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This is to certify that the

thesis entitled

ISOLATION AND CHARACTERIZATION OF THE
GLYOXYSOMAL B-OXIDATION SYSTEM FROM
GERMINATING CASTOR BEAN ENDOSPERM

presented by

John Walter Uhlig

has been accepted towards fulfillment
of the requirements for

Ph . D. degree in Biochemistry

 

 

77.3%

 

 

Major professor

Date % 45/ /7C?/

0-7639

 

 
 
  
     

‘ LUBE ~
, ‘ Michigan 3ch
I University

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charge fro. circulation records

 

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.9: v‘ .;

 

 

 

 

ISOLATION AND CHARACTERIZATION OF THE GLYOXYSOMAL B-OXIDATION
SYSTEM FROM GERMINATING CASTOR BEAN ENDOSPERM

By

John Walter Uhlig

A DISSERTATION

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

DOCTOR OF PHILOSOPHY

Department of Biochemistry

1981

ABSTRACT

ISOLATION AND CHARACTERIZATION OF THE GLYOXYSOMAL B-OXIDATION
SYSTEM FROM GERMINATING CASTOR BEAN ENDOSPERM

By

John Halter Uhlig

B-Oxidation of fatty acids was found only in the glyoxysomes from

the endosperm of germinating castor bean seeds (Ricinus communis).

 

B-Oxidation and thiolase activities were not found in the mitochondrial
fraction. A complex of the glyoxysomal enzymes for B-oxidation was
purified 41 fold as a complex consisting of acyl-CoA oxidase, enoyl-CoA
hydratase, 3-hydroxyacyl-COA dehydrogenase, 3-ketoacyl-CoA thiolase and
possibly D,L-3-hydroxyacyl-C0A epimerase and cis-3,trans-2-enoyl-C0A
isomerase. These enzymes were considered as a complex because of their
co-purification during ammoniun sulfate fractionation and co-chromatog-
raphy on hydroxyapatite, Biogel A 0.5 and carboxymethyl Sephadex 6-25.
The association of the six enzymes seems analogous to the B-oxidation
complex in g, £911. The enzymes from the castor bean co-sedimented on
sucrose gradients, but smeared during migration on polyacrylamide gels,

even in the presence of stabilizing reagents. Polyacrylamide gel

John Walter Uhlig

indicated a high degree of purification, but, due to dissociation,
absolute purity was not proven. _Molecular sieving and sucrose gradient
sedimentation indicated an unrealistically low molecular weight for the
complex. The apparent low molecular weight could have been due to the
presence of the neutral lipids in the purified preparations.

The overall s-oxidation activity was unstable, but could be main-
tained for several days at 4°C with the incorporation of 10 mM dithio-
threitol and 25 to 50% (v/v) glycerol or other polyhydric alcohols into
the buffers. The logarithum of the half life of a-oxidation activity
was proportional to the glycerol concentration. A deviation from the
relationship occurred when the glycerol concentration and temperature
were simultaneously lowered, perhaps due to a cold inactivation.
Glycerol also reduced a loss of specific activity which occurred at low
protein concentrations. B-Oxidation was completely inhibited at 50 pH
p-chloromercuribenzoate.

B-Oxidation activity was measured both as oxygen uptake and as NAD
reduction. The pH Optimum was between 8 and 9, which is characteristic
of peroxisomal enzymes. Activity required the presence of CoA, NAD,
oxygen, acyl-CoA substrate and enzyme. NADP did not substitute for
NAD, nor did NADP inhibit the B-oxidation process. The apparent Kms of
the B-oxidation complex were 72 uM for oxygen, 0.15 pm for FAD, 79 uM
for NAD, 71 pH for butyryl-CoA, 44 uM for octanoyl-CoA, 1 to 10 uM for
long chain acyl-CoA substrates and approximately 4 uM for CoA. B-Oxi-
dation activity was stimulated by added FAD, but not by added FMN. The
only flavin isolated from the purified complex was FAD. Acetyl-CoA and
NADH caused no product inhibition of B-oxidation, whereas the longer

chain acyl-CoA substrates at higher concentrations were severly

John Walter Uhlig

Bovine serum albumin reduced long chain acyl-CoA inhibition, but it
also inhibited B-oxidation activity when substrate concentrations were
lower. Detergents were also inhibitory and the magnesium ion caused
50% inhibition at 4 mM.

The stoichiometry of the oxidation of palmitoyl-CoA was 7 moles
NAD reduced per mole of substrate added in the presence of saturating
amounts of NAD and CoA. Various other fatty acyl-CoA substrates were
also completely oxidized. The presence of the isomerase and epimerase
activities would account for the systems ability to completely oxidize
unsaturated fatty acids. The complete oxidation of both saturated and
unsaturated fatty acids to acetyl4CoA would be consistent with the
efficient conversion of fats to carbohydrates in the germinating seed.
Based on NAD reduction, ricinoleoyl-CoA was oxidized by glyoxysomes and
the purified complex only to the 4-hydroxydecanoyl-C0A intermediate.

In the absence of CoA, 1 mole of NAD was reduced per mole of palmitoyl-
CoA added indicating the complete oxidation of the substrate to the
3-keto intermediate.

The chain length specificity of the glyoxysomal B-oxidation system
favored the longer chain length acyl-CoA substrates. Shorter chain
length substrates were oxidized with a lower affinity (higher Km) and
lower reaction velocity. The chain length specificity was similar to
that of the rat liver peroxisomal acyl-CoA oxidase and the B-oxidation
system. Thus, the liver peroxisomal and castor bean glyoxysomal B-oxi-
dation systems seem to be very similar, yet a difference in the
processing of the shorter chain substrates may exist. The castor bean
glyoxysomal system completely degrades all fatty acyl-CoA substrates to

acetyl-CoA, whereas the liver peroxisomal system degrades fatty

John Walter Uhlig

acyl-CoA substrates only to about decanoyl-CoA. The difference may be
due to the presence in‘the liver peroxisomal systen of a mediun chain
length carnitine acyl transferase which competitively converts acyl-CoA
to the acyl carnitine derivative.. Acetyl- and octanoyl-CoA
transferases were absent in seed glyoxysomes, so the medium chain

length acyl-CoA intermediates can only be further oxidized.

To My Parents and My Wife

ii

ACKNOWLEDGEMENTS

I would like to thank Dr. N.E. Tolbert for his guidance and
support during the course of this research. I appreciate the counsel-
ing and guidance given me by my committee members; Dr. L. Bieber, Dr.
N. Good, Dr. W. Smith, and Dr. W. Wood. My thanks also goes to Dr. R.
Gee for his invaluable assistance on the flavin characterization
studies and to Dr. J. Krahling for both his time and effort during the
study of e-ketoacyl-COA thiolase.

Special appreciation goes to my wife, Lois, who supported and

encouraged me during the preparation of this dissertation.

iii

TABLE OF CONTENTS
Page
LIST OF TABLES ooooooooooooooooooooooooooooo o oooooooooooooooo oooooo TV

LIST OF FIGURESOOOO ooooooooooooo o oooooo o ooooooooo oooooooooooo ooooo Vii
LIST OF ABBREVIATIONS ................. . .......... ........ ..... .... TX
CHAPTER I LITERATURE SURVEY. ......... .. ..... ......... ..... ........ 1
IntrOdUCtionooooooooooooooooooooooooooooooooooooooooooooooooooo
Synopsis of Mitochondrial Fatty Acid Oxidation.................
B-OXTdatTOH in PIBHtS....-.....................................

MicrObOdEESOOOOOOOO0.0.0.0....O....0.COO...OOOOOOOOOOOOOOOOOOOO
B'OXTdation in the MicrObOdyOOOOOOOOOOOOOO0.0... ....... 00...... 1

r—IKOO‘VwH

CHAPTER II MATERIALS AND h1ETHODSOOOO0.0.0.00.0.0...OOOOOOOOOOOOOOO 20

Materials............................................ ........ .. 20
Methods........................................................ 20
Purification of the Enzyme Complex.......................... 20
Enzyme Assays............................................... 22
Polyacrylamide Gel Electrophoresis: SDS and Native.......... 26
Polyacrylamide Gel Staining................................. 27
Sedimentation Velocity on Sucrose Density Gradients......... 27
Subcellular Fractionation of Castor Bean Endosperm
Tissue by Isopynic Sucrose Gradient....................... 28
Flavin Purification......................................... z8
Flavin Extraction from the Castor Bean e-oxidation
Complex................................................... 29
Flavin Chromatography....................................... 30
Protein Determination....................................... 31
Phosphate Determination.............. ..... .................. 31
Acyl-CoA Thioester Synthesis................................ 32
Granular Hydroxyapatite..................................... 34

CHAPTER III GLYOXYSOMAL 5-0XIDATION: PURIFICATION AND
PHYSICAL PROPERTIESOOOOOOOOOOOOO0.00.00.00.00.0.00.00.00.00. 35

Subcellular Localization of B-oxidation Activity............ 36
Purification of the B-Oxidation System...................... 39
Yields of Enzyme Activity................................... 46
Polyacrylamide Gel Electrophoresis.......................... 47
Molecular Weight Determination of Sieving Columns........... 49
Sedimentation Velocity of Sucrose Density Gradients......... 49
Lipids...................................................... 52
Charge Characterization..................................... S3
Stability of a-Oxidation Activity........................... 54

iv

Page
CHAPTER IV CHARACTERISTICS OF B-OXIDATION ACTIVITY................ 74

The Effect of pH on Activity................................... 74
The Effect of Incubation Time and Protein Concentration

on Activity........... ..... .................................. 77
Rate and Stoichiometry of NAD Reduction........................ 82
Assay Components............................................... 86
Salt Requirements.............................................. 90
Flavin Identification and Stimulation of B-Oxidation........... 99

CHAPTER V SUBSTRATE UTILIZATION ........ .. ........ ................. 105
OXTdatTOH Of Palmitoyl'COAoooooooooooooooooo oooooooo 0000000000. 107
Km vaIUES...................................................... 112
SUbStrate SpeCTfTCTtyoooooooooooooooooooooooooooooooooooooooooo 119
CHAPTER VI SUMMATION ..... . ......... ............................... 133

BIBLIOGRAPHY. .............. ....................................... 143

LIST OF TABLES

Table Page
1 Purification of the 3-0xidation System from the
Glyoxysomes of Castor Bean Endosperm.................... 40
2 Yields of the B-Oxidation Enzymes from Castor Bean

G]yoxysome50oooooooooooooooooooooooooooooooooooooooooooo 44

3 Stability of the B-Oxidation Enzymes in Glycerol
and Dithiothre‘itOEOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO. 69

4 The Effect of the Deletion of Standard Assay
Components on B-Oxidation............................... 87

5 The Effect of Various Salts on the Reaction
VETOCTty 0f 8-0Xidation00000000000000...... ....... 0.000. 98

6 Rf Values for the Flavin Moiety Extracted from
Purified Preparations of the B-Oxidation Complex........ 105

7 Stoichiometry of Acyl-CoA 0xidation....................... 110

8 Km and Relative Vmax Values for Various Acyl-CoA
SUbStrateSOOOI...00.......0...OOOOOOOOOOOOOOOOOOO..0000.128

vi

LIST OF FIGURES
Figure Page
1 Compartmentation and Interrelationship of the
B-Oxidation Pathway (43), Malate-Aspartate Shuttle

(153), and the Glyoxylate Pathway (38) in the
caStor Bean EndospermOOOOOOOOOOOOOOOOOOOOOOOOIOOOO00.... 12

2 Subcellular Fractionation of Castor Bean Endosperm
by Isopynic centrifugationooOI...OOOCOOOOIOOOOOOOOOOOOOO 37

3 Elution Profiles of Typical Hydroxyapatite and
Carboxymethyl Sephadex-ZS columns....................... 42

4 Sedimentation of the B-Oxidation Enzymes on Sucrose
DenSity Gradients...0.0.000...00.......OOOCOOOOOOOOOOOOO 50

5 Stability of B-Oxidation Activity in the Presence
of Various Concentrations of Glycerol at 22°C........... 55

6 Stability of a-Oxidation Activity in the Presence
of Various Concentrations of Glycerol at 4°C............ 57

7 The Effect of Glycerol on the Half Life of
B-Oxidation Activity at 4°C and 22°C.................... 59

8 The Effect of Various Polyhydric Alcohols on
the Stability of B-Oxidation Activity...... ......... .... 61

9 Heat Inactivation in the Presence and Absence
Of G]ycerO]OOOOOOOOOOOOOOOOOOOO...OOOOOOOOOOOOOOOOOOOOOO 63

10 The Effect of Glycerol and Dithiothreitol on
B‘OXidation ACtiVityOOOOOOO0.00.00.00.00...OOOOOOOOOOOO. 66

11 The Effect of Parachloromercuribenzoate and
Arsenite on 8-Oxidation Activity........................ 72

12 The Effect of pH on B-Oxidation Activity.................. 75

13 The Effect of Enzyme Concentration on the Rate
Of NADH FormationOOOOOOOOOOOOOOOOOOOOCOOOOOOOOOOOOOOOOOO 78

14 The Effect of Glycerol Concentration on the Rate
Of NADH FormationOO0.00...OO...OOOOOOOOOOOOOOOOOOOOOOOOO 8O

15 Schematic Representation of NADH Formation During
8-0x1dation000000OOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 83

\I'I‘I

Figure Page

16 The Effect of Detergents on B-Oxidation Activity.......... 88
17 The Effect of Bovine Serum Albumin on the Rate of

PaImitoy'l-COA OXidationOOOIOOOOOOOOOOOOOOOOOOOOOOOOOO0.0 91
18 The Effect of Bovine Serum Albumin on the Reaction

Velocity of B-Oxidation as a Function of
Palmitoy]-COA concentrationOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 93

19 B-Oxidation Activity as a Function of Potassium

Chloride and Magnesium Chloride Concentrations.......... 95
20 Flavin Stimulation of B-Oxidation Activity................ 100
21 Inhibition of B-Dxidation Activity by the Flavin

Analog, AtebrinOOOOO.0.0.00.00...OOOOOOOOOOOOOOOOOOOOOO.102
22 Palmitoyl-COA 0Xid6t10n00000000000OOOIOOOOOOOOOOOOOOOOOOOO 108
23 The Effect of Oxygen Concentration on Activity of

the Isolated Castor Bean a-Oxidation System............. 113
24 Reaction Velocity of s-Oxidation as a Function of

NAD ConcentrationOI0.0.0.0000...OIOOOOOOOOOOOOOOOOOOO... 115
25 Reaction Velocity of s-Oxidation as a Function of

Coenzyme A Concentration................................ 117
26 Reaction Velocity of 3-0xidation as a Function of

palmitoyl-COA concentrationOOOOOOIOOOOOOOOOOOOOOOOOOOOO.120
27 Chain Length Specificity for the 3-0xidation System

from Castor Bean GlyoxysomeSOOOOOIOOOOOOOOOOOOOOOOOOOOOO 1'22
28 Chain Length Specificity for 3-Ketoacyl-C0A Thiolase

Activity of the Isolated B-Oxidation System............. 124
29 Comparison of Chain Length Specificity Curves............. 129
30 Reaction Velocity of Thiolase as a Function of

B-KEtO-TEtradecanoyI-COA concentrationoooooooooooooooooo 131

viii

Bicine
CoA
DEAE-
DTT
EDTA
QAE-

S

SP-
Tricine

Tris

LIST OF ABBREVIATIONS

N,N-bis(2-Hydroxyethyl)glycine
Coenzyme A

Dithylaminoethyl-

Dithiothreitol
(Ethylenedinitrilo)tetraacetic acid
Diethyl-(Z-Hydroxypropyl)-aminoethyl-

Svedberg unit (1 x 10‘13

seconds)
Sulfopropyl-
N-tris(Hydroxymethyl)methyl glycine

tris-(Hydroxymethyl)aminomethane

ix

CHAPTER I

LITERATURE SURVEY

Introduction

Fatty acids constitute an important concentrated source of energy
in higher organisms. Their degradation has been studied since the turn
of the century and is presently an active area of research. B-Oxida-
tion, the principle mode of fatty acid degradation was believed to
exist solely in the mitochondria of eukaryotes until a decade ago when
it was observed in another organelle, the microbody. Localization of
B-oxidation in these two organelles is dependent on the tissue, the
tissues stage of development and on the presence of inducers. The
cellular role of the degradation of fats seems to have also affected
the evolutionary localization of this oxidative pathway.

The chemical mechanism of degradation of fatty acids appears to be
identical in the mitochondria and the microbody. The catalytic con-
stituents of the parallel pathways are not isoenzymes, but the enzymes
differ in their molecular weights, number of subunits and kinetic
characteristics. The most pronounced difference lies in the first
oxidation step. A dehydrogenase desaturates the fatty acids in the
mitochondria and transfers the electrons to the mitochondrial electron

transport chain via an electron transfer flavoprotein. The microbody

contains an oxidase which transfers the reducing equivalents directly
to molecular oxygen producing hydrogen peroxide which is eliminated by
the organelles complement of catalase. The energy of this step is thus
conserved in the mitochondria and is lost as heat in the microbody.

B-Oxidation in the micorbody has been documented from various
sources such as Euglena, Tetrahymena, yeast, fatty seeds, toad bladder
and mouse, rat and human liver. The characteristics of this metabolic
pathway have been thoroughly investigated in mammalian liver. Sub-
strate specificity favors the long chain fatty acids (Clo’C18)
with little or no activity being observed for the short chain fatty
acids (C4-C3). The shortened chain length intermediates and
acetyl-CoA are presumed to be transported to the mitochondria for
further degradation. Information on fatty acid oxidation in plants is
limited. B-Oxidation in the germinating fatty seeds exists solely in
the microbody and no build up of short chain length fatty acid inter-
mediates have been observed. This system is believed to completely
oxidize fatty acids to acetyl-CoA, but no thorough analysis of this
system has been made. Fatty acid degradation in the microbody plays
either a catabolic role where the product of B-oxidation (acetyl-CoA)
is further oxidized by the mitochondrial tricarboxylic acid cycle or is
anabolic and the acetyl-CoA enters the gluconeogenic glyoxylate
pathway.

