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THESIS

 

 

This is to certify that the

thesis entitled

MITOCHONDRIAL CREATINE KINASE IN NORMAL AND

DYSTROPHIC CHICKEN SKELETAL MUSCLE: FUNCTION

IN THE CREATINE PHOSPHATE SHUTTLE, PURIFICATION
AND EXPRESSION IN MUSCLE CELL CULTURES

presented by

VICKI E DEE BENNETT

has been accepted towards fulfillment
of the requirements for

PH.D. BIOCHEMISTRY

degree in

 

 

Major professcr

Date ogiCL/Wtflau V7; Wigs}?

 

0-7639 MSU is an Affirmative Action/Equal Opportunity lnstitulimr

 

RETURNING MATERIALS:
Place in book drop to
remove this checkout from
your record. FINES will
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MITOCHONDRIAL CREATINE KINASE IN NORMAL AND DYSTROPHIC CHICKEN
SKELETAL MUSCLE: FUNCTION IN THE CREATINE PHOSPHATE SHUTTLE,
PURIFICATION AND EXPRESSION IN MUSCLE CELL CULTURES

By

Vickie Dee Bennett

A DISSERTATION

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

DOCTOR OF PHILOSOPHY

Department of Biochemistry

1983

ABSTRACT

MITOCHONDRIAL CREATINE KINASE IN NORMAL AND DYSTROPHIC CHICKEN
SKELETAL MUSCLE: FUNCTION IN THE CREATINE PHOSPHATE SHUTTLE,
PURIFICATION AND EXPRESSION IN MUSCLE CELL CULTURES

By

Vickie Dee Bennett

A study of the levels of mitochondrial creatine kinase (mt-CK)
activity in muscle mitochondria from normal and dystrophic chickens
reveals decreases in mt-CK activity in dystrOphic chicken breast and
heart muscle compared to normal muscle. Oxygen consumption measurements
in the presence of creatine, creatine phosphate production rates and the
maximal rates of ATP synthesis in normal and dystrOphic chicken breast
and heart muscle mitochondria all suggest low levels of mt-CK activity
in dystrophic breast muscle cause a loss of creatine regulation in the
creatine phosphate shuttle. The abnormal function of the creatine
phosphate shuttle may then explain the loss of function of the white
fiber muscle in the dystrophic condition.

Since future studies of mt-CK in normal and dystrophic muscle

require homogeneous mt-CK, the development of a purification procedure

 

 

for mt-CK from chicken breast muscle is necessary. A new purification

procedure involves isolation of mitochondria, mitoplast preparation,
phosphate solubilization of mt-CK from mitoplasts, Procion Red-agarose
chromatography and agarose-hexane-ATP affinity chromatography and yields
two distinct forms of mt-CK. Cellulose-acetate electrophoresis shows a
fast-moving cathodal form purified to 90% of homogeneity with a specific
activity of 272 IU/mg and a slow—moving cathodal form only 40-50% pure
with a specific activity of 123 IU/mg.

Primary muscle cell cultures incubated at 37°C in serum-containing
medium do not contain mt-CK activities detectable by cellulose acetate
electrophoresis. Thus, improvements in available muscle cell culture
techniques to achieve cultures expressing detectable activities of mt-CK
also became necessary. Increases in the incubation temperature of
muscle cultures to 41°C, chicken body temperature, result in higher
creatine kinase (CK) specific activity and a greater percentage of the
muscle specific CK isozyme (MM-CK); mt-CK activity is not detected at
41°C. However, the use of a chemically defined muscle cell culture
medium containing insulin, conalbumin, fibroblast growth factor,
fibronectin and retinoic acid in muscle cell cultures incubated at 41°C
results in the expression of 9% of the total CK activity as mt-CK.

These improvements in muscle cell culture techniques should provide a

basic system for future studies of mt—CK in culture.

 

To Howard

with all my love

for his constant love, devotion and encouragement

and

To
MOm and Dad
for their love, guidance, encouragement and support

throughout my life and educational years

ACKNOWLEDGEMENTS

I would like to express my sincere gratitude to Dr. Clarence Suelter
for his guidance, advice, patience and good humor throughout my entire
graduate career.

I would like to thank all the past and present members of my
graduate committee: Dr. Pamela Fraker, Dr. Arnold Revzin, Dr. William
Hells, Dr. James Asher and Dr. Ronald Young for their faithful service.

I would like to thank Dr. Marlene DeLuca and Dr. Gordon Sato from
the University of California, San Diego for the opportunity to work on
aspects of this project in their respective laboratories during
Dr. Suelter's six month sabbatical in La Jolla, California. In
addition, I would like to extend special thanks to Dr. Norman Hall and
Dr. Donald McClure for acquainting me with laboratory procedures and
techniques in these laboratories and for many stimulating discussions.
I would like to extend warmest wishes and friendship to Ms. Brigitte
Feucht who shared her home with me during my stay in La Jolla.

Many thanks are also in order for individuals in the laboratory:
Dave Husic, Tom Carlson, Jeff Baxter, Stephen Brooks and Peter Toth and
to the friends in the laboratory next door: Dr. Shelagh
Ferguson-Miller, Debra Thompson, Maria Suarez and Jerome Hochman with
whom I have shared stimulating discussions, many extracurricular

activities and good friendships. Also, I would like to thank Kim

 

Hildebrandt for her friendship and encouragement as a friend in the

department and as a roommate.
Finally, many thanks to Theresa Fillwock for the hard work and

patience necessary for the typing of this dissertation.

TABLE OF CONTENTS

Page
LIST OF TABLES. . . . . . . . . . . . . . . . . . ...... . viii
LIST OF FIGURES . . . ...... . . . . . . . . ....... ix
LIST OF ABBREVIATIONS . ........... . . . . ..... xi
CHAPTER 1. LITERATURE REVIEW . . . . . . . . ......... 1
Human Muscular Dystrophies . . . . . . . . ........ 1
Muscular Dystrophy of the Chicken. . . . . . . ...... 6
Creatine Kinase Isozymes . . . . . . . . . ....... 8
Creatine Kinase Isozymes During Muscle Devel0pment
Ifl_Vivo ........................ 12
Creatine Kinase Isozymes During Muscle Development
In Vitro. . . . . . . . . . . . . . . 13
Role of Creatine, Creatine Phosphate and Creatine
Kinase Isozymes in Muscle Energy Metabolism ...... 16
Statement of the Problem . . . . . . . . . . ....... 20
List of References . . . . . . . . . . . . ........ 21
CHAPTER II. DECREASED MITOCHONDRIAL CREATINE KIANSE ACTIVITY
ALTERS THE NORMAL FUNCTION OF THE CREATINE PHOSPHATE
SHUTTLE IN DYSTROPHIC CHICKEN BREAST MUSCLE ........ 32
Introduction . . . . . . . . . . . . . . . . ...... 33
Materials and Methods ..... . . . . . . . . . . . . . . 35
Materials . . . . . . . . . ..... 35

Isolation of Breast and Leg Muscle Mitochondria . . . . 35
Isolation of Heart Mitochondria . . . . . . . . . . . . 36

Enzyme Assays and Protein Determination . . ...... 37
Cellulose Acetate Electrophoresis ........... 37
Determination of Mitochondrial Creatine Kinase Levels . 38
Mitochondrial Oxygen Consumption. . . . . . ...... 38
Creatine Phosphate Assays ..... . . . . . ..... 39
Determination of the Rate of the Maximal

Mitochondrial ATP Synthesis. . . . ........ . 4O
Mitoplast Preparation . . . . . . ......... 41

Effect of Phosphate on the Interaction of
Mitochondrial Creatine Kinase with Mitoplasts. . . . 42
Results. . . . . . . . . . . . . . . . . . . . . . . . 43
Levels of Mitochondrial Creatine Kinase in Normal

and Dystrophic Muscles ............... 43
Mitochondrial Respiration in the Presence and
Absence of Creatine ....... . ......... 46

Page

Creatine Phosphate Production in Respiring

Mitochondria ....... . . . 58
Maximal ATP Synthesis Rates in Normal and Dystrophic
Breast Muscle Mitochondria .......... . . 61

Effect of Inorganic Phosphate on the Interaction of
Mitochondrial Creatine Kinase with Normal and

Dystrophic Breast Muscle Mitoplasts ......... 63
Discussion ........................ 67
References ............. . ........ . . 78

CHAPTER III. PURIFICATION OF TWO ELECTROPHORETICALLY DISTINCT
MITOCHONDRIAL CREATINE KINASES FROM CHICKEN BREAST MUSCLE

USING DYE-LIGAND AND ATP—AFFINITY CHROMATOGRAPHY ..... 81
Introduction . . .................. . . . 82
Materials and Methods ................... 84
Materials ....................... 84
Enzyme Assays and Protein Determination ........ 84
Cellulose Acetate Electrophoresis ........... 85
Polyacrylamide Gel Electrophoresis ........... 85
Purification Procedure .................. 87
Isolation of Mitochondria ............... 87
Mitoplast Preparation ................. 90
Solubilization of Mitochondrial Creatine Kinase . . . . 90
Procion Red-Agarose Chromatography ........... 92
Agarose-Hexane-ATP Affinity Chromatography ....... 92
Concentration and Storage of the Enzyme ........ 97
Discussion ........................ 100
Phosphate Solubilization of Mitochondrial Creatine
Kinase . ...................... 100
Procion Red-Agarose Chromatography ........... 100
Agarose—Hexane-ATP Affinity Chromatography ....... 102
Purity and Molecular Weight Analysis of the Two
Forms of Mitochondrial Creatine Kinase ....... 102
Partial Hydrophobic Nature of Mitochondrial Creatine
Kinase . ...................... 105
Aggregation Phenomenon ................ . 106
Behavior of the Two Forms of Mitochondrial Creatine
Kinase in 50% Glycerol ............... 106
Estimation of the Amount of Mitochondrial Creatine
Kinase in Chicken Breast Muscle ........... 107
References ........................ 109

CHAPTER IV. MUSCLE CELL CULTURES FROM CHICKEN BREAST MUSCLE
HAVE INCREASED ACTIVITIES OF ADULT MUSCLE ENZYMES WHEN

INCUBATED AT 41°C COMPARED TO 37°C ............ 112
Introduction . ...................... 113
Materials and Methods ................... 115
Preparation of Primary Muscle Cell Cultures ...... 115
Preparation of Chick Embryo Skin Fibroblast Cultures. . 116
Preparation of Cell Culture Homogenates ........ 116

vi

Page
Evalution of Cell Fusion ................ 117
Preparation of Crude Muscle Extracts ...... . . . . 117
Enzyme Assays and Protein Determination ........ 118
Cellulose Acetate Electrophoresis ........... 118
Results and Discussion .................. 119
References ........................ 134
CHAPTER V. DEVELOPMENT OF A CHEMICALLY DEFINED MEDIUM FOR
PRIMARY CHICK MUSCLE CELL CULTURES ............ 137
Introduction ....................... 138
Materials and Methods. .................. 141
Media Preparation ................... 141
Preparation of Primary Muscle Cell Cultures in
Chemically Defined Medium .............. 141
Preparation of Cell Culture Homogenates ........ 142
Evaluation of Cell Fusion ............... 143
Enzyme Assays and Protein Determination ........ 143
Cellulose Acetate Electrophoresis ........... 143
Results and Discussion .................. 145
Effect of Defined Medium for Muscle and Nerve Cells
(DMN) on Primary Chick Muscle Cell Cultures ..... 145
Effects of Other Additives to DMN .......... . 146
Replacement of Human Transferrin with Conalbumin
in DMN ...................... . 147

Effect of 1 uM Retinoic Acid on Muscle Cell
Cultures Grown in Chemically Defined Medium. . . . . 149
References ........................ 152

SUMMARY ........................... . 154

vii

LIST OF TABLES

CHAPTER I.
Table I. Muscular Dystrophies. . . . . . . . . . . . . . . 2
CHAPTER II.

Table I. Maximal Rates of ATP Synthesis in Mitochondria
From Normal and Dystrophic Muscle ....... . 62

CHAPTER III.

Table I. Purification of Mitochondrial Creatine Kinase
From Chicken Breast Muscle ........... . 91

CHAPTER IV.

Table I. Isozyme Distribution During Time Course in
Cultures Incubated at 37°C or 41°C ..... . . . 128

CHAPTER V.

Table 1. Fusion Levels, Creatine Kinase Specific
Activity and Creatine Kinase Isozyme
Distribution in Seven-Day Muscle Cell
Cultures Incubated in Various Media Types
at 37°C and 41°C ..... . ........... 148

viii

CHAPTER I.
Figure
CHAPTER II.

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

Figure

CHAPTER III.

Figure

Figure

Figure

LIST OF FIGURES

1. Creatine phosphate shuttle mechanism .......

1. Levels of mitochondrial creatine kinase as a

N

function of age for normal and dystrophic
breast (B), leg (L), and heart (H) muscle . . . .

. Levels of mitochondrial creatine kinase as a

function of age for normal and dystrophic
breast and heart muscle ....... . . . .

. Oxygen consumption traces for normal and.

dystrOphic breast muscle mitochondria ......

. Effect of phosphate concentration on the rates of

oxygen consumption by normal and dystrophic
breast muscle mitochondria from chickens 12-20
days ex ovo . . . . . ..... .

. Effect of phosphate concentration on the rates

of oxygen consumption by normal and dystrophic
breast muscle mitochondria from chickens 5 days
ex ovo. ................

. Effect of phosphate concentration on the rates

of oxygen consumption of normal and dystrophic
heart mitochondria. ..............

. Creatine phosphate synthesis in normal and

dystrophic breast and heart muscle mitochondria .
Effect of phosphate on the solubilization of
mitochondrial creatine kinase from normal and
dystrophic breast muscle mitoplasts ......

. Creatine phosphate shuttle mechanism. ......

. Cellulose acetate electrophoresis of creatine

kinase isozymes at various stages in the
purification. . ....... . ........

. Elution profile of creatine kinase activity

and protein during Procion Red- -agarose
Chromatography. O O O O O O O O O 0 O O O

. Elution profile of creatine kinase activity

and protein during agarose- -hexane- ATP
affinity chromatography .............

ix

Page

18

44

47

49

52

54

56
59

64
69

88

93

95

Figure

Figure

CHAPTERIV.

Figure

Figure

Figure

2.

 

Page

Cellulose acetate electrOphoresis of the

two forms of mitochondrial creatine kinase

after agarose-hexane-ATP affinity

chromatography. . . . . . . . . . . . . . . . . 98
SDS-polyacrylamide gel of Pool I and Pool

II mt-CK from agarose-hexane-ATP affinity
chromatography. ................ 103

Creatine kinase specific activities, percent

nuclei in myotubes and total protein per

plate from a representative 13-day time

course experiment for muscle cell cultures

incubated at 37°C and 41°C. . . . . . . . . . . 120
The average fold increases of CK specific

activity in cultures incubated at 41°C

compared to 37°C from three identical

13-day time course. . . . . . . . . . ..... 123
Densitometer scans of creatine kinase

isozymes separated by cellullose acetate
electrOphoresis . . . . . . . . . . . . . . . . 126

 

AMP

AMPDA

ATP

BB-CK

BSA

BSS

CK

Cr

Creatine-P (CrP)
DMD

DME

DMN

DNA

EDTA

EGF

EMEM

F12

Glucose—6-P (Glc-6-P)
HDL

HEPES

LIST OF ABBREVIATIONS

Adenosine 5'-diphosphate

Adenosine 5'-monophosphate

AMP deaminase

Adenosine 5'-triphosphate

Brain type creatine kinase

Bovine serum albumin

Buffered salt solution

Creatine kinase

Creatine

Creatine phosphate

Duchenne muscular dystrophy
Dulbecco's Modified Eagle's medium
Defined medium for muscle and nerve cells
Deoxyribonucleic acid
Ethylenediaminetetraacetic acid
Epidermal growth factor

Eagle's minimal essential medium
Ham's Nutrient Mixture F-12
Glucose—6-Phosphate

High density lipoprotein
N-2-hydroxyethylpiperizine-N'-2-ethanesulfonic

acid

xi

HK
MB-CK
MES
MM-CK
mRNA
mt-CK
NADP
PBS
P1
RCR
SDS
SDS-PAGE

SuccinateleT Reductase

TRIS

 

Hexokinase

Hybrid creatine kinase

2(N-morpholino)ethanesulfonic acid

Muscle type creatine kinase

Messenger RNA (ribonucleic acid)

Mitochondrial Creatine kinase

Nicotinamide adenine dinucleotide phosphate

Phosphate buffered saline

Inorganic phosphate

Respiratory control ratio

Sodium dodecyl sulfate

Sodium dodecyl sulfate-polyacrylamide gel
electrophoresis

Succinate:p-iodonitrotetrazolium reductase

Tris(hydroxymethyl)aminomethane

 

CHAPTER I

LITERATURE REVIEW

Human Muscular Dystrophies

The muscular dystrophies represent a heterogeneous group of
genetically determined disorders characterized by muscle dysfunction
and degeneration (1,2). The clinical manifestations of these muscular
disorders differ in age of onset, severity, affected muscles and the
type of inheritance pattern involved (3). Table I (2) reviews the
major human muscular dystrophies for comparison of their clinical
characteristics. Two other types of human muscular dystrophies, not
shown in Table I, include myasthenia gravis and the various myopathies.
The general causes of these two types of muscular dystrophy are known.
Myasthenia gravis results from an autoimmune attack against
acetylcholine receptors in the neuromuscular junction. Antibody
binding to these receptors effectively reduces the number of available
receptors for neuromuscular transmission and thus causes muscle
weakness and fatigue (4). The muscular myopathies appear as disorders
of glycogen metabolism or glycolysis resulting from deficiencies of
various enzymes involved in these processes (5). The primary defects
of the other major muscular dystrophies remain elusive, even after

almost thirty years of biochemical study (6).

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Duchenne muscular dystrophy (DMD), the most studied and debilitat-
ing of the muscular dystrophies, is a sex-linked recessive disease (7)
affecting the white-fiber muscle of male children. The incidence of
DMD is approximately 1 out of 4800 male births (8) with an estimated
spontaneous mutation rate of 10.5 x 10'5 genes per generation (9).

The clinical, genetic, pathological and biochemical homogeneity of DM0
allows for accurate diagnosis and prediction of the clinical symptoms
as the disease progresses (10). Shortly after birth, symptoms appear
as a difficulty in sitting, standing, or walking. Progressive muscle
weakness soon affects the arms, as well as the legs, and confines the
child to a wheelchair by nine to ten years of age. Muscle hypertrophy
also characterizes the early symptoms of DMD. Progressive motor
impairment eventually leads to respiratory difficulties resulting in
death around the age of twenty. Diagnosis of the disease generally
involves observation of elevated serum levels of various muscle enzymes
and the histological appearance of muscle biopsies.

Histochemical examinations of DMD muscle reveal: 1) large
hyalinized fibers, 2) small muscle fiber groups at the same stage of
regeneration and degeneration, 3) proliferation of endomysial and peri-
mysial connective tissue, 4) an increase in intracellular and extra-
cellular lipid, 5) fiber splitting and disorganization, 6) variation of
fiber size, 7) internalization and proliferation of nuclei, 8)
myofibrillar necrosis and phagocytosis and 9) the appearance of fat
cells between the disintegrating fibers to produce the characteristic
hypertrophy (10,11).

Several major theories currently exist to explain the basic cause

of muscle degeneration apparent from these histological

characteristics. The three most prominent theories include: 1) an
abnormal vascular supply to muscle, 2) abnormal nerve-muscle
interactions or 3) a genetic mutation leading to leaky membranes (12).
Two of these, the vascular and neurogenic theories, are losing support
even though some prominent investigators still support them (12,13).
The membrane defect theory, however, remains p0pular (For review, see
6).

Biochemical evidence in support of the membrane theory began with
the observation of increased aldolase activity in the serum of boys
with DMD (14,15,16). Subsequent studies revealed elevated serum levels
of several enzymes such as creatine kinase (17) and pyruvate kinase
(17,18,19). (See 20 for a more complete list of enzymes elevated in
the serum of DMD patients.) Generally, these elevated serum activities
are due to the muscle (M) isozymic form of the enzyme (21) as is the
case for aldolase (22,23), lactate dehydrogenase (24,25,26) and
creatine kinase (21,27,28,29).

Serum creatine kinase activities are the most popular of these
enzymes as diagnostic tests of muscular disease for several reasons.
First, this activity is more dramatically elevated than the other
muscle enzymes in the serum of DMD patients (30,31). Second, serum
creatine kinase is increased at birth before the onset of muscular
weakness (32,33,34) and is elevated in some affected fetuses
(35,36,37). Third, elevated serum creatine kinase activities occur in
as many as 80% of the female carriers of DMD even though these women
are usually asymptomatic and have little necrosis of muscle fibers

(38,39).

 

The increased serum activities of creatine kinase and other muscle

enzymes eventually accompanies a decrease of the activities of these
enzymes in the muscle (40,41). The membrane defect theory of muscular
dystrophy suggests this phenomenon may be the result of enzyme leakage
from muscle (6,41,42). Furthermore, isozyme patterns of creatine kinase
(43,44,45), lactate dehydrogenase (44,46), aldolase (47), acetylcho—
linesterase (48) and hexokinase (49) from adult dystrophic muscle
contain isozymes normally absent from muscle after embryonic develop-
ment, even though the adult muscle isozyme still predominates. These
observations led to the hypothesis that DMD may be a developmental
deficiency resulting in incomplete differentiation of the muscle
(43,50). An alternative hypothesis suggests the presence of embryonic
isozymes may result from the high number of regenerating muscle fibers
in DMD muscle (11).

