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A Study On The Loss Of Uricolytic Activity During Prieate

Evolution 1. Silencing 0f Urate Duidase In A Hoeinoid Ancestor.

bY

George Enrique Polanco

A dissertation submitted in partial fulfillment of the
requireeents for the degree of Master of Science (Zoology) at

Hichigan State University 1985.

Coeeittee eenbersa

Professor Thoeas B. Friedean, Chairean
Professor Buy L. Bush

Associate Professor Karen L. Kloeoarens
Associate Professor Donald 0. Straney

Acknowledgments
Abstract

Introduction
Materials and Methods
Results

Discussion

Tables

Figures

Appendix

References

TABLE OF CONTENTS

 

 

 

 

 

 

 

 

 

 

GUI“

l3
I4
22
30
34
38

To my parents,
Jorge E. Polanco

Haria 8. Polanco

Acknowledgeeents

I wish to thank Dr. Thomas D. Friedman for his guidance,
encourageeent and friendship throughout the course of this
study. Dr. Leos S. Kral, Dr. Daniel H. Johnson and Dr. Jack H.
Schlein for contributing to ey developeent as a scientist. 1 ae
deeply grateful for their advice and friendship. I also thank ey
coeeittee eeobers, Dr. Karen L. Kloeparens, Dr. Buy L. Bush and
Dr. Donald 0. Straney for their critical review of this
eanuscript. Special thanks to Dawn H. Lounsbury for her
companionship, patience and assistance in the preparation of this
eanuscript. I thank God for giving ee the strength to continue

when I could no longer do so.

Financial support during ey studies at Hichigan State University
was provided by the Zoology Departeent, the Center for Electron
Optics and the Minority Fellowship Prograe. without the
cooperation and generosity of investigators at the prieate
research centers listed in table 1 this study could not have been

accoeplished.

Abstract
A study on the loss of uricolytic activity during prieate
evolution. 1. Silencing of urate oxidase in a howinoid ancestor.

By
George Enrique Polanco

This study extends previous findings on the presence, absence and
stability ,of prisate hepatic urate oxidase activity and
clarifies conflicting published data. Urate oxidase
[E.C.l.7.3.3.l, a liver specific enzyee, catalyzes the oxidation
of uric acid to allantoin, carbon dioxide and hydrogen peroxide.
Urate oxidase activity assays used in earlier studies eeasured
the rate of disappearance of uric acid or monitored the
consueption of oxygen during the oxidation of uric acid and
therefore did not exclude non-specific oxidation of uric acid by
other enzyees or non-enzysatic degradation of uric acid. In this
study a definitive and ultrasensative nicroradiochenical assay
for urate oxidase activity which measures the production of
(14CJallantoin from [14CJuric acid was used (Friedean and Herril,
1973; Friednan and Johnson, 1977).

The results of this study indicate that: 1. All the New florid
and Old World eonkeys exaeined exhibit stable and easily
detectable hepatic urate oxidase activity 2. Prieate hepatic
urate oxidase has a lower specific activity than eouse and rabbit
hepatic urate oxidase 3. Representative species of the five
genera of hoeinoids all lack hepatic urate oxidase activity 4.
No detectable activator or inhibitor in whole liver hoeogenates

of urate oxidase activity is present in hoeinoids. New world

monkeys and Old world monkeys 5. There exist no evidence for
intermediate steps in the loss of urate oxidase activity in the
hominoids 6. Urate oxidase activity was silenced in an ancestor
of the five present day genera of hominoids after the ancestor
diverged from the Old Norld monkeys.

Christen and coworkers (1970a, 1970b) and Hacker (1970) claimed
without published data that urate oxidase activity is highly
labile in some New World and Old Horld monkeys. This assertion is
the main premise of a model proposed to explain the loss of urate
oxidase activity during primate evolution. No evidence of highly

labile urate oxidase activity was observed in this study.

Introduction-

For many mammalian species, the terminal step in the metabolic
degradation of purines is the oxidation of uric acid by urate
oxidase (E.C.1.7.3.3J to the excretory product allantoin . The
principle subcellular location of urate oxidase is the peroxisome
of hepatic tissue<Essner, 1969; Masters and Holmes, 1977; Smith,
1979). Human disorders associated with elevated uric acid levels
have prompted examination of serum uric acid levels and urate
oxidase activity in a variety of vertebrates including nonhuman
primates.

A survey of the literature reveals that hepatic urate oxidase
activity levels have been examined in a few New Horld monkeys,
Old world monkeys and hominoids (Hells, 1909; Hells and Caldwell,
1914; Sorensen,1959; de la lglesia et al.. 1966; Christen et
al., 1970a and 1970b; Nakajima and Bourne, 1970). Past studies
have employed urate oxidase activity assays which measured the
rate of disappearance of uric acid or which monitored the
consumption of oxygen during the enzymatic oxidation of uric
acid. These assays are not particularly sensitive to low levels
of urate oxidase. Often proper controls were not included and
therefore these studies could not distinguish between urate
oxidase activity and non-specific oxidation of uric acid. In
this study an ultrasensitive microradiochemical assay measured
the rate of synthesis of [14C1allantoin from (14C1uric acid and
consequently non-specific oxidation of uric acid can be ruled
out (Friedman and Herril, 1973; Friedman and Johnson, 1977).
Furthermore, appropriate controls were used to assure that tissue

samples were stored properly for maintenance of enzymatic

activity.

At present, the limited data available on humans. orangutans, and
chimpanzees indicates that no detectable hepatic urate oxidase
activity is found in these hominoids (Wells and Cadwell, 1914;
Sorensen, 1959). Urate oxidase activity levels were measured in
five different primate species by Christen and coworkers (1970a
and 1970b). They reported that two species of New World monkeys
completely lacked urate oxidase activity and that one New World
and on- Old World mk'Y! 9.9.!!! 3121592.!!! Md assess aegis;
respectively, had highly labile urate oxidase activity although
the data was not provided. The unpublished observations on the
lability of urate oxidase in 993;; and flaggga formed the basis of

postulated multiple steps in the loss of urate oxidase activty

during primate evolution.

