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This is to certify that the

thesis entitled

 

Microsomal mixed—function oxidases of the house
fly, Musca domestica (L): Endogenous factors
affecting their isolation and stability

 

presented by

Daune L. Crankshaw

has been accepted towards fulfillment
of the requirements for

PhD degree in Entomology

60‘

Major rofessor

' Date Ma 14 1975

0-7639

 

 

 

 

 

ABSTRACT

MICROSOMAL MIXED—FUNCTION OXIDASES
OF THE HOUSE FLY, MUSCA DOMESTICA (L): ENDOGENOUS
FACTORS AFFECTING THEIR ISOLATION AND STABILITY

 

By

Daune L. Crankshaw

The mixed-function oxidase NADPH-cytochrome c reductase has been
shown to be tightly bound to the mammalian microsomal memgrane. House
fly microsomal NADPH—cytochrome c reductase activity was extensively
solubilized during the isolation of the microsomal fraction from mortar-
and-pestle homogenates of whole house flies. In 23 isolations, only
20 i 24.5% 5.0. of the microsomal and soluble reductase activity was
recovered in the pellet. Phenylmethylsulfonylfluoride, a protease
inhibitor, and EDTA did not decrease the observed solubilization. The
specific activity of the reductase was not significantly altered by these
treatments and was 0.025 i 0.008 pMOIES 8.0. of cytochrome c reduced/min/
mg protein.

Microsomal fractions isolated from 30 abdominal homogenates prepared
by glass-Teflon homogenization contained 80.3 i 21.3% 8.0. of the micro—
somal and soluble reductase activity recovered in the 105,000 xg pellet.
The specific activity of the reductase for these isolations was 0.050 t
0.025 moles 3.0. of cytochrome c reduced/min/mg protein. The above dif-

ferences obtained with the two methods of homogenization were significant

Daune L. Crankshaw

at the 0.0l and 0.05 level for the percentage of the reductase isolated
in the microsomal fraction and its specific activity, respectively, when
subjected to the Student's t-test. The aldrin epoxidase activities of
the homogenates and microsomal fractions from glass-Teflon preparations
were unstable. The homogenates lost 68% of their initial epoxidase ac-
tivity in 30 minutes and the frozen microsomal fractions lost 50 to 60%
of their activity within 24 hours.

Since the phenol oxidase complex has been implicated in the loss of
aldrin epoxidase activity when whole southern armyworm homogenates were
used, the effect of this system on rat liver microsomal mixed-function
oxidases was examined. Phenol oxidase and its substrates are present
in house fly homogenates and microsomal fractions, but have not been de-
tected in rat liver microsomes. The incubation of rat liver phenobarbital-
induced microsomes with tyrosinase and catechol at 4° C caused a signifi-
cant decrease of cytochrome P-450 content, aldrin epoxidation, and NADPH—
cytochrome c reductase activity. When rat liver microsomes were incubated
with either cyanide-inhibited tyrosinase and catechol, tyrosinase alone,
or catechol alone, the same enzymatic activity and cytochrome P-450 con-
tent as the untreated control was observed. Erythrocyte lysis,used as
an indicator of the occurrence of free radical reactions, occurred when
red blood cells were incubated with tyrosinase and catechol at 4° C. There
was no lysis when they were incubated in the presence of tyrosinase alone
or catechol alone. Sodium dodecyl sulfate polyacrylamide gel electrophor-
esis of rat liver phenobarbital-induced microsomes incubated with tyrosin-
ase and catechol indicated that cross-linkage of the proteins had occurred,
since much of the sample failed to migrate into the gel. When a sample

of microsomes incubated with cyanide-inhibited tyrosinase was electrophoresed,

 

Daune L. Crankshaw

it gave the same banding pattern as the untreated control.

The addition of catechol, a tyrosinase substrate, to the 10,000 xg
supernatant of house fly homogenate gave a microsomal fraction without
detectable cytochrome P-450 or P—420. Addition of cyanide to house fly
homogenate significantly increased the cytochrome P-450 and aldrin epoxi—
dase activity of the microsomal fraction. The aldrin epoxidation activity
of the cyanide—treated microsomal preparation decreased 22% in 24 hours
compared to 56% for the catechol—treated and untreated microsomal frac-
tions.

The glass—Teflon homogenization method is superior to the mortar—and—
pestle method for isolating house fly microsomes because of the extensive
solubilization of the NADPH—cytochrome c reductase that occurred with
mortar-and-pestle preparations. Microsomal aldrin epoxidation activity
in house fly preparations obtained by glass-Teflon homogenization was
unstable. The effect of tyrosinase and catechol on the rat liver micro-
somal mixed-function oxidases and of catechol and cyanide on house fly
microsomal preparations indicate that the catechol oxidase complex may
be an important factor involved in the instability of house fly micro—
somal mixed-function oxidases. The use of inhibitors and polymers to
eliminate the effect of this complex should be a key factor in obtaining

stable microsomal mixed-function oxidases from house flies.

 

MICROSOMAL MIXED—FUNCTION OXIDASES
OF THE HOUSE FLY, MUSCA DOMESTICA (L): ENDOGENOUS
FACTORS AFFECTING THEIR ISOLATION AND STABILITY

By

\L‘ ‘

Daune L? Crankshaw

A DISSERTATION

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

DOCTOR OF PHILOSOPHY
Department of Entomology

T975

 

To my wife, Jacqueline
and our daughters,

Carolyn and Nadia

 

ACKNOWLEDGEMENTS

Looking back over the time that I have spent at MSU, there are many
people who have helped me during my doctoral program and deserve special
thanks. Dr. Norman Leeling advised me during the first part of my re-
search and also provided financial support for which I am grateful. My
major advisor, Dr. Matthew Zabik, generously supported the latter part
of my research. A special thanks goes to Dr. Stephen Aust, Biochemistry
Department, for tolerating a “super fly” buzzing about his lab. His
advice and encouragement helped me through the difficult and sometimes
discouraging parts of my research. My association with the members of
Dr. Aust's laboratory was a stimulating and helpful experience. The
members of my advisory committee, Drs. A.w.A. Brown, Roger Hoopingarner
and Robert Cook gave freely of their time and I appreciate their advice
and help. Drs. Gordon Guyer and James Bath, the former and present
chairmen, respectively, of the Department of Entomology, provided funds
for assistantships and research for which I am grateful.

Research that utilizes house flies as the experimental animal gen—
erates a great deal of routine work in maintaining the colony and pre—
paring them for use in experiments. The cheerful and conscientious
assistance in such work of Mr. David Cushman, Miss Joella Herwaldt,

Mrs. Belinda Giessel, Miss Barbara Goelling, Beth Hockman, Carolyn
Crankshaw and Judy Herwaldt is deeply appreciated. Mrs. Janise Ehmann
earned a special thanks by being a good critic while this dissertation

iii

was being written. The invaluable help of the Pesticide Research Center
secretaries, Mrs. Rose Magistro and Mrs. Phillis Stasik is deeply appre-
ciated. Mrs. Jane Fortman deserves a special thanks for donating her
expertise in the typing of the final copy of this dissertation. Without
the love and support of my wife and daughters the completion of this
degree would not have been possible and I appreciate their encouragement
very much. These individuals and many others have become our friends,
given generously of their time, and made our stay in East Lansing a

richly rewarding one.

iv

 

TABLE OF CONTENTS

Page
INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
LITERATURE REVIEW . . . . . . . . . . . . . . . . . . . . . . . . 4
Components of the Microsomal Mixed-Function Oxidase System 5
Induction of the Microsomal Mixed-Function Oxidases . . . . . . 7
Fractionation of Tissue Homogenates . . . . . . . . . . . 8
Effect of Isolation Procedure . . . . . . . . 9
Endogenous Inhibitors of Insect In Vitro Preparations . . . . . ll
Biochemistry of Catechol Oxidase— . . . . . . . . . . . . l5
Phenols and Quinones in Biological Systems . . . . . . . . . . l8
The Effect of Free Radicals in Biological Systems . . . . . . . 20
MATERIALS AND METHODS . . . . . . . . . . . . . . . . . . . . . . 23
The House Fly Strain and its Culture . . 23
Isolation of the Microsomal Fraction from House Fly Homogenates 25
Distribution Studies of Marker Enzymes in MP Homogenates . . . 25
Sucrose Density Gradient Centrifugation . . . . . . . . . 26
Isolation of the Microsomal Fraction from Abdominal
GT Homogenates . . . . . . . . . . . . . . 27
Enzyme Assays and Solution Preparation . . . . . . . . . . 28
Assays of NADPH- Cytochrome c Reductase Activity . . . . . . . . 28
Assays of Cytochrome Oxidase Activity . ..... . . . . . . . 28
Microsomal Pesticide Metabolism Assay . . . . . . . . . . . . . 28
Extraction of Pesticides . . . . . . . . . . . . . . . . 29
Gas Liquid Chromatographic Analysis . . . . . . . . . . . . . . 29
Microsomal Cytochrome P- 450 Analysis . . . . . . . . . . . . . 3O
Assay of Tyrosinase Activity . . . 30
Treatment of House Fly Homogenates with Catechol and Cyanide . 31
Incubation of Rat Liver Microsomes with Tyrosinase . . . . . . 3l
Erythrocyte Hemolysis . . . . . . . . . . . . . . . . . . . . . 32
Other Methods . . . . . . . . . . . . . . . . . . . . . . . . 32
RESULTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33
Marker Enzyme Distribution Patterns of Mortar-and-Pestle
Homogenates . . . . 33
Density Gradient Centrifugation of Mortar— Pestle Homogenates . 40
Inhibition of Enzyme Solubilization in Mortar Homogenates . . . 42
Glass- Teflon Homogenization and Solubilization . 48

Effect of Endogenous Tyrosinase on House Fly Microsomal Enzymes 56
Effect of Mushroom Tyrosinase on Rat Liver Microsomal Enzymes . 57

V

 

DISCUSSION . . . . . .
SUMMARY .......
BIBLIOGRAPHY .....

............
............

............

vi

........

 

Page

7]
88
91

Table

 

LIST OF TABLES

Comparison of NADPH-cytochrome c reductase activity
in microsomal fractions from mortar-pestle
homogenates of abdomens and whole flies . . . .

NADPH-cytochrome c reductase activity in microsomal
fractions from whole house fly mortar-pestle
homogenates of the larval and adult stages .....

The distribution of NADPH-cytochrome c reductase
activity following sucrose density gradient
centrifugation of the 800 xg supernatant

of MP homogenate ...................

The effect of isolation conditions on the percent
of NADPH-cytochrome c reductase activity found

in the microsomal fraction from house fly homogenates . .

The effect of isolation conditions on the specific
activity of NADPH-cytochrome c reductase in
the microsomal fraction from house fly homogenates

Individual isolation of the microsomal fraction
from MP homogenates of whole house flies. The
effect of PMSF, DTT, and EDTA on microsomal
NADPH-cytochrome c reductase activity .....

The effect of the homogenization method on the
percent of the microsomal and soluble
NADPH-cytochrome c reductase activity detected
in the microsomal fraction .........

The effect of the homogenization method used to prepare
house fly homogenates on the specific activity
of NADPH-cytochrome c reductase in the
microsomal fraction ..........

The effect of the protease inhibitor, DFP, on
the NADPH-cytochrome c reductase activity in the
microsomal fractipn of house flies using
the glass-Teflon homogenization method ......

Page

34

35

43

44

45

46

49

50

52

Table

10. Mixed-function oxidase activity in microsomal
fractions isolated from house fly homogenates
treated with either catechol or cyanide .

ll. Effect of tyrosinase on cytochrome P-450 and

enzymatic functions of liver microsomes from
phenobarbital—pretreated rats . . . . .

viii

Page

55

64

LIST OF FIGURES

Figure

l. The mixed-function oxidase electron transport system
2. Reactions of tyrosinase complex . .

3. Distribution of cytochrome oxidase in subcellular

fractions of rat liver and whole house fly homogenates .

4. Distribution of marker enzymes in subcellular

fractions of rat liver and whole house fly homogenates .

5. The distribution of NADPH-cytochrome c reductase
activity and protein profile following sucrose
density gradient centrifugation of the 800 xg
supernatant of MP homogenate .

6. Effect of homogenization method on the percent of
NADPH-cytochrome c reductase activity found in the
microsomal fraction of house fly homogenates . .

7. The stability of aldrin epoxidase activity in
glass-Teflon homogenates of house fly abdomens .

8. The effect of tyrosinase concentration on NADPH-
cytochrome c reductase activity of rat liver
microsomes . . . . . . . . . . . . . . .

9. The effect of tyrosinase plus catechol on rat liver
microsomal NADPH— —cytochrome c reductase activity:
effect of tyrosinase concentration . . . . . .

TO. The effect of tyrosinase plus catechol on rat liver
microsomal NADPH— ~cytochrome c reductase activity:
rate of inhibition . . . . . . . . . . . .

ll. Effect of mushroom tyrosinase on cytochrome P—450 of
liver microsomes from phenobarbital—pretreated rats

l2. The effect of tyrosinase and catechol treatment on the
SDS- polyacrylamide gel electrophoresis protein profile
of the liver microsomal fraction from PB- pretreated
rats . . . . . . . . . . . . . .

Page

36

38

4T

5T

54

58

6O

62

67

69

INTRODUCTION

The role played by mixed-function oxidases in the detoxification
of pesticides has become apparent only in the last decade. Insect de-
toxification systems are of special interest to man because they enable
pest species to circumvent control measures and continue to compete with
man for a limited food supply. The importance of microsomal mixed-
function oxidases in pesticide detoxification is still being defined as
more basic knowledge of this system accumulates.

Insecticide resistance is a complex phenomenon with factors, both
enzymatic and non-enzymatic, contributing to the overall resistance of
a species. Genetic analysis of susceptible and resistant insect strains
has helped to characterize the factors involved. In_vitrg_experiments
employed to elucidate specific enzymatic detoxification mechanisms uti-
lize subcellular fractions prepared from homogenates of insects. In
addition, comparative studies using enzyme preparations from insect and
mammalian sources can detect similarities and differences in the mech-
anisms utilized. Information of this nature aids in the design and
deveTOpment of pesticides that are less toxic to non-target organisms.

The importance of developing less toxic compounds lies not only in
the fact that the use of pesticides has been growing nearly l0% a year,
but in that some of the newer compounds being utilized are considerably
more toxic to humans. With the recent banning of several persistent

pesticides, such as DDT, there has been an increase in the use of the

less persistent organophosphorus compounds. These, although environ-
mentally less persistent, are general biocides with some of the most
widely used ones, such as parathion and azinphosmethyl, being quite
toxic to man and other vertebrates.

The occurrence of resistance has been detected throughout the world
and has appeared in most pest species of insects and acarines. Resistance
has been demonstrated for each category of pesticides used to control
these species. The recent development of insect hormone mimics as con-
trol agents was stimulated by the ubiquitous occurrence of insecticide
resistance. It was hoped that the utilization of compounds which mimicked
the actions of naturally occurring growth regulators would circumvent the
problem of resistance. Such compounds would also be quite selective for
the target organism. Research in this field has focused on the growth
regulator juvenile hormone and its mimics. Evidence is beginning to ac-
cumulate that insecticide resistant species show cross-tolerance to juve—
nile hormone and its analogues. There are also reports of non-insecticide
resistant strains developing tolerance to these compounds. The exact role
that microsomal mixed—function oxidases play in the metabolism of juvenile
hormone mimics is still unclear. The problem is complicated by the fact
that esterases also have the capacity of deactivating these chemicals.

