Nagoya J. Med. Sci. 54. xx - xx, 1992

ISOLATION AND SOME BIOCHEMICAL PROPERTIESOF PORCINE PANCREAS MITOCHONDRIA
Yuzo SAKAI l , KAyO ADACHe, TETSUO HAYAKAWA 1 and TAKASHI WAKABAYASHl 2]2nd Department of Internal Medicine and 2 Department of Anatomy,
Nagoya University School of Medicine, Nagoya 466, Japan

ABSTRACT
Morphologically and functionally intact mitochondria were isolated from the porcine pancreas using a
conventional, differential centrifugation method. The homogenate of the porcine pancreas made in a medium containing 0.25 M sucrose, 10 mM Tris-CI, pH 7.4, 0.5% bovine serum albumin and 1 mM EDTA, inthe presence or in the absence of dibucaine and trypsin inhibitor, was centrifuged for 10 min at 700xg. The
supernatant was centrifuged for 10 min at 7000xg, and the pellet was washed three times. Trypsin activity of
mitochondria isolated in the absence of dibucaine and trypsin inhibitor was as low as that of mitochondria
isolated in the presence of dibucaine and trypsin inhibitor, and a major part of the activity remained inactivated. Phospholipase A2 activity of the former mitochondria was as low as that of the latter, and remained
unchanged up to 8-10 hr at 4'C. The presence of bovine serum albumin and EDTA in the respiration medium was absolutely required to obtain good respiratory controls of those mitochondria. These data suggestthat well-coupled mitochondria can be obtained from the pancreas by a conventional isolation procedure
without activating the major part of trypsin.
Key Words: Pancreas; Mitochondria; Dibucaine; Trypsin inhibitor; Phospholipase A2 ; Bovine serum albumin

INTRODUCTION
The role of mitochondria in the pathogenesis of various pancreatic diseases largely remains tobe solved. Data to correlate abnormal mitochondrial functions to pancreatic pathologic conditions are scanty. It has been suggested that abnormal mitochondrial functions may account foralcoholic fatty liver!) or alcoholic pancreatic steatosis 2 ), although controversial data have beenpresented by other 3). In necrotizing pancreatitis induced experimentally by the administration oflysine, the earliest change in acinar cells is the swelling of mitochondria 4). Taking into accountthe central role of mitochondria in the energy metabolism of the cell, it is reasonable to assume
that disturbances in the function of mitochondria are directly or indirectly related to various
pathologic states of the pancreas. Thus, it is urgent to establish the isolation procedure for pancreas mitochondia. However, a survey of the literature has revealed that data on pancreas mitochondria are surprisingly few. Sardesai et al. 5) could not observe oxygen uptake of mitochondria
isolated from the rat pancreas, and suggested the presence of some inhibitory substance on mitochondrial respiration. Recently, Wilson et al. 3 ) have developed a quick method for the isolation of intact mitochondria from the rat pancreas. Using their method, they obtained ADP/O
ratios of 2.6 ± 0.1 and 1.4 ± 0.2 when oxidizable substrates were glutamate (+ malate) andsuccinate, respectively. The isolation medium they used contained dibucaine to inhibit

Correspondence: Dr. Takashi Wakabayashi, Department of Anatomy, Nagoya University School of Medicine,Showa-ku, Nagoya 466, Japan
Received for Publication in March 14, 1991
41

42
Yuzo Sakai et al.

phospholipase A z activity, and soybean trypsin inhibitor to prevent the activation of trypsinogen.
In the present study we demonstrate that tightly coupled pancreas mitochondria can be obtained by a routine isolation procedure for liver mitochondria without using dibucaine and trypsin inhibitor. Instead, bovine serum albumin (BSA) and EDTA are absolute requirements forthe isolation of intact mitochondria from the pancreas.

MATERIALS AND METHODS
Isolation of mitochondria from porcine pancreas
Porcine pancreatic glands obtained from a slaughter house within one hr after the death of
the animal were trimmed of covering fat and finely minced with a single-edged razor blade.
They were homogenized in a medium containing 250 mM sucrose, 10 mM Tris-Cl, pH 7.4, 1
mM EDTA, 0.5% fatty acid free bovine serum albumin (BSA) (Sigma Chemical Co., St.
Louis), 0.25 mg/ml soybean trypsin inhibitor (Sigma Chemical Co., St. Louis) and 400 ~M
dibucaine according to the method of Wilson et at. 3 ). The homogenization of the tissues was carried out in a Teflon-glass homogenizer using a Labo-stirrer (type LR-51B, Yamato ScientificCo., Tokyo) with five up-and-down strokes at 800 rpm. In the control experiments, either dibucaine or trypsin inhibitor or both were omitted from the medium described above. Furthermore,a routinely used isolation medium for liver mitochondria in our laboratory was also used insome experiments. The medium contained 2 mM Hepes, pH 7.4, 70 mM sucrose, 220 mM
mannitol, 0.05% BSA and 0.1 mM EDT A. The mitochondiral fraction was obtained by the following two differential centrifugation methods: 1) The method of Wilson et at. 3): The homogenate made in various isolation media, described above, was centrifuged at 2000xg for 7 min.
The residue consisted of two layers. A thin, surface layer was pale brown indicating that a partof the mitochondria were sedimented. The inner layer of the residue was whitish indicating thatthe zymogen granules were largely sedimented in addition to nuclei and cell debris. The resultant supernatant was centrifuged at 6000xg for 10 min to sediment mitochondria. The pellet
thus obtained was again composed of two layers. The upper layer was brownish and the lower
layer was whitish. The upper layer was collected carefully leaving the lower layer to avoid thecontamination of zymogen granules, and it was suspended in a small amount of the medium.
This is the mitochondrial fraction that Wilson et a/. 3 ) developed, and it will be designated as the
2000-6000xg' R z fraction hereafter; 2)The homogenated made in various isolation media wascentrifuged at 700xg for 10 min. The supernatant thus obtained was centrifuged at 7000xg for
10 min to sediment mitochondria. This will be designated as the 700-7000xg' R z fraction hereafter. The R z fraction, obtained either by the 2000-6000xg centrifugation method or by the
700-7000xg centrifugation method, was washed three times at the same speed at which it was
sedimented. The fraction thus obtained was designated as the 2000-6000xg' Rt or the
700-7000xg' R z"' fraction. Protein concentrations were determined by the method of Lowry etal.(,).

Measurement of oxygen uptake
Respiration of mitochondria was measured in the medium described by Wilson et al. 3 ).
Namely, the medium contained 250 mM sucrose, 22 mM KCI, 22 mM triethanolamine, pH 7.4,
1 mM EDTA, and 0.5% fatty acid free BSA (medium A). In some experiments, the respiration
medium routinely used in our laboratory was also tested. It contained 70 mM sucrose, 220 mM
mannitol, 5 mM Tris-CI, pH 7.4 and 0.1 mM EDTA (medium B). The rate of oxygen

43
ISOLATION OF PANCREAS MITOCHONDRIA

consumption was measured at 30T with a Clark-type oxygen electrode (Beckman Co., Fullerton, California). Oxidizable substrates were 5 mM glutamate (+ 5 mM malate) or 5 mM succinate. State 3 respiration was measured by the addition of ADP 200-300 nmoles to the reaction
mixture in the presence of substrate and Pi (5 mM). State 4 respiration was defined as the respiratory rate after the expenditure of ADP. ADP/O ratios and respiratory control index (RCI)
were calculated according to the method of Estabrook 7 ).