Originally microbody B-oxidation was observed in the germinating
castor bean seed, but the main focus of research since, has been on the
characterization of the enzymes from rat liver induced with hypoli-
pidemic drugs and on yeast grown in a hydrocarbon environment. Since

little has been done on the system from germinating plant seeds, the

research aims in this thesis were toward the further characterization
of the B-oxidation system from the endOSpenn of the germinating castor
bean seed.

Chapter I describes the purification, stability, and partial
physical characterization of the B-oxidation system. Chapter II
characterizes some of the factors affecting the reaction velocity and
Chapter III dwells on the stoichiometry and substrate specificity of

the system.

Synopsis of Mitochondrial Fatty Acid Oxidation

Oxidation of the fatty acyl beta carbon was first proposed by the
German chemist F. Knoop in 1904 (81) based on studies of the breakdown
products of derivatized fatty acids. Prior to this time intermediates
of fatty acid oxidation could not be detected because the substrates
under investigation were either completely oxidized or were excreted
unaltered by the experimental animal. Kn00p, by attaching a benzene
group to the terminal end of various fatty acids and collecting their
residual catabolites, found that odd chain length fatty acids yielded
phenylacetate while even chain length fatty acids yielded benzoic acid.
From this, Knoop hypothesized that fatty acids are oxidized at carbon 3
(beta carbon) producing an acetate residue and a fatty acid shortened
by two carbons. This process was proposed to cycle until the fatty
acid was completely oxidized or a noncatabolic intermediate was
obtained. Between these initial experiments on fatty acid degradation
and the first cell free preparation that exhibited e-oxidation
(93,120), much work was done on liver slices and perfused livers and

was reviewed by Deuel (31) and Lynen (99).

Lehninger in 1945 demonstrated that B-oxidation resided in a
particulate cell fraction (91) and later with the development of
centrifugational separation techniques, localized it in the
mitochondria (76,77). Further research indicated the B-oxidation was
closely associated with the mitochondrial reSpiration and oxidative
phosphorylation and that an 'activation' with ATP or NADH was required
in order for fatty acid oxidation to occur (31). The final
achievements in the explanation of the B-oxidation system were based on
Lipmann's isolation of CoA (94) and Lynen's discovery that the ATP
dependent 'activation' was the esterification of the fatty acids to the
thiol group of CoA (159,97).

As the study continued and armed with the 'activated' substrates
and B-oxidation intermediates, the enzymes of fatty acid degradation
were isolated and characterized. These accomplishments, by the
research groups of Ochoa, Lynen, Green and Beinert have been thoroughly
reviewed (166,189,78,98,99,165,90,51). The present picture of the

mitochondrial B-oxidation pathway is as follows:

Activation:

Acyl-CoA Synthetase (Thiokinase)
fatty acid + CoA + ATP acyl-CoA + AMP + PPi

Carnitine Acyl-CoA Transferase
acyl-CoA + carnitine acyl-carnitine + CoA

Oxidation Cycle:

Acyl-CoA Dehydrogenase
acyl-CoA + FAD trans-Z-enoyl-CoA + FADHZ
FADHz + ETF FAD + ETFHz

Enoyl-CoA Hydratase (Crotonase)
trans-Z-enoyl-CoA + H20 L-(+)-3-hydroxyacyl-COA
cis-Z-enoyl-CoA + H20 D-(-)-3-hydroxyacyl-COA

L-(+)-3-Hydroxyacyl-COA Dehydrogenase
L-3-hydroxyacyl-C0A + NAD 3-ketoacyl-COA + NADH + H+

3-Ketoacyl-COA Thiolase (Acetyl-Acyl-CoA Transacetylase)
3-ketoacyl-COA + CoA acyl-CoA + acetyl-CoA

Auxillary Enzymes:

Cis-3,trans-2-enoyl-COA Isomerase

cis or trans-3-enoyl-COA trans-Z-enoyl-CoA
D-(-), L-(+)-3-Hydroxyacyl-C0A Epimerase
D-3-hydroxyacyl-COA L-3-hydroxyacyl-CoA

Fatty acids are activated by esterification with CoA by carrier
exchange or by direct ATP dependent ester synthesis (90). Carnitine
functions as a carrier of acyl groups from the extramitochondrial space
where the fatty acids are activated to the intramitochondrial space
where they are oxidized. The transfer of the acyl ester is accom-
plished by carnitine acyl-00A transferase. This step is believed to be
the rate limiting reaction of mitochondrial B-oxidation (70). The
fatty acyl carnitine is then shuttled across the membrane into the
matrix. This shuttle is one point of control of mitochondrial fatty
acid oxidation and was recently reviewed by McGary and Foster (107).
Inside the mitochondrial matrix, CoA is exchanged back and the acyl-CoA
then enters the oxidative cycle.

Three separate acyl-CoA dehydrogenases with different Specifi-
cities for varying chain lengths, desaturate a fatty acid between
carbons 2 and 3 producing trans-Z-enoyl-CoA. The electrons removed
from the fatty acids are transferred from the enzymes FAD moiety to a
flavoprotein known as ETF (electron transfer flavoprotein). ETF
shuttles the electrons to coenzyme Q and on through the mitochondrial

electron transport chain to oxygen. Enoyl-CoA is hydrated by enoyl-CoA

hydratase to the L-isomer of 3-hydroxyacyl-C0A. This compound is
dehydrogenated by the NAD dependent L-3-hydroxyacyl-COA dehydrogenase
to the 3-keto intermedate. This intermediate, in the final step,
undergoes thiolytic cleavage by 3-ketoacyl-C0A thiolase producing a
fatty acyl-CoA shortened by two carbons and acetyl-CoA. The B-oxida-
tion cycle continues until the fatty acid is completely oxidized. Odd
chain length, unsaturated and unusual fatty acid oxidation have been
reviewed (90,51). Present studies on hepatic mitochondrial B-oxidation
revolve around the enzymology of its various constituents (42,41,35,52,
89,130, 12), the oxidation of various substrates (33,86,11) and its
relationship to the B-oxidation pathway found in 1976 in a subcellular

organelle known as the peroxisome (175,84,138).

B-Oxidation in Plants

 

Little evidence was available until the early 1950's supporting
the existence of B-oxidation in plants. Fawcett gt al. (35,36) showed
that omega-phenoxy fatty acids fed to flax seedlings exhibited similar
results as that observed by Knoop (81) on experimental animals. Even
chain length fatty acids gave rise to appreciable amounts of phenol.
Odd chain length fatty acids yielded only traces of phenol which were
attributed to onega-oxidation (omega and alpha oxidation have been
reviewed in 166,189,78,90,51).

Newcomb and Stumpf (122) two years earlier were able to obtain
cell free preparations fran germinating peanut cotyledons. This tissue
was capable of converting labeled palmitic acid to labeled carbon
dioxide. Subsequent studies by Stumpf and Barber (168) in 1956 tenta-

tively localized B-oxidation in the mitochondrial fraction of peanut

cotyledon extracts. Similar mitochondrial systems were reported to
exist in avocado mesocarp, lupin and pine seedlings (167).

Rebeiz and Castelfranco (150,151) reexamined the system from
germinating peanut cotyledons in the mid 1960's and found a consider-
able proportion of the B-oxidation activity to be localized in the
soluble protein fraction. Similar dual localization was observed by
Yamada and Stumpf in their studies of B-oxidation in the castor bean
seed (185,184). They observed that the majority of the fatty acid
degradation occurred in the nonparticulate fraction of the endosperm
extracts.

Extramitochondrial B-oxidation was not finally localized (65,26)
until two years after Breidenbach and Beevers (18) demonstrated by
isopynic banding on sucrose gradients that the enzymes of the glyoxy-
late pathway (gluconeogenic pathway for acetyl-CoA (8)) of germinating
castor bean endosperm were found in a new organelle which they called a
glyoxysome. Dual localization of fatty acid oxidation in the mitochon-
dria and microbody were also reported in rat liver (88), pine seeds
(21), Tetrahymena (17), Euglena (50), mango (3) and corn seeds (95).
The residence of B-oxidation in the two compartments has been well

documented in the mammalian system (84,87,136) and in Tetrahymena (58)

 

but few papers have confirmed the earlier findings for the plant
tissues. Reexamination and further studies on various systems has
shown that B-oxidation activity resided only in the glyoxysomes of pine
seeds (61), castor bean seeds (26), jojoba seeds (117), cotton seeds
(112), cucumber seeds (82) and yeast (74). The initial observation of
dual localization could be attributed to (a) incomplete separation of

mitochondria and the microbodies or (b) from damaged microbodies

banding in close proximity to the mitochondria (60,176). This does not
preclude the existence of the fatty acid oxidizing system in the
mitochondria.

Avacodo mesocarp (144) and pea cotyledons (171,108) seem to be
exceptions to a hypothesis that B-oxidation in plant tissues resides
predominantly if not solely in the glyosysome. L-Carnitine dependent
palmitate oxidation was observed in crude mitochondrial preparations
in both tissues. This activity was attributed to the mitochondria
since peroxisomal fatty acid oxidation is carnitine independent.
Panter and Mudd (144) proposed that at least two systems exists in
higher plants for the oxidation of fatty acids; the carnitine asso-
ciated system localized in the mitochondria (as in the animal system)
and a glyoxylate pathway associated systen found in the glyoxysome.
The mitochondrial system exists in non-fatty seeds where starch (e.g.
peas) or possibly protein is the major food reserve. These tissues
contain carnitine (145) and the microbodies appear to have a more
limited enzyme makeup relative to the specialized glyoxysome (61).
Tissues which are rich in fats and carnitine but lack microbodies such
as the avacado mesocarp, also have the B-oxidation pathway localized
within their mitochondria. Oil seeds, such as the castor bean seed
(26) and cotton seed (112), solely utilize fats through the pathway in
the glyoxysomes, thus directing the major energy and carbon resource
into carbohydrate synthesis. These tissues lack carnitine and mito-
chondrial B-oxidation (26,112,145). Hence where fats seem to play a
gluconeogenic role, B-oxidation seems to reside in the glyoxysome, and
where this function does not appear to exist, the mitochondrial

B-oxidation system functions.

Microbodies

Microbodies (glyoxysomes and peroxisomes) are fragile organelles
consisting of a finely granular matrix surrounded by a single tripar-
tite limiting membrane with a diameter of 0.1 to 0.7 uM. Microbodies
sediment to a density of 1.23 to 1.25 g/cm3 on sucrose isopynic
gradients. Electron dense cores and internal structures have been
observed from some sources, but their composition have not yet been
elucidated. Electron micrographs have shown microbodies in close
proximitry to lipid bodies, mitochondria, chloroplasts and endoplasmic
reticulun (the latter being the proposed site of origin of the
organelle). Microbodies were first observed in animals in 1954, later
in plants (176) and were isolated and characterized from rat liver by
de Duve's group (29,92,6) in the 1960's. Subsequently, they were
isolated from protozoa (56,119), Euglena (50), yeast (169), various
plant tissues (55,112,174,18,62,61) and seem to be a general phenomenon
of eukaryotic cells. Microbodies have many properties in common
regardless of the source (175,176).

The term microbody has been used by microscopists as a morpholog-
ical description and more recently by biochemists as a general tenn for
peroxisomes, glyoxysomes and those similar organelles that have not
been biochemically characterized. Peroxisomes, a named proposed by de
Duve (30), contain at least one flavin containing oxidase which produce
hydrogen peroxide and catalase which eliminates this strong oxidant
from the organelle. Glyoxysomes, as described by Breidenbach and
Beevers (18), are peroxisomes which also contain isocitrate lyase and
malate synthase of the glyoxylate pathway. These two enzymes have been

well characterized from plant tissues, Tetrahymena, Euglena, and yeast.

10

Recently these enzymes have been reported in fetal guinea pig liver
(73) and in toad urinary bladder epithelial cell peroxisomes (49). The
latter findings have not yet been confirmed by other laboratories. If
true, these findings would support the ubiquitous character of the
microbody enzymes and would disrupt the dogma of the nongluconeogenic
character of lipids in animal tissues.

The variability of the enzyme constituency in microbodies is
dependent not only on the tissue being studied and its stage of
development, but also on substrate availability and chemical induce-
ment. Glyoxysomes and glyoxysomal B-oxidation proliferate during the
germination of the castor bean and cotton seed, then rapidly decline as
the lipid supplies are depleted. The gluconeogenic glyoxysome of
germinating fatty seeds give way to the photorespiratory leaf peroxi-
some, coinciding with changes in the stage of development and enzyme
composition of the microbodies. Yeast grown on various substrates have
shown different enzymological makeup of its microbodies. Sugars are
observed to repress microbody development, methanol induces the forma-
tion of microbodies with alcohol oxidase (46), while malate induces
glyoxysomal proliferation. Alkanes and fatty acids are known inducers

of microbody B-oxidation in yeast, Tetrahymena, and Euglena (74,50,17,

 

135, 45). The administration of different hypolipidemic drugs (88,181)
or plastizers (137,140), along with starvation (70) and high fat diets
(71,121) increase B-oxidation in hepatic peroxisomes of the rat.
Gibberellic acid has been observed to stimulate the development of the
enzyme activities of isocitrate lyase and 3-hydroxyacyl-CoA dehydro-

genase in the develOping castor bean seed (102).

11

The pathways of the microbody include the photorespiratory
glycolate and glycerate pathways, ureide metabolism, s-oxidation and
the glyoxylate pathway (175). The pr0perties and physiological role of

microbodies have not yet been fully elucidated.

B-Oxidation in the Microbody

B-Oxidation is a catabolic pathway by which fatty acids are
degraded yielding reducing equivalents and acetyl-CoA. The
intermediates of this pathway in the mitochondria and in the
peroxisomes appear to be identical but the amount of recoverable
reducing equivalents and the enzymes responsible for the degradation
are different. Also the extent of fatty acid oxidation seems to differ
between these two organelles, as do their substrate specificities.
B-Oxidation and the utilization of its products in the endospenn of
germinating castor bean seed is illustrated in Figure 1.

Intracellular fatty acids from lipid body degradation (128,129) or
fatty acids from the extracellular environment must be activated before
undergoing oxidation. Various ATP dependent acyl-CoA synthetases have
been characterized from microbodies (158,24,112,85,187), however, the
synthetases have not been localized within the peroxisomes. A
peroxisomal fatty acid carrier protein has been observed in the rat
liver (2) but its substrate preference (free fatty acid or CoA
thioester) and the presence of any synthetase activity were not
demonstrated. The lack of a carnitine requirement for peroxisomal
B-oxidation (112,137,59,26,88) and the lack of a palmitoyl-CoA
carnitine acyl-transferase (112,101,100) supports the hypothesis that

fatty acids or their CoA thioesters traverse the peroxisomal membrane

12

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14

membrane by a carnitine independent process. The mechanism of
transport and localization of fatty acid activation has not been
determined.

The first degradative step of B-oxidation in the mitochondria is
catalyzed one of three chain length Specific acyl-CoA dehydrogenases
(tetramer MH-172,000 (42,172)) producing trans-Z-enoyl-CoA. The
energy fran this reaction is conserved by the transfer of the reducing
equivalents via ETF to the electron transport chain, yielding 2 ATPs
and the reduction of oxygen to water. Acyl-CoA oxidase catalyzes the
reaction in the peroxisome. This flavoprotein (dimer MN-140,000
(68,69,142)) transfers the electrons directly to molecular oxygen
producing hydrogen peroxide (26,88). As with all peroxide produced by
peroxisomal oxidases, it is eliminated by the organelles complement of
catalase and the energy is lost as heat. Since the direct coupling of
the reducing equivalents to oxygen in the peroxisome does not involve
the elctron transport system it is insensitive to cyanide poisoning,
which is observed in the mitochondria (88,49).

The next two steps in the degradation of fatty acids are catalyzed
by one bifunctional enzyme (monomer Mw-80,000 (43,141,47,139,44)) in
the peroxisome whereas two enzymes (crotonase, hexamer MN-155,000--
161,000 (47,40) and 3-hydroxyacyl-CoA dehydrogenase, dimer Mw-64,000
(141)) are required in the mitochondria. The hydratase is Specific for
the 2-enoyl-CoA intermediate with the cis and trans configurations
yielding the D- and L-isomers, respectively. The dehydrogenase step is
specific for L-3-hydroxyacyl-CoA and NAD producing 3-ketoacyl-CoA and
NADH (156,141). The hydratase and dehydrogenase activities can be

measured separately in the peroxisomal system. The final step of the

15

oxidative cycle is the thiolytic cleavage of acetyl-CoA. Rat liver
peroxisomal, mitochondrial and cytoplastmic thiolases are probably not
isoenzymes since they exhibit different sulfhydryl activations and were
immunologically distinguishable (115). Little is known about the
thiolase from plant glyoxysomes. Kindl (43) has shown the thiolase
from cucumber cotyledon glyoxysomes to be a dimer with a molecular
weight of 90,000 (43).

The peroxisomal B-oxidation system is most active with the long
chain fatty acyl-CoA substrates. Degradation is reported for 610
to 622 substrates with maximun activity being observed for the
614 to 615 acyl-CoAs (19,26,87,59). Rat liver peroxisomes
oxidize fatty acids only to the 012 to Cg acyl-CoA intermediates.

