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CIRCULATORY CLEARANCE OF MUSCLE ENZYMES
IN NORMAL AND DYSTROPHIC CHICKENS
By
Harold David Husic
A DISSERTATION
Submitted to
Michigan State University
in partial fquiIIment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY
Department of Biochemistry
1982
ABSTRACT
CIRCULATORY CLEARANCE OF MUSCLE ENZYMES
IN NORMAL AND DYSTROPHIC CHICKENS
By
Harold David Husic
The most widely used test for the early diagnosis of Duchenne muscu-
lar dystrophy in humans, and for the detection of the X-linked trait in
female carriers, is the determination of the levels of some muscle
enzymes in the circulation. The activities of creatine kinase and muscle
pyruvate kinase are also markedly elevated in the circulation of dystro-
phic chickens compared to normal chickens. However, the activities of
some other abundant muscle proteins including AMR aminohydrolase (AMPAH)
and adenylate kinase are not elevated. Some of the factors which deter-
mine the serum levels of these muscle enzymes in dystrophic chickens were
investigated.
The circulatory clearance rates of several muscle enzymes were
measured in normal and dystrophic chickens after the intravenous injec-
tion of the purified enzymes. AMPAH and adenylate kinase are cleared
rapidly with half-lives of only a few minutes. However, the circulatory
half-lives of creatine kinase and pyruvate kinase are several hours.
Thus, those enzymes that are rapidly cleared are not elevated in the
circulation of dystrophic chickens. However, based on the activities
of these enzymes in muscle press juices, AMPAH is extensively associated
with intracellular structures and may not be released into the circula-
tion.
The rapid circulatory clearance of AMPAH was studied in detail.
AMPAH is cleared primarily by the spleen and the parenchymal cells of the
liver, where the enzyme is internalized and degraded in lysosomes. The
rapid clearance is inhibited by the intravenous injection of heparin and
other sulfated polysaccharides.
AMPAH binds to hepatocyte monolayers in vitro. This binding to the
cell surface of hepatocytes is inhibited by effectors of AMPAH activity,
heparin, and other sulfated polysaccharides. The bound enzyme is inter-
nalized and degraded. AMPAH also binds to heparin. This may explain the
heparin-induced release of AMPAH bound to hepatocyte monolayers.
These experiments describe the circulatory clearance rates of muscle
proteins. The effect of clearance rates on the observed levels of these
proteins in the circulation of dystrophic chickens are discussed. The in
vivo and in vitro characteristics of the mechanism for the rapid circula-
tory clearance of AMPAH are also described.
to
my Mom and Dad
for their love and encouragement
and
to all of those with whom I have ever shared a tune
ii
ACKNOWLEDGEMENTS
I wish to express my deepest gratification to Dr. Clarence Suelter
for allowing me to pursue those ideas in which I was the most interested,
for always finding a way to provide me with financial support, and for
always keeping his wit and composure under any and all circumstances.
I also wish to thank those faculty members who served on my graduate
committee: Dr. James Fairley, Dr. David McConnell, Dr. Allan Morris,
Dr. Ronald Young, and Dr. George Ristow. Special thanks are due to
Dr. Ristow who stepped in to replace Dr. Young so late in my graduate
career.
I would also like to thank the Department of Biochemistry, The
Muscular Dystrophy Association of America, and the National Institutes of
Health for providing financial support.
Many thanks are in order to all of those individuals with whom I have
shared the laboratory, discussions, and good times; including Gerry
Oakley, Dave June, Mary Pearce, Debra Thompson, Jeff Baxter, Tom Carlson,
Vickie Bennett and Krystian Kaletha. Many thanks also to my other
friends in the Department especially Sally Ann Camper Lyons, to Theresa
Fillwock for working so hard on typing this thesis, and to Joyce Urso for
numerous shared lunches and for not crashing my thesis defense seminar.
TABLE OF CONTENTS
Page
LIST OF TABLES . . . . . . . . . . . . . . . . . . . . . . . . . . vii
LIST OF FIGURES. . . . . . . . . . . . . . . . . . . . . . . . . . ix
LIST OF ABBREVIATIONS. . . . . . . . . . . . . . . . . . . . . . . xii
CHAPTER I. LITERATURE REVIEW. . . . . . . . . . . . . . . . . . . 1
A. Duchenne Muscular Dystrophy. . ......... . . . . . 1
B. Muscular Dystrophy in the Chicken. . . . . . . . . . . . . 12
CHAPTER II. MUSCLE ENZYMES IN THE SERUM OF DYSTROPHIC CHICKENS. . 19
IntrOdUCtion I O O O O O O O O O O O O 0 O O O O O O O O I O O 20
Materials and Methods. . . . . . . . . . . ._. . . . . . . . . 21
Materials. . . . . . . . . . . . . . . . . . . . . . . . . 21
Preparation of Muscle Crude Extracts . . . . . . . . . . . 22
Collection of Blood Samples. . . . . . . . . . . . . . . . 22
Enzyme Assays. . . . . . . . . . . . . . . . . . . . . . . 22
Enzyme Purification. . . . . . . . . . . . . . . . . . . . 23
Radioiodination of Enzymes . . . . . . . . . . . . . . . . 24
Rates of Loss of Enzymes from the Circulation. . . . . . 25
Measurement of the Inactivation of Enzyme Activity in
Serum In Vitro . . . . . . . . . . . . . . . . . . . . 26
DeterminatTEn o? the Tissue Distribution of
ZSI'Adenylate Kinase. 0 o o o o o o o o o o o o o o o 26
Preparation of Muscle Press Juices . . . . . . . . . . . . 27
Resu1t50 O O O O O O O O O O O O O O I I O O O O O O O O O O O 28
Levels of Enzymes in the Muscle and Blood Plasma
of Normal and Dystrophic Chickens. . . . . . . . . . . 28
Pyruvate Kinase Isoenzymes in the Plasma of
Dystrophic Chickens. . . . . . . . . . . . . . . . . . 28
Pyruvate Kinase Clearance and Inactivation . . . . . . . . 30
Creatine Kinase Clearance and Inactivation . . . . . . . . 35
Adenylate Kinase Clearance and Inactivation. . . . . . . . 41
iv
AMP Aminohydrolase Clearance . ....... . . . . . .
Enzyme Activities in Muscle Press Juices . . .
Discussion ......... . ...............
CHAPTER III. CIRCULATORY CLEARANCE, UPTAKE, AND DEGRADATION
0F MUSCLE AMP AMINOHYDROLASE O O O O O O O O O O O O C C O O
IntrOdUCtion O O O O O O O O 0 0 O 0 O O O O 0
Materials and Methods. . . . ..... . . . .
Materials. 0 O O O O O O O O O O O 0 O O 0
Preparation of Radiolabeled Proteins . . .
Clearance and Tissue Distribution of AMPAH.
Gel -Filtration Chromatography of Tissue Extracts
Sucrose Density Gradient Sedimentation . . . . .
Liver Perfusion and Separation of Parenchymal and
Nonparenchymal Cells . . . . . . . . .
Effect of Compounds on AMPAH Clearance . .
Release of Cleared AMPAH by Heparin. . . .
ReSUItSo O O O O O O O O O O O O O O O O O O 0
Clearance and Tissue Distribution of AMPAH . . .
Degradation of AMPAH in the Liver and the Spleen
Subcellular Localization of Cleared AMPAH. . . .
Parenchymal and Nonparenchymal Cell Distribution
[14CJSucrose-AMPAH Cleared by the Liver. . .
Inhibition of AMPAH Clearance. . . . . . . . . .
Release of Cleared AMPAH into the Circulation by
DisCUSSion O 0 O O O O O O O C O O O 0 O O O 0
CHAPTER IV. INTERNALIZATION AND DEGRADATION OF AMP
AMINOHYDROLASE BOUND TO HEPATOCYTE MONOLAYERS.
IntrOdUCtion I O O O O O O O O O O O O O O O 0
Materials and Methods. . . . . . . . . . . . .
Material! 5. O O O O O O O O O O O O O 0 0 0
Preparation of Radiolabeled Proteins . . .
Preparation of Monolayer Cultures of Chicken
Parenchymal Liver Cells. . . . . . . . . . . . . . .
Measurement of the Binding of AMPAH to Hepatocyte
Mona] ayers O 0 O O O O O O O O O O O O O O O O O O O O
Bio-Gel P-60 Chromatography in 8 M Urea. . . . . . . . . .
Page
.113
.114
.115
.115
.115
.115
.116
.118
Resu1ts O O O O C O O O O O O O O O O O O O O O O O O 0
Binding of AMPAH to Hepatocyte Monolayers . ...... .
Inhibition of AMPAH Binding and Release of Bound
AMPAH by Heparin. . . .
Binding of 2
Size Distribution of
at 37°C 0 O O O 0
Di SCUSSion ....... O O O ......... O O O 0
CHAPTER V. INVESTIGATIONS INTO THE NATURE OF THE INTERACTION
0F MPAH WITH HEPATOCYTES O O O O O O C O O O O O C I
51-AMPAH and [14cisucroéelAMPAH'to °°°°°°
Hepatocyte Monolaygrs at 4°C and 37°C . . .......
5I-AMPAH Bound to Hepatocytes
Introduction. . . . ...... . . . . . . . . . ......
Materia] s and Methods 0 O I O O C O O O O O O O O O O
Materi als O O O O O O O O O O O O O O O O O O O O O 0
Measurement of the Effect of Compounds on the Binding
of AMPAH to Hepatocyte Monolayers . . . . . . . .
Measurement of Heparin Inhibition of AMPAH Activity .
Binding of AMPAH to Heparin-Sepharose 4B. . . . . . .
RESU] ts O O I O O O O O O O O O O O O O O O .0 O O O O O O O 0
Inhibition of the Interaction of AMPAH with Hepatocyte
Monolayers by Sulfated Polysaccharides and Other
Polyanions. . . . . . . . . . . . . . . . . . . . .
Inhibition of the Interaction of AMPAH with Hepatocytes
by Effectors of AMPAH Enzymatic Activity. . . . . . .
Release of AMPAH Bound to Hepatocyte Monolayers by Salts.