In the step-wise mutational model. a genetic change in a primate
ancestor resulted in reduced and/or labile urate oxidase
activity. An additional mutation or series of mutations which
followed in time. presumably silenced urate oxidase activity
entirely in a hominoid ancestor while the extant genotype that
gives rise to low and/or labile urate oxidase activity can be
observed in some New and Old World monkeys. Furthermore.
additional mutations in some New and Old World monkeys supposedly
resulted in a 1. further reduction in urate oxidase activity, 2.
a more highly labile variant of urate oxidase activity, or 3.
the complete loss of urate oxidase activity. Despite the pausity
of supporting data, this postulate of a step-wise series of

mutations resulting in the loss of hepatic urate oxidase activity

during primate evolution is widely accepted. (Orowan, 1955;
Christen et al.,1970a, 1970b; Wacker, 1970; Kuster et al., 1972;
Logan et al.,1976; Roch-Ramel and Peters, 1978; Ames et al.,
1981).

The genetic events that occured during the evolution of primates
resulting in the loss of urate oxidase activity in hominoids are
.unknown. However, there exists considerable speculation about the
adaptive significance of elevated uric acid levels in primates.
Of special interest are the affects on human development which
may have resulted from the increase in serum uric acid level
which followed the loss of urate oxidase activity. Since uric
acid is structurally similar to caffeine and theobromine, Orowan
(1955) proposed that it may mimic their effect and act as a
cerebral stimulant. According to Orowan, the loss of urate
oxidase activity in primates and the concurrent rise of serum
uric acid levels produced an abrupt increase in intellectual
potential and thus may have been a significant event in man's
evolution. Ames and coworkers (1981) suggested that since uric
acid is a powerful antioxidant it may act as a sink for free
radicals and singlet oxygens present in the serum. Smith (1983)
has demonstrated that uric acid is an effective antioxidant for
unsaturated fatty acids and that it protects lipids of cell
membranes. High serum uric acid levels may have contributed to a
lengthened hominoid life span and a decreased cancer rate
(Proctor, 1970; Ames et al. 1981; Smith, 1983).

This study re-examines and extends previous findings on primate
urate oxidase activity and the stability of primate urate oxidase

activity. Seven species of hominoids, which included gorilla and

gibbon, which had not previously been examined for uricolytic
activity. were assayed for hepatic urate oxidase activity. The
evolutionary relationship among primates and the results. on the
presence and absence of urate oxidase activity, from this study
were compared in order to estimate at which phase during primate

evolution urate oxidase activity was lost.

Materials and Methods

Chemicals

14 14
Xanthine-Z I C] (48mCi/mmole) and uric acid-2t C] (53mCi/mmole)
were acquired from Research Products International, Elk Grove,
Illinois and Amersham, Arlington Heights Illinois, respectively.
Xanthine, uric acid, allantoin and ESTA were purchased from Sigma

and borate from MOB. Polygram cel 300PEI thin layer sheets were

supplied by Brinkman Instuments, Westbury, New York.

Liver Samples

The human liver samples used in this study were obtained from
patients of Drs. J. Mayle and D. Greenbaum (College of Human
Medicine, Michigan State University). Human liver samples were
biopsied by percutaneous needle biopsy from patients requiring
histological examination to diagnose a suspected liver
dysfunction. Only when the amount tissue was in excess of that
required for medical examination was the tissue used for assaying
urate oxidase and xanthine oxidase activity. Permission from
Michigan State University and local hospital research committees
was obtained and the guidelines of these institutions regulating
informed consent were followed.

Human liver samples were immediately frozen on dry ice and
stored at -70°C before being assayed for urate oxidase and
xanthine oxidase activity. Forty to eighty microliters of
deionized water was added to the human liver sample and the
tissue was then disrupted in a Kontes Duall glass/glass

homogenizer. The mean protein concentration was 7.6mg/ml with a

range of 1.1mg/ml to 25mg/ml.

IO

Nonhuman primate liver samples were obtained from six different
primate centers in the United States. Liver samples were obtained
at autopsy or were biopsied from live animals. For nonhuman
primates, depending on the size of the liver sample, deionized
water was added to yield a mean protein concentration of

approximately 168mg/ml with a range of 113mg/ml to 282mg/ml.
Protein concentrations were determined by the Bradford method

(Bradford, 1976) using bovine ...u. albumin as the standard.
The sources and conditions of the primates livers are decribed in

Table 1.

Enzyme Assays

Urate Oxidase Assay

Urate oxidase activity was assayed by modifications of an
ultrasensitive microradiochemical assay procedure which measures
the production of £14C3allantoin from [I‘Cluric acid (Friedman
and Merril, 1973; Friedman and Johnson, 1977). The commercially
prepared [14C1uric acid was further purified on a cellulose-
coated thin layer backed sheet using water as a solvent. This
ultrapure I14CJuric acid was used in cases where tissue was
suspected of having low or no urate oxidase activity. Optimal
assay conditions for urate oxidase activity with respect to EDTA
and borate concentration and optimal buffer pH was determined for
several primate species (Appendix A).

To assay urate oxidase activity 13.0 microliters of 0.01M borate/
15.7mM EDTA pH 9.0 buffer, 2.0 microliters of primate liver

14
homogenate and 10.0 microliters of 0.79mM t CJuric acid were

II

0
mixed and incubated in'a 37 C water bath. At designated times,

5.0 microliter aliquots were removed from the reaction mixture
and spotted on a PEI cellulose thin layer plate. Prior to
spotting the reaction mixture, the PEI plates were washed and
allowed to air dry and lanes were lightly penciled on the
surface. Each sample site was spotted with 5.0 microliters of
16.0 mM allantoin which was visualized with
dimethylamionbenzaldehyde. The allantoin served as a marker for
identifing the location of II‘CJallantoin. One-dimensional
chromatography was carried out in an ascending fashion to a
height of 10 centimeters using 0.15M NaClnlOOX EtOH (431) as a
solvent. [1‘Clallantoin spots were cut out and placed in Nalgene
filmware scintillation bags with 3.0 milliliters of scintillation
cocktail. The scintillation bags were sealed, wiped with 70%
ethanol to eliminate static charges and counted in a Deckman LS

7500 liquid scintillation spectrometer. The average counting

efficiency for all experimental runs was 97%.

