THESIS
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This is to certify that the

thesis entitled

THE PARTIAL PURIFICATION OF PARA-NITROPHENOL:
UDP—GLUCURONYLTRANSFERASE FROM RABBIT
SMALL INTESTIONAL MICROSOMES

presented by
Wanda W. Broderick

has been accepted towards fulfillment
of the requirements for

MS. degree in Biochemistry

MW

Major prgessor

 

 

Date August 13, I982

 

0-7 639

 

 

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THE PARTIAL PURIFICATION OF
PARA-NITROPHENOL: UDP-GLUCURONYLTRANSFERASE
FROM RABBIT SMALL INTESTINAL MICROSOMES

By

Wanda W. Broderick

A THESIS

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

MASTER OF SCIENCE

Department of Biochemistry

1982

Q

I

A,“

0117.

ABSTRACT

THE PARTIAL PURIFICATION OF PARAfNITROPHENOL:
UDP-GLUCYRONYLTRANSFERASE FROM RABBIT SMALL INTESTINAL MICROSOMES.
By
Wanda Woodward Broderick

The purification of rabbit small intestinal microsomal
pfnitrophenol: UPD-glucuronyltransferase might resolve many questions
about the enzyme including its substrate specificty and heterogeneity.
A procedure for the partial purification of pfnitrophenol:
UDP-glucuronyltransferase from rabbit small intestinal microsomes was
established, involving the preparation of intestinal microsomes, the
solubilization of pfnitrophenolz UDP-glucuronyltransferase from these
microsomes, and the DEAE column chromatography of the solubilized
enzyme. This procedure was part of a scheme to purify the enzyme to
homogeneity. Microsomes were obtained from the whole small intestine,
which had an average pfnitrophenol: UDP-glucuronyltransferase activity
of 24 nmoles pfnitrophenol conjugated per minute per mg of protein, and
which were stable for six months when stored at -20'C. The microsomal
solubilization process using octylglucoside increased the apparent
pfnitrophenol: UDP-glucuronyltransferase specific activity
approximately two fold, with a 752 recovery of enzyme activity. DEAE
chromatography of the solubilized protein resulted in a 10 fold
increase in pfnitrophenolz UDP-glucuronyltransferase specific activity

over that of the microsomes.

ACKNOWLEDGMENT

I would like to thank Dr. Steven Aust for his guidance and
support.

TABLE OF CONTENTS

List of Tables . . . . . . . . . . . . . . . . . . . . . .
List of Figures . . . . . . . . . . . . . . . . . . . . .
List of Abbrevations . . . . . . . . . . . . . . . . . . .

INTRODUCTION 0 C O O C O O O O O O O O O O O O O O

The Modulation of UDPGT Activity . . . . . . . . . . .
The Heterogeneity of UDPGT . . . . . . . . . . . . .

METHODS AND MATERIALS 0 O O O O O O O O O C O O O O O O 0

Material Sources . . . . . . . . . . . . . . . . . . .
Animals . . . . . . . . . . . . . . . . . . . . . . .
Isolation of Intestinal Microsomes . . . . . . . . . .
Assay of PNPGT Activity . . . . . . . . . . . . . . .
Assay of Phenolphthalein GT . . . . . . . . . . . . .
Protein and Phosphate Assays . . . . . . . . . . . . .
Solubilization of Microsomes and DEAE Chromatography .

Sodium Dodecylsulfate-Polyacrylamide Gel ElectrOphoresis

Extraction of Microsomal Lipid . . . . . . . . . . . .
Preparation of Liposomes . . . . . . . . . . . . . .
Enzyme Incubation with Liposomes . . . . . . . . . . .

RESIILTS O O O O C O I O O O O O O I O O O O O O O O O O O

Microsomal Preparation . . . . . . . . . . . . . . . .
Assays for PNPGT and Phenolphthalein GT Activity . . .
Solubilization Studies . . . . . . . . . . . . . . . .
DEAE Chromatography . . . . . . . . . . . . . . . .
Properties of PNPGT Eluted from the DEAE Column. .

Gel Electrophoresis . . . . . . . . . . . . . . . . .

DISCUSSION 0 I O O C C O C O O O O O O O O O O O O O O O C
The Purification Scheme . . . . . . . . . . . . . . .
The Yield of PNPGT Activity After DEAE Chromatography

The Elution Profile of UDPGT Activity After DEAE
Chromatography . . . . . . . . . . . . . . . . . . .
Phenolphthalein GT and PNPGT . . . . . . . . . . . . .
SWARY AND co NCLUS IONS O O O O O O O O O O O O O O O O 0

LIST OF REFERENCES . . . . . . . . . . . . . . . . . . . .

ii

Page
iii

vii

\fi

14

14
15
16
16
17
18
18
l9
19
20
20

21
21
29
41
S9
62
76
79

84
85

87
88

89

91

TABLE

10.

11.

12.

13.

LIST OF TABLES

THE EFFECT OF THE GLYCEROL CONCENTRATION OF THE HOMOGENIZING
MEDIUM ON INTESTINAL MICROSOMAL UDP-GLUCURONYLTRANSFERASE
ACTIVITY TOWARDS PfNITROPHENOL . . . . . . . . . . . . . . .

THE LOCALIZATION OF UDP-GLUCURONYLTRANSFERASE ACTIVITY TO-
WARDS PfNITROPHENOL IN RABBIT SMALL INTESTINE . . . . . .

THE GLUCURONIDATION OF PfNITROPHENOL BY INTESTINAL MICRO-
SOMES FROM FED AND FASTED RABBITS . . . . . . . . . . . . .

SEX DIFFERENCES IN THE CONJUGATION OF P-NITROPHENOL BY RABBIT

INTESTINAL MICROSOMAL UDP-GLUCURONYLTRANSFERASE . . . . . .

THE EFFECT OFIG’NAPHTHAFLAVONE OR PBBs ADMINISTRATION ON THE
PNPGT ACTIVITY OF RABBIT INTESTINAL MICROSOMES . . . . . . .

THE EFFECT OF PHENOBARBITAL ADMINISTRATION ON THE PNPGT
ACTIVITY OF RABBIT INTESTINAL MICROSOMES . . . . . . . . . .

THE SOLUBILIZATION OF RABBIT INTESTINAL MICROSOMAL PNPGT WITH

NONIONIC DETERGENTS . . . . . . . . . . . . . . . . . . . .

THE EFFECT OF PROTEIN CONCENTRATION ON THE SOLUBILIZATION OF
RABBIT INTESTINAL MICROSOMAL PNPGT WITH LUBROL WX . . . . .

THE EFFECT OF pH ON THE SOLUBILIZATION OF RABBIT INTESTINAL
MICROSOMAL PNPGT WITH LUBROL WX . . . . . . . . . . . . . .

THE EFFECT OF TEMPERATURE ON THE SOLUBILIZATION OF RABBIT
INTESTINAL MICROSOMAL PNPGT WITH LUBROL WX . . . . . . . . .

THE EFFECT OF IONIC STRENGTH ON THE SOLUBILIZATION OF
INTESTINAL MICROSOMAL PNPGT WITH LUBROL WX . . . . . . . . .

THE EFFECT OF SONICATION ON THE SOLUBILIZATION OF RABBIT

INTESTINAL MICROSOMAL PfNITROPHENOL UDP-GLUCURONYLTRANSFERASE

WITH LUBROL WK 0 O O O I O O O O O O O O O O O O O O O O I 0

THE SOLUBILIZATION OF INTESTINAL MICROSOMAL PNPGT WITH LUBROL

wx AND w ITH 0 CTYLGLUCO S IDE 0 O O O O O O O O O O C O Q C O 0

iii

PAGE

22

23

24

26

27

28

46

48

49

50

51

52

53

LIST OF TABLES (continued)
TABLE pass

14. THE EFFECT OF pH AND IONIC STRENGTH ON THE SOLUBILIZATION OF
RABBIT INTESTINAL MICROSOMAL PNPGT WITH OCTYLGLUCOSIDE . . . 58

15. THE PARTIAL PURIFICATION OF RABBIT SMALL INTESTINAL
UDP-GLUCURONYLTRANSFERASE ACTIVITY TOWARDS fi-NITROPHENOL AS
SUBSTRATE USING SEQUENTIAL DEAE COLUMNS . . . . . . . . . . 61

16. THE PARTIAL PURIFICATION OF RABBIT SMALL INTESTINE
UDP-GLUCURONYLTRANSFERASE ACTIVITY TOWARDS E-NITROPHENOL AS
SUBSTRATE O O O I O O O O O O O O C O O O C O O O O O O I O .67

17. THE PARTIAL PURIFICATION OF RABBIT SMALL INTESTINE

UDP-GLUCURONYLTRANSFERASE ACTIVITY TOWARDS E-NITROPHENOL AS
SUBSTRATE O O O I I O O O I O O O O O O I O O O C O O O O O 68

iv

LIST OF FIGURES

FIGURE PAGE

1. THE DISTRIBUTION OF MICROSOMAL PNPGT ACTIVITY ALONG THE SMALL
INTESTINE OF THE RABBIT . . . . . . . . . . . . . . . . . . 30

2. RABBIT INTESTINAL MICROSOMAL PNPGT ACTIVITY AS A FUNCTION OF
MICROSOMAL PROTEIN IN THE PNPGT ASSAY . . . . . . . . . . . 32

3. THE EFFECT OF FIVE NONIONIC DETERGENTS ON THE PNPGT ACTIVITY
OF RABBIT INTESTINAL MICROSOMES . . . . . . . . . . . . . . 35

4. THE EFFECT OF THE NONIONIC DETERGENTS, RENEX 690 AND TRITON
X-100 , ON THE PNPGT ACTIVITY OF RABBIT INTESTINAL
MICRO SOMES I I I I I I I I I I I I I I I I I I I I I I I I I 37

5. THE EFFECT OF CHOLATE AND DEOXYCHOLATE ON THE PNPGT ACTIVITY
OF RABBIT INTESTINAL MICROSOMES . . . . . . . . . . . . . . 39

6. DOUBLE RECIPROCAL PLOTS INITIAL RATES OF RABBIT INTESTINAL
MICROSOMAL UDP-GLUCURONYLTRANSFERASE AS A FUNCTION OF
VARYING CONCENTRATIONS OF EfNITROPHENOL AT DIFFERENT FIXED
CONCENTRATION UDP-GLUCURONIC ACID . . . . . . . . . . . . . 42

7. DOUBLE RECIPROCOAL PLOTS OF INITIAL VELOCITY AGAINST VARIABLE
UDP-GLUCURONIC ACID CONCENTRATIONS IN RABBIT INTESTINAL
MICRDWMES I I I I I I I I I I I I I I I I I I I I I I I I I 44

8. THE DIALYSIS OF OCTYLGLUCOSIDE . . . . . . . . . . . . . . . 54

9. THE SOLUBILIZATION OF RABBIT INTESTINAL MICROSOMAL
UDP-GLUCURONYLTRANSFERASE ACTIVITY TOWARDS PfNITROPHENOL AT
VARYING CONCENTRATIONS OF OCTYLGLUCOSIDE . . . . . . . . . . 56

10. DEAE-SEPHACEL COLUMN CHROMATOGRAPHY OF RABBIT INTESTINAL
'EfNITROPHENOL UDP-GLUCURONYLTRANSFERASE ACTIVITY AT pH 8.0,
00602 OCTYLGLUCOSIDE I I I I I I I I I I I I I I I I I I I I 63

11. DEAE-SEPHACEL COLUMN CHROMATOGRAPHY OF RABBIT INTESTINAL
PfNITROPHENOL UDP-GLUCURONYLTRANSFERASE ACTIVITY AT pH 8.0,
0.652 OCTYLGLUCOSIDE . . . . . . . . . . . . . . . . . . . . 65

12. DOUBLE RECIPROCAL PLOT WITH INITIAL RATES OF UDP-GLUCURONYL
TRANSFERASE IN THE DEAE-SEPHACEL COLUMN KCI ELUATE AS A
FUNCTION OF VARYING CONCENTRATIONS OF EfNITROPHENOL . . . . 70

13. DOUBLE RECIPROCAL PLOT OF INITIAL RATES OF UDP-GLUCURONYL
TRANSFERASE IN THE DEAE-SEPHACEL COLUMN KCI ELUATE AS A FUNCTION
OF VARYING CONCENTRATIONS OF UDP-GLUCURONIC ACID AT DIFFERENT
FIXED CONCENTRATIONS OF UDP . . . . . . . . . . . 72
v

LIST or FIGURES (continued)

FIGURE PAGE
14. THE INACTIVATION OF UDP-GLUCURONYLTRANSFERASE ACTIVITY 74
TOWARDS EfNITROPHENOL AS A FUNCTION OF THE PROTEIN TO
OCTYLGLUCO 8 IDE RATIO I I I I I I I I I I I I I I I I I

15. SDS DISC PAGE SLAB GEL . . . . . . . . . . . . . . . 77

vi

BHT
.GME
BSA
cpm
DEAE
DTT
EDTA

i.p.
3-MC
PAGE
PB
PBB
PEG
.ENP
PNPGT
SDS
Tris
UDP
UDPGA
UDPGT

LIST OF ABBREVIATIONS

butylated hydroxtoluene
fl-mercaptoethanol

bovine serum albumin

counts per minute

diethylamino ethyl

dithiothreitol

ethylene diamine tetraacetric acid
glucuronyltransferase
intraperitoneal
3-methylcholanthrene
polyacrylamide gel electrophoresis
phenobarbital

polybrominated biphenyls
polyethylene glycol

pfnitrophenol
pfnitrophenol:UDP-glucuronyltransferase
sodium dodecyl sulphate

tris (hydroxymethyl) aminomethane
uridine-S-diphosphate

uridine diphospho-glucuronic acid
UDP glucuronyltransferase

vii

INTRODUCTION

Man is exposed to a wide variety of environmental xenobiotics.