Due to the lack of a standardized method for the preparation of in-
sect subcellular fractions to be used for in_vitrg_studies, greater dif-
ficulties are encountered in trying to understand and interpret the impor—
tance of each detoxification mechanism. The great number of problems en-
countered in preparing enzymatically active fractions from insect homogen—
ates, indicates that an optimum method of preparing these subcellular

fractions is a prerequisite for the most accurate picture of the mechanism

involved. The Optimum isolation procedure devised for each insect must
take into consideration endogenous inhibitors and/or other unresolved
factor(s) known to cause enzyme instability of ifl_vitro_preparations.

The majority of these have yet to be completely identified and controlled
in homogenates of insect tissue. The standard methods of analysis being
used in related mammalian systems that are currently yielding the most
significant information about microsomal enzymes demand stable prepara-
tions. Since insect homogenates lack this stability, the resolution of
this problem is the major challenge facing researchers utilizing insect

microsomal preparations.

LITERATURE REVIEW

The endoplasmic reticulum (ER) of certain tissues, such as liver of
mammals and fat body of insects, contain enzymes that metabolize fatty
acids, steroids and numerous xenobiotics (l). The homogenization of
these tissues disrupts the ER, resulting in the formation of small mem-
branous vesicles which can be isolated from other subcellular components,
eLg;_mitochondria, by differential centrifugation of the homogenate. The
pellet of membranous vesicles obtained by high speed centrifugation of
the microchondrial supernatant is called the microsomal fraction (2).

This fraction contains two electron transport systems. One, com—
posed of NADH-cytochrome b5 reductase and cytochrome b5, is involved in
fatty acid metabolism. The other is a mixed-function oxidase system
which is involved in the detoxification of drugs and pesticides. This
system is the one investigated in the research presented in this disser-
tation. Its composition, as currently understood, consists of NADPH-
cytochrome c reductase and cytochrome P-450. This enzyme system requires
atmospheric oxygen and reducing equivalents from NADPH. The electrons
and oxygen are utilized in the hydroxylation of a wide variety of sub—
strates. The mixed-function oxidases of mammals have been reviewed by
Mason (l,3,4), Gillette (5,6), Orrenius gt_al, (7) and Siekevitz (8),
and of insect systems by Terriere (9), Casida (10), Perry and Agosin (ll),
and Wilkinson and Brattsten (l2). The role of cytochrome P-450 in insecti-

cide resistance (l3) and the induction of insect mixed-function oxidases by

pesticides has also been assessed in recent years (14).

Components of the Microsomal Mixed-Function Oxidase System:

 

The mixed-function oxidase components are diagrammatically represen-
ted in Fig. l (l5). The flavoprotein, NADPH-cytochrome c reductase
(often referred to as the reductase) accepts electrons from NADPH and
reduces the cytochrome P—450. The exact mechanism of electron transfer
is not completely understood. In studies with the purified enzyme, one
electron at a time was transferred (16), however, the transfer mechanism
for the second electron required per mole of substrate hydroxylated is
still to be precisely identified, and the question mark in Fig. l indi-
cates this area of uncertainty. The reductase is tightly bound to the
microsomal membrane and is used as a marker enzyme for the microsomal
fraction (l7). In_yitrg_assays for this enzyme employ electron acceptors
such as cytochrome c and certain dyes.

Klingengberg (l8) and Garfinkel (19) first described a unique cyto-
chrome that appeared in difference spectra of the microsomal fraction of
liver homogenates when it was flushed with carbon monoxide (CO) and re-
duced. Its unique spectrum shows only one, sharp maximum absorbance peak
at 450 nm. Omura and Sato (20) have further characterized this cytochrome
as a hemoprotein with several unusual properties; one property being the
conversion of its typical P-450 form to a spectrally distinct form with
its maxima at 420 nm by several reagents including deoxycholate (21,22).
Conversion of cytochrome P—450 to P—420 was paralleled by the loss of
enzymatic activity with the solubilized P-420 being extremely labile when
reduced in the presence of oxygen. The biological function of this cyto-

chrome was suggested by such observations as the inhibition of the steroid

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reversal of this inhibition by monochromatic light of 450 nm (23).
Cytochrome P-450 has since been shown to be the oxygen-activating com-

ponent of the mixed—function oxidase system (24).

Induction of the Microsomal Mixed-Function Oxidases:

 

The ifl_vivg administration of certain drugs and pesticides such as
phenobarbital and dieldrin elicit an increase in the rate of drug meta-
bolism by the microsomal fraction. A corresponding increase occurs in
the microsomal cytochrome P-450 content and NADPH-cytochrome c reductase
activity (25,26).

This response to certain drugs and pesticides has aided investigators
in elucidating how the system can metabolize a wide variety of substrates.
Administration of phenobarbital (P8) or 3-methylcholanthrene (3-MC) to
a wide variety of animals resulted in microsomes displaying different
spectral forms, cytochrome P-450 and P-448 respectively (27), and dif-
ferent catalytic activities (28). Antisera obtained against partially
purified cytochrome P-448 from the livers of 3—MC-treated animals were
more specific for cytochrome P-448 from the liver microsomal fraction of
3-MC-pretreated animals than for the P-450 of PB-pretreated animals as
determined by quantitative immunoprecipitation and Ouchterlony double
diffusion analysis (29).

Welton (30) has compared the sodium dodecyl sulfate (SDS) polyacryl-
amide gel patterns of liver microsomes from control, PB- and 3-MC—treated
rats. The gel protein and iodination profiles of the induced microsomes
showed significant differences when compared to the control and to each

other. The major differences were in the 43,000 to 53,000 molecular

weight band region of the gels. Gels of microsomes from PB—pretreated

rat livers showed a dramatic increase in concentration and 125

I labelling
of the 45,000 molecular weight band. Treatment with 3-MC induced a sig-
nificant increase of the 53,000 band region in the gel. The major band
in the gels of control microsomes was shown to occur in the region of
50,000 molecular weight (3l).

An antibody obtained against cytochrome P-450 of PB-induced micro—
somes precipitated only the 45,000 molecular weight protein from the de—
oxycholate solubilized untreated, PB, and 3-MC microsomes, as demonstrated
by polyacrylamide gel electrophoresis of the immunoprecipitate (32).
Utilizing the same procedures, an antibody against purified NADPH—cyto—
chrome c reductase from rat liver microsomes demonstrated that the reduc-
tase of control, 3—MC—, and PB—induced microsomes was antigenically and
electrophoretically the same (33). This work explains how the mixed-
function oxidases can metabolize a wide variety of substrates. Substrate
specificity exists in the multiple forms of cytochrome P—450 which are

reduced by the same reductase.

Fractionation of Tissue Homogenates:

 

Quantitative fractionation of the rat liver homogenate shows the above
enzymes to be located almost exclusively within the microsomal fraction
(34,35). Microsomal fractions are usually obtained by high speed cen-
trifugation of the l0,000 xg supernatant and this procedure was employed
in the present studies. The specific activity of rabbit NADPH—cytochrome
c reductase, glucose—6-phosphatase, and aminopyrine demethylase in the
microsomal fractions of rabbit lung and liver was found to be 2-4 times

that in any other fraction. Microsomal glucose-6-phosphatase specific

activity in lungs was found to be about one—fiftieth of that in the liver.
The specific activity of NADPH—cytochrome c reductase in the microsomal
fraction from PB-treated rat liver was eight times the amount found in

the homogenate. In the same study, marker enzymes for mitochondria,
peroxisomes, and plasma membranes had specific activities lower than those
found in the homogenate (36).

In one study of rat liver homogenate, the distribution of 25 enzymes
in fractions obtained by differential centrifugation was examined. Eight
of the enzymes studied, including glucose-6-phosphatase, NADPH—cytochrome
c reductase, and aminopyrine demethylase, had 70% or more of their enzy—
matic activity recovered in the microsomal fraction and had similar dis-
tribution patterns. The microsomal specific activity of these enzymes
was almost four times that found in the homogenate. Contamination of the
microsomal fraction by mitochondria, lysosomes, and peroxisomes was 6% of
the total microsomal protein. The microsomal fraction in this study was
from the 41,000 xg supernatant of the mitochondria plus lysosome fraction
(35).

Ultrastructural studies of the microsomal fractions from rat liver
show that they consist primarily of endoplasmic reticulum and ribosomes.
The microsomal fraction of rabbit lung contained tangles of filamentous
material that trapped vesicle and free ribosomes. Liver microsomal sec-

tions rarely contained such structures (36).

Effect of Isolation Procedure:

 

The method and duration of homogenization, the gravitational forces
used in centrifugation and the homogenization medium all affect the de-

gree to which the microsomal fraction will be contaminated by other

TO

cellular components. Hook et_al, (36) compared four methods of homogeniza-
tion and the effect of the length of homogenization for the four methods
he employed in isolating microsomes from rabbit lung. As the duration
of homogenization increased so did the contamination of the microsomal
fraction by mitochondrial enzymes. The specific activity of microsomal
enzymes also decreased with increased homogenization. By comparing the
cytochrome P-450 concentration, mitochondrial contamination, and the
release of aryl sulfatase from lysosomes, it was demonstrated that the
glass-TeflonR method of homogenization produced a better microsomal
preparation than did the glass—glass homogenizer, Waring BlendorR or
VirTisR homogenizer.

Microsomal fractions from glass—Teflon homogenization of whole house
flies also were shown to have more enzymatic activity than those prepared
by Waring Blendor or Sorvall OmnimixerR homogenization (37). Increasing
the duration of homogenization of house fly abdomens gave preparations
with decreasing enzymatic activity. Four passes with a glass-Teflon
homogenizer was the maximum amount of homogenization attainable without
a decrease in enzymatic activity. A tight-fitting pestle would also de-
crease the activity of the preparation (38).

The effect of centrifugation on the purity of a subcellular fraction
is influenced by the tissue composition. The rate of NADPH oxidation and
the ultrastructure of the microsomal fractions from house fly abdomens
and the gut and fat body of the southern armyworm, Spodoptera eridania
(Cramer), were examined following different centrifugation regimens.
Electron micrographs of the microsomal fractions from the 15 minute
10,000 xg supernatant of house fly abdominal homogenates showed primarily

ribonucleoprotein particles and membranous vesicles. Microsomal pellets

ll

of S, eridania gut prepared with a preliminary l0,000 xg spin for l5 min-
utes contained numerous mitochondria and microorganisms. Increasing the
preliminary spin to l5,000 xg removed the microorganisms and mitochondria.
A preliminary spin of 20,000 xg removed large glycogen clusters. A more
uniform preparation of small membranous vesicles were observed when the
preliminary spin was increased to 30,000 xg. However, the specific ac-
tivity of NADPH oxidase was not significantly reduced when the force of
the preliminary spin was increased (39). The effect of the different
centrifugation rates on microsomal drug metabolism, NADPH-cytochrome c
reductase and cytochrome P-450 content was not examined. Wilkinson
(personal communication, Cornell University) has not observed bacterial
contamination of his microsomal preparation from the midgut of the
armyworm. He suggested that the difference in Ultrastructural observa-
tions might depend on the thoroughness with which the gut contents were
removed by washing prior to homogenization.

Several workers (40 to 42) in assessing the distribution of drug
and pesticide metabolizing activity in fractions of insect homogenates,
have found the greater part of the activity in the microsomal fraction.
The specific activity of the microsomal fraction relative to the total
homogenate activity was reported to be two-fold greater in the house

cricket, Acheta domesticus (L.) (40), l.7-fold in armyworm midgut (4T),

 

and 2.8-fold in caeca of the Madagascar cockroach, Gromphadorina

 

portentosa (Schaum (42).

Endogenous Inhibitors of Insect In Vitro Preparations:

 

Researchers isolating the microsomal fraction from insect tissues

have found numerous endogenous inhibitors of mixed-function oxidases.

12

A potent inhibitor of these enzymes isolated from the gut contents of

the armyworm was identified as a protease (43), that can solubilize the
microsomal NADPH-cytochrome c reductase (44). Ninety to 100% solubili-
zation of this enzyme occurred at low concentrations of the partially
purified inhibitor. Since the amount of protein released during incu—
bations with the inhibitor was only 5% of the total microsomal protein,
this enzyme was very susceptible to proteolysis. Cytochrome P-450 was
apparently not affected by the low c0ncentrations of the partially
purified inhibitor used in the study (44). The gut contents of the

house cricket were also found to contain a protease that inhibited micro-
somal pesticide metabolism (45). Proteolytic activity has been detected
in homogenates of other insect species including the house fly (43). Al-
though every body segment of house flies contains an inhibitor(s) of
mixed-function oxidases (46), the inhibitor from the head has been the
one most extensively characterized.

Matthews and Hodgson (47) found the microsomal fraction from whole
house flies to be inactive unless it had been extensively dialyzed. The
freeze—dried dialysate obtained from the dialysis of the 15,000 xg super-
natant of whole house fly homogenate inhibited the metabolism of p—nitro-
phenyl N,N-dimethylcarbamate by mouse liver microsomes. The inhibitor
was heat stable, water soluble, and insoluble in most organic solvents.
Tsukamoto and Casida (48) reported that fly abdomens gave much greater
mixed-function oxidase activity than whole house flies, heads, or thor—
aces. The homogenates of heads and/or thoraces greatly reduced the ac—
tivity of the abdominal preparation, and the inhibitor was again heat
stable. However, this heat stable inhibitor was absent from the head and

thoraces of house flies with the mutant yellow-eye (38). Schonbrod and

13

Terriere (49) found that single heads of wild-type house fly strains de-
creased microsomal naphthalene hydroxylation by 60 to 65%, whereas heads
from white- or ocra—eyed mutant strains inhibited naphthalene metabolism
by 10% or less. A subsequent report (50) by these workers described the
isolation of the eye pigment, xanthommatin, from fly heads and its inhi—
bition of mixed—function oxidases. The eye pigment acted as an electron
sink by accepting electrons from NADPH—cytochrome c reductase. Concen-

7

trations as low as 5 x 10' M would inhibit aldrin epoxidation. Extra—

polation of their published data suggests that the xanthommatin I of

6

50
M. Wilson and Hodgson (51) demon-

aldrin epoxidation would be 2 x 10—
strated that purified NADPH-cytochrome c reductase from house flies
would reduce xanthommatin, which incidentally stimulated the reduction
of cytochrome c by this enzyme. The pigment prevented the reduction of
cytochrome b5, but did not interact with cytochrome P—450.