Xanthine Oxidase Assay

An ultrasensitive microradiochemical assay which measures the
production of [14CJuric acid from (1‘Clallantoin was used in this
study. The xanthine oxidase assay is similar to the urate oxidase
assay with the following exceptions; Ten microliters of primate
liver homogenate was combined with 5.0 microliters of 0.1 M
cyclohexylaminopropane sulfonic acid buffer (CAPS), pH 11.0 and
10.0 microliters of I14CJxanthine. Five microliters of 2.9mM uric

acid served as a marker and was visualized under short wave

ultraviolet light. The solvent used consisted of

I2

glycerol3isopropano130.15M LiCl in water (139310).

Concurrent Controls

Primate liver homogenates were simultaneously assayed for urate
oxidase and xanthine oxidase. At the same time mouse liver
homogenates were also assayed for urate oxidase and xanthine
oxidase. These two concurrent positive control assays assured
that an absence of urate oxidase activity in a particular primate
liver homogenate could not be attributed to a technical error in
the assay procedure. Primate liver homogenates were also boiled
and assayed for urate oxidase and xanthine oxidase activity.
Assays using boiled homogenates and assays in which primate liver
was omitted from the reaction mixture served as negative

controls.

Stability of Urate Oxidase And Xanthine Oxidase Activity

The stability of primate liver urate oxidase activity was
examined under the following conditions3 1. After maintaining
liver homogenate at 0-4DC for 7.5 and 12 hours, 2. After
maintaining liver homogenate at ream temperature (23°C) for 1 and
2.5 hours, 3. After subjecting the liver homogenate to five and
ten rapid freeze-thaw cycles (-70 to 23°C). Primate liver
xanthine oxidase activity was measured after five freeze-thaw
cycles. Rabbit and mouse liver homogenates were also subjected to
some of the same treatments before being assayed for urate

oxidase and xanthine oxidase activity .

I3

Results

All hominoids, New World and Old World monkeys reported in table
2 had xanthine oxidase activity. Xanthine oxidase activity served
as a control, since the existence of xanthine oxidase in a
primate liver homogenate suggests that the tissue sample was
properly preserved and could be assayed for urate oxidase
activity with confidence. Xanthine oxidase activity is believed
to be present in all primates except humans with xanthinuria, a
rare inherited deficiency (Holmes and Wyngaarden, 1983). When a
primate liver sample exhibited no urate oxidase and no xanthine
oxidase activity, as was the case with one Erythggggbgg 935;;
sample, it suggested that the liver sample was not properly
stored. Another Ergthroceggg ggggg liver sample could not be
obtained to verify the presence of xanthine oxidase and urate
oxidase in this primate species. However, a liver sample from a
closely related genus, fliggighggug talagoin, was assayed and
exhibited easily detectable levels of these two enzyme.

Livers from six humans, three chimpanzees, two orangutans,. two
gibbons and one gorilla were assayed for urate oxidase and
xanthine oxidase activity (Table 2). These represent species from
the five living genera of hominoids. No urate oxidase activity
was detected in any hominoid liver sample (Table 2). Seven
species of Old World monkeys and three species of New World
monkeys all had easily detectable levels of urate oxidase
activity.

When the specific activity of hepatic urate oxidase in New World
and Old World monkeys and that found in rabbit and mouse livers

are compared, a significant quantitative difference is observed

14

(Table 3). The combined average specific activity of urate
oxidase from New World and Old World monkey liver homogenates is
16.5% and 28.9% of the urate oxidase activity observed in rabbit
and in mouse liver, respectively. The observed specific activity
differences could reflect an increased in 21559 lability of New
World and Old World monkey hepatic urate oxidase activity.

The stability of primate urate oxidase activity was investigated
under three different sets of conditions (Table 4 t 5). Urate
oxidase activity from nine species of Old World and three species
of New World monkeys appear to be as stable as urate oxidase
activity from rabbit and mouse liver. For example, after ten
freeze-thaw cycles or after maintaining liver homogenates at 23°C
for 2.5 hours, the percent of initial Macaga gglgtta urate
oxidase activity remaining was nearly identical to the percent of
rabbit or mouse liver urate oxidase activity enduring the same
treatment (Table 4 & 5). Primate xanthine oxidase activity was as
stable as rabbit or mouse liver xanthine oxidase activity (Table
4 & 5). The most distinctive aspect of these results is that

primate liver urate oxidase activity and xanthine oxidase

activity, when present, are stable.

Discussion

There are inconsistencies among published reports on absence or
presence of hepatic urate oxidase activity in different species
of hominoids, New World and Old World monkeys. The following
characteristics of the experimental design used in this study

make the results reported here definitive and unequivoca13

15

1. A direct ultrasensative microradiochemical urate oxidase
activity assay which measures the rate of synthesis of £14Cl
allantoin from I14CJuric acid excludes the possibility of non-
specific oxidation of uric acid by other enzymes or non-enzymatic
degradation of uric acid.

2. To be certain that the primate liver was removed and stored
properly soon after death of the animal, a direct ultrasensitive
microradiochemical xanthine oxidase activity assay which measures
the rate of synthesis of £14CJuric acid from £14Clxanthine was
simultaniously performed on all primate liver homogenates that
were assayed for urate oxidase activity.

3. At the same time that primate liver homogenates were assayed
for urate oxidase and xanthine oxidase activity positive control
assays for these two enzymes were carried out on mouse liver
homogenates; this monitored any variation of urate oxidase
activity during different experimental runs and revealed any
technical error.

4. A small sample of each liver homogenate was boiled and
subsequently assayed for both urate oxidase and xanthine oxidase
activity; these boiled homogenates were used as a negative
control and to establish a background level of [14CJallantoin
since all enzymatic activity is extinguished by the boiling
process.