Despite thirty years of biochemical studies, the primary genetic
lesion of muscular dystrophy remains elusive. However, comparative
biochemical studies have produced some negative clues. For instance,
comparative studies of carbohydrate and lipid metabolism reveal no
evidence for abnormal storage of glycogen, lipid or polysaccharides
(6). In addition, no definite abnormality appears in the muscle
contractile proteins; such as myosin (51), actomyosin (52,53), troponin
or tropomyosin (54,55). Differences in several other enzyme activities
and metabolite levels are not extreme and thus probably represent
secondary effects of the primary lesion. For example, the levels of
adenylate kinase (56), AMP aminohydrolase (57,58) and the enzymes of
glycogenolysis and glycolysis (56,59,60,61) are decreased in DMD muscle

compared to normal muscle; Krebs cycle enzymes (56,59) and lysosomal

enzyme activities (62,63,64) are increased. Metabolite differences
include an increase in muscle lipids (65,66,67) and decreases in muscle
creatine phOSphate (68,69) with increased excretion of creatine and
decreased excretion of creatinine (12). Discussion of the possible
ramifications of the abnormalities in these creatine metabolites will
follow in later paragraphs of this review and throughout this
dissertation.

Muscular Dystrophy of the Chicken

 

The limited availability of large amounts of human dystrophic
tissue favors the use of animals for the study of the muscular
dystrophies. Inherited forms of muscular dystrophy exist in the mouse
(70), chicken (71), hamster (72), mink (73), sheep (74), duck (75), and
turkey (76). Muscular dystrOphy in the chicken appears as an autosomal
recessive trait and affects primarily the white-fiber muscle (77). The
gross and microscopic structural alterations characteristic of
homozygous dystrophic chickens also appear, in milder forms and later
in life, in chickens heterozygous for the dystrophic trait (78).

Our laboratory presently uses normal and dystrophic chickens (line
412 and 413, respectively) as our animal model. Several other
dystrOphic chicken lines are also available (79). Characteristics of
the 413 line include an inability of the chicken to right itself from a
supine position, early onset dystrophy, early hypertrophy of the
pectoral muscle, low fat content and a normal life span (79). The
righting ability test distinguishes between normal line 412 and
dystrophic line 413 birds at approximately ten days ex 919 (79).

The use of the chicken as an animal model for the study of

muscular dystrOphy has several advantages (80,81). First, the large

quantities of muscle tissue necessary for biochemical investigations
are more readily available from chickens than from humans. Second, the
large pectoralis muscle, composed almost entirely of white, glycolytic
muscle fibers, allows convenient biochemical studies on a near
homogeneous tissue. Third, the availability of fertile eggs with a
predicted phenotype allows study of the embryonic development of
muscular dystrophy. Finally, muscular dystrophy of the chicken
exhibits many characteristics similar to those of human muscular
dystrophies (82).

Major similarities of dystrophic chicken muscle to DMD muscle
include histologic signs of muscle fiber degeneration, high serum
activities of several muscle enzymes and abnormal muscle isozyme
patterns (79). Histochemical examinations reveal the following
characteristics similar to those in DMD muscle: 1) evidence of
regenerating muscle fibers, 2) lipid accumulation between muscle
fibers, 3) fiber splitting, 4) heterogeneity of fiber size, 5)
internalization and proliferation of nuclei and 6) myofibrillar
necrosis and phagocytosis (83,84). Unique characteristics of chicken
muscular dystrophy include enlargement of the sarcotubular system (79)
and vacuolization within muscle fibers (85).

In addition, activities of several muscle enzymes are elevated in
the serum of the dystrophic chicken as in dystrophic humans. These
include aldolase (86), creatine kinase (87~92), pyruvate kinase
(88,92,93) and acetylcholinesterase (90,94). Heterozygous carriers of
the dystrophic trait also exhibit elevated serum activities of creatine

kinase (87) and pyruvate kinase (93).

 

Finally, the abnormal enzyme activities and isozyme patterns of
dystrophic chicken muscle appear similar to the characteristic patterns
of DMD muscle. For example, dystrophic chicken muscle contains
increased activities of lysosomal enzymes (95-98) and Krebs cycle
enzymes (85,99). Other enzymes elevated in activity in dystrophic
chicken muscle include hexose monophosphate shunt enzymes (100,101),
acetylcholinesterase (79,102,103) and enzymes involved in cyclic AMP
metabolism (104,105) and purine metabolism (106). Decreases in enzyme
activities of several enzymes such as creatine kinase (71,88,107),
lactate dehydrogenase (108), AMP aminohydrolase (106,109-111), aldolase
(85,112) and other glycolytic enzymes (79,95,88,100, 111,113-115) appear
in dystrophic chicken muscle and agree with observations from DMD
muscle. The isozyme patterns of some of these enzymes reveal isozymes
present only during normal embryonic development. For example, elevated
levels of embryonic forms of creatine kinase (88,107) and lactate
dehydrogenase (108) exist in adult dystrophic muscle. 0n the other
hand, pyruvate kinase (93) and aldolase (112) isozyme profiles of adult
dystrophic muscle appear normal.

Creatine Kinase Isozymes

Creatine kinase (CK; EC.2.7.3.2.) consistently appears in the serum
of both humans and chickens with muscular dystrophy, as well as carriers
of the disease. Generally, this serum CK activity is due to the muscle
(M) isozymic form. Isozyme profiles of adult dystrophic muscle also
reveal isozymes generally present only during embryonic development of
the muscle. These observations have generated interest in creatine
kinase isozymes and their role in the dystrophic process. In turn, this

interest has provided useful clinical applications for diagnosis

 

 

of muscle disease and for carrier identification of these disease

states.

Creatine kinase occurs in several isozymic forms in a variety of
tissues (For review, see 116). The major CK isozymes appear as three
cytoplasmic forms (117,118) and at least one mitochondrial form (119).
In addition, several electrophoretic variants of these forms exist (For
reviews, see 120 and 121).

The literature contains numerous structural, physical, biochemical
and immunological studies of the cytoplasmic CK isozymes; extensive
kinetic and mechanistic studies are also available (For extensive
reviews, see 120 and 122). Briefly, the three cytoplasmic isozymes
exist as dimers composed of two different subunits, the M (muscle) and
B (brain) subunits. These subunits dimerize to form MM-CK (or CK-3),
BB—CK (CK-1) and one hybrid MB-CK (CK-2) (123,124). Eppenberger,
Dawson and Kaplan purified and characterized all three forms from
chicken breast muscle and chicken heart (124-126). Chicken MM-CK has a
molecular weight of 83,700, with subunit molecular weights of 42,000
(125). For comparison, the molecular weight of rabbit MM—CK is 82,600
(127) and values for other purified creatine kinases fall within the
range of 78,500-85,100 (120). The muscle and brain isozymes differ
considerably in amino acid composition, primary amino acid sequence
based on tryptic and peptide map data, and immunological characteris-
tics (124). Immunologically, MM-CK and BB-CK completely lack cross-
reactivity while MB—CK weakly reacts with antibody to either MM-CK or
BB-CK. In addition, catalytic differences also exist between the
muscle and brain isozymes; brain isozymes have lower Krn values for

ADP and creatine phosphate in the forward reaction and a 2-fold lower

- "cfi'a'm' ‘-

W. iinxyruh-"L'vfii u: ." *4. ~'

   

3.. ha..-

 

 

10

Km for creatine in the reverse reaction than muscle isozymes (124).
However, several similarities also exist. For example, modification of
a single reactive sulfhydryl group per subunit generally leads to
complete inactivation of any CK isozyme; this sulfhydryl group is not
essential for catalysis, but may be involved in a conformational change
important to catalysis (For review, see 122). Furthermore, structural,
stereochemical and kinetic evidence, consistent with the data from all
three CK isozymes, points to an ordered addition mechanism for catalysis
(128) involving a direct, in-line transfer of the phosphoryl group
between bound substrates; no phosphorylated enzyme intermediate appears
to exist (129-131).

In addition to the three cytoplasmic isozymes, a mitochondrial
isozyme (mt-CK) also exists in some tissues. Jacobs gt__l, (119) first
reported a CK isozyme associated with mitochondria isolated from rat
heart and brain tissues. This isozyme moves toward the cathode at pH
8.8 during electrOphoresis, whereas the cytoplasmic forms move toward
the anode (132). Significant amounts of mt-CK appear in human, beef and
rat heart mitochondria as well as rat brain, skeletal muscle and
intestinal muscle mitochondria; nominal amounts appear in rat and rabbit
liver, kidney and testis mitochondria (133).

Soon after the identification of a mitochondrially associated
creatine kinase, a controversy began concerning the submitochondrial
localization of this isozyme. First reports identified the location of
mt-CK in the intermembrane space (134,135). However, most
investigators now believe mt-CK is associated with the outer surface of
the inner mitochondrial membrane in all tissues containing the isozyme

(133,136-139).

 

The exact nature of the association of mt-CK with the inner mito-
chondrial membrane remains unclear at present. Jacobus and Lehninger
(133) suggest mt-CK is loosely bound to the membrane because release of
mt-CK in soluble form occurs during incubation of the mitochondria with
isotonic sodium phosphate or sodium acetate. Vial gt_al. (139) suggest
the association is strongly phosphate dependent and is a reversible
phenomenon. Hall and DeLuca support this view since phosphate-
extracted heart and liver mitochondria specifically rebind mt-CK under
low phosphate conditions (140). In addition, Font gt_al. (141) suggest
mitochondrial swelling is a prerequisite, but not a sufficient
condition, for the solubilization of mt-CK; sufficient ionic strength,
or molecules such as negatively charged organomercurials, to induce
conformational changes in the enzyme (141,142) also seem necessary.

Purification procedures for mt-CK from beef heart (143,144) and
dog heart (145) take advantage of the phosphate-dependent solubiliza-
tion. Thus, mt~CK is first released from mitochondria with 100 mM
sodium phosphate and then separated from MM-CK, the major cytoplasmic
isozyme in mammalian hearts, and other proteins by anion exchange
chromatography, gel filtration chromatography and agarose—hexane—ATP
affinity chromatography (144,145). Hall et_al. (143) report purifica-
tion of two homogeneous interconvertible forms with a specific activity
of 111 units/mg from beef heart. During or after concentrating, the m-l
form converts into the m-2 form. Incubation of the m-2 form in
B-mercaptoethanol then causes the conversion to the m-l form. This
interconversion is consistent with reversible oxidation of protein
sulfhydryl groups (143). Sephacryl S—200 chromatography of these two

forms reveals a molecular weight of 65,000 i 2000 for m-1 and

 

12

180,000—190,000 for m-2, whereas SDS-polyacrylamide gel electrophoresis
(SDS—PAGE) indicates subunit molecular weights of both forms are

35,000 i 4000 (144). SOS—PAGE indicates the subunit molecular weight
of dog heart mt-CK is 42,000 and gel filtration chromatography shows a
molecular weight of 84,000 for the dimer (145).

Both beef and dog heart mt-CK contain different amino acid compo-
sitions from the cytoplasmic CK isozymes (144,145). In addition,
hybridization studies reveal mt-CK will not hybridize with any cyto—
plasmic CK isozyme (144,145). Furthermore, antisera to dog mt-CK does
not cross-react with the dog B or M subunits (145). These observations
suggest mt-CK is biochemically distinct from the cytoplasmic isozymes.

Recently, Perryman gt_al. (146) reported differences in the
cell-free translation products of mitochondrial and M creatine kinase
subunits. Translation of both subunits occur in the cytoplasm from
separate nuclear encoded genes. The primary mitochondrial subunit mRNA
translation product is a polypeptide with a molecular weight 6000
greater than the mature mitochondrial subunit, while translation of the
M subunit mRNA results in a polypeptide with a molecular weight
identical to the mature cytoplasmic M subunit. These results are
consistent with proteolytic processing of a precursor molecule during
translocation of mt-CK from the cytoplasm into the mitochondria (146).
Creatine Kinase Isozymes During Muscle Development In Vivo

The distribution of creatine kinase isozymes not only vary with
the tissue, but also with the stage of development. For example, a
change in cytoplasmic CK isozyme patterns accompanies normal develop-
ment and maturation of vertebrate skeletal muscle and mammalian heart

(118). During normal muscle development, CK activity in embryonic

 

13

muscle is initially due to the BB-CK isozyme. As embryonic development
progresses, total CK activity increases dramatically along with the
appearance of MB-CK and finally MM-CK. MM-CK eventually becomes the
major CK isozyme in adult muscle; greater than 95% is MM-CK, a trace is
MB-CK and BB-CK is not found (147,148).

In addition to the transition from predominantly BB-CK in
embryonic muscle to predominantly MM-CK in adult muscle during normal
muscle devel0pment, mitochondrial CK activity appears shortly after
birth and begins to increase to about 9% of total CK activity by 25
days of age in rabbit and mouse cardiac muscle (149,150). Furthermore,
in sheep heart mt-CK represents 5% of total CK activity 28 days before
birth and increases to the adult level of 15% shortly after birth
(151). Thus, the appearance of heart muscle mt-CK represents a CK
isozyme transition during muscle devel0pment.

In contrast to normal tissues, the CK isozyme profiles of
dystrophic muscle resemble fetal profiles; significant quantities of
BB-CK and MB-CK isozymes are present in adult dystrophic muscle
(43,44,45,88,107 and 152). In addition, mitochondrial CK activity in
dystrophic chicken skeletal muscle decreases with increased age (153).
This decrease in mt-CK activity in dystrOphic adult muscle possibly
correlates with the absence of this isozyme in fetal muscle and thus
again suggests a similarity between dystrOphic adult and normal
embryonic muscle.

Creatine Kinase Isozymes During Muscle Development In Vitro

O

Skeletal myogenesis 1

vivo and in vitro normally involves the

 

proliferation of individual mononucleated myoblasts, spontaneous cell

fusion to form multinucleated myotubes and the appearance of muscle

14

specific proteins such as myosin, acetylcholine receptors and the M
subunit of creatine kinase (154-156). The morphological similarities

of muscle cell culture myogenesis to muscle development j__vivo make

 

muscle cell cultures an attractive experimental system for the analysis
of muscle differentiation in normal and diseased states. Therefore,
extensive studies of these processes using chick, quail, and rat
primary cultures, as well as established myoblast cell lines such as L6
and L8, appear in the literature (For reviews, see 157 and 158).
However, muscle differentiation in yitrg does not entirely reflect

muscle devel0pment i vivo (159). For example, normal myogenic chicken

 

breast muscle cells grown in culture have significantly lower specific
activities of creatine kinase and AMP deaminase than the specific

activities of these enzymes j__vivo (111). In addition, these muscle

 

cell cultures do not complete the isozymic shift seen in yiyg (see
preceding section for discussion of the normal CK isozymic shift in_
3119); significant amounts of MB (20-25%) and BB (10-12%) are still
present at 10 days in_yitrg (147,148). The abundance of the brain CK
subunit (B-CK) still found in normal muscle cultures indicates these

cultures are not as differentiated as adult muscle tissue i vivo.

 

Another problem indicative of primary muscle cell cultures
reflects the heterogeneity of the muscle source; muscle tissue is
composed primarily of fibroblasts and myoblasts (159). Thus, inhibi-
tion of fibroblast proliferation becomes crucial for the production of
myoblast enriched cultures since myoblasts stop dividing and fuse
together to form multinucleated myotubes after three days in culture.
Fibroblasts, on the other hand, continue to divide and eventually

overgrow the cultures. Adding DNA synthesis inhibitors, such as

 

15

fluorodeoxyuridine (162) and cytosine arabinoside (147) to the culture
media on Day 3 prevents this fibroblast overgrowth (161). However, an
exogenous drug may exert unknown side effects on the myotubes.
Alternatively, the achievement of myoblast enriched cultures often
involves various preplating techniques (162,163). These methods take
advantage of a collagen (164,165) or fibronectin (166,167) substratum
requirement for myoblast attachment to tissue culture dishes. However,
both of these methods invariably result in the presence of at least
10-15% mononucleated cells; presumably a combination of fibroblasts and
undifferentiated myoblasts. Researchers tolerate this percentage of
mononucleated cells since the fibroblast contribution to CK activity is
minimal; CK specific activities in control fibroblast cultures repre-
sent 1% of the specific activity in Day 7 muscle cultures with 95% of
the fibroblast CK activity due to BB-CK (147). Even though this
contribution is minimal, a significant number of contaminating fibro-
blasts decreases the overall specific activity of CK and the percentage
of MM-CK in the cultures. However, 10% contamination of fibroblasts
does not account for the 10-20% BB-CK and 20-25% MB-CK present in 10
day muscle cultures. Thus, differentiated myotubes in culture still
produce significant B-CK subunit (147,148,169).

The characteristic decrease in total CK specific activity and the
increased percentage of BB-CK and MB-CK of adult dystrophic muscle may
not be readily apparent in dystrophic muscle cultures since normal
chicken muscle cultures exhibit similar properties. Presently, some
controversy exists as to whether these CK properties characteristic of

the dystrophic process jg_vivo actually occur in dystrophic cultures.

 

For example, Ionasescu gt al. (169) show a 30% decrease in CK specific

16

activity in the cells accompanied by increased CK activity in the
culture medium of dystrophic chicken cultures. Weinstock and Jones
(172) also report an increased CK activity in the culture medium of
dystrophic chicken muscle cultures. In contrast, Young gt_al, (111)
show no differences in CK specific activity of normal and dystrophic
chicken muscle cell cultures. These discrepancies may be due to the
use of different dystrophic chicken lines. Therefore, more highly
differentiated muscle cultures may be necessary for the consistent
expression of dystrophic characteristics in vitrg.

Role of Creatine, Creatine Phosphate and Creatine Kinase Isozymes in

Muscle Energy Metabolism

 

The elucidation of the biochemical and enzymatic steps involved in
the coordination of energy production for muscle contraction has long
been of major interest in the field of muscle metabolism. Over the
years, changes in our understanding of the roles of ATP, creatine,
creatine phosphate and the creatine kinase isozymes in muscle energy
production and utilization have changed our models for this process
(173). The identification of creatine phosphate (172-174) and its role
as a high energy phosphate metabolite (175,176) in 1927 led to the
first model of muscle energy transport for muscle contraction. This
model suggests the hydrolysis of creatine phosphate by creatine kinase
directly supplies the necessary energy for muscle contraction (177).
However, in 1962, Cain and Davies showed the inhibition of muscle
creatine kinase with fluorodinitrobenzene results in a decline of ATP
during muscle contraction while creatine phosphate levels remain
constant (178). These data implicated ATP as the primary energy source

for contraction and shifted creatine phosphate to a secondary energy

17

"reservoir" for maintaining ATP levels during high energy demand. This
model assumes the adenylate energy charge (179) of the cell is the
important regulatory parameter in maintaining proper ATP levels.

Finally, in 1966, Bessman and Fonyo (180) observed a creatine-
regulated respiration in pigeon breast muscle mitochondria and linked
this result to the presence of a mitochondrial creatine kinase isozyme
(119). Around the same time, experiments with heart cells and ischemic
dog hearts revealed heart contraction stops 1 minute after ischemia
induction. At this time, only 20% of the available ATP is depleted,
yet 70% of the creatine phosphate is hydrolyzed; creatine phosphate
correlates with contractility much better than ATP (181-184). The
combination of these experiments led to widespread acceptance of the
creatine-creatine phosphate shuttle mechanism for muscle energy
transport (133,185-188).

The creatine phosphate shuttle, outlined in Figure 1, links
oxidative phosphorylation to muscle contraction and revolves around the
compartmentation of two creatine kinase isozymes, mt-CK and cytoplasmic
CK. The mitochondrial isozyme is bound to the outer surface of the
inner mitochondrial membrane (133,136-139) presumably functionally
coupled to the adenine nucleotide translocase (133,180,189-194). This
bound mt-CK preferentially utilizes the ATP produced during oxidative
phosphorylation to phosphorylate creatine and regenerate ADP. This
regeneration of ADP causes the post-ADP state 4 rate to continue at the
state 3 rate during oxygen consumption measurements. The resulting
creatine phosphate from the mt-CK reaction then diffuses from the
mitochondrial intermembrane space to be utilized by MM-CK, the other

isozyme of skeletal and mammalian heart muscle, to phOSphorylate ADP.

 

18

Figure 1. Creatine phosphate shuttle mechanism as described by

Bessman (188).

 

19

 

ab .6
mo_a€@\ .
n56. / Eco

288623

5

92.
Jon
_nto \ //n_o<

E8325

 

 

20

Since MM-CK binds to myosin, presumably near the myosin head group
(195), the CK produced ATP is preferentially utilized for muscle
contraction (196). Thus, creatine, the end product of muscle contrac-
tion, rather than ADP, serves a regulatory role in the control of
muscle energy production.

Creatine may also regulate muscle protein synthesis and thus
explain the correlation of muscle growth with increased muscular
activity (For review, see 186). Creatine is a unique product of muscle
contraction as a result of the reverse CK reaction, creatine phosphate
+ ADP-EEe>creatine + ATP, needed to produce ATP for muscle
contraction (133,186-188). Therefore, transient increases in creatine
concentration occur during muscle contraction. Several experiments
with heart and skeletal muscle cells in culture suggest creatine
stimulates the synthesis of contractile proteins such as myosin
(197,198), actin (198) and creatine kinase (199,200). In addition,
some investigators demonstrate a stimulation of the rate and extent of
the fusion of cultured skeletal muscle myoblasts into myotubes
(201,202) in the presence of creatine. Thus, creatine may regulate
muscle differentiation and muscle growth by stimulation of
muscle-specific protein synthesis.

Statement of the Problem

 

Mitochondrial creatine kinase (mt—CK) is an important component of
the creatine phosphate shuttle. This dissertation discusses the role
of mt-CK in normal and dystrOphic chicken skeletal muscle. Specifi-
cally, investigation of its function in the creatine phosphate shuttle,
its purification from chicken breast muscle and its expression in

muscle cell cultures appear in the following four chapters.