The shortened carbon chains are then transferred to carnitine by the
peroxisomal octanoyl-CoA transferase and presumably they move to the
mitochondria for completion of the oxidation cycle. Mitochondrial
B-oxidation in the dual localization systems oxidizes short chain fatty
acids as well as long chain acids. In the germinating fatty seeds
mitochondrial B-oxidation has not been reported and the accumulation
short chain fatty acids in the seeds have not been observed. The
results of studies on cotton and castor bean seeds indicate the ability
of the glyoxysome to completely oxidize fatty acids to acetyl-CoA
(26,9,112).

The stoichiometry of B-oxidation during a given cycle of fatty
acid degradation in the microbody has been shown to be the same from
all systems studied (58,26,157,74). For each acetyl-CoA produced, one
molecule oxygen is converted to hydrogen peroxide, one NAD is reduced,
and one molecule CoA and water are consumed (as shown in Figure 1).

The regeneration of NAD and 60A is required for sustained B-oxidation

16

in the castor bean glyoxysome. The nelate-aspartate shuttle (110) and
the glyoxylate pathway (18,25,118) provide this essential function.
Oxaloacetate (0AA), the initial substrate for both pathways, is
produced in the glyoxysome by the transamination of aspartate and
a-ketoglutarate from the cytosolic pools. NADH is oxidized to NAD by
malate dehydrogenase during 0AA reduction. The reducing equivalents
are shuttled, with malate as the carrier, to the mitochondria where
malate is oxidized back to 0AA and transmination to aspartate occurs.
Aspartate reenters the cytosolic pool, balancing the carbon flow in the
regeneration of NAD (Figure 1).

Oxaloacetic acid is condensed with acetyl-CoA by citrate synthase
in the first step of the glyoxylate pathway. Aconitase interconverts
citrate to isocitrate which is cleaved into succinate and glyoxylate by
isocitrate lyase, the unique enzyme of the glyoxylate pathway.
Succinate is transported to the mitochondria where it undergoes
successive oxidations to 0AA. Transamination again occurs balancing
the carbon flow. Glyoxylate and another acetyl-CoA are condensed to
form malate by malate synthase. Malate enters the cyt0plasmic malate
pool and provides the carbon skeleton needed for sucrose synthesis.

The regeneration of NAD in yeast and rat liver peroxisomes occurs
by a different shuttle mechanism (48,45) than that found in the castor
bean. Both systems utilize a NAD linked glycerol-B-phosphate'
dehydrogenase in the peroxisome associated with a FAD linked
glycerol-3-phosphate dehydrogenase in the mitochondria. This forms the
glycerol-3-phosphate/dihydroxyacetone phosphate shuttle with glycerol

phosphate acting as the carrier for the reducing equivalences.

17

The recovery of 60A in the rat liver peroxisome occurs by the
exchange of 60A with carnitine by peroxisomal transferases. The ace-
tate and short chain fatty acid units are transported out of the
peroxisome for utilization and further degradation. The recovery of
00A in yeast possessing the B-oxidation cycle and the glyoxylate
pathway is similar to that found in the castor bean with one exception
(45). The glyoxylate pathway in yeast is divided between the peroxi-
some and the mitochondria, requiring part of the acetyl-CoA units to be
transferred to the mitochondria. This is accomplished by the exchange
of carnitine for CoA as in the rat liver and subsequent reexchange back
to CoA in the mitochondria. The castor bean, rat liver, and yeast
peroxisomal systems seem to contain their own complement of NAD and
CoA.

Various unsaturated and derivatized fatty acids occur in nature.
Odd-numbered carbon trans and cis configurations and even-numbered
carbon cis configurations are barriers to the B-oxidation system. Two
enzymes, D-(-),L-(+)-3-hydroxyacyl-C0A epimerase and cis-3,trans-2--
enoyl-CoA isomerase, were discovered by Stoffel et al. (163) that
circumvent these obstacles. Unsaturated fatty acids with a cis config-
uration on an even carbon, such as petroselinic acid (cis-6-octa-
decenoic acid), are degraded to a cis-Z-enoyl-CoA intermediate.
Enoyl-CoA hydratase catalyzes its hydration to the D-isomer. This
isomer can not be used by the stereospecific L-3-hydroxyacyl-CoA
dehydrogenase. The epimerase isomerizes the hydroxyl group in a
reversible reaction and allows the continued oxidation of the fatty
acid. Acyl-CoA oxidase and acyl-CoA dehydrogenase produce the trans-2
configuration which is hydrated to the utilizable L-3-hydroxy isomer

(162).

18

Unsaturated fatty acids containing a double bond on an odd-num-
bered carbon are oxidized to 3-enoyl-CoA. This intermediate can not be
utilized by enoyl-CoA hydratase. Cis-3,trans-2-enoyl-CoA isomerase
makes this intermediate available for further oxidation by the isomeri-
zation to the trans-2 configuration (163). The isomerase has been
found in both the plant and animal systems (64,112,163) and shows a
specificity for shorter chain length fatty acyl-CoAs and a preference
for the cis configuration (164,86). The enzyme also seems to maintain
conjugation during the isomerization of polyunsaturated fatty acids
(33).

Many fatty acids require isomerization during their degradation,
such as oleic, linoleic, arachidonic, vaccenic, erucic and ricinoleic
acids. Ricinoleic acid is of some interest since about 90% of the
castor beans fat content consists of this acid (185,64). Degradation
of ricinoleic acid (12-D-hydroxy-9-cis-octadecenoic acid) via the
B-oxidation pathway occurs until 4-hydroxydecanoyl-CoA is formed.
Oxidation does not proceed beyond this point in §;_ggli (126), rats
(177), pea cotyledons (64) or yeast (125). The hydroxyl group in the
even-numbered position can not be utilized by the B-oxidation system
forming a metabolic block. Hutton and Stumpf (64) proposed a mechanism
by which the castor bean endosperm is capable of utilizing this unusual
fatty acid intermediate. The 4-hydroxydecanoyl-CoA intermediate was
proposed to be oxidized via two pathways in the glyoxysome to 2-keto-
octanoyl-CoA which then undergoes alpha-oxidation to 002 and
heptanoyl-CoA. B-Oxidation then completed the degradation process.

Studies on the interrelationships of the fatty acid oxidizing

enzymes have been studied only in the prokaryote, E; coli (124,39,148,

19

7,146,23). A complex of five enzymes was purified with a molecular
weight of about 250,000-300,000 (146,39,148,124). The complex consists
of 3-ketoacyl-CoA thiolase, enoyl-CoA hydratase, 3-hydroxyacyl-CoA
dehydrogenase, 3-hydroxyacyl-CoA epimerase and cis-3,trans-2-enoyl-CoA
isomerase (acyl-CoA dehydrogenase does not copurify with the complex
under the purification procedures used). The existence of an
association in eukaryotes has not been investigated, but has been
proposed in rat liver and in another prokaryote, Mycobacterium
smegmatis (58,69,156). Extended associations of the oxidative cycle
enzymes during purification has been the basis of the proposition

(68,69,156).

CHAPTER 11
MATERIALS AND METHODS
Materials

Castor Beans (Ricinus communis) were purchased from Herbst

 

Brothers, 1000 N Main Street, Brewster, New York. Trans-3-hexenoic
acid, 3-dimethylaminobenzoic acid and 3-methyl-2-benzothialinone
hydrazone°HCl were obtained from Aldrich. The ketoacyl-CoA

compounds were gifts from Jeff Krahling, Department of Biochemistry,
Michigan State University. Some of the acyl-CoA substrates were gifts
from L. Bieber, Department of Biochemistry, Michigan State University.
Other chemicals used were obtained from commercial sources and were of

reagent grade.

Methods
Purification of the Enzyme Complex
Germination. The fatty acyl B-oxidation system was isolated from

germinating castor bean endosperm (Ricinus communis). Seeds were

 

imbibed overnight in either aeriated water or under running tap water.
The seeds were then sown on a moist bed of perlite/vermiculite and
germinated at 30°C in the dark for six days. Fungal contamination was
minimized by proper ventilation of the germination bed and by minimal

watering.

20

21

Extraction. The seed coat and primary root were removed and
discarded. All subsequent steps were carried out at 4°C. The endo-
sperm and primary leaves were washed, dried on a towel, weighed and
diced in an onion chopper in three volumes of buffer consisting of 500
mM ultrapure sucrose (Schwatz/Mann), 10 mM EDTA and 200 mM potassium
phosphate at pH 8.0. The slurry was passed through a meat grinder and
filtered through eight layers of cheese cloth. The pulp was homo-
genized in two volumes of the same buffer using a Tekmar 'Ultra-Turrax'
tissumizer (18N generator, setting 25). The homogenized tissue was
filtered and the milky extract was centrifuged at 10009 for 15 minutes
to remove the debris. The supernatant was recentrifuged at 11,0009 for
60 minutes. The pellet, containing the glyoxysomes and light mitochon-
dria, was resuspended in 100 mM potassium phosphate pH 7.8 buffer using
the tissumizer with a 10N generator.

Ammonium Sulfate Fractionation. The suspension was made up to 2%
(w/w) Triton X-100 and was fractionated with saturated (0°C) ammonium
sulfate (neutralized) from 30% to 50% of saturation. The pellet after
centrifugation was resuspended in 5lnM potassium phosphate buffer, pH
7.8 containing 50% (v/v) glycerol. The preparation was dialyzed
against the same buffer overnight at 4°C. This fraction was considered
the ammonium sulfate fraction and was stored at -20°C.

Hydroxyapatite Chromatography. The ammonium sulfate fraction was
diluted 50% with water and applied to a granular hydroxyapatite (105)
column equilibrated at 4°C with 5 mM potassium phosphate and 25% (v/v)
glycerol at pH 7.8. The column was washed with equilibrium buffer and
then with 40 M phosphate (25% glycerol) buffer at pH 7.8. The

B-oxidation activity was eluted with 100 mM phosphate and 25% glycerol

at pH 7.8. The active fractions were combined and concentrated by
dialysis against ammonium sulfate and centrifugation of the precipi-
tate. The active fractions were also concentrated by ultrafiltration
(Amicon XM-lDOA). Both concentrated active fractions were dialyzed
against 5 mM potassium phosphate and 50% glycerol buffer at pH 7.8.

Carboxymethyl Sephadex-25 Chromatography. The dialyzed sample
containing B-oxidation activity was applied to a carboxymethyl
Sephadex-25 column equilibrated with 5 mM potassium phosphate and 25%
glycerol buffer at pH 7.8. The column was washed with 40 mM phosphate
and the B-oxidation activity eluted with 110 mM potassium phoshate and
25% glycerol buffer at pH 7.8. The active fractions were concentrated
by ultrafiltration using XM-lOOA membranes, made to 50% (v/v) glycerol
and stored at —20°C.

Enzyme Assays

All assays were carried out at 25°C.

B-Dxidation activity was measured by palmitoyl-CoA dependent
oxygen uptake and/or NAD reduction. The assay consisted of 100 mM
glycylglycine buffer at pH 9.1, 500 uM NAD, 200 uM CoA, 25 uM FAD and
50 uM palmitoyl-CoA. Assays were initiated with the addition of the
substrate. Oxygen uptake was neasured in a 1 ml volume using a Y.S.I.
Clark-type oxygen electrode that was calibrated against air saturated
glass distilled water at 25°C. An oxygen content of 236.1 nmol 02
per ml was used (54). NADH formation was followed spectrophoto-
metrically at 340 nm (e = 6.22 x 103 M"1 cm'l) using a

Gilford 2400 recording spectrophotometer and 1 ml cuvettes.

23

Acyl-CoA oxidase activity was measured using a modified peroxidase
assay (123). The assay consisted of 50 mM sodium phosphate buffer at
pH 7.8 containing 10 mM dimethylaminobenzoic acid, 200 uM 3-methyl-2-
benzothiazolinone hydrazone'HCl monohydrate, 25 uM FAD, 250 ug/ml
horseradish peroxidase and 50 uM palmitoyl-CoA. The change in absor-
bance at 590 nm was followed and an extinction coefficient of 18.38 x
103 M"1 cm‘1 was used. The extinction coefficient was
calculated from the change in A590 when standardized hydrogen
peroxide was added to the basic assay medium. The peroxide concentra-
tion was determined using a extinction coeffient of 36 M"1 cm"1
at 240 nm. Acyl-CoA oxidase/peroxidase assay was severely inhibited by
sulfhydryl compounds. Diethiothreitol (4 mM) and CoA (200 uM)
inhibited activity 99.97% and 84% respectively and were subsequently
omitted from all analysis. Hence, peroxide formation could not be used
as a separate means of measuring B-oxidation.

Enoyl-CoA hydratase was assayed in 100 mM glycylglycine buffer at
pH 9.1 containing 21.5 uM crotonyl-CoA as substrate. The loss of
absorbance at 263 nm was followed and the rate of activity was
calculated using an extinction coefficient of 6.7 x 103 M"1
cm'l. The measurement of enoyl-CoA hydratase activity was severely
impaired by the addition of dithiothreitol. Loss of activity seemed to
be due to a reaction or an association of oxidized dithiothreitol with
enoyl-CoA. An increase in A263 had been observed upon the
addition of sulfhydryl reagent which increased with the pH of the
buffer system (pH 7.7-9.1). The cyclic disulfide product of dithio-
threitol oxidation has a maximal absorbance at 283 nm (27) which would

account for this change in 0.0. Addition of crotonyl-CoA to these

24

solution (in the absence of enzyme) caused a decrease in A263'
The rate of this reaction was proportional to the initial change in
absorbance due to the sulfhydryl reagent. No change in absorbance was
noted for either crotonyl-CoA or dithiothreitol, except for the initial
changes due the double bond and oxidation, respectively.
3-Hydroxyacyl-CoA dehydrogenase was measured at 340 nm in a 100 mM
glycylglycine buffer at pH 9.1 containing 21.5 uM crotonyl—CoA and 400
uM NAD. The reaction was initiated with NAD after a one minute incuba-
tion period (c = 5.22 x 103 M-1 cm-l). The observed rates
did not vary when enzyme, NAD, or crotonyl-CoA was used to initiate the
reaction. No lag in activity was observed when crotonyl-CoA was used
for initiation. Dithiothreitol was observed to inhibit the activity
greatly if allowed to incubate with crotonyl-CoA. If the enzyme and
substrate were incubated together so that 3-hydroxybutyryl-CoA was
formed before the addition of the sulfhydryl reagent no inhibition was
observed. This would support the proposition that dithiothreitol was
reacting with crotonyl-CoA, most likely across the double bond.
Thiolase activity was measured spectrOphotometrically at 303 nm by
observing the disappearance of a magnesium enolate complex (87). The
assay consisted of 100 mM Tris'Cl at pH 8.0, 50 mM KCl, 25 mM
MgCl2, 200 uM CoA, 4 mM dithiothreitol and 10 pH 3-ketoacyl-CoA. The
extinction coefficients were 16.9 x 103 M"1 cm"1 for acetoacetyl-CoA,
15.6 x 103 M“1 cm'1 for 3-ketohexanoyl-CoA, 14.4 x 103 M'1 cm'1 for
3-ketooctanoyl-CoA, 13.5 x 103 W1 cm'1 for 3-ketodecanoyl-CoA, 11.4 x
103 M"1 cm‘1 for 3-ketododecanoyl-CoA, 11.2 x 103 M'lcm'1 for 3-keto-
3 1

tetradecanoyl-CoA, 9.6 x 10 M.1 cm-

3 1

for 3-ketohexadecanoyl-C0A and

17.4 x 10 W1 cm' for 3-ketooctadecanoyl-C0A. The 3-ketoacyl-CoA

25

compounds were synthesized and the extinction coefficients determined
by Jeff Krahling, Department of Biochemistry, Michigan State Univer-
sity.

3-Hydroxyacyl-CoA epimerase was measured spectrophotometrically at
340 nm (a: = 6.22 x 103 M"1 cm'l. The epimerase dependent
formation of the L-3-hydroxyacyl-C0A intermediate of petroselinoyl-CoA
(cis-6-octadecenoyl-CoA) and subsequent B-oxidation (NADH formation)
was followed. Rates were expressed as the final rate of NADH formation
after the epimeration step, and the ratio (f/i) of the epimerase
dependent rate to the epimerase independent rate of NADH formation.

The assay conditions were identical to those used for the determination
of s-oxidation but with the substitution of 23 uM petroselinoyl-CoA as
substrate.

Cis-3,trans-2-enoyl-CoA isomerase activity was followed based on
the formation of NADH using 3-transhexenoyl-CoA as substrate. The
assay conditions were the same as those used to determine B-oxidation
with 46 uM 3-transhexenoyl-CoA as substrate.

Catalase activity was measured at 240 nm in 100 mM potassium
phosphate buffer at pH 7.5 containing hydrogen peroxide (17 mM). The
reaction was initiated with enzyme and an extinction coefficient of 36
M"1cm'1 was used.

Lactate dehydrogenase was measured by the loss in absorbance at
340 nm (e = 6.22 x 103 M"1 cm'l) upon initiation of the
reaction with pyruvate. The reaction mixture consisted of 50 mM
potassium phosphate at pH 7.0, 250 uM NADH, 10 mM pyruvate (potassium

salt) and enzyme.

26

Cytochrome c oxidase activity was measured by the loss in absor-
bance at 550 nm using an extinction coefficient of 21.1 x 103 M'1
cm'l. The enzyme was first incubated with 25 ul digitonin
(saturated solution 1%) for one minute. This mixture was then diluted
with 100 mM potassium phosphate buffer at pH 7.0 and the reaction
initiated with 1 mg cytochrome c (reduced with dithionite). Total

assay volume was 1 ml.