The cytotoxicity of a xenobiotic depends in part on its metabolism.
Since many xenobiotics are nonpolar and lipid soluble, it is
advantageous to convert such xenobiotics into more water soluble
compounds, thus facilitating their excretion and limiting the duration
of their effect. In order to make them less lipid soluble, many
xenobiotics are oxidized, reduced, or hydrolyzed by mammalian enzyme
systems. The altered compounds may then be acted upon by transferases
which catalyze the conjugation of these compounds with glucuronic acid,
glycine, glutathione, or sulfate. Since the resulting conjugates are
acidic and are ionized at physiological pH, they are water soluble and
readily excreted into the urine and bile.

The most common types of conjugates formed with xenobiotics are
B-glucuronides. Uridine diphosphate glucuronyl-transferase
(UDP-glucuronate-glucuronosyltransferase [acceptor unspecific]
E.C.2.4.l.l7) catalyzes the formation of B-glucuronides by transferring
the D-glucuronic acid of uridine diphospho-glucuronic acid (UDPGA) to
many structurally different aglycones such as phenols and alcohols (l),
carboxylic acids (2), amines (3), thiol derivatives (4), and acetylenic
derivatives (5). Endogenous compounds, such as bile acids (6), many

steriod hormones (7-9), thyroxine (10), and catecholamines (11) are

2
also glucuronidated. The anionic carboxylic acid group of the
resulting conjugate facilitates excretion.

Some substrates of UDP-glucuronlytransferase (UDPGT) are the
products of the mixed-function oxidase systems. These electron
transport systems, located in the endoplasmic reticulum, are
responsible for the oxidative metabolism of steroids and fatty acids,
as well as xenobiotics. They consist of NADPH-cytochrome P-4SO
reductase, lipid, and multiple forms of the terminal oxidase,
cytochrome P-450. Many compounds, once oxidized to hydroxylated
derivatives by these systems, may then serve as substrates for UDPGT.
Furthermore, epoxides, sometimes formed by the mixed-function oxidase
system, may be converted to hydroxy compounds by epoxide hydrolase.
While the hydroxylated compounds are usually more water soluble than
their parent compound, glucuronidation of these hydroxlated derivatives
greatly increases their solubility. Glucuronidation almost always
results in detoxification and may be of great importance in the removal
of carcinogenic, mutagenic, and toxic intermediates (12).

While in most instances glucuronidation results in the
detoxification and excretion of xenobiotics, there are a few instances
where glucuronidation results in more reactive xenobiotics. For
example, phenacetin (13), and the carcinogens, safrole
(4-allyl-1,2-methylenedioxybenzene) (14), and 2-acetyl-aminofluorene
(15), are hydroxylated and then glucuronidated. The glucuronides of
these compounds bind covalently to tissue macromolecules more readily

than their unconjugated parent compounds.

3

Mammalian UDP-glucuronyltransferase activity is located in the
endoplasmic reticulum of most tissues of the body including kidney
(16), lung (17), skin (18), spleen, brain, heart, thymus (19), and the
gastrointestinal mucosa (20). After homogenization, activity is
recovered in the microsomal fraction. UDPGT activity has also been
found in the nuclear envelope (21). While the liver plays an important
role in glucuronidation, intestinal glucuronidation is important as
well because the intestine is one of the sites of entry of many
xenobiotics into the body. UDPGT and the other xenobiotic metabolizing
enzymes found in the intestine, serve as a first line of defense
against xenobiotics in the body.

Intestinal glucuronidation may have a significant effect on the
overall metabolism, disposition, and pharmacokinetics of many
xenobiotiotic and endogenous compounds metabolized by the liver. The
fate of these compounds is influenced by the intestinal metabolism
through the enterohepatic circulation. Hepatocytes excrete
glucuronides of xenobiotic and endogenous compounds into the blood or
the bile. Glucuronides in the bile enter the intestinal lumen where
they may be hydrolyzed by bacterial S-glucuronidase. These
metabolites, which have access to the large surface area of the
intestine, may once again become glucuronidated. This process in which
glucuronides are broken down and reformed may have a pronounced effect
on the reactivity and half-life of many xenobiotics and their
metabolites (22). For instance, after an intraperitoneal injection of

phenobarbital or progesterone, they are excreted in the bile as

4
glucuronides. Saccharolactone, a potent inhibitor of B-glucuronidase,
has been shown to shorten the pharmacological action of these drugs
(23).

The intestinal microsomal mixed-function oxidase systems which are
instrumental in xenobiotic metabolism are similar in structure to those
in the liver. Cytochrome P-450 dependent polycyclic aromatic
hydrocarbon hydroxylase activity has been demonstrated in the
gastrointestinal wall of man, rabbit, rat, guinea pig, mouse, and
hamster. Wattenberg's extensive work on benzo(a)pyrene hydroxylase
activity in rat indicated that the intestinal mixed-function oxidase
system plays an important role in the detoxification of
arylhydrocarbons (24-28). Chhabra and Fouts (29) demonstrated both
biphenyl and benzo(a)pyrene hydroxylase activities in the intestinal
microsomes of rabbit, guinea pig, rat, and mouse. In some species,
cytochrome P-450, and NADPH-cytochrome P-450 reductase were found in
relatively high levels. In rabbit intestinal microsomes the cytochrome
P-450 content per mg of microsomal protein was 332 that of the liver,
while the NADPH-cytochrome P-450 reductase specific activity was 75%
that of the liver (29).

Some of the hydroxylated aromatic products of the intestinal
mixed-function oxidase system have been shown to be glucuronidated by
the intestinal mucosa (30). Naphthalene, for instance, is converted by
the mixed-function oxidase system to l-naphthol, which in turn is
rapidly glucuronidated (31). Aniline is hydroxylated by the

mixed-function oxidase system to 27aminophenol which may may then serve

S

as a substrate for intestinal UDPGT (32). Hydroxlylated metobolites of
7-ethoxycoumarin (33), perazine (34), and biphenyl (35) have also been
shown to be effectively glucuronidated in the small intestine.

Glucuronidation in the small intestine is also significant because
it reduces the effect of many orally administered drugs. As an
example, morphine, when administered orally, has diminished analgesic
effect when compared to its effect when administered by another route
(36). Morphine and several other phenolic drugs have been reported to
be poorly absorbed when taken orally while their gfmethylated congeners
are absorbed to a greater extent (37).

The Modulation of UDPGT Activity:

 

Many questions about UDPGT, both ighzi!2_and in_yi££2, remain to
be resolved. Among these questions is how UDPGT activity is modulated
awandmmo

It is not known how UDPGT activity is regulated in 3112.
Experiments comparing the $2 lilo with the ighziggg hepatic or
intestinal microsomal activity indicate that for all species studied,
UDPGT activity is not fully expressed (38-40). "Latent" is an
operational term used to describe the native form of the enzyme; that
form found in_yiyg or found in tissue slices which have not been
exposed to membrane perturbants. The activity of UDPGT in tissue
slices may be increased by exposure to UDP-N-acetylglucosamine which
enhances the affinity and specificity of the enzyme for UDPGA, thus

making the enzyme more efficient (41). It has not been determined

6
whether or not UDP-N-acetylglucosamine regulates enzyme activity in
1332.

The mechanism by which UDPGT activity is enhanced in zitgg is
unknown. This enhancement of UDPGT activity ignziggg by mechanical or
chemical means is termed ”activation". The activation of UDPGT by
membrane perturbing agents is most commonly explained by assuming the
breakdown of permeability barriers in the modified membranes (42-44).
Vessey and Zakim proposed that activation might also arise from changes
in the lipid-protein interaction (45).

The activity of hepatic microsomal UDPGT in ziggg reflects the
manner in which the microsomes are treated. A variety of conditions
and agents which effect the integrity of the microsomal membrane to
which UDPGT is bound have a pronounced effect on the activity of the
enzyme. Studies on the effects of phospholipases, detergents, trypsin,
freezing, and ultrasonication indicate that altering the microsomal
environment has a marked effect on hepatic UDPGT activity. With
membrane disruption, a biphasic activation-inactivation of enzyme
activity is often observed. Zakim and Vessey (46) were able to
activate rat, guinea pig, and rabbit microsomal UDPGT with
phospholipase A. Continuation of phospholipase A treatment, after peak
activity was reached, resulted in a decline in UDPGT activity. While
ultrasonication of guinea pig microsomes initially activated UDPGT,
prolonged ultrasonication produced a decrease in UDPGT activity (47).
Low levels of the anionic detergent, deoxycholate, and the nonionic

detergent, Triton X-100, activate guinea pig, mouse, and rat microsomal

7
UDPGT (48-49), although loss of activity is observed at high
concentations of detergent.

UDPGT activity may vary not only with the way in which microsomes
are treated, but may also vary with the organ from which the microsomes
are obtained. Comparative studies on naphthol glucuronidation with
intestinal loops $2.3l22.and with mucosal homogenates innzi££g_suggests
that intestinal UDPGT is latent in the intact tissue, as is the liver
enzyme, and can be effected by a variety of membrane perturbing
treatments (50). Aitio (51) studied the effect of phospholipase C,
digitonin, and trypsin on UDPGT activity in rat liver and small
intestinal microsomes. Digitonin had a negligible effect on intestinal
UDPGT while it greatly enhanced liver activity. Although hepatic UDPGT
was activated by trypsin and phospholipase C treatments, intestinal
UDPGT activity was depressed. Del Villar g£_al3 (52) demonstrated a
difference in response of intestinal and hepatic microsomes to Triton
X-100. The intestinal microsomal UDPGT might be activated to a greater
extent than the hepatic enzyme and therefore respond differently to
various membrane perturbing treatments. Since trypsin, phospholipase
C, and surface active agents, like bile salts, are constituents of the
small intestinal lumen, UDPGT might be spontaneously activated during
the microsomal isolation procedure by these endogenous surface active
agents (50).

Zakim and Vessey (S3) and Wisnes (54) investigated the kinetic
properties of hepatic pfnitrophenol:glucuronyltransferase (PNGT)

activated by various membrane perturbing treatments. The activated

8
form of the enzyme was inhibited by UDP and UDP-sugars, had a decreased
affinity for UDPGA and pfnitrophenol, and was insensitive to
UDP-N-acetylglucosamine.

It has been proposed (55) that the altered kinetic properties of
PNPGT, after treatment with membrane perturbants, are due to an altered
enzyme conformation brought about by changes in the lipid environment.
When Gorski and Kasper (56) separated 982 of the phospholipid from rat
liver microsomal protein, using gel filtration, PNPGT activity was
reduced to 0-62 of its original activity. By incubating the lipid
depleted PNPGT with liposomes derived from microsomes, 30 to 442 of the
original activity was restored. Besides mixed microsomal lipid,
phosphatidylcholines, both synthetic and natural, restored PNPGT
activity. Tukey SE E£° (57) found that phosphatidylcholine and
lysophosphatidylcholine were most effective in restoring the activity
of partially purified rabbit liver PNPGT which had been depleted of
lipid. Burchell and Hallinan (58) were able to activate rat liver
PNPGT, purified to homogeneity and containing very little phospholipid,
40-1002 by incubation with phosphatidyl - and lysophosphatidyl -
choline. With the addition of phosphatidyl - and lysophospha -
tidyl-choline to lipid depleted guinea pig liver PNPGT, Erickson g£_al.
(59) achieved a 50 fold stimulation of PNPGT activity. The length and
degree of unsaturation of the acyl chains appeared to have a
significant influence on the reactivation of PNPGT. Phosphatidylserine

and phosphatidylethanolamine did not reactivate the enzyme.

