) 1-14
"MICROSOMES"
IN
HEART-MUSCLE
HOMOGENATES
7
essentially only the ali-esterase and the aromatic esterase are measured ~. It is clear from the results shown in Table I I that the esterase activity is much higher (about 2u-fold) in liver than in heart muscle. In both tissues, the highest specific activity was found in the P fraction. A much greater fraction of the activity was found in the supernatant fraction of heart muscle than of liver, although the specific activity was no greater. No difference between the esterases in the P and S fractions of heart muscle could be found by use of the inhibitors neostygmine (6-8 % inhibition by o.oi mg/ml) or diisopropylfluorophosphate (47-50 % inhibition by 0.5 mM). It is possible that serum esterase is responsible for the activity in the supernatant. DPNH-cytochrome c reductase. Liver homogenates appear to contain three systems catalysing the reduction of added ferricytochrome c by added D P N H , viz. (I) the antimycin- and Amytal-sensitive system present in the mitochondria; (2) the antimycin- and Amytal-insensitive reaction catalysed by the microsomes; and (3) an obscure pathway, also insensitive to antimycin and Amytal, which appears to be present on the exterior of the mitochondria 29. Table I I I shows that the highest activity of the antimycin-insensitive D P N H cytochrome c reductase of heart-muscle homogenates is found in the P fraction. TABLE III DISTRIBUTION OF ANTIMYCIN-INSENSITIVE DPNH--CYTOCHROME C REDUCTASE ACTIVITY IN F R A C T I O N S S E P A R A T E D BY D I F F E R E N T I A L C E N T R I F U G A T I O N OF H O M O G E N A T E S OF R A T - H E A R T M U S C L E
The values are for the same fractionations with heart muscle as in Table I. Fraction
A A 550 m#lmin/mg protein
°/o of total activity in fraction*
N E M
o.o63 o.264 0.224
21.8
P
o.816
29. 5
S M+P+S
0.055
35-7 64.3
6.0 57.3
* Expressed as percentage of that found in N fraction + E fraction. D P N + pyrophosphorylase. In agreement with HOGEBOOM AND SCHNEIDERs°, DPN + pyrophosphorylase was found to be concentrated in the nuclear fraction isolated from rat-liver homogenates. However, no activity of this enzyme could be found in any of the fractions separated from the heart-muscle homogenate. (Furthermore, no activity could be found in brain nuclei.) D P N + nucleosidase. This enzyme is localized in the microsome fraction of rat-liver homogenatesZl, 3~. Table IV shows that the P fraction of heart-muscle homogenates also contains the greatest activity. Nucleic acids. It is long known from histochemical studies that DNA is exclusively located in the cell nucleus (see ref. 33) and this was confirmed for liver homogenates by HOGEBOOMAND SCHNEIDER34. Table V shows a comparison between the distribution of DNA in the fractions isolated from rat heart and rat liver in our studies. The liver contained rather more DNA than the heart. In both tissues the DNA is largely confined to the N fraction, but an appreciable amount was found in the heart M Biochim. Biophys. Acta, 54 (196I) 1-14
8
H.A.M.
HULSMANS
TABLE IV DISTRIBUTION
OF
BY DIFFERENTIAL
DPN +
NUCLEOSIDASE
CENTRIFUGATION
ACTIVITY IN
OF H O M O G E N A T E S
FRACTIONS OF
SEPARATED
RAT-HEART
MUSCLE
T h e v a l u e s g i v e n are t h e m e a n s of 3 f r a e t i o n a t i o n s n o t i d e n t i c a l w i t h t h o s e s h o w n in T a b l e I, Protein Fraction
D P N + nucleosidase i~mole D P N + split/rag protein/h
% of total protein in fraction*
N E M P S M+P+S
66 34
o.525 o.546 o.496 1.341 0.238
19.4 3.2 lO. 7
% of total activity in fraction*
65 35 18.o 8.1 4.9
33.3
31
* E x p r e s s e d as p e r c e n t a g e of t h a t f o u n d in N f r a c t i o n + E fraction. TABLE V DISTRIBUTION
OF DNA
IN
FRACTIONS
OF HOMOGENATES
SEPARATED
OF RAT
HEART
BY DIFFERENTIAL MUSCLE
CENTRIFUGATION
AND LIVER
T h e v a l u e s g i v e n for h e a r t m u s c l e are t h e m e a n s of 3 e x p e r i m e n t s (the s a m e f r a c t i o n a t i o n s as i n T a b l e I), t h o s e for l i v e r are for one e x p e r i m e n t (one of t h o s e s h o w n in T a b l e I).