Polyacrylamide Gel Electrophoresis: SDS and Native

Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis
was carried out using the system of Weber and Osborn (182). The
resolving gel consisted of 10% (w/v) acrylamide, 0.27% (w/V) methylene-
bisacrylamide, 0.1% (w/v) SDS and 100 mM sodium phosphate buffer at pH
7.2. Polymerization was initiated with 0.15% (v/v) N,N,N',N'-tetra-
methylethylenediamine and 0.075% (w/v) ammonium persulfate. The reser-
voir buffer consisted of 100 mM sodium phosphate buffer at pH 7.2 and
0.1% (w/v) SDS. Samples for electrophoresis were prepared by injecting
a 100 pl aliquot of enzyme into a solution (100°C) consisting of 15 ul
0.02% (w/v) bromphenol blue, 10 ul 2-mercaptoethanol, 50 ul glycerol
and 100 pl 200 mM sodium phosphate at pH 7.2, 0.2% (w/v) SDS buffer.
Electrophoresis was performed at 8 milliamps per tube at room tempera-
ture. Standards used were bovine serum albumin (68,000), catalase
(60,000), ovalbumin (43,000), carbonic anhydrase (29,000), myoglobin
(17,800) and cytochrome c (11,700). Standards were treated identically
to the unknown. ‘

Native polyacrylamide gels were prepared as described by Weber and

Osborn (182) minus the SDS and by a procedure described by R. Burgess,

27

Harvard University (unpublished). The resolving gel in the second
procedure consisted of 10% (w/v) acrylamide with 0.13% (w/v) methyl-
bisacrylamide or 7.5% (w/v) acrylamide with 0.20% (w/v) nethylbisacryl-
amide in 375 mM Tris-Cl buffer pH 8.9. Polymerization was initiated
with 0.058% (v/v) N,N,N',N'-tetramethylethylenediamine and 0.07% (w/v)
ammonium persulfate. The stacking gel consisted of 2.5% (w/v) acryla-
mide, 0.62% (w/v) methylenebisacrylamide, 0.37% (v/v) N,N,N',N'-tetra-
methylethylenediamine and 0.035% (w/v) ammonium persulfate in 30 mM
Tris-phosphate buffered at pH 6.9. The reservoir buffer consisted to
Illnfl Tris, 77 mM glycine buffer at pH 8.3. The gels were electro-

phoresed at 2.5 to 3.5 milliamps per tube.

Polyacrylamide Gel Staining
Native and SDS polyacrylamide gels were stained in Coumassie
Brilliant R 250. The stain consisted of 0.25% Coumassie Brillian Blue
in 50% (v/v) methanol and 10% (v/v) glacial acetic acid. The gels were
stained for four hours and then destained in 25% (v/v) methanol in 10%
(v/v) glacial acetic acid in the presence of an activated charcoal

absorbant.

Sedimentation Velocity on Sucrose Density Gradients
The techniques of Martin and Ames (109) were followed. Sucrose
gradients (60 ml) were prepared in 10 mM glycylglycine, 100 mM
potassium chloride bffer at pH 8.0 from 5 to 25% (w/w) sucrose and
allowed to equilibrate overnight. The gradients, prepared in 5% steps,
were shown to be linear by refractometry. Solutions of lactate

dehydrogenase (rabbit muscle) and hydroxyapatite purified B-oxidation

28

system were applied to the gradients in combination and individually.
The sucrose gradients were run in a Beckman SH-25.2 swinging bucket
rotor in a Beckman LC—2 ultracentrifuge at 5°C for 50 hours at 25,000
rpm. The gradients were fractionated in 1 ml aliquots from the top of
the gradient using a ISCO density gradient fractionator (model 185) at

a rate of 0.75 ml per minute.

Subcellular Fractionation of Castor Bean Endosperm
Tissue by Isopynic Sucrose Gradient
Castor bean endosperm (38 g) extracted as described in Methods
(purification of the enzyme complex) was centrifuged at 2709 for 10
minutes. The supernatant was centrifuged for 20 minutes at 10,0009 and
the organelle pellet was carefully resuspended in 8 ml 20% (w/w)
sucrose with 1 mM EDTA pH 7.5 buffer. Approximately 2 ml of the
resuspended pellet was layered onto a continuous sucrose gradient. The
gradient consisted of a 5 ml pad of 56% sucrose with a 50 ml 56-20%
(w/w) sucrose gradient (in 1 mM EDTA pH 7.5) layered on top. The
gradients were centrifuged for three hours at 25,000 rpm in a Beckman
SH-25.2 swinging bucket rotor with a Beckman LC-2 ultracentrifuge at
5°C. The gradients were fractionated from the top using an 1500

gradient fractionator.

Flavin Purification
Flavins (FAD and FMN) purchased from Sigma were observed to be
impure by thin layer chromatography (TLC). They were purified by a
modified procedure of Fazekas and Kokai (37). FAD or FMN was applied

to a Sephadex G-25 column equilibrated with glass distilled water.

29

Both flavins eluted first, followed by impurities. Each flavin peak
was concentrated by lyophilization, redissolved in glass distilled
water and applied to a cellulose column (Brinkmann MN-300) equilibrated
and eluted with glass distilled water. The flavins again eluted first
and were concentrated by lyophilization. The flavins were finally
chromatographed on a Brinkmann preparative MN-300 cellulose TLC plate
in 5% NazHPO4. FAD and FMN purified by this procedure were shown

to be pure by twelve chromatographic systems (see Methods - Flavin
Flavin Chromatography). All steps were carried out in the dark to

minimize photodegradation.

Flavin Extraction from the Castor Bean e-Oxidation Complex

Two extraction procedures were used to characterize the flavin
composition of the partially purified B-oxidation system. The enzyme
samples used were prepared fresh and in the dark to minimize photo-
degradation. The first method employed was a modification of the
trichloroacetic acid extraction procedure of Mayhew and Massey (104).
A sample (1.08 ml) containing 32.4 mg protein was mixed with 0.12 ml
50% (w/w) trichloroacetic acid (TCA) at 0°C. The sample was centri-
fuged at 10,0009 for 10 minute after a 5 minute incubation. The super-
natant was removed and the pellet was resuspended in 1.2 ml 5% (w/v)
TCA and recentifuged as before. The combined supernatants were
neutralized with 1.2 ml 1 M KH2P04 and the solution made up to 4 ml
with water. All of the above steps were done at 0 to 4°C and in the
dark. This sample was chromatographed but proved to contain too high
of a salt concentration for some chromatographic systems. The
remainder of this sample was extracted with phenol and treated as

described in the second procedure.

30

The second method employed was that of Fazehas and Kokai (37). A
sample (1.0 ml) containing 30 mg protein was heated in a boiling water
bath for three minutes then cooled and centrifuged at 90009 for five
minutes. The supernatant was removed, the pellet resuspended in
distilled water and recentrifuged as before. The combined supernatants
were extracted twice with 2 ml of 80% (w/w) phenol. The combined
phenolic upper phases were centrifuged for 5 minutes at 15009 to break
up the emulsion. The phenolic upper phase was then removed and mixed
with an equal volume of diethyl ether. This solution was extracted
twice with 2 ml of distilled water. The flavin containing water
fractions were combined, filtered and were taken to dryness under
reduced pressure at 60°C, then lyophilized overnight to remove the
final traces of phenol. All steps in both procedures were carried out
in the dark. The lyophilized samples were resuspended in distilled

water and chromatographed.

Flavin Chromatography

Twelve separate chromatographic systems (six solvents and two
supports) were used to determine the purity and identification of FMN
and FAD. The supports used were Brinkman (Merck) MN-300 cellulose and
silica gel-60 thin layer chromatography plates (20 cm x 20 cm). The
chromatograms were run in the dark and the flavins visualized with a UV
lamp (wavelength 366 nm). The solvent systems employed were:

1) 80% phenol

2) 5% NazHP04

3) 3% sodium borate (10 H20)

4) water saturated with iso-amyl alcohol

31

5) propanol/water (3:1) with 1% ammoniun hydroxide
6) butanol/propionic acid/water (47:22:31)
(references 5,104,37).

Protein Determination

Protein concentration was determined by a modified Lowry procedure
(11), using fatty acid poor bovine albumin as the standard. This
procedure was used to remove interfering compounds such as phenolics,
sugars, sulfhydryl reagents, ammonium sulfate and some buffers. An
aliquot of an unknown containing approximately 15 to 20 micrograms
of protein was added to 1.5 ml of water. To this was added 25 ul 1%
sodium deoxycholate and the sample was incubated for 15 minutes.
Trichloroacetic acid (500 pl 24% w/v) was mixed in and the samples
centrifuged in a swinging bucket rotor at 10°C for 2 hours at 10009.
The supernatant was removed by aspiration and the pellets dissolved in
500 pl 1.2% NaOH. To this was added 1 ml of 3% Na2003, 0.03%
sodium tartrate, 0.0075% copper sulfate (5 H20) solution. Color was
developed with the addition of 300 pl Folin-Ciocalteau Reagent (diluted
4 fold) and the absorbance was measured at 660 nm exactly 30 minutes
after the initiation of color development. Standards were treated

identically with the unknowns.

Phosphate Determination
Phosphate concentration was determined by a modified
Fiske-Subbarow assay (1). An aliquot of 0.5 ml sample containing up to
500 nmol phosphate was added to 4 ml molybdate reagent and then mixed

with 0.5 ml elon reagent. The sample was left to stand for 30 minutes

32

for color development and was read at 660 nm. The standard used was 1
mM sodium monophosphate. The molybdate reagent consisted of 3.1 g/l
ammonium molybdate-4H20 in 0.625 N sulfuric acid. The elon reagent
consisted of 3 g sodium bisulfite and 1 g elon (p-methylaminophenol
sulfate) in 100 ml water. The molybdate reagent was stable
indefinitely at room temperature whereas the elon reagent was unstable

and hence was kept refrigerated in the dark.

Acyl-CoA Thioester Synthesis

Palmitoyl-CoA was synthesized by the procedure of Seubert (154)
and purified by the procedure of Pullman (149). Fifty mg of CoA (61
nmol lithium salt) was dissolved in 12 ml water and 25 nmol mercap-
toethanol was added. The solution was flushed with argon and 25 ml
tetrahydrofuran freshly distilled over sodium borohydrate was added.
Palmitoyl chloride (400 mg) dissolved in 10 ml tetrahydrofuran was
added over a thirty minute period with continuous stirring. The pH was
maintained between 7.5 and 7.8 during the course of the synthesis with
1 N NaOH. The reaction was allowed to continue for four hours with the
addition of tetrahydrofuran to maintain a homogeneous solution.
Perchloric acid (7%) was then added to give a pH of 3 and the tetrahy-
drofuran was removed under a stream of argon or by vacuum distilled.
The solution was heated to 80°C for two minutes and then lyophilized to
dryness. The residue was extracted twice with acetone (15 ml), 3 times
with diethyl ether (15 ml) and twice with 0.8% (v/v) perchloric acid
(10 ml). The produce was resuspended in a small volume of water and
any precipitate removed by centrifugation. The palmitoyl—CoA was

precipitated with perchloric acid (2% final concentration) and washed

33

with 0.8% perchloric acid, acetone and finally diethyl ether.
Ricinoleoyl-CoA and 3-transhexenoyl-CoA were synthesized by the mixed
anhydride method as described by Barber et al. (4). Petroselinoyl-CoA
was prepared from its acyl-chloride by the procedure used for
palmitoyl-CoA.

Purity was determined by thin layer chromatography on Avicell
cellulose plates developed in butanol/acetic/acid water (5:2:3) and by
the absorbance ratios of 250/260, 280/260 and 232/260 (16). Concentra-
tion determination were based on absorption at 259 nm at pH 7.0 (s =
15.4 x 103 M'1 cm'l) (16) or by thioester analysis (DTNB).

Sulfhydryl concentration was determined before and after alkaline
hydrolysis (1 minutes at 95°C in 0.1N NaOH) (22) by the addition of a
aliquot of the sample to a mixture containing 100 mM Tris'Cl buffer

at pH 8.0 and 250 u“ DTNB (5,5'-dithio-bis-(Z-nitrobenzoic acid)). An
absorbance change was monitored at 412 nm and an extinction coefficient
of 13.6 x 103 M"1 cm"1 was used (147). Sulfhydryl compounds

on thin layer plates were visualized using a nitroprusside reagent
(173) before and after thioester hydrolysis with 2% (w/v) NaOH in 95%
methanol. The nitroprusside reagent was prepared by dissolving 1.5 g
nitroprusside (sodium nitroferricyanide) in 5 ml 2 N sulfuric acid.
This was added to 95 ml methanol and 10 ml concentrated ammonium
hydroxide (28%) and filtered. All other thioesters used were obtained
from Sigma or PL Biochemicals and were used without further

purification.

34

Granular Hydroxyapatite

Granular hydroxyapatite was prepared by the method of Mazin et al.
(105). One gram silica gel type H (Sigma) was swelled and washed three
times with distilled water. The gel was resuspended in 200 ml
distilled water in a.two liter beaker. Half molar solutions of calcium
chloride and dibasic sodium phosphate were added slowly to the silica
gel using a peristaltic pump (approx. 0.7 ml/min). The silica gel
suspension was continuously and slowly stirred. The calcium phosphate
that formed was washed four times with distilled water, then incubated
in a solution of 1% (w/v) sodium hydroxide (100°C) which was maintained
at 100°C for one hour with occasional mild resuspension of the
precipitate. The NaOH solution was decanted and the fragile
hydroxyapatite washed with distilled water until a pH of 8 was
achieved. The hydroxyapatite was then washed four times with 10 mM
potassium phosphate at pH 7.8 (100°C) and stored at 4°C in 5 mM

potassium phosphate buffer at pH 7.8.

CHAPTER III

GLYOXYSOMAL B-OXIDATION: PURIFICATION AND PHYSICAL PROPERTIES

Since the discovery of glyoxysomal B-oxidation in the castor bean
by Cooper and Beevers in 1969 (26), much work has been done on the
purification and characterization of the B-oxidation enzymes from rat
liver and yeast (175,45,176). Acyl-CoA oxidase has been isolated by
Osumi and Hashimoto (142,136), Shimzu et al. (157) and Inestrosa et al.
(68,69,66). The protein with a dual function for enoyl-CoA hydratase
and hydroxyacyl-CoA dehydrogenase has been purified from rat liver
(139,141) and from cucumber cotyledons (44). Thiolase, the last enzyme
of the fatty acid oxidizing cycle, has been characterized by Miyazawa
et al. (115), Frevert et al. (43) and Krahling and Tolbert (in press)
in this laboratory. Since little research has been reported on the
B-oxidation enzymes from plants, an attempt at the isolation of the
components of the fatty acid oxidizing system from castor bean
endosperm was carried out.

Studies on the association as a complex of the enzymes constitu-
ting B-oxidation have been studied only in §;_ggli (124,39,148,7).
Little is known about the possible association of the enzymes from
eukaryotic peroxisomes, but it has been proposed for the rat liver
system (58,69). During the initial work on the isolation of the
B-oxidation system from the castor bean, c0purification of the

B-oxidation enzymes was observed. The goal of the research was subse-

36

quently shifted from the isolation of the components to the isolation
of the associated aggregate.

Subcellular Localization of B-Oxidation Activity

 

Organelles from extracts of castor bean endosperm were separated
by isopynic density gradient centrifugation and were used to survey the
intracellular distribution of fatty acid degradation. The profiles of
catalase (glyoxysomal marker), cytochrome c oxidase (mitochondrial
marker), B-oxidation and thiolase indicate that all B-oxidation activ-
ity was associated with the glyoxysomal fractions at a density of 1.237
g/cm3 (Figure 2). The mitochondrial marker banded at 1.185 g/cm3.

A shoulder of catalase, thiolase and B-oxidation activities were
observed at 1.204 g/cm3. This shoulder of activity is similar to

that observed by Huang and Beevers for ruptured glyoxysomes (60). The
small amounts of thiolase and catalase activity at the top of the
gradient was due to breakage of organelles during resuspension. The
absence of B-oxidation in the mitochondria of germinating fatty seeds
has been observed by others (26,112,61,117,82). Thiolase activity for
both C4 and C12 3-ketoacyl-CoA substrates was absent in the mito-
chondrial peak (Figure 2), precluding the inability of measuring mito-
chondrial B-oxidation activity or the existence of a chain length
specific fatty acid oxidizing system in the mitochondria. There is
little likelihood then that the B-oxidation system isolated from crude
organelle pellets could be mitochondrial in origin or be contaminated
with the mitochondrial system, as could be the case with the purifica—
tion of the peroxisomal B-oxidation system from rat liver. B-Oxidation
activity was insensitive to antimycin A, rotenone and cyanide, potent

inhibitors of the mitochondrial electron transport chain and thus an

Figure 2.

37

Subcellular fractionation of castor bean endosperm by
isopynic centrifugation. The figures contain the
distribution of B-oxidation and thiolase activities
relative to the mitochondrial (cytochrome c oxidase) and
glyoxysomal (catalase) markers on 20 to 56% (w/w sucrose

gradients.

38

 

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39

inhibitor of the B-oxidation in rat liver mitochondria (84,134,88,116,
134).

Purification of the s-Oxidation System

 

The purification of the B-oxidation system from germinating castor
bean endosperm is summarized in Table 1. The loss of activity was
probably due to the instability of the individual enzymes as well as to
the gradual separation of the components during the purification
process. Activity in the crude homogenates was not measurable due to
suspended fat and particulate matter. Differential centrifugation of
the endosperm extract (line 2, Table 1) separated out a significant
portion of the suspended fat and the heavy mitochondrial fraction (118)
after which assays were more feasible. The isolation of organelles
(line 3, Table 1) rather than using whole tissue extraction provided an
excellent purification step, even though loss of activity occurred from
glyoxysomal breakage during homogenization.

