THE EFFECT OF A SERIES OF AROMATIC AMIDINES AND SODIUM NUCLEATE ON ARGINASE ACTIVITY IN VITRO

142 Pages·07.092 MB·English

by  MOSS, SAMUEL

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Xerox University Microfilms
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•G7 Moss, Samuel, 1914“ , 5 T r ' U
1950 The e ffe c t o f a series of arom atic
.M7 amidines and sodium nucleate on
arginase a c tiv ity in v itro .
Hew YorlC, 1950#
[l4.],103 ty pew ritten leaves,
charts, ta b le s. 29cm«
Thesis (Ph.D.) - New York Univer­
sity , Graduate School, 195>0.
Bibliography: p .91- 98*
C57517
Xerox University Microfilms, Ann Arbor, Michigan 48106
THIS DISSERTATION HAS BEEN MICROFILMED EXACTLY AS RECEIVED.
LIBRARY OF
HEW TORI UNIVERSITY
UNIVERSITY ALIGHT?
THE EFFECT OF A SERIES OF AROMATIC AMIDIHES AND
SODIUM NUCLEATE ON ARGINASE ACTIVITY
IN VITRO
b?
Samuel Moss
A pril 1950
A d isse rta tio n In the department of biology subm itted
In p a rtia l fu lfillm en t of the requirem ent for the degree
of Doctor of Doctor of Philosophy a t New York U niversity,
ACKNOWLEDGMENTS
The author wishes to express sincere appreciation
to Professor M. J. Kophc fo r his inspired guidance
and invaluable help in the execution of this
problem. Sincere appreciation is also expressed
to Professor H. A. Charipper fo r fa c ilita tin g th is
work.
In addition the author wishes to thank Dr. S. Ratner
for her valuable suggestions In the preparation of
the enzyme and Mrs. S. Moss for her help in the
preparation of the m anuscript.
TABLE OF CONTENTS
Page
I. Introduction .......................................................................................... 1
A. Phenomena of enzyme in h ib itio n ............................. 1
B. Aromatic amidines ............................................................... 5
1. Development of the arom atic amidines
as therapeutic agents ........................................ 5
2. Mechanism of action of aromatic
amidines ......................................................................... 8
3- Search for nev compounds ............................... 16
G. The arginase system .......................................................... 18
1. Arginase in neoplasms ....................................... 18
2. Methods of arginase preparations .......... 19
3 . Methods of arginase a c tiv ity
measurements ...................................................... 21
D. Purpose of th is in v estig atio n ................................. 23
II. M aterials and methods ................................................................. 26
A. P reparation of arginase ................................................. 26
1. Preparation of Enzyme I .................................. 26
a. Reagents .................................................................... 26
b. Procedure .................................................................. 27
2. P reparation of Enzyme I I ................................ 27
a. Reagents .................................................................... 28
b. Procedure .................................................................. 28
B. Arginase a c tiv ity determ inations ........................ 30
1. Experimental conditions .................................. 30
a. Enzyme d ilu tio n .................................................. 30
b. S ubstrate concentration ............................. 31
c. pH of reaction mixture ........................
d. Incubation tem perature ........................
e. Reaction period ..........................................
2. Enzyme reaction and urea determ ina­
tio n ...........................................................................
a. Reagents ...........................................................
b. Apparatus .........................................................
c. Procedure .........................................................
d. C alibration of reference curve ..
3- Potency determ ination of arginase
p rep aratio n s.........................................................
a. Arginase unit determ ination ...........
b. P rotein determ ination ..........................
1) Reagents ....................................................
2) Apparatus .................................................
3) Procedure .................................................
c. C alculations .................................................
D. M odifications of arginase a c tiv ity
1. Reagents .................................................................
2. Procedure ...............................................................
Experimental resu lts ........................................................
A. In h ib itio n studies of Enzyme I .......................
1. O utline of typical experiment .............
2. Tabulation of Enzyme I in h ib itio n
studies .....................................................................
B. In h ib itio n studies of Enzyme II ..................
1. Aromatic amidine in h ib itio n of
Enzyme II ...................................................................... 55
2. Sodium nucleate in h ib itio n of
Enzyme II ...................................................................... 60
3* R ev ersib ility studies of arginase
in h ib itio n ................................................................... 64
C. M odification of the d ilu tio n effec t ............... 67