Enzyme assays
Cytochrome oxidase (EC 1.9.3.1) activity was measured using a Clark-type electrode as described before Rl • The reaction medium contained 10 mM Tris-Cl, pH 7.4, 2 mM ascorbate andcytochrome c (0.5 mg/ml) (type IV, from horse heart, Sigma Chemical Co., St. Louis). Ascorbate was prepared freshly prior to use. Lysophosphatidylcholine (1-2 mg/ mg mitochondrial
protein) was added to the reaction mixture as a solubilizing agent to obtain full activity. The rateof oxygen consumption during the oxidation of reduced cytochrome c in the presence of ascorbate indicated the cytochrome oxidase activity.
Mg2+-activated ATPase (EC 3.6.1.3): The activity of mitochondria was measured in thepresence of 2,4-dinitrophenoI 9l . The activity was measured in a medium containing 250 mM
sucrose, 10 mM Tri-Cl, pH 7.4, 3 mM MgCl 2 and 0.1 mM EDTA. A 100 ~l portion of 100
mM ATP (pH 7.4) was added to 900 ul of the medium containing 0.50 mg of mitochondria at
30°C. After 10 min, 1 ml of 16% perchloric acid was added to the incubation mixture. The reaction mixture was then centrifuged at 8000xg for 10 min. The inorganic phosphate released tothe supernatant was spectrophotometrically measured according to the method of Fiske andSubbarow lO ) as described before 9 ).
Trypsin activity of tissue homogenate or mitochondria was determined spectrophotometrically by the method of Hayakawa et at. ll ) using a-N/benzoyl-DL-arginine p-nitroanilide HCl(BAPNA) (Sigma Chemica!. Co., St. Louis) as the substrate with or without activation by enterokinase. Aqueous stock solution of BAPNA (1 mg/ml) was prepared by heating to 8YC andthen cooling on ice. It was diluted at equal volumes with 0.1 M Tris-Cl, pH 8.0, containing 0.04
M CaCl 2 just prior to use. Trypsin (2x crystallized, sigma) was dissolved in 0.0025 N HCl as astock solution (1 mg/ml). To obtain a standard curve for trypsin, the stock solution of trypsin
was diluted to appropriate concentrations with 0.025 N HCl containing BSA at a concentrationof 1 mg/m!. Enterokinase (EC 3.4.21.9) (Sigma) was dissolved in 0.2 M Tris-Cl, pH 8.0 at aconcentration of 10 mg/m!. Protein concentration of a mitochondrial fraction or homogenate
was brougth to 5-10 mg/ml with 0.2 M Tris-Cl, pH 8.0. A 10 III portion of the sample was put
into test and reference tubes containing 2 ml of BAPNA solution (0.5 mg/ml) and 2ml of acetic
acid (30%, v/v), respectively. They were incubated for 10 min at 3TC, and then a 2 ml portionof acetic acid and a 2 ml portion of BAPNA solution were put into test and reference cuvettes,
respectively. Difference in absorbance between the test and the reference cuvettes was read at
410 nm using Hitachi Model 100-40 spectrophotometer. Similarly, the standard curve for trypsin was obtained using BAPNA. Trypsin activity of the sample was calculated from absorbancechanges of the sample and the trypsin standard, and expressed as ug trypsin/mg protein. To activate trypsinogen by enterokinase, an equal volume of a solution of enterokinase was added toa sample and the mixture was incubated for 1 hr at 4T. A 10 ~l portion of the mixture waswithdrawn, and trypsin activity was measured as described above.
Phospholipase A 2 (EC 3.1.1.4) activity of a mitochondrial fraction or homogenate wasmeasured essentially according to the method of Tamiya-Koizumi et a/. 12 ) The homogenate(0.2-0.7 ~g protein) or mitochondria (0.4-3.0 ~g protein) was incubated for 60 min at 37Twith 3 nmoles of l-acyl-2-( 1_ 14 C) arachidonyl phosphatidylethanolamine (specific activity,

44
Yuzo Sakai et af.

10,000 cpm/nmol) in a 200-!!1 system containting 100 mM Tris-acetate, pH 9.0 and 10 mM
CaCI 2 • The reaction was terminated by adding 4 !!l of acetic acid and 800 !!l of chloroform/
methanol (2: 1, vIv) containing 20 !!g of arachidonic acid. The reaction mixture was centrifuged,
and the lower phase of the supernatant was collected and concentrated. The concentrated
sample was then chromatographed on plastic sheets of silica gel 60 (Kanto Chemical Co.,Tokyo) with chloroform/methanol/water (65:25:3, v/v/v/). The spot corresponding to arachidonic acid was cut out and its radioactivity was measured using a toluene scintillator with aliquid scintillation spectrometer. The reactions were linear at concentrations of protein ranging
0.1-1.0 !!g and 0.4-3.0 !!g for homogenate and mitochondria, respectively.
A TP synthesis
ATP synthesis of mitochondria was measured by the luciferin-luciferase method!3) as described before!4). This method of ATP analysis is based on the linear luminescence response of
firefly luciferin to added ATP. Luciferin is converted into oxyluciferin by luciferase in thepresence of ATP, Mg2+ and oxygen. Light generated during the reaction is measured with aphotomultiplier Mitochondria were preincubated for 10 min at 30'C with 5 ml of medium A(1.2 mg/ml). Then, 5 mM sodium glutamate,S mM sodium malate,S mM potassium phosphate, and 500 nmoles of ADP were added to the reaction mixture. After 0, 0.5, 1, 1.5 and 2
min, a 20 !!l portion of the sample was withdrawn and put into a test tube containing 980 !!l of
the ice-cold medium A. The sample (100 !!I) was immediately put into a cuvette containing 100!!l of ATP releasing agent (Labo Science, Tokyo). The cuvette was mounted into a TD-4000
lumiphotometer (Labo Science, Tokyo) and a 100-!!1 portion of luciferin-luciferase (EC1.14.14.3) (Labo Science, Tokyo) was added to the cuvette. To correlate light intensity with theconcentration of ATP, light intensity was plotted against standard ATP solutions (1 X 1O-L 2.5
X 10-6 M). The light intensity occurring after the addition of luciferin-luciferase to the cuvettecontaining an unknown concentration of ATP was thus related to the concentration of ATP.
Cytochrome contents
Cytochrome contents of mitochondria were measured spectrophotometrically according to
the method of Williams 15 ) as described before!6). Mitochondria (10 mg) dissolved in 900 !!l of
100 mM phosphate buffer, pH 7.4, containing 2.2% deoxycholic acid were put into two cuvettes, respectively. A 100 !!l portion of 0.05 M K3Fe(CN)6 as the oxidizing agent was added to
one cuvette, and 100 !!I of 50 mM ascorbate as the reducing agent was added to the other cuvette. A few grains of sodium hydrosulfite were put into the latter cuvette. The reducedoxidized difference visible spectra of the cytochromes were obtained using a Hitachi U-3200
spectrphotometer.
Electron microscopy
Mitochondrial suspensions were fixed in 2% glutaraldehyde containing 250 mM sucrose and
50 mM sodium cacodylate, pH 7.4, and processed for electron microscopy as described before!?). Thin sections were cut on a Reichert ULTRACUT N ultramicrotome, stained with leadcitrate and examined in a Hitachi H-800 electron microscope operated at 100 kV.