The Heterogeneity of UDPGT:

 

There are several indications that UDPGT consists of a
heterogeneous group of enzymes. Some glucuronyltransferase activities
have been separated from each other during the course of purification
(60-64). Recent work suggests that at least two forms of
glucuronyltransferase exist in the rat which differ in tissue
distribution, inducibility by xenobiotics and glucocorticoids,
substrate specificity and perinatal deveIOpment.

Hepatic glycuronyltransferase activity towards several groups of
substrates is differentially induced. Many microsomal drug
metabolizing enzymes are induced preferentially by the model inducer,
3-methylcholanthrene, while other enzymes are induced preferentially by
another model inducer, phenobarbital. Glucuronyltransferase activity
toward one group of substrates, which includes pfnitrOphenol,
l-naphthol, and 2-aminophenol, is induced by 3-MC (65).
Glucuronidation of another group of substrates, which includes
morphine, phenolphthalein, and chloramphenicol, is induced by PB
(65-66). Neither PB or 3-MC induce steriod glucuronidation (61).

Hepatic glucuronidation of different substrates follows different
developmental patterns (67-68). The activity towards that group of
substrates which is induced by 3-MC reaches adult levels during the
late fetal stage of development, while that activity which is induced
by PB, does not reach adult levels until 2 days after birth. Wishart
(67) has termed these two groups of substrates "late fetal" and
"neonatal". The "late fetal" substrates include l-naphthol,

2-aminophenol, and E-nitrophenol, corresponding to that group of

10
substrates whose conjugation is induced by 3-MC; while the "neonatal"
group inculdes phenolphthalein, morphine, bilirubin, and estradiol.
Conjugation of this group of substrates is induced by PB. Having
demonstrated that the synthetic glucocorticoid, dexamethasome,
precociously induces transferase activity towards the ”late fetal"
group, but not to the "neonatal" group, Wishart proposed that fetal
glucocorticoids may induce transferase activity towards the "late
fetal” group. The activity towards the ”neonatal" group may be
unresponsive to glucocorticoids or may require an additional endogenous
agent(s) for induction to occur.

The "neonatal" and ”late fetal" group of substrates have have
different tissue distribution (69). Transferase activity towards the
"late foetal" group of substrates is ubiquitous, being found in such
tissues as the liver, kidney, small intestine, lung, skin, and spleen,
while activity towards the ”neonatal" group of substrates is found
primarily in the liver and intestine, being undetectable or barely
detectable in other tissues.

It has been proposed that the substrate specificity of the various
forms of transferase is based, in part, on the size and shape of the
substate (66). According to the hypothesis developed by Wishart 35
3%,, that form of glucuronyltransferase, inducible by 3-MC, whose
activity reaches adult levels at the late foetal stage of development,
is specific for substrates which are planar. That

glucuronyltransferase inducible by PB, whose activity reaches adult

11
levels neonatally, is specific towards phenolic compounds with bulky
£251 substituents.

This proposal is supported by Wishart's studies on the perinatal
development of the glucuronidation of a series of phenols substituted
with alkyl groups in the £251 position. The glucuronidation of phenols
which had relatively small alkyl groups in the p§£a_position attained
adult levels during the late foetal stage of development, while
glucuronidation of those phenols which had bulky alkyl groups attained
adult levels neonatally. The same developmental pattern was
demonstrated for a series of phenols with alkyl substituents in the
9323 or in the EEEBE position.

While studies on the perinatal development, induction, and tissue
distribution of rat liver glucuronyltransferase have provided evidence
for the functional heterogeneity of UDPGT, evidence for the true
molecular heterogeneity of UDPGT has been provided by the purification
of UDPGT. Early attempts at the purification of UDPGT from liver
microsomes were hindered by enzyme instability and inadequate
solubilization, but recent techniques have been more successful in
overcoming these problems. Using DEAE- and UDP-affinity
chromatography, in the presence of nonionic detergents, several
investigators have been able to separate and purify to apparent
homogeneity a hepatic glucuronyltransferase with activity towards small
phenolic substrates from a glucuronytransferase with activity towards
bulky substrates. Burchell g£_gl. have purified rat liver bilirubin GT

from rat liver p-nitrophenol GT (62). The purified rat liver PNPGT had

12
activity towards I-naphthol,'p-nitrophenol, and gfaminophenol in
approximately the same ratio as in the liver homogenate suggesting that
a single microsomal enzyme is responsible for the glucuronidation of
these substrates (70-71). This enzyme also had slight activity towards
morphine. Bock EEHELB have purified morphine GT and PNPGT from the rat
liver (61). While the purified PNPGT had no detectable activity
towards morphine and testosterone, the purified morphine GT had no
detectable activity towards pfnitrophenol. Tukey 25 al. have purified,
to apparent homogeneity, estrone GT and p-nitrophenol GT, from the
rabbit liver (63-64). The purified estrone CT showed no activity
towards PNPGT while the PNPGT had some activity towards estrone.
Recently rat liver testosterone CT was purified by Matern 25 al. This
purified transferase had no detectable activity towards estrone,
bilirubin, p-nitrophenol or morphine (72).

Tukey g£_gl, concluded that the estrone and p-nitrophenol
glucuronytransferases were definitely different proteins based on their
physical properties (64). Both enzymes had subunit molecular weights
of 57,000 and gel chromatography indicated that both enzymes existed as
tetramers with an apparent molecular weight of 230,000. However, with
polyacrylamide isoelectric focusing, PNPGT had an isoelectric point of
6.8, while estrone GT had an isoelectric point of 7.6. Amino acid
analysis of the purified enzymes revealed that PNPGT contained 53%
hydrophobic amino acids, while estrone GT contained 602 hydrophobic

amino acids. Furthermore, limited proteolysis, in the presence of SDS,

13
produced distinct differences in the peptide map composition of the two
enzymes.

Richard Jagger and Steven Aust at Michigan State University have
studied the activity of rabbit intestinal UDP-glucuronyltranferases
towards several substrates. At this time, it is not known whether one
transferase or several transferases are responsible for the
glucuronidation of these substrates. Nor is it known whether the
intestinal transferase(s) and the liver transferases which
glucuronidate these substrates, are identical proteins.

Knowledge of substrate specificity would enhance the understanding
of drug interactions and of the toxicity of xenobiotics. For instance,
ingestion of significant amounts of a substance which is a substrate
for PNPGT might limit the glucuronidation of other PNPGT substrates.
Such substrates might remain in the intestinal lumen longer than they
would otherwise. Glucuronidation of substrates brought to and from the
intestine through enterohepatic circulation might also be effected.

Little is known on the substrate specificity of intestinal
PNPGT(S) nor is it known whether liver and intestinal PNPGT are
identical proteins. Purifying PNPGT(s) from rabbit intestinal
microsomes would help clarify these and other questions.

UDPGT has yet to be purified from the small intestine.

The work in Dr. Aust's lab was undertaken to establish procedures
for the purification of rabbit intestinal microsomal PNPGT. The
purification of PNPGT requires a procedure for the large scale

preparation of rabbit intestinal microsomes with stable PNPGT activity,

14
as well as a non-denaturing procedure for the solubilization of PNPGT.
Once PNPGT is purified to homogeneity, its kinetic properties, physical

properties, and substrate specificity can be better studied.

METHODS AND MATERIALS

Material Sources:

 

B-Glucuronidase, DEAE-Sephacel,.p-nitrophenol (spectrophotomeric
grade), phenophthalein, UDP (sodium salt), UDPGA (ammonium salt, 98%
grade), UDP-N-acetylglucosamine, butylated hydroxytoluenefl3
-mercaptoethanol, heparin (soduim salt, Sigma Grade I), chicken
ovomucoid trypsin inhibitor (Type II-O), egg yolk phosphatidylcholine
crude soybean phoshatidylcholine, chlorhexidine, Dextran Blue 2000,
Brilliant Blue R, sodium dodecyl sulfate, phenylmethylsulfonylfluoride,
polyethylene glycol (approximate weight 400), bovine serum albumin (98%
fatty acid free) Lubrol WX, and Tris base were obtained from Sigma
Chemical Company, St. Louis, Missouri. B-Naphthaflavone was obtained
from Aldrich Chemical Company, Milwaukee, Wisconsin. Phenobarbital
(sodium U.S.P.) was purchased from Merck and Company Inc., Rahway, New
Jersey. Firemasters PBB, manufactured by Michigan Chemical
Corporation, St. Louis, Michigan, was obtained from the Michigan
Department of Agriculture, Lansing, Michigan. 3-MC, acrylamide,
methylenebisacrylamide and tetramethylenediamine were purchased from
Eastman Organic Chemicals, Rochester, New York. Octyl-B
-D-glucopyranoside, and saccharo-1,4-lactone were obtained from
Calbiochem-Behring Corporation, La Jolla, California. Triton X-100
(scintillation grade) and Aquasol 2 were purchased from New England
Nuclear, Boston, Massachusetts. C14octylglucoside was synthesized

15

16
by Paul Rosevear of Michigan State University. Emulgen 911 was a gift
from the Kao-Alto Company, Tokyo, Japan. Renex 690 was a gift from
Janice Lacedo of ICI, Wilmington, Delaware. Cholate and deoxycholate
were recrystalized from ethanol.

All other chemicals were reagent grade.

Animals

Animals, outbread male Sprague-Dawley rats and New Zealand White
rabbits were purchased from Spartan Research Animals, Inc., Haslett,
Michigan. The rabbits ranged in weight from 2 to 5 kilograms and the
rats from 225 to 250 grams. Water and Purina chow were given ad
libitum.

For enzyme induction studies, rabbits were pretreated with PBBS,
or with B-naphithaflavone, or with phenobarbital.

B-Naphthaflavone was dissolved in polyethylene glycol (MW 400) at
37°C, with vigorous stirring, to a concentration of 60mg/ml. The 37°C
solution was immediately injected into the rabbits. Rabbits were
injected i.p. with 80 mg/kg. Control rabbits were injected with
polyethylene glycol. The rabbits were sacrificed 40 hours after they
were injected.

Firemasters PBBs were dissolved in polyethylene glycol (MW 400) in
a boiling water bath for several hours, to a concentration of 45mg/ml.
The solution was equilibrated to ambient temperature. Rats and rabbits
were injected with 90 mg/kg 7 days prior to sacrifice. Control rabbits

and rats were injected with 2 ml/kg polyethylene glycol.

17
A 0.2% solution of phenobarbital in distilled water was adjusted
to pH 7.5-7.8 with concentrated HCl. The solution was diluted 1:1 with
tap water and given to rabbits ad libitum for 7 days~prior to
sacrifice.

Isolation of Intestinal Microsomes

 

Rat and rabbit microsomes were isolated by a method modified from
that of Stohs 35 El“ (73). Rabbits were sacrified by a blow to the
head, rats by decapitation. The rabbit small intestine was immediately
excised from the pylorus to 5 cm proximal to the ileocecal valve. The
rat small intestine was excised to the first 25 cm of the duodenum.

The intestinal lumen was washed vigorously with an ice cold solution
containing 20 mM Tris-HCI pH 7.4, 20% glycerol, 154 mM KCl, and 5 mM
EDTA. Excess fat was trimmed from the outer intestinal wall and the
intestine was then placed in the above solution. Four ml of this
solution was used per gm wet weight of small intestine. To this
solution was added chicken ovomucoid trypsin inhibitor (6 mg/gm wet wt.
small intestine), and 5 U/ml heparin. The intestine was minced with
scissors and homogenized in a glass Teflon Potter-Elvehjem homogenizer
at 500 rpm and immediately centrifuged at 10,000 g for 20 minutes. The
supernatant was poured through four layers of cheese cloth and
centrifuged at 105,000 g for 90 minutes. The microsomal pellet was
washed once by suspension and recentrifugation in three volumes of
isotonic KCl. The final pellet was suspended in 50 mM Tris-HCl, 50%
glycerol, 2uM butylated hydroxytoluene pH7.4, to a protein

concentration of 40 to 50 mg/ml and stored under argon at -20°C.