 

 

 

 

 

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CHAPTER II

Decreased Mitochondrial Creatine Kinase Activity Alters the Function of

the Creatine Phosphate Shuttle in DystrOphic Chicken Breast Muscle

Vickie 0. Bennett, Norman HallT, Marlene DeLuca+

and Clarence H. Suelter

From the Department of Biochemistry, Michigan State University, E.
Lansing, MI 48824 and the +Departments of Medicine and Chemistry,
University of California, San Diego, La Jolla, CA 92093

Running Title: Mitochondrial Creatine Kinase Activity in Dystrophic

Muscle

32

INTRODUCTION

Creatine kinase (adenosine-S' triphosphate creatine
phosphotransferase; EC 2.7.3.2) exists in several isozymic forms.
Eppenberger (I) first reported the existence of three cytoplasmic forms
composed of the M(muscle-type) or B(brain-type) subunits. These
subunits dimerize to form MM, BB and hybrid MB creatine kinase
isozymes. The MM creatine kinase predominates in mature skeletal
muscle of birds and mammals and mammalian myocardium whereas BB
creatine kinase predominates in brain, neural tissue and embryonic
skeletal muscle of mammals and myocardial muscle of birds. Hybrid MB
creatine kinase also appears in mammalian heart and skeletal muscle.
(See (2) for review of localization of various forms). Jacobs (3)
reported the existence of an additional creatine kinase isozyme located
on the outer surface of the inner mitochondrial membrane (4,5). This
mitochondrial isozyme differs from the cyt0plasmic isozymes in amino
acid composition, electrOphoretic mobility, and immunological
properties; it does not hybridize with the cytoplasmic isozyme subunits
(6,7).

In addition to these apparent structural differences between mito-
chondrial creatine kinase and the cyt0plasmic forms, intracellular
compartmentation also creates functional differences. Jacobus and

Lehninger (5) attribute an increase in the post-ADP state 4 respiration

33

34

rate of rat heart mitochondria in the presence of creatine to
mitochondrial creatine kinase activity. In the presence of creatine,
the mitochondrial isozyme regenerates ADP from the ATP produced during
oxidative phosphorylation. Moreadith and Jacobus (8) and Gallerich and
Saks (9) report additional evidence to support a functional coupling
between mitochondrial creatine kinase and the adenine nucleotide
translocase (5,lO-l4). Bessman (l5) discusses these results in terms
of a creatine phosphate (creatine—P) shuttle where creatine plays an
important regulatory role in intracellular energy transport of muscle.
Based on the involvement of mitochondrial creatine kinase in the
creatine—P shuttle and thus its importance in intracellular energy
transport, we initiated a study of this creatine kinase isozyme and its
function in mitochondria from normal and dystrophic avian pectoralis
muscle. Previously, Mahler (l6) reported a progressive decrease of
mitochondrial creatine kinase activity per mg of mitochondrial protein
in the skeletal muscle of dystrophic chickens with increased age
compared to normal controls. We confirm Mahler's observation using
genetically-related, age-matched normal and dystrophic chickens; lines
412 and 413 (l7), respectively. In addition, we report a loss of
regulation by creatine on the respiration of mitochondria from

dystrophic chicken breast muscle.

 

 

MATERIALS AND METHODS

Materials

All biochemicals, enzymes, and creatine kinase assay kits were
obtained from Sigma Chemical Co. (St. Louis, MO) unless otherwise
specified. Creatine and rabbit muscle creatine kinase were purchased
from Calbiochem—Behring Corporation (La Jolla, CA). Sephraphore III
cellulose acetate electrophoresis strips were obtained from Gelman
Sciences, Inc. (Ann Arbor, MI). Millipore filters were purchased from
Millipore Corp. (Bedford, MA). Common laboratory chemicals were
reagent grade or better. Solutions were prepared in distilled and
deionized water. Normal (line 4l2) and dystrophic (line 413) chickens
were obtained from the Department of Avian Sciences, University of
California, Davis, California.

Isolation of Breast and Leg Muscle Mitochondria

 

Preparation of mitochondria from the pectoralis and the primarily
red fiber leg muscles of normal and dystrophic chickens involved the
basic procedure described by Lee §t_gl. (l8) substituting Sigma
protease Type VII (l mg/g muscle) for Nagarse (3 mg/g muscle).
Mitochondrial yields based on the recovery of
succinate:p-iodonitrotetrazolium reductase (succinate INT reductase), a

mitochondrial marker, in the final mitochondrial suspension ranged from

l5-30% for mitochondria isolated from chickens between the ages of

35

36

12-20 days g§.gyp; 10% for mitochondria isolated from chickens 5 days
.g§.pyp. Respiratory control ratios (RCR) at 25°C for normal and
dystrophic breast muscle mitochondria from chickens 12-20 days gyhpyp
were 3.4 and 2.4, respectively and ADP:0 ratios were 1.5 and 1.2,
respectively using succinate as substrate in the presence of rotenone.
RCR values at 25°C for normal and dystrophic breast muscle mitochondria
from chickens 5 days g5 pyg were 1.5 and 1.2, respectively and ADP:0
ratios were 1.4 and 1.3, respectively. The addition of 4 pM
atractyloside after the onset of the post-ADP state 4 rate causes this
rate to immediately decrease to less than or equal to the initial state
4 rate. Since atractyloside blocks entrance of ADP via the adenine
nucleotide translocase, our skeletal muscle mitochondrial preparations
appear intact and free of mitochondrial ATPase activity; ADP is not
available for oxidative phosphorylation from any other source.

Isolation of Heart Mitochondria

 

Normal and dystrOphic chicken hearts were weighed, washed and
homogenized in cold 0.21 M mannitol, 70 nM sucrose, 0.l mM EDTA, l0 mM
TRIS-HCl, pH 7.4 buffer and isolated by differential centrifugation as
previously described (l9). Mitochondrial yields based on the recovery
of succinatezINT reductase in the final mitochondrial suspension ranged
from 20-40%. Respiratory control ratios for normal and dystrophic
heart mitochondria at 25°C were l.8 and 1.6, respectively using
succinate as substrate in the presence of rotenone. ADP:0 ratios were
l.l and l.2, respectively. The addition of 4 uM atractyloside after
the onset of the post-ADP state 4 rate causes this rate to immediately

decrease to less than or equal to the initial state 4 rate. Thus, our

 

 

37

heart mitochondrial preparations appear intact and free of
mitochondrial ATPase activity.
Enzyme Assays and Protein Determination

Total creatine kinase activity in the protease-treated heart,
breast muscle and leg muscle mitochondria was solubilized by suitably
diluting a portion of the final mitochondrial suspension into 0.l M
sodium phosphate, pH 6.5, 25 mM B-mercaptoethanol, 1 nM EDTA and l%
Triton X-lOO to give a final activity of 1-4 IU/ml. This dilution is
generally 10—fold; 20-fold for normal breast muscle mitochondria at
older ages. Creatine kinase activity was determined spectrophotome—
trically at 340 nm at 30°C using the creatine kinase assay mix
from Sigma (20,21). The mitochondrial marker, succinate:INT reductase,
was assayed by an endpoint assay following 10-12 minute incubations
using the conditions described previously (22). Mitochondrial pellet
protein was determined by the Lowry method (23) following a 1:1
solubilization in l N NaOH for one hour. Bovine serum albumin was used
as a standard. Mitochondrial yield is based on the fraction of total
succinate:INT reductase activity recovered in the final mitochondrial
suspension.

Cellulose Acetate Electrophoresis

 

Creatine kinase isozymes were separated on 2.5 x l7 cm Gelman
Sephraphore III cellulose polyacetate electrophoresis strips in 0.06 M
TRIS-barbital, pH 8.8, 25 mM B-mercaptoethanol as previously described
(24). The electrophoresis buffer contained l% Triton X-lOO to allow
creatine kinase associated with mitochondria to migrate from the
origin. Electrophoresis proceeded for two hours at 300 V at 6°C.

Following electrophoresis, the strips were stained for creatine kinase

 

 

38

isozymes as previously described (25) and scanned on a Gelman ACD-l8
automatic computing densitometer.

Isozyme positions were identified by comparing sample mobilities
with the migration of purified fractions of NM, MB, BB or mitochondrial
creatine kinase on independent electrophoretic strips. In control
electrophoretic strips, creatine—P was omitted from the assay mix to
check for interference by adenylate kinase.

Determination of Mitochondrial Creatine Kinase Levels

 

The proportion of mitochondrial creatine kinase in final
mitochondrial suspensions was determined from densitometer scans of the
electrophoretically separated isozymes. The percent mitochondrial
creatine kinase calculated from relative peak areas of the densitometer
scans was multiplied by the activity of total creatine kinase in the
final mitochondrial suspensions to obtain the activity of mitochondrial
creatine kinase alone. Since the creatine kinase assay mix is
optimized for MM creatine kinase and these conditions may vary somewhat
for mitochondrial creatine kinase there may be some error in the final
mitochondrial creatine kinase activity calculation. Mitochondrial
creatine kinase activity was then normalized to the amount of
mitochondria in each sample by dividing by the activity of
succinate:INT reductase in the sample.

Mitochondrial Oxygen Consumption

Mitochondrial respiration was monitored at 25°C on a Gilson Model
K-IC oxygraph equipped with a Yellow Springs Instruments oxygen elec—
trode and a high sensitivity Teflon membrane (Yellow Springs Instru—

ments). Standard skeletal muscle mitochondria buffer contained l50 mM

 

 

39

sucrose, l mM EDTA, 2 mg/ml BSA and 25 mM TRIS—HCl, pH 7.5. Standard
heart muscle mitochondria buffer contained 0.2l M mannitol, 70 mM
sucrose, l nM EDTA, 2 mg/ml BSA and l0 mM TRIS-HCl, pH 7.4. Breast and
leg muscle mitochondria (50 ul of 20-30 mg protein/ml) were incubated
in l.75 ml medium containing standard muscle mitochondria buffer, 5 mM
succinate, l0 uM rotenone and 5 mM M9504 in addition to 2.5, 5, l0,

20, 50, 75 or 98 nM potassium phosphate and 0 or 20 mM creatine. Heart
mitochondria were incubated under the same conditions except in medium
containing standard heart mitochondria buffer and 0.5, l, 2.5, 5 or l0
mM potassium phosphate. Addition of 20 pl 10 mM ADP to each incubation
mixture initiated State 3 respiration. The solubility of oxygen in
buffered medium at 25°C was taken to be 240 nmoles Oz/ml (26). All
oxygen consumption measurements were made on mitochondria isolated from
chickens between the ages of l2-20 days 35 gyg unless otherwise
specified due to increasing difficulty of isolating coupled
mitochondria from breast muscle of older chickens.

Creatine Phosphate Assays

 

Breast muscle mitochondria (l25 pl of 20—30 mg protein/ml) were
incubated at 25°C in 5 ml reaction mixtures containing standard
skeletal muscle mitochondria buffer, 5 mM succinate, l0 uM rotenone, 5
mM MgSO4, 20 mM creatine and 2.5, 5, l0, 20, 50 and 98 mM potassium
phosphate. Heart mitochondria were incubated under the same conditions
except in medium containing standard heart mitochondria buffer and 0.5,
l, 2.5, 5, ID or 20 nM phosphate (Pi). Samples (0.5 ml) were withdrawn
from the reaction chamber at 0, l, 2, 3, 4, 5, 6 and 7 minutes after
addition of 50 pl 10 mM ADP and immediately added to 0.5 ml 30 mg/ml

activated charcoal to remove ATP. The charcoal suspension was filtered

 

 

40

through Millipore filters (GSWP, 0.22 pm pore size) as quickly as
possible. A portion (200 pl) from each filtered sample was added to
each of two tubes containing l ml of l.2 mM ADP, 3.7 mM AMP, l4 mM
glucose, 9 mM magnesium acetate, l.8 mM NADP, 4 mM dithiothreitol, l.67
units/ml glucose-6-phosphate dehydrogenase and l.67 units/ml hexokinase
in 20 mM HEPES, pH 7.4. Rabbit muscle creatine kinase (10 pl of 2
mg/ml) was added to one of the two tubes for each sample. All tubes
were incubated at room temperature for 60 minutes and the absorbance
read at 340 nm. The difference in absorbance between the samples with
and without creatine kinase was plotted against creatine phosphate
concentrations in standard solutions. The resulting standard curve was
used to determine the concentration of creatine-P in each sample. The
slopes from a graph of concentration versus sampling time resulted in
production rates of creatine-P at each Pi concentration.

Determination of the Rate of Maximal Mitochondrial ATP Synthesis

 

Breast muscle mitochondria (125 pl of 20-30 mg protein/ml) were
incubated at 25°C in 5 ml reaction mixtures containing standard
skeletal muscle mitochondria buffer, 5 mM succinate, 10 pM rotenone, 5
mM MgS04, 20 mM creatine, 5 mM glucose, 60 units hexokinase and 5 or
50 mM potassium phosphate. Samples (0.5 ml) were withdrawn from the
reaction chamber at 0, 1, 2, 3, 4, 5, 6 and 7 minutes after addition of
50 pl 10 mM ADP and immediately added to 0.5 ml 30 mg/ml activated
charcoal to remove ATP. The charcoal suspension was filtered through
Millipore filters (GSWP, 0.22 pm pore size) as quickly as possible. A
portion (200 pl) from each filtered sample was added to individual
plastic cuvettes containing 1 ml of 1.2 mM ADP, 3.7 mM AMP, 14 mM

glucose, 9 mM magnesium acetate, 1.8 mM NADP, 4 mM dithiothreitol, 1.67

 

 

41

units/ml hexokinase (HK) in 20 mM HEPES, pH 7.4. The absorbance was
read at 340 nm after a 30 minute incubation period at room temperature.
Glucose-6-phosphate dehydrogenase (1.67 units/ml) was then added to
each sample. The absorbance was read at 340 nm following another 30
minute incubation period. Next, rabbit muscle creatine kinase (10 pl
of 2 mg/ml) was added to each cuvette, the samples allowed to incubate
for another 30 minutes and the absorbance read again at 340 nm.

The difference in absorbance between the initial reading and the
reading after the addition of glucose-6-P dehydrogenase was used to
determine the concentration of glucose-6-P in each sample. The slope
from a graph of concentration versus sampling time resulted in
production rates of glucose-6-P. Next, the difference in absorbance
between the reading after the addition of glucose-6-P dehydrogenase and
the reading after the addition of creatine kinase was plotted against
creatine-P concentrations in standard solutions. The resulting
standard curve was used to determine the concentration of creatine-P in
each sample. The slopes from a graph of concentration versus sampling
time resulted in production rates of creatine phosphate. Finally, the
rate of total mitochondrial ATP synthesis was taken as the total of the
rates of glucose-6-P and creatine-P.

Mitoplast Preparation

 

Mitoplasts from normal and dystrophic breast muscle were prepared
by first swelling the outer mitochondrial membrane in a hypotonic
solution of IO mM Tris-HCl, pH 7.5 at 0° for 5 minutes. Addition of a
hypertonic solution (l.75 M sucrose, 2 mM ATP and 2 mM MgSO4) to the

previous suspension at 0°C for 5 minutes caused shrinking of the inner

 

42

membrane and matrix. This osmotic treatment allowed removal of the
outer membrane by mild sonication (Branson Sonifier at 3 amperes for 20
seconds). Mitoplasts were then sedimented at 20,000 x g x 20 minutes.

Effect of Phosphate on the Interaction of Mitochondrial Creatine Kinase

 

with Mitoplasts
Mitoplasts (30 pl aliquots) were incubated with shaking in 750 pl

solution containing 0.2l M mannitol, 70 nM sucrose, 0.1 nM EDTA and 10
mM TRIS-HCl, pH 7.4 and increasing concentrations of potassium
phosphate at pH 7.4 for 2 hours at 6°C. The mitoplast suspensions were
then centrifuged in an Eppendorf centrifuge for l0 minutes and the
supernatants assayed for creatine kinase activity to determine the
percent of total mitochondrial creatine kinase released from the

mitoplast membrane.

 

RESULTS

Levels of Mitochondrial Creatine Kinase in Normal and Dystrophic

 

Muscles

Figure I shows the levels of mitochondrial creatine kinase
(expressed as units mitochondrial creatine kinase per unit of
succinate:INT reductase, a mitochondrial marker) as a function of age
for normal and dystrophic breast (B), leg (L) and heart (H) muscle.
Normal breast muscle mitochondria contain the highest levels of
mitochondrial creatine kinase at all ages; this level increases as the
chicken ages reaching a plateau around ll days. Dystrophic breast
muscle mitochondria contain somewhat lower but approximately the same
level of mitochondrial creatine kinase until about ll days gx_pyp when
these levels begin to decrease with increased age. Normal and
dystrophic leg muscle mitochondrial creatine kinase levels remain
relatively constant throughout this time period. Levels in heart
muscle are again relatively constant at all ages but are significantly
lower than the levels in breast and leg muscle. Mitochondria from
both leg and heart muscle of dystrophic chickens contain lower levels
of mitochondrial creatine kinase than the levels found in their
respective controls although these differences are not as large as
those between normal and dystrophic breast muscle mitochondria. The

activity of succinate:INT reductase per gram of muscle is not

43

 

Figure 1.

44

Levels of mitochondrial creatine kinase (expressed as units
mt-CK per unit of succinate:INT reductase, a mitochondrial
marker) as a function of age for normal and dystrophic
breast (B), leg (L) and heart (H) muscle. Open areas
represent the average level of mt-CK in each type of muscle
mitochondria from two normal birds at each age, while the
shaded areas represent the average levels in each muscle
type from two dystrophic birds. Error bars represent

variation from the mean.

 

45

 

 

 

 

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46

significantly different between normal and dystrophic breast muscle
(0.5 i 0.2 and 0.6 I 0.2, respectively) or normal and dystrophic heart
(4.l 1 1.0 and 5.0 t 0.8, respectively) at the ages in this study.

The data in Figure 2 shows the units of mitochondrial creatine
kinase per gram of normal and dystrophic heart and skeletal muscle.
Again the data show differences between normal and dystrophic skeletal
and heart muscle as already evident in Figure 1. However, we wish to
emphasize one additional point, namely, that normal heart and skeletal
muscle contain approximately the same number of units of mitochondrial
creatine kinase per 9 muscle; dystrophic heart and skeletal muscle also
contain the same level of mitochondrial creatine kinase per g muscle.
In addition, the level of mitochondrial creatine kinase per 9 muscle in
these dystrophic muscles is lower than the levels in their normal
counterparts. From l8 to 26 days, both dystrophic tissues contain from
60 to 70% of the units of mitochondrial creatine kinase per 9 muscle as
normal tissue. Since leg and heart muscle are not visibly affected by
dystrophy in the chicken, we became interested in the effect this
decreased level may have on the respiration of dystrophic breast muscle
mitochondria.

Mitochondrial Respiration in the Presence and Absence of Creatine

 

Oxygen consumption traces in Figure 3 show normal and dystrophic
breast muscle mitochondrial respiration in the absence and presence of
20 mM creatine. In the absence of creatine, the initial state 4
respiration of both normal and dystrophic breast muscle mitochondria
increases upon addition of ADP to the state 3 respiration before
returning to approximately the state 4 rate after exhaustion of ADP.

In the presence of 20 mM creatine, the post-ADP state 4 respiration of

 

Figure 2.

47

Levels of mitochondrial creatine kinase (expressed as units
mt-CK per gram of muscle) as a function of age for normal
and dystrophic breast and heart muscle. Each point
represents the average from two birds for each muscle type.
(a) normal breast muscle; (0) dystrophic breast muscle; (A)

normal heart; (A) dystrophic heart.

48

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Figure 3.

 

49

Oxygen consumption traces for normal (A) and dystrophic (B)
breast muscle mitochondria. Mitochondria were added to

standard breast muscle mitochondria buffer containing 20 mM
phosphate and either no creatine or 20 mM creatine at 25°C.
Addition of ADP initiated state 3 respiration. The numbers

in parentheses represent nmoles 02 consumed per minute.

 

 

50

 

A. Normal Breast Muscle

Mlios
I

(10) ADP
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No Creatine 20 mM Creatine
35 20 mM Phosphate 20 mM Phosphate
nmoles

I min

 

B. Dystrophic Breast Muscle

Miios
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No Creatine 20 mM Creatine
35
nmoles 20 "1M PhOSPhaie 20 mM Phosphate
02

I min

 

 

 

51

normal breast muscle mitochondria continues at the state 3 rate (Figure
3A). However, the post-ADP state 4 respiration of dystrophic breast
muscle mitochondria slows to a rate twice the post-ADP rate in the
absence of creatine (Figure 3B).

Figure 4 shows the initial state 4, state 3 and post-ADP state 4
respiratory rates (expressed as pmoles 02°min-1(IU succinate:INT
reductase’l) for normal and dystrophic breast muscle mitochondria
from chickens 12-20 days gx_pyp_in the absence and presence of creatine
at various Pi concentrations. Normal and dystrophic breast muscle
mitochondria in the absence of creatine (Figure 4A and 4C, respective-
ly) show similar patterns for all three rates at various Pi concentra-
tions indicating dystrophic mitochondria function like normal mitochon-
dria under these conditions. However, a major difference arises in the
presence of creatine. Normal breast muscle mitochondria in the
presence of creatine (Figure 4B) continue to respire at the state 3
rate after presumed exhaustion of the added ADP for all Pi concentra-
tions below 98 mM. 0n the other hand, in dystrophic breast muscle
mitochondria (Figure 40) as little as l0 mM Pi causes the appearance of
a post-ADP state 4 rate separate from the state 3 rate. This rate
remains nearly constant from 20 to 98 mM Pi but about l.5 times the
post-ADP state 4 rate in the absence of creatine.

An identical experiment with normal and dystrophic breast muscle
mitochondria from chickens 5 days gy_pyp reveals creatine has no
visible effect on the post-ADP state 4 rate of normal as well as
dystrophic mitochondria at this young age (Figure 5). In addition, the
same experiment with normal and dystrophic heart mitochondria in the

presence and absence of creatine (Figure 6) also indicates creatine

 

Figure 4.