RESULTS
Yields of mitochondrial proteins obtained by various isolation procedures
When the 2000-6000xg R 2 fraction obtained in the presence (the mitochondrial fraction of

45
ISOLATION OF PANCREAS MITOCHONDRIA

Fig. 1 Electron micrographs of pancreatic mitochondrial fractions obtained by various isolation
procedures. For details, see Materials and Methods.
A. A 2000-6000xg' R 2 fraction isolated in the presence of dibucaine and trypsin inhibitor.
X13700.
B. A 2000-6000xg' R"" 2 fraction isolated in the presence of dibucaine and trypsin inhibitor. X 13700.
C. A 700-7000xg' Rm 2 fraction isolated in the presence of dibucaine and trypsin inhibitor.
X 13700.
D. A 700-7000xg' R m 2 fraction isolated in the absence of dibucaine and trypsin inhibitor.
X13700.

46
Yuzo Sakai et al.

Wilson et al. 3») or absence of dibucaine and trypsin inhibitor was washed three times, the yieldof the 2000-6000xg' R"' z fraction thus obtained to the homogenate was decreased to about
30% of that of the 2000-6000xg' R z fraction to the homogenate. As was expected, the yield of
the 700-7000xg' R z fraction obtained in the presence or absence of dibucaine and trypsin inhibitor to the homogenate was larger than that of the 2000-6000xg' R z fraction to the homogenate. When the homogenate was centrifuged either at 2000xg for 7 min or at 700xg for 10 min,the residue was made of two layers: an upper layer, which was pale brown and relatively rich inmitochondria, and a lower layer, which was whitish and rich in zymogen granules. The twolayers were not well demarcated. In the present study special care was taken to collect only theupper layer leaving the whitish layer attached to the bottom of the centrifuge tube. The R z fraction thus obtained by the 2000-6000xg centrifugation method was the mitochondrial fractionused by Wilson et al. 3 ) (Fig. lA). The higher yield of R z fraction obtained by the 700-7000xg
centrifugation method than by the 2000-6000xg centrifugation method was, as expected, due
to severer contaminations with zymogen granules and membrane fragments mainly composed ofendoplasmic reticulum. When the R z fraction obtained by either 2000-6000xg or 700-7000xg
was washed three times, a fraction rich in mitochondria (R'" z fraction) was obtained (Figs.
lB-IO). The yield of the R"'z fraction to the homogenate obtained by the 700-7000xg centrifugation method tended to be slightly higher than that of the R"' z fraction obtained by the
2000-6000xg centrifugation method (Table 1). The yield of the pancreatic mitochondrial fraction (R"' 2) to the homogenate or the tissue was extremely low when it was compared with that of
the liver. On the other hand, a ratio of the homogenate to the wet weight of the pancreas basedon the amount of protein was larger than that of the liver. One of the reasons for this may be asmaller population of mitochondria per one acinar cell compared with that of the hepatic parenchymal cell since pancreatic acinar cells are occupied by abundant rough-surfaced endoplasmic
reticulum and zymogen granules. Another possible reason may be that porcine pancreas is rich

Table I.

Yield of pancreatic mitochondrial proteins obtained by various isolation procedures"·h2000-6000xg'

700-7000xg"+Dib., +T.!.

-Dib., -T.!.

+Dib., +T.1.

-Dib., -T.!.
5.96±0.34-

Liver

R/Homogenate (%)

4.55 ±0.28

4.51 ±0.03

5.77 ± 0.38-

R/Wet weight of the tissue (%)

1.14 ± 0.07

1.09 ± 0.08

1.44 ± 0.11*

1.47 ± 0.09-

R-/Homogenate (%)

1.32 ± 0.22

1.39±0.12

1.56 ± 0.36

1.65 ± 0.31

8.16±0.21

R-/Wet weight of the tissue (%)

0.33 ±0.05

0.34 ± 0.03

0.39 ±0.080040 ± 0.06

1.61 ± 0.09

25.04 ± 1.25

24.21 ±0.82

24.92±0.87

24.71 ± 1.16

20.Q3 ± 0.63

Homogenate/Wet weight of
the tissue (%)- - - - - - - - - - - _..

_--_ . . - - - -a Basic isolation medium contained 10 mM Tris-C1, pH7A, 0.25M sucrose, 1 mM EDTA, 0.5% BSA in thepresence or absence of 0.25 mg/ml soybean trypsin inhibitor (T.!.) and 400 uM dibucaine (Dib.).
b Data are the average of four different experiments (mean ± SE). -Statistically differnet from the corresponding
data obtained by 200-6000xg (0.02 < P < 0.05).
c Homogenate was centrifuged at 2000xg for 7 min. The resultant supernatant was centrifuged at 6000xg for 10
min to sediment the crude mitochondrial pellet (R,). The R, fraction was obtained by washing R, fraction threetimes.
d Homogenate was centrifuged at 700 xg for 10 min. The resultant supernatant was centrifuged at 7000xg for 10
min. The three-time-washed R 2 fraction was designated as the R, fraction.

47
ISOLATION OF PANCREAS MITOCHONDRIA

Table 2.

Contents of cytochromes ion pancreas mitochondria obtained by various isolation procedures"
Cytochromes (nmol/mg protein)Centrifugation
MethodFraction

a+aJ

b

c,

c

R2

0.133 ± 0.004

0.lZ7±0.01l

R- 2

700-7000xg

R2
R- 2

0.Z07 ± 0.016
0.lZ4±0.01Z
0.Z70 ± 0.010*

0.171 ±O.OlZ
0.1l8±0.013
0.Z18 ± O.OlZ**

Liver

R- 2

0.196±0.004

0.190 ± 0.006

ZOOO-6000xg
ZOOO-6000xg
700-7000xg

c+c,---_.-"._----_._----

0.lZ7±0.015

0.076 ± 0.013

0.054 ± 0.004
0.079 ± 0.006
0.056 ± 0.004
0.089 ± 0.005

0.128±0.004

0.191 ±0.010

0.324 ± 0.014

0.073 ± 0.0 lZ
0.088 ± 0.007
0.054 ± 0.003

0.167±0.01O
0.110±0.005
0.164±0.016

a Homogenate of porcine pancreas was centrifuged either for 7 min at 2000xg or for 10 min at 700xg. The supernatant was centrifuged for 10 min either at 6000xg or at 7000xg. The residue (R 2 ) was washed three times (R'· 2)'
The isolation medium contained 10 mM Tris-C1, pH 7.4, 0.Z5M sucrose, 1 mM EDTA, 0.5% BSA, soybean
trypsin inhibitor (0.25 mg/ml) and 400 !-1M dibucaine. Data are the average of six different experiments(mean±SE). Comparison was made between 2000-6000xg' R2 and 700-7000xg' R 2, and between
ZOOO-6000xg' R- 2 and 700-7000xg' W 2'
*0.001 <p<0.01; **0.01 <p<0.02