Other inhibitors present in house flies have not been characterized.
Jordan and Smith (38) described two factors in house fly abdomen homo-
genates that decreased the mixed-function oxidase activity. One was
called the linearity factor because it limited the time over which the
reaction proceeded at a constant rate. The rate of biphenyl hydroxyla-
tion was not proportional to the amount of enzyme used when the homogen-
ate concentration exceeded two abdomens/ml. Including bovine serum al-
bumen (BSA) in the assay increased the time over which the reaction was
linear. The second factor was called the decay factor, which reduced
the hydroxylase activity of an improperly made preparation to negligible
activity within 30 minutes after homogenization. Homogenates containing
25 abdomens/m1 lost 80% of their hydroxylating activity in 30 minutes.

Those containing 5 abdomens/ml decayed more slowly but still lost about

14

50% of their activity in 30 minutes. The rate of decay was essentially
the same at 0° and 20° C. Substrates, BSA, and KCN prevented the loss
of activity. BSA would restore the activity lost in a ”decayed" prepar-
ation, and enhanced the enzymatic activity of the fresh homogenate. KCN
increased the reaction rate but not the amount of time over which the
reaction was linear.

Both of these factors were heat labile, proportional to the concen-
tration of enzyme used, and were not removed by centrifugation at 10,000
xg. The release of these factors was also affected by the method of pre-
paration. Prolonged homogenization, 14e43 more than four strokes in a
glass-Teflon homogenizer, and a tight—fitting Teflon pestle caused a
decrease in the stability of the preparation.

Another factor that has been implicated in the instability of in-
sect microsomal preparations is the polyphenol oxidase complex. William-
son and Schecter (52) found that the 12,000 xg filtered supernatant of
lepidopterous larvae darkened rapidly on exposure to air. They attri-
buted this to phenol oxidase activity. Ascorbic acid and phenylthiourea,
both inhibitors of this enzyme, prevented the melanization of the super-
natant. An increase in the concentration of the supernatant increased
the degree of darkening and also significantly reduced its aldrin epox—
idase activity. Increasing the supernatant concentration with phenyl-
thiourea present gave an increase in the amount of dieldrin produced.

The microsomal fraction from the lepidopterous preparations darken-
ed only over an extended period of time. Adding BSA to the microsomal
incubation mixture nearly doubled the activity. Addition of KCN to the
microsomal fraction caused a decrease in aldrin epoxidation activity

which was the opposite of what occurred for the 12,000 xg supernatant.

15

Drug metabolism by rat liver microsomes is decreased by the addition of
KCN (53). A KCN-binding protein has been isolated from rat liver micro—
somes (54,55). Addition of KCN to the microsomal fraction of house fly
homogenates, however, increases the amount of pesticide metabolized (56,
57,58). Cyanide is known to inhibit a large number of enzymes (59) thus
the stimulation of activity in house fly preparations by KCN may involve
other factor(s) in addition to tyrosinase. This was suggested by the
following observations of Krieger and Wilkinson (60); they bubbled air
through the soluble fraction of southern armyworm homogenates to stimu—
late tyrosinase activity and found that the darkened soluble fraction
when added to the incubation media sharply reduced aldrin epoxidation.
Neither BSA nor KCN prevented the inhibition by the darkened supernatant.
They concluded that it was the products of the tyrosinase activity that

caused the inhibition.

Biochemistry of Catechol Oxidase:

 

The phenol oxidase complex (EC 1.10.3.1) (61 thru 65), also called
polyphenol oxidase, catechol oxidase or tyrosinase, plays an important
role in the process of cuticle hardening and tanning in arthropods (66).
The purified enzyme appears to have two metabolic functions, namely, the
o—hydroxylation of phenols and the oxidation of the phenols to the corres—
ponding quinones (61,65). Tyrosine hydroxylation by this enzyme has
an initial lag phase that can be eliminated by catalytic amounts of cate—
chol. This has indicated to some workers that the enzyme has, in ad-
dition to the catalytic sites, a third modifier site (67). The function
of tyrosinase in the process of cuticle formation (68,69) is shown in

Figure 2. Since the enzyme incorporates one atom of oxygen into the

 

16

OH 0H 0H
[::::] Tyrosinase 'OH 'OH
————————9
02, H+
‘1“2 ‘5 2 5
H N-CH H N-CH . (R ‘ H)
2 I 2 1 Tyros1nase
coon coo 1 o
3 2
I R

N

O
1 ” ‘70
HCNH + [::::] + H 0
l 2
R

on
'OH
,0

[a 5::::9
OH
I
OH ’0 0“

Red color

CROSS-LINKING OF PROTEINS

 

Figure 2. REACTIONS OF THE TYROSINASE COMPLEX

l7

substrate and reduces the other atom to water, it is a mixed-function
oxidase (3). The reducing equivalents may come from the oxidation of
the phenols or such compounds as NADH, NADPH, cytochrome and FMN (61,
70). Tyrosinase is a copper-containing enzyme and is inhibited by KCN,
phenylthiourea, glutathione, 2,3-dimercapt0propanol (BAL), and borate
(63,71).

In insects, this enzyme has been found in the hemolymph and cuticle
and is easily extracted from the cuticle by cold water (72). Tyrosinase
activity in the soluble fraction of adult house fly homogenates was 6
times that detected in the microsomal fraction (58). The levels of the
enzyme activity vary during the different stages of an insect's life
cycle. In several insects there is a peak of tyrosinase activity just
before pupation, followed by a dramatic decrease. In dipterous insects,
the enzyme appears to be initially a proenzyme which is thought to be
activated by a protease (73). Other workers postulate that the activa-
tion is an autocatalytic process (66).

Ohnishi (74) found the activation of tyrosinase to be inhibited by
a NaCl concentration of 0.25 M or greater, and other salts had a similar
affect. Since 0.25 to 1.0 M sucrose did not interfere with the activa-
tion he concluded that the inhibition observed was a function of ionic
strength. Schweiger and Karlson (73) saw a similar inhibition by in-
creased ionic strength of prophenoloxidase activation in samples from
Calliphora larvae.

Mushroom tyrosinase has been observed to catalyze the oxidation of
tyrosine residues in several proteins. Alcohol dehydrogenase lost 50%,
and aldolase 82%, of their enzymatic activity when incubated with tyrosin-

ase. Aldolase incubated in the presence of cyanide-inhibited tyrosinase

18

maintained control levels of activity. One of the other proteins whose

tyrosine residues were oxidized by tyrosinase was BSA (75,76).

Phenols and Quinones in Biological Systems:

 

The quinones produced by tyrosinase are very reactive compounds
(61,77,78). Webb (77) lists 63 enzymes that are inhibited by various
quinones. The list included cholinesterase, cytochrome oxidase, deoxy-
ribonuclease, d0pa decarboxylase, NADH-tetrazolium oxidoreductase, pro-
teinase and succinate dehydrogenase. The more common mechanisms by which
quinones are known to inhibit enzymes are:

1) oxidation of enzyme groups
2) reaction with enzyme sulfhydryl groups
3

complexing with metal ions of enzymes

reactions with substrates or cofactors

0301b

)
l
) production of hydrogen peroxide
) nonspecific binding through aromatic rings
)

7 competition with quinonoid or polyphenolic substrates
It has been observed that the interaction of phenols with proteins caused
marked changes in their structure and properties (79).

The reactivity of quinones lies in their ability to participate in
oxidation-reduction reactions. Within the pH range of biological systems,
the oxidation reduction potentials (E6) of most quinones fall between

that of the cytochromes and the flavins, as follows:

E6 (pH + 7.0)
o—quinone + 0.378 v.
p—quinone + 0.284 v.
cytochrome b + 0.770 v.
cytochrome c + 0.250 v.
flavins - 0.210 v.
pyridine nucleotides (NAD) - 0.320 v.
thiols — 0.350 v.

These values (77) indicate that quinones could interact with electron
transport systems. Webb lists the following ways in which they could
bring this about:

1) directly inhibit enzymes of the sequence

2) compete with or displace natural or endogenous quinones
3) serve as simple electron donors or acceptors

4) establish alternate or bypass pathways for electron flow
5) block specific site in the electron transfer sequence

The oxidation-reduction reactions of most quinones are thought to
involve the free—radical (semiquinone) intermediate. Miller and Rapp
(80) used electron spin resonance spectroscopy to observe the photochem—
ical and enzymatic production of the semiquinone from catechols and the
oxidation of ferrocytochrome c by the radical. The formation of a stable
complex between the catechol semiquinone and the copper of superoxide
dismutase has been observed by Rapp and co-workers (81). The develop-
ment of electron spin resonance (ESR) signals was detected in cuticles

of Calliphora erythrocephala as the white cuticle darkened during the

 

process of melanization (82). The development of an ESR signal was also

20

observed in cultures of melanoma cells following the introduction of
tyrosinase and catechol. Severe alteration in cellular morphology such
as the deterioration of membranes was observed within 24 hours after the
chemicals were introduced (83).

Several phenolic compounds have been isolated from insect homogen—
ates. The compounds identified thus far have the catechol nucleus sub-

stituted with different groups at the 4 position of the ring (69).

The Effect of Free Radicals in Biological Systems:

 

The effects of free radical reactions occurring within biological
systems has drawn increased attention recently. It has been proposed that
these reactions are the mechanism through which several xenobiotics exert
their toxic and carcinogenic effects (84). Lipid peroxidation is a free
radical process that is thought to play a role in several diseases and
in the aging process (85).

The process of lipid peroxidation can be described as follows (86):

Initiation: Rh ................. R-
Propagation: R- + 02 ............ R00-
Termination: 2 R00- ------------ stable products + 0

2
The reaction can be stimulated by catalytic amounts of transition metal

ions such as iron which promotes the formation of radicals. It can be
inhibited by antioxidants such as vitamin E and butylated hydroxytoluene
(BHT) which trap the radicals thus preventing propagation. Lipid peroxi-
dation has been demonstrated by oxygen consumption, NADPH oxidation and
malondialdehyde formation (87). The NADPH-cytochrome c reductase of rat
liver microsomes has been shown to be an enzymatic component of NADPH—

dependent lipid peroxidation (88).

21

Malondialdehyde is a product of lipid peroxidation only when fatty
acids having three or more methylene-interrupted olefin bonds are sub-
strates. It can be detected in nanomolar quantities by the formation of
a red colored chromagen in the presence of thiobarbituric acid (TBA) (89).
Though lipid peroxidation has been demonstrated to occur in rat and goat
liver microsomes, it has not been detected in microsomal fractions of in—
sects. The addition of the house fly microsomal or soluble fraction to
goat liver microsomes completely inhibited the formation of the TBA chroma-
gen, and presumably, lipid peroxidation. House fly microsomes (heat
killed), tyrosinase plus tyrosine and dihydroxyphenylalanine also in-
hibited the reaction. The addition of either tyrosine, tyrosinase or
tyrosinase (heat killed) plus tyrosine did not prevent lipid peroxidation
(57). Tyrosinase is known to be present in the house fly microsomal and
soluble fraction (58,91). The endogenous substrates of tyrosinase have
been shown to act as lipid antioxidants (90). The above data would indi-
cate that the failure to detect lipid peroxidation in housefly microsomal
preparations is because of the presence of this system.

Lipid peroxidation of microsomes causes the degradation of membrane
structure and a decrease in enzymatic activity. A visible clarification
of the normal turbidity of the microsomal suspension also-occurs (92).
Bidlack and Tappel found that in one hour lipid peroxidation caused a
50% loss of NADPH-cytochrome c reductase and a 90% decrease in glucose-6-
phosphatase activity. Solubilization of protein from the microsomal
membrane also occurred. Enzyme inhibition and protein solubilization
was significantly reduced when an antioxidant was present during incu—
bation (93). Cytochrome P-450 was also degraded during lipid peroxidation

(92). The spectral shift from cytochrome P-450 to P-420 during the

22

peroxidation process was different from that seen during the incubation
of microsomes with trypsin (94). The CO-difference spectra of trypsin-
treated microsomes showed a parallel shift of the absorbance at 450 nm
to 420 nm when the amount of protease was increased. An increase in

lipid peroxidation resulted in a decrease of absorbance at 450 nm with-

out a corresponding increase in the absorbance at 420 nm (92).

MATERIALS AND METHODS

The Fc strain of house flies used in this research was obtained
from Dr. F. W. Plapp, Jr., Texas A & M University, College Station,
Texas. The rat liver microsomes were supplied by Dr. S. 0. Aust, Depart—
ment of Biochemistry, Michigan State University.

NADP-isocitrate dehydrogenase (Sigma type IV), cytochrome c (Sigma
type VI), bovine serum albumen, fraction V, NADP+, NADPH, DL-sodium iso-
citrate, mushroom tyrosinase grade III, phenylmethyl-sulfonylfluoride,
diisopropylfluorophosphate, dithiothreitol, EDTA, Tris base and Tris-HCl
were all obtained from the Sigma Chemical Company, St. Louis, Missouri.
Catechol was obtained from the Aldrich Chemical Company, Milwaukee, Wis-
consin. Aldrin and dieldrin recrystallized, were obtained from Shell
Chemical Company, New York, N.Y. Mushroom tyrosinase, B grade, was ob—
tained from Calbiochem, La Jolla, California. All other reagents used
were analytical grade. All aqueous solutions were prepared with distil-
led water that had been passed through charcoal and a mixed-bed resin

column.

The House Fly Strain and its Culture:

 

The PC strain of house flies has a high mixed-function oxidase ac-
tivity when compared to susceptible strains and it is resistant to sev-
eral pesticides such as DDT and dieldrin (95). The adults were reared

under a 16:8 photoperiod at 80° C and 55% relative humidity. They were

23

24

fed a diet consisting of a 1:1 mixture of lowfat powdered milk and pow-
dered sugar with tap water ag_libitum. Every few months, the adults
were exposed to 100 ppm of 001 in their food, in order to maintain the
resistance level of the colony. The DDT was dissolved in acetone and
mixed thoroughly with the mixture of ground sugar and powdered milk;
the acetone was allowed to evaporate thoroughly before the mixture

was fed to the flies.

Eggs were obtained by exposing the adults to NH4C03 solution impreg-
nated in a cloth, which was stretched over the wet CSMA medium employed
to rear the larvae. The eggs were suspended in distilled water at 21° C
and allowed to settle, and the water was decanted, the process being re-
peated 2 more times. Lots of approximately 300 eggs were then aspirated
with a suction bulb into a calibrated glass tube for transferring to the
larvae medium.

The CSMA larval medium (2400 ml) was mixed with malt syrup (25 ml),
5% yeast solution (75 m1), and lukewarm tap water (1200 ml). This mix-
ture was then transferred into a No. 10 fruit can, to which the eggs
were added, covered with paper toweling, and kept in a growth chamber at
80° F and 70% relative humidity. During the 2nd and 3rd days of larval
development, a total of 250 mls of tap water was added to each can. In
order to prevent the growth of mold which would interfere with complete
larval development, it was necessary to stir the media on the 3rd day of
larval develOpment.

Under these controlled conditions, the larva pupated on the seventh
day and were transferred into aluminum pans. Prior to their emergence on
the 10th day of development, the pans were placed in cages. Occasionally,

the adults were egged for maintenance of the colony. It was also observed

25

that after the 6th day following emergence the adults in the cage con-
sisted almost entirely of females. Except for the study concerning the
effect of age on enzymatic activity, the flies collected consisted pri—

marily of 8 to 10 day old females.