5. In primate liver homogenates where little or no urate activity
was expected the commercially prepared I1‘Cluric acid was further
purified to remove trace contaminants which comigrate with Ii‘C]
allantoin; this procedure. reduced the background counts of

14
[ Clallantoin and increased the sensitivity of the assay.

16

The results indicate that representatives of the five living
genera of hominoids, adult human, gorilla, chimpanzee, orangutan
and gibbon, all lack urate oxidase activity. Seven species of Old
World monkeys and three species of New World monkeys have easily
detectable urate oxidase activity which are as stable as the
urate oxidase activity found in rabbits and mice (table 4 & 5).
These results are not in agreement with the unpublished
observations made by Christen and coworkers (1970a and 1970b) and
Wacker (1970). Christen and cowokers claimed that 5959;; 59133;;

0
and 89322 tgivirggtgg liver homogenates stored at -20 C for one

day lost a substantial amount of the total urate oxidase activity
which was previously observed. Wacker (1970) reported, presumably
based on the same unpublished observation, that no urate oxidase
activity could be detected in liver homogenates from Magggg and
5953; after storage at -20°C for 24 hours. Based on their
unpublished observation, these investigators erroneously
concluded that urate oxidase activity from M3535; and 3933; was
highly labile. Despite the lack of any supporting evidence, the
notion that urate oxidase activity is highly labile in primates
is often sited. As the underlying premise to the popular
multiple step mutational model for the gradual evolutionary
silencing of urate oxidase activity in primates, the stability of
urate oxidase must be carefully examined.

Christen and coworkers' (1970a and 1970b) postulate on the loss
of urate oxidase activity, attributes the increased lability of

primate urate oxidase to a mutational event. This first mutation

was believed to have occured preceeding the divergence of

l7

hominoids form the Old and New World monkeys. A testable
prediction from this hypothesis is that this first mutational
event, and therefore its' effect, can be found in present day Old
and New World monkeys. The hypothesis further states that the
manner by which urate oxidase activity was lost in the different
primate species may have been completely independent. Also, Old
and New World monkeys which now 'have different genetic
backgrounds, might have different degrees of urate oxidase
lability.

This study found no hint, by three criteria, of highly labile
urate oxidase activity or xanthine oxidase activity in nine
species of Old World monkeys and in three species of New World
monkeys (Table 4 & 5). Old World and New World monkey livers
stored at -70°C for greater than 52 months and 46 months
respectivly, have easily detectable urate oxidase and xanthine
oxidase activity (Table 1). The rate of loss, after treatment

(Table 4 t 5), of urate oxidase and xanthine oxidase in a whole

liver homogenate of Macaca mglatta and Cebu; agella was not

 

significantly different from the rate of loss of mouse or rabbit
liver urate oxidase and xanthine oxidase activity. The observed
variation in the rates of loss of urate oxidase activity, after
the various treatments among the different Old and New World
monkey liver homogenates, is not significantly different either
(Table 4 I: 5).

No evidence of highly labile urate oxidase activity was observed
in Old and New World monkeys. Furthermore, urate oxidase actvity
of all primates appears to be equally stable (Table 1, 4 b 5) and

also as stable as urate oxidase from rabbit and mice liver

18

homogenates. The presence or absence of primate urate oxidase
activity was easily demonstrated in liver homogenate preparation
and the results were unambiguous and definative. On the basis of
the results from this study and with particular consideration of
the evolutinary relationship among primates, as Judged by
morphological, chromosomal, biochemical and molecular evidence
(Templeton, 1983), it appears as if a single mutational event
silenced urate oxidase activity in a homonoid ancestor after
divergence from the Old World monkeys (Fig. 1).

flagggg mglgttg hepatic urate oxidase is sufficiently stable to be
purified to apparent homogeneity as evidenced by a single major
silver stained peptide band in an SDS polyacrylamide gel of a
high urate oxidase activity fraction obtained by conventional
affinity chromatography procedures (Fig. 2). The evidence
presented in this study suggests that primate urate oxidase
activity, when present, is stable. The observed variation in
specific activity of urate oxidase in the different primate liver
homogenates (Table 3) may be due to the differences between and
among species, time and temperature of storage of liver tissues
prior to assaying, age and sex of the animal (Townsend and Lata,
1969) and medical history and diet of the animal.

Endogenous levels of unknown competitive or non-competitive
inhibitors of urate oxidase activity may also modify the Ln,xitpg,
urate oxidase activity levels. When liver homogenates from
primates with and without urate oxidase activity were mixed there
was no detectable inhibition or activation of urate oxidase

activity in either homogenate in the mixture. The predicted

19

values of urate oxidase activity in the mixtures closely
resembled those observed (Table 6).

The specific activity of urate oxidase of primates is lower than
that of other mammals, such as the mouse or rabbit (Table 3).
Rabbit liver urate oxidase specific activity levels are 4.5 and
18.1 times greater than the specific activity observed in liver
homogenates from 9.93; gngllg and fling; glniiggng, respectively.
Using Christen and coworkers' (1978a and 1978b) data, similar
differences in specific activities can be calculated. The ratio
of the urate oxidase activity of rabbits to anug aggllg and
genus glbij;ggg_ calculated from Christen and coworkers' data is
9.67 and 7.25, respectively. Christen and coworkers (1978a and
1978b) misinterpreted their own findings when they concluded that
urate oxidase activity was absent in 912219 Based on the results
from this study and Christen and coworkers' (1978a and 1978b)
findings, Clhfll. gngLL._ and Clhfll. glnifingnl_ have easily
detectable levels of stable liver urate oxidase activities and
that the specific activities are approximately 21% and 18%,
respectively, of that observed in rabbits.

Christen and coworkers (1978a and 1978b) multiple step mutational
model for the gradual evolutionary elimination of urate oxidase
activity in a hominoid ancestor is not refuted by the results of
this study herein reported. However, since no evidence for a
highly labile urate oxidase was found in any primates tested
(Table 1, 4 k 5 ; Fig. 2) such a complex explanation without any
data for intermidiate steps in the loss of detectable urate must
be questioned. The simplest explanation, which is consistent with

the data , on the presence, absence and stability of urate

20

oxidase activity and which is in accordance with the genealogical
relationships among primates, is that a single mutational event
in a common hominoid ancester resulted in the loss of urate
oxidase activity. This single mutational event occurred in a
hominoid ancestor after the divergence of hominoids from the Old
World monkeys (Fig 1).