both in intra- and interlobular fibrous connective tissue lowering the ratio of parenchymatous
acinar cells to the homogenate or the tissue.
Contents of cytochromes in pancreatic mitochondria
There was statistically no difference in the content of cytochromes between the
2000-6000xg' R z fraction and the 700-7000xg' R z fraction. However, the content of cytochromes in the latter fraction became much larger than that in the former fraction after both fractions were washed three times (Table 2). This was due to remarkable increases in cytochromesa+a) and b in the latter fraction. The content of cytochromes a+a) and b in the
700-7000xg' R m z fraction was higher than that of rat liver mitochondria whereas that of cytochrome c+c) was approximately half of the latter mitochondria.
Coupling efficiency ofpancreas mitochondria obtained by various isolation procedures
Before we measured the coupling efficiency of mitochondria obtained by various isolation
procedures, we compared two different respiration media: medium A, according to Wilson etal. )); and medium B, routinely used in our laboratory for liver mitochondria. In Fig. 2, typicalexamples of oxygen traces of a 700-7000xg' R z fraction obtained in the above-described two
different media are shown. The rate of oxygen consumption in state 3 respiration after the addition of ADP was definitely lower in medium B than in medium A resulting in lower repiratory
control in the former especially when glutamate was the oxidizable substrate. Thus, we employed medium A as a respiration medium thereafter. However, we found later that medium A
could be replaced by medium B as long as the concentrations of BSA and EDTA of the latter
were as high as those of the former. The rates of oxygen consumption both in state 3 and state 4(after the conversion of externally added ADP to ATP) respiration were low in the
2000-6000xg' R2 fraction compared with those in the 2000-6000xg' Rm ~ fraction or in the
700-7000xg' R"z fraction (Table 3). Lower rates of oxygen consumption of the R~ fraction
corresponded well to the fact that the fraction was severely contaminated with zymogen granules

48
Yuzo Sakai et al.

Medium A

Medium B

Medium A

Meclum B

Mit.
MIt.
~ Glut.+Mal. ~

/

ADP,

ADP,
300 nmoles

200 nmoles

ADP,"300 nmoles\100 "tom, a-RCI-4.81
ADP/Q-1.96

1 min

Fug. 2.

Rate of oxygen consumption of pancreas mitochondria measured in different respiration media. The rateof oxygen consumption of mitochondria was measured at 30T in mediums A and B, respectively, using a
700-7000xg' R 2 fraction isolated in the presence of dibucaine and trypsin inhibitor. Final concentrationsof reagents added were Pi,S mM; succinate,S mM; glutamate,S mM; malate,S mM; and DNP, 25 lAM.
Numbers in the figures indicate the amount of oxygen (natoms) consumed/min/mg protein. Medium A
contained 250 mM sucrose, 22 mM KC!, 22 mM triethanolamine, pH 7.4, 1 mM EDTA and 0.5% BSA.
Medium B contained 70 mM sucrose, 220 mM mannitol,S mM Tris-C!, pH 7.4, and 0.1 mM EDTA.

Table 3.

Coupling efficiency of porcine pancreas mitochondria obtained by various isolation procedures'

FractionCentrifugation

Isolation Medium

R2

2000-6000xg

R"2
R"' 2

2000-6000xg
700-7000xg
700-7000xg

STBE+Dib.+T.I.
STBE+Dib.+T.I.

~

'"c
'u
u

::l
Vl

R"' 2

-"'s,Jg"

,,~

'"

'"
-~

..2+

c~

R2
R- 2
R"' 2
R"'2

2000-6000xg
2000-6000xg
700-7000xg

700-7000xg

STBE+Dib.+T.I.
STBE-Dib.-T.I.
STBE+Dib.+T.I.
STBE+Dib.+T.I.
STBE+Dib.+T.I.
STBE-Dib.-T.I.

State 4
State 3
(natoms O/mg/min)
58.9t 12.0
89.0 t 11.9
93.7 t 8.8*
91.4 t 12.4*
40.6±9.762.3±6.5
69.0 ± 6.0*
70.1 ± 3.4*

Rei

ADP/O ratio

13.3 t 1.8
20.4 t 5.2
21.7 t 3.9*

4.36tO.34
4.70 t 1.05
4.50tO,82

1.70 to.24
2.08 t 0.11 *20.6±2.6*

4.58 ± 1.00

8.3 ± 1.0
1O.7±3.3
14.1 t 3.1*

4.13 ± 0.44

16.6 t 2.7*

6.60 ± 1.91
5.12tO.79
4.36tO.61

1.91 to.19
1.92±O,142.86±0.16
2.90tO.17
2.96±0.08
3.00 ± 0.16

a Porcine pancreas was homogenized in a medium containing 10 mM Tris-C!, pH 7.4, 0.25 M sucrose, 0.5% BSA
and 1 mM EDTA (STBE) in the presence or absence of 400 lAM dibucaine (Dib.) and 0.25 mg/ml tryspin inhibitor (T.I.). The homogenate was centrifuged for 7 min at 2000xg or for 10 min at 700xg. The resultant pellet
was suspended in the appropriate medium, and centrifuged for 10 min at 6000xg or 7000xg. The pellet (R 2) waswashed three times (R- 2)' Data are the average of three different experiments (mean ± SE).
* Statistically different from data obtained by the 2000-6000xg . R 2 fraction at 0.02 < P < 0.05.

49
ISOLATION OF PANCREAS MITOCHONDRIA

2000-6000xg R2
STBE+Dlb+TI
Mit.
~ Glut.+Mal.

I

2000-6000xg R~ 2
STBE+Dlb+TI
MIt.

l./ ADP,,

700-7000xg R- 2
STBE+Dlb+TI
MIt.
Glut.+Mal.

l

l

I

I

__ ADP,

300 nmo es-

300 nmoles

ADP,
ADP,

700-7000xg R~ 2
STBE-Dlb-TI
Mit.

/'300 nmoles

ADP,/300 nmoles

ADP,; / 300 nmoles/300 nmoles
RCI=5.08 60.0

ADP/O=2.92

1100 natoms 0

-

1 min

Fig. 3.

\

Coupling efficiency of pancreatic mitochondrial fractions obtained by various isolation procedures. The
respiration of mitochondria was measured in medium A. Isolation medium contained 250 mM sucrose, 10
mM Tris-C1, pH 7.4, I mM EDTA and 0.5% BSA (STBE) in the presence and absence of 400 J.lM dibucaine (Dib.) and 0.25 mg/ml soybean trypsin inhibitor (T.!.).

and microsomes as shown in the previous section. Another point to be stressed here is the effectof the composition of the isolation medium on the coupling efficiency of those mitochondria.
The coupling efficiency of mitochondria obtained in the absence of both dibucaine and trypsin
inhibitor in the isolation medium was unexpectedly as good as that of mitochondria isolated in
the presence of both dibucaine and trypsin inhibitor. Typical examples of oxygen traces of suchmitochondria are shown in Fig. 3. Moreover, the coupling efficiency of mitochondria isolated in
the absence of dibucaine and trypsin inhibitor was well maintained for at least 8-10 hr after
they were isolated. A tightly coupled mitochondrial fraction was obtained also from the rat pancreas using a medium not containing dibucaine and trypsin inhibitor. These results strongly suggest that enzymes of zymogen granules including trypsinogen are not easily activated during theisolation procedure of pancreas mitochondria. Data on trypsin and phospholipase A 2 activitiesof pancreas mitochondria will be presented later.