Isolation of the Microsomal Fraction from House Fly Homogenates:

 

The isolation of the microsomal fraction from whole house flies or
their abdomens from mortar-and-pestle (MP) homogenates followed the pro—
cedure outlined by Morello et a1. (96). The flies were anesthetized in
a walk-in freezer at —20° C, collected in a pre—chilled flask, and kept
on ice. A buffer volume, 4 times the weight of the flies, was poured
into a pre-chilled mortar and homogenized by gentle strokes of the pes-
tle which was continued until a brei was obtained, 2 to 3 minutes later.
The brei was then filtered through a double layer of cheesecloth into a
cold flask and transferred into centrifuge tubes. The buffer used dur-
ing the preliminary and aging studies was 0.15 M phosphate buffer that
ranged from pH 7.4 to 7.8. The homogenate was then centrifuged at 10,000
xg for 20 minutes, followed by 105,000 xg for 90 minutes, in order to
isolate the microsomal fraction. The microsomal pellet was then homo-
genized gently in a 1:1 ratio of 0.05 M Tris buffer and glycerol at pH

7.5 and stored under nitrogen at -20° C if not used immediately.

Distribution Studies of Marker Enzymes in MP Homogenates:

 

The MP method of homogenization was the same as used above, with the
changes being made only in the buffer parameters and centrifugation regi-
men. Sucrose of 0.32 M concentration, isotonic to insect hemolymph, was
employed to help preserve the integrity of mitochondria during the isola—

tion. After filtering the first MP homogenate, it was then rehomogenized

26

by 2 strokes in a glass-Teflon homogenizer (GT) to insure a more complete
disruption of the cells. The differential centrifugation of the homogen-
ate was done at 800 and then 5,000 xg, each for 10 minutes, followed by
10,000 and then 20,000 xg, each for 20 minutes, and 105,000 xg for 90
minutes. The centrifugation of 20,000 xg and below were done in a Sorval
RC2 refrigerated centrifuge using the GSA and SS-l rotors. The centrifu-
gation at 105,000 xg was performed in the Beckman model L ultracentrifuge
with type 30 and Ti 50 rotors.

The pellets obtained from the 800 xg through the 20,000 xg centrifu—
gations were resuspended in Medium A (see below). The microsomal fraction
from the 105,000 xg centrifugation was resuspended in 0.05 M Tris plus
glycerol, 1:1, at a pH of 7.5. The volumes for each fraction were record-
ed, and protein assays were made by the Lowry method (97). The distribu-
tion patterns of marker enzymes were calculated by the method of de Duve

(98).

Sucrose Density Gradient Centrifugation:

 

The density gradient centrifugation method utilized a discontinuous
gradient composed of 8 ml each of 10, 20, 41.5, and 50% sucrose. A 5 m1
sample of the 800 xg supernatant in 0.32 M sucrose layered between the
10 and 20% layers of the gradient. The preparation was centrifuged for
2.5 hours at 24,000 rpm in the Beckman model L ultracentrifuge with a SW
27.1 rotor. The density gradient columns were monitored by the use of
an ISCO density gradient fractionator coupled to a UA-2 ultraviolet ana-
lyzer set at 254 nm. Two-ml fractions were collected during the monitor—
ing, kept at 4° C, and assayed for cytochrome oxidase and NADPH-cytochrome

c reductase activity.

27

Isolation of the Microsomal Fraction from Abdominal GT Homogenates:

 

Adult house flies, 7 to 10 days after emergence, were anesthetized
in a walk-in freezer, -20° C, and kept on ice. The whole flies were then
transferred into a 2-2 Erlenmyer flask containing 6 mm glass beads that
had been pre-chilled in a dry—ice acetone bath. During the following 20
minutes, the flask was turned several times to ensure the thorough freez—
ing of the flies, after which it was shaken rapidly by hand for 30 sec-
onds and replaced in the dry-ice-acetone bath. The flask was rotated in
the bath for 2 minutes and then the shaking was repeated. Three cycles
of shaking and freezing were required to separate the heads, abdomens,
and thoraces from each other. The separated segments were replaced in
the ice bath for 2 minutes and then emptied into a set of sieves held at
-20° C. The beads were retained by the first sieve (US Standard Sieve
Series, Tyler equivalent 10 mesh), the abdomens and thoraces by the sec-
ond sieve (Tyler equivalent 6 mesh), and the heads were collected in a
pan. The thoraces and abdomens were kept frozen and the abdomens sepa-
rated from the thoraces by an aspiration procedure.

The abdomens were rinsed once in a buffer volume 4 times the sample
weight (4° C) and homogenized in the same buffer volume using 2 strokes
of the GT homogenizer with a loose-fitting pestle. The homogenate was
filtered through 2 layers of cheesecloth into a chilled flask and cen-
trifuged at 10,000 xg for 20 minutes. The 10,000 xg supernatant was
centrifuged at 105,000 xg for 90 minutes. The resulting pellet was sus-
pended by gently homogenizing it with a GT homogenizer in 0.2 M KZHPO4
and 50% glycerol at pH 8.0. If not used immediately, it was stored un-

der N2 at -20° C.

28

Enzyme Assays and Solution Preparation:

 

All solutions were prepared in deionized distilled water. The phos-
phate buffers were filtered through a 5 u Millipore filter and then pas-

sed over a Dowex chelating column to remove impurities.

Assays of NADPH-Cytochrome c Reductase Activity:

 

The reduction of cytochrome c was measured by following the increase
of absorbance at 550 nm with a Beckman DB spectrophotometer. The assays
were made with 35 OM of cytochrome c and 0.16 mM of NADPH in 0.15 M phos-
phate buffer at pH 7.5 or 8.0. The rate of activity is expressed in
umoles of cytochrome c reduced/min/mg protein, using an extinction coef-

4-1-1

ficient of 2.10 x 10- M cm (99).

Assay of Cytochrome Oxidase Activity:

 

The oxidized form of cytochrome c at a concentration of 0.175 mM
in medium A was reduced with sodium dithionite crystals. Air was passed
over a 10% NaOH solution and bubbled gently through the cytochrome c
solution for 15 minutes. Medium A was composed of 15 mM KCl, 5 mM MgC12,
50 mM Tris, 2 mM EDTA, and 35 mM phosphate (100). The assays for the oxi—
dation of the reduced cytochrome c were made with 17.5 ON of reduced cyto-
chrome c and medium A at pH 7.5. The oxidation of reduced cytochrome c
was measured by following the decrease in absorbance at 550 nm with a

Beckman DB spectrophotometer.

Microsomal Pesticide Metabolism Assay:

 

The epoxidation of aldrin to dieldrin was used to assay the mixed—
function oxidase activity of the microsomal preparations (101). The 5—ml

reaction mixture contained microsomes or the homogenate, 0.2 M KZHPO4 at

29

pH 8.0, and a NADPH-generating system that consisted of 2 mM DL-isocit-
rate, 7 mM MgCl, 0.1 mM NADP+, and 0.05 units of NADP-isocitrate dehydro-
genase per ml. The mixture was pre-incubated in a water-bath shaker at
30° C for 5 minutes, and then 27.5 uM of aldrin was added in 0.1 m1 etha-
nol. The dieldrin production was measured at various time intervals by
pipetting l-ml samples from the reaction mixture and mixing the sample

immediately with 15 mls ethanol-acetone-water (2:1:1).

Extraction of Pesticides:

 

After the protein had precipitated, the sample was poured into a
60-ml separatory funnel and extracted 3 times with 10 mls redistilled
n-hexane. The combined extracts were backwashed 3 times with approxi-
mately 30 mls deionized distilled water, and the heXane was then dried
over anhydrous Na2504. A stream of N2 was used to reduce the volume of
the house fly extracts to 2 ml. The efficiency of this extraction pro-

cedure averaged 82% in its recovery of dieldrin. A 2-A sample of the

hexane extract was injected into the gas chromatograph for quantitation.

Gas Liquid Chromatographic Analysis:

 

A Packard 834 Gas Chromatograph equipped with a tritium-foil elec-
tron-capture detector was employed to determine the amount of dieldrin
present in each sample. It was fitted with a 6 foot x 2 mm 1.0. silaniz-
ed glass column, packed with 3% XE-60 on 60/80 mesh Gas Chrom 0 (Applied
Science Laboratories). The instrument was operated at a column temper-

ature of 200° C and a N flow of 50 ml/min. The injection port temper-

2
ature was 220° C and the detector temperature 210° C.
Standards of aldrin and dieldrin were injected at the beginning and

end of each run, with intervening check being added during long runs.

30

The linearity of the detector was determined and only the samples injec-
ted within that range were taken for determination of the dieldrin concen-
trations. The retention times of aldrin and dieldrin under the conditions
stated above were about 1.0 and 3.5 minutes respectively. The height and
area of the peaks were measured and were then cut out and weighed; the
figures obtained were similar and congruent with the computer deter-
minations of the peak areas. The rate of dieldrin production was ex—

pressed as nmoles/min per mg protein.

Microsomal Cytochrome P—450 Analysis:

 

The cytochrome P—450 spectra were obtained by the method of Omura
and Sato (20). The microsomal preparations, 1.5 to 4.0 mg protein per
ml, were divided between two cuvettes. Carbon monoxide was bubbled gent-
ly through the sample cuvette for one minute. A few crystals of sodium
dithionite were added to the sample and reference cuvettes and mixed
gently until dissolved. The sample was scanned twice in a Beckman DB
spectrometer. The scan was repeated a third time following the addition
of several crystals of sodium dithionite making certain that the sample
was fully reduced. The cytochrome content was calculated on the dif-
ference in absorbance at 490 and 450 nm and was expressed in nmoles/mg

protein using an extinction coefficient of 91 mM_] cm'] (21).

Assay of Tyrosinase Activity:

 

The tyrosinase activity of microsomal preparations and mushroom ty—
rosinase were assayed using 5 mM catechol in a 0.1 M phosphate buffer at
pH 7.0. The oxidation of catechol was measured by following the increase

in absorbance at 470 nm (102) with a Beckman DB spectrophotometer.

31

Treatment of House Fly Homogenates with Catechol and Cyanide:

 

Equal quantities of house fly abdomens were homogenized with the GT
homogenizer in 0.2 M K2HP04 at pH 8.0. The buffer for the cyanide-inhib-
ited preparation contained 10 mM KCN. Catechol was added to the homogen-
ate without KCN after the 10,000 xg centrifugation to give a final concen-
tration of 0.38 mM. The microsomal fractions were isolated from the
10,000 xg supernatants by centrifuging at 105,000 xg for 90 minutes, re-
suspending in 0.2 M K2HP04 plus 50% glycerol at pH 8.0, and then assess—
ing the aldrin epoxidase activity, NADPH-cytochrome c reductase activity,
and cytochrome P—450. The microsomes not used in the above assays were

stored under N2 at -20° C and assayed the following day.

Incubation of Rat Liver Microsomes with Tyrosinase:

 

The incubations for determining the effects of tyrosinase on micro—
somal mixed-function oxidases were made with rat liver microsomes iso-
lated from control and phenobarbital treated rats. The incubation mix-
ture contained 5 to 6.5 mg microsomal protein per ml, 0.1 M KZHP04,

(pH 7.0), 2.8 to 5 mM catechol, and 0.31 to 2.5 pg tyrosinase per mg
microsomal protein. The incubations were initiated with tyrosinase and
kept on ice during the incubation, except for the studies made at 13° C.
When tyrosinase was inhibited with KCN, they were incubated together at
21° C for 5 minutes before being added to the microsomal incubation mix-
ture. Samples were removed at various times after the initiation of the
reaction for the determination of cytochrome P—450 spectra, NADPH—cyto—
chrome c reductase activity and aldrin epoxidation. The NADPH-cytochrome
c reductase activity was usually expressed as the percent of the original

reductase activity remaining at the time of sampling.

32

Erythrocyte Hemolysis:

 

The occurrence of free—radical intermediates have been detected by
their ability to cause hemolysis of red blood cells (103). Sterile sheep
erythrocytes were washed 3 times in isotonic saline and packed by centri-
fugation. One ml of the packed cells was diluted in 19 mls of isotonic
saline. Two mls of this suspension were used to determine if hemolysis
occurred during the oxidation of catechol by mushroom tyrosinase. After
a 15 minute incubation of erythrocytes with tyrosinase alone, catechol
alone, and tyrosinase plus catechol, on ice, the mixtures were centri—
fuged at 2000 rpm for 10 minutes. The supernatants were carefully as-
pirated and their absorbance at 541 nm was determined with a Beckman DB
spectrophotometer. The results were expressed as the percent increase
in the absorbance of the tyrosinase plus catechol supernatant over that

of the treatments with catechol alone and tyrosinase alone.

Other Methods:

The determination of protein concentration was by the method of
Lowry gt_al, (97) using dilutions of BSA solutions (1 mg/ml) for stan-
dardization. The sodium dodecyl sulfate polyacrylamide gel electro-
phoresis was done as described by Dr. Ann Welton (15) employing a modi-
fication of the method used by Fairbanks et_al, (104). The polyacryl-

amide gels were stained with Coumassie blue.

RESULTS

The microsomal NADPH—cytochrome c reductase activity from mortar—
and-pestle (MP) homogenates of whole flies was compared to that of ab-
dominal MP preparations when first beginning this research. The micro-
somal reductase activity of the abdominal preparations in 2 isolations
was slightly higher than that found for the whole house fly isolations
(Table 1). Subsequent isolations of microsomal fractions from MP homo-
genates also showed wide variation in their enzymatic activity, not
only for the adult but also for the larval homogenates. For example,
Table 2, the NADPH-cytochrome c reductase activity for the 10 day old
adult ranged from 0.5 to 50.0 nmoles of cytochrome c reduced/min/mg
protein. This variation initiated a series of centrifugation studies

designed to characterize the isolated microsomes.

Marker Enzyme Distribution Patterns of Mortar—and—Pestle Homogenates:

 

The marker enzyme distribution patterns of mitochondrial cytochrome
oxidase and microsomal NADPH-cytochrome c reductase obtained after dif—
ferential centrifugation of the MP whole house fly homogenates are shown
in Figs. 3 and 4. Also shown for comparison are the patterns of mito-
chondrial and microsomal marker enzymes as found with rat liver homo—
genates (105). The distribution of the mitochondrial marker, cytochrome
oxidase, differs significantly from that obtained with the rat liver pre—

parations. Over 20% of the rat liver total nitrogen is located in the

33

34

Table l
COMPARISON OF NADPH-CYTOCHROME C REDUCTASE ACTIVITY IN MICROSOMAL
FRACTIONS FROM MORTAR-PESTLE HOMOGENATES OF ABDOMENS AND WHOLE FLIES
Homogenates were prepared from equal weights of abdomens and
whole flies by the mortar-pestle technique. The 10,000 xg supernatant
of each homogenate was centrifuged at 105,000 xg to obtain the micro»
somal fraction for the NADPH-cytochrome c reductase determinations.

This was done with two groups of adult flies 7 and 10 days old.