To successfully understand the genetic mechanism by which urate
oxidase activity was lost in a hominoid ancestor, several
plausible explanations must be considered. A mutation in the
primate urate oxidase structural gene as well as a mutation in
any cis-acting regulatory regions may result in absence of urate
oxidase activity. Furthermore, in chgggnilg, the presence of
urate oxidase activity has been shown to be repressed by the
steroid hormone ecdysone (Kral et al. 1982). This finding suggest
that the absence of urate oxidase in primates could result from a
mutation in any one of several different genes whose product acts
upon the primate urate oxidase structural gene or directly on the
primate enzyme. Other events which may account for the loss of
urate oxidase activity are 1. A chromosomal break involving the
urate oxidase structural gene giving rise to an inversion, a
deletion or a translocation, 2. An insertion element integrating
into a region necessary for the expression of urate oxidase
activity.

A polyclonal antibody to flg;.;._mu1‘ttg urate oxidase and a
cloned gene for M.§.§g_urate oxidase could be used in experiments
designed to distinguish between the various mutational events

which may account for the loss of urate oxidase activity in

21

hominoids.

Recently, Martin and coworkers (1983), examined the molecular
basis for the inactivation of the delta globin gene in Old World
monkeys. The delta globin gene is present in hominoids, Old World
and New World monkeys but is no longer expressed in Old World
monkeys. A molecular comparison of the delta globin gene of
Mggggg, and legggg disclosed three 5' point mutations that are
shared by these two Old World monkeys and several other mutations
found exclusively in one monkey or the other but not in both.
Martin and coworkers (1983) reasoned that one of the three point
mutations shared by Magic; and legbgg,was responsible for the
silencing of the delta globin gene while the mutations unique to
each species occurred after the gene was silenced.

The results of the study reported herein indicate that all .five
genera of hominoids are completely devoid of urate oxidase
activity. In addition, all New World and Old World monkeys
examined had stable and easily detectable urate oxidase activity.
The mutational event which silenced urate oxidase activity
occurred in a hominoid ancestor after it diverged from the Old

World monkeys (Fig 1).

22

TABLE 1. SOURCE RND CONDITION OF HEPATIC TISSUE FOR URATE OXIDASE AND XANTHINE

OXIDASE ASSAYS

IEQLQIIQI All

SEEQIE
EQUIUQIQQ

Oil! lllLlOl

Ban iconloixias

Ban oanisus

Bongo oxoaasui axooaani

3921113 9951111

leohaisi lac

leolsiss console:
QEBCQEIIUEQQIQI

title cioocsonalus soutii

fliilii isssisulacia

flasasa colitis

basics neasstcina

nioooinssui islaaoio

Ecssthii sniillui

9919131 9915111
ELAIXBBUIUI

Cilia 111111

11111 altiicooi

$111151!!! splash

nus auscului (8-1-- ICR)

Qtifllillilll EUCLELUI
(Nee Zelano white rabbit)

SIQBRQE EQIQIIIQIEIZL

Lrlg/

Lrlggl
or/3123/42.3/
or/3123/35.3/
DF/STZI/lB
Lr/gg/
or/sr7a/23.3/

OF/8T7I/IZ.2I
DF/3T7I/I3.6/
DF/8T7I/I.4l
DF/ST7I/O.4I
DF/ST7I/52.5l
DF/3T7I/16.3/

DF/8T7I/46.I/

DF/8T7I/35.7l
DF/8T7I/I.Sl

DF/8T7I/46.3/

L191

LF/8T7I/I.3/

‘IQUBQEIIL

Hichigan State University Clinical
Center, Bparrox Hospital lnghae
County Hospital

Prieate Research Institute Holloean
Air Force Base, 3

Yerkeys Regional Prieate Research
Center/Eeory University, 6

Yerkeys Regional Prieate Research
Center/Eeory University, d

Yerkeys Regional Prieate Research
Center/Eeory University, c

Yerkeys Regional Prieate Research
Center/Eeory University, c

National Zoological Park, Departeent
of Pathology, f

Regional Prieate Research
Center/University of Nashington, b
Regional Prieate Research
Center/University of Nashington, b
Regional Prieate Research
Center/University of Haehington, b
Regional Prieate Research
Center/University of Nashington, b
University of California, Berkley
Dr. L. Broeley, c

University of California, Berkley
Dr. P. Dohlinox, c

National Zoological Park, Departeent
of Pathology, f

University of California, Berkley, c
Oregon Regional Prieate Research
Center. e

National Zoological Park, Departeent
of Pathology, 4

Harlan Inc.

Bailey lnc. Michigan

23

Table Legend 1.

(I)

(2)

The following persons provided liver saeples.

a. Dr. C.E. Brahae and Dr. L.B. Cueeins

b. Dr. J.A. Johnson

c. B. Stewart and Dr. A.C. Hilson

d. Dr. H.H. HcClure and Dr. E. Lockwood

e. Sharon Haher

f. Dr. J. Lagenberg. Dr. R. Rontali and R. Freeean

119113192 10! fiifitlfll CODILSIQO 991!

L I Reeoved froe a live anieal, hoeogenized ieeediately and assayed for
urate oxidase and xanthine oxidase activity.

LF I Reeoved froe a living organise, frozen on dry ice, thawed, hoeogenized
and assayed for urate oxidase and xanthine oxidase activity.

ST7I - Stored at -7B C
3120 - Stored at -2| C

The second set of two digits following the teeperature of storage are the
nueber of eonths of storage at the designated teeperature prior to
assaying. A 1 indicates tissue hoeogeniaed and used within one hour of
biopsie. [1 indicates tissue frozen on dry ice ieeediately after biopsie,
thawed, hoeogenized and assayed within 24 hours of biopsie. D and DF are
identical conditions to L and LF except the tissue was reeoved froe a
recently deceased or sacrificed anieal. The nuebers following D and D! are
explained above. The sea of aniealis) is indicated.