Fraction
Heart
N E M P S M + P + S
DNA
Protein (% of total protein in fraction*) Liver
7° 3° 12 3.7 13. 5
25 75 13 27 34
29.2
Heart pmoles Pig protein
29-4 5.5 9-3 4.5 1.6
74
Liver
% of total amount in fraction*
92.7 7.3 5 .1 0.8 0.9 6.8
#mole Pig protein
12.7 8 4 IO 4
% of total amount in fraction*
83.2 16.8 1-4 6.9 3.5 11.8
* E x p r e s s e d as p e r c e n t a g e of t h a t found in N f r a c t i o n + E fraction.
fraction and in the liver P fraction, indicating contamination of these fractions b y small amounts of nuclei or nuclear fragments. This was confirmed microscopically (Feulgen staining) for the heart M fraction. CLAUDE7 and SCHNEIDER84, 35 have shown that the highest concentration of RNA is found in the microsome fraction of liver homogenates, although this is not the only cell component containing RNA. Table VI shows that liver homogenates contain very much more RNA than heart homogenates. In both cases, b y far the highest concentration was found in the P fraction. DISCUSSION
Clearly the fractions as separated by differential centrifugation are very impure, especially those obtained from the heart-muscle homogenate. The relative proportions of the various fractions reflect rather poorly the proportions of the various cell Biochim. Biophys. Acta, 54 (1961) 1-14
"MICROSOMES" IN HEART-MUSCLE HOMOGENATES TABLE
9
VI
DISTRIBUTION OF R N A IN FRACTIONS SEPARATED BY DIFFERENTIAL CENTRIFUGATION OF HOMOGENATES OF RAT HEART MUSCLE AND LIVER
T h e v a l u e s g i v e n are for t h e s a m e f r a e t i o n a t i o n s as s h o w n in Table V. RNA Heart
Fraction
Liver
I~mole Pig
% of total amount
t~moleP/g
% of total amount
protein
in fraction*
protein
in fraction*
N
25
70
69
15.8
E
24
3°
122
84.2
M
23
12.1
26
3.1
P
51
7.6
254
62.5
S
io
5.2
41
13.o
M + P + S
78.6
24.9
* E x p r e s s e d as p e r c e n t a g e of t h a t f o u n d in N fraction + E fraction.
TABLE
VII
PROPORTION OF CELL COMPONENTS IN HOMOGENATES OF RAT HEART MUSCLE AND LIVER
Calculated f r o m t h e d a t a in Tables I and II, on the basis of a s s u m p t i o n s given in t h e text. Celt component
% of total protein Heart
Nuclei, etc. * Mitochondria Microsomes "Soluble" p r o t e i n
Liver
49
15
22
25
15
3i
13
32
99
lO3
* I n c l u d i n g u n b r o k e n cells, cell debris, e r y t h r o c y t e s and, in t h e case of heart, myofibrils.