Inhibition of the Interaction of AMPAH with Hepatocytes
by Carbohydrates. . . . . . . . . . . . . . . . . .
Inhibition of AMPAH Activity by Heparin and Other
POIyanionSO O O O O O O O O O O O O O O O O O O O
Interactions of AMPAH with Heparin-Sepharose 4B . . .
01 SCUSSIOno O O O O O O O O 0 O O O O O O O O O O O 0
SUMMARY AND DISCUSSION. I O O C O O O O O O O O O O O O O 0
APPENDIX: Papers, Abstracts, and Manuscripts in Preparation.
LIST OF REFERENCES ....... . . . . . . ............
vi
Page
119
119
119
125
133
139
142
143
144
144
144
145
146
146
150
150
154
158
160
169
172
180
Table
Table
Table
Table
Table
Table
Table
Table
Table
Table
Table
Table
1.
5.
6.
9.
10.
11.
LIST OF TABLES
Some Properties of Muscle Proteins and the Extent of
Elevation in DMD. . . . . . . . . . . . . . . . . . .
The Activities of Several Enzymes in the Blood Plasma
and Breast Muscle of Normal and Dystrophic Chickens .
Characterization of Pyruvate Kinase Isoenzymes in
the Serum and Tissues of Normal and Dystrophic
Chickens by Activation with Fructose-1,6-Bisph05phate
(Fru-1,6-P2). . . . . . . . . . . . . . . . . . . . .
Rates of Loss of Pyruvate Kinase Activity and
I-Pyruvate Kinase from the Circulation of
Normal and DystrOphic Chickens. . .g. . . . . . . . .
Rates of Loss of Creatine Kinase Activity and
5I—Creatine Kinase from the Circulation of
Norma] and DYStrOph'IC ChICkeflS. o o o o o o o o o o '0
Rates of Loss of Adenylate Kinase Activity and
25I-Adenylate Kinase from the Circulation
of Normal and Dystrophic Chickens . . . . . . . . . .
Tissue Distribution of 125I-Adenylate Kinase
30 Minutes After Intravenous Injection. . . . . . . .
Recovery of Adenylate Kinase (AK) Activity in the
Liver and Spleen 40 Minutes After the Intravenous
Injection of Adenylate Kinase . . . . . . . . . . . .
Rate of Loss of AMP Aminohydrolase Activity and
25I-AMP Aminohydrolase from the Circulation
of Normal and Dystrophic Chickens . . . . . . . . . .
Enzyme Activities of Press Juices and Crude Extracts
from Normal and Dystrophic Chicken Breast Muscle. . .
Rate Constants for the Distribution of Creatine
Kinase and Pyruvate Kinase After Intravenous
Injection. 0 O O O O O O O O O 0 O O O 0 O O 0 O 0 0
Recovery of AMPAH Activity, 1251, or 14c in the
Spleen and Liver BO Minutes After the Injection of
Unlabeled AMPAH, 251-AMPAH or [14C]Sucrose-
Page
29
31
34
4O
46
52
53
57
6O
71
AMPAH O O O O O O O O O O O O O O O O O O O O ..... 88
vii
Table
Table
Table
Table
Table
Table
'Table
Table
Table
13.
14.
15.
16.
17.
18.
19.
20.
21.
Distribution of 14C in Parenchymal Cells (PC)
and Nonparenchymal Cells £NPC) of the Liver 4 Hours
After the Injection of [1
Release of 1251 from the Liver and Spleen
into the Circulation by Heparin Injection 30
Minutes After ZSI-AMPAH Injection. 0 O O O O O O 0
Characterization of the Binding of AMPAH to
Hepatocyte Monolayers . . . . . . . . . . . . . . . .
Inhibition by Unlabeled AMPAH of the Binding of
25I-AMPAH and [1 CJSucrose-AMPAH to
Hepatocyte Monolayers . . . . . . . . . . . . . . .
Effect of Heparin on the Binding of 125I-AMPAH to
Hegatocyte Monolayers and on the Release of Bound
IZI'AMPAHQ00000000000000.0000.
Inhibition of lzsl-AMPAH Binding and Release
of 125I-AMPAH Bound to Hepatocytes by Sulfated
Polysaccharides and Other Polyanions. . . . . . . . .
Inhibition of 125I-AMPAH Binding and Release of
125I-AMPAH Bound to Hepatocytes by Allosteric
Effectors of AMPAH Activity . . . . . . . . . . . . ' .
Inhibition of IZSI-AMPAH Binding and Release
of 125I-AMPAH Bound to Hepatocytes by
Carbohydrates . . . . . . . . . . . . . . . . . . .
Polyanion Inhibition of AMPAH Activity. . . . . . .
viii
CJSucrose-AMPAH . . . . . .
Page
100
106
122
123
124
147
151
157
159
LIST OF FIGURES
Page
Figure 1. The Loss of Intravenously Injected Pyruvate Kinase
Activity from the Circulation of Normal and Dystrophic
Ch1CkenS. O O O O O O O O O O O O O O O O O O O 0 O O O 33
Figure 2. The Loss of Radioactivity from the Circulation after
the Intravenous Injection of 125I-Pyruvate
Kinase in Normal and Dystrophic Chickens. . . . . . . . 37
Figure 3. The Loss of Intravenously Injected Creatine Kinase
Activity from the Circulation of Normal and Dystrophic
ChiCkenSO I O O O O 0 O O O O O O O O O O O O O O O O O 39
Figure 4. The Loss of Radioactivity from the Circulation After
the Intravenous Injection of 125I-Creatine
Kinase in Normal and Dystrophic Chickens. . . . . . . . 43
Figure 5. The Loss of Intravenously Injected Adenylate Kinase
Activity from the Circulation of Normal and Dystrophic
ChiCkens. O O O O O O O O O I O O O O O O O O O O O O 0 45
Figure 6. The Loss of Adenylate Kinase Activity in Chicken Serum
1E Vitro at 41°C. 0 O O O O O O O O O O O O O 0 O O O O 48
Figure 7. The Loss of Radioactivity from the Circulation After
the Intravenous Injection of 125I-Adenylate
Kinase in Normal and Dystrophic Chickens. . . . . . . . 51
Figure 8. The Loss of Intravenously Injected AMP Aminohydrolase
Activity from the Circulation of Normal and Dystrophic
ChICkenSo O O O O O O O O O O O O O O O 0 O O O O O O O 55
Figure 9. The Loss of Radioactivity from the Circulation After
the Intravenous Injection of 125I-AMP
Aminohydrolase in Normal and Dystrophic Chickens. . . . 59
Figure 10. A Hypothetical Model for the Distribution of Enzymes
Between BOdy F] U1 ds 0 O O O O O O O 0 O O O O 0 O O O 0 68
Figure 11. The Loss of AMPAH Activity and 14C from the
Circulation After Intravenous Injection of AMPAH or
[4C]Sucrose-mpAHooooooooooooooooooo87
ix
Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure
Figure
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
Recovery of 14C and 1251 in the Liver and
Spleen at Various Times After the Injection of
[ 4C]Sucrose-AMPAH or IZSI-AMPAH. . . .
Bio-Gel P-6O Elution Profiles Showing the Size
Distribution of 125I in the Liver Spleen, and
Excrement After the Injection of 125I-AMPAH . . . . . .
Bio-Gel P-60 Elutlon Profiles Showing the Size
Distribution of C in the leer and Spleen
4 Hours After Injection of [ CJSucrose-AMPAH . . . . .
Sucrose Density Gradient Sedimentation Profiles
of Homogenates from the Liver and Spleen 7 Hours
After the Injection of [ CJSucrose-AMPAH . . . . . . .
The Effects of Several Compounds on the Loss of
AMPAH Activity from the Circulation . . . . . . . . . .
Release of AMPAH Activity into the Circulation by
Heparin Injection After the Clearance of
Intravenously Injected AMPAH. . . . . . . . . . . . . .
The Release of AMPAH Activity or 14C into
the Circulation by Heparin Injection at Several
Times after the Injection of Unlabeled AMPAH
or [ 4C]Sucrose-AMPAH . . . . . . . . . . . . ... . . .
Concentration Dependent Binding of AMPAH to
Chicken Hepatocyte Monolayers . . . . . . . . . . . . .
Time Course for the Binding of 125I-AMPAH to
Hepatocyte Monolayers at 4°C and 37°C . . . . . . . . .
Concentration Dependent Binding of
Heparin-Releasable 25I-AMPAH to Hepatocyte
Monolayers at 4°C and 37°C. . . . . . . . . . . . . . .
Time Course for the Binding of
[ CJSucrose-AMPAH and [14C]Sucrose-BSA to
Hepatocyte Monolayers at 37°C . . . . . . . . . . . .
Size Distribution of Heparin-Releasable and
Heparin-Resistant Radioactivity after Hepatocytes
are Incubated at 37°C with Radiolabeled AMPAH . . . . .
Release of Low Molecular Weight 1251 into
the Media After the Binding of 125I-AMPAH to
Hepatocyte Monolayers . . . . . . . . . . . . . . . . .
ancentration Dependence for the Release of
1 5I-AMPAH Bound to Hepatocytes by Sulfated
Polysaccharides . . . . . . . . . . . . . . . . . . .
X
Page
93
96
98
102
104
108
121
127
130
. 132
135
138
. 149
Figure 26.
Figure 27.
Figure 28.
Figure 29.
Figure 30.
Figure 31.