52

Effect of phosphate concentration on the rates of oxygen
consumption by normal and dystrophic breast muscle
mitochondria from chickens 12-20 days p5_pyp. Conditions
were as described for Figure 3, with the indicated phosphate
concentrations for normal breast muscle mitochondria with no
creatine (A) and 20 mM creatine (B) and dystrophic breast
muscle mitochondria with no creatine (C) and 20 mM creatine
(D). Respiration rates are expressed as pmoles 02

consumed per minute per IU succinate:INT reductase. The
data in A and B, and C and D, were each obtained in a
separate experiment utilizing a single mitochondrial
preparation. Three additional experiments for both normal
and dystrophic mitochondria show similar results. (0)
Initial state 4 respiration; (0) state 3 respiration; (A)

post-ADP state 4 respiration.

 

53

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Figure 5.

54

Effect of phosphate concentration on the rates of oxygen
consumption by normal and dystrophic breast muscle
mitochondria from chickens 5 days gx_pyp. Conditions were
as described for Figure 3, with the indicated phosphate
concentrations for normal breast muscle mitochondria with no
creatine (A) and 20 mM creatine (B) and dystrophic breast
muscle mitochondria with no creatine (C) and 20 mM creatine
(D). Respiration rates are expressed as pmoles 02

consumed per minute per IU succinate:INT reductase. The
data in A and B, and C and D, were each obtained in a
separate experiment utilizing a single mitochondrial
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and dystrophic mitochondria show similar results. (0)
Initial state 4 respiration; (0) state 3 respiration; (A)

post-ADP state 4 respiration.

 

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56

Effect of phosphate concentration on the rates of oxygen
consumption of normal and dystrophic heart mitochondria.
Conditions were as described in Figure 3, with the indicated
phosphate concentrations for heart mitochondria with no
creatine (A) and 20 mM creatine (B) and dystrophic heart
mitochondria with no creatine (C) and 20 mM creatine (D).
Respiration rates are expressed as pmoles 02 consumed per
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and C and D, were each obtained in a separate experiment
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does not affect the post-ADP state 4 rate of normal and dystrophic
heart mitochondria; Pi from 0.5 to 10 mM has no effect either.
Therefore, in the presence of creatine both normal and dystrophic heart
mitochondria from chickens at any age, as well as both normal and
dystrophic breast muscle mitochondria from 5-day-old birds, function
like dystrophic breast muscle mitochondria from older birds at Pi
concentrations greater than 10 mM. In addition, normal and dystrophic
leg muscle mitochondria behave like normal and dystrophic heart muscle
mitochondria (data not shown).

Creatine Phosphate Production in Respiring Mitochondria

 

Creatine-P production rates in respiring mitochondria confirms that
the effects observed during oxygen consumption measurements in the
presence of creatine in both normal and dystrophic heart and breast
muscle mitochondria are due to a perturbation of the mitochondrial
creatine kinase function. A decrease in mitochondrial creatine kinase
function, possibly due to release of mitochondrial creatine kinase
from the inner mitochondrial membrane, should reduce the rate of
creatine-P production. Figure 7 shows creatine-P production rates
(nmoles creatine-P produced min‘l (IU succinate:INT
reductase)'1) at various Pi concentrations for respiring breast and
heart muscle mitochondria from both normal and dystrophic chickens.
The inset in Figure 7 shows creatine-P concentration as a function of
time after initiation of mitochondrial respiration. Slapes of these
lines for each Pi concentration (after the initial lag period)
determine the creatine-P production rates. Normal breast muscle
mitochondria maintain high, nearly constant rates at all Pi

concentrations from 5 to 98 mM corresponding to the oxygen consumption

 

 

Figure 7.

Creatine phosphate synthesis in normal and dystrophic breast
and heart muscle mitochondria. Mitochondria were added to 5
ml reaction medium (See Materials and Methods) containing
various concentrations of phosphate and 20 mM creatine at
25°C. Removal of a 0.5 ml fraction for a zero time sample
was followed by addition of 50 pl of l0 mM ADP to initiate
the reaction. At l minute intervals, 0.5 ml samples were
removed for creatine phosphate assay (See Materials and
Methods). The figure inset shows nmoles creatine phosphate
formed by dystrophic breast muscle mitochondria at each time
point for the indicated phosphate concentration. The slopes
from this analysis result in the production rates of
creatine phosphate at each phosphate concentration. Figure
7 shows these production rates expressed as nmoles creatine
phosphate produced per minute per IU succinate:INT reductase
as a function of phOSphate concentration for the indicated
muscle type. Data are the average rates from two
independent experiments each utilizing a single

mitochondrial preparation from each muscle type.

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measurements where post-ADP state 4 rates continued at the state 3 rate
(Figure 4B) for all Pi concentrations up to 98 mM Pi. In contrast to
normal breast muscle mitochondria, dystrOphic breast muscle
mitochondria exhibit a sharp decline in the rate of creatine-P
production after l0 mM Pi. This decline in production rate parallels
oxygen consumption measurements; Pi concentrations greater than 10 mM
allow return of a distinct post-ADP state 4 rate (Figure 40) indicating
less conversion of ATP to creatine-P. Heart muscle mitochondria from
both normal and dystrOphic chickens have significantly slower
creatine-P production rates than normal or dystrophic breast muscle
mitochondria. These very low creatine—P production rates are
consistent with the lack of either a creatine or Pi effect on oxygen
consumption rates during respiration of normal and dystrophic heart
mitochondria (Figure 6). Thus, the creatine-P production rates in
respiring mitochondria from various muscle types are consistent with a
loss of mitochondrial creatine kinase activity in dystrOphic breast
muscle mitochondria.

Maximal ATP Synthesis Rates in Normal and Dystrophic Breast Muscle

Mitochondria

 

Table I shows the maximal ATP synthesis rate, or the sum of the
rates of glucose-6-P and creatine-P production in the presence of
excess hexokinase and glucose in the reaction mixture, in normal and
dystrOphic breast muscle mitochondria in the presence of 5 and 50 mM
P1. (See Materials and Methods for exact calculation procedures).
Increasing the Pi concentration from 5 to 50 mM has little effect on
the total rate of mitochondrial ATP synthesis in normal and dystrOphic

breast muscle mitochondria; a 14% increase and an 11% decrease,

_62

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63

respectively. Furthermore, mitochondria from dystrophic muscle produce
ATP as effectively as those from normal muscle; in fact, the total rate
of ATP synthesis in mitochondria from dystrophic muscle is greater than
the rate in normal mitochondria (1100 and 750 nmoles ATP produced
min“1 unit"1 succinate:INT reductase in dystrophic and normal
mitochondria, respectively). The data in Table I also show that the
rates of creatine-P production in the presence of excess creatine when
the post-ADP state 4 rate is identical to the state 3 rate is 80% of
the maximal rate of ATP synthesis at 5mM Pi; at 50mM Pi, only 53% of
the ATP produced during oxidative phosphorylation is trapped as
creatine-P. Similar calculations with data obtained with mitochondria
from dystrophic muscle are not valid since creatine does not maintain
post-ADP state 4 rates identical to state 3 rates.
Effect of Inorganic Phosphate on the Interaction of Mitochondrial
Creatine Kinase with Normal and Dystrophic Breast Muscle Mitoplasts

The effect of Pi on the interaction of mitochondrial creatine
kinase with normal and dystrOphic breast muscle mit0plasts is shown in
Figure 8. Increasing concentrations of Pi release increasing amounts
of mitochondrial creatine kinase into the incubation medium until 60 mM
P1 when 85% of the total mitochondrial creatine kinase is released.
K0.5 (concentration of phosphate required to release 50% of total
mt-CK) is 26.5 and 30 mM Pi for normal and dystrophic breast muscle
mitoplasts, respectively; no significant difference exists between the
concentrations of Pi required to release mt-CK from normal and
dystrophic mitoplasts. The shape of the curves are very similar to the

phosphate solubilization curve for mt-CK reported by Vial gt_al (27)

 

 

Figure 8:

64

Effect of phosphate on the solubilization of mitochondrial
creatine kinase (mt-CK) from normal and dystrophic breast
muscle mitoplasts. Aliquots (30 ul) of isolated mit0plasts
(See Materials and Methods) were incubated at 6°C for two
hours in 750 pl standard breast muscle mitochondria buffer
(See Materials and Methods) and the indicated concentrations
of potassium phosphate. After centrifugation, the
supernatants were assayed for CK activity. The percent of
total mt-CK in the supernatant, expressed as % soluble
mt-CK, as a function of phosphate concentration is shown for
normal (0) and dystrophic (x) breast muscle mitoplasts.

Each point represents the average of data from four

independent determinations : standard deviation.

% Soluble Mi-CK

IOO~
90-
80— .. l//
/ d
701'
I,

60— I1.

I
my— I

I

I
40— I

I
— I ' 1"?— Normal
3Q /
20 l I ,l/ ..;L_£. Dystrophic

/

.. 1 I
Q 1 1 1 1 1 1 4 1 1
002030405060708090l00

 

 

 

 

 

 

 

 

[Phosphate], mM

66

for pig heart mitochondria, but the K0.5 values are higher for

chicken breast muscle mitochondria.

 

 

DISCUSSION

A comparison of mitochondrial creatine kinase levels in various
muscle types from normal and dystrOphic chickens (Figure 1) basically
confirms Mahler's observation (16) that mitochondrial creatine kinase
activity is decreased in dystrophic breast muscle compared to normal
age-matched controls with increased age. However, Mahler used chickens
at 44, 91, 200 and 566 days ex_919, when the dystrophic condition is in
its advanced stages. Since the dystrophic characteristic becomes
visibly apparent by 9 days §x_gvg_in the 413 line of chickens (l7), we
felt the need to examine levels of mitochondrial creatine kinase early
in the onset of the dystrophic symptoms. Thus, our study of younger
ages reveals a closer correlation with the decrease in mitochondrial
creatine kinase level and the deterioration of dystrophic muscle. In
addition, we express the levels of mitochondrial creatine kinase in
terms of a specific mitochondrial marker enzyme, succinate:INT
reductase, rather than per mg of mitochondrial protein. We consider
this difference in reporting procedure to be important when comparing
normal and dystrophic nfitochondria since the dystrophic mitochondrial
pellet may contain extraneous protein because of the increased
connective tissue in dystrophic muscle (see (28) for review).

Of particular interest in this study is the relatively high level

of mitochondrial creatine kinase per unit of succinate:INT reductase in

67

68

normal chicken breast muscle compared to chicken heart and leg muscle
(Figure 1). When expressed per unit of succinate:INT reductase, the
chicken heart muscle contains less than 10% of the level of
mitochondrial creatine kinase in the essentially pure white fiber,
fast-twitch, glycolytic breast muscle. The leg muscle, essentially a
red fiber muscle, contains intermediate levels. The low level of
mitochondrial creatine kinase in chicken heart muscle contrasts

with the generally accepted view that heart muscle mitochondria contain
significant amounts of this isozyme. When the levels of mitochondrial
creatine kinase are expressed per 9 muscle (Figure 2) rather than per
unit of succinate:INT reductase, we again see that dystrophic muscle
contains less mitochondrial creatine kinase activity than normal
muscle. However, both chicken heart and breast muscle contain roughly
the same number of units of mitochondrial creatine kinase per g of
muscle; dystrOphic heart and breast muscle also contain the same number
of units per 9 muscle but only 60-70% of their normal counterparts. To
our knowledge, this is the first demonstration of a biochemical
difference between normal and dystrOphic chicken heart. Yet the
dystrophic chicken lives a normal life span (l7); the disease affects
primarily the fast twitch white muscle fibers which are ultimately
destroyed.

A consideration of the physiological effects of reduced levels of
mitochondrial creatine kinase brings us to the creatine-P shuttle (for
review see 15). This shuttle including its composition and importance
evolved in recent years basically from the initial work of Bessman and
colleagues (l0,29) and Jacobus and Lehninger (5). The shuttle,

outlined in Figure 9, revolves around the compartmentation of two

69

Figure 9: Creatine phosphate shuttle mechanism as described by Bessman

(15).

..-_ -0--.- -.._ . - ._ _fi ' " '

7O

 

N

 

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0:. 0o
)\ ..

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(
00<\ /_0001

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71

creatine kinase isozymes. One isozyme, the mitochondrial one, is bound
to the outer surface of the inner mitochondrial membrane (4,5)
presumably functionally coupled to the adenine nucleotide translocase
(5,8-l4). This bound mitochondrial creatine kinase preferentially
utilizes the ATP produced during oxidative phosphorylation to
phosphorylate creatine and regenerate ADP. This regeneration of ADP
causes the post-ADP state 4 rate to continue at the state 3 rate during
oxygen consumption measurements. The resulting creatine-P from the
mitochondrial creatine kinase reaction then diffuses from the
mitochondrial intermembrane space to be utilized by MM creatine kinase,
the other isozyme of skeletal muscle, to phosphorylate ADP. Since MM
creatine kinase binds to myosin, presumably near the myosin head group
(30), the creatine kinase produced ATP is preferentially utilized for
muscle contraction (31). Thus, creatine in conjunction with
mitochondrial creatine kinase has an important regulatory role in
muscle respiration by participating in the creatine-P shuttle.

When breast muscle mitochondria contain their full compliment of
mitochondrial creatine kinase (at approximately 11 days §x_gyg),
creatine functions effectively to maintain the post-ADP state 4 rate
equivalent to the state 3 rate at Pi concentrations from 5 to 98 mM
(Figure 48). Yet only 80% of the maximal ATP produced during oxidative
phosphorylation is trapped as creatine-P. When muscle mitochondria
contain approximately 50 units of mitochondrial creatine kinase per
unit of succinate:INT reductase then at Pi concentrations exceeding l0
mM, creatine no longer functions effectively in maintaining the

post-ADP state 4 rate equivalent to the state 3 rate (Figure 40). At

72

Pi concentrations at l0 mM and below, the post-ADP state 4 rate in the
presence of creatine is nearly equivalent to the state 3 rate.

As the level of mitochondrial creatine kinase decreases in
mitochondria from dystrophic breast muscle with increasing age, the
post-ADP state 4 rate slows and approaches the initial state 4 rate in
the presence of creatine at lower P, concentrations. For example, 20
mM creatine in the presence of 5mM Pi no longer maintains the post-

ADP state 4 rate equivalent to the state 3 rate in breast muscle
mitochondria from dystrophic chicken at 19-20 days of age, whereas 20
mM creatine does maintain the post ADP state 4 rate equivalent to the
state 3 rate in mitochondria from dystrophic chickens at 11-12 days of
age even in 10 mM Pi (data not shown). In addition, breast muscle
mitochondria from 5 day old normal chickens have not reached their full
compliment of mitochondrial creatine kinase (Figure 1) and thus are
unable to maintain the post-ADP state 4 rate at the state 3 rate in the
presence of ZOmM creatine and 5mM Pi (Figure 58). In this case, normal
breast muscle mitochondria with a low level of mitochondrial creatine
kinase do not maintain state 3 respiration in the presence of creatine
in a manner similar to dystrophic breast muscle mitochondria (Figure 58
and 5D).

The results with heart mitochondria, containing less than 5 units
of mitochondrial creatine kinase per unit of succinate:INT reductase
(Figure 1) basically confirm the behavior of dystrophic breast muscle
mitochondria and mitochondria from 5 day old normal muscle. As with
mitochondria from 5 day old birds, 20 mM creatine in the presence of
5mM Pi, has little or no effect on the post-ADP state 4 rate (Figure 6B

and 6D). Leg muscle mitochondria behave similarly; leg muscle

73

mitochondria from 11-19 day old birds contains approximately 25 units
mitochondrial creatine kinase per unit of succinate INT reductase.
Thus, whether the mitochondria are obtained from normal or dystrophic
muscle, when the level of mitochondrial creatine kinase is below 25
units per unit of succinate:INT reductase, 20mM creatine in the
presence of 5mM Pi has little or no effect on the post ADP state 4
rate.

The rates of creatine-P production in respiring mitochondria
isolated from heart and breast muscle (Figure 7) strengthen the
viewpoint that the level of mitochondrial creatine kinase in normal and
dystrophic breast muscle accounts for the differences of creatine and
Pi on oxygen consumption measurements with these two mitochondria.
Normal breast muscle mitochondria maintain a high, nearly constant rate
of production of creatine-P at all Pi concentrations consistent with
the presence of large amounts of mitochondrial creatine kinase. The
sharp decline in the rate of creatine-P production around l0 mM Pi in
dystrophic breast muscle mitochondria is consistent with the dr0p in
post-ADP state 4 rates of oxygen consumption when Pi concentrations
exceed l0 mM. Heart muscle mitochondria produce creatine-P at
significantly lower rates than normal and dystrOphic breast muscle
mitochondria again consistent with the lack of an appreciable effect of
creatine on the post-ADP state 4 rate.

While the creatine phosphate production rates for each of the
mitochondrial types shown in Figure 7 indicates their relative
efficiency for trapping ATP as creatine-P as a function of Pi
concentration, these experiments do not rule out possible defects in

ATP production as an explanation for the decreased creatine-P

 

74

production rates at phosphate concentrations greater than 10 mM. Thus,
we measured the total rate of mitochondrial ATP synthesis in both
normal and dystrophic breast muscle mitochondria at low and high Pi
concentrations.

The addition of hexokinase and glucose to the incubation mixture
traps ATP exiting from the mitochondria as glucose-6-P. Since the
hexokinase reaction is an essentially irreversible reaction,
equilibrium conditions in the intermembrane space then favor the
reverse mitochondrial creatine kinase reaction and glucose-6-P becomes
a sink for most mitochondrial ATP. (The establishment of these
equilibrium conditions accounts for the decreased rate of creatine-P
production in the presence of hexokinase and glucose compared to the
rate in the absence of hexokinase (Table 1)). Therefore, the sum of
the glucose-6-P and creatine-P production rates allows calculation of
the maximal rate of mitochondrial ATP synthesis. Table I shows little
effect of 50 mM Pi on the total rate of mitochondrial ATP synthesis in
normal and dystrophic breast muscle mitochondria. In addition, the
higher rate of total ATP synthesis for dystrOphic mitochondria compared
to normal mitochondria indicates the absence of a defect in producing
ATP in dystrOphic mitochondria, and may in fact represent compensation
for the decreased efficiency of creatine phosphate synthesis.

The results in Figure 8 indicate that mitochondrial creatine kinase
and the outer surface of the inner mitochondrial membrane are not
altered in dystrophic muscle; the concentration of Pi required to
release mt-CK from the mitoplast membrane is the same for both normal
and dystrophic mitochondria. In terms of the creatine-P shuttle any

displacement by Pi of mitochondrial creatine kinase from the inner

 

 

75

mitochondrial membrane would reduce preferential utilization of ATP
exiting from the translocase. However, when mitochondria contain a
large excess of mitochondrial creatine kinase, that is, around lOO
units per unit of succinate:INT reductase, release of the enzyme from
the membrane has little effect on the ability of mitochondrial creatine
kinase to trap ATP since the enzyme would still be maintained at high
concentrations in the intermembrane space. Thus, the enzyme remains in
sufficiently high concentration in normal chicken breast muscle
mitochondria to trap the ATP before it diffuses into the cytosol. 0n
the other hand, in chicken heart mitochondria, the level of
mitochondrial creatine kinase is too low to trap all the ATP exiting
from the translocase even at Pi concentrations where most, if not all,
mitochondrial creatine kinase remains bound to the membrane. An ideal
situation for observing the phosphate effect occurs in dystrOphic
chicken breast muscle mitochondria where the intermediate level of
mitochondrial creatine kinase allows one to observe displacement of the
enzyme from the membrane by phosphate. At concentrations of IO mM Pi
or below, sufficient mitochondrial creatine kinase remains bound to the
membrane to trap a significant percentage of the ATP exiting from the
mitochondria. However, when Pi concentrations exceed l0 mM,
insufficient mitochondrial creatine kinase remains bound to the
membrane and the concentration of displaced enzyme in the intermembrane
space is too low to trap a significant percentage of the ATP before it
diffuses out of the mitochondria.

Evidence showing that patients with Duchenne's muscular dystrophy
excrete much smaller amounts of creatinine than normal individuals (see

(33) for review) is consistent with diminished mitochondrial creatine

 

 

76

kinase activities in dystrophic muscle. Creatinine arises from
creatine-P by a lst order nonenzymatic cyclization reaction. Thus, a
diminished excretion of creatinine is consistent with a reduced steady
state concentration of creatine-P in dystrophic muscles as recently
reported by Newman gt_al_(34) in the muscles of Duchenne patients. In
addition, a variety of isotopic exchange studies of creatine metabolism
in patients with Duchenne's muscular dystrophy and in dystrophic mice
indicate a defect in creatine trapping in muscle cells by
phosphorylation; there is no evidence for an active transport mechanism
(33). Furthermore, studies in single frog muscle fibers support the
idea that reduced levels of creatine-P may be responsible for loss of
muscle function (35). In these studies, stimulation of the frog
semitendinosus muscle for l00-l50 sec results in a prolonged fatigue
that could not be overcome by l sec of rest. Muscle fiber creatine-P
levels fell logarithmically from a mean of l2l i 8 nmoles/mg protein to
l0% of this value. Fiber glycogen was not significantly affected; ATP
levels dropped by at most l5%. Whatever the cause of the fatigue, it
is characterized by decreased concentrations of creatine-P in the
muscle. The decreased levels of mitochondrial creatine kinase in
dystrophic muscle are consistent with a reduced effectiveness of the
creatine-P shuttle and thus account for the inability to trap creatine
and the reduced levels of creatine-P.