components present in the homogenate. However, by making certain assumptions, it is possible to obtain a much more accurate, although still approximate, picture. The assumptions are: (I) cytochrome oxidase is present only in the mitochondria; (2) the M and P fractions consist only of mitochondria and microsomes; (3) the S fraction is not contaminated by any appreciable amount of microsomes; (4) "soluble" protein is present only in the S fraction; (5) there are both a microsomal-bound and a soluble esterase. On the basis of these assumptions Table VII can be derived from the data given in Tables I and II (see APPENDIX). About one half of the protein of the heart-muscle homogenate consists of the so-called nuclear fraction, which in fact is largely made up of myofibrils. This fraction constitutes only 15 % of the liver homogenate. In both tissues, mitochondria make up about one quarter of the protein. The higher respiratory activity of heart is due to the greater concentration of the cytochrome system in heart mitochondria ~. When microsomal contamination is allowed for, the heart sarcosomes have 3.5 times the Biochim. Biophys. A c t a , 54 (1961) 1-14-
I0
H.A.M.
HULSMANS
cytochrome oxidase activity of fiver mitochondria. More than 6o % of the total protein of a liver homogenate is made up of the microsomes (31%) and "soluble" protein (3z %). These components form a considerably smaller proportion of a heartmuscle homogenate. In Table VIII are shown the compositions of the various fractions isolated by differential centrifugation. The N fraction is heavily contaminated with mitochondria (especially in liver) and with microsomes. The M fraction contains appreciable TABLE
VIII
COMPOSITION OF FRACTIONS SEPARATED BY DIFFERENTIAL CENTRIFUGATION" OF HOMOGENATES OF RAT HEART MUSCLE AND LIVER Calculated from the data in Tables I and II, on the basis of assumptions
given in the text.
Composition of reactions Cell component
N
M
heart
liver
P
heart
liver
Nuclei, etc. *
7o
54
.
14
38
86
91
42
5
Microsomes
I6
8
14
9
58
95
"Soluble" protein
. IOO
* Including unbroken
. IOO
.
S liver
Mitochondria
.
.
heart
. IOO
.
TABLE
.
IOO
liver
.
.
IOO
cells, cell d e b r i s , e r y t h r o c y t e s
.
heart
IOO
I
I
--
--
99
99
IOO
IOO
a n d , i n t h e c a s e of h e a r t , u l y o f i b r i l s .
IX
DISTRIBUTION OF MITOCHONDRIA AND MICROSOMES IN VARIOUS FRACTIONS C a l c u l a t e d f r o m t h e d a t a i n p r e v i o u s T a b l e s . T h e v a l u e s g i v e n r e p r e s e n t t h e p e r c e n t a g e s o f t h e cell constituent or component found in the various fractious.
Fr~t~n
Mitochondrm
Mwrosomcs
DPNHcytochromec reduct~e
DPN+ n~leosidase
RNA
Heart N
46
75
36
65
7o
M
47
II
22
18
I2
P
7
I4
3o
8
8
S
0. 4
--
6
5
5
IOO
94
96
95
16
lOO. 4 Liver N
4°
7
--
--
M
52
4
--
--
P
6
88
--
---
62
S
1.6
--
---
--
13
99.6
IOO
3
94
B i o c h i m . B i o p h y s . d c t a , 54 (1961) 1 - 1 4
"MICROSOMES" 1N HEART-MUSCLE HOMOGENATES
II
amounts of microsomes, especially that obtained from heart. The P fraction isolated from liver consists of 95 % microsomes, while that isolated from heart is heavily (42 %1 contaminated with mitochondria. The reason for this is t h a t the amount of microsomes in the P fraction in heart is only 7.7 % of the corresponding value in liver, so that a similar contamination with the mitochondria (the amounts of mitochondria in the P fractions from the two tissues are in fact identical) has relatively a much greater effect in the case of the heart. The distribution of the mitochondria and microsomes in the various fractions is given in Table IX, which also shows for comparison with the microsomes the data on the distribution of the antimycin-resistant D P N H - c y t o c h r o m e c reductase, the DPN+ nucleosidase and the RNA, taken from Tables III, IV and VI, respectively. With both heart and liver, only about one-half of the mitochondria are obtained in the M fraction, most of the remainder being sedimented with the nuclei and myofibrils in the N fraction. The distribution of the microsomes presents a very different picture in the two tissues. Most (88 °/o) of the liver microsomes were obtained in the P fraction, but only 14 °/o of the heart microsomes were found in this fraction. Most (75 %) were sedimented with the myofibrils and nuclei which strongly suggests