18

Assay of PNPGT Activity

 

Rabbit intestinal PNPGT activity, unless otherwise stated was
assayed by a modification of Lucier's colorimetric assay (74). From
0.25 to 1.0 mg of protein was assayed in 1.4 ml of an assay mixture
containing 0.15 M Tris-H01, 2.0 mM p-nitrophenol, 10 mM MgC12, and
3 mM UDPGA. When assaying microsomes, the assay mixture also contained
0.013% (w/v) Triton X-100 per mg of protein. The assay mixture was
preincubated for 5 minutes at 37°C. With the addition of UDPGA, the
reaction was initiated. Substrate dissappearance was monitored
continuously at 425 nm with a Cary 219 double beam spectrophotometer on
the reverse beam mode with a 2.0 nm slit width and using cuvettes 0.2
cm wide. The reference cuvette contained the assay mixture without
UDPGA. The extintion coefficient for p-nitrophenol at 425 nm in 0.15 M
Tris-HCl, 10 mM MgC12, 3 mM UDPGA, pH 7.4 at 37°C is 9.5
uM'lcm'l.

Rat intestinal PNPGT activity was assayed by a modification of the
colorimetric method developed by Grote ggngl. (75). 1.5 mg of protein
was assayed in 0.5 ml of an assay mixture containing 0.05 M Tris-HCl pH
7.4, 0.15 M KCl, 0.05% Triton X-100, 10 mM MgC12, 0.6 mM
'p-nitrophenol, and 5.0 mM UDPGA. After preincubation at 37°C, the
reaction was started by adding UDPGA to the reaction mixture and
stopped by adding 50u1 aliquots of the reaction mixture to 1.0 ml of 2%
trichloroacetic acid (w/v). After standing in trichloroacetic acid for

10 minutes, the assay mixture was centrifuged for 10 minutes at 2000 g

l9
and the supernatant made more alkaline with the addition of 50ul of 5 N
KDH. The absorbance was measured at 403 nm.

Assay of Phenolphthalein GT

 

Rabbit intestinal phenophthalein GT activity was assayed by a
modification of a method developed by Wisnes (76). One mg of protein
was assayed in 0.5 ml of an assay mixture containing 75 mM Tris-Maleate
pH 7.4, 20 mg/ml BSA, 10 mM MgC12, 0.45 mM phenolphthalein and 4.3 mM
UDPGA. After preincubation at 37°C, the reaction was started by adding
UDPGA to the reaction mixture and stOpped by adding 100 ul aliquots of
the reaction mixture to 400 ul of 0.5 M glycine buffer pH 10.4. The
reduction in absorbance at 550 nm was read immediately with a Cary 219
double beam spectrometer on the reverse beam mode with a 0.75 nm slit
width. The extinction coefficient for phenolphthalein at 550 nm in
0.5M glycine buffer pH 10.4 is 1.3 x loZuM'lcm'l.

Protein and Phosphate Assays:

 

Protein concentrations were estimated as described by Lowry §£_§l.
(77), using BSA standards.

Phosphate determinations were performed by the method of Bartlett
(78).

Solubilization of Microsomes and DEAE Chromathography:

 

All steps were performed at 4°C. All buffers were deaerated and
flushed with argon.

Solubilization of Intestinal Microsomes:

 

Unless otherwise stated, the rabbit intestinal microsomes were

solubilized using B-D-octylglucoside. Microsomes were suspended to a

20
protein concentration of 10 mg/ml in 50 mM Tris-HCl pH 8.0, 1%
octylglucoside, 20% glycerol, 100 mM KCl, 1 mM EDTA, 1 mM 8MB, and
0.002% (v/v) Chlorhexidine. The suspension was sonicated, (4 x 20 s.)
using a Branson sonifier with a microtip at a power setting of 6 (7 1/2
amperes), and centrifuged at 105,000 g for 2 1/2 hours.

DEAE-Sephacel Chromatography:

 

The 105,000 g supernatant was diluted 1:2, stirring gently but
constantly, with 20% glycerol, 0.4% octylglucoside, and applied
to a DEAE-Sephacel column (1:20, width:height) previously equilibrated
with 50 mM Tris-HCl pH 8.0, 0.6% octylglucoside, 20% glycerol, 1 mM
EDTA, 1 mM BME, and 0.002% Chlorhexidine. Five mg of protein was
applied per ml of DEAE-Sephacel at a flow rate of 12 m1/h. The column
was then washed with 2 column volumes of equilibration buffer. PNPGT
activity was eluted with a linear 0 to 0.05 M KCl gradient in 4 volumes
of equilibration buffer. The void volume was determined using Dextran
Blue 2000.

Sodium Dodecylsulfate-Polyacrylamide Gel Electrophoresis:

Discontinous sodium dodecylsulfate-polyacrylamide slab gel
electrophoresis was performed according to the method of O'Farrell
(79). A 4.75% stacking gel and a 7.5% running gel was used. After
electrophoresis, gels were stained with Brillian Blue R. The molecular
weight markers run in parallel with all membrane protein samples were

ovalbumin, glutamate dehydrogenase, catalase, and bovine serum albumin.

21

Extraction of Microsomal Lipid:

 

The extraction of lipid from intestinal microsomes was performed
by the method of Folch ggugl. (80). All steps were carried out at 4°C.
All buffers were flushed with nitrogen. Three grams of intestinal
microsomes were extracted with 200 ml of 2:1 chloroform-methanol (v/v)
and the insoluble material removed by filtration. The lipid extract
was washed four times with a solution containing chloroform, methanol,
and Folch salt solution in respective proportions of 3:48:47 by volume.
The upper phases from the first three washes were discarded. The
washed lipid extract was dried by rotary vacuum evaporation. The lipid
was dissolved in 5 ml of 2:1 chloroform methanol and stored at -20°C
under argon.

Preparation of Liposomes:

Aqueous suspensions of lipid were prepared by sonication at 4°C.
An aliquot of lipid solution was transferred to a thin walled plastic
tube and dried under nitrogen. Deaerated 0.15 M Tris-H01 pH 7.4 was
added to the lipid, the tube capped under argon, and the suspension
vigorously vortexed. The tube was then placed in an opening of a 3
necked vessel filled with water. The microtip of the Branson sonifier
was placed in another opening of the vessel. The lipid suspension was
sonicated (4x20 3) at a power setting of 6 (7 1/2 amperes).

Enzyme Incubation with Liposomes:

Fractions from the DEAE-Sephacel column were added to the
liposomal suspensions with vigorous vortexing and left standing for six
hours at 4°C. The protein-lipid preparations were then assayed for

PNPGT activity by a method modified from that of Lucier 25 El. (74).

RESULTS

Microsomal Preparation:

 

The procedure used for the preparation of rabbit microsomes was a
modification of the method developed by Stohs g£_al, (73) for the
isolation of rat intestinal microsomes with stable cytochrome P-450.

Rabbit intestinal microsomes, prepared using the modified method,
had a specific activity of 24 112.1 nmoles p-nitrophenol conjugated per
minute per mg of microsomal protein, with a range in specific activity
of 21 to 27. The average microsomal protein yield from one small
intestine was 320 mg (6mg/g wet weight), to give an average of 7680
total units of PNPGT activity.

Microsomal PNPGT activity was stable for at least 6 months, when
the microsomes were stored under agron at -20°C.

The effects of changing various aspects of Stohs' isolation
procedure on the yield of PNPGT activity was studied. Increasing the
concentration of glycerol in the homogenizing medium above the 20%
(v/v) used in Stohs' method, decreased the total units of activity
(Table 1). In Stohs' procedure, microsomes are isolated from the upper
villous layer of the mucosa. A comparision between microsomes taken
from the whole wall, with those from the mucosal lining, showed that
mucosal microsomes had a 30% higher specific activity. However 20%
more total units of PNPGT activity were obtained by isolating
microsomes from the whole wall (Table 2). Stohs derived microsomes
from rats which were not fasted. When rabbits were fasted for 24 hours

22

23

TABLE 1. THE EFFECT OF THE GLYCEROL CONCENTRATION OF THE
HOMOGENIZING MEDIUM 0N INTESTINAL MICROSOMAL
UDP-GLUCURONYLTRANSFERASE ACTIVITY TOWARDS p-NITROPHENOL.

 

 

% Glycerol PNPGT Specific Activity Total PNPGT Activity
(v/v) nmole p-intropenol con- nmole p-nitrophenol
jugated/min per mg conjugated/min
0 9.1 2000
10 9.6 1860
20 9.1 1640
30 10.2 1470

 

PNPGT activity was assayed by the method of Lucier g£_§l. (74) as
described under "Methods".

24

TABLE 2. THE LOCALIZATION OF UDP-GLUCURONYLTRANSFERASE ACTIVITY
TOWARDS E-NITROPHENOL IN RABBIT SMALL INTESTINE.

 

PNPGT Specific Activity
nmole p-nitropenol con-

Total PNPGT Activity
nmole p-nitrophenol

 

jugated/min per mg conjugated/min
Whole Intestine 9.13 i 1.8 1100 j: 70
Mucosal Lining 13.4 _-+_- 2.1 950 j; 172

 

The mucosal lining of the small intestine was obtained by
scraping the everted small intestine with the edge of a
glass slide. PNPGT activity was assayed by the method of
Lucier g£_§l. (74) as described under "Methods”.

25
prior to sacrifice these rabbits had slightly higher PNPGT specific
activities than microsomes from nonfasted rabbits (Table 3), but more
total units of microsomal PNPGT activity were obtained from nonfasted
rabbits. Stohs only used rats which were male. With rabbits,
microsomes had a similar PNPGT specific activity whether derived from
male or female animals (Table 4).

In an effort to increase the specific activity and yield of PNPGT,
rabbits were treated with several chemicals, known to induce enzymes
for the metabolism of xenobiotics, prior to sacrifice. Pretreatment of
rabbits with 3-MC type inducersf3-naphthaflavone or P888 did not
increase microsomal PNPGT specific activity or yield (Table 5).
Likewise, pretreatment of rabbits with phenobarbital did not increase
yield (Table 6). Unlike rabbits, pretreatment of rats with P383

resulted in increased PNPGT specific activity. Rat microsomes,
obtained from the 25 cm distal to the stomach pylous of intestines of
rats not pretreated with PBBS, had a PNPGT specific activity of 25
nmoles p-nitrophenol conjugated per minute per mg of microsomal
protein, while microsomes obtained from rats which were pretreated had
a specific activity of 42.

The effect of changing several chemicals in the homogenizing
medium used in Stohs' isolation procedure on the stability of
microsomal PNPGT was examined. Stohs' method used soybean trypsin
inhibitor to increase the stability of rat cytrochome P-450 during the
microsomal preparation. In the of intestinal microsomes, it was found

that the addition of chicken ovomucoid trypsin inhibitor, along with

26

TABLE 3. THE GLUCURONIDATION OF‘BfNITROPHENOL BY INTESTINAL
MICROSOMES FROM FED AND FASTED RABBITS.

 

Treatment PNPGT Specific Activity
nmole p-intropenol con-

Total PNPGT Activity
nmole pfnitrophenol

 

jugated/min per mg conjugated/min
Fasted 9.6 1210
Fed 8.2 1600

 

Fasted rabbits were given water §d_libitum but no food for 24
hours prior to sacrifice. PNPGT activity was assayed by the
method of Lucier 32.2}! (74) as described under "Methods."

27

TABLE 4. SEX DIFFERENCES IN THE CONJUGATION OF RfNITROPHENOL BY
INTESTINAL MICROSOMAL UDP-GLUCURONYLTRANSFERASE.

 

 

Sex PNPGT Specific Activity Total PNPGT Activity
nmole p-intropenol con- nmole pfnitrophenol
jugated/min per mg conjugated/min

Female 13.0 i 1.2 1320 i 92

Male 11.2 + 0.8 1010 i 81

 

PNPGT activity was assayed by the method of Lucier SE 31. (74) as
described under "Methods".

28

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30
EDTA, to the homogenizing medium increased the Stability of rabbit
intestinal microsomal PNPGT. Microsomes prepared with a homogenizing
medium containing 0.1 mM phenylmethysulfonylfluoride had half the
specific activity of those microsomes prepared using chicken ovomucoid
trypsin inhibitor.

The distribution of PNPGT activity of microsomes obtained from
rabbit small intestine is shown in figure 1. The duodenum had the
highest specific PNPGT activity while microsomes obtained from the
ileum had one third the activity of microsomes obtained from the
duodenum.

Rabbit small intestinal microsomes had an average phenolphthalein
GT activity of 0.14 nmoles phenophthalein conjugated per minute per mg
of microsomal protein.

Assays for PNPGT and Phenolphthalein GT Activity:

 

The enzyme assay for rat intestinal PNPGT modified from Grote's
assay for rat liver microsomal PNPGT, was apparently linear for 10
minutes when 5.0 mM UDPGA was used.