Extraction of the fatty acid oxidizing activity from organelle
pellets by high salt lysis or osmotic shock (60,112,15,14) was effec-
tive in the extraction of dilute solutions, but yields were substan-
tially decreased in concentrated resuspensions. Solubilization with
detergent and fractionation with ammonium sulfate was used as a quick
and more effective method of extraction of concentrated solutions. The
range of fractionation by amnonium sulfate was similar to that observed
for acyl-CoA oxidase (136,69,142) and enoyl-CoA hydratase/hydroxyacyl-
CoA dehydrogenase (139). Resuspended ammonium sulfate pellets were
generally dialyzed against 5 MM potassium phosphate buffer in 50% (v/v)

glycerol and were used in many experiments in this thesis (ammonium

40

Table 1. Purification of the B-oxidation system from the glyoxysomes
of castor bean endosperm. Endosperm (3839) was homogenized,
filtered and purified as described in Methods. B-Oxidation
activity (NADH formation) was expressed in units with one
unit equaling 1 nmol NADH formed/minute.

 

 

TOTAL TOTAL SPECIFIC
PURIFICATION ACTIVITY PROTEIN ACTIVITY
STEPS (units) (mg) (units/mg)
Filtered Homogenate -- 11,201 --
10009-15 Minute
Supernatant 280.0 9,374 .030
11,0009-60 Minute
Pellet 256.7 1,136 .226
Dialyzed 30-50%
Ammonium Sulfate
Fraction 159.7 343 .466
Hydroxyapatite Column 83.1 67.5 1.231

Carboxymethyl
Sephadex G-25 14.1 14.5 .971

 

41

sulfate fraction). Yields from ammonium sulfate fractionation was
usually 90-100%.

Heat denaturation has been used in the purification of the §;_ggli,
B-oxidation complex (38), acyl-CoA oxidase (142,69) and enoyl-CoA
hydratase/hydroxyacyl-CoA dehydrogenase (44). Attempts to use this
procedure on castor bean preparations have failed. Heat denaturation
in the absence of glycerol above 40°C resulted in substantial losses of
activity (Figure 9) and in the presence of glycerol little precipitate
was formed. The proper conditions for a heat denaturation step were
not found.

The use of hydroxyapatite (line 5, Table 1) in the purification of
the e-oxidation enzymes proved to be an excellent chromatographic step.
The major drawback in the use of this material is its tendency to pack
during use and the need to include glycerol to stabilize enzyme
activity only compounded this problem. Spheroidal hydroxyapatite and
hydroxyapatite mixed with cellulose have been used to improve flow
rates, but their use has been limited to lower protein concentration
and low viscosity buffers. Hydroxyapatite prepared in the presence of
crystallization centers (105) provided a chromatographic material with
excellent flow rates and high capacities. A precaution must be
observed when using hydroxyapatite, buffers and chelating reagents that
bind calcium (e.g. Bicine, Tricine, EDTA) greatly reduce the binding
capacity of this material.

The fatty acid oxidizing enzymes eluted from hydroxyapatite at
about 70 mM phosphate in a symmetrical yellowish-brown peak (Figure
3A). Similar elution profiles were observed with linear phosphate

gradients. Active fractions were combined and concentrated by dialysis

Figure 3.

42

Elution profiles of typical hydroxyapatite and
carboxymethyl Sephadex-25 columns. Granular hydroxyapatite
chromatography (A) of the B-oxidation system was run after
ammonium sulfate precipitation and dialysis (Table 1). The
column was equilibrated with 5 mM phosphate pH 7.8 buffer
and 25% (v/v) glycerol at 4°C. The eluted B-oxidation peak
was concentrated and dialyzed against the equilibrium
buffer before being applied to a carboxymethyl Sephadex-25
column (B). Column conditions were identical to those in

the hydroxyapatite chromatography.

43

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Table 2. Yields of the a-oxidation enzymes from castor bean
glyoxysomes. Assays employed were described in Methods with
the substitution of 33 uM acetoacetyl-CoA as the substrate
for the thiolase assay. Percent activity is in parenthesis.

Ammonium Hydroxy- Carboxymethyl
Organelle Sulfate apatite Sephadex

Enzymes Pellet Fraction Fraction Fraction

8-oxidation 257 (100) 160 (62) 83 (32) 14 (5)

Acyl-CoA Oxidase 268 (100) 153 (57) 63 (24) 10 (4)

Enoyl-CoA

Hydratase 6381 (100) 6504 (102) 3145 (49) 1574 (25)

Hydroxyacyl-CoA

Dehydrogenase 328 (100) 410 (125) 299 (91) 80 (24)

Thiolase 211 (100) 174 (82) 4.9 (2) .14 (.01)

Isomerase 69 (100) 21 (30) .10 (0.1) .12 (0.2)

Epimerase 30 (100) 4.6 (15) 1.1 (4) 0 (0)

f/i .272 .112 .029 0

 

45

against saturated ammonium sulfate. The presence of glycerol in the
buffer, resulted in lower yields when standard ammonium sulfate
fractionation procedures were used. High concentrations of ammonium
sulfate were required in order to overcome the stabilizing effect of
glycerol, resulting in excessive volumes and loss of B-oxidation
activity. Dilution of the sample to reduce the glycerol concentration
was unsuccessful since it also produced excessive volumes and loss of
activity. Dialysis against ammonium sulfate provided good recovery and
minimal dilution of the sample. The dialysate was subsequently
centrifuged, the pellet resuspended and dialyzed against resuspension
buffer to remove traces of ammonium sulfate.

Hydroxyapatite purification of enoyl-CoA hydratase/hydroxyacyl-CoA
dehydrogenase from cucumber cotyledons (44) has been observed to elute
at a much high salt concentration than that required for the associated
castor bean system. The harsh nature of acetone precipitation used in
the initial purification of the bifunctional protein could have readily
destabilized an associated system and thus allowed the individual
enzymes to be purified. The association of the B-oxidation system
observed in the castor bean was not as strong as that observed in E;
9911 (39,148,7) but careful handling allowed for c0purification of the
B-oxidation enzymes.

Chromatography on carboxymethyl Sephadex-25 (CM-25) yielded
variable results, but as shown in Figure 3B the protein peak eluted
coincidently with the B-oxidation peak. A low yield of B-oxidation
activity was obtained from CM-25 and a retention on the column was
observed after an eluting phosphate concentration was achieved.

Retention of the partially purified enzyme system on Sephadex,

46

acrylamide, and agarose columns was also observed but the cause has
not been elucidated. Chromatography of crude enzyme preparations gave
better yields and the absence of retention on the CM-25 column.

The decrease in the specific activity after chromatography on
CM-25 was not necessarily an indication of a reduction in purity. The
instability of the overall B-oxidation activity even in the presence of
50% glycerol (Figure 5 and 6) could account for much of this loss
(estimated 33 to 50%). Also the effects of retention on the column
could have contributed to this decrease in specific activity.
Carboxymethyl Sephadex chromatography was not routinely used in the
preparation of the B-oxidation system.

The presence of glycerol in the buffers effected the
chromatographic behavior on hydroxyapatite and CM-25. Originally
purification was run at room temperature using 50% glycerol and 50 mM
phosphate was required for elution from hydroxyapatite and 33 mM
phosphate from CM-25. When the columns were run at 4°C the viscosity
of the buffer increased and the glycerol concentration had to be
reduced to 25%. The phosphate elution concentration increased to 70 mM
for hydroxyapatite and 110 mM for CM-25. This effect was not further
investigated but the cause could result from a change in the water
structure around the B-oxidation system due to the temperature change
or the change in the glycerol concentration (72,155).

Yields of Enzyme Activity

Yields of the various activities of the fatty acid oxidizing cycle
are presented in Table 2. The total activities and the percent
yields are given for the fractions from the isolated organelles to the

carboxymethyl Sephadex-25 peak (Table 1). Comparative analysis of the

47

individual activities should be undertaken with some caution. The
assay conditions and the substrate chain lengths used were very
different, having a potentially great effect on the relative rates.
Some have used this method of analysis to show that acyl-CoA oxidase
is the rate limiting step of B-oxidation (58,114,140). Though this nay
be the case such analysis should be viewed with some reservation.

Parallel losses in activity were evident between B-oxidation and
acyl-CoA oxidase and between enoyl-CoA hydratase and 3-hydroxyacyl-CoA
dehydrogenase. The latter case could readily be explained, since it
has been shown that these two activities are due to a single protein in
other peroxisomal systems (139,44). The increase in activities of the
hydratase and dehydrogenase after ammonium sulfate fractionation could
be attributed to the removal of particulate matter which had a great
effect on the observed rates. The parallel loss of s-oxidation
activity and acyl-CoA oxidase would support the proposition that
acyl-CoA oxidase is the rate limiting step of B-oxidation. A great
loss of thiolase activity occurred during the chromatographic steps.
This suggested a very high specific activity or an excess of thiolase
present in the glyoxysome, since the 90% loss had no discernable effect
on the total rate of B-oxidation.
Polyacrylamide Gel Electrophoresis

Attempts at determining the purity and the molecular weight of the
B-oxidation system by polyacrylamide gel electrophoresis were unsuc-
cessful. Glycerol, which has been used to stabilize the B-oxidation
complex fron1§¢_gglj_on polyacrylamide gels (23), was incorporated into
7.5% and 10% acrylamide gels at 0, 25 and 50% (v/v) concentrations.

The gels were characterized by a few faint bands and by a smear in the

48

upper third of the gel after coumassie brilliant blue staining.
Glycerol did not have a stabilizing effect on the B-oxidation enzymes
from castor bean glyoxysomes. Catalase, BSA, ovalbumin, cytochrome c,
carbonic anhydrase and myoglobin were used as standards and were
observed as distinct bands. No conclusions could be made on the
molecular weight of the B—oxidation system based on these findings.

SDS polyacrylamide gel electrophoresis gave similar results.
Acrylamide gels (10%) prepared by the procedure of Weber and Osborn
(182) exhibited five major and six minor bands after staining. The
identification of the bands could only be spectulative since the purity
of the preparation was unknown. Standards (See Methods) allowed for
tentative identification of three of the major bands based on the
reported molecular weights of the oxidase, thiolase, and hydratase/
dehydrogenase (47,43,141,139,44,68,69,142).

Purified rat liver acyl-CoA oxidase (142) and enoyl-CoA hydra-
tase/hydroxyacyl-CoA dehydrogenase (47) electrophoresed on SDS poly-
acrylamide gels exhibited extra bands due to the proteolytic cleavage
of the proteins. This may account for four additional bands on the SDS
gels of the castor bean B-oxidation system. The proteolytically
cleaved enzymes were reported to maintain activity, making such a pro-
position feasible. The presence of isomerase and epimerase activities
in the samples electrophoresed could account for additional bands,
however, the characteristics of these enzymes are not known. Based on
native and SDS polyacrylamide gels the purity of the preparations could
not be ascertained. The lack of major definable bands on native gels

would suggest a relatively pure preparation.

49

Molecular Weight Determination byASieving Columns

Molecular sieving on Sepharose 6B, Sepharose 4B, Sephacryl-ZOO and
Biogel A-0.5 showed inconsistent and very low values in the estimated
molecular weight of the B-oxidation system. Buffers consisting of 25
mM glycylglycine and 100 mM potassium phosphate (pH 8.2 and 7.7,
respectively) were used in the presence and absence of 20% (v/v)
glycerol, in attempts to vary the chromatographic characteristics of
the B-oxidation system. Chromatography of hydroxyapatite purified
enzymes on Biogel A-0.5 resulted in an estimated molecular weight
between cytochrome c and glucose. This value was similar to those
observed on other molecular sieving materials. Fractionation of
organelle extracts on Sepharose 6B exhibited three peaks of activity.
One peak eluted with the voided material while another eluted near the
included materials. The final peak of B-oxidation activity came off
the column on the leading edge of the catalase peak, suggesting a
possible association of the two activities. A small peak of catalase
activity was also observed to elute at the leading edge of the included
B-oxidation activity. No conditions were found that would allow for a
molecular weight determination on sieving gels. The cause of these
chromatographic characteristics is not known.
Sedimentation Velocity on Sucrose Density Gradients

Since attempts at the determination of molecular weights by molec-
ular sieving proved to be unsuccessful, a nonsupport method was tried.
Sucrose gradients prepared as described by Martin and Ames (109) were
employed to obtain a sedimentation coefficient and an estimation of the
molecular weight. e-Oxidation activity co-migrated with rabbit muscle
lactate dehydrogenase (Figure 4). This identical migration was not due

to an interaction between the two systems, since both the B-oxidation

Figure 4.

50

Sedimentation of the B-oxidation enzymes on sucrose density
gradients. Sucrose gradients, 5 to 25% (w/w), were
prepared according to Martin and Ames (109). Rabbit muscle
lactate dehydrogenase and the B-oxidation system were

were applied to the gradients separately and in combination
and centrifuged for 50 hours (5°C) in a Beckman SH-25.2
swinging bucket rotor at 25,000 rpm. Gradients were

fractionated and assayed as described in Methods.

51

cakeeseéc 84288930 3454.. o
I 8 4

 

 

 

   

 

 

 

_ m _
m
n
m .m o
9 .m 4
ON. Mu. m
bl r x
I Va M
C." .
08 0U
LD 3N
IIIII I an
: n
’lmm
F.
.m
. p _ o
m 2. o. o

0
2:25:22 .25: 29295-:

52

activity and lactate dehydrogenase alone sedimented to identical
positions.

Lactate dehydrogenase from rabbit muscle has a molecular weight of
140,000 (28) and a sedimentation coefficient (520,”) of 7.2 (183).

The sedimented B-oxidation peak showed cycling of the acyl-CoA
substrate indicating the presence of all three B-oxidation enzymes:
thiolase (90,000 MN), acyl-CoA oxidase (140,000) and enoyl-CoA
hydratase/hydroxyacyl-CoA dehydrogenase (77,000 MN). The calculated
molecular weight from sedimentation on sucrose gradients (109) of
140,000 and the molecular weight estimated from the known values of the
components of the B-oxidation system (306,000 MN) is not easily
reconcilable. Lipids and carbohydrates associated with a protein can
reduce its sedimentation by reducing the proteins partial specific
volume. Lipids have been found to be associated with the B-oxidation
complex isolated from §;_ggli (23,124).

one;

The lipid content of the B-oxidation preparation purified through
the hydroxyapatite step was determined by a modified procedure of
Donaldson (32). An enzyme sample was exhaustively dialysed in five
changes of buffer (4°C) containing 5 nM glycylglycine pH 8.0. The
preparation was then extracted with ten volumes of chloroform/methanol
(2:1, v/v), centrifuged and filtered. The residue was washed with four
volumes of the same solvent. The extracted lipids were washed three
times with 8 ml of 0.1% (w/v) MgClz. The aqueous upper phase was
discarded and the lower chloroform phase evaporated to dryness. The
residue was redissolved in a small volume of chloroform and spotted on

a silica gel G-60 (Brinkmann) thin layer plate. The plate was then

53

chromatographed in two dimensions using chloroform/methanol/7N (NH )OH
(65:25:4 by volume) in the first direction and chloroform/methanol/
acetic acid/water (170:15:15:2 by volume) in the second direction (51).
The separated materials were detected by spraying with 50% sulfuric
acid and charring. A major spot tentatively identified as neutral
lipids was observed with traces of two unidentified compounds. Whether
the neutral lipid was associated with the B-oxidation system is not
known but it could acocunt for the observed low estimated molecular
weight observed on the sucrose sedimentation gradient by reducing the
partial specific volume of the B-oxidation system.

Charge Characterization

 

The manner in which a protein binds to hydroxyapatite can be used
in the characterization of its overall charge (13). Proteins can be
adsorbed to the phosphate sites or to the calcium sites of
hydroxyapatite. Acidic and neutral proteins tend to bind to the
calcium noiety and are easily eluted with low phosphate or very high
sodium chloride (molar) concentrations, but not with calcium chloride
solutions. Basic proteins on the other hand, bind to the phosphate
sites of hydroxyapatite and require very low calcium chloride
concentrations (millimolar) or molar solutions of phosphate or sodium
chloride.

The B-oxidation enzymes were bound to hydroxyapatite and the
absorbant washed with 25 nM calcium chloride, 250 M potassium chloride
and 50 mM potassium phosphate at pH 7.8 (no glycerol present).
Phosphate was the only solute to elute any of the B-oxidation activity
from hydroxyapatite. This indicated a neutral to acidic characteristic

at pH 7.8 for the B-oxidation system which was supported by the

54

observed isoelectric points of the oxidase pI 9.2 (142), thiolase pI
9.2 (115) and hyratase/dehydrogenase pI 9.5-9.8 (44,141). An acidic
nature at pH 7.8 was also supported by the observation that the
associated system binds to SP-Sephadex-25 and carboxymethyl-Sephadex-25
but voided on QAE and DEAE Sephadexes.

Stability of a-Oxidation Activity

 

During the initial studies of the glyoxysomal s-oxidation system,
loss of activity was substantial. Attempts at stabilization with azide
(0.02%), thioglycerol (10 mM), potassium chloride (100, 200 and 400 mM)
and pH 6.5, 7.5 and 8.5) proved to be ineffective. Glycerol had been
used to stabilize the B-oxidation activity in §;_ggli (7,124,38) and
was tested on the system from the castor bean (Figures 5 and 6).
Stabilization was both temperature and glycerol concentration depen-
dent. The half life (tl/z) of B-oxidation activity increased from
less than a day to 2.7 days with 50% glycerol at room temperature and
to 11.7 days in 50% glycerol at 4°C.

The logarithum of the half life of B-oxidation activity was pro-
portional to the glycerol concentration (Figure 7). At 22°C the
increase in t1/2 was directly related to the change in the
glycerol content. Reducing the storage temperature to 4°C increased
the t1/2 in the presence of glycerol. The change in the half life
paralleled that at room temperature except at glycerol concentrations
below 20%. The cause of this deviation is now known but could have
been due to a cold inactivation that was reduced by low levels of
glycerol (72) and was eliminated at glycerol concentrations above 20%.
Storage of the system with other polyhydric compounds showed no strong

preference (Figure 8).