that they were not fragmented and set free in the original homogenization. If they are in close association with the myofibrilss, they will be protected from fragmentation during homogenization. I t is clear that a different technique should be used to obtain a good yield of reasonably pure microsomes from a heart-muscle homogenate. The value (75 %) given for the percentage of the heart microsomes present in the N fraction is based on the particulate esterase activity, on the assumption that this is confined to the microsomes. This assumption is supported by the distributions of the DPN+ nucleosidase and RNA, which are quite similar to that of the particulate esterase. Thus, it appears reasonable to conclude that heart-muscle homogenates contain microsomes characterized like liver microsomes b y the localization therein of all the particulate esterase, the RNA and the DPN+ nucleosidase. Any one of these three components can be used to follow the distribution of the microsomes. The antimycin-resistant D P N H - c y t o c h r o m e c reductase is much less suitable for this purpose since, although the highest specific activity of this enzyme was also present in the P fraction, much less of the enzyme was found in the N fraction, and more in the M and P fractions than would be expected from the other distributions. I t appears that the little-understood antimycin-resistant D P N H - c y t o c h r o m e c reductase in mitochondria (the so-called "external pathway") is confusing the picture (cf. ref. 26). The possible presence in heart muscle of DE DUVE'S lysosomes 1° has been ignored in the above treatment. In fact, small activities of acid phosphatase and RNAase, typical lysosomal enzymes in liver, were found in heart-muscle homogenates. The highest specific activity was obtained in a combined (M + P) fraction. The activities of the two enzymes were distributed over the N, (M + P) and S fractions in a very similar manner. From these results, it seems rather likely that some lysosomes are present in heart muscle. However, this cannot be stated with certainty since no increase in the activity of these enzymes was obtained after hypotonic or sonic treatment. This is one of the most important characteristics of lysosomal enzymes in liver. I t is possible that the lysosomes are disrupted during homogenization of the heart muscle. This could also be the reason why 16-2o °/o of the enzyme activities were found in the S fraction. Biochim. Biophys. Acta, 54 (1961) l-14
12
H.A.M.
HULSMANS
In most tissues, the endoplasmic reticulum, from which the microsome fraction is derived, consists of lipoprotein membranes and RNA-containing granules. In heart mucle, however, these granules are lacking s. Nevertheless, the RNA appears to be associated with the microsome fraction in this tissue also. If the concentrations of RNA in the P fractions (Table VI) are corrected for the mitochondria in these fractions (Table VIII), and assuming that the mitochondria contain no RNA, it can be calculated that heart microsomes contain 87 and liver microsomes 267 ~moles RNA-P/g protein, i.e. a difference of a factor of 3. ACKNOWLEDGEMENTS
This work was supported in part by a grant from the Foundation "De Drie Lichten". I wish to thank Professor E. C. SLATERfor his interest and advice, and Dr. P. BORST and Dr. C. VEEGER for their help given in many time-consuming experiments. The help given by Mr. F. AMONS and the skilful technical assistance of Miss M. VAN U F F E L E N , Miss A. SEARLEand Mr. A. KOEKEBAKKERare also gratefully acknowledged.
APPENDIX
Calculation of the proportion of the various cell components in homogenates of rat heart muscle and liver The following assumptions are made: (I) Cytochrome oxidase is present only in the mitochondria. (2) The M and P fractions contain only mitochondria and microsomes. (3) The S fraction does not contain any microsomes. (4) Soluble protein is present only in the S fraction. (5) Esterase is present both in the microsomes and the "soluble" fraction. Suppose that the M fraction consists of x % mitochondria and ( I O O - x ) % microsomes and that the P fraction consists of y ~o mitochondria and (IOO - - y ) % microsomes. Then the Qo2 (cytochrome oxidase) of pure mitocbondria equals (IOO/X) ((202 of M fraction) = (IOO/y) (Qo2 of P fraction) x
i.e .