Page
ancentration Dependence for the Release of
1 5I-AMPAH Bound to Hepatocytes by Effectors
Of AMPAH ACtiVity O 0 I O O O O O I O O O O O 00000 153
Concentration Dependence for the Release of
25I-AMPAH Bound to Hepatocytes by Salts. . . . . . . . 156
The Effect of KCl on the Inhibition of AMPAH
ACtiVity by Heparin I O O O O O O O O O O O O O O O O O 162
Effect of Heparin on AMPAH Kinetic Parameters . . . . . 164
Dixon Plot for the Inhibition of AMPAH by Heparin . . . 166
Elution of AMPAH from Heparin-Sepharose 4B. . . . . . . 168
xi
ADP
AK
AMP
AMPAH
ATP
ATPase
BSA
Ci
cpm
CTP
DMD
EDTA
Fru-1,6-P2
GTP
HEPES
IMP
ITP
MES
MIT
NADH
NADP
LIST OF ABBREVIATIONS
Adenosine 5'-diphosphate
Adenylate kinase
Adenosine 5'-monophosphate
Adenosine 5'-monophosphate aminohydrolase (AMP deaminase)
Adenosine 5'-triphosphate
Adenosine S'-triphosphatase
Bovine serum albumin
Curies
Counts per minute
Cytosine 5'-triphosphate
Duchenne muscular dystrophy
Ethylenediaminetetraacetic acid
Fructose-1,6-bisphosphate
guanosine 5'-triph05phate
N-2-hydroxyethylpiperizine-N'-2-ethanesulfonic acid
Inosine 5'-monophosphate
Inosine 5'-triphosphate
2(N-morpholino)ethanesulfonic acid
monoiodotyrosine
nicotinamide adenine dinucleotide
nicotinamide adenine dinucleotide phOSphate
xii
NPC
PBS
PC
PPi
RNA
t1/2
TRIS
Nonparenchymal cells
Phosphate-buffered saline
Parenchymal cells
Orthophosphate
Pyrophosphate
Ribonucleic acid
Half-life
Tris(hydroxymethyl)aminomethane
xiii
CHAPTER I
LITERATURE REVIEW
A. DUCHENNE MUSCULAR DYSTROPHY
Muscular dystrophies are disorders that result in the dysfunction and
degeneration of muscle. There are numerous muscular disorders that
differ markedly in their clinical characteristics including degree of
severity, age of onset, affected muscles, and role of inheritance in the
transmission of the disorder. The primary causes for only a few muscular
diseases are known. Myasthenia gravis which results in muscle weakness
and fatigue, is an autoimmune disorder. These patients produce
antibodies which bind to the acetylcholine receptor at the neuromuscular
junction (1). Myopathies are associated with deficiencies of enzymes
which metabolize glycogen (2), phosphofructokinase (3), AMP aminohydro-
lase (4), carnitine palmitoyl transferase (5), and carnitine (6).
However, for other common muscular disorders the primary causes are not
known and effective modes of treatment are not available.
The most common and widely studied of the neuronuscular diseases is
Duchenne muscular dystrophy (DMD). In contrast to many other myopathies,
the clinical characteristics are predictable and largely invariant fran
one patient to the next (7). The disease is genetically transmitted,
follows a preditable mode of X-linked inheritance, and on the average
affects one out of every 4800 male children (8). Symptoms usually occur
1
2
before three years of age and include initial weakness of the legs
followed by weakness of the arms, and muscle hypertrOphy early in the
disease followed by atrOphy at later stages. By adolescence, patients
are wheelchair bound, and death from complications generally occurs by 25
years of age.
Clinical diagnosis of DMD is generally based on the examination of
the serum levels of muscle enzymes and histochemical examination of
muscle biopsies. Elevated levels of some muscle enzymes are invariably
observed in DMD patients. This will be discussed in detail later in this
review. Histochemical examination of DMD muscle biopsies shows: 1) large
hyalinized muscle fibers; 2) small groups of fibers at the same stage of
regeneration or degeneration; 3) proliferation of endomysial and
perimysial connective tissue; 4) increase in intracellular and
extracellular lipid; 5) fiber Splitting; 6) heterogeneity of fiber
diameter; 7) centrally placed nuclei; 8) nuclear proliferation; and 9)
myofibrillar necrosis (7). Histochemical stains for enzyme activities
reflect the pattern of enzyme activity differences in muscle homogenates
discussed below.
Despite considerable research, the molecular basis for the primary
defect in DMD continues to elude investigators. As with any genetically
transmitted disorder the defect must be due to the absence, reduced
amounts, or reduced functional activity of a protein that is necessary
for normal cellular function.
If the disorder in DMD is due to the reduced activity of an enzyme,
the defect could be detected by noting markedly reduced levels of the
enzyme activity or the abnormal levels of a metabolite. Altered levels
of many enzyme activities are observed in biopsies of DMD muscle,
3
however, the differences are not extreme and probably are secondary
effects to the primary lesion. The levels of creatine kinase (9), adeny-
late kinase (9), AMP aminohydrolase (10,11), and the enzymes of glyco-
genolysis and glycolysis (9,12-14) are decreased in muscle biopsies from
DMD patients compared to normal patients. 0n the other hand there are
increased levels of enzymes of the citric acid cycle (9,12) and lysosomal
enzymes (15-18). Differences in the levels of several metabolites are
observed in the muscle of DMD patients compared to normal patients.
Increased levels of muscle lipids are observed (19-21); ATP levels in the
muscle of DMD patients are either not reduced (22) or slightly reduced
(23,24); phosphorylcreatine and glycogen levels are slightly decreased
(22). Because ATP and phosphorylcreatine levels are not markedly reduced
in DMD muscle, it is clear that the decreased levels of glycolytic and
glycogenolytic enzymes do not compromise the ability of DMD muscle to
prbduce ATP and phosphorylcreatine. Therefore, the primary defect in DMD
is not likely a defect in energy metabolism. Furthermore, the symptoms
of DMD are more severe than those disorders in which there is an impaired
ability to utilize carbohydrates for energy production as in the glycogen
storage diseases (2) and phosphofructokinase deficiency (3).
The relative levels of enzymes involved in glycolytic and oxidative
metabolism in muscle from DMD patients are similar to those observed in
embryonic muscle tissue; it has been suggested that DMD may be a develop-
mental disorder that results in the incomplete differentiation of muscle
tissue (25,26). This suggestion is further supported by studies which
show that adult muscle from DMD patients contains isoenzymes not normally
found in muscle after embryonic development. The isoenzyme patterns of
lactate dehydrogenase (27,28), creatine kinase (26,27,29), aldolase (30),
4
acetylcholinesterase (31), and hexokinase (32) in DMD muscle show measur-
able levels of isoenzymes characteristic of embryonic muscle, though the
normal adult isoenzyme predominates in all cases. The presence of
embryonic isoenzymes may be due to the high number of regenerating muscle
fibers that are observed in DMD muscle tissue (33).
Several investigators have examined the properties of structural
proteins of the contractile apparatus and other proteins in search for
the primary lesion in DMD. Biochemical studies of myoglobin (34), myosin
(35), actomyosin (36), and tropomyosin (36) show no evidence for altera-
tions in the properties of these proteins isolated from DMD muscle.
Others have suggested that the defect in DMD results in the dysfunc-
tion of muscle membranes (36-38). Electron—microscopic investigations of
DMD muscle biopsies show focal lesions of the sarcolemma (39-41) and an
abnormal distribution and increased number of intramembranous particles
(42,43). Muscle damage may be due to high intracellular concentratibns
of calcium caused by either an increased influx of calcium at the plasma
membrane or impaired uptake of calcium by the sarcoplasmic reticulum
(44). High intracellular concentrations of calcium could activate a
neutral calcium-activated proteinase in skeletal muscle (45) and result
in degradation of muscle tissue. Decreased levels of Mg-ATPase,
Na/K-ATPase, and Ca-ATPase were observed in isolated sarcolemma from DMD
muscle in one report (46), but not in another (47). Compared to nonnal
muscle, the membrane bound enzyme adenylate cyclase is relatively
unresponsive to stimulation by epinephrine or sodiun fluoride in DMD
sarcolemma (38,48). One report showed an altered distribution of
concanavalin A binding sites in sarcolemma from DMD patients (49). 4A
difference in the types of phospholipids associated with DMD membranes is
observed (21).
Tissues other than muscle from DMD patients have also been studied
extensively. Numerous reports show alterations in the characteristics of
erythrocyte membranes from DMD patients; however, as reviewed by Rowland
(38) most of the observed abnormalities are either contested or uncon-
firmed. Studies of the capping characteristics of DMD lymphocytes
(50-52) and the growth patterns of cultured skin fibroblasts from DMD
patients (53-55) have also produced controversial results.
The first observations that implicated defective muscle membranes in
DMD were those which showed marked increases in the levels of some muscle
enzymes in the serum of DMD patients. The early observation that
aldolase activity is elevated in the serum of boys with DMD (56,57) was
followed by reports of increased serum levels of creatine kinase (58),
pyruvate kinase (58-60), glutamate-oxaloacetate transaminase (61),
phosphoglucomutase (62), aspartate aminotransferase (62), alanine amino-
transferase (62), glucosephosphate isomerase (62), triosephosphate
isomerase (62), malate dehydrogenase (62), and myoblobin (63-66).
Slightly increased levels of adenylate kinase (67,68) are reported in
some patients. Of the serum enzymes studied, creatine kinase activity is
the most widely used diagnostic test of muscular disease because the
activity is more dramatically elevated than other muscle enzymes in the
serum of DMD patients. Creatine kinase activity is often elevated at
birth before the onset of muscular weakness in DMD patients (69-71), and
is elevated in as many as 80% of the female carriers of the x-linked
trait (72). The activity of creatine kinase is elevated in the blood of
some affected fetuses, though assay of creatine kinase in fetal blood is
not sufficiently reliable to be used for prenatal diagnosis of DMD
(73-75). Increased serum levels of muscle enzymes are also observed in
patients with other myopathies though the elevation is usually not as
dramatic as in DMD (76).
It is apparent that the enzymes which are elevated in the blood of
DMD patients are from muscle tissue. The most convincing evidence is
that the isoenzyme patterns of the enzymes found in the blood are similar
to those in muscle. Aldolase activity in the serum of DMD patients is
the muscle isoenzyme (77,78). Furthermore, arteriovenous differences in
the activity of aldolase across the foreann of a DMD patient indicate
release from muscle tissue (79). There are several isoenzymes of
pyruvate kinase in human tissues and the increased sermn activity in DMD
patients is the muscle isoenzyme (59). The isoenzyme patterns of lactate
dehydrogenase (27,80-83) and creatine kinase (27,83-86) also reflect the
isoenzyme patterns observed in DMD muscle tissue. The levels of
adenylate kinase are slightly increased in the serum of some DMD patients
(67,68) and the serum enzyme is reported to be an "aberrant" fonm of the
muscle isoenzyme (68). Also, in DMD patients the serum activity of
muscle enzymes decreases and approaches normal levels at late stages of
the disease when muscle mass is greatly reduced (87-89).