24'

TABLE 2. PRESENCE_OF URATE OXIDASE AND XANTHINE OXIDASE ACTIVITY IN PRIHATES

itiili unlit QIIDSSE 11112111 lillfllli Qillili Sillllli
Present or absentid) Present or absentid)
unoiuoin§<a»
Hose IIBLIDS(6) - .
Bio tooaloixtiitzl - .
tin aaoicuatl) - .
tense 11111!!! etasieuit2) - +
flotills oecillsill - +
EXLQliLii lacil) - .
dilatitii ceocolocii) - .
QESQQEIIHEQQIDS‘U)
E111! cioeciitilui soutii<ll + +
Eiitil isicisulaciiill + .
Oliiii sulsttsl3) + .
nacica osssatciosil) + +
dieaetbacui tiliuoioill , .
toastitii sotiilui<2> + .
Colitis iuiciiilll + .
ELSIYMUIHU c )
Q1211 111L11‘1’ * +
91111 iltiicooiill + .
Qallicitus aolecbil) + .
QIUEB daddaLi
Uni ausculuitfl + .
Qcctitolaaui cuoitlusill + +

The nueber in parenthesis following the species naee is the nueber of liver
biopsies froe different anieals which were assayed for urate oxidase and
xanthine oxidase activity.

a) great apes

b) Old Rorld eonkeys

c) New Horld eonkeys

d) . I easily detectable levels of activity; - I no detectable activity

25

TABLE 3. URATE OXIDASE AND XANTHINE OXIDASE SPECIFIC ACTIVITY IN PRIHATES

SBECIE UBSIE 0111885 6911211! lellfllflfi QllleSE 1911211!
Specific activityid) Specific activityie)
Unfillnllfiill
dose lilLlniibl 3 596
Bad tcoaleixtestzi 3 :37
Bio oaoicuitii 3 13,7e3
Boone excesses eiisiiuitzi 3 12.133
flotilla lQLLLLt‘l) 3 12.933
dilatatas Llcii) 3 1.350
Uzietatcs inniolecil) 3 3.430
QEBQQEIIUEQQIQSib)
Rania cioecsntslui soutiiill 64.300 3.233
licaci iaiciculiciiill 39.6OI 2.85.
fiifiiii aulattaiS) 28.9II 4.950
OIESEI elasticioiill 34.900 10.55!
dioiottacus talscoioil) 36.80! 4.650
Bcsstxtis intilluii2> 32.050 2.90!
QQLRIII ausciiiill 28.900 o.3|I
BLSIIBBululic)
Qghgg gggLLgill 53,323 3.933
91111 altlicoosill 23.55I 6.150
Callicatui aslesblll 34.110 11.940
QIUEB ESUUSLS
fig; guggglggif) 135.65! 10,600
Qcctitoliaui cuoiciuiill 237.550 8.950

The nueber in parenthesis following the species naee is the nueber of liver
biopsies froe different anieals which were assayed for urate oxidase and
xanthine oxidase activity.

a) great apes

b) Old Horld eonkeys

c) New Horld eonkeys

d) Specific activity is the initial rate of synthesis of 14 C-allantoin froe
if C-uric acid expressed as the change in CPR per einute of reaction tiee
divided by protein concentration in eilligraes per eilliliter. At 951
counting efficiency, 112 CPH equals 1 x iii-6) u soles of if C-allantoin.

e) Specific activity is the initial rate of synthesis of if C-uric acid froe
if C-xanthine expressed as the change in CPR per einute of reaction tiee
divided by protein concentration in eilligraes per eilliliter. At 951
counting efficiency. 120 CPR equals 1 x lSi-ol u soles of if C-uric acid.

TABLE 4.

§EEQIE
QEBQQEIIUEEQLQS

C1112 EZOQEIDOILEE 182111
OIEIEI {QEELEULIELE
OIEIEQ 511111113)

OIEIEQ DESIIIELOI
OLQDQEOIEII 11112218
CEEEEIELI IOIILLQE
QQLQIBE 111L111

ELSIISSHIUI
91111 111111
11111 111111111
1111111111 111111

11111 1111111

111 11111111
111111111111 11111111

One freeze thaw cycle I freezing hoeogenate at
thawing saeple at rooe temperature, waiting one einute and freezing at

27'

EEBQEBI CE 9861
89112111 BEDSIU
IBESIDEU

E11111 111!
11

31.4
688‘
82.8
32.1
36"
67.3

47.1
52.6
84..

STABILITY OF PRIHATE URATE OXIDAS

5
I!
I

J

14.7
58.9
77.6
38.8
2983

32.1

-78 C

4‘09
39.8
41.7
61.6

for 10 einutes, coepletely
-70 C.

a) Data is the eean of two independant detereinations froe hepatic tissue of two

different anieals.

28

TABLE 5. STABILITY OF PRIHATE URATE OXIDABE ACTIVITY AND XANTHINE OXIDASE ACTIVITY

EEBQENI DE 11111 OXIDASE 1911211!
851611181 $1158 131111111

11111 11 1 1 11111 11 2} 1
111111 11: BLIL 12 8511 1 D511 21: BEIL
11111111111111:
11111 111111111111 111111 60-5 27-7 67.7 71.9
111111 111111111111 64-3 58.3 45.9 39.1
Q1111; guLattg(a) 62.8 53.1 94.2 79.6
111111 1111111111 75-6 73.2 -- --
11111111111 11111111 42.1 19.1 66.1 31.2
111111111 11111111 45-8 42-9 28-4. 27~6
1111111 1111111 -- -- -- --
11111111111
11111 111111 31.3 51.9 49.1 --
11111 111111111 -- 47.9 -- --
1111111111 111111 -- -- -- --
11111 1111111
111 11111111 -- 71.5 -- --
111111111111 11111111 -- 69.7 -- --

1) Data is the eean of two independant detereinations froe hepatic tissue of two
different anioais.