Figure 5.

55

Stability of B-oxidation activity in the presence of
various concentrations of glycerol at 22°C. The ammonium
sulfate fraction in 25 mM Bicine, 1 mM EDTA pH 7.8 was
stored in 0 to 50% (v/v) glycerol and four assays were made
at each time point. The average standard deviation

observed was 1.7%.

56

 

IOO

 

50

50 °/o

5
l

 

 

ACTIVITY (70)

(II
I

O °/e |O°/o

 

 

 

 

5
DAYS

Figure 6. Stability of B-oxidation activity in the presence of
various concentrations of glycerol at 4°C. Conditions were
identical to those in Figure 6. Four assays were run at
each time point and an average standard deviation of 1.7%

was observed.

58

 

 

IO.

50%

 

40%

30%

20%

 

l0%

0%

 

 

20

W L5.

>t>_._.o< e\o

I5

l0

DAYS

59

Figure 7. The effect of glycerol on the half life of B-oxidation
activity at 4° and 22°C. The half life of the B-oxidation
activity from each curve in Figures 5 and 6 were plotted

against the corresponding glycerol concentration.

60

 

l0

 

 

4°C

5 O
’5.
§ 2.5 A’
I: 22°91:
.J ,’
u. I ,A
.J A I
<1 /
I ,’

.5 9’

I
I
_ 1 1 1 1 1
0 IO

20 30 40
GLYCEROL my

 

Figure 8.

61

The effect of various polyhydric alcohols on the stability
of B-oxidation activity. The ammonium sulfate fraction in
100 mM potassium phosphate buffer pH 7.8 was diluted two
fold and made up to 25% (w/v) with the various polyhydric
alcohols indicated or with 25% (w/v) DMSO or 2 mM NAD.
Each data point represents four assays. Fractions were
left unsealed at 4°C. 0n the ninth day each sample was

made to 10 mM dithiothreitol and assayed the next day.

IOO

75

mmqu)
0

N
0'

O 2

62

 

A- Glycerol
" B'Control G
C'Ethylene glycol
D-Sorbiiol

E- Sucrose
F- DMSO

 

6" NAD
I

  
   

4— IO mM DTT

 

 

J 1
4mm6

ID

63

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65

Glycerol was also effective at protecting the enzyme complex
during heat inactivation (Figure 9) (72). The temperature at which 50%
of the activity was lost in ten minutes time dropped from 64°C in the
presence of glycerol to below 40°C in the absence of glycerol.
Glycerol has now been used to stabilize all of the enzymes involved in
the peroxisomal fatty acid oxidizing system (40,44,115,57,46) as well
as other enzymes involved in fat metabolism (79,12,53,80). It was
routinely used to stabilize the B-oxidation activity from castor bean
endosperm. Very little loss in activity was observed upon storage at
-20°C in 50% glycerol. Polyhydric alcohols have been reported to
stabilize oligomeric protein systems by reducing subunit dissociation
and by protecting against denaturation (155,186,72).

The addition of 10 mM dithiothreitol (DTT) after an extended
incubation in various polyhydric alcohols (Figure 8) resulted in a
substantial recovery of activity. Control samples and NAD containing
samples showed low sulfhydryl activation suggesting that two forms of
inactivation might be occurring. The first could be an irreversible
inactivation that is slowed or eliminated by polyhydric alcohols. The
second looked like an inactivation which was reversed by incubation
with DTT.

The effect of DTT on stability in the presence and absence of
glycerol is shown in Figure 10. Incubation with the sulfhydryl reagent
for several hours before the initial assays induced an increase in
activity up to 80%. Assays of the control sample in which DTT, up to
20 mM, had been added just prior to the initiation of the assay showed
little or no increase in activity. Activation under these conditions

were observed only with older preparations or when longer incubation

66

Figure 10. The effect of glycerol and dithiothreitol on B-oxidation
activity. Preparation and analysis of samples were

identical to that in Figure 8.

0.8

pmol NADH/min/ml
,o
.b

O
N

67

 

 

 

Glycerol + l0 mM DTT

  
   

Contro

+—-IOmM DTT

 

NI—

DAYS

 

68

periods were used. The results suggest that slow inactivation and slow
activation processes were occurring.

Dithiothreitol by itself (1 and 10 mM) did not stabilize
B-oxidation activity, for the percentage loss was similar to that
observed in the control (Figure 10). Protection against irreversible
inactivation similar to that provided by glycerol was observed with DTT
which allowed some recovery of activity after incubation with addi-
tional DTT. The use of the sulfhydryl reagent in the presence of
glycerol, however, provided maximum stability and activation greater
than from either substance alone (especially with 10 mM DTT). Enzyme
activity was slowly lost with time, perhaps as the DTT was oxidized by
the air. The exact nature of inactivation and activation are not
discernable without comparable enzyme analysis of each component in the
B-oxidation system.

A relative analysis after a fourteen day incubation period in
sealed containers to eliminate air oxidation is shown in Table 3.
Maximal B-oxidation activity was again found in the presence of
glycerol and DTT as observed in Figure 10. The sealed samples contain-
ing glycerol showed no loss of activity whereas substantial losses
occurred in its absence. This indicated that some component(s) of the
B-oxidation system was stabilized by glycerol with DTT stimulating
(activity, presumably by reducing already oxidized sulfhydryl groups.
The second form of inactivation was due to oxidation from the exposure
to air (glycerol, sealed and unsealed). Activation by DTT seemed to be
a slow process and the mechanism of its effect are not completely

understood.

69

Table 3. Stability of the B-oxidation enzymes in glycerol and
dithiothreitol. The B-oxidation system, purified by hydroxy-
apatite chromatography, was incubated in sealed containers
with 50% (v/v) glycerol and/or 4.nM DTT for fourteen days at
4°C. Thiolase was assayed using B-ketododecanoyl-CoA. Value
in parenthesis are percent of original activity.

 

 

 

a-Oxidation Thiolase Enoyl-CoA Hydroxyacyl-CoA Acyl-CoA
Hydratase Dehydrogenase Oxidase
,pmol/minlmg
Control 0.014 (12) 12.32 30 5.06 .093
DTT 0.766 (38) 16.32 128 9.12 --
Glycerol 1.921 (101) 8.90 151 9.92 1.342

Glycerol
DTT 3.565 (119) 17.90 222 10.61 --

 

70

Acyl-CoA oxidase in the seed B-oxidation system was shown to be

greatly stabilized by glycerol, as has been observed in Tetrahymena

 

(57). The levels of B-oxidation activity and acyl-CoA oxidase activity
were very similar in the control and glycerol containing samples. The
loss in oxidase activity could cause the loss of total B-oxidation
activity, but as stated earlier, a pr0per comparative analysis must be
done under identical conditions using the same chain length substrates
in order to determine the rate limiting step. The effect of DTT on the
activity of acyl-CoA oxidase was not determinable, since sulfhydryl
compounds interfered with that assay (Methods). Enoyl-CoA hydratase/-
hydroxyacyl-CoA dehydrogenase activities were effected by glycerol and
DTT to different degrees. If it is assumed that these two activities.
are due to a single protein (44,139) (although this has not yet been
shown to be the case for the castor bean system) the differential
effect observed could suggest a separate site for each reaction. This
was recently supported by the work of Furuta et al. (47). They
isolated enoyl-CoA hydratase/hydroxyacyl-CoA dehydrogenase from rat
liver and showed that the two activities were differentially affected
by salts, acetoacetyl-CoA, proteolytic inactivation and sulfhydryl
titration. From these data they proposed that the two activity centers
were located at different sites on the protein.

The stability of the thiolase activity was not altered over time
(data not shown), but the sulfhydryl reagent in the assay had a signif-
icant effect on the relative activities. Omission of DTT from the
assay mixture caused a large decrease in the observed rate of trans-

acetylation. The long term effect of DTT incubation was not studied,

71

but thiolase could be the component that was oxidized by air and
reactivated by DTT.

Further characterization of the sulfhydryl sensitivity of
B-oxidation is shown in Figure 11. Parachloromercuribenzoate (pCMB)
gave a more complete inhibition of the B-oxidation activity than
arsenite, which was used by Rein et al. (153) to inhibit thiolase
and s-oxidation in rat liver mitochondria. Incubation with pCMB (50
pH) resulted in activity of short duration and rate curves were similar
to those when CoA or thiolase were absent from the assay (Figure 15).
The results support the observation that sulfhydryl groups of thiolase
were readily oxidized. Incubation with pCMB (500 uM) completely
eliminated NADH formation. This would indicate a less sensitive
sulfhydryl group(s) located elsewhere in the fatty acid oxidizing cycle
and might account for the slow DTT activation. Acyl-CoA oxidase from
yeast has been shown to be sensitive to sulfhydryl reagents such as
pCMB, mercuric acetate, silver nitrate and mercuric chloride (157).
Hepatic peroxisomal enoyl-CoA hydratase/hydroxyacyl-CoA dehydrogenase

were sensitive to DTNB titration (47).

Figure 11.

72

The effect of parachloromercuribenzoate and arsenite on
B-oxidation activity. Parachloromercuribenzoate and
arsenite were incubated 10 and 5 minutes respectively with
ammonium sulfate fraction and assayed by NADH formation.
Cofactors were added after incubation with the sulfhydryl
reagents. The standard deviation of four assays are

shown.

73

 

IOO

 

 

 

 

 

 

 

 

75-—
l
A 50* pCMB
a“:
>—
L".
>
F: Arsenite
‘4’ \
75‘
l
O i 7 a f) .
O 25 50 500

SULFHYDRYL REAGENT (pM-pCMB,mM-Arseniie)

CHAPTER IV
CHARACTERISTICS OF a-OXIDATION ACTIVITY
The characterization of an enzymatic activity by the variation of
the assay parameters and the addition of various effectors is standard
procedure in the study of a newly isolated system. Cofactor require-
ments, optimal assay conditions, enzyme concentration linearity and
other parameters were evaluted.

The Effect ofng on Activity

 

The effect of pH on the velocity of B-oxidation is characteristic
of most peroxisomal enzymes (175). As shown in Figure 12, the pH
optimum was 8.4 to 9.2 for crude glyoxysomal extracts as measured by
NAD reduction, but shifted to a slightly more alkaline profile after
ammonium sulfate fractionation. The pH optimum based on 02 consump-
tion yielded similar results. The optimal pH reported for B-oxidation
in other peroxisomal systems is between 8.0 and 9.0 (170,58), similar
to that observed for acyl-CoA oxidase (59,157.67). Enoyl-CoA hydra-
tase/hydroxyacyl-CoA dehydrogenase has a more alkaline range of 9 to 10
(156,141,161,180,87). The shift in the pH optimum could be correlated
with the relative losses in activity during purification of the
B-oxidation enzymes (Table 2), but confirmation of such a proposal
would require analysis of pH curves of the enzymes in question, using

identical assay conditions and chain length substrates.

74

75

Figure 12. The effect of pH on B-oxidation activity. The buffer
consisted of 100 mM glycylglycine and 100 mM glycine.
Curve A represents the effect of pH on a crude glyoxysome
extract. Curve 8 represents the effect on the ammonium

sulfate fraction.

76

 

 

 

 

 

 

 

 

IQO

 

 

4.
_e\=_e\zo<z as:

2.

c’20

77

The Effect of Incubation Time and Protein Concentration on Activity

 

Prior incubation of the enzyme preparation in the standard assay
mixture had no affect on the rate of NADH formation. Incubation
periods up to thirty minutes at 25°C were examined. The results
indicate that dilute solutions of the enzyme system at this temperature
and duration were not notably unstable.

The effect of protein concentration on activity is shown in Figure
13. The specific activity of the B-oxidation system declined as the
protein concentration in the assay was reduced below 80 ug. Such an
effect could be attributed to the instability of the enzyme association
or to an unstable component in very dilute solutions. The build up of
an intermediate in the e-oxidation pathway would have been expressed in
the form of an extended lag period before maximal rates were achieved,
but the specific activity (controlled by the limiting step) should have
been constant for all protein concentrations. The stimulation of
activity by FAD (acyl-CoA oxidase cofactor) increased the rates 1.5
fold but did not effect the loss of specific activity at low protein
concentrations. Glycerol which was used to stabilize activity reduced
the deviation at low protein concentrations, but did not eliminate the
loss. Hhether glycerol stabilized the configuration of the B-oxidation
system or stabilized an individual enzyme from inactivation by dilution
is not known.

The addition of variuos amounts of glycerol to the assay medium in
the linear protein range for maximal specific activity resulted in an
inhibitory effect above 10% (Figure 14). The inhibition is believed
due to the decrease in the rate of diffusion caused by the increase in

the viscosity of the buffer. This effect any account for the 25%

78

Figure 13. The effect of enzyme concentration on the rate of NADH
formation. Glycerol (25%) and/or 10 pH FAD were added to
varying amounts of enzyme and assayed by monitoring NAD

reduction. Data is expressed as specific activity.

79

 

n
.0
O)
T

NADH/nin/gig protoi
2b
l

pmol p
'l’

 

 

 

 

FAD ,fi f
Glycerol t FAD

G erol .
lye ‘

II

 

 

3F

If

PROTEIN (pql'

 

80

Figure 14. The effect of glycerol concentration on the rate of NADH
formation. Standard spectrophotometric assay and ammonium

sulfate fraction were used with a flavin concentration of

10 pH.

81

 

 

_

40

J
30

20
GLYCEROL ("la in assay)

1
IO

 

 

8.
0

w
522.. 9.55.5754

_
4.
0
z .08:

0

82

reduction in the activity in the presence of FAD and glycerol (Figure
13). A similar inhibition by glycerol was not observed in the absence
of FAD.

Rate and Stoichiometry of NAD Reduction

The typical assay rates observed during the study of the
B-oxidation system are schematically illustrated in Figure 15. Curve
'A' represents the normal rate of NADH formation in the complete assay
mix (saturating NAD and CoA). Rates of NADH formation gradually
decreased, probably due to the accumulation of short chain length
acyl-CoAs for which the B-oxidation system had a lower affinity.
Curves similar to '8' occurred when CoA or thiolase were limiting. The
secondary slope was used as the measure of fatty acid oxidation for
enzyme preparations that exhibited such reaction curves. Under
conditions where either CoA or thiolase were totally absent from the
assay medium, curve ‘C' resulted. The shoulder of activity observed
during purification of the B-oxidation system on hydroxyapatite (Figure
3A) consisted predominantly of curve 'C' type reaction rates. No
thiolase activity was found in this activity peak but there was a fair
rate of initial oxidation indicating the presence of acyl-CoA oxidase,
enoyl-CoA hydratase and hydroxyacyl-CoA dehydrogenase.

The initial rates of NAD reduction when CoA or thiolase were
limiting or missing (curves '8' and 'C') probably reflected the
oxidation of palmitoyl-CoA to its 3-keto intermediate. The total NADH
produced under conditions where CoA or thiolase were absent was always
equal to or less than the amount of substrate added. This also seemed
true for the NADH produced up to the inflection point on curve '8'.

Unity between NAD produced and palmitoyl-CoA added was only observed

83

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84

 

 

 

 

 

 

 

 

85

when high levels of enzyme activity were present, suggesting a product
inhibition that was overcome by enzyme saturation. 3-Ketohexadecanoyl-
CoA inhibition of acyl-CoA oxidase (Ki = 0.47 pH) has been reported

for the purified enzyme from rat liver (142), as has a chain length
specific inhibition of rat liver enoyl-CoA hydratase by 3-ketoacyl-CoA
compounds (47). Endogenous or background activity was eliminated when
the fatty acid oxidation system was purified beyond the organelle
extract. The lag periods illustrated in Figure 15 were inversely
related to e-oxidation activity.

Crude glyoxysomal extracts exhibited a loss of NADH that was due
to a palmitoyl-CoA dependent NADH oxidation. The subcellular localiza-
tion of this system for NADH oxidation is not known, but this reaction
was separated from the fatty acid oxidizing system upon further purifi-
cation. The oxidation of NADH was not due to diaphorase activity. The
actual substrate for NADH oxidation could have been either acyl-CoA,
enoyl-CoA or 3-hydroxyacyl-CoA since the B-oxidation enzymes were
present. Various chain length substrates tested supported NADH
oxidation except acetyl-CoA. Acetyl-CoA cannot produce the enoyl and
hydroxy intermediates that were most suspect as the actual substrate.
The rate of NADH oxidation was inhibited by concentrations of
palmitoyl-CoA greater than 20 uM. The activity was destroyed by
heating in a boiling water bath, but was not effected by detergents and
could not be removed by centrifugation at 100,DODg for 90 minutes. In
one preparation, the rate of NADH oxidation was 25% of the rate of NAD
reduction due to B-oxidation. This caused severe underestimation of
both rate and stoichiometric measurements. The ratio of NADH oxidized
to palmitoyl-CoA added was 5.8. The function of this phenomenon and

the further characterization of the system was not undertaken.

86

Assay Components

 

The effect of deleting components from the standard assay on
a-oxidation activity is illustrated in Table 4. Removal of FAD reduced
the reaction velocity 23%. The requirement for FAD was dependent on
the age and the previous treatment of the enzyme preparation.
Elimination of CoA decreased activity 64%. The activity observed with
the removal of CoA was of short duration and was identical to the rate
profile illustrated by curve 'C' of Figure 15. Removal of the protein,
substrate or oxygen eliminated activity completely. The effect of NAD
elimination was studied by the polarographic assay, since the deletion
would have been meaningless in the spectrophotometric assay based on
nucleotide reduction. As with the removal of CoA, a reduced rate of
short duration was observed. Similar results were noted with the
acyl-CoA oxidase/peroxidase assay.

