}·' '.
•, ,·'
,·· - ;.
~ .' - ' ',.- . :··,· DISTRIBUTION. OF ARYLAMIDASE IN SOME .SELECTED BACTERIA
1.·:·: ''ij • ':!. '.)'.
';:
;·. •· ''· '·• i'• . :..... '[', '''I '! 1 ~~
,, ,, ··.·
'by
·, : ' ~ ·Elizabeth Bell Lumpkin
"'·. ·.··: . .
Submitted to the Faculty of the Graduate School
of ·the. ~edical College of Georgia in Partial Fulfillment
.f' .··.: of the .Requirements for the Degree of ;_,_· ···-:·-' Master of Science
'May
1967 ··.... ~,· . . . ..
·-·:·· THE DISTRIBUTION OF ARYLAMIDASE IN SOME SELECTED BACTERIA
· This thesis submitted by Elizabeth Bell Lumpkin has been
examined and approved by an appointed committee of the faculty
of the School of Graduate Studies of the Medical College of
Georgia.
The signatures which appear below verify the fact that all \ required changes have been incorporated and that the thesis
has received final approval with reference to content, form'
and accuracy of presen.ta tion.
This thesis is therefore accepted in partial fulfillment
of the requiremetits for the degree of Master 6f Science~
/.
lViAY 3 1 1967
Date·
f c' . __ ....Gi, ...... ··-·.. ··-...... :------....- ... __ ...... ,.,..._. ·- ~;_;,;:,.,._ -~--~·.-· ____ .__ ~ jA<:JO()h t-915~~
; ..
I'' •, '' . ··')
~ I ,' ;
',. .-!. ;;;· .. . ,, ACKNOWLEDGEMENTS ·,lo
I am grateful to my advisor Dr. Francis·J. Behal for his
guidance and encouragement.
To my fellow student and laboratory co-worker, James
Folds, I owe much. He willingly and unselfi~hly gave of
. ,:)- ..:,his time and knowledge helping. to successfully complete this
project. Mr. Folds has to his credit much of the subsequent
work done on arylamidase in.this laboratory.
I wish to, acknowledge the valuable. assistance of Dr. J.
Warren Banister in obtaining and. maintaining cultures. '.;·
r: ' -.. ' ~ ' .
:., .~ - .
1__ ._,. :ii~_:L~-~ .·. ·j' Ao.IOO~ \ ..... ~
•• ,;1 ·· .. :,_··
. :.
' .
. TABLE:OF'CONTENTS •: ! ' ,' ~ •,!
page .. !· ..
METHODS AND PROCEEDURES
I. Arylamidase assay.· 11 16 ... ·II. Protein ~ete~mination ,.i.
.. ~. ' ; .-. 'III. Chromatographic fractionation of cell free
.. ,• ,, extracts 16 •J
: .. IV. Starch gel electrophoresis 17 v. Substrate specificity 19
'. . ' •'·. VI. Metal ion requirements 19 VII. Inhibitors 19 :RESULTS
' ' ·. -... ·~ I~ Electrophoresis 20
' ~' l . II. DEAE-ce1lulose chromatography 25 ':,, ... III.- Properties of the enzyme 25
,.- ·_;. DISCUSSION 37
·. ',·, BIBLIOGRAPHY 43
: _:
,. ',· ,· ·.•. :•,
LIST OF TABLES
-'··· '! l
•(, .. :TABLE PAGE ·, ~
Arylamidase activity on the subst~ate L-leucine
beta-naphthylamide from cell free extracts of
several organisms. 21
·~rylamidase activity .. of cell free extracts of
several organisms on several amino acid beta- 'naphthylamide s. 30
·:,···~Ilia Effect of inhibitors on arylamidase activity··
of the enzyme from several organisms 32
. . ~IV.· Effect of metal ions on arylamidase from
several organisms 33
.. ~.
... ~ : : ' ' ·, . ~ .,.-
.;_,'} -· '··,·,
LIST OF FIGURES
·.; .' ··._FIGURE PAGE ,. , ·1.· Reactions for arylamidase .assay· 12 ·f
·:. :·:::· 2·. Standard curve for determination of liberated ~ ': ·,\· i:: be ta-naph thylamide .. '·' 14
.. ;\ ; : ~· Elution system for DEAE-cellulose chromatography 18
Starch_gel electrophoresis of cell free extracts
·.of~ catarrhalis, Sh. -Flexneri., Sh. dysente+iae
and Sh.. sonnei 22 . -- Starch gel elettrophoresis of cell free extracts
of only N. catarrhalis and a'mixture of equal parts·
of N. catarrhalis and Sh. sonnei 22
6. Starch gel electrophoresis of arylamidase from cell
free extracts of N. catarrhalis, N. perflava,
~· lysodeikticus and Alcaligenes faecalis. 23
7~ Starch gel electr9phoresis of cell free extracts
· of.N. catarrhalis, Proteus vulgaris, Aerobacter~·
and E.· coli. · - --- 23· Strach gel electrophoresis of cell free extracts 'r .' ~._ .
of N. catar.rhalis, E. ~' Alcaligenes faecalis, 23 and Sarcina lutea.