A continuous enzyme assay was developed for rabbit intestinal
PNPGT by modifying Lucier's discontinuous PNPGT assay (74). When
rabbit intestinal microsomes were assayed by Lucier's unmodified
method, the apparent PNPGT specific activity, was half that seen when
these microsomes were assayed by the modified method which uses higher
concentrations of UDPGA and pfnitrophenol. The modified enzyme assay
was linear for 15 minutes and between 0.25 to 2 mg of microsomal

protein (Figure 2) per 1.4 ml of incubation mixture. In the absence of

Figure 1.

31

THE DISTRIBUTION OF MICROSOMAL PNPGT ACTIVITY ALONG THE

SMALL INTESTINE OF THE RABBIT.

Rabbit small intestines were cut into 50 cm segments and
microsomes were derived from these segments as described
under "Methods." PNPGT activity was assayed by the

unmodified method of Lucier 25 El' (74).

32

 

 

 

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Distance from Pyloric Valve (cm)

33

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Protein (mg)

35
MgClz, the enzyme reaction was approximately half as fast.

The PNPGT assay was a spectrophotometric assay which involved
monitoring the disappearance of the chromophore, pfnitrophenol, at 425
nm with the formation pfnitrophenol-glucuronide. The addition of
saccharo-l,4-1actone, a potent inhibitor of intestinal B-glucuronidase,
an enzyme which hydrolyzes p-nitrOphenol-glucuronide, to the enzyme
assay mixture, at concentrations of 0.5, 1.0, and 5.0 mM did not
increase the apparent PNPGT specific activity.

In order to confirm that the decrease in absorbance at 425 nm was
due to the formation of pfnitrophenol-glucuronide,£3-glucuronidase was
added to the incubation mixture after the reaction had proceeded for an
hour. The addition of -glucuronidase resulted in a rapid increase in
the absorbance at 425 nm.

The effect of incubating microsomes with various detergents on the
activity of PNPGT was examined. The enzyme was activated by Lubrol WX,
Emulgen 911, octylglucoside, Renex 690, and inhibited by them at high
concentrations (Figures 3 and 4). Cholate, Brij 56, and dexycholate
were inhibitory even at low concentrations (Figure 5).

Wisnes developed a discontinuous enzyme assay for phenolphthalein
GT (76) in rat liver homogenate. The assay was a spectrophotometric
assay which involved monitoring the disappearance of the chromophore,
phenolphthalein, at 555nm. In assaying for rabbit intestinal
microsomal phenolphthalien GT, this assay was modified and was
apparently linear for 10 minutes and between 0.5 to 2 mg of microsomal

protein per ml of incubation mixture. The addition of 10 mM MgC12 to

36

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38

Figure 4. THE EFFECT OF THE NONIONIC DETERGENTS, RENEX 690 AND TRITON
X-100, ON THE PNPGT ACTIVITY OF RABBIT INTESTINAL

MICROSOMES.

Intestinal microsomes were assayed by the method of Lucier
35 El. (74) as described under "Methods", except that the
indicated type and final concentration of detergent were

used.

200

150 ‘

100

"la Relative PNPGT Activity
01
O

O

39

WP

  
 

/ Renex 690

#1

a
l I

5 15 25

 

0

Concentration (w/v) at Detergent (%)110'3

Figure 5.

40

THE EFFECT OF THE CHOLATE AND DEOXYCHOLATE ON THE PNPGT

ACTIVITY OF RABBIT INTESTINAL MICROSOMES.

Intestinal microsomes were assayed as described under
"Methods", except that the indicated type and concentration

of detergent were used.

-v-‘u-u--u - .‘I U-

°/o Relative PNPGT Actlvtty

100

41

 

 

 

Concentration (w/v) ot Detergent (7o)

42
the incubation mixture had no effect on the reaction rate.

The effect of nonionic detergents on the apparent activity of
phenolphthalein GT differed from their effect on the activity of PNPGT.
Unlike PNPGT, phenolphthalein CT was not activated by relatively low
levels of octylglucoside. At levels greater than 0.55% (w/v), the rate
of change of absorbance at 555 nm decreased. Phenolphthalein was
insoluble when concentrations of Triton X-100 as low as 0.001% (w/v)
were added to the incubation mixture.

The kinetic properties of rabbit intestinal microsomal PNPGT were
studied. The apparent Km of microsomal PNPGT for p-nitrophenol was
0.3 mM at a UDP-glucuronic acid concentration of 3 mM, (Figure 6) and
for UDP-glucuronic acid was 0.2 mM at a ErnitrOphenol concentration of
2 mM (Figure 7). Five mM UDP-N-acetylglucosamine had no effect on the
detergent activated enzyme.

Solubilization Studies:

 

Because the instability of glucuronyltransferase hindered its
characterization, the solubilization of rabbit intestinal PNPGT with
the relatively mild nonionic detergents - Lubrol WX, Emulgen 911, and
octylglucoside - was examined. In order to solubilize microsomal
protein, buffered detergent was added to the microsomes and the
resulting suspension was sonicated and centrifuged.

Lubrol WX and Emulgen 911 were used in the initial solubilization
studies. Table 7 shows the result of this experiment. After treatment
with 0.5% Lubrol WX, 19% of the original PNPGT activity was detected in

the 2 1/2 h, 180,000 x g supernatant. Increasing the Lubrol WX

43

4:23 2:. A325” .8325; .on same

one: meow ouaounosawlmaa mo unequmuuaooaoo as? .afiououa
Hmaomouoaa no we won :«a use woumwsficou Hoaonaouufich.

mo moaoac mm nowmouaxo can :mvozuoz: Home: wonfiuommv

mm moaomouofia aw huu>wuum ommuowmamuuHhaousozawimas

mo :ofiumcfiaumumv mum» Hmfiuucw co comma ma ucaoa comm

.QHU< UHzomDUDAUImQD mo monH<MHzmuzoo meHm
HzmxmmmHa H< AOZMEmomHHZLm.mO monH<mHzmozou UZHNM<> ho
ZOHHUZDm < m4 mm<¢mmmz<mewzomaoaqolmas A<zomomon A<ZHHmMHzH

HHmm<m mo mm9<d A<HHHZH mo mHOAm A<UommHomm mumaon

.c ouswfim

44

1pc

 

ATEEV

T—n—ZM:

.u-N

 

45

.28 o.~ was Hocmnaouuach.mo cofiumuucooaoo

one .awououa Hmaououowa «0 ma you :«a non woumwancoo
Hoconaonufictm.wo moHoa: mm commouaxo cam :mvonumz:
nova: monauomov mm hufi>uuom cannonwcmuuazaousozawlmaa

mo coaumcfiauouov oumu Hmauwca no women ma uaaoa comm

.mmzomOMUHz
A<zHHmm92H HHmm<m zH mon9<mHzmozoo QHU< UHZOMDUDAQImQ:

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.N muswfim

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47

.33 .mMmM .833 we venues 2: .3 3.33m 33

Homzm .musoz ~\~ N you w x oco.om~ um nodumwsmmuucooauuas an vflwaaoua one: m:0muomum
A.uamv oumuwamooua new ~.uoa:mv ucmumauoaam .uaowumumv + H8: zam.o .aouoUAHw
A>\>V Nmm .o.s In o<lmmue :5 cm mo as u cw Doe um vmxma an: amououa amaomouome we o~

 

 

ma ma ea c ~ o Noo.~
mm ofi mu ¢~ N m Nom.o
me n Re C a o No~.o
on m fix 0 o o Nmo.o
-a zmoqszm
mm o~ On 0 a o Noo.~
mm o~ Ne o~ m Nd Nom.o
on a ~w o o o No~.o
es a ~m o e o Nmo.c
x3 gamma;
mu m mm o m o mzoz
.umm .ummmm .uma .ummnm .umm .uoanm
cmououa HmuOB xum>wuon
amEOmouomE Humzm ~muou ngOm we won :«E\ oumwsm
~mcmwwuo uo N Iouoma dmcmwmuo mo N Icoo fioconaouuwcl egos:
ammuoum ~QBOH >um>muo< Humzm deuOH >um>muo< omumooam Homzm ucmwuouoo

 

.mHzmummHmo

OHZOHZOZ 18H: Homzm A<20momuH2 A<zHHmmHzH HHmm<m mo onH<NHAHmsqom MIR .h m4m<fi

48
concentration to 12 did not increase the amount of enzyme activity in
the supernatant. Using Emulgen 911, instead of Lubrol WX, resulted in
a lower recovery of total PNPGT activity. This lower recovery might
have been due to the presence of peroxides in the Emulgen. The 1.0%
Emulgen solution was found to be more than 3 mM in peroxide.

The effect of protein concentration, pH, ionic strength, and
temperature on the solubilization of PNPGT activity with Lubrol WX was
examined. Lowering the protein to detergent ratio did not increase the
percentage of initial enzyme activity recovered in the supernatant.

At protein concentrations of 2.5 mg/ml (0.5% ( ) Lubrol WX), PNPGT
activity was not detected in the supernatant (Table 8). Increasing the
pH of the detergent solution, while keeping the protein concentration
at 10 mg/ml, did not increase the supernatant recovery of PNPGT
activity (Table 9). No increase in the recovery of PNPGT activity in
the supernatant was observed after incubating microsomes for half an
hour in detergent solution at either 10°C or 22°C (Table 10).
Increasing the ionic strength of the solubilization buffer did not
improve solubilization (Table 11).

Mild sonication, however improved the recovery of PNPGT activity
in the supernatant. If the microsomal detergent suspension was
sonicated with a Bronson sonifier (4x20 sec) with a microtip set at 7
1/2 amperes, and centrifuged for 2 1/2 hours at 180,000 x g, 63% of the
initial activity was recovered in the supernatant (Table 12).

Under similar conditions, with either Lubrol WX, or with

octylglucoside, about 50-60Z of the microsomal PNPGT activity was

.33 am Mm H383 mo 852. 9: .3 8.352. was 982m .33: N: N you w x 80.9: on.
:owumwSMMmucmomuuH: an vmumaouq mum: acouuomum A.udmv mumuaaaomua can A.uon=mv unaumCuonsm .xz downs;

Nm.o + .esa :5 m.o .Houmoxaw A>\>v Nmm .o.~ ma u<umfiue :8 on an ace um conga was :fimuoud Hmaomouofiz

 

 

«a ma ms 0 n.a o x3 downs; Nm.o
No 0 me o m.m o 0:02 m.~
m” 00 ON mm o o.m o x: Hounsq Nm.o
mg m no 0 m.m o wcoz o.m
om om mm 0N w.¢ o.m x3 Hogan; Nm.o
mu m ~c o «.5 o mcoz o~
.umm .ummnm .mmm .ummom .umm .ummsm
:Hmuoua Hmuou xuu>quom
Hmaomouoaa Humzm Hmuou Hmaom we you :Ha\vmumw:n AHa\wav
Hmcuwfiuo mo N Iouoaa Hmcwwuuo mo N Icoo Hocwsaouuuotm moaoac coaumuucwocoo
samuoum Hmuoe >ua>auo< Homzm Hmuoe zuw>wuo< owwwomam Humzm ucwwumuwn aaououm Hmauaam

 

.x3 Joanna IHH3 Homzm
A<Zomoxoaz A<zHHmmHzH HHmm<M mo ZOHH<NHAHmDJOm are 20 onH<tzmozoo ZHMHomm mo Hommmm MIR .w m4m<fi

.Aqmv .mm MM wagon; mo conume

ocu an voxmmmm was Homzm .00: um muaon ~\~ N new w x ooo.ow~ um coaummsmfiuucmomuuaa hp cmumamua

mums meowuomum A.udav oumuuaaomuq new A.uoa=mv ucmumcumqam .x: Hounaq Nm.o “H.990 28 m.o .Houooaaw
A>\>v wa :Q vmumowccfi mnu mo Humans o<tmau9 :5 on 6% now um kuuaum was :wmuoua Hmaomouofia Ha\ws oq

 

 

mm mm on ¢~ ~.o~ «.0 x3 downs; nm.o
me Q me o m.m o «:02 m.w
mm cm mm ad o.a a.o x3 downs; Nm.o
:04 we m me o o6 o 952 o6
do mm mm om o.m m.w x3 Hounsg nm.o
me n ~m o ¢.o~ o 0:02 0.5
.umm .ummsm .umm .ummam .umm‘ .uommm
:Hmuoua awuou zuw>wuom
Hmsomouofia Homzm Amuou Hmaom we mom :«a vmumwsn
Hmcawauo mo N Iouoaa Hmcawfiuo mo N Icoo HocwcaoHUficl moHoa:
:Hmuoum sauce aua>auo< yoazm Hades auu>quo< oamauoam eoazm ucmwuwuon u<umaue co ma