A TPase and cytochrome oxidase activities of pancreas mitochondria obtained by various isolation procedures
In the previous section we have shown that mitochondria isolated in the absence of dibucaine
and trypsin inhibitor are well coupled. To analyze further the effect of the composition of isolation medium on mitochondrial functions, we also measured ATPase and cytochrome oxidase activities of those mitochondria (Table 4). It is clear from the table that the absence of dibucaine
and trypsin inhibior did not affect the activities of either enzyme.

50
Yuzo Sakai el al.

Table 4.

Cytochrome oxidase and ATPase activities of pancreas mitochondria obtained by various isolation
procedures'

Centrifugation

ATPasec

Cytochrome oxidase(natoms Glmg/min)(nmol Pilmg/min)
99.2±2.0
191.2±8.6*254.2± 17.8*
261.4 ± 11.2*

Isolation Medium

Fraction"

STBE+Dib.+TI.

R2

STBE+Dib.+TI.
STBE+Dib.+T.I.
STBE-Dib.-TL

R"'2
Rm 2

109.8± 5.0
338.0 ± 27.0*
356.9 ± 11.9*

R"' 2

353.7 ±6.0*----

2000-6000xg
2000-6000xg
700-7000xg
700-7000xg

a Isolation medium for porcine pancreas contained 10 mM Tris-Cl, pH 7.4, 0.25 M sucrose, I mM EDTA, and
0.5% BSA (STBE) in the presence or absence of 400 fAM dibucaine (Dib.) and 0.25 mg/ml soybean trypsin inhibitor (T.L). Data are the average of three different experiments (mean ± SE).
b The crude mitochondrial fraction (R 2 ) was washed three times either at 6000xg or at 7000xg (R"' 2)'
C MgH-activated ATPase activity was measured using frozen samples in the presence of DNP to obtain full activity.
* Statistically different from the 2000-6000xg' R 2 fraction at p < 0.001.

100..-------.,.-------"""T"""""""'1

l ::

Q)

o

'-

COl

E
( J)
Q)

(5

E

l::

o
Fig. 4.

1
2Time After Addition of ADP (min)Ability of pancreatic mitochondrial fractions obtained by various isolation procedures to synthesize ATP.
For details, see Materials and Methods. A 2000-6000xg' R 2 fraction isolated in a medium containing 250
mM sucrose, 10 mM Tris-Cl, pH 7.4, 1 mM EDTA, 0.5% BSA in the presence (L'.) or the absence (.A) of
400 fAM dibucaine and 0.25 mg/ml soybean trypsin inhibitor; a 700-7000xg' R 2 fraction isolated in a
medium containing 250 mM sucrose, 10 mM Tris-CI, pH 7.4, I mM EDTA, 0.5% BSA in the presence(0) or absence (e) of 400 fAM dibucaine and 0.25 mg/ml soybean tryspin inhibitor.
m

51
ISOLATION OF PANCREAS MITOCHONDRIAThe ability ofpancreas mitochondria to synthesize A TP
In the present study the ability of mitochondria to synthesize ATP was also tested in addition
to polarographic measurements of oxygen uptake of mitochondria to evaluate their phosphorylating capacities (Fig. 4). The 2000-6000xg' R 2 fraction converted about 84.8% of externallyadded ADP into ATP. On the other hand, the 700-7000xg' R'" 2 fraction obtained in thepresence or absence of dibucaine and trypsin inhibitor converted about 97.1 % and 99.2% of
externally added ADP into ATP after 2 min, respectively. These data again suggest that it is notessential to add dibucaine and trypsin inhibitor to the isolation medium to obtain well-coupledmitochondria from the pancreas.
Activities of trypsin and phospholipase A 2 in pancreas mitochondria obtained by various isolation
procedures
Wilson et al. 3 ) have paid special attention to avoiding the unfavorable effects of trypsin on theprocess of the isolation of pancreas mitochondria. Their isolation medium contained trypsin inhibitor and dibucaine to prevent trypsin and phospholipase A 2 activities. However, the present
data have shown that mitochondria isolated in the absence of soybean trypsin inhibitor anddibucaine maintain a high coupling efficiency even for 8-10 hr after they are isolated. Thus, wemeasured trypsin and phospholipase A 2 activities in pancreas mitochondria (Table 5 & 6). Trypsin activity in the 2000-6000xg' R 2 fraction isolated in a medium containing dibucaine andtrypsin inhibitor was definitely higher than that in the 700-7000xg' R'" 2 fraction isolated in a
medium with or without dibucaine and trypsin inhibitor, and yet the activity was as low as 5 [!glmg protein in the absence of enterokinase (Table 5). When the R 2 and R'" 2 fractions weretreated with enterokinase, a potent activator of trypsinogen, trypsin activities of those fractions
were remarkably enhanced. These data indicate that the R 2 fraction obtained in the present
study is far more contaminated with zymogen granules than the R'" 2 fraction, and yet a major
portion of the enzymes remains inactivated. In the present study we have also measured phospholipase A 2 activities of pancreas mitochondria (Table 6). If trypsin activity of a mitochondrial
fraction obtained in the absence of trypsin inhibitor is as low as that of a mitochondrial fraction
obtained in the presence of trypsin inhibitor, phospholipase A 2 activity of the former might be
as low as the latter, since phospholipase A 2 is activated by trypsin. Actually, there was no difference in phospholipase A 2 activities between the R 2 fraction isolated in the presence of trypsin
inhibitor and dibucaine and that isolated in the absence of trypsin inhibitor and dibucaine. There
was also no difference in the enzyme activity between the R2 and R'" 2 fractions. Moreover, phospholipase A 2 activities in the R 2 and R'" 2 fractions isolated in the absence of EDTA, an inhibitorof phospholipase A 2 activity 18.19), remained almost unchanged for up to 8-10 hr of aging, as
logn as these fractions were kept at 4'C. All these data strongly suggest that dibucaine and trypsin inhibitor could be omitted from the isolation medium to obtain intact mitochondria from thepancreas. These data suggest at the same time that trypsin activities found in these fractions
might not be high enough to be injurious to mitochondrial functions. Thus, further studies wereconducted to examine the effect of trypsin or dibucaine on the coupling efficiency of pancreas
mitochondria. In Fig. 5A a R'" 2 fraction isolated in the absence of trypsin inhibitor and dibucaine was preincubated with trypsin (150 [!g/mg protein) for 5 min at 30°C and then the rate ofoxygen consumption of mitochondria was measured. The rate of oxygen consumption in state 3
respiration was unchanged while that in state 4 respiration was slightly increased. Trypsin atconcentrations up to 100 [!g/mg protein had no effect on mitochondrial respiration. Similar experiments were performed using rat liver mitochondria (Fig. 5B). In the case of liver mitochondria, effects of trypsin on mitochondrial respiration were not evident when its concentration
was less than 100 [!gl mg protein, as in the case of pancreas mitochondria, but at higher

52
Yuzo Sakai et af.

Table 5.