Finally, with regard to the chicken heart function, this muscle has
a much greater capacity to generate ATP by oxidative phosphorylation
than white fiber muscle. This increased oxidative capacity may offset
the effects of a mitochondrial creatine kinase deficiency and thus the

dystrOphic chicken lives a normal life span (17). Also, and perhaps

77

just as important, the dominant cytoplasmic creatine kinase isozyme in
chicken heart is BB rather than MM creatine kinase found in mammalian
hearts (36). A similar isozyme pattern exists in the embryonic muscle
of various species. The BB isozyme predominates in embryonic chicken
breast muscle (37) as well as other mammalian embryonic muscles where
mitochondrial creatine kinase is apparently absent or very low (38).
When this pattern exists in the embryonic chicken breast muscle, the
dystrophic condition is not yet evident (l7).

We do not suggest that the decreased levels of mitochondrial
creatine kinase found in dystrophic breast muscle is the primary lesion
in muscular dystrophy, but rather that this decrease may be of primary
importance in causing the loss of function of white fiber muscle by
decreasing the efficiency of trapping available mitochondrial ATP as
creatine-P, the high energy storage molecule for muscle contraction

energy.

10.

REFERENCES

Eppenberger, H.M., Dawson, D., and Kaplan, N.O. (l967) J. Biol.
Chem. 242, 204-209.

Neumeier, D. (l98l) in Creatine Kinase Isoenzymes (Lang, H., ed.)

 

pp. 85-l09, Springer-Verlag, New York.
Jacobs, H., Heldt, H.W., and Klingenberg, M. (I964) Biochem.
Biophys. Res. Comm., l6, 5l6-52l.

 

Scholte, H.R., Weigers, P.J., and Witpeeters, E.M. (l973) Biochem.
Biophys. Acta 29l, 764-773.

 

Jacobus, W.E., and Lehninger, A.L. (l973) J. Biol. Chem. 248,

 

4803-4810.

Hall, N., Addis, P., and DeLuca, M. (l979) Biochemistry l8,

 

l745-l75l.

Roberts, R., and Grace, A.M. (l980) J. Biol. Chem. 225,

 

2870-2877.
Moreadith, R.W., and Jacobus, W.E. (l982) J. Biol. Chem. 257,

 

899-905.
Gellerich, F., and Saks, V.A. (l982) Biochem. Bi0phys. Res.

 

Commun. l05, l473-l481.

Bessman, S.P., and Fonyo, A. (T966) Biochem. Biophys. Res. Commun.

 

22, 597-602.

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1].

l2.

l3.

14.

15.

l6.

l7.

l8.

19.

20.

21.

22.

23.

24.

 

79

Saks, V.A., Lipina, N.V., Smirnov, V.N., and Chazov, E.I. (T976)
Arch. Biochem. Biophys. l73, 34-4l.

 

Saks, V.A., Kupriyanov, V.V., Elizaroua, G.V., and Jacobus, W.E.

(1980) J. Biol. Chem. 255, 755-763.

 

Yang, W.C.T., Geiger, P.J., Bessman, S.P., and Borrebaek, B.
(l977) Biochem. Biophys. Res. Commun. 76, 882-887.

Defuria, R.A., Ingwall, J.S., Fossell, E.T. and Dygert, M.K.
(l980) in Heart Creatine Kinase: The Integration of Isozymes for

Energy Distribution (Jacobus, W.E., and Ingwall, J.S., eds) pp

 

l35-l4l, Williams and Wilkins, Baltimore.
Bessman, S.P., and Geiger, P.J. (l98l) Science 2ll, 448-452.
Mahler, M. (l979) Biochem. Biophys. Res. Commun. 88, 895-906.

 

Wilson, B.W., Randall, W.R., Patterson, G.T., and Entriken, R.R.
(1979) Ann. N.Y. Acad. Sci. 3l7, 224-246.

 

Lee, C.P., Martens, M.E., Jankulovska, L., and Neymark, M.A.
(l979) Muscle and Nerve 2, 340-348.

 

Pande, S.V., and Blanchaer, M.C. (l97l) J. Biol. Chem. 246,

 

402-4ll.
Oliver, I.T. (l955) Biochem. J. 6l, ll6-l22.

 

Rosalki, 5.8. (1967) J. Lab. Clin. Med. 69, 696-705.

 

Baxter, J.H., and Suelter, C.H. (l983) Muscle and Nerve, 6,

 

l87-l94.
Lowry, P.H., Rosebrough, N.J., Farr, A.O., and Randall, R.J.
(l95l) J. Biol. Chem. l93, 265-275.

 

Hall, N., Addis, P., and DeLuca, M. (l977) Biochem. Biophys. Res.

 

Commun. 76, 950-956.

25.

26.

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28.

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30.

31.

32.

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34.

35.

36.

37.

38.

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Hall, N., and DeLuca, M. (l976) Anal. Biochem. 76, 56l-567.

 

Chappell, J.B. (l964) Biochem. J. 90, 225-237.

 

Vial, C., Font, B., Goldschmidt, D., and Gautheron, D.C. (l979)

Biochem. Biophys. Res. Commun. 88, l352-l359.

 

Sweeny, P.R., and Brown, R.G. (l98l) Comp. Biochem. Physiol. 708,

 

27-33.
Bessman, S.P. (1966) Am. J. Med. 40, 740.

 

Turner, D.C., Wallimann, T., and Eppenberger, H.M. (1973) Proc.
Nat]. Acad. 8C1. UoSvo _Z_0_, 702-7050

 

Bessman, S.P., Yang, W.C.T., Geiger, P.J., and Erickson-Viitanen,

S. (l980) Biochem. Biophys. Res. Commun. 96, l4l4-l420.

 

Ernster, L., and Nordenbrand, I. (1967) in Methods in Enzymology

 

(Estrabrook, R.W. and Pullman, M.E., eds.), Vol. 10, pp. 86-94,
Academic Press, New York.

Fitch, C.D. (l977) in Pathogenesis of Human Muscular Dystrophies

 

(Rowland, L.P., ed.) pp. 328-336, Excerpta Medica,
Amsterdam-Oxford.

Newman, R.J., Bore, P.J., Chan, L., Gadian, D.G., Styles, P.,
Taylor, D., and Radda, G.K. (l982) British Med. J. 284,

 

l072-l074.
Nassar-Gentina, V., Passonneau, J.V., Vergara, J.L., and Rapoport,

5.1. (1978) J. Gen. Physiol. 72, 593-606.

 

Eppenberger, H.M., Dawson, D.M., and Kaplan, N.0. (l967) J. Biol.
Chem; 242, 204—209.

Eppenberger, H.M., Eppenberger, M., Richterich, C., and Aebi, H.
(l964) Develop. Biol. l0:l-l6.

 

Hall, N., and DeLuca, M. (1975) Biochem. Biophys. Res. Commun. 66,

 

988-994.

CHAPTER III

Purification of Two ElectrOphoretically Distinct Mitochondrial
Creatine Kinases from Chicken Breast Muscle Using

Dye-Ligand and ATP-Affinity Chromatography

Vickie D. Bennett and Clarence H. Suelter

From the Department of Biochemistry, Michigan State University,

East Lansing, Michigan 48824

Running Title: Two Mitochondrial Creatine Kinases

81

INTRODUCTION

Creatine kinase (EC 2.7.3.2) exists in several isozymic forms. The
M (muscle type) and B (brain type) subunits dimerize to form the three
cytoplasmic isozymes, MM, BB and hybrid MB (1). The MM creatine kinase
predominates in mature skeletal muscle and mammalian myocardium whereas
BB creatine kinase predominates in mammalian brain, neural tissue and
embryonic skeletal muscle as well as myocardial muscle of birds (2).
Hybrid MB creatine kinase appears in mammalian heart and skeletal muscle
(2).

Jacobs gt_al. (3) first reported the existence of an additional CK
isozyme associated with mitochondria isolated from rat heart and brain.
This isozyme moves toward the cathode at pH 8.8 during cellulose acetate
electrophoresis, whereas the cytoplasmic forms move toward the anode
(4). Significant amounts of this mitochondrial creatine kinase (mt-CK)
appear in human, beef and rat heart as well as rat brain, skeletal
muscle and intestinal muscle mitochondria; nominal amounts appear in rat
and rabbit liver, kidney and testis mitochondria (5). Mt-CK appears to
be associated with the outer surface of the inner mitochondrial membrane
in all tissues containing the isozyme (5-9).

Purification procedures for mt-CK from bovine heart (10,11) and
canine heart (12,13) take advantage of a phosphate-dependent release of

mt-CK from the inner mitochondrial membrane (9,14). These procedures

82

 

 

83

involve solubilization of mt-CK from mitochondria with 100 mM sodium
phosphate and then separation of the isozyme from MM-CK, the major
cytoplasmic isozyme in mammalian hearts, and other proteins by anion
exchange chromatography, gel filtration chromatography and ATP-affinity
chromatography (IO-13). Both bovine and canine heart mt-CK contain
different amino acid compositions than the cytoplasmic CK isozymes
(11,12). In addition, mt-CK subunits will not hybridize with
cytoplasmic CK subunits (11,12) and antisera to canine mt-CK does not
cross-react with the canine B or M subunits (12,13). These observations
suggest mt-CK is structurally distinct from the cytoplasmic isozymes.

In this paper, we present a two column procedure involving
dye-ligand and ATP-affinity chromatography for the isolation and
purification of two forms of mt-CK from chicken breast muscle. These
two mitochondrial forms migrate with slightly different cathodal

mobilities during cellulose acetate electrophoresis at pH 8.8.

 

 

 

 

 

MATERIALS AND METHODS

Materials

All biochemicals, enzymes and creatine kinase assay kits were
obtained from Sigma Chemical Co. (St. Louis, MO) unless otherwise
specified. Matrix Gel Red A (Procion Red-agarose) was purchased from
Amicon Corp. (Lexington, MA). Adenosine 5' triphosphate and
agarose-hexane-adenosine 5' triphosphate, type 3 (agarose-hexane-ATP)
was obtained from P.L. Biochemicals, Inc. (Milwaukee, WI). Sephraphore
III cellulose acetate electrophoresis strips were purchased from Gelman
Sciences, Inc. (Ann Arbor, MI). Common laboratory chemicals were
reagent grade or better. Solutions were prepared in deionized distilled
water. Frozen chicken pectoralis muscle was obtained from Pel Freez
Inc. (Rogers, AK).

Enzyme Assays and Protein Determination

 

Creatine kinase activity was determined spectrophotometrically at
340 nm at 30°C using the creatine kinase assay mix from Sigma (15,16).
The mitochondrial marker, succinatezp-iodonitrotetrazolium reductase
(succinate:INT reductase) was assayed by an endpoint assay following
10-12 minute incubations using the conditions described previously (17).
Mitochondrial yield is based on the fraction of total succinate:INT
reductase activity in the crude homogenate recovered in the final

mitochondrial suspension.

84

85

Protein concentrations in all samples from supernatants and washes
during the mitochondria/mitoplast isolations, mt-CK solubilization from
mitOplasts and ATP-affinity chromatography steps were determined by
fluorescamine assays (18); protein concentrations in samples from the
procion red-agarose chromatography step were determined by a modified
Lowry method (19). Protein in the final mitoplast suspension was also
determined by fluorescamine assay following a 1:1 solubilization in IN
NaOH for one hour followed by a 10-fold dilution. Bovine serum albumin
in the appropriate buffer was used as a standard for all protein
determinations.

Cellulose Acetate Electrophoresis

 

Creatine kinase isozymes were separated on 2.5 x 17 cm Gelman
Sephraphore III cellulose polyacetate electrophoresis strips in 0.06 M
TRIS-barbital, pH 8.8, 25 mM e-mercaptoethanol as previously described
(10). The electrophoresis buffer contained 1% Triton X-100 to allow
creatine kinase associated with mitochondria to migrate from the origin.
Electrophoresis proceeded for two hours at 300 V at 6°C. Following
electrophoresis, the strips were stained for creatine kinase isozymes as
previously described (20) and scanned on a Gelman ACD-18 automatic
computing densitometer.

Polyacrylamide Gel Electrophoresis

 

Homogeneity and subunit molecular weights of the two pools of CK
activity were determined using sodium dodecyl sulfate-polyacrylamide gel
(10% polyacrylamide, 0.27% bisacrylamide) electrophoresis (SDS-PAGE) in
B-mercaptoethanol according to the method of Laemmli (21). Molecular
weight standards were a mixture of phosphorylase 8 (94,000), bovine

serum albumin (68,000), catalase (58,000), fumarase (50,000), aldolase

86

(40,000), malate dehydrogenase (34,000), carbonic anhydrase (29,000) and

soybean trypsin inhibitor (21,000). Gels were stained overnight in 0.2%

Coomassie brillant blue dissolved in 95% ethanol, acetic acid and water

(50:10:40) and destained with 7% acetic acid.

PURIFICATION PROCEDURE

The pH of all buffers was measured at 20°C. All purification steps
were performed at 4°C unless otherwise noted.

Isolation of Mitochondria

 

Frozen chicken breast muscle (500 g) was thawed for 24 hours at
4°C. Large pieces of fat and connective tissue were removed and the
muscle chopped into approximately 1 inch cubes. The muscle was weighed
and blended in a Haring blender at low speed for three 15-second
intervals in 3000 ml 250 mM sucrose, 10 mM EDTA, 60 mM KCl, 25 mM
B-mercaptoethanol and 40 mM imidazole-propionate, pH 7.0 (homogenization
relaxing buffer). The low-speed supernatant, resulting from
sedimentation of the muscle debris at 600 xg x 10 minutes, was poured
through two layers of cheese cloth to remove lipids and loose cell
debris. Centrifugation of this supernatant at 20,000 xg x 10 minutes
resulted in mitochondria-enriched pellets. These pellets were
resuspended in 500 ml homogenization relaxing buffer and recentrifuged
at 20,000 xg x 10 minutes. Two additional washes with 250 and 125 ml
relaxing buffer, respectively, resulted in a mitochondrial pellet with a
wet weight of approximately 30 grams. Cellulose acetate electrophoresis
of samples from the low-speed centrifugation and mitochondrial washes
indicates only MM-CK is solubilized during homogenization, mitochondria

isolation and further washes of the mitochondria; no detectable release

87

Figure 1.

88

Cellulose acetate electrophoresis of creatine kinase isozymes
at various stages in the purification of mitochondrial CK
from chicken breast muscle. Samples were from A) the
low-speed supernatant, B) the last mitochondrial wash with
homogenization relaxing buffer, C) the supernatant from the
initial mitoplast sedimentation, D) the phosphate solubilized
extract, E) the pool of nonbound CK activity during Procion
Red-agarose chromatography and F) the pool of CK activity
eluting from the Procion Red-agarose column with 2M NaCl.

BB, MB, MM and mt indicate the position of brain type, hybrid
brain and muscle type, muscle type and mitochondrial CK

isozymes.

 

89

BBMB lVlVl MT

 

90

of mt-CK from mitochondria occurs during these procedures (Figures 1A
and 1B).

Mitoplast Preparation

 

The preparation of mitoplasts involved removal of the outer mito-
chondrial membrane by first swelling the outer membrane in a hypotonic
solution of 10 mM TRIS-HCl, pH 7.5 at 0°C for 5 minutes. Addition of a
hypertonic solution (1.75 M sucrose, 2 mM ATP and 2 mM M9504) to the
swollen mitochondria at 0°C for 5 minutes caused shrinkage of the inner
membrane and matrix. This osmotic treatment then allowed removal of the
outer membrane by mild sonication (Branson Sonifier at 3 amperes for 20
seconds). Mitoplasts were then sedimented at 20,000 xg x 20 minutes.
Cellulose acetate electrophoresis of a sample of the resulting superna-
tant indicates no detectable release of mt-CK during removal of the
outer mitochondrial membrane; MM-CK is the only isozyme detected on
electrophoretic strips (Figure 1C).

Solubilization of Mitochondrial Creatine Kinase

 

The mitoplasts were resuspended in 75 ml 100 mM sodium phOSphate,
pH 6.5 containing 1 mM EDTA and 25 mM B-mercaptoethanol. The suspension
was stirred slowly overnight at 4°C to allow solubilization of mitochon-
drial creatine kinase before centrifugation at 20,000 xg x 20 minutes to
remove mit0plasts. The resulting supernatant containing mt-CK was
saved, the pellet resuspended in in 50 ml 100 mM sodium phosphate buffer
and the suspension recentrifuged to recover remaining mt-CK. These two
supernatants were combined, the pH adjusted to 8.4 and the solution made
0.25% in Triton X-100 and 1% in sodium deoxycholate. Mt-CK accounts for
75% of the total CK activity in the phosphate solubilized extract
(Figure 10); the extract has a specific activity of 21 IU/mg protein
(Table I).

 

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Procion Red-Agarose Chromatography

 

The phosphate solubilized extract was loaded directly onto a 9 ml
column of procion red-agarose equilibrated with 50 mM TRIS-HCl, pH 8.4,
1 mM EDTA, 25 mM B-mercaptoethanol, 0.25% (w/v) Triton X-100 and 1%
(w/v) sodium deoxycholate. A representative elution profile of CK
activity from a column of Procion Red-agarose in 0.25% Triton X-100 and
1% deoxycholate appears in Figure 2. Washing the loaded column with two
column volumes of equilibration buffer removes contaminating MM-CK
(Figure 1E) and other non-bound proteins (Figure 2). When CK activity
is no longer detected in the eluant, mt-CK is eluted with equilibration
buffer containing 2M NaCl. Fractions containing the peak of CK activity
were pooled and dialyzed against two to three changes of 20 mM HEPES, pH
7.5, 1 mM EDTA, 25 mM s-mercaptoethanol and 0.25% Triton X-100 for at
least 24 hours to remove deoxycholate and NaCl. The pool of CK activity
from the Procion Red-agarose column contains only the mt-CK isozyme
(Figure 1F) with a specific activity of 35 IU/mg (Table I).

Agarose-Hexane-ATP Affinity Chromatography

 

Following dialysis, the pool of mt-CK activity was applied to a 2
ml column of agarose-hexane-adenosine 5' triphosphate equilibrated with
the previously described HEPES buffer. The column was washed with 25-30
ml buffer to remove non-bound proteins and then was eluted while
collecting 0.5 ml fractions with a 40 ml gradient of 0-50 mM sodium
phosphate in 20 mM HEPES, pH 7.5, 1 mM EDTA, 25 mM s-mercaptoethanol and
0.25% Triton X-100 to remove the first pool of mt-CK activity. This
gradient was immediately followed by another 40 ml gradient of 50—200 mM
sodium phosphate in the same buffer to remove the second pool of mt-CK

activity. The result of such an elution appears in Figure 3. Samples

Figure 2.

93

Elution profile of creatine kinase activity and protein
during Procion Red-agarose chromatography. Cellulose acetate
electrophoresis shows the pool of nonbound CK activity
contains MM-CK and some mt—CK; while the pool of CK activity
eluting from the column at approximately 1M NaCl is mt-CK.
(o———o) represent CK activity, (k———A) is protein and (o———o)

is NaCl concentration during a 0-2M NaCl gradient.

94

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95

Elution profile of creatine kinase activity and protein
during agarose-hexane-ATP affinity chromatography. Cellulose
acetate electrOphoresis shows the first pool of CK activity
eluting from the column at approximately 48 mM sodium
phosphate is the slow-moving cathodal form of mt-CK; the
second pool of CK activity elutes at 110 mM sodium phosphate
and is the fast-moving cathodal form of mt-CK. (o———o)
represents CK activity and (-—-—-) is the sodium phosphate
concentration during 0-50 mM and 50-200 mM sodium phosphate

gradients.

 

 

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97

were taken at various critical locations in the elution profile for
isozyme analysis by cellulose acetate electrophoresis. The two peaks of
activity elute from the column at approximately 48 and 110 mM phosphate
and correspond to the slow and fast-migrating cathodal forms of mt-CK,
respectively, on cellulose acetate electrophoresis strips (Figure 4).
The resulting specific activities of the two forms are 123 IU/mg and 276
IU/mg, respectively; the slow-moving form represents 10-20% of total CK
activity and the fast-moving form represents 80-90% (Table I).

Concentration and Storage of the Enzyme

 

A convenient method for concentrating enzyme and replacing the
Triton X-100 with 50% glycerol for long-term storage involves separate
reapplication of each form to a 1 ml agarose-hexane-ATP column. After
binding the enzyme to the affinity column in the presence of Triton, the
column was washed with 25 ml of 20 mM HEPES, pH 7.5, 1 mM EDTA and 25 mM
B-mercaptoethanol to the column removes Triton X-100 (less than 1%
remains). Reequilibration of the column with HEPES buffer containing
50% glycerol and elution of the enzyme from the column with
HEPES-glycerol buffer containing 200 mM sodium phosphate concentrates
the enzyme 20-fold. Dialysis of the concentrated enzyme against the
HEPES-glycerol buffer removes the sodium phosphate with only about 10%
loss of activity. Storage of the enzyme in this form at -20°C for
several months minimizes the loss of activity over time. Both forms of

mt-CK may be concentrated and stored in this manner.

 

Figure 4.

98

Cellulose acetate electrophoresis of the two forms of
mitochondrial creatine kinase after agarose-hexane-ATP
affinity chromatography. Samples were from A) Pool 1, B)

Pool 11 and C) a mixture of Pool I and II.

 

99

 

DISCUSSION

Phosphate Solubilization of Mitochondrial Creatine Kinase

Incubation of mit0plasts with 100 mM sodium phosphate releases
mt-CK. This result agrees with several reports showing the association
of mt-CK with the inner mitochondrial membrane and inorganic phosphate
release of mt-CK in a reversible process (5, 9 and 14). Thus,
purification procedures (10-13) take advantage of this phosphate-
dependent solubilization to selectively extract mt-CK with minimal
contamination of many mitochondrial proteins.

Procion Red-Agarose Chromatography

 

Dye-ligand chromatography represents a relatively new approach to
the purification of proteins. A major advantage of dye-ligand
chromatography over traditional affinity chromatography derives from the
nonbiodegradable nature of the dyes cross-linked to the support medium
(22). This feature allows direct application of crude extracts to the
columns and thus dye-ligand chromatography serves as an excellent
initial purification step for many proteins. Furthermore, the use of
increased ionic strength or a specific bioligand for elution of the
desired protein from the column creates versatility in the method's
specificity for the protein of choice (23). Thus, dye-ligand
chromatography has become a successful variant of affinity

chromatography.