 

x3
40mm34 :HHZ Homzm A<zomoxUHz A<zHHmmHzH HHmm<x mo ZOHH<NHAHmDAOm mzh 20 ca mo Powhmm axe .m mqm<e

51

.Ach

N you w x ooo.ow~

 

.Hm mm.uwaosq mo conuoa mnu an vmxwmmm «ma Homzm .ooc um muao; ~\—
um coaummswwuucoomuuas an voumqouq mum: mooauowuw A.uanv manuuauooua

van A.uma=mv ucmuwcumdsm .x3 Nouns; Nom.o H .89D :8 n.o .Houmuzaw A>\>v NmN .o.m In
o<ImHuH 25 on :a mouaumumaaou voumoavcu mnu um wouuqum mm: casuoud Hmaomououa Ha\wa OH

 

 

mm ma we ad c.w c.- comm
~m m~ Nn ed m.w ~.n 000‘
mm w~ mm 0“ m.m m.N 00¢
.umm\ .ummzm .umm .ummsw .umm .ummam
cumuoum Hmuou >ua>auow .
amaomouowa Humzm Hmuou Hmaom we won :Na\voumw2n
Hmcwwauo no N Iououa Hmafimwuo «0 N Icoo Hocwnaouuwan.oHoac
:aououm deuce aua>fiuu< aomzm Hmuos Nua>fiuu< uouaumam somzm unassumaaua

 

4<zHHmMHzH

.x: Joana; thz Homzm A<zomomouz
HHmm<x mo onH<NHAHm34om mzh zo mmDH<xmmzme mo Powhmm axe .ou m4m<9

52

 

.AQNV .Hm mm.uo«osa mo vozuma mcu an vommmmm mos
Humzm .oos um mason N\~ N you w x ooo.om~ um couumwsmuuucmomuuas ma umumnmuq mums
mcoauomuw A.uamv oumuaafiomum vcm A.umasmv ucmumcumqsm .x: Hounsq Nm.o.H..HHQ ZS m.o

.Houmoxaw A>\>v NmN .c.~ :a o<lmfiue

ooe um umuufium mm: cqmuoua Hmaowouuaa Ha\wa ON

 

 

cm mm mm mm m.¢ o.m z o~.o
mm mm ~m NN m.m m.a z o~.o
om Nu on mm o~ m.m z no.0
.umm .ummsm .umm .ummsm .umm .uomsm
:uououa Hmuou Nufi>fiuow
Hmaomouuwa Homzm Hmuou Hmaom we won :wa\voummsfi
Hmcumuuo mo N Iououa Hmcwwauo mo N Icoo HocmsqouuNCLm.mHoac uwuwsmlo< mums
seasons Hmuoe aufi>fiuu< eoaza dance mua>fiuu< camuomam yuazm mo muaumfioz

 

A<zHHmmHZH mo ZOHB<NHAHmDAOm

.x: AOMQDA IHHB Homzm Adzomomon
MIR zo :Hozmmfim UHZOH mo Pomumm are .- mqm<H

53

.33 .W W .333 «o cofioa

ozu ma cozmmmm mma Homzm .Uoc um mason N\~ N you w x ooo.om~ um cowumwsmfiuucmomuuaa
an voumaouq mums meowuomuw A.uaav mumuuauouoa cam A.uwasmv ucmumcuoaam

m nag: uofimwcom comcmum a mafia: AmONxcv .kumoucom mm: coamcoamsm ucwwumumvlamaomouoae
one .x: Hoops; Nm.o .eea :9 n.o .Houmoaam A>\>v Nmu .e.N ma u<umaue :5 on

ooc um voxaa mm: .- mo >ua>uuom owwuomam Homzm m :UHB :Nmuouq Hmaomouowa Ha\wa OH

 

 

 

oq we 0N no a.q w.m~ cofiumowcom
mm mN mm ~N m.m o.m coaumoucom oz
.uam .ummmm .umm .ummsm .umm\ .ummam
cumuoud Hmuou >uu>auom
Amaomouowa Humzm Amoco HmEOm we mod :Na\vmumw:fi
amcuwwuo no N Iouofia Hmcfiwauo «o N Icoo HocmnaouufiaLN mace:
:«muoum Hmuoe Nua>wuu< Homzm Houoe Nuq>auo< ouwaooam Homzm osmsummua
.x: Joanna =HH2 mm<¢mmmz<meqwzom203401ma= aozmzmomHHZLm.
4<20momon 4<2HemmazH mo onH<NHAHngom mzh zo zc~9<o~zom mo Homamm wry .N~ m4m<9

54
recovered in the supernatant (Table 13). Octylglucoside was used in
subsequent solubilization studies because it was readily dialyzable
(Figure 8), had a defined composition, had a reduced absorption at
280nm allowing for more sensitive uv monitoring of protein, and it gave
more consistent results than Lubrol WX. Fifty percent of the
microsomal PNPGT activity was recovered in the supernatant when
microsomes were treated with 1.0% (34 mM) octylglucoside in this buffer
at pH 7.5 (Figure 9). The recovery of PNPGT activity was improved by
treating the microsomes with a more alkaline octylglucoside solution
(Table 14). An even greater recovery of PNPGT activity was obtained by
increasing both the pH and the ionic strength of the octylglucoside
solution (Table 14). With 34 mM (1.0%) octylglucoside in 50 mM
Tris-HCl (pH 8.0), 100 mM KCl, 70% of the original microsomal PNPGT
activity (Figure 9) and 31% of the original microsomal protein was
recovered in the supernatant. The PNPGT activity, in a supernatant
containing EDTA and ME was stable for 12 hours at 4°C and had a half
life of 3 days. In the absence of EDTA, PNPGT activity had a half life
of 12 hours.

Less than half the initial microsomal phenolphthalein GT activity
was detected in the 34 mM octylglucoside microsomal suspension. After
centrifugation, little or no activity was seen in the supernatant or
the pellet.

DEAE Chromatography:

 

In preliminary experiments, the binding of octylglucoside

solubilized PNPGT to DEAE-Sephacel at pH 7.5 was studied. At this pH,

55

 

.Aqmv .Hm.mm weaved mo

vogume one NA cmzwmmm mm: Humzm .musos N\~ N you w x ooc.ow~ um eowumwemuuuewomuuae an
commaoud who: meowuomuw A.uaav manuamuomun new A.umaamv ucwumcumqam .mmumaem N\~ N am
aquouoae w :uN3 AoomOquv uofimaeom eomcmum m wean: .voumoueom was eonemawem uemwumumv
Hmaomouuaa one .ucmwumumu .eeo 2a m.o .Houmoefiw A>\>v New .m.~ ea Hozumfiue 2a om

Doc um vmxfie mm: .- mo >uw>uuom Homzm ofimwomam m nu“: :Nmuoud Hmeomouofie He\we ON

 

 

 

mm as ON on q me No.2 mvamoosawfiacoo
on «e w~ No a md Nom.o x3 Nouns;
.umm .uwmsm .umm .ummem .umm .ummim
cwwuoua Hmuou muw>fiuom
Hmeomouoqe Humzm Hmuou Hmeom we use e«e\kumw:n
Nmerauo «o N louowe Hmeawuuo «0 N Icoo HocmnaouuweLm.mHoee
cfimuoua Aesop sufi>auu< amaze sauce >u3>auu< ofiufiomam amaze samwumuma
.maumousgquhoo

024 N3 Joanna =9H3 Homzm A<zomomUHz 4<2HemmHzH mo onH<NHAHmDJOw Mme .m~ m4m<fi

56

Figure 8. THE DIALYSIS OF OCTYLGLUCOSIDE.

10 mg/ml of microsomal protein in a Tris buffer containing
1.0% octylgucoside was dialyzed against 500 volumes of the

same buffer without octylglucoside

57

 

 

 

 

 

 

18 24 30 36

Time of Dialysis (hours)

12

 

 

 

 

 

 

 

 

 

 

 

20.0
17.5
15.0

g s
10.0

1

23230 5 3.3039200 3.9::

Figure 9.

58

THE SOLUBILIZATION OF RABBIT INTESTINAL MICROSOMAL
UDP-GLUCURONYLTRANSFERASE ACTIVITY TOWARDS ETNITROPHENOL AT

VARYING CONCENTRATIONS 0F OCTYLGLUCOSIDE.

10 mg per ml microsomal protein was mixed at 4°C in
varying concentrations of octylglucoside and 50 mM Tris-HCl
pH 7.5, 20% (v/v) glycerol (0) or SOmM Tris-HCl pH 8.0,
100mM kCl, 20% (v/v) glycerol (0) as described under
"Methods". Supernatant (super) fractions were prepared by

centrifugation at 105,000 X g for 2 1/2 hours.

 

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60

mam: meoauomum uemumaquem

.:mvoSuoz: wave: wonauommv mm efimuoua use aua>auom
Huezm now vohmmmm use mueos N\~ N you w x coo.mo~ um cowumwSNNuuemu as vwamamua

.mvamooeawdzuuo No.~ cam .oeavwxmnuoazo NNoo.o .Hox zeoo~

H..m: zea .aouooaaw A>\>v NON .Huxlmwua 2e om uoq um vmxwe was we awe :He\vmumw=neou
HoemnmoauNeLm.mmHoee «N No >uu>auom oawfiooqm Homzm m nu“: cumuoua Hmeomouowe He\we 0a

 

 

 

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me mm mm m.a za Hmzu

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se mm an s.~ ze amzu

cfimuoam Hauou Nua>auom

Hmeomououe Hoezm Hmuou Hmeom we use cae\voumw=m
Hecawaao mo N Ioaoae Hmcawaao «o N Icoo Hocozdouuaclm.maoec

:ewuoae emuoa eue>auu< amaze amuoa eua>auu< oeeeumem amaze ucwaumwaa

moumooaqoaweoc 29H: Hoeze Adzowomouz A<zHHmmezH
a~mm<x mo ona<NHAHmsqom mzh 2o zeozmmem onoH az< me no Homeem mmh .ea mqm<9

61
(Table 14). With 34 mM (1.0%) octylglucoside in 50 mM Tris-HCl (pH
8.0), 100 mM KCl, 70% of the original microsomal PNPGT activity (Figure
9) and 31% of the original microsomal protein was recovered in the
supernatant. The PNPGT activity, in a supernatant containing EDTA and
ME was stable for 12 hours at 4°C and had a half life of 3 days. In
the absence of EDTA, PNPGT activity had a half life of 12 hours.

Less than half the initial microsomal phenolphthalein GT activity
was detected in the 34 mM octylglucoside microsomal suspension. After
centrifugation, little or no activity was seen in the supernatant or
the pellet.

DEAE Chromatography:

 

In preliminary experiments, the binding of octylglucoside
solubilized PNPGT to DEAE-Sephacel at pH 7.5 was studied. At this pH,
with a 50 mM Tris-HCl column buffer, 15% of the original PNPGT activity
applied to the column was eluted immediately after the void volume and
15% of the activity was eluted with a linear 0 to 0.25 M KCl gradient.
Column fractions immediately after the void volume, had apparent PNPGT
specific activities higher than that of the solubilized protein applied
to the column. Changing the pH of the column buffers from pH 7.5 to
7.4 or to pH 7.6 did not increase the PNPGT specific activity or yield
of column fractions immediately after the void volume. To determine if
the activity of column fractions eluted immediately after the void
volume could be attributed to overloading the column, those fractions
containing PNPGT activity were applied to another DEAE-Sephacel column

equilibrated with 50 mM Tris-HCl pH 7.5. Activity was eluted

62
immediately after the void volume which contained the highest PNPGT
activity were pooled and applied to a second DEAE-Sephacel column
equilibrated with 20 mM Tris-HCL pH 7.5. Forty percent of the PNPGT
applied to this column was eluted with a 0 to 0.15 M KCl gradient. The
pooled column fractions from the KCl eluate had an apparent PNPGT
specific activity 10 fold higher than that of the original microsomes,
with an overall yield of 3% (Table 15).

In subsequent experiments octylglucoside solubilized rabbit
intestinal microsomal protein was applied to a DEAE-Sephacel column
equilibrated with 50 mM Tris-HCl pH 8.0. Rabbit intestinal microsomes
were solubilized in octylglucoside buffered with 50 mM Tris-RC1 pH 8.0,
containing 100 mM KCl, and centrifuged at 105,000 xg for 2 1/2 hours.
The chloride concentration of the 105,000 x g supernatant was
approximately 145 mM. At this chloride concentration, PNPGT does not
bind to DEAE-Sephacel equilibrated to pH 8.0. However, PNPGT does bind
to DEAE-Sephacel at 45 mM chloride. Therefore, the supernatant was
diluted to lower the ionic strength. When the 105,000 x g supernatant
was diluted 1:2 with 20% glycerol to allow for the binding of PNPGT to
DEAE, the solution became turbid, though there was no apparent loss in
PNPGT activity. After centrigation of this solution for 2 1/2 hours,
at 105,000 xg, 48% of the original PNPGT activity was in the pellet.
Therefore, after microsomes were treated with octylglucoside, sonicated
and centrifuged, the resulting supernatant was diluted 1:2 with 20%

(v/v) glycerl containing enough octylglucoside to prevent turbidity.