 

NADPH-cytochrome c reductasea
Homogenization method

 

 

Isolation 1 Isolation 2
Whole flies 0.078 0.054
Abdomens 0.090 0.043

 

a pmoles of cytochrome c reduced/min/mg protein

35

Table 2
NADPH-CYTOCHROME C REDUCTASE ACTIVITY IN MICROSOMAL FRACTIONS FROM
WHOLE HOUSE FLY MORTAR-PESTLE HOMOGENATES OF THE LARVAL AND ADULT
STAGES
Whole larvae or adults were homogenized with a mortar and pestle

and the microsomal NADPH-cytochrome c reductase activity was determined.

 

NADPH-cytochrome c reductasea

 

Isolation Larvab Adultb
4 5 5 10
1 0.002 0.001 0.006 0.0005
2 0.009 0.013 0.018 0.010
3 0.019 0.013 0.033 0.052
4 0.033 0.056 0.060

5 0.039

 

a pmoles of cytochrome c reduced/min/mg protein

b Age of larval and adult stages in days

 

Figure 3.

36

DISTRIBUTION OF CYTOCHROME OXIDASE IN SUBCELLULAR
FRACTIONS OF RAT LIVER AND WHOLE HOUSE FLY HOMOGENATES.

Fractionation of whole house fly mortar-and-pestle
homogenates in 0.32 M sucrose, pH 7.5, was done by dif-
ferential centrifugation. The fractions isolated were
the 0 to 800 xg nuclear (N), the 800 to 5,000 xg heavy
mitochondrial (M2), the 10,000 to 20,000 xg light mito-
chondrial (L), the 20,000 to 105,000 xg microsomal (P),
and the 105,000 xg supernatant (S). The fractions are
represented along the abscissa in the order in which
they were isolated. The distribution patterns of rat
liver marker enzymes are from studies done by de Duve
and co-workers (105). The rat liver fractions, repre-
sented along the abscissa in the order they were iso-
lated, are: nuclear (N), heavy mitochondrial (M), light
mitochondrial (L), microsomal (P), and supernatant (S).
The relative specific activity is the percent of the
total enzyme activity in a fraction divided by the
percent of the total nitrogen in the case of rat liver
or for the house fly studies by percent of the total

protein.

 

Relative Specific Activity

37

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

RAT LIVER
4
3 ~ M
J;
21
l
P
’1 S
0 20 4O 6O 80 100
% of total nitrogen
CYTOCHROME OXIDASE
HOUSE FLY
4
“.2.
3.
2. N
L
11 M1
ngA s
T I L I
0 20 40 60 80 100

% of total protein
CYTOCHROME OXIDASE

Figure 3

Figure 4.

38

DISTRIBUTION OF MARKER ENZYMES IN SUBCELLULAR FRACTIONS
OF RAT LIVER AND WHOLE HOUSE FLY HOMOGENATES

The distribution patterns of rat liver glucose-6-
phosphatase and house fly NADPH-cytochrome c reductase
are for the fractions of rat liver and house fly homo—

genates listed in Fig. 3.

Relative Specific Activity

39

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

RAT LIVER
4 P
3..
2‘
L
14
M s
o 20 40 60 so 100
% of total nitrogen
GLUCOSE-6-PHOSPHATASE
HOUSE FLY
4
3 s
3.
LI
2.
M2
N
1 M1 I“
o 26 4o 60 so 100

% of total protein
NADPH-CYTOCHROME C REDUCTASE

Figure 4

4o

fraction containing the highest relative specific activity of the mito—
chondrial marker. The mitochondrial fractions of whole house fly homo-
genates with the highest relative specific activity has about 3% of the
total protein. There also was activity in the soluble fraction which
was not detected in the rat liver preparations.

The distribution of microsomal NADPH—cytochrome c reductase from
the house fly homogenate also differed significantly from that of the
rat liver microsomal marker enzyme, glucose-6-phosphatase (105). Both
of these enzymes have been shown to have the same microsomal distribu—
tion pattern in subcellular fractions of rat liver (35). The relative
specific activity of the house fly soluble fraction was nearly as high
as the activity in the microsomal fraction. The amount of reductase
activity recovered was only 63% of the total homogenate activity. Based
on the amount recovered in the different fractions, 89% of the reductase
activity was in the soluble fraction. Since this is the opposite of
the results obtained with glass—Teflon (GT) homogenates (see below),
it is evident that solubilization of the reductase occurred during the

centrifugation of MP homogenates.

Density Gradient Centrifugation of Mortar—Pestle Homogenates:

 

Sucrose density gradient centrifugation of the 800 xg supernatant
confirmed the solubilization of NADPH-cytochrome c reductase seen in
the differential centrifugation studies. The distribution of reductase
activity in fractions collected from discontinuous sucrose gradient and
the 254 nm absorbance profile are shown in Fig. 5. Two major peaks of
reductase activity were found in the gradient with 67% of the reductase

activity in peak I and 31.6% in peak II (Table 3). The distribution of

 

 

 

 

41

 

 

 

Z

1—4

E

g

m

U

:3

Q

E

o E

g I:
<r

e - 1 n

:x:

U I

0 e.

S o

0

PM

o

m

a:

8

E:

:1

O . -r ; O
l 03 l 08 1.19 1.23
DENSITY
Figure 5. THE DISTRIBUTION OF NADPH-CYTOCHROME C REDUCTASE ACTIVITY

AND PROTEIN PROFILE FOLLOWING SUCROSE DENSITY GRADIENT
CENTRIFUGATION OF THE 800 XG SUPERNATANT 0F MP HOMOGENATE

Whole house flies were homogenized with the mortar—
and—pestle in 0.32 M sucrose and 10 mM EDTA, pH 7.5. The
800 xg supernatant (5 ml) was layered on a discontinuous
gradient and centrifuged for 2.5 hours at 127,000 xg.
Successive 2 m1 fractions were assayed for NADPH-cytochrome
c reductase. The dots on the broken line show the activity
of each fraction. The solid line indicates the 254 nm

absorbance profile of the gradient.

 

42

enzymatic activity in the fractions closely followed the protein distri—
bution in the gradient. The fraction with highest activity occurred at
the interface of the top sucrose layer and the slightly heavier layer
of the 800 xg supernatant sample that formed the second layer in the
gradient. Thus activity and protein migrated up the gradient as well

as down. The second peak sedimented to a density of 1.19, which is low-
er than that of 1.14 observed for rat liver microsomes in a linear suc-
rose gradient(106). Peak II also had in the same fraction the highest
cytochrome oxidase activity, which was negligible throughout the rest

of the gradient.

 

Inhibition of Enzyme Solubilization in Mortar Homogenates:

Attempts to prevent the solubilization of the membrane-bound reduc-
tase in MP homogenates by using inhibitors of calcium-dependent phos-
pholipases and proteases were unsuccessful. The addition of EDTA to
chelate divalent metals and so inhibit phospholipases did not affect
the percent of reductase retained in the microsomal fraction (Table 4)
or its specific activity (Table 5). The same results were obtained when
the protease inhibitor, phenylmethylsulfonylfluoride (PMSF), was used
(Tables 4, 5). Table 6 shows the results of 3 isolations in which PMSF
was added to the buffer to inhibit proteases, EDTA to inhibit phospholi-
pases, and dithiothriotol (011) to protect against oxidation of enzyme
sulfhydryl groups in the combinations listed. The first isolation in
which PMSF was used gave 80% of the reductase in the microsomal pellet
of the treated sample. This suggested that endogenous proteases solu—
bilized the enzyme during the isolation (isolation A of Table 6). The

results of two subsequent isolations failed to confirm this (isolations

43

Table 3
THE DISTRIBUTION OF NADPH-CYTOCHROME C REDUCTASE ACTIVITY FOLLOWING
SUCROSE DENSITY GRADIENT CENTRIFUGATION OF THE 800 XG SUPERNATANT
0F MP HOMOGENATE
The fractions collected from the gradient represented in Fig. 5
were combined into samples representing peak I and II. The NADPH-
cytochrome c reductase activity of the samples were assayed and is

expressed as a percentage of the total activity recovered from the

 

 

 

gradient.

Total Activity Peak I Peak 11 Pellet Total
Total Activitya 10.66 5.04 0.24 15.94
Percentb 67.0 31.6 1.4 100

 

a Total activity = umoles of cytochrome c reduced/min

b activity recovered in fract1on x 100

total recovered activity

 

Percent =

 

 

 

44

Table 4
THE EFFECT OF ISOLATION CONDITIONS ON THE PERCENT OF NADPH-CYTOCHROME
C REDUCTASE ACTIVITY FOUND IN THE MICROSOMAL FRACTION FROM HOUSE FLY
HOMOGENATES
The isolations were performed as described in the Materials and
Methods. The percent activity was calculated from the reductase

activity in the microsomal pellet and supernatant retained in the

 

 

 

 

 

 

pellet.
Method of Homogenization Number of Percenta
Homogenization Medium Isolations Activity ’
0.32 M Sucrose 8 18.7:7.3 a
Mortar-pestle 0.32 M Sucrose + EDTA 4 8.0:l.3 a
0.32 M Sucrose + PMSF 3 32.3223.8 a
0.2 M KZHPO4 9 63.5:9.l b
R 0.32 M Sucrose 3 79.4:5.4 b
Glass—Teflon
0.57 M Tris 3 93.7:8.3 b
0.2 M KZHPO4 + DFP 3 88.9:4.5 b
a % Activity = units 1n m1crosoma1 pellet x 100 t SE

units in pellet + units in supernatant
units = nmoles cytochrome c reduced/min/mg protein x mg
protein in fraction

b Pairs of means followed by the same letter were not significantly
different at the 5% level using the Student's t—test

 

 

 

45

Table 5
THE EFFECT OF ISOLATION CONDITIONS ON THE SPECIFIC ACTIVITY OF NADPH—
CYTOCHROME C REDUCTASE IN THE MICROSOMAL FRACTION FROM HOUSE FLY
HOMOGENATES

The isolations were performed as described in the Materials and

 

 

 

Methods.
Method of Homogenization Number of Specifica
Homogenization Medium Isolations Activity ’
0.34 M Sucrose 9 0.023i0.008 a
Mortar-pestle 0.34 Sucrose + EDTA 5 0.027:0.007 a
0.34 M Sucrose + PMSF 3 0.023:0.010 a
0.2 M KZHPO4 10 0.061:0.033 b
R 0.032 M Sucrose 3 0.040i0.019 b
Glass-Teflon
0.057 M Tris 3 0.033:0.005 b
0.2 M KZHPO4 3 0.068:0.010 b

 

a Specific activity = pmoles cytochrome c reduced/min/mg protein t SE

b Pairs of means followed by the same letter were not significantly

different at the 5% level using Student's t-test

 

 

 

 

46

Table 6

INDIVIDUAL ISOLATION OF THE MICROSOMAL FRACTION FROM MP HOMOGENATES
0F WHOLE HOUSE FLIES. THE EFFECT OF PMSF, DTT, AND EDTA 0N MICRO—
SOMAL NADPH-CYTOCHROME C REDUCTASE ACTIVITY

For each isolation, whole house flies were divided into equal
groups for subsequent homogenization in one of the cold homogeniza—
tion media listed below. After obtaining the microsomal fraction
by differential centrifugation, the percent of reductase activity
retained in the pellet and its specific activity were determined.
The treatments were 0.32 M sucrose, pH 7.5 plus additive(s).

l. 0.32 M Sucrose, pH 7.5

2. 0.32 M Sucrose, 0.1 mM DTT

3. 0.32 M Sucrose, 0.2 mM PMSF

4. 0.32 M Sucrose, 0.2 mM PMSF, 10 mM EDTA

5. 0.32 M Sucrose, 0.4 mM DTT, 0.4 mM PMSF

6. 0.32 M Sucrose, 0.4 mM DTT, 0.4 mM PMSF, 10 mM EDTA

 

47

 

 

 

 

Table 6
Isolation Treatment % Activitya Specific activityb
1 26 0.011
2 56 0.013
A
3 80 0.011
4 79 0.019
1 13 0.023
3 10 0.027
B
5 16 0.025
6 9 0.026
1 9.9 0.029
3 7.5 0.030
C
5 7.5 0.032
6 8.5 0.037

 

Enzyme units in microsomal pellet X 100
units in pellet + units in supernatant

b Specific activity = uMoles of cytochrome c reduced/min/mg protein

 

a % activity =

 

 

 

48

B & C of Table 6). The percentage of reductase activity in the treated
microsomal fractions of isolations B and C did not differ from that of
the controls. The average of the specific activities in isolation A,
however, was less than half of those obtained in B and C. Since the
specific activities of the microsomal fractions in isolation A were

the lowest of all the 25 MP isolations obtained it is probable that in
this isolation some endogenous inhibitor had been released during the

homogenization.

Glass-Teflon Homogenization and Solubilization:

 

In contrast to the solubilization of the reductase observed during
the isolation of the microsomal fraction from MP whole fly homogenates,
the GT method, which used only abdomens, gave preparations with most of
the reductase activity in the microsomal pellet (Table 7). This method
gave an average of 80.3 i 21.3% of the reductase activity in the micro—
somal fraction as compared to 20.1 i 24.5% for the MP method. When the
differences were assessed for significance by Student's t-test they were
significant at the 0.01 probability level. The specific activity of the
reductase in the microsomal fractions obtained by the two methods was
significantly different at the 0.05 probability level. The GT method
gave an average specific activity twice that of the MP preparations
(Table 8). The percent of NADPH—cytochrome c reductase activity recover—
ed in the microsomal fraction with each method is shown in Fig. 6.

Microsomes isolated in the presence of EDTA or diisopropylfluoro-

phofiiphate (DFP), a protease inhibitor, were not significantly different
from “the controls with respect to either percent of reductase activity

recoveered in the microsomal fraction of its specific activity (Tables 4, 5).

 

 

 

49

Table 7
THE EFFECT OF THE HOMOGENIZATION METHOD ON THE PERCENT OF THE
MICROSOMAL AND SOLUBLE NADPH—CYTOCHROME C REDUCTASE ACTIVITY
DETECTED IN THE MICROSOMAL FRACTION
The homogenizations and isolations were performed as described

in the Materials and Methods.

 

Method of Number of

 

Homogenization Isolations % Activitya’b
Mortar-pestle 23 20.1:5.l a
Glass-Teflon R 30 80.3:3.9 b

 

 

Enzyme un1ts 1n m1crosomal pellet X 100 i SE

a . - _
% acthltY ' units in pellet + units in supernatant

units = umoles of cytochrome c reduced/min/mg x mg protein in fraction
b Pairs of means followed by the same letter were not significant at

the 5% level using Student's t-test

50

Table 8
THE EFFECT OF THE HOMOGENIZATION METHOD USED TO PREPARE HOUSE FLY
HOMOGENATES ON THE SPECIFIC ACTIVITY OF NADPH-CYTOCHROME C REDUCTASE
IN THE MICROSOMAL FRACTION
The homogenizations and isolations were performed as described

in the Materials and Methods.

 

 

Method of Number of Specifica b
Homogenization Isolations Activity ’

Mortar-pestle 25 0.025:0.002 a
Glass-TeflonR 31 0.05020.004 b

 

a Specific activity = umoles of cytochrome c reduced/min/mg protein i SE
b Pairs of means followed by the same letter were not significant at the

5% level using Student's t-test

 

NUMBER OF ISOLATIONS

Figure 6.