Acetyl-CoA, the product of B-oxidation, had no effect on activity
when concentrations as high as 800 uM were added. This indicated the
lack of feedback inhibition in the associated system. Acetyl-CoA
inhibits the mitochondrial thiolase from pig heart (127) and the
peroxisomal enoyl-CoA hydratase from rat liver (47). The other product
of B-oxidation, NADH, showed no inhibition by concentrations up to 300
uM. NADP would not substitute for NAD. NADP from 0 to 3.8 mM did not
inhibit NAD reduction.

The use of detergents to eliminate enzyme latency in organelle
associated systems has been used in the study of B-oxidation in the
microbody (68,59,19,88,137,84,71). However, detergents severely
inhibited a-oxidation activity of the isolated system as shown in

Figure 16. The concentration usually employed (0-01%) cause a 15%

87

Table 4. The effect of the deletion of standard assay components on
B-oxidation. Components of the NAD assay were omitted. The
decreased rates are discussed in the text. Oxygen was
eliminated by exhaustive flushing of a Thunberg cuvette with

nitrogen.

 

Deletion

Activity

umol/min/mg protein i standard deviation

 

Control

-FAD

-CoA

-Protein
~Palmitoyl-CoA
-0xygen

-Protein + boiled enzyme

0.721 _ 0.028

+

0.551 0.014

|+

0.254 _ 0.007

4.

GOOD

 

88

Figure 16. The effect of detergents on B-oxidation activity. The
ammonium sulfate fraction was assayed in varying amounts

of Triton X-100 and deoxycholate.

89

 

 

-§ 0'6 Triton x-IOO
‘5
8-
O.
O
E
2 0.4
E Dooxycholato
5 "i
f 0.2 4
O
E
1
o L 1 l » ,
0 0.025 0.050” OJO

 

 

DETERGENT (% in assay)

90

inhibition for Triton X-100 and 55% inhibition for deoxycholate.

Bovine serum albumin (BSA) has been added to the assay mix to improve
rates of B-oxidation in isolated organelles (50,88,69,19,26,67,137,70).
It has also been incorporated in the assay of acyl-CoA oxidase (68,59).
The effects on the B-oxidation system from the castor bean is shown in
Figure 17. Similar results were observed with glyoxysomal preparations
from sucrose gradients and from differential centrifugation of crude
endosperm extracts. The concentration range of BSA generally employed,
150 ug/ml to 300 ug/ml, had little effect on the rate of palmitoyl-CoA
oxidation under Optimal assay conditions, but concentrations of 400
ug/ml or higher were inhibitory. BSA has been reported to inhibit
peroxisomal fatty acid oxidation in rat liver, with 50% inhibition
being observed at 1500 ug/ml (158). This is approximately twice that
required for 50% inhibition of the castor bean system. BSA probably
exerts its inhibitory effect through the adsorption of the acyl-CoA
substrates. The effect of BSA on palmitoyl-CoA oxidation at varying
concentrations is illustrated in Figure 18. BSA reduced substrate
inhibition at higher acyl-CoA concentrations but its presence also
caused sigmoidal kinetics at the lower substrate concentrations. The
maximal rate of oxidation was not effected, only the substrate concen-
tration changed at which the optimal rate was observed. Similar
effects of BSA have been observed with s-oxidation (26) and palmitoyl--
CoA hydrolase (12).

Salt Requirements

 

s-Oxidation of fatty acid by the isolated system was not inhibited
by EDTA. Most divalent metals tested (Ba++, Mn++, ZnTT,

Mg++) either caused precipitation of the protein or severely

91

Figure 17. The effect of bovine serum albumin on the rate of
palmitoyl-CoA oxidation. Essentially fatty acid free BSA
was added to the B-oxidation assay containing the ammonium
sulfate fraction. Data is presented with its standard

deviation.

92

 

 

l
600

460
BSA (pg/ml)

I
200

 

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inhibited activity. The effect of the Mg++ ion is shown in Figure

19. Addition of EDTA eliminated the inhibitory effect. Divalent
cation inhibition could have been due to an affinity for the pyrophos-
phate moiety of CoA making it unavailable for some step in the pathway,
or it could have been due to a direct inhibition of a component(s) of
the B-oxidation system.

Potassium chloride from 50 to 250 mM stimulated B-oxidation
(Figure 19), but higher concentrations were inhibitory. These effects
have been attributed to salting in (electrostatic stabilization) and
salting out (hydrophobic interactions) effects (109). Various other
anions and cations at 200 uM ionic strength were tested to note if
there was a correlation of the effect of salts on B-oxidation activity
to the lyotropic series (Table 5), but no consistent relationship was
observed. Nhen enzyme levels were used below the linear range for
maximal specific activity (Figure 13), no correlation was observed, but
there was greater stimulation of activity relative to the control.
Cyanate and thiocyanate were inhibitory, but the actual cause was not
further studied. Furuta et al. (47) observed similar salt effects with
the rat liver peroxisomal enoyl-CoA hydratase. The same laboratory
also observed an anion inhibition of acyl-CoA oxidase from rat liver
peroxisomes that paralleled the lyotropic series (142). The difference
between the rat liver and the castor bean system could be due to the
tissue source, the assay conditions or to a possible difference between
the expressed characteristics of an individual component and the

component functioning in the associated system.

96

Figure 19. e-Oxidation activity as a function of potassium chloride
and magnesium chloride concentrations. KCl and MgClz

were added to the standard assay mixture and the rate of

NADH formation was recorded.

97

 

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98

Table 5. The effect of various salts on the reaction velocity of
B-oxidation. Salt solutions were added to the standard assay
mix to a final ionic strength of 200 uM. Activities and the
standard deviation are given.

 

 

Salts Activity
u = 200 uM umoles NADH/min/ml
Control 0.521 t 0.027
KCl 0.534 t 0.020
NaCl 0.511 t 0.036
LiCl 0.438 1 0.024
NH4Cl 0.554 t 0.014
KBr 0.592 t 0.024
KI 0.528 t 0.027
KF 0.472 t 0.027
KN03 0.596 t 0.032
K2504 0.566 1 0.024
KOCN 0.044 t 0.011
KSCN 0.263 t 0.044

 

99

Flavin Identification and Stimulation of 5-0xidation

Stimulation of peroxisomal B-oxidation activity by FAD has been
previously demonstrated (58,68,69,157,170,19,59,67). The effect of
purified FMN and FAD on the B-oxidation system from the castor bean is
shown in Figure 20. Maximal stimulation was observed with about 10 pH
FAD, while there was little stimulation with FMN. The increase in
activity of purified enzyme fractions by FAD addition ranged from 1.3
to 5.1 fold. FAD was incorporated as a standard component of the assay
mixture to maximize the observed rates.

Inhibition of palmitoyl-CoA oxidation by the flavin analog atebrin
(quinacrine) is shown in Figure 21A. Atebrin inhibition was not
reduced by the addition of FAD (Figure 218). An unexplained phenomenon
was observed when B-oxidation was inhibited by atebrin in the presence
of increasing amount of FAD. Instead of recovery of activity or no
effect, greater inhibition was observed. At 500 uM FAD in the presence
of'l nM atebrin, all activity was lost. The same amount of FAD in the
absence of inhibitor had the same stimulatory effect as 25 pH FAD. A
similar effect was observed when FMN was added to the standard assay
containing FAD. An inhibition occurred that was not present when
either flavin was analyzed alone.

The initial oxidative step in peroxisomal B-oxidation is a FAD
mediated reaction in yeast and rat liver (142,157). The flavin
cofactor has not been identified in the castor bean seed where this
flavin mediated step was originally proposed (26). It was deemed
desirable to identify the flavin component present in the semi-purified
castor bean B-oxidation system. Purification of the B-oxidation system

through the hydroxyapatite step was carried out in the dark to minimize

100

Figure 20. Flavin stimulation of B-oxidation activity. FMN and FAD

were purified to homogeneity as described in Methods.

lOl

 

l.5 -

 

 

 

 

 

 

 

 

FAD J; . a
C

"" I.O
E
.E
a 4
5
3 0.5-
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1

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O 20 46' 6O IZO

FLAVIN (yM)

Figure 21.

102

Inhibition of B-oxidation activity by the flavin analog,
atebrin. Atebrin was incubated with the B-oxidation
enzymes with and without added FAD for five minutes prior
to initiation of activity with palmitoyl-CoA. Both the
effect of atebrin on B-oxidation (A) and the effect of

flavin 0n atebrin inhibition (B) were studied.

103

 

    
 

 

 

 

 

 

 

 

 

 

 

 

0.6
. +25 pM FAD
0.4-
+
c ‘5
'5 0.2 -
. W
a .
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2 DEV-M 4.—
2 1‘
'25
E
1 0.4
0‘ it 1 l I"
o 50 I00 150 ' 500

FAD (pM)

 

104

flavin photo-oxidation. The system was extracted by two techniques as
described in Methods to isolate the flavin components. The extracts
were then chromatographed in eight separate systems and in every case
the flavin component had the Rf of FAD (Table 6). No other flavins
were observed except for traces of lumiflavin. Lumiflavin is the
photodegradation product of flavins stored in an alkaline environment.
Since the enzyme preparation was not shown to be homogeneous, the
absolute source of FAD could not be determined. The FAD was attributed
to the B-oxidation system, in part, since the system was stimulated
only by FAD and every peroxisomal system studied to date either

contains or has been stimulated by FAD.

105

 

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CHAPTER V
SUBSTRATE UTILIZATION
The stoichiometry of the peroxisomal s-oxidation system has been

shown to be the same in rat liver, yeast, Tetrahymena and castor bean

 

(26,67,136,157,74,87,57). For each turn of the cycle one acyl-CoA is
shortened by two carbons yielding one acetyl-CoA, one reduced
nucleotide and one equivalent of hydrogen peroxide. The cycling of
palmitoyl-CoA through the fatty acid oxidizing system has varied from
two to five times (26,87,132,88) resulting in the accumulation of C5
to C12 acyl-CoA intermediates (75,132). This correlates well with
the much lower rates of B-oxidation and acyl-CoA oxidase activity
observed for the shorter chain length substrates (19,67,87,136). Most
of the research on substrate specificity has been done on rat liver
peroxisomes and recently on the isolated enzymes for that e-oxidation
system.

The peroxisomal B-oxidation system from both plant and animals
were believed to be the same, however, an inconsistency was apparent
with the known utilization of fatty acids. The shorter chain length
intermediates in the liver are believed to be transported out of the
peroxisome to be utilized in the cytoplasm or oxidized further by the
mitochondria. In the germinating seed, no mitochondrial fatty acid

oxidation has been observed. Since the fatty acids were totally

106

107

oxidized it has been assumed that the B-oxidation system in the glyoxy-
somes utilized both long and short chain substrates. The kinetics of
the utilization of fatty acids of different chain lengths by the castor
bean glyoxysome were studied to explain the inconsistency.

Oxidation of Palmitoyl-CoA

 

Palmitoyl-00A (hexadecanoyl-CoA) can theoretically cycle seven
times through the peroxisomal B-oxidation pathway, producing 8 acetyl-
CoA, 7 NADH and 7 H202. The number of cycles (expressed as the
ratio of NADH produced to the amount of substrate added) has been
reported to be between two and five for palmitoyl-CoA, corresponding to
the production of dodecanoyl-CoA'and hexaonyl-CoA (87,88,26,132). The
results of palmitoyl-CoA oxidation by the isolated castor bean B-oxida-
tion system under saturating NAD and CoA conditions is shown in Figure
22. The amount of nucleotide reduced was proportional to the substrate
added. Palmitoyl-CoA cycled 7.3 times through the s-oxidation system
in the presence of CoA. This value is expected for the complete oxida-
tion of palmitoyl-CoA, and supports the proposition that the glyoxy-
somal system from castor bean has the ability to completely oxidize
fatty acids. The use of organelle extracts resulted in fewer observed
cycles, because of the unexplained palmitoyl-CoA dependent NADH oxida-
tion. Lower enzyme levels resulted in a stoichiometric yield similar
to that observed in rat liver, but the exact cause was not determined.
The number of cycles observed was 1.09 in the absence of CoA, repre-
senting the oxidation of palmitoyl-CoA only to its B-keto
intermediate.

The degree of oxidation of other acyl-CoA substrates was also
studied. The values in Table 7 represent the percentage of the total

oxidation (the ratio of the total NADH produced to the theoretical

Figure 22.

108

Palmitoyl-CoA oxidation. Total NADH produced from varying
amounts of palmitoyl-CoA were determined with saturating
NAD and CoA concentrations. High enzyme activities of
0.43 units per assay were used of preparations after
hydroxyapatite chromatography. Assays were run in both
the presence (cyclic oxidation) and absence (noncyclic

oxidation) of CoA.

109

 

200

NADH (nmol)
o
o

50

 

 

IO 20
PALMITOYL-CoA (nmol)

 

110

Table 7. Stoichiometry of acyl-CoA oxidation. Various acyl-CoA
substrates were oxidized as in Figure 22. The percentage
oxidation represents the ratio of the total NADH produced to
the theoretical value for the complete oxidation of the given
substrate (x100). Accuracy was limited by the technical
character of the assay and the accuracy of the determination
of substrate concentration.

 

Percent Oxidation

 

Substrate 1 standard deviation
Palmitoyl-CoA 104 1 11
Butyryl-CoA 122 1 9
Octanoyl-CoA 91 1 3
Tridecanoyl-CoA 116 1 9
Stearoyl-CoA 85 1 4
Oleoyl-CoA 84 1 7
Arachondenoyl-CoA ' 97 1 2
Petroselinoyl-CoA 87 1 15

Ricineoyl-CoA 49 1 2

 

111

yield for the complete oxidation of the given substrate, times 100) of
the fatty acid by the B-oxidation system after purification through
either the ammonium sulfate fractionation or hydroxyapatite
chromatography. Saturated fatty acids were completely oxidized within
the error of the assay and of the determination of substrate
concentration. The unsaturated fatty acids were oxidized to varying
degrees which corresponded with the known enzyme complement of the
preparations. Substrates requiring the auxillary isomerizing and
epimerizing enzymes should exhibit in their absence reduced levels of
oxidation. The degree of oxidation is a constant and is dependent on
the location of the B-oxidation block. The values should be 37.5% for
oleoyl-CoA and ricinoleoyl-CoA and 11.1% for arachidonoyl-CoA in the
absence of cis-3,trans-2-enoyl-C0A isomerase, while petroselinoyl-CoA
would have a value of 25% and arachidonoyl-CoA a value of 33.3% in the
absence of D,L-3-hydroxyacyl-COA epimerase. These values were observed
only a few times with old enzyme preparations and in preparations
purified through the carboxymethyl Sephadex step. Values more
consistent with complete oxidation were obtained (Table 7) with fresher
preparations. This indicated that these two activities nay be
components of the e-oxidation system as is found in E; 9011 (148).

The hydroxyl group at carbon twelve of ricinoleic acid was found
to be a barrier to the B-oxidation systems in yeast (125), rat liver
(177), pea (64), and §;_ggli (126). Ricinoloeyl-CoA was not oxidized
to completion by any of the enzyme preparation from castor bean.
Isolated glyoxysomal organelle suspensions were also unable to oxidize
completely ricinoloeyl-CoA. Hutton and Stumpf (64) had reported that

an a-oxidation system that circumvents this metabolic block was

112

associated with the castor bean glyoxysome. Since the observed
oxidation of ricinoloeyl-CoA was 49% (Table 7) which correlated well to
the formation of 4-hydroxydecanoyl-COA as was reported to accumulate in
germinating pea seeds (64), it was assumed no a-oxidation had taken
place. This could have been due to assay conditions, to the lack of
required cofactors or to previous treatments that could have
inactivated the a-oxidation system.

Km Values

The kinetic parameters of fatty acid degradation were determined
by measuring the complete B-oxidation system rather than the individual
enzymes. The Kms are subsequently reported as the apparent rather than
the actual values.

The apparent Kd for FAD was estimated from the FAD stimulation
curve (Figure 20) to be 0.15 uM. The flavin content of the enzyme
preparation was small relative to the amount of flavin added and the
effect of the endogenous FAD on the estimation of the Kd was believed
to be minimal. The Kd observed for the castor bean system was
similar to the value reported (0.6 uM) for the rat liver acyl-CoA
oxidase (69).

The apparent Km for oxygen was 72 uM and for NAD the value was 79
uM (Figures 23 and 24). The reported Kms for oxygen (5 uM) (142) and
NAD (20 pH) (141) for the purified rat liver enzymes were lower than
those found for the glyoxysomal e-oxidation system. The Km for CoA
measured for rat liver peroxisomal thiolase (65 uM) (unpublished data,
Jeff Krahling) was 15 times greater than the estimated apparent Km (4
uM) observed for the castor bean B-oxidation system (Figure 25). It is

not known whether these differences in Kms were due to the tissue

113

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115

Reaction velocity of B-oxidation as a function of NAD
concentration. NADH formation was measured
spectrophotometrically as described in Methods. NAD
concentration was determined by absorption at 259 nm
(178). Data is shown with its standard deviation and the

double reciprocal plot is in the insert.

116

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119

source, the means of analysis, or to the fact that apparent Kms rather
than the actual Kms were measured.