.
Y
Qo~ of M fraction
. . Qo~ of p fraction
/1
(I)
Similarly, Qco2 (esterase) of pure microsomes equals ElOO/(ioo- x)]Qcoz of M fraction = [ioo/(ioo--Y)] Qco2 of P fraction IOO
-
-
i.e. i o o - - ~
x
Qco~ of M fraction Qco~ of p fraction
= [2
(2)
The solution of equations (I) and (2) gives y
IOO --
IOO [2
[t--t2
;x =]Iy Biochim. Biophys. Acta, 54 (1961) I - I 4
13
"M1CROSOMES" 1N HEART-MUSCLE HOMOGENATES
The values in Tables I and I I give ]1 [2 11 - - [2 I 0 0 - - I 0 0 [2
Y x Qo2
( c y t o c h r o m e oxidase) pure mitochondria Qco2 (esterase) pure microsomes
Heart
Liver
2.02 0.24 1.7 8 75.8 42.5 86
17. 5 0,I I7. 4 89.8 5.2 90.5
3020
906
118o
14 500
The distribution of mitochondria in the various fractions can be calculated from the data of Table I Heart Fraction
protein N M P S
Liver
%
7° 12 3.7 13. 5
o;
OO~ 43 ° 26oo 129o 20
99.2
(0
(2)
(3)
protein
Q02
(t)
(2)
(3)
o.142 o.86 0.425 0.007
IO.O lO. 3 1.57 0.09
45.6 47.o 7.2 0. 4
27 14. 5 29 32.5
34 ° 820 47 io
0.376 0.905 o.o52 O.Oli
lO.2 13.i 1.5 0. 4
40.5 52.o 5.9 1.6
~'(2) = 21.96
lOO.2
lO3.O
~(2) = 25.2
IOO.O
( I ) = p r o p o r t i o n of p r o t e i n in f r a c t i o n c o n s i s t i n g of m i t o c h o n d r i a = Qoz/(Qo2 of p u r e m i t o chondria). (2) = % of t o t a l p r o t e i n in t h i s f r a c t i o n c o n s i s t i n g of m i t o c h o n d r i a = (i) × (% p r o t e i n ) . I00
(3) = % of t o t a l m i t o c h o n d r i a p r e s e n t in f r a c t i o n -- -
-
X (2)
21(2)
Similarly, the distribution of the microsomes in the various fractions can be calculated from the data of Table I I Heart Fraction
N M P
%
Liver
%
protein
QCO~
(~)
(2)
(3)
protein
7° 12.o 3.7
186 164 678
o.158 o.139 0.575
11.o 7 1.67 2.13
74.6 11.2 14. 4
27.0
~7(2) = 14.87
lOO.2
14. 5 29.0
QCO2 1221 1398 13 745
(#
(2)
(,1)
0.084 0.097 0.948
2. 3 1.4 27-5
7.4 4-5 88.4
~'(2) = 31.2
lOO.3
The composition of the N fraction is then % mitochondria % microsomes
% nuclei, etc. % of t o t a l p r o t e i n found in N f r a c t i o n % of t o t a l p r o t e i n c o n s i s t i n g of nuclei, etc.
Heart
Liver
I4.2 15.8
37-6 8.4
30.0
46.o
7 °.0
54.o
7°
27
49
14.6
Biochim. Biophys. dcta, 54 (1961) t - I 4
14
H. A. M. HULSMANS
The composition of the S fraction is % mitochondria ~o " s o l u b l e " % of total protein found in S fraction % of total protein consisting of "soluble" protein
Heaet
Liver
0. 7 99.3
I.I 98.9
13. 5
32.5
13. 4
32.2
The results of these calculations are summarized in Tables V I I - I X .
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Biochim. Biophys..4cta, 54 (1961) 1-14