100

 

 

101

Much speculation concerning the nature of the protein-dye
interaction exists in the literature. Thompson gt a1. (24) originally
concluded from kinetic and spectral studies that the dyes may interact
with a dinucleotide fold present in the majority of bound proteins.
However, many proteins such as interferon (25,26) and human serum
albumin (27,28) lack this specific nucleotide-binding domain, yet also
interact with the dyes; thus, this concept represents an
oversimplification. Present evidence indicates the dyes, containing
multiple aromatic and sulphonate groups (28), interact with a
hydrophobic domain on a protein in the vicinity of positively charged
residues (28,29). These features include, but are not exclusive to,
nucleotide requiring enzymes.

Chromatography of mt-CK using dye-ligand chromatography in the
presence of Triton X-100 requires certain modifications of conventional
methods because mt-CK fails to bind to Procion Red-agarose in 0.25%
Triton X-100. However, consistent with the thesis proposed by
Stellwagon (30), addition of 1% deoxycholate to the phosphate solubi-
lized extract allows binding of mt-CK to the Procion Red-agarose; MM-CK
does not bind. Presumably, deoxycholate forms mixed, charged micelles
with the Triton X-100 to reduce the affinity of the mixed micelle for
the dye and allows binding of the dye to the hydrophobic domain of the
protein (30). Thus, Procion Red-agarose chromatography affords a
convenient method for separating MM-CK from mt-CK even though only 42%
of the estimated mt-CK activity in the phosphate solubilized extract is
recovered in the peak of activity. The presence of some mt-CK in wash
fractions (Figure 1E) and the recovery of at least 90% of the CK

activity loaded onto the column in all fractions suggests not all mt-CK

102

binds to the column. We cannot distinguish from cellulose acetate
electrophoresis whether preferential loss of one form or the other
occurs during this step. Figure 2 and Table I present data from two
different preparations of mt-CK. Thus, the lack of total mt-CK binding
may be the result of column overloading for the preparation of mt-CK in
Table I because little mt-CK was lost during the Procion Red-agarose
step from the preparation shown in Figure 2.

The elution pattern of protein from the Procion Red—agarose column
(Figure 2) shows a group of proteins in the phosphate-solubilized
extract does not bind the Procion Red-agarose, whereas another group
binds and coelutes with the peak of CK activity during the NaCl
gradient. Since minimal protein elutes outside the peak of activity,
batch elution of mt-CK from the column with 2M NaCl, rather than a 0-2 M
NaCl gradient, provides a simple and quick method of elution. Ne
presently use this batch elution procedure for all mt-CK preparations.

Agarose-Hexane—ATP Affinity Chromatography

 

Hall §t_al, (10,11) originally used agarose-hexane-ATP affinity
chromatography for the purification of mt-CK from bovine heart by
elution of mt-CK from the column with an ATP-NaCl gradient. Because
phOSphate is particularly effective in solubilizing mt-CK from mitochon-
dria, a phosphate gradient at pH 7.5 was used instead of an ATP-NaCl
gradient. This procedure results in the separation of two mt-CK forms
(Figure 3 and 4).

Purity and Molecular Weight Analysis of the Two Forms of Mitochondrial

 

Creatine Kinase

 

The SDS-PAGE of Pool I (lanes 1-2) and Pool II (lanes 3-5) along

with low molecular weight standards (lane 6) are shown in Figure 5.

Figure 5.

103

SDS-polyacrylamide gel of Pool I (lanes 1 and 2) and Pool II
(lanes 3—5) mt-CK from agarose-hexane-ATP affinity chromatog-
raphy. Approximately 15 pg of protein was subjected to
electrophoresis using a 10% acrylamide, 0.27% bisacrylamide
gel, 0.75 mm thick. Molecular weight standards (lane 6) were
a mixture of phosphorylase B (94,000), bovine serum albumin
(68,000), catalase (58,000), fumarase (50,000), aldolase
(40,000), malate dehydrogenase (34,000), carbonic anhydrase
(29,000) and soybean trypsin inhibitor (21,000).

104

LANE 1 2

 

105

Pool II contains one protein band, essentially 90% pure, with a subunit
molecular weight of 46,500. Oh the other hand, Pool I contains two
major protein bands and some minor bands. Other preparations of Pool I
contain different proportions of contaminating proteins (data not
shown). The major protein in most Pool I preparations has an Rf value
equivalent to the band of protein in Pool 11; the two forms of mt-CK
probably have the same molecular weight. The mt-CK specific activity of
Pool 1, 123 IU/mg, represents only 44% of the specific activity of Pool
II, 276 IU/mg. This result is consistent with the SDS-PAGE data for
Pool I, unless the mt-CK form in Pool I is a less active form of the
enzyme. Therefore, the presence of contaminating protein bands in
SDS-PAGE gels and the relatively low specific activity of Pool I indi-
cates an additional purification step is necessary to completely purify
to homogeneity the CK activity in Pool I. Hall et_al, (11) report
homogeneous preparations of two forms of mt-CK from bovine heart with
specific activities of 97 and 113 IU/mg and subunit molecular weights of
35,000 i 4000. Canine heart mt-CK preparations of a single form result
in a specific activity of 310 IU/mg with a subunit molecular weight of
about 42,000. Species variations may account for these discrepancies.

Partial Hydrophobic Nature of Mitochondrial Creatine Kinase

 

Chicken muscle mt-CK appears to have a substantial hydrophobic
domain. We base this conclusion on differences in behavior of mt-CK
in the absence and presence of the non-ionic detergent Triton X-100.
First, 0.25% Triton X-100 in all buffers prevents excessive loss of
enzyme activity during a purification or dialysis step, presumably due
to hydrophobic interactions allowing the enzyme to adhere to column

materials and other surfaces in the absence of Triton. Second, a salt

106

gradient from 0-3 M NaCl will not release all the bound mt-CK from a
Procion Red-agarose chromatography column (data not shown), whereas 1%
Triton X-100 in a 0-2 M NaCl gradient elutes all CK activity from the
column in one peak of approximately 1 M salt. Because of these
observations, we include Triton X-100 in all buffers during the
purification procedure.

Aggregation Phenomenon

 

Hall gt_al. (10) suggest that two mt-CK forms in bovine heart
result from the reversible oxidation of protein sulfhydryl groups as
well as aggregate formation of the more oxidized form. The two forms of
mt-CK in human heart may also represent differences in the state of
aggregation (32). However, our preparations utilize buffers containing
25 mM s-mercaptoethanol and 0.25% Triton X-100. The increase in the
concentration of B-mercaptoethanol from 10 mM (10) to 25 mM should
effectively eliminate oxidation of the sulfhydryl groups. In fact, we
observe no interconversion of forms with the addition of fresh
B-mercaptoethanol to either of the pools of mt-CK activity. In
addition, 0.25% Triton X-100 should reduce protein-protein hydrophobic
interactions to prevent aggregate formation.

Behavior of the Two Forms of Mitochondrial Creatine Kinase in 50%
Glycerol

The reapplication of each mt-CK form to the agarose-hexane-ATP
column, substitution of the HEPES buffer containing 0.25% Triton X-100
with HEPES buffer containing 50% glycerol and elution from the column
with 200 mM sodium phosphate concentrates both forms approximately
20-fold and replaces Triton X-100 with a stabilizing buffer for

long-term storage.

107

However, the behaviors of the two forms of mt-CK change as a result
of the replacement of Triton X-100 with 50% glycerol and storage at
-20°C. For example, cellulose acetate electrophoresis of a mixture of
the two forms results in only one band of mt-CK activity. We can not
distinguish whether the electrophoretic mobilities of each separate form
change with the replacement of Triton X-100 with 50% glycerol. In
addition, reapplication of a mixture of the two forms on
agarose-hexane-ATP and elution of the mt-CK activity from the column
with the consecutive 0-50 mM and 50-200 mM sodium phosphate gradients,
containing 0.25% Triton X-100, results in a single peak of activity
eluting at approximately 70 mM sodium phosphate; previous elution of
Pool I and II from the column before replacement of Triton with 50%
glycerol occurred at 48 and 110 mM phosphate, respectively.

Estimation of the Amount of Mitochondrial Creatine Kinase in Chicken

 

Breast Muscle

 

The data in Table I show the purification of the two forms of
mitochondrial creatine kinase results in a 49% recovery during the steps
following phosphate solubilization of mt-CK from mitoplasts. The
recovery of succinate:INT reductase, a mitochondrial marker enzyme, in
the final mitoplast pellet ranges from 5-10% of the total succinate:INT
reductase in the crude homogenate for most of our preparations. The
representative experiment outlined in Table I results in a 4% recovery
of succinate:INT reductase from 487 grams of muscle containing 1.33 x
106 IU total CK activity. Thus, total mt-CK activity in 487 g of
muscle is 1.88 x 104 IU and represents 1.4% of the total CK activity
in muscle or 38.5 10 mt-CK per gram muscle. The major loss of

mitochondria and thus mt-CK activity, occurs during the low-speed

108

centrifugation; only 10-20% of total succinate:INT reductase is in the
low-speed supernatant and suggests 80-90% of the mitochondria remain
associated with the muscle debris.

These experiments provide a method to separate two electrophore-
tically distinct pools of mt-CK activity from chicken breast muscle
mitochondria. One pool of activity corresponds to the fast-moving
cathodal form (Pool II) during cellulose acetate electrophoresis and is
90% homogeneous enzyme with a specific activity of 276 IU/mg. The pool
corresponding to the slow-moving cathodal form (Pool 1) remains only

40-50% pure with a specific activity of 123 IU/mg.

10.

11.

REFERENCES

Eppenberger, H.M., Dawson, 0., and Kaplan, N.0. (1967) J. Biol.
Chem. 242, 204-209.

Neumeier, D. (1981) in Creatine Kinase Isoenzymes (Lang, H., ed.),

 

Springer-Verlag, New York, pp. 85-109.
Jacobs, H., Heldt, H.W., and Klingenberg, M. (1964) Biochem.

Biophys. Res. Commun. 16, 516-521.

 

Sobel, B.E., Shell, M.E., and Klein, M.S. (1972) J. Mol. Cell.

 

Cardiol. 4, 367-380.

Jacobus, W.E., and Lehninger, A.L. (1973) J. Biol. Chem. 248,

 

4803-4810.
Scholte, H.R., Weijers, P.J., and Wit-Peeters, E.M. (1973) Biochim.

Biophys. Acta 291, 764-773.

 

Baba, N., Kim, S., and Farrell, E.C. (1976) J. Mol. Cell. Cardiol.

 

8, 599-617.
Ogunro, E.A., Peters, T.J., Wells, 0., and Hearse, D.J. (1979)

Cardiovascular Res. 13, 562-567.

 

Vial, C., Font, B., Goldschmidt, D., and Gautheron, D.C. (1979)
Biochem. Biophys. Res. Commun. 88, 1352-1359.

 

Hall, N., Addis, P., and DeLuca, M. (1977) Biochem. Biophys. Res.

 

Commun. 76, 950-956.

Hall, N., Addis, P., and DeLuca, M. (1979) Biochemistry 18,

 

1745-1751.

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2]..

22.

23.
24.

25.

26.

27.

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Roberts, R., and Grace, A.M. (1980) J. Biol. Chem. 255, 2870-2877.

 

Roberts, R. (1980) Experientia 36, 632-634.

 

Hall, N., and DeLuca, M. (1980) Arch. Biochem. Biophys. 201,

 

674-677.
Oliver, I.T. (1955) Biochem. J. 61, 116-122.

 

Rosalki, S.B. (1967) J. Lab. Clin. Med. 69, 696-705.

 

Baxter, J.H., and Suelter, C.H. (1983) Muscle and Nerve 6,

 

187-194.

Udenfriend, S., Stein, S., Bohlem, P., and Dairman, w. (1982)
Science 178, 871-872.

Markwell, M.A.K., Haas, S.M., Tolbert, N.E., and Bieber, L.L.

(1981) in Methods in Enzymol. (Lowenstein, J.M., ed.), Vol. 72,

 

Academic Press, New York, pp. 296-303.
Hall, N., and DeLuca, M. (1976) Anal. Biochem. 76, 561-567.

 

Laemmli, U.K. (1970) Nature(London) 227, 680-685.

 

Tucker, R.E., Babul, J., and Stellwagon, E. (1981) J. Biol. Chem.

 

256, 10993-10998.
Turner, A.J. (1981) Trends in Biol. Sci. 6, 171-173.

 

Thompson, S.T., Cass, K.H., and Stellwagon, E. (1975) Proc. Natl.

 

Acad. Sci. USA 72, 669-672.

 

Maeyer-Guignard, J., Thang, N.Y., and Maeyer, M.A. (1977) Proc.
Natl. Acad. Sci. USA 74, 3787-3790.

 

Jankowski, H.J., Ansen, H.H., Sulkowski, E., and Carter, M.A.

(1976) Biochemistry 15, 5182-5187.

 

Travis, J., Brown, J., Tewksbury, D., Johnson, D., and Pannell, R.

(1976) Biochem. J. 157, 301-306.

 

28.

29.

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32.

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27-34.

Beissner, R.S., and Rudolph, F.B. (1978) Arch. Biochem. Biophys.
189, 76-80.

Robinson, J.B., Jr., Storttmann, J.M., and Stellwagon, E. (1981)

Biochemistry 78, 2287-2291.

 

Robinson, J.B., Strottmann, J.M., Hick, D.G., and Stellwagon, E.
(1980) Proc. Natl. Acad. USA 77, 5847-5851.

 

Kanemitsu, F., Kawanishi, I., and Mizushima, J. (1982) Clin. Chim.

Acta 119, 307-317.

CHAPTER IV

Muscle Cell Cultures from Chicken Breast Muscle have
Increased Activities of Adult Muscle Enzymes when

Incubated at 4l°C Compared to 37°C

Vickie 0. Bennett, H. David Husic, Jeffrey H. Baxter

and Clarence H. Suelter.

Department of Biochemistry
Michigan State University

E. Lansing, Michigan 48824

Running Title: Myoblast Incubation Temperature

112

INTRODUCTION

Myogenic cells from developing skeletal muscle of avian and
mammalian embryos differentiate jn_yjt£g_in the absence of neuronal and
circulatory influences to form multinucleated, cross-striated,
contractile muscle fibers (Holtzer, Jones and Yaffe, l975). These

morphological similarities to muscle development_in vivo make muscle

 

cell culture an attractive experimental system for the analysis of
muscle differentiation in normal and diseased states. However, muscle
differentiation in yitrg does not entirely reflect muscle development
in vivo. For example, normal chick muscle cells in culture have
significantly lower specific activities of creatine kinase (E.C.
2.7.3.2) and AMP deaminase (E.C. 3.5.4.6) than the specific activities
of these enzymes in yjy9_(Young gt_al, l981). In addition, the
transition of creatine kinase from the embryonic isozyme to the isozyme
characteristic of adult muscle remains incomplete in yitrg (Hauschka,
1972). Early chicken embryonic muscle contains only brain-type
creatine kinase isozyme (BB-CK). As development progresses, total
creatine kinase activity increases. This increase is principally due
to the adult muscle type isozyme (MM-CK) and levels of the BB-CK
isozyme decrease. In adult muscle, greater than 95% of the creatine
kinase is MM-CK, a trace is MB-CK (hybrid isozymic form); BB-CK is not

found (Lough and Bischoff, I977 and Perriard §t_al,, 1978). However,

113

114

myogenic chicken breast muscle cells grown in culture do not complete
the isozymic shift seen in vivo; significant amounts of MB (20-25%) and
BB (TO-12%) are still present at TD days Em (Lough and Bischoff,
l977 and Perriard §t_al,, 1978). The abundance of the brain CK subunit
(B-CK) still found in muscle cultures indicates these cultures are not
as differentiated as adult muscle tissue in yiyg. Since highly
differentiated muscle cultures may be necessary for the expression of
characteristics of muscular diseases in yitrg, we began an examination
of other culture conditions which might yield more highly
differentiated muscle cells in vitro.

A review of the muscle cell culture literature reveals that chicken
muscle cell cultures are routinely incubated at 37°C. Yet chicken body
temperature is 4l°C (King and Farner, l96l). Thus we reasoned that
myogenic cultures might differentiate to a state more reflective of

normal adult muscle in vivo if they were grown at the natural body

 

temperature of the chicken. In this communication, a 13-day time
course evaluating total CK specific activity, isozyme distribution and
fusion levels reveals cultures incubated at 41°C differentiate more
rapidly and attain a higher level of differentiation by these criteria

than cultures incubated at 37°C.

MATERIALS AND METHODS

Preparation of Primary Muscle Cell Cultures

 

Breast muscles were dissected from ll-l2 day normal chicken embryos
(White Leghorn, Omega Chicks, Haslett, M1) or chicken embryos from
normal (line 412) and dystrOphic (line 413) chicken lines (Department
of Avian Sciences, University of California, Davis California). The
muscles were placed in Eagle's minimal essential medium (GIBCO, EMEM)
supplemented with 2.0 ug/ml amphotericin B (GIBCO), lOO units/ml
penicillin, lOO ug/ml streptomycin (GIBCO, penicillin-streptomycin
solution), 0.2 ug/ml insulin (Sigma) and 25 ug/ml conalbumin (Sigma,
Type II).

Myoblasts were dissociated mechanically and filtered through a 35 u
nylon filter to remove bones and clumps of cells. The resulting muscle
cell suspension was preplated at a density of 1.0 x l07 cells per l00
mm plastic tissue culture dish (Corning). Cultures were incubated for
two hours in an atmosphere containing 5% C02/95% air at 90% relative
humidity at 37°C. After two hours of incubation, the medium containing
the unattached myoblasts from the preplates (Richler and Yaffe, l970)
were decanted in sets of three and combined into one culture dish.
Aliquots of 0.5 ml were withdrawn and placed into 35 mm culture dishes
coated with 6.0 ug/cm2 collagen (GIBCO, purified) (Young, Goll and

Stromer, T975) and containing l.5 ml of the culture medium as described

115

 

116

above supplemented to a final concentration of lO% (v/v) with horse
serum (GIBCO) (complete EMEM) to promote myoblast attachment (Puri and
Turner, T978). These subcultures were incubated at either 37°C or 4l°C

with daily media changes.

Preparation of Chick Embryo Skin Fibroblast Cultures

 

The skin layer from the back of ll-l2 day chicken embryos was
removed and placed into a 10 ml phosphate buffered saline (PBS)
solution (0.2 g/l KCl, 0.2 g/l KCl, 0.2 g/l KH2P04, 8.0 g/l NaCl,

0.78 g/l NazHPO4 adjusted to pH 7.35 with NaOH) containing l mg/ml
collagenase (T70 units/mg Sigma), l00 ul 2.5 mg/ml DNAse I
(Worthington) in l0‘3M HCl and lOO ui 0.4 M glucose. The cells

were dissociated by shaking for l hour in a 37°C room and then filtered
through a 35 u nylon filter. The filtrate was centrifuged at full
speed in a clinical centrifuge for 5 minutes. The supernatant was
discarded and the pellet resuspended in 5 ml complete EMEM as described
above. The resulting cell suspension was plated at a density of 2.0 x
l06 cells per lOO mm plastic tissue culture dish (Corning) in 4 ml
medium. Cultures were incubated at either 37°C or 4l°C in an
atmosphere containing 5% C02/95% air at 90% relative humidity with
daily media changes. Cells reached confluency by three days in

culture.

Preparation of Cell Culture Homogenates

 

Three culture dishes from each set of five culture dishes grown at
37°C or 4l°C were rinsed twice with cold buffered salt solution. (B55;

137 mM NaCl, 2.7 mM KCl, 1.0 mM CaClZ, 1.0 mM NaH2P04, 1.36 mM

117

Na2HP04, 6.0 mM NaHC03 and 5.5 mM glucose adjusted to pH 7.4).

The cells from the three combined plates were suspended into 0.3 ml
total extraction buffer (0.18 M KCl, 0.054 M KH2P04, 0.035 M

KZHP04, pH 6.5) with a plastic spatula and were homogenized with 20
strokes in a l ml ground glass Ten-Broeck homogenizer. Homogenates
were centrifuged for l0 min in an Eppendorf centrifuge. Enzyme
activities and protein concentrations were then determined as described
below. Each time point of the time course contained four sets of five

culture dishes.

Evaluation of Cell Fusion

 

The remaining two culture dishes from each set of five dishes at
each incubation temperature were rinsed twice with cold BSS, fixed in
methanol and stained with Giemsa stain as previously described (Young,
Goll and Stromer, l975). The total number of nuclei and the number of
nuclei found in myotubes were counted microscopically at l00x
magnification. Percent fusion is defined as the percent of total
nuclei in myotubes and was determined in ten randomly chosen fields on

each culture dish.

Preparation of Crude Muscle Extracts

 

Crude muscle extracts were prepared by homogenizing breast muscle
from l4 day ex gyg chickens in a Waring blender using 3.3 ml extraction
buffer per gram tissue. The homogenate was centrifuged at 6,000 x g x
20 min. Enzyme activities and protein concentrations were then

determined as described below.

118

Enzyme Assays and Protein Determination

 

Creatine kinase activity was determined spectrophotometrically at
340 nm at 30°C using the CK assay mix from Sigma Chemical Company.
Total AMP deaminase (AMPDA) was determined spectrophotometrically by
following the change in absorbance at 290 nm as AMP is deaminated to
form IMP (Smiley, Berry and Suelter, 1967). The assay mixture contains
5.0 mM AMP, 0.l5 M KCl and 50 mM 2(N-morpholino)ethanesulfonic acid
(MES) titrated to pH 6.5 with tris (hydroxy-methyl)aminomethane (TRIS).
Protein was determined by the method of Lowry (Lowry gt 21°, l95l)

using bovine serum albumin (Sigma) as a standard.