Various determinations of the apparent Km for palmitoyl-CoA have
ranged from 1.6 to 9.4 pM. Similar Km values have been reported for
rat liver peroxisomal B-oxidation (8.6 uM) (170) and for its isolated
acyl-CoA oxidase (11.6 0M) (142). The reaction velocity as a function
of palmitoyl-CoA revealed severe substrate inhibition (Figure 18 and
26). This inhibition was found to be inversely related to the chain
length of the acyl-CoA substrate. No inhibition was observed with 84
uM octanoyl-CoA or 180 uM butyryl-CoA, but the inhibition became
increasingly apparent with longer chain length substrates. Various
groups have published substrate concentration curves that have
exhibited this form of inhibition (26,142,141,58). Inhibition is
believed to be caused by micelle formation by the fatty acyl-CoA
substrate. It could also be caused by a direct chain length dependent
inhibition of one or more components of the B-oxidation system.

Substrate Specificity

 

The fatty acid oxidizing system was found to be capable of
oxidizing palmitoyl-CoA completely (Figure 22). The reaction velocity
was observed to diminish after approximately 40% of the theoretical
yield was produced. The reduced rate of NADH formation suggest a lower
specificity for the shorter chain length fatty acids. This reduced
activity with the shorter acyl-CoA substrates was observed by both the
NAD and polargraphic assays (Figure 27). The same general specificity
for long chain fatty acids was observed for the thiolase activity

(Figure 28).

 

120

 

 

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122

Figure 27. Chain length specificity for the B-oxidation sytem from
castor bean glyoxysomes. Acyl-CoA substrates (25 0M) were
used in the polarographic assay and 30 uM substrate

concentrations were used in the NAD reduction assay.

123

 

pmol NADH or Ozlmin/ml

\
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CARBON CHAIN LENGTH

124

Figure 28. Chain length specificity for 3-ketoacyl-C0A thiolase
activity of the isolated B-oxidation system. Assays were
performed as described in Methods except 40 0M substrate
concentrations were used. Hydroxyapatite purified complex

was used.

125

 

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THIOLASE (pmgl/min/ml)
iv

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126

The substrate specificities found with the isolated castor bean
glyoxysomal system were similar to that observed for the peroxisomal
B-oxidation system (67,170,87) and for acyl-00A oxidase (136,157) from
other tissues. These combined data suggest that the peroxisomal system
from castor bean, rat liver or yeast should be capable of total oxida-

tion of acyl-CoA substrates under the proper conditions. The actual

 

extent of oxidation would depend on the presence of other enzymes which
compete for the shorter chain length acyl-CoA intermediates such as the
carnitine octanoyl-CoA and carnitine acyl-CoA transferases of the liver

peroxisome (75,101,100). These enzymes are not present in glyoxysomes

 

par-Ia.

from germinating seeds and the B-oxidation system continues to degrade
the fatty acids to acetyl-CoA. Another competing enzyme could be
acyl-CoA hydrolase (132) but their presence in peroxisomes can not be
confirmed (L. Bieber, J. Krahling, P. Clark, K. Valkner, N. Tolbert,
unpublished).

Great precaution should be maintained when analyzing and comparing
substrate specificities, for many factors can affect the relative rates
of B-oxidation. High concentrations of the longer chain acyl-CoA
substrates can inhibit B-oxidation activity. This inhibition may be
due to micelle formation by the substrate, which is effected by
dependent on the assay conditions (BSA, protein, pH, substrate chain
length). The inhibition will skew the observed substrate preference
toward the shorter chain lengths and is likely the cause of the
difference in the specificities observed by Hryb (59) and Osumi (136)
for the B-oxidation system in the rat liver peroxisome. Another factor
that influences the observed reaction velocity for B-oxidation of

different substrates can be the substrate concentration. The Km for

127

acyl-CoA substrates increases with the decrease in the chain length
(Table 8). At concentrations where the longer chain substrates are not
inhibitory, the concentrations of the shorter chain length fatty acids
are below their Km value. Substrate specificity curves have been
reevaluated for the castor bean s-oxidation system (Figure 29) to show
a major difference between an analysis at a constant substrate concen-
tration and one at maximal concentrations. The B-oxidation system with
sufficient added substrate has considerable activity with the shorter
chain length substrates. Analysis with limiting and excess substrate
presents two means of substrate utilization analysis. The constant
concentration method provides a good indication of utilization rates
for substrates in vivo, while the optimal substrate method shows the
substrate handling capability of the system in question. Neither form
of analysis, in all likelihood, characterized the B-oxidation system in
the microbody in vivo.

The maximal rates of fatty acid oxidation and the affinity of the
system for various acyl-CoA substrates is dependent on the individual
kinetic parameters of the enzymes comprising the B-oxidation system and
how they interrelate as a unit. A greater affinity for the longer
chain substrates was observed for the glyoxysomal system (Table 8), as
was found for acyl-CoA oxidase, enoyl-CoA hydratase and hydroxyacyl-CoA
dehydrogenase from rat liver (47,164). Thiolase also exhibits a pre-
ference for the longer chain substrates (Figure 29) (see also liver
peroxisomes, J. Krahling and N.E. Tolber, in press). Reported Km
values for thiolase are 1.7 pH for 3-ketohexadecanoyl-C0A (142), 21 uM
for 3-ketotetradecanoyl-C0A (Figure 30) and 87 0M for acetoacetyl-CoA
(26). Maximal velocities for the various enzymes did not exhibit the

same chain length profiles as was observed for the substrate.

 

128

Table 8. Km and relative Vmax values for various acyl-CoA substrates.
The assay for NADH formation was used as described in

 

 

Methods.
Substrate Km (uM) relative Vmax (%)
Palmitoyl-CoA 6.3 100
0leoyl-C0A 1.8 95
Stearoyl-CoA 2.6 61
Octanoyl-CoA 43.7 54
Butyryl-CoA 71.1 19
Petroselinoyl-CoA 7.8 65
Ricinoleoyl-CoA 1.6 40

Arachidonoyl-CoA 7.7 66

 

 

Figure 29.

129

Comparison of chain length specificity curves. Chain
length specificity at optimal substrate concentrations and
at a fixed substrate concentration were compared. Curve A
represents the maximal velocities (optimal substrate) for
each saturated fatty acid in Table 8. Curve B is the
specificity curve of Figure 28 (NADH formation)

representing a fixed substrate level (30 pH).

130

 

IOO ‘

1
m
o
f

ACIIVITY (%
OF

N
O
T

 

K

  

 

4

8
CARBON CHAIN LENGTH

I8

 

131

Figure 30. Reaction velocity of thiolase as a function of
B-keto-tetradecanoyl-CoA concentration. Thiolase assays
were carried out as described in Methods. Standard

deviations and double reciprocal plots are shown.

132

 

 

0.2

pmol/min/ml
,<_>

  

 

 

 

 

1 1
.05 .l0
1* .
0 20 40 60

B-KETOTETRADECANOYL-CoA (pM)

 

 

CHAPTER VI
SUMMATION

Systems for the degradation of fatty acids by the B-oxidation
pathway have been found in two different subcellular organelles. The
mitochondrial system is well reviewed by textbooks. It is composed of
enzymes with multiple subunits and has a close association with the
electron transport chain for the utilization of reduced FAD and NAD
formed during B-oxidation. The peroxisomal system, which was dis-
covered in the past decade, is now under detailed analysis. It has
been characterized by the absence of a requirement for carnitine, the
presence of monomeric and dimeric subunit enzymes, the presence of a
dual-function protein for the enoyl-CoA hydratase and 3-hydroxyacyl-COA
dehydrogenase reactions and by the loss of 40% (in systems containing
the malate shuttle, e.g. castor bean glyoxysomes) or 60% (in systems
containing the glycerol phosphate shuttle, e.g. rat liver peroxisomes)
of the reducing potential through the formation of hydrogen peroxide by
acyl-CoA oxidase and the elimination of the hydrogen peroxide by the
organelles complement of catalase.

My initial research was on the isolation and characterization of
the glyoxysomal flavoprotein, acyl-CoA oxidase. During the initial
experiments at isolating this enzyme, it was observed that the B-oxida-
tion system (acyl-CoA oxidase, enoyl-CoA hydratase, 3-hydroxyacyl-CoA

dehydrogenase and thiolase) tended to copurify. Since no interactions

133

 

 

PLEASE NOTE:

Page 134 is missing in
number only as text follows

UNIVERSITY MICROFILMS INTERNATIONAL

 

 

135

of the B-oxidation enzymes had been noted in eukaryotes, the research
goals became the isolation and stabilization the associated system.
The specific activity of the B-oxidation system was increased more than
41 fold (Table 1), but it was impossible to ascertain the homogeneity
of the final preparation by the methods employed.

An association of the B—oxidation enzymes has been shown to exist
I".§;.EQli (39) and has subsequently been proposed for the rat liver
peroxisomal system (58,69). Several observations were made that indi-

cated the existence of an associated system or complex of the B-oxida-

tion enzymes in castor bean glyoxysomes. The first observation was the

 

In Max-‘31

tendency of the enzymes to copurify through the various steps employed.
Another was the presence of a shoulder of activity that was consistent-
ly observed during hydroxyapatite chromatography (Figure 3A). The
shoulder varied in size and contained acyl-CoA oxidase, enoyl-CoA
hydratase and 3-hydroxyacyl-C0A dehydrogenase. These three activities
had separated from the complete B-oxidation system suggesting a change
in chromatographic behavior in the absence of thiolase or the coinci-
dental elution of a small population of the enzymes at a lower phos-
phate concentration. Third, the retention of the e-oxidation system on
carboxymethyl Sephadex (Figure 3B) and on various sieving columns under
different conditions is hard to explain without proposing the existence
of a complex.

The known molecular weights of the isolated enzymes from other
tissues indicated that a separation of the proteins should have
occurred on the sieving columns, resulting in the loss of B-oxidation
activity. Recovery of B-oxidation activity from these columns was
generally greater than 50%. The normal migration of standards ruled

out anomalies due to the column.

136

Polyacrylamide gel electrophoresis exhibited no prominent protein
bands even at high protein applications (200 pg). The proteins tended
to smear on the gels under all conditions employed, suggesting
protein-protein interactions. Standards showed no unusual patterns on
the gels even in combination with the B-oxidation enzymes. Glycerol,
which was observed to stabilize the §;_goli B-oxidation complex on
polyacrylamide gels (39), had no effect on the stability of the system
from castor bean glyoxysomes.

The sedimentation of the B-oxidation system on sucrose gradients

produced a single sharp peak corresponding to a molecular weight of

 

(am-1x _

140,000 and a sedimentation coefficient of 7.2 5 (Figure 4). The known
differences in the molecular weight for the individual enzymes from
other tissues would have again predicted a separation of the proteins,
if they did not associate as a complex, resulting in the subsequent
loss of B-oxidation activity. The molecular weight of 140,000 measured
on the sucrose gradient is far too low for an associated system of the
B-oxidation enzymes. The neutral lipid found in the enzyme
preparations could have been the cause of the low estimated molecular
weight if it were associated with the complex. The lipid would have
change the partial specific volume of the complex, reducing the
sedimentation of the B-oxidation system on the sucrose gradient.
Finally, the concentration of dilute solutions of the B-oxidation
system using Amicon Diaflow XM-lOOA ultrafiltration membrane always
resulted in complete recovery of activity. The membrane is rated for
greater than 90% retention of globular proteins of molecular weights of
more than 100,000. Both thiolase and enoyl-CoA hydratase/3-hydroxy-

acyl-CoA dehydrogenase from cucumber (43) and rat liver (141) have

molecular weights below this value and should have passed through the
membrane if they were not part of a complex.

These observations do not prove the existence of a complex, but as
a whole, provide good evidence for it. Further work on the nature of
the complex from the castor bean must be done as well as examining the
possible existence of a similar association in rat liver peroxisomes.

The complex isolated from the castor bean glyoxysomes consisted of
the fatty acid oxidizing cycle enzymes and possibly

cis-3,trans-2—enoyl-C0A isomerase and D,L-3-hydroxyacyl-CoA epimerase.

4.0-15. r.—
I

The exact complement of the enzymes was hard to determine since

 

differential inactivation and dissociation during the purification
process could account for the variations in the degree of purification
observed (Table 2). All five enzyme activities were detected in
variously purified preparations suggesting an system analogous to that
found in §;_ggli. Similar differential losses of enzyme activities
were also reported by Binstock (39) and O'Brien (124) with the §;_gglj_
B-oxidation complex.

The fatty acid oxidizing system was found to be localized
exclusively within the glyoxysomes of castor bean endosperm (Figure 2)
with no activity in the mitochondria. The same observation has been
reported by Cooper and Beevers (26). The pH optimum (Figure 12) was
characteristic of the other enzymes, particularly the oxidases, found
in this organelle (175). The activity proved to be very unstable with
a substantial loss occurring in a day (Figure 5 and 6). High concen-

trations of glycerol and the presence of dithiothreitol were used to

maintain activity (Figures 7 and 10, Table 3). The effect of these

138

compounds was extensively studied and from the data and recent litera-
ture, the effects of glycerol and dithiothreitol appeared to be multi-
faceted. Glycerol stabilized the overall s-oxidation activity of the
complex and reduced the loss of specific activity observed at low pro-
tein concentrations (Figure 13). The main component of the B-oxidation
system stabilized by glycerol was acyl-CoA oxidase (Table 3). The
mechanism of stabilization by glycerol way be through the direct inter-
action of the polyhydric alcohol with the proteins or by the modifica-
tion of the structure of the hydration sphere and the alteration of the
proteins electrostatic and hydrophobic character (155,186,72). Dithio-
threitol predominantly affected 3-ketoacyl-C0A thiolase (Table 3).

Kinetic analysis of the substrates and cofactors of the B-oxida-
tion complex provided apparent Kms for oxygen (72 uM), NAD (79 0M), CoA
(approximately 4 pM), palmitoyl-CoA (6 0M) and FAD (0.15 uM). Acetyl--
CoA and NADH exhibited no product inhibition. The longer chain
acyl-CoA substrates severely inhibited B-oxidation concentrations near
the level needed for maximal activity (Figure 26). This inhibition was
attributed to micelle formation by the substrate. It could also be due
to detergent effects of the alkyl side chain. Detergents were found to
be inhibitory. This inhibition could have been due to a direct disso-
ciation of the B-oxidation system by disruption of protein-protein or
protein-lipid associations or by the sequestering of the fatty acyl-CoA
substrate. Detergent inhibition has been reported for e-oxidation in
rat liver peroxisomes (170).

The e-oxidation complex was specific for NAD and would not use nor
was it inhibited by NADP. FAD has been found to be the flavin cofactor

of acyl-CoA oxidase in rat liver and yeast (157,142). In the castor

 

 

139

bean, FAD could not be replaced by FMN (Figure 20) and FAD was the only
flavin found in active B-oxidation samples (Table 6).

Several long chain saturated and unsaturated fatty acids were
found to be completely degraded by the B-oxidation complex (Table 7).
The activities of D,L-3-hydroxyacyl-C0A epimerase and cis-3,trans-2--
enoyl-CoA isomerase were both found in the preparations of the glyoxy-
somal B-oxidation complex and would account for the glyoxysomes ability
to oxidize unsaturated fatty acids. The system described by Hutton and
Stumpf (64) to eliminate the hydroxyl group of ricinoleic acid could
not be detected. The ability to completely oxidize fatty acids
accounts for the lack of the build up of intermediates and the effi-
cient conversion of fatty acids to carbohydrates via the glyoxylate and
gluconeogenic pathways in germinating fatty acids. In comparison, the
peroxisomal system in rat liver degrades fatty acyl-CoA substrates only
to octanoyl-CoA and dodecanoyl-CoA. These intermediates are believed
to be oxidized further by the mitochondrial B-oxidation system.

The chain length specificity of the rat liver system indicates a
lower reaction velocity for the shorter chain acyl-CoAs as compared to
the longer chain acyl-CoAs. A uniform specificity was expected for the
plant system since palmitoyl-CoA was observed to be completely oxidized
and a quantitative conversion of fats to sugars had been reported (9).
The chain length specificity for the castor bean system (Figure 27) was
found to be very similar to the rat liver system. The lower affinity
for the shorter chain length fatty acyl-CoA substrates was the probable
cause for their lower rate of utilization. The rat liver peroxisomal

B-oxidation system should be capable of the total oxidation of fatty

140

acids under the proper conditions based on the similarities of the rat
liver and the castor bean systems.

The major difference in the glyoxysomal and peroxisomal B—oxida-
tion systems is the utilization of the shorter chain acyl-CoA sub-
strates. Both acetyl-CoA and the medium chain acyl-CoAs in liver
peroxisomes can be converted to their carnitine derivatives by the
peroxisomes complement of carnitine acetyl-CoA transferase and
carnitine octanoyl-CoA transferase. The acyl-carnitines diffuse out of
the organelle to be utilized further by the cytoplasm and the mitochon-
dria. The medium chain fatty acyl-CoA intermediates in the microbodies
of yeast and germinating fatty seeds are degraded completely to
acetyl-CoA because of the lack of the competing carnitine octanoyl-CoA
transferase.

Acetyl-CoA, in seeds and yeast, is utilized by the glyoxysomal
glyoxylate pathway. The castor bean glyoxysome contains the complete
glyoxylate pathway, and has been found devoid of carnitine acyl-CoA
transferases. Yeast contain the peroxisomal carnitine acetyl-CoA
transferase (75), believed due to the compartmentalization of part of
the glyoxylate pathway in the mitochondria. Since no B-oxidation has
been observed in the yeast mitochondria (74) the transferase could
provide a shuttle for acetate units to the mitochondria for utilization
by the tricarboxylic acid cycle and for synthesis of isocitrate needed
by the glyoxylate pathway in the microbody.

The results of this study on the s-oxidation system from the
germinating castor bean seed indicates that: the B-oxidation system in
microbodies seems to be very similar regardless of the tissue source,

the utilization of the products by this system is dependent on the

.L‘Jllr ' hr “.1

 

141

metabolic role of the tissue, and the level of degradation of fatty
acids seems to be more a function of competing pathways for the medium

chain intermediates than the system's ability to degrade them.

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