':.- 9. Column. chromatographic profile of arylamidase act-· ! ••.. ··
ivity separat~d by DEAE-cellulose chromatography
. ·: : .,_ ' from the cell free extract of N. catarrhalis 26
-_:lO~Cdlumn chromatographic profile of arylamida$e act-
.. ' ' . ·.··i ivity separated O? DEAE-cellulose from the cell free .i extract of Sh. flexneri. 26 __ ,'.l, ·--.
\ .... :,.· ......
.· ';r" ' . 11. _Column chromatographic profile of arylamidase act-;::· . , ."- ·' ! ivi ty separated on DEAE-cellulose from the '·lCell free . ! \ ; . extract of Sh. dysenteriae.
12~ Column chromatographic profile of arylamidase act-'.
ivity separated on DEAE-cellulose from the cell free
. . .: ' :-· ··.· extract of Sh. sonnei .. 27
·: .. ": .. 13. · Column chroma to graphic profile of aryl amidase act-·. · ivity separated on DEAE-cellulose from the cell free
·.extract of Proteus vulgaris~ 28
.. 14. Column chroma to graphic profile of arylamidase act-·
.ivity separated on DEAE~cellulose from the cell free
extract of E. coli. 28
:.·_: 15. Column chroma to graphic profile of arylamidase act-. ivity separated on DEAE-cellulose from the cell free
,-·.·-·. extract of Alcaligenes faecalis. 29 16. Column chromatographic profile of arylamidase act-
ivity seoarated on DEAE-cellulose from the cell free
extract of Sarcina lutea~ .29
17. Michaelis constant comparison between activity of the
a-enzyme of Sarcina lutea on the substrates leucine-
,.. . . . ' BNA and alanine-BNA.
~·. 18. Michaelis constant study showing the non-competitive
~ •, : - .
.·.' inhibition by puro~ycin of arylamidase from B-enzyme
of Sarcina lutea • .. · . .' ... ·· 19. Michaelis constant study showing the non-competitive_
inhi·bi tion of arylamidase from the. a-enzyme. of
Sarcina lutea caused b~ puromycin. - --'- b:.:..=-'"'--~.- .. '....=.. {~-i:~~], - ....~;:;,;=-·
,T-, ··:.1·· '·;·:.
INTRODUCTION '' ' ' '
The purpose of this investigation is to study_the distri-
bution of arylamidase in representative bacteria~ Interest.
·_in this problem grew because . of the lack of information avail-
able on bacterial arylamidases. One objective of this study t:.. 'was to find out whether there appeared to be many different aryi-
amidases in bacteria or whether bacterial arylamidases are gen- ; ·' . ,,'t.:. •'' ·erally similar. '·,, l ·An arylamidase ·is an enzyme which hydrolyzes amino acyl·_·
'l ._' '•, ,;_ amides, ·in'this case amino acyl beta-naphthylamides. A free
amino group is required for hydrolysis.
In the late 1920s a leucyl peptidase was described from
hog erepsin.· This enzyme, now called leucine am~nopeptidase~
was later purified to a high degree and many of its properties:·'
determined. Later there appeared artificial chromogenic sub--
strates which supposedly were hydrolyzed by leucine aminopeptdase·
and which made assay for leucine aminopeptidase considerably
easier. Since the advent of these artificial substrates, it
has been determined that, although leucine aminopeptidase will
····hydrolyze them, other enzymes are responsible for the hydro-·
'' :.:· .lysis of these amino acyl beta-naphthylamides; these are now called arylamidases. ·
The arylamidase of Neisseria catarrhalis has been studied
extensively in our l~boratory. N. catarrhalis seems to be an
. extremely rich source of this enzyme. It now has become necessary
to know to wll,at extent arylamidase is p'resent in other micro- ,.: . . :_.2 ~~~~.