Several studies show that those enzymes which are elevated in the
serum of DMD patients are often present in decreased levels in muscle
tissue; it has been suggested that the decreased muscle activity of these
enzymes is due to leakage from the muscle to the circulation (38,88,90).
However, based on the rates of circulatory clearance of creatine kinase,
and the muscle and plasma levels of creatine kinase, Pennington (76)
estimated that less than 1% of the total muscle creatine kinase would
need to be released from the muscle daily to the circulation to maintain
the observed serum levels of the enzyme. This estimate is based on the
7
rate of clearance of creatine kinase after myocardial infarction.
However, the major creatine kinase isoenzyme in the heart is the MB
isoenzyme (91), and may be cleared at a different rate than the MM
isoenzyme which predominates in muscle (26). Furthermore, whether the
release of enzymes from the heart after myocardial infarction stops
gradually or abruptly is not known. If the release stops gradually the
actual rates of clearance of the enzymes may be more rapid than
measured.
There are two theories which attempt to explain the pathological
release of muscle enzymes into the circulation of DMD patients (38,76).
The first is that the sarcolemma contains large physical interuptions
that occur before actual necrosis of the muscle cell. Presumably,
lesions of this type would render the sarcolemma permeable to all soluble
.sarcoplasmic constituents and extensive cellular degeneration and
necrosis would soon follow. The second theory is that the sarcolemma of
DMD patients is abnormally permeable to some macromolecules, and some or
all soluble muscle enzymes are released from the sarcoplasm of viable
muscle cells into the circulation. The evidence for these theories is
discussed in an eXcellent review by Rowland (38).
The evidence that the release of muscle enzymes is through large
physical interuptions in the sarcolemma is based primarily on electron
microscopic studies. There is evidence of physical interuptions in the
sarcolemma from DMD muscle that precedes muscle cell necrosis (39,41).
The release of mitochondrial enzymes into the serum of DMD patients
(83,92) is consistent with degenerate mitochondria at the site of the
lesions in the sarcolemma (39). It seems likely that the release of
muscle enzymes into the serum of DMD patients is at least in part due to
physical interuptions in the sarcolemma.
Several lines of evidence suggest that the release of muscle enzymes
into the circulation of DMD patients is due primarily to the increased
permeability of the sarcolemma to muscle proteins. Elevated levels of
serum enzymes are usually observed in the early stages of DMD and in
female carriers of the X-linked trait, often when there is little or no
evidence of sarcolemmal lesions or cell necrosis in muscle biopsies (93).
A decrease in the serum levels of muscle enzymes are observed when DMD
patients are treated over a period of time with drugs known to influence
membrane properties. Prednisolone (94,95) and diethylstilbesterol
(96-98) reduce the serum levels of creatine kinase in DMD patients.
However, it can not be readily determined if the effect of these drugs is
due to the alteration of membrane properties or due to the effect of
these drugs on other cellular properties. The altered permeability of
the sarcolemma may be in part determined by the metabolic state of the
cell. For example, the release of muscle enzymes from normal human
muscle after strenuous exercise (99,100) may be related to decreased ATP
levels in the muscle (99).
If the release of muscle enzymes into the circulation of DMD patients
is by passage through large lesions in the sarcolemma, it would be
expected that all soluble muscle proteins would be released from muscle
tissue at similar rates and that similar levels of increased activity of
different muscle enzymes would be observed. However, the extent of
elevation of different abundant muscle enzymes in the serum of DMD
patients differs considerably (Table 1). Creatine kinase is the most
markedly elevated enzyme in the serum of DMD patients; levels nearly 400
times that of the serum of normal controls are observed. Aldolase,
.mcmszs can» cmguo mmpomam ccw_uesme cow mew mangmz copaumpoza
.umuomuou no: u ecu
AONHV ee -- afiecfiv so" Ammv e: oboePSoxoz
AONHV no» -- efimfifiv 0mm ANOH.omV one oboe_xouo=eeoeemoed
-- -- afiefifiv mme-oe~ afioflv m.H omepxeoedmoee
Aomfiv oe -- AMHHV m.H~ Awe.eov m.H omee_¥ obe_»eoe<
AeNH.m-v no» -. eANAHV mew ANov e.fi omo_oee»;oe_e< az<
-- e; ea efiflfifiv Ne. Amev H.m omoeoaoeeAeoo ooe_ez
AONHV no» -- AOHHV emfl Auov 8.” omoeoeomc opeeomoeeomooepo
mmmcmmoevxsmc
Aomfiv o: -- namOSV em” Away e.m ooeeemoed-m-oeseoePeeooa_o
-- -- nfieofiv mfifi Awev m omeeocmeeeooece< oe.=e_<
AONHV no» -- afieofiv co Away m omoeocmeoeooepe< ooeoeoom<
ANNSV no» t; cc afieocv Nefi Awov m.e omeeoaoeexeoo obeooeb
-- -- BAAHHV NH . Amev NH eceopmosz
ANNH-0~#v no» 2; HN namOSV oefi ANov mu omopoe_<
AONHV no» -- eroHV emu Ammv KN omoecx opossexd.
-- -- Adofiv om Away em omeeaeoo=Pmoeomoee
Amfifiv no» 2; mH Amcfiv em ANGV mmm omee.¥ oewoeoeu
mmczuozcum AmHflv cowuucaycn choupcu ioH x v escmm ozo c. :pmuogm
empappmumcucfi puvugcuoxz agmpmz empauopoz cowum>mpm upon
new; :orumwoomm< Levee mozocom—u
eoc ooeoe_>m toe oc._-c.oz
.mamoggcmcca a? =w>Fm mmucacmwoc as“
sect umFPaeoo mco open .929 a. cowu~>wpu mo acmuxm msu use mcpouoco mpomaz to mmvucoaoca meow .H w4mvuupuomqmmc
.mcmewgo owsqocumxv use Peace: cw a um van a mm to agm_mz mpumas umomcn mo mmzpm> vocammms
mewma .Ape mHV a omm co peace: aeoo oeo co no.e no oe:_o> mamopd one aecooecomo en eooe_=o.euo
.Au_;aocum»uv nae m=_p mmum:m_mmu a new A—uscocv Nae m:_P mapm:m_mmu za
.mmmmgucmcoa :_
woumwu mcmxu_;o we Lassa: use co» copuow>wv ucaucmum A moaco>o us» we ummmmcaxm ace mapsmwc mgha
ec.o Aev H n mH Rev Noo.o n moo.c a
do.o Aev m n on Amy ~oo.o n mo¢.o z eomo_oee»eoe_e< dz<
oe.o Rev ab 4 em Rev No.8 n e~.o o
H.H Aev em n ma va so.o n mH.o z omeepx oeo_»eoe<
m.m Aev mmH 4 won Amy e.~ n ~.e o
om.o Rev RAH n moo Amy ~.o n m.o z omeeex obesacxd
e.~ Rev one n owe” .OSV e.e n m.m a
NH.o Aev Gem 4 can” Amv ~.o h e.o z omeecx oe_booeu
«puma: umamcm mare: Faucw mpumaz ummmcm osmmFa acmxu_go msx~=m
onmofi x came_d have: .ouoe sm\mocea _e\ao.e=
amzmxu_;u u_;aocumzo
ecu Poscoz we mpumsz ummmcm new osmupa noopm on» cw moexncm pocm>om co mmwup>_uu< use .~ m4m
wm wee—e> emece>e one see» eeue_ee_ee mewpec es» sage apagmw—m ceee_e mes sews:
.ezezm meeFe> we» e>wm ea eemece>e ece xpeueceeem eFeEem seem so» eeuepeepee wee: mewuec emezhe
.epeEem FE
cee euecwe Lee a: oem we eeeeecemee e_ eeeege on» we eemmecexe ece eupemmm .Neie.aiecd 25 o.~ Any
weezuwz Le A+V new: :5 H.o we: epe>ecaepecoegemeze ueeexm avenue: mg» er eeewcemee me ace: mzemm_e e_e5eoz
e.e -- e.H~ e.m Age oeemae oexooeeeaee e_e5eoz
Ace mueemmese:
me.e n ee.e -- e.e n ~.e ~.e n m.e opens: emeoee ooeeeoeemxe
Amy eueceaeee:
Ne.e n ee.e -- e.m n e.- e.e n e.H~ opomaz emeoee o_e5eoz
me.e n mH.H e.e n e.e ~.e n e.m~ e.m n e.me Ace seeom ooeeeoeemxe
eh.e n Ne.m m.e n m.e e.e n e.~ He.e h me.e Ree saeom oPBELoz
A-e »e_>coo< A-e »e_>.eo< A+e A-e
Awe awe>_eo< -A+e ae.>_eo< »e_>_eo< auc>eeo<
eAmeie.Hiecev muesemegemwmie.Hiemeueacm saw: cewue>¢ae< ze meexewgu u_;eecumao
nee Peace: we meemmwh eee Eecwm mg» cw mesxuceeme emeepx ege>=cze we :ePQeNPLeueecesu .m mem<~
32
.HHH :ewueecm mcwme
meexewge weELe: Ecew xuw>wuee emeewx eue>=c>¢ we eeeeceepe esp cew e epeew cw ee>wm mceuesecee
esp secw eeuepeewee we; e>c=e es» .ecwuuepe ecewee meewe> ppm eecw eeuuecueem we: :ewueemew
eu cewce eEmePe esp cw xuw>wwee emeewx eue>ecwm .meeguez esp cw eeewcemee me eeewELepee
we: mcexewse on ewgeecumxe ecu Awaoe we meow mew .meexewge ewgeecumze
eee Peace: we :ewuepeecwe esp secw apw>wpee emecwx mae>=c>e eeueenew xpmeece>ecuew we mmew mew
.H ecemww
33
EIV 29.—.092. .mmzha H2;
o¢ on ON 0. 0
Au — q d _
VWS‘V'ld WNW"
.fl mcemwm
34
.msesewse ewes» cw xuw>wuee emesws
eee>esae eseecmsees saws ms» cw msewueeuueww ea wee arenasmmce Fewmmeeeem ecu; meesewse
ewseesumxe cw aew>wuee emesws eae>ecwa we eeseseewu es» mewsemeos museswceexe use we esezs
.eses szesm museumsee mums use we sewuewee—ee ms» cw
eeeepesw we: use: use Emceese smueeEee mswuuwwie>c=u seeswwieee use xe uww es e» eeceuueem
eeu use: museswsmexe pese>em Eesw name use .co>ezes .meesewse uwseecpmxe e use Peace: e sw
eeswscmuee we: emesws eue>=caeiHmNH use emesws mae>=c>e we museseepe esp .memesusecee
cw meesewse we Lessee esu sew sewaew>ee eceeceum.« ewese>e use me eemmecexe ese muwememe
mHHe me.He
HH n He NHe.e h eee.e e~.e H ee.e see oweeosemae omeewx beesseze-HmNH
mNHs He.~s
e n ee mHe.e n eme.e eH.e h mm.e Hes Hesse: omeews obessexe-Hm~H
i- ii ii sacs ewseecumao emeswx eue>=sxm
meHe m~.~e
mH n ee mee.e e Hme.e eH.e e Hm.e Hes Hesse: oboewx obeseewe
emese eweem mMssv «\Huu mMcsv «\wuu waxy sesewsu meausm
:H umos u Hussy Nx Hussy x
emsesewse ewseesumxo use Hessez we sewueweeswu
esp Eecw emeewx eue>es>miHmNH ece auw>wue< emecwx eue>=sae we wees we moves .e msm=czaism~H
we eeseseeHe ms» sew s ewsew sw se>wm mseueEecee use we emece>e esp Eesw ueuepeewee we:
e>see esw .muesuez esa sw ueswcemeu me ueswsseeeu we: msesewse on ewseecumau use Aev Peace:
Eesw zuw>wueeewues we mmew esw .msexewse ewseesumAu use Hesse: sw emesws eue>=sxeiHmNH
we sewueensw meese>esusw esu Loewe sewueweeswo as» Eesw zpw>wueeewues we mmew esw
.N esemws
37
on
:17
E; zo_._.owuz_ amend us:
e.v em ow. . e. _
d
8 8
M) ed
VWSV‘Id Idos/Wdo
§
' B
..~ mszmws
Figure 3.