10

51

 

 

 

 

 

 

 

 

 

 

 

 

 

 

1 I _l I 25222:
20 3

O 10
% ACTIVITY IN MICROSOMAL FRACTION

EFFECT OF HOMOGENIZATION METHOD ON THE PERCENT OF NADPH-
CYTOCHROME C REDUCTASE ACTIVITY FOUND IN THE MICROSOMAL
FRACTION OF HOUSE FLY HOMOGENATES

The percent of reductase activity in the micro-
somal fraction was calculated on the total amount of
enzyme activity in the microsomal pellet and super-
natant. The percent activity in each pellet was
placed in the corresponding 5% interval. These results
are from 23 mortar-pestle (E23) and 30 glass-teflon (in!)

isolations.

 

 

 

52

Table 9
THE EFFECT OF THE PROTEASE INHIBITOR, DFP, ON THE NADPH—CYTOCHROME C
REDUCTASE ACTIVITY IN THE MICROSOMAL FRACTION OF HOUSE FLIES USING

THE GLASS-TEFLON R

HOMOGENIZATION METHOD

In each isolation house fly abdomens were separated into two
equal groups. One group was homogenized in 0.57 M Tris and the
other in 0.57 M Tris with 1 mM DFP, pH 8.0. When the 105,000 xg
centrifugation was completed, an aliquot of the supernatant was
carefully pipetted from the centrifuge tube. This aliquot was

used for the determination of the supernatant reductase activity.

The pellets were carefully drained before resuspending in Tris-

 

 

glycerol.
Isolation Treatmenta % Activityb Specific ActivityC
Tris 100 0.033
1
DFP 100 0.031
Tris 81 0.029
2
DFP 85 0.028
Tris 100 0.038
3
DFP 100 0.032

 

a
Treatment = 0.57 M Tris i 1 mM DFP

Enzyme units in microsomal pellet

units in pellet + units in supernatant X 100

be
4 Activity =

c
SDeeific Activity = umoles of cytochrome c reduced/min/mg protein

 

 

 

53

This is further illustrated by the results of three isolations made to
determine the effect of DFP on the percent of the reductase retained
in the pellet and its specific activity (Table 9). The conditions were
the same for the control and the DFP-treated homogenate in each isola-
tion. Two of the isolations had 100% of the reductase activity in the
microsomal pellet, for both the treated and control, while the third
isolation showed respectively, 85% and 80% of it in the pellet. This
was typical of the results obtained with the GT method using two other
buffers with or without DFP (Tables 4, 5). Evidently, the protease
inhibitor did not significantly increase the amount of reductase retain-
ed in the microsomal fraction or its specific activity.

From the above results, it can be concluded that under the condi-
tions utilized for each method, GT homogenization gave preparations
with much more of the reductase activity in the microsomal fraction.
Treatment with phospholipase or protease inhibitors did not give re-
sults that differed significantly from those obtained with the controls.
This was true for each method. However, even though microsomal fractions
from the GT preparations retained an average of 80% of the NADPH-cyto-
chrome c reductase activity in the microsomal pellet and supernatant,
the aldrin epoxidase activity of the homogenate and microsomal fraction
was not stable. The homogenate lost 68% of its original aldrin epoxi-
dase activity within 30 minutes after homogenization. The addition of
DFP, a protease inhibitor, inhibited aldrin epoxidation by 62%, but the
activity of the DFP-treated homogenate showed little change during the
60-minute sampling period (Fig. 7). The microsomal fraction, when stored
as specified under Materials and Methods, lost 50% or more of the aldrin

epoxidase activity within approximately 24 hours (Table 10).

 

 

 

 

Figure 7.

54

125

 

100‘
>.,
E‘
P—i
>
H
E3
.< 75‘
LL]
(I)
<
G
1—4
><
O
0..
LL]
2. 50 4
1—4
CZ
Q
._.1
<
25 4
O v v .

 

 

0 15 30 45
MINUTES AFTER HOMOGENIZATION

THE STABILITY OF ALDRIN EPOXIDASE ACTIVITY IN GLASS-TEFLON
HOMOGENATES OF HOUSE FLY ABDOMENS

Equal quantities of the abdomens were homogenized in
0.2 M K2HPO4,
plus 1 mM DFP ( ------- ). Aliquots for aldrin epoxidation

 

pH 8.0 (- . ) and 0.2 M K2HP04, pH 8.0
assays were taken after homogenization at the times in-
dicated and preincubated at 210 C for 5 minutes in the

assay mixture. Aldrin was added to initiate the reaction.

 

 

 

 

 

55

Table 10
MIXED-FUNCTION OXIDASE ACTIVITY IN MICROSOMAL FRACTIONS ISOLATED FROM
HOUSE FLY HOMOGENATES TREATED WITH EITHER CATECHOL OR CYANIDE
House fly abdomens were homogenized by the glass-Teflon method in
either 0.2 M K HPO

pH 8.0 or in 0.2 M KZHPO with 10 mM KCN, pH 8.0.

 

 

2 4’ 4
The preparation treated with catechol was homogenized in 0.2 M KZHP04,
pH 8.0, with catechol being added to the 10,000 xg supernatant.

NADPH-cytochrgme Cytochrome Aldrin

Treatment c reductase P-450b epoxidationC
CONTROL 0.095 0.197 0.046e 0.020f
Catechol 0.081 NDd 0.067e 0.029f
KCN 0.124 0.397 0.237e 0.185f

 

a umoles of cytochrome c reduced/min/mg protein

b nmoles of cutochrome P-450/mg protein

c nmoles of dieldrin produced min/mg protein
d Not detected

e Assays done as soon as the microsomal fraction was isolated

f Assays done the day following the isolation

 

 

 

56

Effect of Endogenous Tyrosinase on House Fly Microsomal Enzymes:

One isolation was made to see if the stimulation and inhibition of
tyrosinase activity in house fly homogenates would affect the activity
on stability of microsomal enzymes from the house fly. Catechol was
added to the 10,000 xg supernatant of a glass-Teflon abdominal homo-
genate, since it is known to stimulate the hydroxylation of tyrosinase,
and cyanide, an inhibitor of tyrosinase, was added to another (Table 10).
Addition of catechol reduced two of the parameters assayed when compared
to the control. NADPH-cytochrome c reductase activity was reduced by
15%, and the preparation had no detectable cytochrome P-450 content.
Aldrin epoxidation was slightly higher than in the control. The NADPH-
cytochrome c reductase activity in the cyanide-treated preparation was
30% more than in the control and 35% more than that observed in the
catechol-treated preparation. The aldrin epoxidation of the cyanide—
treated preparation was 5 times that detected in the control and 3.5
times that of the catechol-treated isolation.

Of significant interest was the fact that the control and catechol-
treated isolations lost 56% of their aldrin epoxidation activity by the
next day. For the cyanide treatment the loss of activity in one day
was 22%. Assays made 3 weeks later gave the same amount of aldrin
epoxidation as observed the day after the isolation. These results
suggest that the phenol oxidase complex did decrease the activity of

microsomal enzymes.

 

 

57

Effect of Mushroom Tyrosinase on Rat Liver Microsomal Enzymes:

 

Using catechol as the substrate tyrosinase activity was detected
in house fly microsomes but not in the rat liver microsomal prepar-
ation. The effect of mushroom tyrosinase plus catechol on rat liver
microsomes was examined to see what effect exogenous tyrosinase would
have on their enzymatic activities.

Looking first at NADPH-cytochrome c reductase, increasing concen-
trations of tyrosinase, in the presence of catechol, gave a correspon-
ding decrease in reductase activity (Fig. 8). Activity decreased with
time following an initial lag period (Fig. 9), with the lower concen-
tration of tyrosinase used in the incubations. The rate at which ac-
tivity was lost also increased with a rise in temperature (Fig. 10).
Whereas at 4° C 10% of the activity was lost in 20 minutes, at 13° C
almost 50% of the original reductase activity was lost in 10 minutes.

The effect of tyrosinase on different microsomal parameters fol-
lowing a 22 hour incubation on ice is shown in Table 11. The reduction
of cytochrome c by NADPH-cytochrome c reductase was not affected by
catechol alone, but it was stimulated 12% by tyrosinase, when compared
to the control. Potassium cyanide reduced the reductase activity by
17%. In the presence of tyrosinase and catechol, the activity was
reduced from 0.204 down to 0.026 umoles of cytochrome c reduced per
minute per mg protein. The reductase activity was 26% greater than the
control for microsomes treated with cyanide-inhibited tyrosinase plus
catechol. The Carbon monoxide difference spectra were essentially
the same for all treatments except for the tyrosinase plus catechol
(Fig. 11). This treatment reduced the cytochrome P-450 to 36% of the

control value (Table 11). The difference spectrum of the tyrosinase

 

 

 

Figure 8.

58

THE EFFECT OF TYROSINASE CONCENTRATION 0N NADPH-CYTOCHROME
C REDUCTASE ACTIVITY OF RAT LIVER MICROSOMES

Liver microsomes (6.0 mg/ml) from PB-pretreated
rats were suspended in 0.1 M KZHPO4 with 2.5 mM cate-
chol and incubated on ice for 15 minutes. At the end
of the incubation period, the reductase activity was
assayed. The following concentrations of tyrosinase
were used:

1. 0 pg tyrosinase/mg microsomal protein

2. 0.31 pg tyrosinase/mg microsomal protein

3. 0.62 pg tyrosinase/mg microsomal protein

4. 1.25 pg tyrosinase/mg microsomal protein

5. 2.50 pg tyrosinase/mg microsomal protein

 

 

59

100

 

 

 

m0 mu mu mu

8 r0 4 2
pr>wuom chwmwco Go pcmogma

xpw>wuom wmmpusuwc o meoczoouxonzao<z

2.5

.5 2.0

1

1.0

0.5

pg TYROSINASE/MG RAT LIVER

MICROSOMAL PROTEIN

Figure 8

 

 

 

 

 

Figure 9.

60

THE EFFECT OF TYROSINASE PLUS CATECHOL 0N RAT LIVER
MICROSOMAL NADPH-CYTOCHROME C REDUCTASE ACTIVITY:
EFFECT OF TYROSINASE CONCENTRATION

Two levels of tyrosinase, 0.31 and 0.62 pg/mg
microsomal protein, were added to liver microsomes
HPO

2 4
with catechol (5mM), pH 7.0. The rate of inhibition

(6.0 mg/ml) from PB—pretreated rats in 0.1 M K

was followed by assaying the reductase activity at

five or ten minute intervals.

 

61

 

0.31 pg

 

 

g
u
0
A
g
M.
2
6
0
q a q q
0 0 0 0 O
O 8 6 4 2
I

xpw>wpom chwmwgo we pcmocma
xpw>wpom mmmposumc o mecc200p>ouzao<z

60

45

3O

15

MINUTES

Figure 9

 

 

   

 

Figure 10.

62

THE EFFECT OF TYROSINASE PLUS CATECHOL ON RAT LIVER
MICROSOMAL NADPH-CYTOCHROME C REDUCTASE ACTIVITY:
RATE OF INHIBITION

Liver microsomes (6 mg/ml) from PB-treated

rats were suspended in 0.1 M KZHPO with 5 mM

4
catechol, pH 7.0. They were preincubated for

10 minutes at 4° C and 13° C before adding

tyrosinase (0.31 pg/mg microsomal protein). The
activity of the reductase was assayed at 5 or 10
minute intervals. Reductase activity did not decrease

when microsomes were incubated alone, with catechol

or with tyrosinase.

 

 

63

4° C

13° C

 

 

100‘

i
0
8

20<

C 1
0 0
6 4

xuv>wuom _mcwmwco we pcwocma
zu_>_pua mmmposuwc u mEog;ooy>o-xmm<z

6O

45

3O

15

MINUTES

Figure 10

.AzE w.mv Fogumumu vcm ACmeoga
_maomogure me\m: Pv mmmCPmogzp umpwnwccw Ase w.ov mvvcmzu mng mco :F m< .0
.A25 w.Nv _ocomumo ncm Acwmpoca FmEomoLqu me\ma Fv mmmcwwocxp mspa mco :F m< .m
.Aze m.ov muwcmxu Ezwmmmpoa m3_a wco :F m< .v
.Azs w.Nv Foguwpmu m:_a mco :_ m< .m
.AchpoLa FmEOmogowE me\m1 Fv wmmcwmoccp mDFa mco CF m< .N
.Azm &_oo.o meccwmucou 0.4 In .qoaxmx z ”.0 E2 A_E\me m.ov mmEOmocuwz .—

.:oTpomm muocumz use _mwcwpmz mcu :F um_w

64

.?qum we mcou mew: mzwmm< .mczo: mm um umCWmuno mmosu mew mew; czozm mp_:mmc
one .uowcma COApmn:OCw msu mcwczv mme?p pcmcmm$wv “a uwxmmmm mew; cowpmu
-wxoam crew—m new xpw>wpum mmmposumc u mEoc:uoa>u-:ao<z .Om¢-m msoczuouau
.08? co meson mm Low umpmnzu:_ new 0.x In
.qoaImx z F.o Cw umucmamsm mew: mua; umwmmcpmca-ma Eogw mmEOmocowE Lm>w4
mk<m omp<mmkmmm-4<HHmm<mozmza zomm mmzom
-oaqu mm>H4 no monHuzzm UHH<2>sz oz< omqum mzomIUOH>u zo mm<szom>H mo Hummum .FF OanH

 

65

ewepeee mE\cwE\eee:eeLe :wLe—ewe we meweEc

 

 

e

cwmuece mE\eEeL:eeu>e we mewesc e

ewepeee mE\:wE\eee:eec e meecceepae we mewese e

N._ _.N wmm.e zex + weeeeeme + emmcwmecse .e

e m.e eme.e _e;eeeee + eme:_meexe ..m

w.w _.N om_.o zux .e

w.m N.N eom.o weceeuee .m

N.N m.m mmm.o emecwmecxu .N

_.N N.N eom.o weeeeee eepeeepc: ._
Usfiwmgu gamma . gammmmfiez :39:

 

FF m_nww

66

plus catechol-treated microsomes had a small shoulder at 420 nm in 30
minutes, but with time it did not increase in proportion to the decrease
of absorbance observed at 450 nm, as it had been reported to do in the
presence of proteolytic enzymes (94).

Aldrin epoxidation was the same for control, tyrosinase-treated, and
catechol-treated microsomes (Table 11). The cyanide and cyanide-
tyrosinase plus catechol treatments reduced the epoxidation from 2.1
down to 1.7 nmoles of dieldrin produced per minute per mg protein,

a 20% decrease. Tyrosinase plus catechol completely inhibited aldrin
epoxidation.

Another effect of tyrosinase on membranes was observed in an ex-
periment using erythrocyte lysis as an indicator of the occurrence of
free radical reactions. The 1200 xg supernatant of washed red blood
cells had a 3.5-fold more hemoglobin than red blood cells alone or red
blood cells plus tyrosinase or catechol, following centrifugation after
a 15-minute incubation with tyrosinase and catechol at 0 to 5° C. SDS
polyacrylamide gel electrophoresis of rat liver PB induced microsomes
incubated with tyrosinase indicated that cross linkage of the proteins
had occurred, since much of the sample failed to migrate into the gel
(Fig. 12). A sample of microsomes incubated with cyanide-inhibited
tyrosinase when electrophoresed gave the same banding pattern as un-

treated rat liver PB microsomes.