63

.uesmvsuwluass New 2 m~.clo ussema s wemsa uswmsn eoNusunmumsvs

uo mseeao> easaoo n sums vsusas as: eua>muos Hueze ssh .uswmsn acmusunmdmsvs

mo mse3~o> m sum: vssmms as: ceeaoo ssh .n\~e a susu 30am s as .A>\>v .scmpmxsnuo~;o
Neom.m .mz zau .«aem 2a ne.m .A>\>e uoamueuw ume .oeuaooaumueuuo Nu.m .m.a ea umeuauua
:e ON "gum: vsusunmdwsvs casaoo Asosnesmlm<mn AEs m.o x Bow.wv sch .:\NE a mo susu
soue a as acezuxmsuoueo uaom.o .<amm zau .A>\>v ecumoeum Noe .meeaooaumueuuo um.m .m.a
me Nomlmmua tacm " sums vsusunmamavs Masosnesm ae\emsuoue we n.nv ee:a0o Nsossesmum<mo
Naow.o Nam m.w~v s cu vsmde s as: emsuoue assomouome vsnmamnaaos ssh .muso:

N\a N uow w x ooc.mc~ us vswsmmuucso was vsusomeom ass acmucsemsu ssh .m.N me No: «mus
ze on em vsvcsessm sus3 «sacmouomz .mflmwymwucm NasEs uannsu N\~ _ mo uesas>azcs ssu Eouw
pscmsuno sus: caonm muaamsm .Asm .as us usmuaq mo voguse snu an ushsmms as: >ua>muos
Hueze .ewe use stuom svmcousosa docsneouumehm saoee a suesssuesu zua>muos mo awe: as

 

m «nu ma mau u cusses umz
m.a za amenaaua 29mm

 

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w owq w.N an Na nus: senaoo
m.e za umeuaeua 25mm
umuanammuu<em .m
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acmusumamn5~0m
svmmoseawaxuso .N
ooa owNm o.~ Na oqs :oNuosum
asEOsouomz .~
sAmuNesv Asmsuoue we\mumc:v
ANV eua>aum< coausmauause eum>muo< Awev esum
lewMV‘ Huuoh uesusee< ommmosem ewwwoun kuoP. acmusommmuee

 

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64

The diluted supernatant was applied to a DEAE-Sephacel column
equilibrated with 50 mM Tris-H01 buffer pH 8.0. Figure 10 shows the
elution profile of this column. When DEAE column was equilibrated with
a buffer containing 0.60% (w/v) octylglucoside, no PNPGT activity was
detected immediately after the void volume. PNPGT activity was
detected after the void volume when the column was equilibrated to
0.65% (w/v) octylglucoside (Figure 11, Table 16). With a linear O to
0.05 M KCl gradient, at pH 8.0, approximately 40% of the transferase
activity applied to the column was eluted. The greatest total PNPGT
activity was eluted with 0.02 M KCl. Fractions, which had an apparent
PNPGT specific activity greater than 200 nmoles pfnitrophenol cojugated
per minute per mg protein, were pooled. The PNPGT activity of the
pool from the column equilibrated to 0.60% (w/v) octylglucoside, when
stored at 4°C, had a half life of about two weeks. The pool from the
column equilibrated to 0.65% (w/v) octylglucoside lost 25% of its
original PNPGT activity in 24 hours.

No phenolphthalein GT activity was detected in the pooled
fractions of the KCl eluate for the DEAE-Sephacel columns equilibrated
to 0.60% (w/v) octylglucoside.

Table 15 shows the results of the solubilization and chromato-
graphy procedure.

Properties of PNPGT Eluted from the DEAE Column:

The kinetic properties of the PNPGT in the pooled fractions of the

KCl eluate from the DEAE column, which was equilibrated to pH 8.0, were

examined. Kinetic analyses yielded an apparent V max of 280 nmoles

65

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.on HoesseouuNeLm.svum3ou euN>Nuus smsuswsesuuaeeousozaw
lea: uom usessms was n\He Na «0 susu Boam

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A<zHHmmezH HHmm<m mo rmmddooe<zom=0 223400 amo<=mmmlm<mn .O~ suewfim

on some);

66
PNPGT Activity

(nmoles pNP conjugated/min per ml)
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68
PNPGT Activity

(nmoles _p_NP conjugated/min per ml)
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69

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70
pfnitrophenol conjugated per minute per mg of protein and an apparent
Km for PNP of 0.2 mM at a UDPGA concentration of 3 mM (Figure 12).
An apparent Km of 0.4 mM was obtained for UDPGA at a 2 mM PNP
concentration (Figure 13). UDP had a strong inhibitory effect on PNPGT
activity. When UDPGA was the variable substrate and the PNP
concentration was fixed at 2.0 mM, UDP at concentrations between 0.5
and 10 mM produced inhibition competitive with UDPGA (Figure 13).

A sample of the pooled fractions, from the DEAE column KCl eluate,
was tested with liposomes to determine if the addition of lipid would
enhance the PNPGT activity. Neither intestinal liposomes nor egg yolk
phosphatidylcholine in lipid to protein (w/w) ratios of 0.2, 1.2, 2.3,
5.8 and 12 had an effect on PNPGT specific activity. Crude soybean
phospolipid at lipid to protein ratios of 0.5 to 15 also had no effect
on PNPGT specific activity. After centrifugation of the
phosphatidylcholine - enzyme suspension for one hour at 105,000 x g,
78% of the PNPGT activity was associated with the lipid pellet.

Egg yolk phosphatidylcholine liposomes did not increase the PNPGT
activity of the DEAE column fractions immediately after the void
volume.

Changes in the protein to octylglucoside ratio produced pronounced
effects on the PNPGT activity of the pooled fractions immediately after
the void volume of the DEAE column. At protein to octylglucoside

ratios (w/w) below 0.03 PNPGT was completely inactivated (Figure 14).

71

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72

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74

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76

 

 

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77

When the protein to octylglucoside ratio of the pool of the KCl
eluate from the DEAE column was increased two fold by ultrafiltration
the solution became turbid. After centrifugation at 150,000 x g for 1
1/2 hours, the pellet had 37% of the original PNPGT activity while the
supernatant had 7% of the original activity.

The pool of KCl eluate from the DEAE column could be diluted with
equilibration buffer with no octylglucoside, without apparent turbidity
or an apparent loss in PNPGT activity.

Gel Electrophoresis:

 

Figure 15 shows the polypeptides which were stained after the gel
electorphoresis of various fractions from the purification procedures.
The pattern produced from the supernatant of the solubilization process
lacked two major bands which were in the patterns produced from the
intesinal microsomes. Most of the polypeptide bands with molecular
weights greater than 65,000 in the pattern from the intestinal
microsomes were absent or not as darkly stained as those in the pattern
produced from the supernatant. The pattern produced on the slab gel,
from the pooled fractions of the DEAE column eluate had 5 major
polypeptide bands. By comparison with standard proteins of known
subunit molecular weight, these bands exhibited molecular weights
estimated at 60,000, 55,000, 48,000 and 46,000, with a very broad band
at 52,000. The pattern produced from the pooled fractions lacked most
of the polypeptide bands with molecular weights greater than 65,000
present in the pattern from the intestinal microsomes. Polypeptide

bands with estimated molecular weights of 60,000, 55,000 and 48,000

 

78

mmm.mm

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.m

79

 

 

80
were darker in the pattern produced from the pooled fractions than in
that produced from the supernatant. In turn, these bands were darker
in the pattern produced from the supernatant than that from the

microsomes.

DISCUSSION

The present work was undertaken to establish procedures for the
purification of PNPGT from the rabbit small intestine. The first step
in the purification scheme is the preparation of intestinal microsomes.
A relatively simple procedure was developed for the large scale
preparation of intestinal microsomes with stable PNPGT activity.

In these studies it was found that the activity of intestinal
microsomal PNPGT measured in a particular assay mixture reflects the
concentration and type of detergent in the assay mixture. From figures
3-5, it is apparent that a slight change in the concentration of a
detergent can result in a dramatic change in intestinal microsomal
PNPGT activity. When compared to other detergents, at the various
concentrations studied, Triton X-100, Renex 690, and octylglucoside
activated PNPGT to the greatest extent.

Illing and House (81) studied the effect of several detergents on
rat liver UDP-glucuronyltransferase activity towards fixed
concentrations of five phenolic acceptor substrates with widely varying
octanol buffer (pH 7.4) partition coefficents. These phenolic
substrates have been postulated to be conjugated by one
UDP-glucuronyltransferase, "GT1” (82). With some detergents the
extent of activation was dependent on the substrate partition
coefficient. While other detergents, such as Triton X-100, acted
independently of this coefficient. Based on these findings, Illing and
House (81) speculated that Triton Xv100 might affect the active site of

81

82
"GT1" (PNPGT) and thus might not be suitable as a solubilizing
agent.

Intestinal microsomes with relatively high PNPGT specific activity
were obtained from the duodenum. Intestinal microsomal PNPGT activity
decreased rapidly from the duodenum to the ileum. This is in agreement
with the findings of Aitio 3£_3l3 (83). On the other hand, Lasker (84)
reported that the capacity for the glucuronidation of the artificial
steroid, diethylstilbesterol, was uniformily distributed throughout the
small intestine. In light of Tukey's conclusion that in the liver the
form of UDPGT which metabolizes the steroid, estrone, is distinct from
the form which metabolizes pfnitrOphenol, (64) it is possible that
these two distinct forms of UDPGT exist in the intestine as well. The
form which conjugates estrone might also conjugate diethyestilbesterol,
and have a distribution pattern along the small intestine distinct from
that of PNPGT.

Since the duodenum is the region of the small intestine in which
the greatest absorption of dietary xenobiotics occurs, there would be a
physiological need for greater xenobiotic metabolizing activity in the
duodenum. Several enzymes, which like PNPGT are instrumental in the
metabolism of xenobiotics, have a distribution along the length of the
intestine, similar to that of PNPGT. Cytochrome P-450 dependent mixed
function oxidase activity has been found to be substantially higher in
the duodenum than in the ileum (85). Epoxide hyclrolase was reported
to have a relatively higher activity in the duodenum (86). The

activity of the cytoplasmic enzyme, glutathione-S-transferase, which

83

conjugates many xenobiotics with glutathione, was shown to be four fold
higher in the duodenum than in the ileum (87). Even though the highest
PNPGT specific activity is found in the duodenum, the entire length of
the rabbit small intestine was used to obtain microsomes in order to

increase the total enzyme yield. The specific activity of PNPGT
was found to be greater in microsomes derived from the intestinal
mucosa than those derived from the whole intestine. These findings are
in agreement with the work of several investigators who by using
mucosal scrapings of the small intestine have shown that the epthelium
was the principal site for intestinal drug metabolism (88,89). Hoensch
ggngl. found that the specific activity of the drug metabolizing
microsomal mixed function oxidase system derived from the intestinal
mucosa was higher in the upper villous cells than in the epithelial
crypt cells (90). Although the specific activity of PNPGT was higher
in microsomes obtained from the mucosa, in the partial purification of
rabbit intestinal PNPGT, microsomes were obtained from the whole
intestine. Microsomal preparations using mucosal scrapings were time
consuming and yielded less PNPGT activity. For studies not involving
the purification of a microsomal enzyme, the microsomal preparation
procedure developed by Shirkey £5.21: might be suitable (91).

It was found that fasting rabbits for 24 hours resulted in a
decrease in the total microsomal PNPGT activity, although the specific
activity was slightly increased. Similar results were reported by

Marselos £5.31: (92).

84

One common means of increasing the total activity of microsomal
enzymes is to treat animals with certain chemicals, such as
phenobarbital or 3-methylcholanthree, which induce the synthesis of the
enzymes. Phenobarbital is one of many drugs that stimulate various
pathways of metabolism in liver microsomes, whereas polycyclic aromatic
hydrocarbons, such as 3-MC, stimulate a more limited number of enzymes
(93). An inducer which functions about the same as 3-MC, but which is
not a carcinogen, is B-naphthaflavone. An inducer with properties of
both penobarbital and 3-MC is the Firemaster mixture of PBBs (94).
Unlike phenobarbital whose inductive effect may be restriced to the
liver (95), 3-MC has been shown to be a good inducer of rat duodenal
PNPGT activity (96).