38
The loss of intravenously injected creatine kinase activity
from the circulation of normal and dystrophic chickens. The
loss of activity from normal (A) and dystrophic (O) chickens
was determined as described in the Methods. Creatine kinase
activity in the plasma prior to injection was subtracted from
all values before plotting. The curve was calculated from the
average of the parameters given in Table 5 using the equation
[1].
39
' -7
N
9
UNITS! ML PLASMA
o
'01"
A
A
1
at;
0
;§ _9\
.9 “3: A
00A A
g A
<9 A .
8’ A -7 0°
i—- .4. Q
A A
ti. £5§S§
O A - A.
I All"
hfibt"
l l 1 l L l
200 400 600 800 1000 l200
TIME AFTER INJECTION (MIN)
Figure 3.
4o
.memesuseses
sH msesewsu we Losses esp sew seweew>eu useuseum N emese>e use me uemmesexe use muwemese
me.ms mNe.ee
e N NN He.e h NH.e ee.e n Ne.H Hes owesoeemwe omeess oeweeoee-HmNH
ne.me Hee.es
N N eN He.e h eH.e mH.e e em.H Hes Hesse: ooee_s oeweeete-HmNH
ne.es mee.ee
HN n ee Hme.e h ewe.e e.e e e.H Hes owesoeemwe omeews oeweeoee
”NHH me.Hs
eH 4 He mNe.e n Nee.e NN.e e Hm.e Hes Heeeoz omeews oeweeoee
obese essem ”Megs N\Hes flMees N\Hee ease eesowee oswuee
eH HmoH a H-ees NH H-ees Hs
emsesewsu ewseesumxa use Pessez we sewaeweuswu
esp Eesw emeswx eswueeLUiHmNH use auw>wpe< emeswx eswueesu we ewes we wages .m msmeu useuseum « emese>e use me uemmmsexe use muwemese
N\H He.Ns
e 4 ee 2; H x H ee.e h eN.e Hes owesoeemse omeews BEBE.HmNH
N\H mH.Ne
H N Ne 2; H A H NH.e n em.e see Hestoz onesws oeeHaeoe<-HmNH
mm.ms
eeH -- Ne.e h eH.e Hes owssosemxs owesws oHeHHeoe<
me.ee
eeH -- Ne.e H mH.e -es Hesse: omeews oHeeroee
emess uwses mMswsv «\Huu mexw sesewsu meansm
sH HmoH n N; H-ewss Hx
emsexewse ewssesumxe use Hessez we sewueweeswe esp
eesw emeswx eeewzseuwue< emeswx epewzseu< we «mes we meues .e msme es» sesw ueuewee—eu we: e>c=e esw .muesuez esp sw ueswsemeu me uesweLeueu
me: msesewse on ewssesumxu use Aev wesses eesw wuw>wuee we weep esw .msesewse ewseesumau use
Hesse: we sewueweecwe es» secw xew>wuee emewesuAseswse sz< ueueensw awmeese>esusw we meow esw
.m esemws
55
2:): ZOFUMuZ. mmrz m2_._.
.m esemws
ILLI/SIIUTI
VWSV'Td
56
in normal and dystrOphic chickens, respectively (Table 9). A small
percentage of the activity is lost in a slower phase with a half-life
greater than one hour. The clearance of 125I-AMP aminohydrolase
(Figure 9) has a half-life of 3.0 and 3.5 min in normal and dystrophic
chickens, respectively (Table 9). Not apparent in Figures 8 and 9 is
that about 95% of the injected enzyme activity or radioactivity is lost
from the circulation prior to the collection of the first blood sample 4
to 5 min after injection. This is estimated based on the amount,of
enzyme injected assuming a plasma volume of 6.0% of the body weight
(226). Extrapolation of the data shown of Figures 8 and 9 to the time of
injection using the rate constants in Table 9 indicates less than 10% of
the injected enzyme is lost at the measured rates. Thus, the clearance
of AMP aminohydrolase is accomplished by essentially a single pass
through the site(s) of clearance. Incubation of AMP aminohydrolase in
chicken serum or heparinized whole blood at 41°C at concentrations
similar to those expected after intravenous injection results in no
measurable loss of enzyme activity fOr up to 2 hr. The details of the
rapid clearance of AMP aminohydrolase are discussed in Chapter III.
Enzyme Activities in Muscle Press Juices
To estimate the extent of association of several muscle enzymes with
intracellular structures, the activities of these enzymes were measured
in breast muscle press juices and compared to the total enzyme activities
in high ionic strength crude breast muscle extracts (Table 10). The
centrifugation method for the preparation of muscle press juices (122)
results in the disruption of the sarcolemma, and the resulting press
juice presumably contains those proteins free in the sarcoplasm.
Myofibrillar proteins and those proteins associated with the myofibrils
57
.eme—ecuaseswae szwuueewues
azcemse esp e» aweseewwwsmwm
ueueswsusee use sewueweecwe esp Eecw ue>esec xwzewm esez sewueseeese emewesuxseswse
Wm es» sw museswseusee esem emeeees uesweseueu we: we: emess uwses
e
Pecuaseswse szwuue ewe—esuxseswse sz< we eeseseewe we emese 3ewm esu we ewes esws
msexewse we senses es» sew sewuew>eu useuseum « emece>e es» me uem
.memespseses sw ueemeu
mesexe ece mu—emec eswe
Hm.ms
-- ee.e h eN.e Ame owssocemwe omewocexsoewee sz<-HmNH
he.mH
-- mH.e A NN.e Hes Hescoz emeHoeewsoewe< ss<-HmNH
HH.es
we He.e A NH.e Hes owssocemse omeHocewsoewe< Ase
Hm.Ne
ea me.e h 4N.e Hes Hesse: umeHocewsoeHse Ase
semess uwses nMswsV «\Hug eexw sexuwsu eE>~sm
eH emoH a H-e_es Hs
emsesewsu ewseesumxo use peace: we sewue—eeswu esp eecw
emeHoeeasoewE< ss<-HmNH eee sewsweo< omeHocessoewss Ass we mmoH we emcee .e eeeew
58
.szesm meewe> esp e>wo ep uepeecpsem sees e>es sewpeweeswe
esp sw sewpeseeess ewe—esuxseswEe szwpeeewuec
we sewpeswspsee esw .muespez esp sw ueswsemeu me ueswscepeu me: msesewse woe ewssecpmau use
fies Peaces Eesw zpw>wpeeewues we ewe. esw .msexewse ewseespmxu use wesses sw emewesuxseswse
szecpsw esp sepwe sewpeweecwe esp sesw xpw>wpeeewues we mmew esw
.m essmws
59
.m exemws
2.2. zoneeez. seen: ”.5:
Ga 0. o
_
1
(D
g;
VWSV'ld "109/de
ICON
60
.msesewse mps esww ewseespmau mepesmwmeu s .msesewse «He esww peace: wepesmwmeu zs
.epsep mwsp sw
se>wm ewemee use eewen mmese esp sw mewpw>wpee eaese>e esp.sesw uepepeepee ww ueswepse mewpes esp eesw
appsawwm sewwwu awwesewmeooe sews: .s3esm meewe> esp e>wm ep uemece>e use sesewse seee sew awepecesem
uepeweewee ese: ewomes sw pesp ep eewen mmess esp sw xpw>wpee eexnse we mewpes esw .memespsesee
sw msesewse we senses esp sew sewpew>eu useusepm w emese>e esp we uemmesexe ece mppemes eswe
we.e « mH.o mN.o A Np.~ oH.e p Hm.e Np.o p m~.e . HN « ww s.m « e.s~ Ass s emewecuzs
HH.e e eN.e eH.H e em.N He.e h ee.H mH.e H em.e HN h NeN em 4 eeH see 2 -oewe< sz<
~.o « e.H e.H p e.e H.H « m.e e.o A w.p mmp p mom sup H see Ass s emeswx
N.e h e.H m.N e e.eH e.m n H.eH e.e A e.N NNH n mee one o eNeH Ame z opesscss
mH.o p pm.o m.e p H.Nm H.m « m.m~ e.o « m.p ems p seep sop « emwp Ass s emeswx
oH.e N ee.e N.s p o.w~ w.s p s.o~ ~.o w e.H ecu A epep esp « seep Amy 2 eswpeece
e.e p e.H mw.o p so.~ mp.e p ~m.~ s.e p e.H mp « em He « pep Ass s emeswx
N.e n H.H mm.e n Hm.H NH.e e em.H m.e n H.N eN « me eN u eeH see 2 opepaeoes
Aewemeev sewemeev Aeewen mmesss Aewemesv Aewumeev Aeowen mmesev sesws esz~sm
swepecs swepese swepese mxmpwse a\mpws= _e\mpws= sesewse
me\mpwse ms\mpwse me\mpws= Neewewmmesev
awewewmmessv ws\mpws=
swepese
ms\mpws=
eepumez
pmeesm sesewsu ewseespmzs use pessez secw mpeespxm euesu use meeweu mmess we mewpw>wpe< esaesw .op msmwpue emesws epe>esxs we weep esp sew ueswssepeu es pes upeee
mpsepmsee epes eeswm emeswx epe>=sxs uewese—iHmNH we weep esp sew ueswssepeu
we: msesewse ewssespmxu sw emeswx epe>=sws we sewpeswspmwu esp sew mpsepmseu epese
.omximmxiwx+ax u meu
. . .emess upses
esp sw pme— esxuse esp we sewpeesw esp mw < eses: ms + Awsipsv< u muse
.mNs\NHHs n emse
.memespseses sw msesewse
we sesEes esp sew sewpew>eu useusepm H emese>e esp me uemmessxe ese mppemes eswe
eee.e H Hee.e eH.e H mm.e ee.e H eH.e uses owssospmse onesws opesssss
mee.e H mse.e eN.e H eN.e mee.e H eme.e see Hesse: emesws opesssss
eH.e H He.e ee.e H Nm.e se.e H eH.e Ass owssospmse owesws eswpeose
NH.e H eH.e NH.e H eN.e me.e H HH.e see Hesse: onesws uswpeose
esH-ses NNH osH-sss «NH esH-sss ems eeHH eosowse ossNee
esewpoewsw meese>espsH sepwe
emesws epe>=sas use emeswx eswpeese we sewpeswspmwe esp sew mpsepmsee epes .HH mpme esp mp
pswes seem .muespez esp sw ueswsemeu we ueswssepeu ese: Ass see—em esp use Aev se>w— esp sw
iv 53 use AS e3 we ase>eees esw sews—2-52 se sww esp sw HmNH se esp we »s
w
pm we
>eoes
.NH assess
91
ps.