Figure 11.

67

EFFECT OF MUSHROOM TYROSINASE 0N CYTOCHROME P-450 0F
LIVER MICROSOMES FROM PHENOBARBITAL-PRETREATED RATS
Liver microsomes from PB-pretreated rats were

suspended in 0.0 M KZHP04, pH 7.0 and incubated for

22 hours on ice. Microsomal samples, 1.3 mg protein,
were flushed with carbon monoxide, reduced with sodium
dithionite, and scanned at different times during the
incubation. The treatments are the same as l and 5
listed in Table 11. The other treatments did not dif-
fer significantly from the spectrum of 1 during the

incubation period.

ABSORBANCE

68

 

 

 

 

450 nm 390 nm 450 nm 390 nm
- 0.2 OD
--0.1
0 HOURS
0
T
- 0.2
0.5 HOURS -0.1
V
- 0.2
22 HOURS
- 0.1
0
1.-
MICROSOMES ALONE MICROSOMES + CATECHOL

+ TYROSINASE

Figure 11

 

 

69

.ewesem empeecpce one we eEem esp ewe; meeemecewe eepeeepnweceepee use
-emeCwmeexu mew .Aomv cepwez he eeeweemee we mwmegeseecueewe Fem eewEe
-wxceexwee-mom Lew emceeege ewe; mmwesem ecw .u oe we mepecwe mp Lew
A22 m.mv wegeepee Le\ece Aswepece wesemegews ms\mn Fm.ov emeCwmecxp
new; eeueeeecw wee; Awe\me 0.0V mesemecewe eeeeecw1me Le>ww pea
mw<m omw<mmwmmeume
some zowwo<md e<zomomowz mm>He mzw no meweome szHome mwmmmozeomwumem
ewe mowz<e>mu<>eoe-mom mzw zo wzmzw<mmw eozuuw<o az< mm<zwmoa>w do womeam mzw

.N_ eagewa

70

N_ eE:e_e

Azee mez<wmee zeew<meez

 

mmpm<

 

macemm

 

 

 

 

 

rm.o

 

 

o.—

( 0'0) mu 039 19 aoueqaosqv

DISCUSSION

The methods of quantitative cell fractionation introduced by
Albert Claude (107) represented a fundamental change in the prepar-
ative fractionation of tissue homogenates. It shifted the emphasis
from the purification of a given visible intracellular entity to a
quantitative analysis of the enzymatic activities present in the ori-
ginal homogenate. This approach focused on the actual objects of
analysis, the enzymes or other biochemical components, and examined
the manner in which these entities were distributed between all the
fractions separated from the homogenate. The focus on enzymatic ac-
tivities and their quantitative recoveries provided a powerful tool
for the study of any such cellular component not irretrievably lost
in the initial homogenization of the cells.

Even though the fractionation procedures allow an almost infin—
ite number of systems and enzymes to be studied, it is not easily
adapted to a diffuse variety of tissues. The majority of this work
has been focused on mammalian tissues, such as the liver. The wealth
of information obtained by the fractionation of liver homogenates has
enticed researchers to apply cell fractionation to a wide variety of
other tissues and systems. It is apparent from the literature that
the analysis of insect preparations lacks the thoroughness of the
mammalian studies. Consequently, assumptions have been made concern-

ing the stability and homogeneity of insect preparations which cannot

71

72

be supported by this worker's research on house fly in_vjtrg_preparations.

When this research was first begun, Morello and co-workers (96) pub-
lished one of the first articles to deal extensively with microsomal cyto-
chrome P-450 of house flies and in this they advocated homogenizing house
flies by the mortar-and-pestle (MP) technique when isolating the micro-
somal fraction. This method, according to these workers, gave the highest
microsomal naphthalene hydroxylation activity per nmole of cytochrome
P-450 hitherto reported, and less contamination of the microsomal fraction.
The MP method gave less than half of the microsomal protein per 10 g of
flies as compared to microsomal fractions prepared from SorvallR —homogen-
ized whole flies. This result was interpreted by these workers as indi-
cating the attainment of "clean" microsomes, free from the sarcosomal
contamination which would interfere with the analysis of cytochrome P-450.
This technique allowed the use of whole flies, without the necessity of
separating the abdomens.

The NADPH-cytochrome c reductase activity of the microsomal fractions
from homogenates prepared by glass-Teflon (GT) homogenization of abdomens
was compared by this investigator with that of the MP whole house fly
preparations. In two isolations, the specific activity of microsomal
NADPH-cytochrome c reductase average was slightly lower for the MP micro-
somal fractions at 0.060 pmoles of cytochrome c reduced/min/mg protein,
than that of the GT preparations at 0.072 pmoles (Table 1). This dif-
ference did not seem great enough to justify the three to four hours
required to separate and collect the large numbers of abdomens required
for each isolation.

Having made this assumption, a number of isolations were made with

house flies at various developmental stages using the MP method to see

73

if the reductase activity followed the same fluctuations within these
various stages as was shown for naphthalene hydroxylation. Naphthalene
hydroxylation by house fly microsomal fractions showed a peak of activ-
ity just prior to larval pupation. The level of activity was very low
during the pupal stage and increased again after ecdysis, with the high-
est level of enzymatic activity occurring 10 to 12 days later (108). It
had been thought that the last larval instar might provide a good source
of enzyme activity, thereby eliminating the time-consuming process of
rearing the flies to their adult stage. However, the microsomal NADPH-
cytochrome c reductase specific activity from 94 isolations ranged as
much as 10-fold for a specific age (Table 1). Since these preparations
were made from whole insects, it was then thought that a cleaner micro-
somal fraction would give more consistent results.

At this time, differential and density gradient centrifugation
studies were conducted in this laboratory to see if a combination of
these methods might reduce the observed variability. They revealed
that most of the reductase was being solubilized during the isolation
procedure. When the use of phospholipase and protease inhibitors
failed to prevent solubilization, it was realized that a new isolation
procedure would have to be established in order to isolate the house
fly microsomal fractions. This procedure utilized the GT homogeniza-
tion of abdomens only (46), and buffers of 0.57 to 0.60 ionic strength
(109). The results obtained by this method contradicted those obtained
with the MP method (Fig. 6, Table 7).

In establishing a reliable isolation procedure, the heterogeneity
of the mixture subjected to further fractionation must be given careful

Consideration. A homogeneous tissue possesses a cell population which

74

contains a variety of intracellular particulate entities that form a
number of distinct populations upon homogenization. Heterogeneous
tissues normally composed of many cell types multiply the complexities
of cellular fractionation. Whole or abdominal house fly homogenates
contain greater numbers of complex elements not present in liver homo-
genates, yet to date the isolation procedure for house fly microsomes
has remained essentially the same as that used in the isolation of

liver microsomal fractions. Although there are vast differences in
composition between the insect and liver homogenates this does not mean
a_prigri_that the procedure will differ, but careful quantitative frac—
tionation studies are needed to determine whether or not the established
procedure used to isolate liver microsomes is the correct one for insects.

The term microsome is a biochemical one designating the subcellular
fraction of membranes Obtained by high speed centrifugation of the mito-
chondrial supernatant. It does not refer to a specific organelle as do
the terms mitochondria or lysosome. Rather, the microsomal fraction con—
tains membranous elements such as endoplasmic reticulum and ribosomes,
originating from a heterogeneous population of cells with the quantity
of each component determined by the isolation procedure. Therefore, the
optimal conditions for isolating the microsomal fraction from each type
of tissue and/or insect must be thoroughly established, since each may
contain elements that uniquely affect the isolation of this fraction.

The analytical procedure that defines the most optimal isolation
conditions involves the quantitative analysis of marker enzymes and
their distribution patterns in the fractions obtained by differential
Centrifugation of the homogenate. The procedure resulting from such

Studies incorporates individual methods that give the best overall

75

results for enzymatic stability, purity and yield. The distribution
study of marker enzymes in fractions from rabbit lung and liver il-
lustrates how such results can aid in establishing a procedure that
gives the fraction possessing the greatest specific activity of micro-
somal enzymes and the least amount of contamination by other enzymes
or cellular components (36). In isolating the microsomal fractions
by differential centrifugation from house cricket Malpighian tubules,
it was found that most of the microsomal activity was obtained at
less than 12,000 xg. A two—step density gradient procedure was found
to provide a means of isolating a fraction containing most of the
pesticide metabolizing activity. However, this isolated fraction was
unstable and rapidly lost its enzymatic activity (40).

Though several excellent studies have been made on the enzymatic
distribution patterns using marker enzymes and taking into account
morphological characteristics of subcellular fractions from rat liver
homogenates (107), quantitative studies of this type have yet to be
done for insect preparations. In the few studies that have analyzed the
enzyme distribution in subcellular fractions of insects, pesticide meta-
bolism alone has been used as the sole indicator of the distribution of
microsomal enzymes. These studies assume that the mixed-function oxi-
dase complex was a complete and functional unit whenever present in a
fraction. Pesticide metabolism, which serves as the primary indicator
of the distribution of insect microsomal enzymes, fails to detect the
location of membranous fragments containing an incomplete complement
0f the mixed-function oxidase system. Therefore, the use of a complex
System drastically reduces the resolving power that could be attained

My using a marker enzyme, consisting of one functional component. Even

76

when several single enzyme species are employed as markers to assess the
composition of each fraction, great caution must be exercised in evalua-
ting their distribution patterns, since the resolving power of the
techniques used is rarely commensurate with the complexity of the mater-
ial being analyzed.

The resolving power of the separation procedures presently available
greatly depends on differences in such physical properties as particle
size, shape, density, and the resulting sedimentation coefficient. In
some instances, factors such as electrical charge or solvent affinity
can even influence the isolation of a component. These isolation pro-
cedures are severely limited, however, by the necessity of preserving
the structural and biochemical integrity of the components during the
utilization of these methods. They are further complicated by the fact
that the populations of these particles are not composed of identical
individual members as is the case in isolating a single species from
a chemical mixture.

Several artifacts may occur during the isolation of subcellular
fractions and can arise from the disruption of cellular organelles,
the adsorption of soluble material onto the organelles and cellular
particles, and enzyme inhibition or activation. The use of complex
enzymatic systems to determine the distribution pattern of an enzyme
can also distort the results. Quantitation of the fractionation pro—
cedure keeps a complete record of the original level of enzymatic
initivities, their distribution patterns and their recoveries enabling
thee investigator to detect any of the problems mentioned above. When
inflibition or activation does occur, the recombination of fractions

car. assist in determining if the inhibitor or activator is associated

 

 

 

77

with an organelle or is soluble.

The presence of endogenous factors causing loss of enzymatic activ-
ity can also be detected by this procedure. The occurrence of adsorption,
inhibition, solubilization, and/or decay of enzymatic activity can alter
the apparent distribution pattern of an enzyme so that the major propor-
tion of its activity is mistakenly associated with the wrong fraction.
Fig. 4 shows how solubilization can alter the distribution pattern of
an enzyme. The recovery of 89% of the house fly NADPH-cytochrome c reduc—
tase activity in the soluble fraction of MP homogenates suggested that
this enzyme in house flies is a soluble one. The rat liver reductase,
however, is tightly bound to the microsomal membrane with 70% or more of
the total homogenate activity recovered in the microsomal fraction, simi-
lar to the distribution pattern of glucose-6-phosphatase (35). The dis-
tribution pattern of the house fly reductase was assumed to be an arti-
fact of the MP homogenization method; the results from GT isolations
confirmed this assumption (Fig. 6, Table 7).

Removal of an organ that contains the desired enzyme(s) prior to
homogenization is a major purification step in the isolation procedure.

In the case of insects, their small size usually prevents this simple
step. The studies conducted with the southern armyworm and the house
cricket illustrate how the utilization of a single organ, as opposed to
the whole insect, increased the enzyme activity of the preparation. The
\Nhole larval armyworm and cricket homogenates had no detectable epoxida-
tion activity. Homogenates of the fat body, Malpighian tubules and washed
gut all epoxidized aldrin, with the highest activity being found in the
Inicrosomal fractions of the armyworm midgut and cricket Malpighian tubules.

Careful evaluation of the isolation procedure is necessary because

 

 

 

 

78

the procedure can have the effect of combining subcellular components as
well as fractionating them. Subfractionation of the rat liver mitochondrial
fraction has revealed two distinct types of minor components, lysosomes
and microbodies. The result of this subfractionation was that only 7 of
the 18 enzymes previously thought to be mitochondrial were actually assoc-
iated with that fraction (105). The association of mitochondrial with
microsomal activity in peak II of Fig. 3 may have been a result of the
densities employed in the discontinuous sucrose gradient.

Homogenization of the tissue followed by centrifugation produces
an unnatural environment for the cellular components, as well as the
mechanical stress of centrifugation. These conditions often result in
the leakage of soluble entities from their organelles followed by their
readsorption onto the surface of other cellular particulates. Whenever
tissues are homogenized, all the barriers within a cell that separates
the cellular constituents are destroyed, placing the enzyme(s) in what
may be a distinctly foreign or hostile environment. This is certainly
true when whole insects or their body parts are homogenized, as indi-
cated by the numerous reports concerning the endogenous inhibitors of
insect microsomal preparations and their instability. Microsomal
fractions from whole armyworm and cricket homogenates failed to metabo—
lize pesticides; this was considered to be due to the solubilization of
the NADPH-cytochrome c reductase by an endogenous protease (43,44). In
homogenates of the house fly abdomens containing 1 g of abdomens per
4 m1 of buffer, the mixed-function oxidase activity decreased by 80% in
3OIninutes at 00 C (38). The exact factors causing this decay have yet
to be isolated and identified. Results from the MP isolations reported

here demonstrated the solubilization of NADPH-cytochrome C reductase

 

 

 

 

 

79

by a factor(s) which also have yet to be identified.

The distribution of NADPH and oxygen-requiring DDT metabolism was
examined in what is apparently the only study published showing the
distribution of enzyme activity in subcellular fractions from MP homo-

genates (110). Whole German cockroaches, Blattella germanica (L.),

 

were homogenized with a mortar-and-pestle, and the subcellular fractions
were obtained by differential centrifugation. Forty-five percent of the
enzymatic activity was found in the microsomal fraction and the remaining
34% of the enzymatic activity in the nuclear and mitochondrial fractions.
Since the activity recovered in the microsomal fraction was only 11%
more than that recovered in the other two fractions, the procedure failed
to fractionate the desired enzymatic activity into an enriched fraction.
In assaying for a complex enzyme system, the procedure also failed to
indicate the complete distribution of microsomal membranes.