Trypsin activities in pancreatic mitochondria obtained by various isolation procedures". _ - - - - _ .. _..•. Isolation Medium

Activity (~g trypsin/mg)-Enterokinase+Enterokinase

Centrifugation

Fraction

2000-6000xg

R2
Homogenate. ------_. " . - - . _ ..-STBE+Dib.+T.I.
STBE+Dib.+TI.
STBE-Dib. - TI.

700-7000xg
700-7000xg

5.01 ±0.41

123.36 ± 6.30

3.14±0.32

205.33 ± 48.49

R~2

1.22 ± 0.18'

36.62 ± 4.88'

Homogenate

3.15 ± 0.36

200.91 ± 28.50

R~2

1.44 ± 0.34"

40.69 ± 2.12'

Homogenate

3.89 ± O.OS'"

222.92 ± 66.96

a Isolation medium contained 0.25M sucrose, 10 mM Tris-C1, pH 7.4, 1 mM EDTA (STBE) in the presence orabsence of 400 ~M dibucaine (Dib.) and 0.25 mg/ml tryspin inhibitor (TI.). Data are the average of three different cxperiments (mean ± SE). Statistically different from the 200-6000xg' R 2 fraction and the homogenate
using STBE+Dib.+T.I. at; " p < 0.001; ", 0.001 < P < 0.01; "',0.01 < P < 0.02.

Table 6.

Phospholipase A 2 activities in pancreas mitochondria obtained by various isolation procedures"

Isolation Medium

Centrifugation

Fraction

STB+Dib.+TI.+EDTA

2000-6000xg

R2
Homogenate

STB+Dib.+T.I.+EDTA

700-7000xg

R~

Activity (nmol PE/mg/min)--------

STB-Dib.-TI.+EDTA
STB-Dib.-TI.-EDTA

700-7000xg
2000-6000xg
700-7000xg

10.7 ± 2.2
15.7 ± 1.3
9.0± 2.8

2
Homogenate

14.5 ±0.2

R~2

10.5 ± 0.2

Homogenate

15.6 ± 1.0

R213.9±0.3 (13.6± 1.4)

R~2

12.6± 1.5 (13.0± 1.8)

Homogenate

16.71 I.S (17.21 1.6)

a Isolation medium contained 10 mM Tris-C1, pH 7.4, 0.25M sucrose, 0.5% BSA (STB) in the presence or absence of 400 ~M dibucaine (Dib.), 0.25 mg/ml soybean trypsin inhibitor (TI.) and 1 mM EDTA. Data are theaverage of three different experiments (mean ± SE). Comparisons were made between the R 2 fraction obtainedby a 2000-6000xg centrifugation method and the homogenate using STB+Dib.+TI.+EDTA as the control.
Data on phospholipase A 2 activities of samples aged for 10 hr at 4T are shown in parentheses. PE: phosphatidyl
ethanolamine

concentrations such as 150 I1g/mg protein, the rate of oxygen consumption in state 3 respiration
was decreased remarkably, with increases in state 4 respiration. On the other hand, the coupling
efficiency of mitochondria treated with trypsin for 3 hr at ooe at a concentration of 200 I1g/mg
protein was well maintained. These data strongly suggest that trypsin at concentrations detectedin various mitochondrial fractions obtained from the pancreas in the present study would not affect the coupling efficiency of these mitochondria.
Effects of dibucaine on pancreas mitochondria were also tested. As was already demonstrated, there were not differences in the rate of state 3 and state 4 respirations and those isolated in the presence of dibucaine and those isolated in the absence of dibucaine (Table 3).
However, rat liver mitochondria isolated in the presence of dibucaine at a concentration of

53
ISOLATION OF PANCREAS MITOCHONDRIA

Control

MILl,TrYPSln~.-Glut.+Mal.

_

ADP,300 nmoles./ADP,300 nmoles
RCI-S.48

76.7

ADP/O~2.87

RCI~3.99
ADP/O~2.87

1100 natoms 0

-

1 min

Control
Mit.
Glut.+Mal.

l

Trypsin-treated
Mit.
Glut.+Mal.

l

I

ControlMit.

I

trypsin./Glut.+Mal.

~I.-ADP, 300 nmoles

ADP, 300 nmoles

I
ADP, 300 nmoles,/
RCI~7.88

RCI=10.70

AOP, 300 nmoles

I

AOP/O=2.72

ADP/Os2.78

1100 natoms 0

-

1 min

Fig. 5.

Effect of externally added trypsin on mitochondrial respiration.
Pancreas mitochondria (a 700-7000xg' R- 2 fraction isolated in the absence of dibucaine and trypsin
inhibitor) was pretreated for 5 min at 30'C with trypsin at a concentration of 150 Ilg/mg protein, andthen the coupling efficiency of mitochondria was measured using glutamate (+ malate) as oxidizable
substrate (on the right). As the control experiment, mitochondria were preincubated for 5 min at
30'C, and then the coupling efficiency of mitochondria was measured (on the left).
B. Rate of oxygen consumption of rat liver mitochondria, pretreated for 3 hr at O'C by trypsin at a concentration of 200 Ilg/mg protein, was almost unchanged (middle) compared with that of the control
mitochondria (left). The rate of oxygen consumption of mitochondria in state 3 respiration pretreatedwith trypsin for 5 min at 30'C at a concentration of 150 Ilg/mg protein was decreased, while that instate 4 respiration was increased resulting in a low respiratory control index (RCI) (right).
A.

54

Yuzo Sakai et al.

B.Dlb.-treated C.Control
D.Dlb.-treated E.Control
A Control
Mit.
Mit
Mit.
MiL
Mit.
I
I Glu,t.+Mal. I GluL+MaJ. I DibucaIne, 300}lM~ Succ.
Succ.
~
,
ADP,
...-ADP,
300 nmoles
300 nmoles

,

I

,

'I

'/

I

54.0

89.3

1100 .alom, a
RCI=12.40

RCI=4.65

ADP/O=1.92

ADP/O=2.82~ mi~Fig. 6.

Effects of dibucaine on coupling efficiency of rat liver mitochondria. The rate of oxygen consumption of
mitochondria in state 3 respiration isolated in a medium containing 400 ~M dibucaine was decreased whenoxidizable substrate was succinate (8) or glutamate (+ malate) (D) compared with that of the control mitochondria isolated in the absence of dibucaine (A and C). Control mitochondria preincubated for 5 min
at 30T with dibucaine at a concentration of 300 ~M were uncoupled (E).

400 IlM (the concentration employed for the isolation of pancreas mitochondria) showed distinct decreases in the rate of state 3 respiration when either succinate or glutamate was used as
substrate (Fig. 6).