.0 I...
-in
«1-
~10
-N
i3 do LO 4 N e0
i u
D «40
1 in
q.
~10
-N
s is: s: 8 co
" anssu NI oauanooae
ALIAILOVOICIVH OBLOEI‘NI :IO .LNBOHBd
TIME AFTER INJECTION (HR)
Figure 12.
92
Figure 13. Bio-Gel P-60 elution profiles showing the size distribution
- of 1251 in the liver, spleen, and excrement after the
injection of 1251-AMPAM. (a) Liver 30 min after
injection; (b) liver 2 hr after injection; (c) spleen 30 min
after injection; (d) spleen 2 hr after injection; (e)
combined excrement collected 7 hrs after injection. MIT =
monoiodotyrosine. '
93
0 5 )0 IS 20 25 30
FRACTION NUMBER
Figure 13.
94
cleared by the kidneys and excreted in the urine. Seven hours after the
injection of [14C]sucrose-AMPAH most of the radioactivity in the
liver and the Spleen eluteS in the low molecular weight region of the
gel-filtration profile (Figure 14). The position of elution of 14C
on the gel-filtration column indicates the size of this material is
larger than [14C]sucrose and is probably [14C]sucrose attached to
an amino acid or small peptide. This material would not likely be able
to diffuse through the lysosomal membrane. This is consistent with the
data in Figure 12 that show 14C is retained in the liver and the
Spleen after the intravenous injection of [14C]sucrose-AMPAH.
Subcellular Localization of Cleared AMPAH
To determine the subcellular localization of the degradation products
of [14C]Sucrose-AMPAH, homogenates of liver and spleen were fraction-
ated on sucrose density gradients 7 hr after the injection of the radio-
labeled enzyme. The distribution of 14C and the lysosomal marker
N-acetyl-8-D-glucosaminidase was determined (Figure 15). The 14C
profile coincides with the N-acetyl-B-D-glucosaminidase profile in both
the liver and the spleen. The 14C and N-acetyl-B-D-glucosaminidase
activity at the top of the gradient is probably from lysosomes broken
during the homogenization procedure. A similar experiment with
125I-AMPAH resulted in only a small amount of 1251 sedimenting
with lysosomes. The remainder of the radioactivity was low molecular
weight radioactivity at the top of the gradient and was probably low
molecular weight degradation products of the 125I-AMPAH that
diffused out of the lysosomes during the preparation procedures.
Figure 14.
95
Bio-Eel P-60 elution profiles showing the Size distribution
of 1 in the liver and Spleen four hours after injection
of [1 C]sucrose-AMPAH. Tissue extraction and Bio-Gel
P-6O chromatography of samples from the Spleen (a) and.liver
(b) were as described in the Methods.
96
. AMPAH [4CJSUCROSE
l 1 1
O 5 IO I5 20 25 30
FRACTION NUMBER
Figure 14.
.HsoH osH oH as HHeoseese esp so sop esp .ses eouHsH esp use see sost
esp eesw mepesemeEes we mpsewuesm sw o epesws see mpseee Axv HAemme emeuwswsemeeewm
m“ ioieiwxpeeeiz Eesw eeseemeseeww e>wpepes was .:wp esp we mepesemeses we wepwwese sewpepseewuem psewuesm zpwmseu emeseem .mp esemws
98
- 300
«250
200
1150
4 100
- so
(-O-) BONBOSBHOO'H BNLV'BH
.13
x—
"8 e810 5 P 0
20
I5
5
l0
FRACTION NUVIBER
IS 20
I0
FRACTION NUVIBER
o “ ‘0
______.....x'
8 in o in Q ID 0
N N
.(-O-) BONBOSBHFIO'H EALLV'BU
Figure 15.
99
Parenchymal and Nonparenchymal Cell Distribution of [14C]Sucrose-
AMPAH Cleared by the Liver
The results in Table 13 Show that most of the 14C is recovered in
the parenchymal cell fraction of the liver when liver cells are
fractionated after the clearance of [14C]sucrose-AMPAH from the
blood. The recovery of 14C per mg cell protein in parenchymal cells
is 2.3 times that of nonparenchymal cells. These results demonstrate
that the parenchymal cells are primarily responsible for the clearance of
AMPAH by the liver.
Inhibition of AMPAH Clearance
To investigate the process involved in the rapid clearance of AMPAH,
the effect of various compounds on the rate of clearance was examined.
The compounds were injected intravenously 5 min before the injection of
AMPAH and the clearance of AMPAH was monitored. Agalactofetuin,
N-acetylglucosamine, yeast mannan, mannose-6-phosphate, heparin,
chondroitin sulfate, and dextran sulfate were tested for reasons that '
will be outlined in the Discussion. Of these compounds, only the
sulfated polysaccharides heparin, chondroitin sulfate, and dextran
Sulfate inhibit the clearance of AMPAH activity (Figure 16).
Release of Cleared AMPAH into the Circulation by Heparin
In addition to the inhibition of AMPAH clearance by heparin,
injection of heparin releases AMPAH into the circulation after the enzyme
is cleared (Figure 17). The activity of AMPAH in the circulation 5 min
after heparin injection iS higher than that observed only 5 min after
AMPAH is injected. Little or no AMPAH activity is released into the
circulation by heparin injection into chickens that had not previously
received AMPAH injections.
100
TABLE 13. Distribution of 14C in Parenchymal Cells (PC)
and Nonparenchymal Cells (NPC) 2f the Liver Four
Hours After the Injection of [1 C]Sucrose-AMPAH.
These results are the average 1 standard deviation
for four determinations.
cpm PC/mg protein PC
cpm PC/cpm NPCa cpm NPC/mg protein NPC
8.5 t 2.8 2.3 t 0.3
aThe cpm in each cell fraction was corrected for the
recovery of cells in the final fraction compared to the
initial cell suspension.
Figure 16.
101
The effects of several compounds on the loss of AMPAH
activity from the circulation. The compounds were injected 5
minutes before AMPAH injection and the loss of AMPAH activity
was monitored. The compounds injected were: (0) none; (0)
20 mg N-acetylglucosamine; (A) 10 mg agalactofetuin; (E3) 10
mg mannose-6-phosphate; (*) 15 mg yeast mannan; (I) 30 mg
heparin; (X) 30 mg dextran sulfate; (A) 30 mg chondroitin
sulfate C; all in 0.5 to 1.0 ml PBS.
102
aim
a ..- “MW mmmmmw
mm
0.. RN
.. Em
oeeem
so- we
imT
‘A0
s s s .L. ..
20:44:01.0 m1... 2. >._._>_._.o<
14.1.26. omhomuZ mo .58me
Figure 16.
103
Figure 17. Release of AMPAH activity into the circulation by heparin
injection after the clearance of intravenously injected
AMPAH. This experiment is representative 0 3 similar
experiments. ~
104
._. I\)
0‘ O
I
E
—
O
'—
~.——C—I—h___
PERCENT OF INJECTED AMPAH
REMAINING IN THE CIRCULATION
C) L, I L I l I l
0 IO 20304050607080
TIME AFTER AMPAH
INJECTION (MIN)
Figure 17.
105
The results in Table 14 show that AMPAH released into the circulation
by heparin after clearance of the enzyme is primarily from the spleen and
the liver. The increase in circulatory 1251 when heparin is
injected 30 min after 125I-AMPAH injection is entirely attributable
to the loss of 125Iifrom the liver and the spleen. The 1251
remaining in these tissues after heparin injection is probably due to
internalized 125I-AMPAH and in part due to the 125I present in
the blood within these tissues. It is possible that some 125I-AMPAH
iS bound in a manner that is resistant to release by heparin.