29’

TABLE 6. HIIINO LIVER HOHOBENATES TO TEST FOR DIFFUSIBLE INHIBITORS OR ACTIVATORO
OF URATE OXIDASE ACTIVITY IN HOHINOIDS. OLD NORLD AND NEH NORLD HONKEYB

Exp-ri-Int 01' canon oxidant lit-d with dicaitbacua titaaoio

E'P'TIO'Dt .2! CID llOLlSll 0".“ “It" Elli! [LILLLQOI

Soociao Urato oxidalo no protoin Soociaa Urato oxidaao uq protoin totai no
A activity(a1 in assay 8 activity(a) in assay protain
22999 0 I bionitbssni 6691 +263 435 ‘35
I ieI 435 615
I 45I 433 III
I 72I 435 1155
BIO I a ggng. 4679 +199 335 so:
I 143 395 328
I 358 385 743
I 572 388 937
RESULTS:
2 Urate oxidase activity 1 Urata oxidasa activity Urato oxidasa activity
oxpactod if no inhibitor oblorvod in aixturo(c) of oixturoia)
or activator in oixturaib)
100 100 -
71 79 5294
49 53 3353
38 37 2487
100 100 -
73 61 2866
52 4B 2238
4I 32 1512

(a) Urato oxidaaa activity is equal to CPH of 14 C-aliantoin producad {rod
14 c-uric acid par ainuto 04 raaction tioo par no of protain.

(b) x Urata oxidaoa activity oxpactod in aouai to ratio o4 no o4 protoin of

species I dividad by total no of protoin.

(c) X Urata oxidaoa activity oboorvad in aouai to urato oxidaoo activity of

aixturo divided by urato oxidase activity of species B.

3O

00¢ m¢<m> “—0 nay—011...:

em on 3

222:: .32
852.50:

 

30236 xv
33:6 355

 

>350: 64:03 9.5
mQOOwIEn—Oommo

 

 

 

>950! 94:03 252
_z=..mm>._.<._a

 

Aeamgv uaoaadz< aza audnom soon
and mount mucouuo>qv 05H .uuuoaqaoz new mvdoousuuaoouoo .«cusouaua—m uo anuwoaazm

J .a:

31

FIG. A URATE OXIDASE ACTIVITY WITH RESPECT TO CONCENTRATION OF

CPM of 1hc allantoin
from 140 uric acid

220"

YLSJ

125‘

705 1

MACACA mulatta LIVER HOMOGENATE

5 4
L51
I T.vfi4r . , r9
0 .1 .2 .3 .I .5 .I .7 .I .9 1
Concentration of Macaca mulatta
liver homogenate (X)

 

(D

C)- CPM after 0 minutes of reaction time
‘A-CPM after 5 minutes of reaction time
[3- CPM after 10 minutes of reation time

32

FIG. B MACACA mulatta URATE OXIDASE ACTIVITY WITH RESPECT TO
I CONCENTRATION OF EDTA

5 of maximal urate
oxidase activity

I“

“3838383

 

 

 

' r ' r V r v I r fl

II 15 20 25 3. I! I I! SO

EDTA final concentration (M x 10'“)

33

FIG. C MACACA mulatta URATE OXIDASE ACTIVITY WITH RESPECT TO
CONCENTRATION OF BORATE

% of maximal urate
oxidase activity

llii

II E

3:13 2:33

20
II

 

 

I' I I I' T I T I l ‘D

I 2 3 I 5 6 7 I 9 19

Borate final concentration (M x 10-3)

34

APPENDIX A
PROTOCOL FOR THE PURIFICATION OF URATE OXIDASE FROM HACACA mulatta
o
1. All operations were carried out at 0-4 C unless otherwise
stated. Two hundred and twenty three grams of flggggg, mgljssg,
liver previously frozen and stored at -70°O was wrapped in
several layers of choeze cloth and aluminum foil and was
fragmented,.with a hammer, into small pieces.
2. The 223 grams of partially thawed liver were added to a total
of 1,858 mls of homogenization buffer (0.25" sucrose, 50 on tris-
HC1,15.7 mfl EDTA pH 7.0) and minced in a Virtis triple blade
homogenizer (at 10,000 RPM x 2 minutes), liquidized in a Tekmar
Polytron Tissuemizer (2 minutes) and reduced into a homogeneous
suspension by a drill driven Teflon Potter-ElveJhen homogeninizer
(one up and down cycle).
3. The whole liver suspension (H) was seperated into 40 ml
aliquots and centrituged (at 750g x 30 minutes) in a Beckman QJ2-
21 centriiuge with a JA 20 rotor. The supernatants (81) wore
saved and the pellets were washed with a small amount of
homogenization buffer, ' rehomogenized in a Tekman Polytron
Tissuemiser as before and centrifuged (at 750g x 30 minutes). The
supernatants of this centrifugation were pooled with supernatant
81, collectively called 91b and had a total volume oI 630 mls.
4. Forty ml aliquots o! 81b were centri‘uged (at 48,000g x 30
minutes) and a total of 545 mls of the supernatant, 82. were

recovered and saved.

35

5. To 82, saturated ammonium sulfate was added until it was 402
saturated. centrifuged at 48,6009 x 30 minutes, saving the
supernatant, to which ammonium sulfate was added to give a final
concentration of 55% and centrifuged at 48.000g x 30 minute,
saving the pellet, P3.

6. P3 was resuspended in 10 mls homogenization buffer and
centrifuged 250,000g x 30 minute. The final supernatant was added
to an affinity column to which xanthine was bound.

7. The column fractions were monitored at 280nm by a Pharmacia
UV*1 ultraviolet analyzer and recorded on a strip chart. The
fractions were assayed for urate oxidase activity by monitoring
the decrease in absorbence of a reaction mixture in a Cary 210
spectrophotometer, at 292nm (Friedman and Baker, 1982).

8. High urate oxidase activity fractions were lyophilized,
resuspended in Laemmli's sample buffer and electrophoresed in a

vertical apparatus (Laemmli, 1970).