Cellulose Acetate Electrophoresis

 

Creatine kinase isozymes were separated on 2.5 x l7 cm Gelman
Sephraphore III cellulose polyacetate electrophoresis strips in 0.06 M
TRIS-barbital, pH 8.8 (Gelman, high resolution), 25 mM
s-mercaptoethanol as previously described (Hall, Addis and DeLuca,
l977). The electrophoresis buffer contained 1% Triton X-l00 to allow
migration of mitochondrial CK from the origin. Electrophoresis was
performed for two hours at 300 V at 6°C. Following electrophoresis the
strips were stained for CK isozymes and scanned as previously described
(Hall and DeLuca, l976).

Isozyme positions were identified by running completely or
partially purified fractions containing only MM, MB, BB or
mitochondrial (mt-CK) independently on electrophoretic strips and
staining for CK activity. In control electrophoretic strips, creatine
phosphate was omitted from the CK assay mix to check for interference

by adenylate kinase.

RESULTS AND DISCUSSION

Figures 1A, 18 and 1C show creatine kinase specific activities, the
percent nuclei in myotubes (fusion levels) and total protein per plate
for cultures incubated at 37°C and 41°C during a 13-day time course
experiment. The data in Figure 1A indicates the 41°C temperature
causes an early increase in the specific activity of creatine kinase.
This increase in CK specific activity peaks on Day 4 in_yitrg_at a
level that is twice the highest CK specific activity found at any time
during the time course in cultures incubated at 37°C. After peaking at
4 days, the CK specific activity in the 41°C cultures begins to fall
and by 8-9 days is less than the specific activity in the 37°
cultures.

The time course in Figures 1A, 18 and 1C is a representative
experiment from a total of three identical experiments. The shapes and
proportions of the curves for this experiment are characteristic of the
data obtained for all three, even though the specific activities in
this experiment are somewhat higher than the other two and a 1.5 to
2.0-fold increase of CK specific activity in the 41°C cultures compared
to the 37°C cultures is generally still apparent on Day 7. Differences
in horse serum lots may contribute to this variability. Thus, to
normalize for the variability from one experiment to another we

determined the average fold increase of CK specific activity in

119

Figure 1.

120

(A) Creatine kinase specific activities, (B) percent nuclei
in myotubes and (C) total protein per plate from a
representative 13-day time course experiment for muscle cell
cultures incubated at 37°C (--—o) and 41°C (-—-). Each
point in Figures 1A and 1C represents the average of data
from four cell culture homogenates. Each homogenate
contains a suspension of cells in extraction buffer (See
Materials and Methods) from three combined culture dishes.
Each point in Figure 18 represents the average of data from

eight culture dishes (See Materials and Methods).

 

121

 

 

 

A

p p -

 

 

 

- h p p

 

 

 

0.
6

b p h
0. O. O 0. 0. CW 0 0 0 0

5 4 a 2 l
323: :23 388 go

I 8 6 4 2
20.3...373232 c. .332 283m

2

m.
2
3

E. 22m com 522.. .20...

8 l2 l6

Days in Culture

4

122

cultures incubated at 41°C compared to 37°C (Figure 2). This normali-
zation shows the CK specific activity in 41°C cultures is greater than
in 37°C cultures until after Day 7 in vitro. By Day 8, the cultures
have approximately equal CK specific activities (fold increase equals
1.0) and at later times the CK specific activity in 41°C cultures is
less than in 37°C cultures.

The data in Figure 1B and 1C may explain in part the decline in
total CK specific activity at later days in the time course. Figure 18
shows a rapid decline in fusion levels over time in the 41°C cultures
compared to the constant high level of fusion in the 37°C cultures.
This result is consistent with an observed increase in the prolifera-
tion rate of mononucleated cells. These mononucleated cells could
include unfused differentiated myoblasts, undifferentiated myoblasts or
contaminating fibroblasts. At this point, we do not know the identity
of the major cell types represented, but we suspect they are fibro-
blasts since the majority of the mononucleated cells attain the three-
pronged star shape generally considered characteristic of fibroblasts.

Figure 1C reveals a decline in fusion level accompanies a rapid and
continual increase in total protein per plate in cultures incubated at
41°C. This result is also consistent with an increase in the
proliferation rate of mononucleated cells at the 41°C incubation
temperature. Fibroblast control cultures in five independent
experiments reveal that the CK specific activity in fibroblasts
incubated at 37°C and 41°C are 0.23 t 0.10 and 0.15 i 0.08 IU/mg,
respectively; 95% is BB-CK. These activities represent about 1% of the
specific activity in Day 7 muscle cultures in agreement with results

obtained by Lough and Bischoff (1977). Thus a significant number of

123

Figure 2. The average fold increases of CK specific activity in
cultures incubated at 41°C compared to 37°C from three

identical 13-day time course experiments.

 

124

 

 

 

 

 

 

3.0 -

p
0.
2

333:. 20.... 3222

l6

l2‘

Days In Culture

125

fibroblasts would decrease the specific activity of CK and the
percentage of MM-CK in the 41°C cultures.

A further explanation for the decline in total CK specific activity
at later days in the tine course may be an increase in the rate of the
muscle cell aging process at 41°C. Observations supporting this
possibility include the relative periods of myotube formation, muscle
contractions and myotube detachment at the two incubation temperatures.
For example, myotube formation occurs approximately one day earlier and
muscle contractions begin approximately two days earlier in muscle
cultures incubated at 41°C than those incubated at 37°C. In addition,
"wotube detachment from the plate is visible by Day 7 in 41°C cultures
whereas myotube detachment does not occur in 37°C cultures until 12-13
days. Thus, an accelerated rate of the senescence process in muscle
cells at 41°C is consistent with the early peak of CK specific activity
at 4 days and the early decline of CK specific activity in cultures
incubated at 41°C compared to the Day 9 peak and later decline of CK
specific activity in cultures incubated at 37°C. Therefore, a
combination of early myotube degeneration and an increase in the
proliferation rate of mononucleated cells at 41°C may account for the
decline in CK specific activity at later days in the time course.
However, the point drawn from these results is the CK specific activity
on Day 4 in the 41°C muscle cell cultures is never attained by the 37°C
cultures at any time during the time course in spite of an early
decline of CK specific activity at 41°C.

The isozyme distributions of CK isozymes (Figure 3) for each day in
culture (Table 1) reveal an increase in the percent of MM-CK in the

41°C cultures compared to 37°C cultures until Day 7. On Day 9, the

Figure 3.

126

Densitometer scans of creatine kinase isozymes spearated by
cellulose acetate electrophoresis. Samples were cell
homogenates from cultures incubated at (a) 37°C or (b) 4l°C.
BB, MB and MM indicate the position of creatine kinase
isozymes brain type, hybrid brain and muscle type and muscle

types, respectively.

Relative Fluorescence

127

 

88 MB MM

 

 

 

 

 

 

l l

l
4 3 2 l O I 2
Centimeters from Origin

128

TABLE 1

ISOZYME DISTRIBUTION DURING TIME COURSE IN CULTURES
INCUBATED AT 37°C or 41°C

 

 

 

 

 

%BB %MB %MM
Day 2 37° 65.8 t 7.4 18.0 i 4.8 16.1 i 6.9
41° 67.6 i 0.1 13.6 t 5.9 18.8 i 6.0
P>.5 P>.5 P>.5
Day 4 37° 52.2 i 14.4 31.6 i 9.1 16.2 i 9.8
41° 18.2 i 9.0 32.2 i 8.5 49.6 i 12.7
P<.001 P>.5 P<.001
Day 7 37° 36.8 i 5.3 32.4 i 1.7 30.7 t 5.3
41° 14.1 i 4.5 28.2 i 5.8 57.7 i 8.2
P<.001 .1<P<.2 P<.001
Day 9 37° 34.7 i 9.4 29.2 i 7.8 36.0 i 9.0
41° 23.4 i 5.5 22.0 i 7.9 54.5 i 7.0
.025<P<.05 .1<P<.2 .001<P<.005
Day 13 37° 31.8 i 2.8 36.3 t 2.6 31.9 i 2.4
41° 48.3 i 6.2 14.2 i 4.7 37.4 i 4.1
P<.001 P<.001 .01<P<.025

 

Note: Creatine kinase isozymes were separated by electrophoresis
after applying 7 milliunits of CK activity onto 2.5 x 17 cm cellu-
lose polyacetate strips. Electrophoresis was performed as
described in Materials and Methods. Following electrophoresis the
strips were stained for CK isozymes and scanned on a densitometer.
Data shown above represents the average of data from six indepen-
dent samples for each incubation temperature.

*Determined by student's t-test for paired values.

129

percent of MM-CK in 37°C cultures continues to increase only slightly.
However, the 58% MM-CK in 41°C cultures on Day 7 is never attained by
our 37°C cultures; the highest value is 36%. The isozyme distributions
of our 37°C cultures differ from the reports of Lough and Bischoff,

(1977) and Perriard §t_ l. (1978). These authors report 70% MM-CK by

Day 7 using ion-exchange chromatography to quantitate the relative
percent of each isozyme and 75% MM-CK by Day 11 using immunoabsorption
chromatography, respectively. We do not know why our culture system
and/or isozyme quantitation technique result in lower percentages of
MM-CK in cultures incubated at 37°C than other systems. However, the
effect of the 41°C incubation temperature on CK differentiation would
presumably still be apparent in the other systems.

The decrease in the percentage of MM-CK in the 41°C cultures
beginning on Day 9 accompanies a significant increase in BB-CK and a
continual decrease in MB-CK. The continual decrease in MB-CK, despite
the increase in BB-CK and the decrease in MM-CK, suggests the presence
of two distinct cell p0pulations. One of these populations may be
responsible for the majority of BB-CK while the other is responsible
for MM-CK. This conclusion is consistent with the fact that control
fibroblast cultures produce 95% BB-CK and no MM-CK. In addition,
visual observation of control fibroblast cultures incubated at both
temperatures supports a more rapid proliferation of fibroblasts at 41°C
than at 37°C. Therefore, the rapid overgrowth of the 41°C cultures
with mononucleated cells is likely due to a more rapid proliferation of
fibroblasts at 41°C compared to 37°C. This result and the accelerated
senescence process of muscle cells in the cultures incubated at 41°C

compared to 37°C accounts for the rapid decline in total CK specific

 

 

 

130

activity as well as the decreased percentage of MM-CK after Day 7 in
the 41°C cultures. In other words, the overgrowth of the 41°C cultures
with mononucleated cells and the accelerated degeneration of myotubes
at later times may mask the temperature effect already evident early
in the time course. Since creatine kinase represents a useful marker
for the level of differentiation of myogenic cells in culture (Morris,
Cooke and Cole, 1972; Turner, Maier and Eppenberger, 1974 and Perriard
_t_al,, 1978) we feel the increase in total CK specific activity and
the increase in MM-CK compared to our 37°C cultures reflects the
achievement of a more highly differentiated state when muscle cultures
are incubated at 41°C rather than 37°C.

Day 7 appears to be a convenient period during the tine course for
spot checks of the effect of incubation temperature on the levels of
other muscle specific enzymes and proteins. At this time, CK specific
activity in the 37°C cultures has almost reached its highest value and
the CK specific activity in 41°C cultures still remains higher than in
37°C cultures. Even though the CK specific activity in 41°C cultures
on Day 7 is decreased from its peak value at Day 4, the Day 7 values
for cultures incubated at 37°C are more indicative of the highest CK
specific activity and percent MM-CK. Thus Day 7 seems to be a
convenient and fair comparison time. For example, the specific
activities of another muscle specific enzyme, AMP deaminase (AMPDA), in
seven-day cultures incubated at 37°C and 41°C appear are 0.05 i 0.07
and 0.23 1 0.16 IU/mg, respectively. The specific activities of AMPDA
is increased about four-fold on Day 7 at 41°C compared to 37°C. Since
AMPDA activity is not detected in fibroblasts (data not shown) and

undifferentiated myoblasts also have low levels of AMPDA (Young gt_al.,

131

1981), a significant number of unfused cells would also decrease the
AMPDA specific activity in 41°C cultures. However, the 25-30% Contami-
nation of muscle cultures by undifferentiated myoblasts or fibroblasts
in Day 7 41°C cultures will not account for approximately ten-fold
lower enzyme activities than jn_yjy9_data at 14 days gx_9yg. Even the
CK specific activities in 41°C cultures on Day 4 represent a five-fold

lower activity than the adult 1

vivo levels. Therefore, even though

 

41°C cultures achieve a higher CK specific activity and percentage
MM-CK than 37°C cultures, they still do not completely differentiate to

the adult level i vivo.

 

All of these experiments represent cultures grown in medium
containing conalbumin rather than chick embryo extract. We also
observed a two-fold average increase in CK specific activity and
similar isozyme distributions for twelve 37-4l° experiments in Day 7
cultures grown in medium containing chick embryo extract (data not
shown). The variability of results obtained from different
preparations of chick embryo extract caused us to explore the report
that sciatin, a glycoprotein purified from chicken sciatic nerves,
(Markelonis and Oh, l979) substitutes for chick embryo extract (Oh and
Markelonis, l980). Sciatin is structurally and functionally similar to
ovotransferrin (Oh and Markelonis, l982) which is available as Type II
conalbumin from Sigma. Conalbumin consistently gave reproducible
results in this study.

Finally, Day 7 muscle cell cultures produced from normal (line 412)
and dystrophic (line 413) 12-day chicken embryos reveal no differences
in CK specific activities and isozyme distributions between normal and

dystrophic muscle cell cultures incubated at 37°C. However, Ionasescu

132

gt_al. (1980) show a 30% decrease in total CK specific activity in
muscle cells accompanied by an increase of CK activity in the culture
medium of dystrophic chicken muscle cultures compared to normal
cultures. These discrepancies may be due to the use of different
dystrophic chicken lines. However, the incubation of dystrophic
cultures at 41°C still does not result in the in yitrg expression of in_
.yin dystrophic characteristics such as a decrease in CK specific
activity and increase in the percentages of BB-CK and MB-CK(Weinstock,
Behrendt and Jones, 1977 and Stewart §t_al, 1981).

These experiments show an increase in CK and AMPDA specific
activities and an increase in the percentage of MM-CK in chicken muscle
cell cultures incubated at 4l°C compared to cultures incubated at 37°C.
However, the increased incubation temperature does not result in enzyme
specific activities as high as those observed in adult muscle tissues,
or in a near complete transition of CK isozymes to the adult MM-CK. A
complete or near-complete transition of CK isozymes may be masked by
the presence of an overgrowth of mononucleated cells and an accelerated
rate of myotube degeneration after Day 7 in culture at 41°C. Yet these
results indicate that muscle cell cultures incubated at the
physiological body temperature of the chicken (4l°C) may be more highly
differentiated than those at 37°C. This effect implies the incubation
temperature likely influences other characteristics of myogenic cells
in yiytrg. We feel that highly differentiated muscle cell cultures nay
be necessary for the in yitrg_expression of the characteristics of
muscle diseases or other unique characteristics of muscle tissue.
However, dystrophic muscle cultures incubated at 41°C still do not

express i vivo dystrophic characteristics. Future experiments will

 

133

examine the effect of these incubation temperatures on other

biochemical characteristics indicative of muscle differentiation.

REFERENCES

Hall, N., Addis, P., and DeLuca, M. (l977). Purification of
mitochondrial creatine kinase: two interconvertible forms of the

active enzyme. Biochem. Biophys. Res. Commun. 76, 950-956.

 

 

Hall, N., and DeLucca, M. (1976). Electrophoretic separation and

quantitation of creatine kinase isozymes. Anal. Biochem. 76,

 

56l-567.

Hauschka, S.D. (l972). Cultivation of Muscle Tissue. In: “Growth,
Nutrition and Metabolism of Cells in Culture", Vol. II (G.H.
Rothblat and V.J. Cristofalo; eds.), pp. 67-l30. Academic Press,
New York.

Holtzer, H., Jones, K.W., and Yaffe, D. (l975). Research group on
neuromuscular diseases: a report on various aspects of myogenic
cell culture with particular reference to studies on the muscular

dystrophies. J, Neurol. Sci. 26, ll5-l24.

 

Ionasescu, V., Ionasescu, R., Witte, D., Feld, R., Cancilla, P.,
Kaeding, L., Kraus, L., and Stern, L. (1980). Altered Protein
Synthesis and Creatine Kinase in Breast Muscle Cell Cultures from

Dystrophic Chick Embryos. J. Neurol. Sci.46, 157-168.

 

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135

King, J.R., and Farner, D.S. (l96l). Energy metabolism,
thermoregulation and body temperture. In: "Biology and
Comparative Physiology of Birds" (A.J. Marshall, ed.) Academic
Press, New York.

Lough, J., and Bischoff, R. (l977). Differentiation of creatine
phosphokinase during myogenesis: quantitative fractionation of

isozymes. Develop. Biol. 57, 330-334.

 

Lowry, 0.H., Rosebrough, N.J., Farr, A.L., and Randall, R.J. (195l).

Protein measurement with the Folin phenol reagent. J, Biol. Chem.

 

193, 265-275.
Markelonis, G., and 0h, T.H. (l979). A sciatic nerve protein has a
trophic effect on development and maintenance of skeletal muscle

cells in culture. Proc. Nat. Acad. Sci. USA 76, 2470-2474.

 

 

Morris, G.E., Cooke, A., and Cole, R.J. (1972) Isoenzymes of Creatine

Phosphokinase During Myogenesis I__Vitro. Exp. Cell Res. 74,

 

 

582-584.
0h, T.H., and Markelonis, G.J. (l980). Dependence of ifl vitro
myogenesis on a trophic protein present in chicken embryo extract.

Proc. Nat. Acad. Sci. gsg 77, 6922-6925.

 

 

0h, T.H., and Markelonis, G.J. (l982). Sciatin is a transferrin-like
polypeptide. J, Neurochem. 39, 3l5-320.

Perriard, J.C., Caravatti, M., Perriard, E.R. and Eppenberger, H.M.
(1978). Quantitation of Creatine Kinase Isoenzyme Transitions in
Differentiating Chicken Embryonic Breast Muscle and Myogenic Cell

Culturies by Immunoadsorption. Arch. Biochem. Biophys. 191,

 

90-100.

136

Puri, E.C., and Turner, D.C. (l978). Serum-free medium allows chicken
myogenic cells to be cultivated in suspension and separated from

attached fibroblasts. Exp. Cell Res. ll5, l59-l73.

 

Richler, C., and Yaffe, D. (l970). The jp_vitro cultivation and

differentiation capacities of myogenic cell lines. Develop. Biol.

 

23, l-22.
Stewart, P.A., Percy, M.E., Chang, L.S., and Thompson, M.W. (1981)
Creatine Kinase Isozyme Transition in Chicks with Hereditary

Muscular Dystrophy. Muscle and Nerve 4, 165-173.

 

Turner, D.C., Maier, V., and Eppenberger, H.M. (1974) Creatine Kinase
and Aldolase Isoenzyme Transitions in Cultures of Chick Skeletal

Muscle Cells. Develop. Biol. 37, 63-89.

 

Weinstock, I.M., Behrendt, J.R., and Jones, K.B.,(1977) Life Sci. 21,
1199-1206.

Young, R.B., Goll, D.E., and Stromer, M.H. (l975). Isolation of
myosin- synthesizing polysomes from cultures of embyonic chicken

myoblasts before fusion. Develop. Biol. 47, 123-T35.

 

Young, R.B., McConnell, D.G., Suelter, C.H. and Phillips, T.A. (l98l).
Normal and dystrophic embryonic chicken pectoralis muscle cultures:
I. Cell differentiation, protein synthesis, and enzyme levels.

Muscle and Nerve 4, ll7-l24.

 

CHAPTER V

Development of a Chemically Defined Medium for

Primary Chick Muscle Cell Cultures

137

INTRODUCTION

Myogenesis ip_yitrp generally requires the use of complex culture
media containing mammalian serum and/or embryo extract (For reviews, see
1-3). The chemically undefined nature of these components often creates
problems in interpretation of data with muscle cell cultures, as well as
other cell types in culture. For example, variability in the concentra-
tions of components in different batches of serum or embryo extract
often cause variability in experimental results. In addition, serum or
embryo extract may contain components such as growth inhibitors or
toxins acting to completely or partially overshadow the effects of
nutrient molecules necessary for growth and/or differentiation of the
cells (4,6). Finally, serum in most primary cell culture media enhances
fibroblast contamination in these cultures since fibroblasts thrive in
serum (6). Thus, Sato and coworkers, as well as other investigators,
have attempted to define hormonal requirements for maximal growth and/or
differentiation of various cell types in culture, including both
established cell lines and primary cultures, to eliminate or diminish
the need for serum and/or embryo extract (4,6-8).

Thus, the use of a group of hormones and/or growth factors to
replace the serum and embryo extract in muscle cell culture medium is
appealing because the culture medium can be optimized to eliminate

variability in experimental results, decrease fibroblast contamination

138

139

and increase growth and differentiation of muscle cells in culture.

With these goals in mind, Florini and Roberts (9) first reported a
serum-free medium for Yaffe's L6 myoblasts and rat primary myoblasts.
This medium (10'5 M fetuin, 10'6 M insulin and 10'7 M dexa-

methasone in Ham's Nutrient F—12 medium) supports proliferation of the
L6 and rat primary myoblasts at rates approximately equal to those in
10% horse serum; both populations fuse to form myoblasts. Another
chemically defined medium, Defined Medium for Muscle and Nerve Cells
(DMN) supports the proliferation and differentiation of trypsinized
primary chick muscle cell cultures for up to 10 days (10). However,
myogenic cells grown in DMN (50 ug/ml fibronectin, 10 ug/ml human trans-
ferrin, 5 ug/ml bovine insulin and 300 ng/ml fibroblast growth factor in
Dulbecco's Modified Eagle's Medium) synthesize and secrete into the
medium a 22,000 molecular weight protein tentatively identified as a
heat-shock protein (11). This result suggests the cells are responding
to unfavorable culturing conditions or "stress“ in DMN and thus the
defined medium may lack a hormone(s) or growth factor(s) normally
supplied by serum.