Figure 18 Shows that there is AMPAH activity, and radioactivity from
[14C]sucrose-AMPAH, released into the circulation by heparin
injection for at least 4 hr after injection of the enzyme. The inset in
Figure 18 Shows that the loss of heparin-releasable AMPAH with time after
AMPAH injection is a first-order process with a rate constant of 0.76
hr-l (t1/2 = 0.98 hr).
106
TABLE 14. Release of 1251 from the Liver and Spleen into
the Circulation by Heparin Injection 30 Minutes After
ZSI-AMPAH Injection
Percent of Injected
Radioactivity Recovered in Tissue
Blood Liver Spleen
Chickens Not Injected 3.2 45.3 10.1
with Heparin '
Chickens Injected With 28.2 23.9 1.8
Heparin
Difference (% of Injected 25.0 21.4 8.3
Released by Heparin)
Figure 18.
107
Release of AMPAH activity or 14C into the circulation by
heparin injection at ieveral times after the injection of
unlabeled AMPAH or [1 C]sucrose-AMPAH. AMPAH activity
(0) and 14C (0) were determined as described in the
Methods. Each point is the average from 2 chickens.
108
6
4
2
TIME AFTER AMPAH INJECTION (HR)
00
_
m c
253nm: >m 29.3.5016 mi... 9.2. omm._..>_._.o< I
mom me Amen mmm memn oz
gmmwnm awn: =_cnnm: crouomn.ppwp ms mgnpxpz m=_n:wm :_
venom znnee u_e_eeem e guy; mma em = :o copumgucmucou
e_eeaoe eo eooceo An .maz<_\~ .ns xeeocpo>\~ ea eo_a peooea_ooe o_e=oo Ac .m.e :a .m_me-mmz
:5 cm ._ux zs mH.o ecu .az< new avenge; eo «copumeucmocou mcwzgm> an muoguw: on» cw umnwgummu
mo mczux_s xmmmc mg» 0» woven mm: : 05 °
._\. ° “— 0 X ex .42
'04" 0 ——=> .58
.03A- 4 .83
~02" ate?
‘0' ‘ arr-24"" 93.539
1 l
-.05 o .05 lo .15
[HEPARIN] ug /ml
Figure 30.
167
.Fux z m~.o emcwmacoo ompm memeyan
cowuspm wpmzamozaoexq new cwgmam; on» .m.w :a .mfimhimmz :5 cm cw __~ .mumzamozqogaa :2
cm Lo .c_gmams Hips me a ._ux z ~.H ._ux z ~.HimH.o we u:m_uoga Fox a emguvm saw: mesmwe
mcu cw cmuoopu:_ mm toga—m use muozumz on» c_ uwnweummu mm on me mmoeugaom vo~wum>_gmu=:
Lo on me wmogmzammicweoaw; on umwpaam mm: zL--9i g2
ldd>l—> 9
in
l L l l
E
g
9:)
Z
Z
(0— “’9
._—
L 1 1956911 2
I
LL.
20
IS
IO
.LNBIOVHE) ION
_ HleNV iw/suun
Figure 31.
169
DISCUSSION
The interaction of 125I-AMPAH with hepatocyte monolayers is
inhibited by sulfated polysaccharides and effectors of AMPAH enzymatic
activity. The interaction is most effectively inhibited by sulfated
polysaccharides with the highest sulfate content. Sulfated polysac-
charides inhibit AMPAH activity, and the interaction of AMPAH with
heparin is further demonstrated by the binding of AMPAH to heparin-
Sepharose 4B. The binding of allosteric effectors and sulfated
polysaccharides to AMPAH may inhibit the interaction of the enzyme with
hepatocytes by decreasing the affinity of AMPAH for cell surface binding
sites. A similar hypothesis has been suggested for the heparin-releas-
able binding of lipoprotein lipase to endothelial cells (252) and low
density lipoprotein to fibroblasts (274). However, the phOSphate-con-
taining polyanions RNA and polyphosphate inhibit AMPAH activity (Table
21), and RNA has been shown to associate with AMPAH (275), yet these
polyanions are relatively poor inhibitors of the interaction of AMPAH
with hepatocytes compared to the sulfated polysaccharides. The sulfated
polysaccharide binding site(s) on AMPAH may be different from that for
phosphate containing polyanions, and the inhibition of the interaction of
AMPAH with hepatocytes may be more marked when sulfated polysaccharides
are bound to the enzyme.
The demonstration that AMPAH has a high affinity for heparin suggests
that the binding of AMPAH to hepatocytes may be due to the binding of
170
AMPAH to cell surface glycosaminoglycans. This is similar to the
mechanism proposed for the binding of lipOprotein lipase to cultured
endothelial cells (254). Ninomiya gt 31. (276) demonstrated that heparan
sulfate is the major component of cell surface glycosaminoglycans
synthesized by cultured rat liver parenchymal cells. Thus, sulfated
polysaccharides may compete directly for the binding of AMPAH.
Furthermore, the release of AMPAH bound to heparin-Sepharose 4B by
pyr0phosphate and KCl suggests that effectors of AMPAH activity and salts
could release AMPAH bound to glycosaminoglycans on the cell surface.
The concentration dependence for the release of AMPAH bound to
hepatocytes by substrate and effectors of AMPAH activity (Figure 26),
shows GTP, ADP, and ATP are more effective than pyr0phosphate and AMP in
releasing AMPAH bound to hepatocytes. Ashby and Frieden (277) have
proposed that AMPAH has three classes of nucleotide binding sites: 1)
a high affinity inhibitory site which binds nucleotide triphosphates and
pyr0phosphate (271); 2) an activating site which binds nucleotide
diphosphates and triphosphates with medium affinity and nucleotide
monophosphates with a lower affinity; and 3) a substrate binding site
which binds AMP. Though speculative, the observation that pyr0phosphate
and AMP are less effective than ATP, ADP, and GTP at releasing bound
AMPAH from hepatocytes suggests that the interaction of nucleotides with
the activating site of AMPAH may be primarily responsible for the release
of bound AMPAH.
The lack of a significant specific inhibition of the binding by the
carbohydrates shown in Table 20 indicates that AMPAH is not bound to
hepatocytes by one of the carbohydrate recognition proteins of the liver,
and that a different system is involved. Because the binding of
171
lipoprotein lipase to endothelial cells and low density lipoprotein to
fibroblasts are also markedly inhibited by heparin and other sulfated
polysaccharides, it would be of interest to determine whether these
enzymes purified from the chicken have an effect on the binding of AMPAH
to hepatocytes.
SUMARY AND DISCUSSION
172
173
SUMMARY AND DISCUSSION
The levels of several muscle enzymes were examined in the blood
plasma from normal and dystrophic chickens. The activities of creatine
kinase and muscle pyruvate kinase are markedly elevated in the
circulation of dystrophic chickens compared to normal chickens. However,
the activities of AMPAH and adenylate kinase are not elevated. This
pattern of elevation is essentially the same as is observed in human DMD
and suggests that the study of those factors which determine the levels
of muscle enzymes in dystrophic chickens may be relevant to the human
condition.
The results in Chapter II demonstrate that AMPAH and adenylate kinase
activity are rapidly lost from the circulation with half-lives of only a
few minutes after the intravenous injection of the enzymes purified from
normal chicken breast muscle. In contrast, the activities of pyruvate
kinase and creatine kinase are lost with half-lives of several hours.
Based on the estimated rate of efflux of pyruvate kinase from dystrophic
muscle tissue, it was determined that the rapid circulatory clearance of
AMPAH and adenylate kinase is sufficient to reduce the circulatory levels
of these enzymes so that the activities are not significantly elevated in
the circulation of dystrophic chickens as are the more slowly cleared
proteins creatine kinase and pyruvate kinase. These results suggest that
the circulatory clearance rates of muscle proteins in the serum may
determine the levels of these proteins in the serum of dystrOphic
174
chickens. It would be of interest to extend these experiments to deter-
mine whether there is a correlation between the circulatory clearance
rates and the extent of elevation of some other muscle enzymes in avian
muscular dystrophy.
Though these experiments demonstrate the ability of chickens to
rapidly remove AMPAH and adenylate kinase fran the circulation, they do
not prove that these enzymes are released into the circulation from
dystrophic muscle tissue as are the enzymes pyruvate kinase and creatine
kinase. The examination of the activities of these enzymes in muscle
press juices compared to muscle homogenates suggests that AMPAH, but not
adenylate kinase, is associated with intracellular components to a
significant extent. This extensive association could result in the
retention of AMPAH within the dystrophic tissue and therefore retard the
release of AMPAH into the circulation. Furthermore, the possibility can
not be eliminated that adenylate kinase and AMPAH are retained in the
muscle tissue because the sarcolemma is not permeable to these two
proteins.
There are several experimental approaches that might verify the
release of AMPAH and adenylate kinase into the circulation of dystrophic
chickens. Using a sensitive AMPAH assay it may be possible to demon-
strate arteriovenous differences in AMPAH activity across the breast
muscle. AMPAH that is cleared from the circulation after intravenous
injection is slowly internalized by the liver and spleen and can be
released from the liver and spleen by heparin injection before internali-
zation. It may be possible to demonstrate increased AMPAH activity in
the circulation of dystrophic chickens after heparin injection that is
due to enzyme which has been released from the dystrophic chicken muscle
175
and has been cleared but not yet internalized. Preliminary experiments
demonstrated no apparent release of AMPAH into the circulation of normal
or dystrophic chickens after heparin injection. However, the high ultra-
violet absorbance of blood plasma at high sample concentrations inter-
feres with the assay of AMPAH which monitors the increase in ultraviolet
absorption at 290 nm or decrease in absorption at 265 nm as AMP is
deaminated to form IMP. A more sensitive assay for AMPAH using radio-
active substrate as described by Maguire and Aronson (278) may be
advantageous to further studies of this type.
In the case of adenylate kinase, the examination of the isoenzyme
profiles of the low levels of adenylate kinase activity in normal and
dystrophic serum might reveal an increase in the level of the muscle
isoenzyme in the serum of dystrophic chickens. The muscle isoenzyme can
be distinguished fran the isoenzymes frun other tissues on the basis of
inactivation of the muscle isoenzyme by sulfhydryl modifying reagents
(222-224), electrophoretic mobility (68,222), or immune precipitation
(68,279).