36

APPENDIX B

808 POLYACRYLAMIDE BEL ELECTROPHORESIS

A 13% separating and a 4X stacking polacylamide slab gel (1.5mm x
100mm x i40xmm) was prepared according to Laemmli (1970). Samples
were boiled in 22 SOS and 0.1M mercaptoethanol for 10 minutes and
electrophoresis was carried out in the presence of 25m" tris,
19.2mH glycine, 1% 808 buffer, pH 8.3, at a constant current of
10 milliamperes for seven hours at 22°C. Proteins were stained
with the modified silver stain procedure of Switzer (Friedman and
Baker, 1982) which is approximately 100-1000 times more sensative
than Coomassie blue R stain. The molecular weight standards were
purchased from Pharmacia Fine Chemicals, prepared according to
the manufactures directions and further diluted 1/5. The
standards and their subunit molecular weights werea (P)
Phosphorylase b, 94,000; (8) Bovine Serum Albumin, 67,000. (O)
Ovalbumin, 43,000; (C) Carbonic Anhydrase, 30.000; (8) Soybean

Trypsin Inhibitor, 20,100; (A) Alpha-Lactalbumin, 14,000.

37

Fig. 2 SDS Gel Electrophoresis Analysis Of 5153;; mglgggg Liver

Lane
Lane

Lane

Lane

Lane

Homogenate

 

" O

1.4.7.

2,3:

5.6:

4...o ‘I’
- --- - C’
- .C -
-
—— -3
.~ A
I
.1
fig .
haw

 

-""P-

11.13: Molecular weight standards
Purified Magaca mulatta urate oxidase (1X)

Partially purified Hacgsg mgiggtg whole liver
homogenate

8,9,10,12: Purified uggjg; mul;§§g urate oxidase (50X)

13,14:

ugsggg mglggt; whole liver homogenate

38

Ames 8. N., Cathcart R., Schwiers E. and Hochstein P. (1981)
Uric acid provides an antioxidant defense in humans
against oxidant- and radical-caused aging and cancer: a

hYPOthfliI- BEDS... [1318... 95%;. 1.1.3.3.. 73. 6959.6362-

Bradford H. H. (1976) A rapid and sensitive method for the
ouantitation of microgram quantities of protein utilizing

the principle of protein-dye binding. englxg&,fliggngmt 72,
248-254.

Christen P., Peacock H. C., Christen A. E. and walker
H. E. C. (1970a) Urate Oxidase in primate phylogenesis.

EHLI. .7... mm. 12. 3-5.

Christen P., Peacock H. C., Christen A. E. and Walker
H. E. C. (1970b) Urate oxidase in primates. Eglia 35133;;
13, 35-39

de la Iglesia F., Porta E. A. and Hartroft H. 8. (1966)
Histochemical urate oxidase activity and microbodies in

nonhuman primate liver. J‘, fliggggngm._cxgg§nggh,14, 685--
687.

Essner E. (1969) Localization of perosidase activity in

microbodies in fetal mouse liver. J;_fl1§§9§h§fi. thnsnlm;
17 ’ 454-466 e

Friedman T. B. and Johnson D. H. (1977) Temporal control of
urate oxidase activity on ncggggnilga evidence of an
autonomous timer in Malpighian tubules. 351335. 197, 477-
479.

Friedman T. 8. and Herril C. R. (1973) A microradiochemical
assay for urate oxidase. englxg& fligsngm&,55, 292-296.

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xmth-nurh- In The metabolic aim of. W 0
(Edited by Stanbury J. 8., Hyngaarden J. 8., Fredrickson
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Kral L. 8., Johnson D. H., Hing H. and Friedman T. 8. (1982)
Temporal control of urate oxidase activity during
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role of 20-hydroxyecdysone. 9.31;,anggL_3, 215-233.

Kuster 8., Shorter R. 9., Dawson 8. and Hallenbeck 8. A.
(1972) Uric acid metabolism in Dalmations and other dogs,

role of the liver. BLED: 133153; uggL,129, 492-496.

39

Logan D. C., Nilson D. 5., Flowers C. H., Sparks P. J. and _
Tyler F. H. (1976) Uric acid catabolism in the woolly
monkey. figtgggligm 25, 517-522.

Hartin S. L., Vincent K. A. and Wilson A. C. (1983) Rise and

fall of the delta globin gene. 1‘. mglgg; Eigl;,164, 513-
528.

Hasters C. and Holmes R. (1977) Peroxisomesa New aspects of
cell physiology and biochemistry. EDXSLQLL leL 57, 816.

NakaJima J. and Bourne 8. H. (1970) Histochemical studies on
urate oxidase in several mammals with special reference to
uricolytic ability of primates. fliggggngmigggx,22, 20-26.

Orowan E. (1955) The origin of man. Ngtgcg 175, 683-684.

Proctor P. (1970) Similar functions of uric acid and
ascorbate in man? uggu;.,223, 868.

Roch-Ramel F. and Peters 8. (1978) Urinary excretion of uric
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by Kelley H. N. and Heiner I. H.), pp. 211-255. Springer,
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J... mien. Ecol... 20. 2-15.

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liver “'9‘. oxidase. EEQSIIQLOQI ELISSEQO BLEEQISQEX
angles! 21 eggcigg Thirty-seventh Annual E. H. S. A.
meeting, 24-25.

Smith R. C. (1983) Antioxidant activity of uric acid and 3-
Ribosyluric acid with unsaturated fatty acids.

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mm 13. 687-703-

Templeton A. R. (1983) Phylogenetic inference from
restriction endonuclease cleavage site maps with
particular reference to the evolution of humans and the

Inc-- Exoluiim 37. 221-244-

Townsend D. and Late 8. F. (1969) Purification of urate
oxidase; a sex dependant enzyme from rat liver. ecgng

31.951110 31.99111]... 135. 166-172-

Nacker N. E. C. (1970) Hana Sapient but gouty. N;_Engl;,l;
ngg.,233, 151-152.

Hells H. 0. (1909) The purine metabolism of the monkey. 4‘,
111.91.. 121m... 7. 171-183-

40

Wells H. 8. and Caldwell S. T. (1914) The purine enzymes of

the orang-utan (8191; gagyggg) and chimpanzee
(W W)- al.. 9.1.9.1... EMS-1a! 157-164-

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