Pretreatment of rabbits with B-naphthaflavone or with Firemaster
PBBs did not increase the total intestinal microsomal PNPGT activity.
SDS polyacrylamide gel electrophoresis revealed that some proteins of
the intestinal microsomes obtained from rabbits pretreated with PBBs
were induced as the polypeptide pattern produced by these microsomes
differed from that produced by the microsomes obtained from untreated
rabbits. The apparent lack of induction might be attributed to species
differences in the stimulation of intestinal enzymes. Treatment of
rats with Firemaster PBBs induced rat duodenal PNPGT activity two fold
but did not induce rabbit PNPGT. Likewise, treatment of rabbits with
3-MC did not increase intestinal mixed function oxidase activity, but
did increase the intestinal mixed function oxidase activity of rats

(97).

85

In these studies, when rabbits were pretreated with phenobarbital,
the intestinal microsomal PNPGT total activity was not increased though
the specific activity increased slightly. This parallels the findings
of Aitio sgflgl., who reported that phenobarbital, whether given in the
drinkng water or given i.p., did not increase UDPGT activity (98), and
parallels the findings of Bock 32 El! (99) as well.

Once a procedure for the isolation of intestinal microsomes had
been established, a procedure was developed for the solubilization of
rabbit intestinal PNPGT from intestinal microsomes. The selection
of a detergent for the solubilization of PNPGT was based in part on its
non-denaturative properties. Because it is a relatively mild
detergent, octylglucoside was chosen for the solubilization of PNPGT.
Another factor in choosing octylglucoside was that octylglucoside was
readily dialyzable in the presence of membrane protein, confirming the
previous work of Baron and Thompson (100). Being readily dialyzable,
octylglucoside could be more readily removed from a purified PNPGT
preparation which might be valuable in reconstitution studies with this
preparation. Other nonionic detergents, such as Lubrol WX, would
probably be more difficult to remove.

Sato 52 El: (101) purified rabbit liver microsomal PNPGT to
apparent homogeneity with an overall yield of 10-15%, using the anionic
detergent, sodium chloate. However, in these studies, sodium cholate
was found to inactivate rabbit intestinal microsomal PNPGT even at very

low concentrations.

86

Emulgen 911, a nonionic polyoxyethylene ether detergent, has been
used for the successful purification of rabbit liver PNPGT (64). In
these studies however, when rabbit intestinal PNPGT was solubilized
with Emulgen 911, all PNPGT activity was lost within 12 hours. Some of
this loss of activity may have been due to the 3 mM peroxide
contamination in the detergent. It was found that 3 mM cumene peroxide
resulted in a complete loss of PNPGT activity after one day. Peroxide
contamination frequently occurs in nonionic polyoxyethylene ether
detergents.

The two fold increase in intestinal PNPGT specific activity that
was found upon solubilization of intestinal microsomes with
octylglucoside indicated that the detergent was slightly selective in
the extraction of PNPGT. This was also indicated by polyacrylamide gel
electrophoresis, as the polypeptide pattern produced from the
supernatant of the solubilization process lacked many of the bands
produced from the unsolubilized intestinal microsomes.

Along with detergents, mechanical means can be used to aid in the
solubilization of membrane proteins. Sonication is widely used to
solubilize microsomal protein, even though it usually produces a
heterogeneous mixture of membrane fragments (102).

The Purification Scheme

 

Successful purifications of rabbit and rat liver PNPGT have
employed UDP-affinity chromatography (60-64, 72, 103). It is likely
that UDP-affinity chromatography could be used successfully for the

purification of rabbit intestinal PNPGT as well. It was found that UDP

87
is strongly inhibitory to rabbit intestinal PNPGT, and therefore it
would be expected that PNPGT could bind well to the UDP meioty of an
affinity resin. In the purifications of liver transferases (60-64, 72,
103), the total detergent solubilized microsomal protein was not
applied to the UDP-affinity resin. Rather, a transferase preparation,
partially purified by DEAE chromatography, was applied to the
UDP-affinity resin. Gorski and Kasper (103) reported that better
results were achieved when a partially purified transferase preparation
was applied to the UDP-affinity resin. They speculated that this may
have been because DEAE chromatography of the solubilized microsomal
protein reduced the amount of nonspecific binding on the affinity gel
and eliminated degradative enzymes which could remove the UDP moiety
from the support. DEAE chromatography might, by separating different
UDP-glucuronyltransferases, reduce the competition between these
transferases for the UDP moiety and result in a more homogeneous
transferase preparation. Thus, the use of DEAE chromatography as one
of the first steps in the purification of rabbit intestinal PNPGT was
studied.

That DEAE-chromatography of octylglucoside solubilized intestinal
microsomes removed many of the contaminating and detrimental proteins
is suggested by the increased apparent PNPGT specific activity, the
enhanced PNPGT stability and the SDS PAGE gel of the pooled fractions

from the DEAE column KCl eluate.

88

The Yield of PNPGT Activity After DEAE Chromatography

 

Although DEAE chromatography has been a useful step in the
purification of rat and rabbit liver PNPGT, the yield of PNPGT activity
has been relatively low. Both Burchell (62) and Tukey 35 El. (57) used
DEAE columns equilibrated with Tris buffer pH 8.0, and eluted PNPGT
with a KCl gradient. In their purification, about 16% of the PNPGT
units applied to the DEAE column were eluted with the pooled fractions
of the KCl eluate,with about a four fold increase in the specific
activity.

In the partial purification of rabbit intestinal PNPGT, about 20%
of the rabbit intestinal PNPGT units applied to a DEAE column
equilibrated to pH 8.0, were eluted with the pooled fractions of the
KCl eluate, with a five and a half fold increase in PNPGT specific
activity.

The low yield of intestinal PNPGT activity in the KCl eluate after
DEAE chromatography, might be due to the degradation, the inactivation,
and/or the inhibition of PNPGT. Inactivation by octylglucoside might
contribute to the low recovery of intestinal PNPGT activity. As shown
in Figure 13, the octylglucoside to protein ratio can have a pronounced
effect on PNPGT activity. Baron and Thompson (100) described that
octylglucoside had a "critical denaturing concentration", a
concentration above which the activity of an enzyme was extensively and
irreversibly lost. Some fractions from the DEAE column had protein to
octylglucoside ratios which could be inactivating. A few column
fractions had protein to octylglucoside ratios less than the 0.03 (w/w)

which was found to be completely inactivating for the PNPGT activity of

89
the fractions immediately after the void volume. Still, other
fractions might have had protein to octylglucoside ratios which, while
not large enough to have caused visible turbidity, could have been
large enough to produce amorphous protein aggregation. Such
aggregation, in turn, might have hindered the resolution of intestinal
PNPGT by DEAE chromatography.

Partial delipidation of intestinal PNPGT might have contributed to
the low yield of PNPGT either by promoting enzyme inactivation or by
diminishing enzyme stability. It is not yet known if delipidation
could lead to the inactivation of rabbit intestinal PNPGT. Rat liver
PNPGT was reported by Jansen £3.2lf to be irreversibly inactivated by
delipidation (104). Several investigators have reported that
delipidation of partially purified liver PNPGT preparations resulted in
a marked diminuation of activity, which was then restored with the
addition of certain classes of phospholipids, phosphatidylcholines
being the most effective in the restoration of activity (105, 106). In
studies with intestinal PNPGT, it was found that incubation of
partially purified intestinal PNPGT with egg yolk phosphatidylcholine,
crude soybean phospholipid, or intestinal microsomal lipid did not
increase PNPGT activity. Still it can not be ruled out that some PNPGT
activity from the DEAE-Sephacel column might have been lost due to
inactiviation resulting from the partial delipidation of the enzyme.

It is not yet known whether or not delipidation could lead to the
diminished stability of rabbit intestinal PNPGT. It was found that the

PNPGT activity of the solubilized intestinal microsomal preparation had

90
a half life of 12 hours. Thus some activity was lost during the
application of the solubilized preparation to the DEAE column.
Delipidation might have contributed to this instability. With rat
liver PNPGT, Gorski and Kasper reported that the delipidation enzyme
showed irreversible inactivation, the rate of which could be slowed by
combining the enzyme with ethylene glycol and/or liposomes (106).

The low yield of rabbit intestinal PNPGT activity might have been
due in part to some of the PNPGT remaining bound to the DEAE column.
After PNPGT activity was eluted with a 0 to 0.05 M KCl gradient, more
PNPGT, with a low specific activity, was eluted with a 0.05 to 0.3 M
KCl gradient.

The Elution Profile of UDPGT Activity After DEAE Column Chromatography:

 

DEAE column chromatography of both liver and intestinal PNPGT
produced similar elution profiles. With DEAE-cellulose column
chromatography (pH 8.0) of both rabbit and rat liver PNPGT (957,62), a
peak of PNPGT activity eluted at low KCl concentrations. These two
peaks exhibited different glucuronidation activities towards alternate
substrates. In the partial purification of rabbit intestinal PNPGT, a
peak of rabbit intestinal PNPGT activity was eluted immediately after
the void volume when the DEAE-column was equilibrated to .60%. The two
peaks of PNPGT activity may be due to two pfnitrophenol:
VDP-glucuronyltransferases which have protein structures which are
distinct from each other or due to two forms of a single PNPGT, such as
an active lipoprotein complex with both size and charge heterogeneity.

Corski and Kasper (106) purified rat liver PNPGT which demonstrated

91
three isoelectric points upon isoelectric focusing but which produced a
single polypeptide pattern upon SDS-polyacrylamide gel
electrophoresis.

Phenolphthalein GT and PNPGT

 

Wishart (67) postulated that in the liver there was a
UDP-glucronyltransferase which conjugated pfnitrophenol which was
distinct from a glucuronyltransferase which conjugated phenolphthalein
GT. During the purification procedure for rabbit intestinal PNPGT,
there were several indications that there may indeed be two distinct
forms of UDPGT. Phenolphthalein CT was inactivated by the
octylglucoside solubilization procedure, while PNPGT was activated. No
phenolphthalein CT was detected in the pooled fractions of the kCl

eluate from the DEAE column which had a high PNPGT specific activity.

SUMMARY

A procedure for the partial purification of PNPGT from rabbit
small intestinal microsomes was developed, which involved the
preparation of intestinal microsomes, the solubilization of PNPGT from
these microsomes and the DEAE column chromatography of the solubilized
enzyme. This procedure was suitable for the first stages of a scheme

to purify the enzyme to homogeneity.

Microsomes obtained from the first 250 cm of the small intestine
distal to the stomach pylorus had an average PNPGT specific activity of
25 nmoles pfnitrophenol conjugated per minute per mg of protein, and

were stable for six months when stored at 20°C.

The distribution of PNPGT in the intestine was studied. The
highest PNPGT specific activity was in the duodenum. Microsomes
obtained from mucosal scraping of the intestine had a higher PNPGT

specific activity than microsomes from the whole intestine.

Microsomes derived from rabbits which were treated with
penobarbital, B-naphthaflavone, or PBBs did not have a higher PNPGT
total or specific activity than microsomes derived from untreated
rabbits.

92

93
Octylglucoside was in the solubilization of microsomal PNPGT as it is a
mild nonionic detergent which is readily dialyzable. Microsomal
solubilization was enhanced by increasing the ionic strength and
alkalinity of the solubilization buffer. With a pH 8.0 solubilization
buffer with 145 mM chloride and 34 mM of the microsomal PNPGT activity
was recovered in the 105,000 x g supernatant, with a two fold increase

in the apparent specific activity.

The actylglucoside-microsomal preparation was diluted to lower
the chloride concentration and applied to a DEAE column equlilibrated
to pH 8.0. When the column was equilibrated to 0.65% octylglucoside,
one peak of PNPGT activity was seen immediately after the void volume
and another peak was eluted with approximately 0.02 MKCI. However,
when the column was equilibrated to 0.60% the peak immediately after
the void volume was not detected. Pooled column fractions from the KCl
eluate had an average specific activity of 250 nmoles pfnitrophenol

conjugated per minute per mg of microsomal protein.

Incubation of the pooled column fractions with
phosphatidylcholine or with intestinal microsomal liposomes did not

increse the PNPGT activity.

The protein to octylglucoside ratio was a critical factor.
Below a certain level, PNPGT was completely inactivated, above a
certain there was visible turbidity and presumably protein
aggregation.

Phenolphthalein GT did not co-purify with PNPGT.

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