~-'. organisms and. if the properties of N. catarrhalis arylamidase· ,, .... ;· . . are the same as aEylamidase isolated from other microorganis~s. 'c .. The. a·pproach to this problem has been to systematic ally isolate·
and characterize enzymes with ary~a~idase activity as ~o (1) :·.. : ',.·
. ··.:; electrophoretic mobility, (2) DEAE-cellulose·chromatography, '(3.) substrate specificity,· (4) metal ion requirements·,. and (5)
>inhibitors.
'. ·.: .. _Early studies of bacterial arylamidase suggested that this
enzyme might be confined to bacteria.: that exhibited a negative ';•i' ·' ·Gram's reaction. With this in mind approximately equal numbers
of both Gram negative and Gram positive organisms have been • ' ~, . : _·. d .... studied. Gram ne.ga tive bacteria, though differing in aryl ami-
dase content seem to be alike insofar as electrophoretic mobil-
'ity of arylamidase is concerned.
The rate of hydrolysis of several amino acid beta-naphthyl-
amines has been determined • It was found that alanine-beta-
. {. ,. anphthylamide was hydorlyzed more rapidly than leucine-beta-
. naphthylamide by the arylamidase from Gram negatiie organismsQ
The metal ion dependence of the aryl~midase activity was
determined by treating the partially purified enzyme fractions
with EDTA. ·upon ion exchange chromatography each of the Gram
negative organis~s·tested thus fa~ contained a single aryl-
amidase component.
In Gram positive organisms there was much less ary~amidase
. '• ... ·- activity when compared to ·arylamidase activity in Gram negative
·_organisms. There seem to be no difference in the arylamidase
in either Gram negative or Gram positive organisms, with one
(2) exception. The arylamidase of SarC?jna lutea has tw-o different
components, one of w-hich is very similiar to the a:rylamicta·se ,··:
component of !!,"' catarrhalis. The second arylamidase componen~
tl '' of Sarcina lutea differs with the first by electroporetic
'•f, mobility, DEAE chromatography, and reaction to certain inhi-
1••• •• ~~ -. '. ·bi tors.
·.
\..
( 3) ~· ·~- ., ' •:.~_ -..;.. ~ ..:..--... .. -~~:1 ---·~ --. ·( ...-~~~() (· [Otl:!IX h . -----:_... .,-.1- ..
Review of the Related Literature
One of the first \vorks on bacterial peptidases in 1vhich '
substrates of known formulae·· (such as synthetic peptides)
were used was published in 1938 by Berger, Johnson~ and.
.· ,·.._ Peterson .. A survey was made of the ~eptida~es in cell,free
• J..' extracts of twelv~ vacterial species, including Escherichia
· ··. ~, Bacillus me ~a teriu.m, Pseudomonas florescens, Bacillus
· ···:· ·· .. mesentericus, Bacillus subtilis, several species of Clostridia,
Lactobacillus pentosus, Propionibacterium pentosaceum and
:. . Phytomonas tumefacien.. The enzyme extraction procedure invol- • ved repeated freezirig and thawing of cells, follow~d by tol- ,·,_ .. vene autolysis. The pH optima was found to be between the values
of pH 8 and 9 using the substrates L-leucylglycine and L-leu-
cylgylcylglycine. Tw"o organisms, how·ever, Lactobacillus pen-
tosus and Propionibacteriu~ 2entosaceum were found to contain
acidopeptidases which split their substrates at pH values of
5.5 to 6.0. In most cases the peptidases were quite stable
at pH 8 and 40°C but the L-leucylglycylglycine-splitting en-
·zymes of the anaerobes tested were extremely unstable under
these conditions. This instability was partly overcome by the
addition of reducing agents such as thioglycolic acid, cysteine~·
hydrosulfuric acid and cyanic_acid.
The specificity of the peptidase systems of Bacillus
:megaterium and Escherichia~ were studied in detail by.
Berger !:..:!?. !!1, (1938). Dipeptides and_ tripeptides l'lere found to
be readily hydrolyzed but not acylated or decarboxylated~ (4) , .. , .. Berger~,~ (1938) studied in some detail the specificity
of the peptidase systems of E.~ and B. me~aterium.· It
was fdund that E. coli peptidases hydrolyzed g~ycolpeptides
more rapidly than leucyl peptides. The reverse was true for._ j: ,,
B~ Mega terium. Decarboxylated peptides \\Tere hydrolyzed only
,very slowly or not- at all while both dipeptides and tripeptides ·,-!' ·'were hydrolyzed readilyo A decrease in hydrolysis could be
affected by substitution of a methyl group for a hydrogen
atom of the free amino group.· A leucyl peptidase-like enzyme
was found in four organisms, _!. ~' :7Proteus vulgaris, ~·
floureacens, and Phytomonas tumefaciens, whose hydrolysis of
L-leucylglycylglycine was activated by 0.003M MgCl.