IV. General D iscussion ...................................................................... 70
A. Arginase preparation ...................................................... 70
B. R elative effectiveness of arginase
in h ib ito rs ................................................................................ 70
C. Significance of sodium nucleate in h ib itio n
of arginase a c tiv ity ....................................................... 78
D. The d ilu tio n effect ........................................................ 83
V. Summary ..................................................................................................... 88
VI. Bibliography ...................................................................................... 91
V II. Appendix ............................................................................................. 99
1
I . INTRODUCTION.
A. Phenomena of Enzyme In h ib itio n .
The phenomena of enzyme in h ib itio n are used in studies
of action of drugs, mechanisms of action of enzymes, and in
general c e llu la r functions. Even the protein chemist makes
use of inform ation obtained from enzyme in h ib itio n phenomena.
This is especially true for inform ation pertaining to the role
of functional groupings, e .g ., -SH and phenolic, in the enzyme
m olecule. Thus the general in te re st in enzyme in h ib itio n is
apparent. Above a ll, however, studies of enzyme in h ib itio n
have developed recently into a fundamental tool in the
search fo r new therapeutic agents. I t is from th is point
of view that th is paper w ill be prim arily concerned.
U ntil recently chemotherapy was based solely on
em pirical findings. Research was conducted on a basis of
testin g the physiological sta te of animals a fte r adm inistra­
tion of compounds which were suspected to have therapeutic
value. Although by th is method some p rac tic al knowledge
was gained, no real ad vane esjw-s^ made in the search fo r new
cAn be
therapeutic agents u n til drug action*wee studied in terms
of c e llu la r metabolism. With the development of the idea
th at c e llu la r metabolism is regulated by a series of in te r­
dependent enzyme systems, Investigators gradually came to
rea liz e th at drug action is somehow related to the enzymatic
a c tiv itie s of the c e ll. Thus as early as 1875 Nasse (68)
investigated the effect of quinine, caffeine, and strychnine
2
on invertase, saliv a, and pancreatic ju ice. Jacoby (43)
published the f i r s t paper on the e ffe ct of an inh ibiting
substance on an iso lated enzyme. He found th at mercuric
chloride inhibited urease in the same way as i t did the
decomposition of urea by b acteria. He also showed th at the
urea sp littin g a c tiv ity was inhibited by sm aller concentra­
tions than those required for k illin g the b acteria, and
th at the reversal of in h ib itio n of the enzyme, as well as
of the urea s p littin g a c tiv ity of b acteria, could be obtained
with potassium cyanide. Jacoby then concluded that the
action of mercuric chloride on liv e b acteria was due to
the in h ib itio n of the urease system in the liv in g c e ll. He
then outlined concepts of drug action which d iffe r l i t t l e
from those we hold today. With the work of Quastel and
Wooldridge (72/73) and Quastel and Wheatley (74), pertaining
to the effect of an tisep tics on the iso lated dehydrogenase
system, the idea became generally accepted th at enzyme
inh ibitory properties of drugs could be studied effectiv ely
as a means of tracing th e ir mechanism of action in the
In tact organism.
The main object of chemotherapy today, however,
is not only to elucidate mechanisms of action of known drugs,
but also to develop compounds of highly specific physio­
lo gical action in fie ld s where no drugs are available and
to replace drugs which are less usefu l. In the search fo r
new therapeutic compounds i t is im portant to re la te stru c ­
3
tu ra l differences w ithin a group of compounds to th eir
differences in physiological action. The overall physiolog­
ic a l a c tiv ity , however, is a property which is related to
a large number of variab les. These involve phases of drug
action such as uptake, d istrib u tio n , metabolism, and the
general to x icity on the anim al. As a re su lt of the v a ri­
a b ility of the effect of a compound on each of these phases,
re
very seldom is*found a close relatio n sh ip between structure
and overall a c tiv ity . Even the m etabolic a c tiv ity by its e lf
involves a large number of v ariab les. Hence a certain
chemical grouping which has an in h ib itin g influence on one
enzyme system, may e ith er in h ib it, have no e ffe ct, or even
activ ate another enzyme system.
Moreover, due to the presence of a large number
of in terferin g substances in the c e ll, the sp ecific action
which is related to a specific chemical grouping might be
masked or m odified. Therefore, the effect of specific
chemical groupings of compounds on th e ir physiological
a c tiv ity can best be understood from studies of th eir
e ffe ct on iso lated enzyme systems.
In sp ite of the need, however, only few studies
have been made of the e ffe ct of a large series of related
compounds on iso lated enzyme system s. Blashko and Duthie
(17) tested a large number of alkylated monojand diam idines,
diguanidines, and diisothioureas as in h ib ito rs of amine
oxidase and showed a relatio n sh ip between degree of in h i­