The rapid circulatory clearance of adenylate kinase activity may be
due to enzyme inactivation caused by the oxidation of essential
sulfhydryl residues of the enzyme. Adenylate kinase activity is rapidly
lost when the enzyme is incubated in serum in vitrg_and the inactivation
is prevented by adding dithiothreitol to the serum. Enzyme activity was
not recovered in the primary tissue sites of clearance of
125I-adenylate kinase shortly after clearance. The rate of
clearance of 125I-adenylate kinase was also rapid, indicating that
the enzyme probably does not remain in the circulation in an inactive
form. It may be possible to demonstrate a prolonged circulatory
176
clearance rate of adenylate kinase by the coinjection of a sulfhydryl
compound such as penicillamine.
The process responsible for the rapid circulatory clearance of AMPAH
was studied in detail. AMPAH activity is cleared with a half-life of
only about 5 min and is recovered primarily by the liver and the Spleen
after intravenous injection of the purified enzyme. Using radioactively
labeled AMPAH it was determined that cleared AMPAH is internalized and
degraded in lysosomes in the liver and the spleen. Clearance is
inhibited by heparin, but not by inhibitors of carbohydrate recognition
systems of the liver which might recognize carbohydrate residues on the
enzyme. Perhaps relevant is the observation that these same
characteristics were observed for the circulatory clearance of
intravenously injected lipoprotein lipase in rats (251).. It would be of
interest to purify lipoprotein lipase fran chickens and to determine if
AMPAH and lipoprotein lipase compete with one another for clearance in
vixg_and/or the binding to hepatocyte monolayers.
I have demonstrated that AMPAH binds to hepatocyte monolayers with a
high affinity. As is observed for the lg_vivg_clearance of AMPAH, the
enzyme is internalized, is degraded, and the binding is inhibited by
sulfated polysaccharides. These results, however, are indirect evidence
that the binding of AMPAH to cultured hepatocytes is by the same process
as that responsible for the rapid clearance of the enzyme in 3139,
Further evidence might be obtained by the purification of the hepatic
component which binds AMPAH at the cell surface. It would be of interest
to determine the effect of the purified hepatic component, or antibodies
against the component if it is a protein, on the binding of AMPAH to
hepatocyte monolayers in vitro. However, initial attempts to
177
purify such a component from chicken liver were not successful. Crude
membrane fractions fron liver did not specifically bind AMPAH, even when
thiol proteinase inhibitors were included during the preparation of the
crude membrane fraction. Further trials under different conditions may
be more successful.
The interaction of AMPAH with hepatocytes is markedly inhibited by
molecules which bind AMPAH including effectors of AMPAH activity and
sulfated polysaccharides. However, some molecules which bind AMPAH are
less effective inhibitors of the interaction of AMPAH with hepatocytes.
Heparin binds AMPAH tightly, as judged by a K; for the inhibitor of
AMPAH activity by heparin of 20 ng ml'l, the association of AMPAH
with heparin-Sepharose 4B, and the release of AMPAH bound to hepatocytes
at heparin concentrations of 10 ug ml"1 or less. It is possible that
AMPAH binds a heparin-like molecule at the cell surface. It has been
demonstrated that heparan sulfate is the major glycosaminoglycan on the
cell surface of cultured rat hepatocytes (276). Recently, Cheng £5 31.
(254) prepared an enzyme from human platelets which specifically degrades
heparin and heparan sulfate. Treatment of endothelial cells with this
preparation abolished the binding of lipoprotein lipase to these cells.
An analogous experiment to determine whether treatment of hepatocytes
with heparinase reduces the binding of AMPAH might help establish the
basis for the binding of AMPAH. A preliminary experiment with a crude
platelet heparinase purified as described by Oldberg gt_al, (280),
resulted in a 35% reduction in the binding of 125I-AMPAH to
hepatocyte monolayers. However, the heparinase activity of this
preparation was low, and some protease activity was apparent in the
preparation.
178
The physiological significance of the rapid circulatory clearance is
not clear. The rapid clearance of AMPAH fron the circulation may be
necessary to remove the enzyme from the circulation so that high circula-
tory levels of AMP are maintained. Though the circulatory levels of AMP
and other nucleotides are low, it has been suggested that circulatory AMP
may be an important source of purines in lymphocytes (281). Furthermore,
nucleotides are potent vasodilators at physiological concentrations (282)
and the relative levels of different nucleotides in circulation may be
important in the regulation of blood flow. It would be of interest to
determine with sensitive radioactive AMPAH assays, whether there is a
steady-state level of AMPAH bound extracellularly that is released into
the circulation by heparin injection that may play a role in the regula-
tion of circulatory levels of AMP.
The results of the press juice experiments in Chapter II demonstrate
that a large percentage of the AMPAH in muscle tissue exists in a bound
form intracellularly and is not free in the cytoplasm of the muscle cell.
If it is assumed that pyruvate kinase exists entirely as a soluble enzyme
in the muscle cell, then by comparison of the ratios of the activities of
these enzymes in the press juice to that in the crude homogenate, 78% of
the AMPAH in the muscle exists in an intracellularly bound fOrm. Ashby
and Frieden (124) estimated that only 5-10% on the AMPAH in muscle exists
in a complex with contractile elements in isolated myofibrils. It is
possible that AMPAH is bound to cellular components other than myofi-
brillar proteins. Preliminary experiments demonstrated that AMPAH binds
not only to hepatocytes in cell culture, but also with chick embryo
fibroblasts and muscle cells. It would be of interest to determine
whether there are binding sites for AMPAH on membranes within the muscle
179
cell analogous to the binding sites responsible for the binding of AMPAH
to the cell surface. This binding might have a marked effect on the
regulation of the activity of the enzyme intracellularly and on the role
of AMPAH in the regulation of metabolism within muscle (283). The
association of AMPAH with membranes within the muscle cell might explain
the high extent of intracellular association of AMPAH. Similarly, Pipoly
.gt.al. (284) have demonstrated that human erythrocyte AMPAH binds to the
cytoplasmic side of erythrocyte membrane ghosts, and that the binding is
inhibited by nucleotide effectors of the enzyme and by salts.
In summary, these results provide for the first time an explanation
based on experimental evidence, for the observations that some muscle
proteins are not elevated in the serum of dystrophic animals. Based on
the rates of circulatory clearance of muscle proteins, it was also
possible to estimate the rate of efflux of creatine kinase and pyruvate
kinase from dystrophic chicken muscle. These results are important in
terms of describing the character of the proposed membrane defect in
muscular dystrophy, and the role of this membrane defect in determining
the serun and muscle levels of enzymes in muscular dystrophy.
The study of the rapid clearance of AMPAH was actively pursued
because it has characteristics which differ from other mechanisms for the
uptake of proteins from the circulation. This may describe a general
mechanism for the specific binding and uptake of some proteins. The
development of an in vitro method to study the binding and uptake of
AMPAH will allow the elucidation of further details of the interaction of
AMPAH with cells.
APPENDIX
2.
4.
6.
180
APPENDIX: Papers, Abstracts, and Manuscripts in Preparation.
Suelter, C.H., Thompson, D., Oakley, G., Pearce, M., Husic, H.D.,
and Brody, M.S. (1979) Comparative Enzymology of 5'-AMP
Aminohydrolase from Normal and Genetically Dystrophic Chicken
Muscle. Biochem. Med. 21, 352-365.
Husic, H.D., Young, R., Suelter, C.H., and McConnell, 0.6. (1979)
Comparative Development of Several Enzymes in Chicken Breast
Muscle in vivo and in Cultured Cells from Breast Muscle of Normal
and Genetically Dystrophic Chickens. Fed. Proc. 38, 667
(Abstract).
Husic, H.D. and Suelter, C.H. (1980) The Rapid Disappearance of
Muscle AMP Aminohydrolase from the Blood Plasma of Normal and
Genetically Dystrophic Chickens. Biochem. Biophys. Res. Commun.
3, 228-235.
Husic, H.D. and Suelter, C.H. (1980) The Rates of Disappearance of
Chicken Breast Muscle AMP Aminohydrolase and Pyruvate Kinase from
the Blood Plasma of Normal and Gentically Dystrophic Chickens.
Fed. Proc. 39, 2171 (Abstract).
Husic, H.D. and Suelter, C.H. (1982) Circulatory Clearance of
Muscle Enzymes from Normal and Dystrophic Chickens. Vth
International Congress on Neuromuscular Diseases, Marseille,
France (Abstract).
Husic, H.D. and Suelter, C.H. (1982) The Levels of Adenylate
Kinase and Creatine Kinase in the Plasma of Dystrophic Chickens
Reflect the Rates of Loss of these Enzymes from the Circulation.
Biochem. Med., submitted for publication.
Husic, H.D. and Suelter, C.H. (1982) Circulatory Clearance, Uptake
and Degradation of Muscle AMP Aminohydrolase. Manuscript in
Preparation.
Husic, H.D. and Suelter, C.H. (1982) Internalization and
Degradation of AMP Aminohydrolase Bound to Hepatocyte Monolayers.
Manuscript in Preparation.
Husic, H.D., Baxter, J.H., Pearce, M., and Suelter, C.H. (1982)
Comparative Enzymology Throughout the Development of Normal and
Genetically Dystrophic Chickens. Manuscript in Preparation.
LI ST 0F REFERENCES
10.
11.
12.
13.
14.
15.
181
LIST OF REFERENCES
Drachman, D.B., Kao, I.
and Hinkelstein, JA. (
Dystrophies (Rowland, L
111-120.
, Pestronk, A., Toyka, K.V., Griffin, D.E.,
1977) in Pathogenesis of Human Muscular
.P., ed.) Excerpta-Medica, Amsterdam, pp.
DiMauro, S., Mehler, M., Arnold, S., and Miranda, M. (1977) in
Pathogenesis of Human Dystrophies (Rowland, L.P., ed.),
Excerpta-Medica, Amsterdam, pp. 506-515.
Layzer, R.B., Rowland, L.P., and Ranney, H.M. (1967) Arch. Neurol.
11, 512-523.
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