The potential for an increased level of muscle differentiation and
decreased fibroblast contamination in muscle cell cultures grown in the
presence of a chemically defined, serum-free medium prompted us to
examine and possibly improve upon DMN described by Dollenmeier §t_al,
(10) for primary chick muscle cell cultures. Thus, we compared cultures
incubated in the presence of DMN to cultures supplemented with various
growth factors, hormones and biochemicals known to promote differentia-
tion in other cell types in culture or muscle development ip_yiyp. The

culture medium developed here contains insulin, conalbumin, fibroblast

140

growth factor, fibronectin and retinoic acid and appears to produce

muscle cell cultures with the most desirable characteristics.

MATERIALS AND METHODS

Media Preparation

 

Three one-liter packages of Dulbecco's Modified Eagle's Medium
(DME) supplemented with glutamine (GIBCO, 430-2100) and one one-liter
package of Ham's Nutrient Mixture F-12 (GIBCO, 430-1700) was prepared in
3500 ml triple glass-distilled water. Sodium bicarbonate, 4.8 g;
histidine, 1.6 g; 1.5 M HEPES buffer, pH 7.9, 40 ml; 1 M 2-aminoethanol
(Sigma), 0.2 ml; and 5.0 x 10'6 M sodium selenite (Difco), 4.0 ml
were added to the media mixture and the final volume and pH adjusted to
4 liters and pH 7.4, respectively. The media, DME:F12 (3:1), was filter
sterilized through 0.2 um Millipore filters and stored up to one month
at 4°C.

Preparation of Primary Muscle Cell Cultures in Chemically Defined Medium

Breast muscles were dissected from 11-12 day chicken embryos (White
Leghorn, Omega Chicks, Haslett, MI) and placed in the above DME:F12
(3:1) mixture with 0.67 ug/ml amphotericin B (GIBCO), 33 units/ml
penicillin, 33 ug/ml streptomycin (GIBCO, penicillin-streptomycin
solution), 5 ug/ml bovine insulin (Sigma) and 25 ug/ml conalbumin
(Sigma, Type II).

Myoblasts were dissociated mechanically and filtered through a 35 u
nylon filter to remove bones and clumps of cells. The resulting muscle

cell suspension was preplated at a density of 1.0 x 107 cells per

141

142

100 mm plastic tissue culture dish (Corning). Cultures were incubated
for two hours in an atmosphere containing 5% C02/95% air at 90%
relative humidity at 37°C.

During this 2 hour incubation period, secondary culture dishes were
prepared. A volume (1.5 ml) of supplemented DME:F12 (3:1) medium was
added to each 35 mm culture dish and followed by the addition of 20
ug/dish fibroblast growth factor (Collaborative Research, Inc.) and 24
ug/dish human fibronectin (Collaborative Research, Inc.) to complete the
Modified-DMN medium. Four sets of four culture dishes were prepared for
each component to be tested and the appropriate concentration of the
component added to these dishes. An additional four sets of culture
dishes were prepared containing Modified-DMN only. The medium in all
culture dishes was then allowed to equilibrate for at least 30 minutes
at 37°C in the C02 incubator.

After the muscle cell suspension in the 100 mm culture dishes
incubated for two hours, the medium containing the unattached myoblasts
from three of these preplates (12,13) were decanted and combined into
one culture dish. Aliquots of 0.5 ml were withdrawn and placed into the
35 mm culture dishes containing Modified-DMN (3:1) plus appropriate
factors as described above. Two sets of four culture dishes for each
combination of factors were incubated at 37°C while the remaining two
sets for each combination of factors were incubated at 41°C for 7 days.

Preparation of Cell Culture Homogenates

 

Three culture dishes from each set of four culture dishes for each
combination of factors and incubation temperature were rinsed twice with
cold buffered salt solution (853; 137 mM NaCl, 2.7 "M KCl, 1.0 mM
CaClz, 1.0 mM NaH2P04, 1.36 mM NaZHPO4, 6.0 mM NaHC03 and

143

5.5 mM glucose adjusted to pH 7.4). The cells from the three combined
plates were then suspended into 0.3 ml total extraction buffer (0.18 M
KCl, 0.054 M KH2P04, 0.035 M KZHPO4, pH 6.5) with a plastic

spatula and were homogenized with 20 strokes in a 1 ml ground glass
Ten-Broeck homogenizer. Homogenates were centrifuged for 10 minutes in
an Eppendorf centrifuge. Enzyme activities and protein concentrations
were then determined as described below.

Evaluation of Cell Fusion

 

The remaining culture dish from each set of four dishes for each
combination of factors and incubation temperature was rinsed twice with
cold BSS, fixed in methanol and stained with Giemsa stain as previously
described (14). The total number of nuclei and the number of nuclei
found in myotubes were determined microscopically by counting nuclei at
100x magnification. Percent fusion is defined as the percent of total
nuclei in myotubes and was determined in ten randomly chosen fields on
each culture dish.

Enzyme Assays and Protein Determination

 

Creatine kinase activity was determined spectrophotometrically at
340 nm at 30°C using the CK assay mix from Sigma Chemical Company.
Protein was determined by the method of Lowry gt_al, (15) using bovine
serum albumin (Sigma) as a standard.

Cellulose Acetate Electrophoresis

 

Creatine kinase isozymes were separated on 2.5 x 17 cm Gelman
Sephraphore III cellulose polyacetate electrophoresis strips in 0.06 M
TRIS-barbital, pH 8.8 (Gelman, high resolution buffer), 25 mM
B-mercaptoethanol as previously described (16). The electrophoresis

buffer contained 1% Triton X-100 to allow migration of mitochondrial CK

 

144

TRIS-barbital, pH 8.8 (Gelman, high resolution buffer), 25 mM
B-mercaptoethanol as previously described (16). The electrophoresis
buffer contained 1% Triton X-100 to allow migration of mitochondrial CK
from the origin. Electrophoresis was performed for two hours at 300 V
at 6°C. Following electrOphoresis the strips were stained for CK
isozymes as previously described (17) and scanned on a Gelman ACD-18
automatic computer densitometer.

Isozyme positions were identified by running completely or
partially purified fractions containing only MM, MB, B8 or mitochondrial

(mt-CK) independently on electrophoretic strips and staining for CK

 

activity. In control electrophoretic strips, creatine phosphate was
omitted from the CK assay mix to check for interference by adenylate

kinase.

RESULTS AND DISCUSSION

Effect of Defined Medium for Muscle and Nerve Cells (DMN) on Primary

 

Chick Muscle Cell Cultures

The incubation of muscle cell cultures, derived from a preplating
procedure to eliminate fibroblasts (see Materials and Methods), with
Defined Medium for Muscle and Nerve Cells (DMN) at 37°C result in muscle
cultures with approximately 10% of total nuclei in myotubes (fusion) by
Day 7 in culture compared to 90% in serum-containing medium. In
contrast, Dollenmeier et_al, report most cells in DMN cultures fuse into

nwotubes by Day 3 1p vitro (10). We believe this discrepancy is a

 

result of the cell populations originally present in the cultures.
Dollenmeier gt_al, use a limited proteolysis treatment of cultures to
obtain a population of cells consisting almost entirely of post-mitotic
myoblasts, or myoblasts destined for terminal differentiation and fusion
into myotubes (10). On the other hand, the preplating procedure used
here most likely selects for a mixture of post-mitotic myoblasts,
presumptive myoblasts and stem cells. The post-mitotic myoblasts and
presumptive myoblasts under apprOpriate conditions, will continue
differentiation through the terminal differentiation event to fonn
highly differentiated myotubes. However, myoblast stem cells may
differentiate into either fibroblasts or presumptive myoblasts. Since

stem cells are so closely related to myoblasts and fibroblasts, the DMN

145

 

I

_r

146

originally employed to support the differentiation of post-mitotic
myoblasts into myotubes may also support growth and proliferation of the
stem cells and fibroblasts. Thus, in the absence of serum or the
chemical message(s) necessary to signal a specific differentation event,
stem cells may continue proliferation without further differentation or
they may randomly choose which differentiation path to follow. In
either case, a mixture of mononucleated, proliferating cells and
terminally differentiated myotubes will exist in the cultures and result
in low fusion levels. Therefore, even though fusion levels are low, the
preplating procedure of preparing primary muscle cell cultures offers a
better test system for determination of the factors necessary for
completion of muscle differentiation from stem cell to differentiated
myotube.

Effects of Other Additives to DMN

 

Since incubation of muscle cell cultures prepared from chick embryo
breast muscle with a preplating procedure in DMN results in low fusion
levels compared to serum-containing medium, a search began for
additional components with a beneficial effect on muscle
differentiation. During initial screening of components, the levels of
fusion and total CK activity in the muscle cell cultures served as the
initial criteria for acceptance or rejection of a potential component.
Several components, including 10 ng/ml epidermal growth factor (EGF),

30 pg/ml high density lipoprotein (HDL), 10 ul per 35 mm culture dish of
a purified urokinase preparation and 5 mM creatine either killed the
cells or had no effect on fusion levels and total CK activity. The
replacement of human transferrin with conalbumin caused a slight

increase in fusion levels and total CK activity. In addition, 1 uM

 

 

147

retinoic acid also improves the chemical defined medium for muscle cell
cultures (see below).
Replacement of Human Transferrin with Conalbumin in DMN

Oh and Markelonis report sciatin, a glyc0protein purified from
chicken sciatic nerves (18) substitutes for chick embryo extract (19).
Sciatin is structurally and functionally similar to ovotransferrin (20)
available from Sigma as Type II conalbumin. The replacement of 10 ug/ml
human transferrin with 25 ug/ml conalbumin in DMN (Modified-DMN) is used
for all experiments discussed throughout the remainder of this chapter.

Table I shows the fusion level, total creatine kinase (CK) specific
activity and CK isozyme distribution for Day 7 cultures grown in the
presence of 10% horse serum, Modified-DMN and Modified-DMN + 1 uM
retinoic acid at 37°C and 41°C. At 37°C, the specific activities and CK
isozyme distributions for cultures grown in Modified-DMN are nearly
identical to those for cultures grown in the presence of horse serum,
even though the fusion level in Modified-DMN cultures (15%) are
drastically reduced compared to the serum cultures (90%). However, the
CK specific activity and %MM-CK in Modified-DMN cultures grown at 41°C
are not as high as those for serum cultures grown at 41°C (Table I).
Previous data indicates muscle cell cultures grown in the presence of
10% horse serum at 41°C, chicken body temperature, more accurately
reflect CK specific activities and CK isozyme distributions of adult
chicken muscle than cultures grown at 37°C (Table I and reference 21).
We do not know why an increase in CK specific activity and %MM-CK does
not occur in Modified-DMN cultures grown at 41°C compared to
Modified-DMN cultures grown at 37°C unless the temperature effect in

serum cultures is either the result of a temperature inactivation of an

 

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149

inhibitor or a temperature activation of a serum component responsible
for the increased synthesis of muscle-specific proteins such as MM-CK.
Effect of 1 uM Retinoic Acid on Muscle Cell Cultures Grown in Chemically

Defined Medium.

 

Retinoic acid, vitamin A acid, exerts several effects on the growth
and differentation processes of all vertebrates through such roles as
epithelial differentiation, maintenance of reproductive capacity and

glycoprotein production (For reviews, see 22 and 23). In addition,

 

retinoic acid stimulates resorption of cultured embryonic chick bone

(24,25) and induces plasminogen activator synthesis in chick embryo

 

fibroblasts (26) and chick muscle cells (27) in culture. The temporal
correlation between the induction of plasminogen activator in the
presence of 1 uM retinoic acid and the completion of myoblast fusion
into myotubes in primary chicken muscle cell cultures suggests a
potential involvement in cell surface structure modification (27).

Thus, the effect of 1 uM retinoic acid on the differentiation and fusion
of chicken muscle cell cultures grown in Modified-DMN was examined.

The effect of 1 uM retinoic acid on the fusion levels, CK specific
activity and CK isozyme distributibn in Modified-DMN cultures incubated
at either 37°C or 41°C is also shown in Table I. Supplementation of
Modified-DMN with retinoic acid doubles the fusion level, as well as CK
specific activity in cultures incubated at either 37°C or 41°C compared
to Modified-DMN cultures incubated at the two temperatures. In fact,
the CK specific activity in muscle cell cultures incubated at 37°C in
Modified-DMN supplemented with retinoic acid are almost 2-fold greater

than the specific activity in cultures incubated in medium containing

150

10% horse serum at 37°C. In addition, the 41°C Modified-DMN-retinoic
acid cultures have CK specific activities equal to CK specific
activities in 41°C serum cultures. The 2-fold increases in fusion
levels in these cultures compared to Modified-DMN cultures at both
incubation temperatures is also an improvement. The only major drawback
to this medium is the decrease in the percentage of MM-CK in the
cultures. This decrease in the percentage of MM-CK in the cultures
incubated at 37°C accompanies a significant increase in the percentage
of BB-CK while the decrease in MM-CK in cultures incubated at 41°C may
partially be due to the appearance of mitochondrial creatine kinase
(mt-CK). For our purposes, the appearance of mt-CK in Modified-DMN
cultures supplemented with retinoic acid and incubated at 41°C
represents a strong advantage for this system since our laboratory is
interested in studying the function of mt-CK in muscle using muscle cell
cultures; no other condition causes the appearance of this isozyme.

In summary, the data in Table I indicate a medium composed of
Modified-DMN and 1 uM retinoic acid provides the most beneficial
chemically defined medium for primary chicken breast muscle cell
cultures. Even though the use of a serum-free medium does not realize
the overall goals of increased levels of differentiation and decreased
fibroblast contamination in chicken breast muscle cell cultures compared
to serum-containing media, the DMN-retinoic acid medium achieves one
clear advantage over serum-containing medium. This advantage is the
appearance of mt-CK in cultures incubated in Modified-DMN-retinoic acid
medium at 41°C to provide a system for the study of mt-CK in culture.

In addition, cultures incubated in this medium at 41°C have a CK

specific activity, CK isozyme distribution and fusion level that most

 

151

closely approximates those found in cultures incubated in

serum-containing media at 41°C.

10.

11.

12.
13.

REFERENCES

Hauschka, 8.0. (1972) in Growth, Nutrition and Metabolism of Cells

 

 

in Culture (Rothblat, G.H. and Cristofalo, V.J., eds.), Vol. II,

 

Academic Press, New York, pp. 67-130.

Holtzer, H., Jones, K.W. and Yaffe, D.J. (1975) J. Neurol. Sci.

 

26:115-124.

Konigsberg, I.R. (1979) in Methods in Enzymology (Jakoby, W.B. and

 

Pastan, I.H., eds.), Vol. 58, Academic Press, New York, pp.
511-527.
Barnes, 0. and Sato, G. (1980) Anal. Biochem. 102:255-270.

 

Dollenmeier, P., Turner, D.C. and Eppenberger, H.M. (1981) Exp.

Cell Res. 135:47-61.

Weinstein, R. (1983) Biotechniques 1:61-64.

 

Sato, G. and Reid, L. (1978) Int. Rev. Biochem. 20:219-251.

 

Mather, J.P. and Sato, G.H. (1979) Exp. Cell Res. 124:215-221.

 

Florini, J.R. and Roberts, S.B. (1979) In Vitro 15:983-992.

Dollenmeier, P., Turner, D.C. and Eppenberger, H.M. (1981) Exp.

Cell Res. 135:47-61.

Dollenmeier, P. and Eppenberger, H.M. (1983) Experientia

 

39:347-350.
Richler, C. and Yaffe, D. (1970) Develop. Biol. 23:1-22.

 

Puri, E.C. and Turner, D.C. (1978) E . Cell Res. 115:159-173.

 

152

 

 

14.

15.

16.

17.
18.

19.

20.
21.

22.

23.
24.

25.
26.
27.

153

Young, R.B., Goll, D.E. and Stromer, M.H. (1975) Develpp. Biol.

 

47:123-135.
Lowry, 0.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951)
J. Biol. Chem. 193:265—275.

 

Hall, N., Addis, P. and DeLuca, M. (1977) Biochem. Biophys. Res.

 

Commun. 76:950-956.

Hall, N. and DeLuca, M. (1976) Anal. Biochem. 76:561-567.

 

Markelonis, G. and 0h, T.H. (1979) Proc. Natl. Acad. USA

 

76:2470-2474.
Oh, T.H. and Markelonis, G.J. (1980) Proc. Natl. Acad. USA

 

77:6922-6925.
0h, T.H. and Markelonis, G.J. (1982) J. Neurochem. 39:315-320.

 

Bennett, V.D., Husic, H.D., Baxter, J.H. and Suelter, C.H. (1983)
In Preparation.

DeLuca, L.M. (1978) in Handbook of Lipid Research (DeLuca, H.F.,

 

ed.), Vol. 2, Plenum Publishing Corp., New York, pp. 1-67.
Lotan, R. (1980) Biochim. Biophys. Acta 605 33-91.

 

Lucy, J.A., Dingle, J.T. and Fell, H.B. (1961) Biochem. J.

 

79:500-508.
Fell, H.B., Dingle, J.T. and Webb, M. (1962) Biochem. J. 83:63-69.

 

Wilson, E.L. and Reich, E. (1978) Cell 15:385-392.
Miskin, R., Easton, T.G. and Reich, E. (1978) Cell 15:1301-1312.

SUMMARY

Mitochondrial creatine kinase (mt-CK) is an important component of
the creatine phosphate shuttle in muscle. A study of the levels of
mt-CK activity in muscle mitochondria from normal and dystrophic
chickens reveal mt-CK activities are decreased in dystrophic chicken
breast and heart muscle compared to normal muscle when mt-CK activities
are expressed as IU mt-CK per IU succinate:INT reductase, a mitochon-
drial marker, or as IU mt-CK per gram of muscle. Results from oxygen
consumption measurements in the presence of creatine, creatine phosphate
production rates and the maximal amount of ATP synthesis in normal and
dystrophic chicken breast muscle mitochondria all suggest this decrease
in mt-CK activity in dystrophic muscle alters normal operation of the
creatine phosphate shuttle to produce the loss of white fiber muscle
function characteristic of dystrophic muscle (Chapter 11). Thus, the
properties and function of mt-CK in muscle as well as its biosynthesis
and degradation are of interest in studies of muscular dystrophy.

Several alterations in normal muscle cell processes could account
for decreases in mt-CK activity in dystrophic muscle. For example,
mRNA synthesis, protein synthesis, protein processing, transport of the
protein from the cytoplasm to the mitochondria, binding of the protein
to the inner mitochondrial membrane, degradation of the protein or

catalytic function of the protein may be altered. Further study of

154

155

mt-CK in any one of these areas requires homogeneous mt-CK and muscle
cell cultures expressing adequate amounts of mt-CK. For example,
homogeneous mt-CK is necessary for comparative binding studies of mt-CK
to the inner mitochondrial membrane of normal and dystrophic muscle
mitochondria, kinetic comparisons of the mt-CK from normal and
dystrophic muscle and the preparation of antibody to mt-CK. Antibody to
mt-CK and a muscle cell culture system expressing high activities of
mt-CK would be useful for cell-free translation, pulse-chase,
protein-labeling and other techniques necessary for study of the
biosynthesis and degradation of mt-CK. However, a procedure did not
exist for the preparation of mt-CK from chicken breast muscle and
available muscle cell culture systems did not result in cultures with
detectable levels of mt-CK activity. Thus, development of a new
purification procedure for mt-CK from chicken breast muscle and
improvements in available muscle cell culture systems for the jp_yit:o
expression of mt-CK are reported here.

The purification procedure for mt-CK from chicken breast muscle
involves the isolation of mitochondria by differential centrifugation,
mitoplast preparation, phosphate solubilization of mt-CK from
mitoplasts, Procion Red-agarose chromatography and agarose-hexane-ATP
affinity chromatography; two distinct forms of mt-CK result. Cellulose
acetate electrophoresis shows a fast-moving cathodal form purified to
90% of homogeneity with a specific activity of 276 IU/mg and a
slow-moving cathodal form only 40-50% pure with a specific activity of
123 IU/mg (Chapter III).

Current muscle cell culture systems do not entirely reflect normal

muscle development in vivo. Thus, attempts were made to improve upon

,... ...-

 

156

current culture methods by increasing the incubation temperature of
muscle cell cultures from 37°C to 41°C, chicken body temperature and by
developing a chemically defined medium to optimize for muscle
differentiation in culture. A higher CK specific activity and a greater
percentage of MM-CK occur in muscle cell cultures incubated at 41°C than
muscle cell cultures incubated at 37°C. Even though muscle cell
cultures incubated at 41°C more closely reflect the CK specific activity
and CK isozyme distribution of adult muscle ipiyiyo_than cultures
incubated at 37°C, CK specific activities are still 5-fold lower than_ip
.yiyo values (Chapter IV). In addition, mt-CK activity is not detected
during cellulose acetate electrophoresis of cell culture homogenates
from cultures incubated at either temperature. However, the use of a
chemically defined muscle cell culture medium containing insulin,
conalbumin, fibroblast growth factor, fibronectin, and retinoic acid in
muscle cell cultures incubated at 41°C results in the expression of 9%
of the total CK activity as mt-CK (Chapter V). These improvements in
muscle cell culture techniques provide a basic system for future studies
of mt-CK in culture.

In summary, decreased mt-CK activity causes an alteration in the
function of the creatine phosphate shuttle in dystrophic chicken breast
muscle. This observation led to the development of a purification
procedure for mt-CK from chicken breast muscle and improvements in
muscle cell culture technology. These new methods will be useful in

future studies of mt-CK in normal and dystrophic muscle.

   

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