DISCUSSION
There are several expected difficulties in isolating functionaly and morphologically intact mitochondria from the pancreas: (1) The pancreas acinar cells are rich in zymogens including trypsinogen and Pro-phospholipase A 2 which may be activated and damage mitochondria when they
are being isolated. (2) The organ is heterogenous in structure. Acinar cells are grouped into lobules by fibrous connective tissues. This causes difficulty in the homogenization. If the tissue ishomogenized too much, mitochondria may be disrupted, while insufficient homogenization of
the tissue may yield a smaller amount of mitochondria. The organ is not only covered with alarge amount of adipose tissue but fatty infiltration is inevitably found within intra- and interlobular spaces. Unsaturated fatty acids are known to swell and uncouple mitochondria. All thesehave made it difficult for us to establish an isolation procedure for intact mitochondria from thepancreas. In the literature, Wilson et al. 3) are the first to report success in isolating intact mitochondria from the pancreas. Their isolation medium contained soybean trypsin inhibitor sincetrypsin is the trigger enzyme responsible for activating all other pancreatic zymogens 19 ). It also
contained dibucaine and 1 mM EDTA to inhibit phospholipase A 2 which might attack membrane phospholipids. A high concentration of BSA (0.5%) was also added to their isolation
medium. However, they did not present data on the question of whether or not trypsin inhibitorand dibucaine were absolutely necessary to obtain functional mitochondria. Two ways of55
ISOLATION OF PANCREAS MITOCHONDRIA

activating trypsinogen to trypsin are known: autoactivation and activation by enterokinase present in the duodenal juice I9 ). Once the pancreas is removed from the animal, there is no chancefor tripsinogen to be activated by enterokinase. Thus, the effects of trypsin produced by the
autoactivation should be taken into account when mitochondria are isolated from the pancreas.
If the rate constant for enterokinase-catalyzed trypsinogen activation exceeds that for the autoactivation by several orders of magnitude I9 ), it is possible that isolated mitochondria are notdamaged by trypsin. The present study has confirmed that the procedure developed by Wilson
et al. 3 ) is useful in detecting phosphorylating capacities of pancreas mitochondria especiallywhen a small amount of tissue is available. However, when it is necessary to look into the detailsof the functional aspects of pancreas mitochondria, such as changes in membrane fluidity, aniontransport, permeability to various drugs, activities of various enzymes, it would be better toavoid the use of dibucaine and trypsin inhibitor in the isolation medium for mitochondria because evidence indicates anesthetics modify the structure and function of biological membrane
systems including mitochondria 20 - 28 ). The present study has shown that intact pancreas mitochondria can be obtained without using either a special isolation medium or a special centrifugation method.

Yield of mitochondria with respect to the wet weight of the pancreas or the homogenate
The present study demonstrated that the yield of mitochondria on the protein basis relative tothe homogenate of the pancreas was extremely low compared with that of the liver although theratio of the homogenate to the wet weight of the tissue was even higher in the former. Meldolesi
et al. 29 ) also obtained a low yield of mitochondria from the guinea pig pancreas although theirisolation procedure for mitochondria was different from that tested in the present study (mitochondria/wet weight of the pancreas, 0.2%, mitochondria/homogenate, 1.39%). Since the ratioof homogenate to the wet weight of the tissue was higher in the pancreas than in the liver, andthe content of proteins per wet weight of the tissue is larger in the former 30•31 ), a smaller contribution of mitochondrial proteins to the total amount of tissue proteins in the pancreas may be
the major reason for obtaining a low yield of pancreas mitochondria. Actually, the number ofmitochondria per acinar cell revealed on a plane section is definitely smaller than that of the hepatocyte when judged on electron micrographs (unpublished obserbvation). Another possible
reason may be that some of the mitochondria were broken and lost during the homogenization
because of the structural heterogeneity described above, despite special care taken when thepancreas was homogenized. If we focus only on the yeild of mitochondria relative to 1 g of thewet weight of the tissue, a total of 3-4 mg for the R'" 2 fraction and 11-15 mg for the R 2 fraction were obtained. When the R 2 fraction is used as a mitochondrial fraction in case, forexample, individual rat pancreas is used as a starting material, a 2000-6000xg centrifugation
method is recommended rather than a 700-7000xg centrifugation method, since the contamination of the latter with other organelles is severer when judged by the content of cytochromes.
When enough of the tissue is available as a starting material a R'H 2 fraction using a 700-7000xg
centrifugation method is recommended rather than that using a 2000-6000xg centrifugation
method based on cytochrome contents, especially of a+a 3 and b (Table 2), and activities of cytochrome oxidase and ATPase (Table 4) indicating that the former is less contaminated withother organelles than the latter.
Correlation between coupling efficiency of pancreas mitochondria and the composition of isolation medium
We have shown in the present study that the coupling efficiency of pancreas mitochondria is
well preserved as long as the isolation medium contains both BSA and EDT A. Rates of oxygen

56
Yuzo Sakai et al.

consumption in state 3 and state 4 respirations of the 2000-6000xg R 2 fraction isolated in thepresence of dibucaine and trypsin inhibitor were definitely low compared with those of the
2000-6000xg' R'" 2 or the 700-7000xg' R'" 2 fraction isolated in the presence or absence ofdibucaine and trypsin inhibitor (Table 3). However, respiratory control ratios of the former wereas high as the latter. These data suggest that the lower rates of respiration of the former fraction
were due to the contamination with other organelles and that neither dibucaine nor trypsin inhibitor was essential to obtain well-coupled mitochondria. Namely, trypsinogen was not activated during the homogenization and the separation of the mitochondrial fraction. Sardesai andProvid0 32 ) also could not detect trypsin activity in their mitochondrial fraction or in the homogenate of the tissue. To summarize, the present results suggest that the presence of dibucaine andtrypsin inhibitor is not an absolute requirement for the isolation of functional mitochondria fromthe pancreas. Finally, we should refer briefly to the cytochrome content of pancreas mitochondria. As already shown in the previous section, we have obtained the highest concentration ofcytochromes in the 700-7000xg' R'" 2 fraction (Table 2). In the present study a differential centrifugation method was applied to the pancreas and further attempts were not made to purify
mitochondria. The averaged concentration of cytochromes obtained in pancreas mitochondria
suggests that they are still contaminated with other organelles to some extent since pancreas mitochondria are almost as rich in the inner membranes as heart mitochondria. Among the data on
cytochrome content of pancreas mitochondria obtained in the presnet study, the highest values
obtained in one experiment were: a+a 3, 0.365; b, 0.296, c J , 0.148 and c, 0.101 (c+c J , 0.249).
In another experiment, a R 2 fraction obtained by a 700-7000xg centrifugation method waswashed 4 times, and the content of cytochromes in the fraction was a+a3 , 0.371; b, 0.351; cJ ,0.136, and c, 0.131 (c+c J , 0.267). Thus, a higher content of cytochromes would be obtained
from pancreas mitochondria if the isolation procedure were improved further.

ACKNOWLEDGEMENTS
We are grateful to Dr. K. Tamiya-Koizumi, Laboratory of Cancer Cell Biology, Research Institute for Disease Mechanism and Control, Nagoya University School of Medicine, for the estimation of phospholipase A 2 activity. We thank Miss K. Hasegawa for the preparation of themanuscript. This work was supported in part by a grant for Intractable Pancreatic Disease from
the Ministry of Welfare of Japan.