The distribution of the marker enzymes, cytochrome oxidase (Fig. 3)
and NADPH-cytochrome c reductase (Fig. 4) in the subcellular fractions
of whole house fly MP homogenates also failed to give an enriched frac-
tion of either enzyme that contained a significant proportion of the total
protein. These results from the cockroach and house fly MP homogenates
differed significantly from the results obtained with rat liver homo-
genates. The distribution of the rat liver microsomal enzymes, NADPH-
cytochrome c reductase, glucose-6-phosphatase and aminopyrine demethylase,
showed 70% of the total homogenate activity in the microsomal fraction
(35). This difference might best be explained as the result of using
whole insects, which would reduce the relative proportion of protein con-
tained in the enriched fraction. However, the obvious solubilization of

NADPH-cytochrome c reductase in MP homogenates in contrast to the GT

 

 

 

 

 

80

preparations, would indicate that the difference is a result of the
method used. The failure to isolate a given enriched fraction contain-
ing the desired enzyme may also be related to aggregation caused by
endogenous tyrosinase and phenols, which have been shown to cross-link
proteins. The association of the microsomal with the mitochondrial
enzymatic activity in the density gradient (Fig. 5) may have resulted
from aggregation.

Another difference between rat liver and insect preparations is
apparent when one considers the amount of initial enzymatic activity
recovered in the fractions. Whereas in the cockroach and house fly
isolations only 79 and 64%, respectively, of the total enzymatic ac-
tivity of the homogenate was recovered, the recoveries for the three
rat liver enzymes mentioned above averaged 100%.

The failure to recover enzyme activity in the insect fractions may
be due to several reasons such as incomplete breakage of cells or loss
of enzymatic activity through inactivation by endogenous factors relea-
sed during homogenization. The decay of enzyme activity detected in
house fly abdominal homogenates (38) (Fig. 7) would account for at least
part of the failure to recover enzymatic activity. The solubilization
of NADPH-cytochrome C reductase is also known to destroy microsomal
drug metabolism (44). If solubilization occurred in the cockroach MP
preparations, as it did in the house fly MP isolations, it could have
resulted in the failure to recover 21% of the total homogenate DDT-
metabolizing activity in the fractions.

Even though eye pigments had been reported previously as endo-
genous inhibitors of mixed-function oxidase in house fly microsomal

preparations (38,49), the high rate of naphthalene hydroxylation by

 

 

81

MP preparations indicated that the pigment was not released during homo-
genization (96). However, subsequent work found that xanthommatin
would inhibit house fly microsomal aldrin epoxidase at concentrations

7 M (50). In this research, MP homogenization of

as low as 5 x 10'
whole flies would occasionally show the red smear of a crushed head

on the side of the mortar. Even though the homogenization was done
gently, there would be isolations when the low speed centrifugation
yielded a pellet containing a zone of red pigment, thus suggesting
that ommatidia had been broken during homogenization. These obser-
vations indicate that another of the difficulties encountered between
one MP preparation and the next was the maintenance of a consistent
degree of homogenization. The solubilization of NADPH-cytochrome c
reductase, the erratic distribution patterns Of marker enzymes from

MP preparations, and the low recovery of enzymatic activity would all
indicate that the MP homogenization technique of whole flies is not

a suitable method for isolation of the house fly microsomal fraction.
These findings also indicate that this isolation procedure had been
adapted for insect studies without the experimental information needed
to properly evaluate its suitability.

The next major problem to be considered was the dramatic loss of
mixed-function oxidase activity within a relatively short period of
time for the homogenate and microsomal fraction of house flies. Be-
cause of this rapid decay of microsomal activity during the isolation
procedure (38), it was not feasible to study the distribution patterns
of marker enzymes for the determination of an optimum isolation pro-

cedure. The microsomal fraction isolated from GT homogenates was

unstable when stored (Table 10) under the conditions listed in the

 

 

 

82

Materials and Methods. Storage of microsomal fractions from house flies
in dilute solutions containing BSA prevented the loss of enzymatic activ-
ity (111). This does not, however, indicate what the factor(s) was that
produced the observed instability. BSA is known to be an alternate pro-
tease substrate, and also protects enzymes from inactivation by lipids
(112) or phenols (113).

Therefore, an important question is: What could be present in
house fly microsomes but absent from rat liver microsomes that would
cause instability? It has been reported that potassium cyanide added
to house fly microsomal incubations stimulated pesticide metabolism
by 15 to 25% (56,57,58). Similar concentrations of KCN inhibit rat
liver microsomal metabolism by 15% (Table 9). Tyrosinase, which is
inhibited by KCN, has not been detected in goat liver microsomes (57)
nor was it detected in rat liver microsomes by this researcher. The
microsomal supernatant of house fly abdominal homogenates, which had
6 times the tyrosinase activity detected in the pellet, inhibited
pesticide metabolism when added to house fly microsomal incubations
(58). As mentioned earlier, the stimulation of this enzyme's activity
decreased aldrin epoxidation in preparations from lepidopterous larvae
(52,60). Jordan and Smith (38) also Observed that KCN prevented the
decay of mixed-function oxidase activity in house fly homogenates.

Even though KCN is known to inhibit a large number of enzymes (59),

an isolation was made to see if the addition of catechol, which stim-
ulates tyrosinase hydroxylation activity, and KCN, which inhibits
tyrosinase, would respectively decrease and increase microsomal enzyme
activity when compared to the control. As seen in Table 9, the results

fulfilled the expectation. These results suggested that catechol

 

 

 

83

oxidase and its substrates adversely affected microsomal enzymes.
Since rat liver microsomes did not contain any detectable tyrosinase
activity, they provided a system for the evaluation of the effects of
exogenous tyrosinase on microsomal mixed—function oxidases.

The effect of tyrosinase and catechol on the three microsomal
constituents examined were quite dramatic. During the incubations
of rat liver microsomes with tyrosinase and catechol, the turbid
microsomal suspension underwent a visible clarification. A decrease
in turbidity of the microsomal suspension has been observed during
lipid peroxidation (92). The loss of NADPH—cytochrome c reductase
activity occurs during lipid peroxidation (93) as well as during the
tyrosinase and catechol treatments. When microsomes were incubated
with a protease, the reductase activity was observed to increase
significantly before showing a decrease. Also during proteolysis,
the cytochrome P—450 peak shifted to 420 nm as the 450 nm peak de-
creased (94). After the tyrosinase plus catechol incubation was
started, cytochrome P-420 began to appear within a few minutes, but
failed to increase as the absorbance at 450 nm decreased. In a study
of cytochrome P-450 degradation during lipid peroxidation (92), the
spectral changes observed with time resembled those observed when
the rat liver microsomes were incubated with tyrosinase and catechol
(Fig. 8).

As reviewed earlier, lipid peroxidation has been shown to be a
free-radical process. It is presently unclear as to whether or not
the semiquinone free radical forms of the phenols are the substrates
for tyrosinase. It has been suggested that semiquinones are involved

in the oxidation-reduction interactions of phenols with quinones

 

 

 

84

(Fig. 2), but this has yet to be rigorously established. The observed
lysis of erythrocytes in the presence of tyrosinase and catechol would
indicate that free radicals are involved in the degradation of micro-
somal enzymes when tyrosinase and the phenol, catechol, are present.
Experiments using red blood cells to detect free radicals, tyrosinase,
and other phenols with more stable semiquinones could more thoroughly
examine the role that free radicals might play in tyrosinase function
and in the oxidation-reduction of phenols and quinones.

Research done on the inhibition of plant enzymes during their
isolation in subcellular fractions by phenols and quinones (114,115)
have indicated some ways that this problem can be eliminated. The uti-
lization of polymers such as polyvinylpyrrolidone to trap quinones
in the plant homogenates gives preparations with much higher enzymatic
activities than those prepared without the polymer (116). Other agents,
such as BSA, which react with quinones also provide preparations with
higher enzymatic activities than those without (115). The inclusion
of reagents that trap quinones during the isolation of phenyl oxidase
from tea leaves, resulted in the enzyme being found only in the soluble
fraction (117). It would appear that the presence of tyrosinase in
other fractions was an artifact resulting from the binding of tyrosinase
to membranes that sedimented in the different fractions. Examination of
this point in future experiments with house fly preparations might yield
the same results. If this should be the case, then the utilization of
reagents to protect enzymatic activities from inactivation by tyrosinase
and phenols would yield a microsomal fraction free from these contaminants.
Such procedures might provide a way to prepare a stable microsomal frac-

tion from house flies. Studies along these lines would be important not

 

 

 

85

only to researchers interested in mixed-function oxidases, but to all
those doing in_vitrg_analysis with insect material.

Obtaining a stable preparation would also permit workers to analyze
the isolation procedure quantitatively, providing important information
basic to the rational use of subcellular fractions from insects. There
are obviously many problems facing the investigator when attempting such
an analysis on homogenates of most insects, but such work is impossible
when the preparations are unstable.

The importance of stability cannot be over emphasized. In prepar-
ations that are not stable, the degree of instability is often variable
depending on the quantity of the unknown factor(s) causing instability
released during each isolation. Under such unstable conditions, studies
to determine the Optimum isolation procedure cannot be undertaken. When
the differential studies of whole house fly MP homogenates were being
made, each isolation varied in the distribution of cytochrome oxidase
and NADPH-cytochrome c reductase.

Welton (15) examined the effect of proteolysis on rat liver pheno-
barbital-induced microsomes in the presence and absence of BHT, an inhibi-
tor of lipid peroxidation. When microsomes were incubated at 25° C for
60 minutes in the presence of trypsin and BHT, approximately 25% of the
microsomal protein was solubilized into 105,000 xg supernatant of the
post-treatment centrifugation. SDS polyacrylamide gels of trypsin-treated

125I-labelled microsomes, centrifuged down after treatment, Showed a $19-

125 e 125

nificant alteration of the protein and I profiles. Th I labelling

had a more dramatic decrease than the protein profile. The destruction

of the microsomes increased when proteolysis was carried out in the ab-

125

sence of BHT with the I remaining bound to the proteins falling

 

 

86

essentially to zero. In another experiment, when the peroxidase label-

ling of rat liver microsomes with 125

I was performed with and without

BHT present, the results showed that the presence of BHT increased by

100% the amount of label incorporated. The BHT also completely inhibited
lipid peroxidation as measured by the malondialdehyde assay. This data
clearly demonstrates the importance of maintaining the integrity of the
microsomal membrane and its enzymatic functions during experimental pro-
cedures. Lipid peroxidation alone decreased iodination by half, and

when allowed to occur during proteolysis completely disrupted the integrity
of the microsomal membrane, as shown by the complete inhibition of
iodination and the severe alteration of the gel protein profile.

At this point, it is uncertain how many degradative and/or inhibitory
factors are present in house fly preparations. The combined effect of
proteolysis and lipid peroxidation on rat liver microsomes should sug-
gest to researchers working with insect preparations that the solution
of one problem may only reveal another. It is obvious that much remains
to be done to characterize the factors affecting the integrity and sta-
bility of insect microsomal fractions.

Armyworm preparations appear to be stable and can be stored without
the loss of aldrin epoxidation activity or degradation of cytochrome
P-450 (118). In this case, the preparations are made from the larval
midgut which reduces the heterogeneity of the homogenate. The abrasive
action of chitin which may also degrade enzymes and which is unavoid-
able in glass-Teflon homogenates of whole or parts of house flies, is
also eliminated. A quantitative analysis of this system using marker
enzymes for the various organelles identified in mammalian systems

would provide information which is clearly absent from insect biochemical

 

 

 

 

87

literature.

The work presented here concerning the effect of tyrosinase plus
catechol on microsomal mixed-function oxidases is only an introduction
into the effect of endogenous tyrosinase, phenols and quinones on the
stability and activity of the enzymes in insect homogenates. If these
compounds are among the primary causes of the notorious instability and
low enzymatic activity of most jn_vjtrg_insect preparations, numerous
avenues are open for a much fuller utilization of insecticide—resistant
strains. Careful experimentation that utilizes the biological differences
of house fly strains should help to further elucidate the molecular mech-
anisms of the mixed-function oxidase complex and its regulation. Such
biological and biochemical information would contribute greatly to our
understanding of this important system and, hopefully, contribute to a

more rational design of pesticides.

 

 

SUMMARY

During the isolation of the microsomal fraction by differential cen-
trifugation from MP homogenates of whole flies, the NADPH-cytochrome c
reductase was extensively solubilized. In 23 isolations, only 20 i 24.5%
5.0. of the 105,000 xg pellet and supernatant reductase activity was in

the pellet. Inhibitors of calcium dependent phospholipases, EDTA, and

 

proteases, phenylmethylsulfonylfluoride, did not decrease the solubiliza-
tion. In 30 isolations obtained from glass-Teflon homogenates of house
fly abdomens, 80.3 i 21.3% 5.0. of the reductase activity was recovered

in the microsomal fraction. The difference in percentage of the reduc-
tase isolated in the microsomal fraction by the two homogenization methods
was significant at the 0.01 level when tested for significance by the
Student's t-test. The specific activity for the MP preparations was

0.025 t 0.008 5.0. of cytochrome c reduced/min/mg protein and for the

GT preparations it was 0.050 t 0.025 pmoles 5.0. of cytochrome c reduced/
min/mg protein. These differences were significant at 0.05 level when
compared by the Student's t-test. Even though microsomal fractions iso—
lated from GT homogenates contained most of the reductase activity, the
aldrin epoxidase activity of the homogenates and microsomal fractions
from GT isolations was unstable. The homogenate lost 69% of its epoxidase
activity in 30 minutes and when stored at -20° C the microsomal fraction

lost 50 to 60% of its activith within 24 hours.

88

 

 

 

89

The catechol oxidase complex has been reported to cause loss of
insect mixed-function oxidase activity. When tyrosinase was inhibited
by cyanide in house fly homogenates, the levels of reductase activity,
aldrin epoxidation, and cytochrome P-450 content was higher than in
untreated preparations. The reductase activity of a catechol-treated
preparation was lower than the untreated control. Aldrin epoxidation
activity was the same as the control, but neither cytochrome P-450 nor
P-420 was detected in the catechol—treated sample. When liver micro-
somes from phenobarbital-induced rats were treated with tyrosinase and
catechol for 22 hours at 4 C, the reductase activity decreased to 13%
of control value, aldrin epoxidation was completely inhibited, and
cytochrome P-450 was reduced to 36% of the concentration detected in
untreated controls. Tyrosinase alone and catechol alone and cyanide-
inhibited tyrosinase and catechol did not degrade microsomal mixed-function
oxidases. The lysis of erythrocytes when incubated with tyrosinase and
catechol suggest that free radicals are involved in the loss of micro-
somal enzymatic activity. Incubation of red blood cells with catechol
alone or tyrosinase alone did not cause lysis. SOS-polyacrylamide gel
electrophoresis of microsomes treated with tyrosinase and catechol
showed that cross—linking of proteins had occurred during the degradation
process. Most of the sample stayed at the origin and failed to migrate
into the gel.

The effect of tyrosinase and catechol on rat liver microsomal mixed—
function oxidases indicates that endogenous tyrosinase complex present
in house fly homogenates causes the loss of enzymatic activity. The
use of inhibitors and polymers should eliminate the effect of tyrosinase,

phenols and quinones thus allowing the isolation of a stable microsomal

 

 

 

 

 

90

fractions from house flies. This will have to be examined carefully as
the resolution of this problem may only reveal that other endogenous
factors, such as proteases, also cause loss of house fly microsomal

mixed-function oxidases.

 

 

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