REFERENCES
1)
2)

3)

4)
5)
6)

Leiber, C.S.: Pathogenesis of alcoholic fatty liver. In Medical disorders of alcoholism. Pathogenesis and treatment, edited by Lieber, C.S., pp. 146-159 (1982), W.B. Saunders Company, Philadelphia.
Noronha, M., Salgadhino, A., Ferreira de Almedia, M.J., Dreiling, D.A. and Bordalo, a.: Alcohol and thepancreas. I. Clinical associations and histopathology of minimal pancreatic inflammation. Am. J. Gastroenterol., 76,114-119 (1981).
Wilson, J.S., Korsten, M.A., Leo, M.A. and Lieber, C.S.: New technique for the isolation of functional rat
pancreatic mitochondria and its application to models of pancreatic injury. J. Lab. Clin. Med., 107, 51-58(1986).
Kishino, Y., Takama, S. and Kitajima, S.: Ultracytochemistry of pancreatic damage induced by excess lysine.
Virchows Arch. Cell Pathol., 52, 153-167 (1986).
Sardesai, Y.M. and Provido, H.S.: The presence of a substance in the pancreas that inhibits mitochondrial
respiration. J. Surg. Res., 35, 176-179 (1983).
Lowry, a.H., Rosebrough, N.J., Farr, L. and Randall, R.J.: Protein measurement with the Folin phenol reagent. J. Bioi. Chem., 193,265-275 (1951).

57
ISOLATION OF PANCREAS MITOCHONDRIA

7)
8)
9)

10)
11)

12)

13)

14)
15)
16)

17)
18)
19)

20)
21)
22)
23)
24)
25)
26)

27)
28)
29)
30)
31)
32)

Estabrook, R.W.: Mitochondrial respiratory control and the polarographic measurement of ADP: 0 ratios.
Methods Enzymol., 10,41-47 (1967).
Wakabayashi, T., Asano, M. and Kawamoto, S.: Induction of megamitochondria in the mouse liver by isonicotinic acid derivatives. Exp. Mol. Pathol., 31, 387-399 (1979).
Wakabayashi, T., Horiuchi, M., Sakaguchi, M., Onda, H. and Misawa, K.: Induction of megamitochondria inthe mouse and rat livers by hydrazine. Exp. Mol. Pathol., 39, 139-153 (1983).
Fiske, CH. and Subbarow, Y: The colorimetric determination of phosphorus. J. BioI. Chern., 66, 375-400(1925).
Hayakawa, T, Kondo, T., Yamazaki, Y., Iinuma, Y. and Mizuno, R.: A simple and specific determination oftrypsin in human duodenal juice. Gastroenterologia Japonica, 15, 135-139 (1980).
Tamiya-Koizumi, K., Vmekawa, H., Yoshida, S., Ishihara, H. and Kojima, K.: A novel phospholipase A 2 associated with nuclear matrix: stimulation of the activity and modulation of the Ca2+ dependency by polyphosphoinositides. Biochim. Biophys. Acta., 1002, 182-188 (1989).
McElory, W.D. and DeLuca, M.: Bioluminescence and chemiluminescence. Basic chemistry an analytical applications. In The chemistry and analytical applications of firefly luminescence. edited by McElroy, W.O. and
DeLuca, M.A., pp. 179-186 (1981), Academic Press, New York.
Takagi, S., Mizuno, N., Adachi, K. and Wakabayashi, T: Effects of demethylchlortetracycline phototoxicity
on the structure and functions of rat liver mitochondria. Photochem. Photobiol., 51, 175-183 (1990).
Williams, J.N., Jr.: A method for the simultaneous quantitative estimation of cytochromes a, b, c, and c inmitochondria. Arch. Biochem. Biophys., 107,537-543 (1964).
Wakabayashi, T., Horiuchi, M., Sakaguchi, M., Misawa, K., Onda, H., lijima, M. and Allmann, D.W.: Mechanism of hepatic megamitochondrial formation by ammonia derivatives. Correlation between structure ofchemicals and their ability to induce the formation of megamitochondria. Eur. J. Biochem., 143, 455-465(1984).
Wakabayashi, T and Green, D.E.: On the mechanism of cuprizone-induced formation of megamitochondria
in mouse liver. Bioenergetics, 6,179-192 (1974).
Tykka, H., Mahlberg, K., Pantzar, P. and Tallberg, T.: Phospholipase A 2 inhibitors and their possible clinicaluse in the treatment of acute pancreatitis. Scand. J. Gastroent., 15,519-528 (1980).
Rinderknecht, H.: Pancreatic secretory enzymes. In The exocrine pancreas: biology, pathology, and diseases.
edited by Go, V.L.W., Books, F.P., DiMagno, E.P., Gardner, J.D., Lebenthal, E. and Scheele, G.A, pp.
163-183 (1986), Ravan Press, New York.
Miller, R.N. and Hunter E.F.JR.: The effect of halothane on electron transport-oxidative phosphorylationand swelling in rat liver mitochondria. Mol. Pharmacol., 6, 67-77 (1970).
Harris, R.A, Munroe, J., Farmer, 8., Kim, K.C and Jenkins, P.: Action of halothane upon mitochondrial
respiration. Arch. Biochem. Biophys., 142,435-444 (1971).
Jain, M.K., Wu, N.Y. and Wray, L.V.: Drug-induced phase change in bilayer as possible mode of action of
membrane expanding drugs. Nature, 225, 494-496 (1975).
Tsong, T.Y, Greenberg, M. and Kanehisa, M.I.: Anesthetic action on membrane lipids. Biochemistry, 16,3115-3121 (1977).
Vanderkooi, J.M., Landesberg, R., Selick. H. and McDonald, G.C: Interaction of general anesthetics withphospholipid vesicles and biological membranes. Biochim. Biophys. Acta., 464,1-16 (1977).
Trudell, JR.: Biophysical concept in molecular mechanism of anesthesia. In Molecular Mechanism of Anesthesia. Progress in Anesthesiology vol.2. edited by Fink, B.R., pp. 267-270 (1980) Raven Press, New York.
Vanderkooi, G., Shaw, J., Storm, S.C, Vennerstrom, R. and Chignell, D.: On the mechanism of action of anesthetics. Direct inhibition of mitochondrial F-ATPase by n-butanol and tetracaine. Biochim. Biophys. Acta,
635,200-203 (1981).
Dluzewski, A.R., Halsey, M.J. and Simmonds, AC: Membrane interactions with general and local anesthetics: A review of molecular hypothesis of anesthesia. Molec. Aspects Med., 6, 459-573 (1983).
Veda, I. and Kawaya, H.: Molecular mechanisms of anesthesia. Anesth. Analg., 63,929-945 (1984).
Meldolesi, 1., Jamieson, J.D. and Palade, G.E.: Composition of the cellular membranes in the pancreas of theguinea pig. I. Isolation of membrane fractions. J. Cell BioI., 40, 109-129 (1971).
Black, D.A.K. and Lumb, G.A.: Chemical composition of the alimentary tract. In Biochemists' Handbook.
edited by Long, C, pp. 675-676 (1961) Richard Clay and Company, Suffolk, England.
Kosterlitz, H.W.: Chemical composition of the liver. In Biochemists' Handbook. edited by Long, C., pp.
675-676 (1961) Richard Clay and Company, Suffolk, England.
Sardesai, V.M. and Provido, H.S.: The presence of a substance in the pancreas that inhibits mitochondrial
respiration. 1. Surgical. Res., 35, 176-179 (1983).