The peptidase systems of Lueconostoc mesenteriodes were
also studied by these same authors (1937). The Leuconostoc _:.peptidase system lvas found to be capable of hydrolyzing both
optical components of the recemic peptides, leucylglycine~
. ~·. - leucylglycylglycine, alanylglycine, and alanylglycylglycine.
· Hydrolysis of DL-leucylglycine and DL-alanylglycylglycine ap
pears to be activated by ~arious metal ioris including Zn++,
Co++, Mn+_+, Sn++, and .Pb++. Th"~s enzyme comp 1 ex appear.e d t o
contain at least two dipeptide-spliting enzymes, and an
'.•., acylase, but no carboxypeptidase.
In 19.37 Berger, J~ohnson, and Peterson published a paper ·
on the proteolytic enzymes of about 30 species of molds. · Molds
of the ..species Asper~illus and ·Penicillium were found to contain
an aminopeptidase as well.·as a dipeptidase and a carboxypep- ·w dasee The peptidases of. Aspergillus parasiticus were also
(5) -- -·---~- ... ------·---- ...... __.- 9.:~-....~:-~:··_ ~~d..:-.:.:.:.~d ------~---~·--~~·-·-·····----- . 2~~ ...... ~~1·\:~:.:,1-'~--- .. -,.• _._:, -- ______..__;,...__,. ______······----- '1 AdOO ~: :1 .J.i'H)O · . ·[~ :_1 O~I:?X
- .. ;, • :' \ ~ , • •r . ' ·,
studied by Johnson and Peterson (~937).
Linderstrom-Lang first described the enzyme aminopep-
tidase in 1929. He studied this enzyme from extracts of swine
intestinal mucosa. rhis enzyme re~idly hydrolyzed leucylgly-
.;, . cine and leucylglycylglycine. For this reason it was called
leucylpeptidase 9 ·Johnson et ~· ( 1936) purified this enzyme to
·some extent and found that it required Mg++ and Mn++ for act- ; •.
·ivation .. In 1941 Smith-and Bergman confirmed th~se results.
Since L-leucineamide as well as-leucine peptides were hyd-
rolyzed, they_ suggested the enzyme aminopeptidase be called
·le1,1cine aminopeptidase .. Al_thou~h it has become C)'1_:>arent .that
hydrolysis is not confined to leucine compounds alone, the
name leucine aminopeptidase has remained. Smith and co-workers
(1952)-clearly demonstrated· that a wide v~riety of synthetic
amides·and peptides were hydrolyzed. Spackman~ al (1955)
described purification proceedures for leucine aminopeptedase
from swine kidney. Smith and Spackman (1955) then sho,v-ed
.that the properties of the highly pruified enzyme were the
same as those of the crude .enzyme preparations obtained ear- • lier from intestinal mucosa. ,'r•. Chromogenic substrates were'developed to demonstrate pep-
tidases·histochemically in human and animal tissues (1959)o
One of the first chromogenic substrates develop~d fo~ this pur
pose was L-leucine-beta nappthylamide. hydrochloriJ@. Develop-
ment of color by azo dye c6upling· takes place at the point of
release of the beta-naphthyl group. This assay is rapid, re-.·
·quires small- amounts of sample, ·-can detect the hydrolysis of (6) only five micrograms of substrate and it forms stable end pro-
ducts.
Nachlas (1955) published a report in 1vhich h'e claimed that
L-leucine-beta-naphthylarnide fulfill~d the specificity'require-
ments of leucine aminopeptidase and that leucine aminopeptidase
alone was able to hydrolzye it. This report lvas later changed
, :,. ~ > · to the admission of the lack of specificity of leucine amino-
peptidase and the posibility of the hydrolysis of·amino acid-
'"'/ .' b~ta-naphthylamides by a leucin~ arylpeptidase (1962).
In 1963 Math~son reported the purification of two amino-
peptidases from E. coli. Matheson. prepared a cell free extract
from E • .£.2.!.!. grown in· broth cultures.. After 24 hours growth
· the cultures were washed· and sonically disrupted. Membrane,
ribosomal, and soluble fr~ctions were obtained from the cell
··free extract by differential centrifugation. To measure the
aminopeptidase of thes fractions the substrate L-leucylglycine
was employed with.the colorimetric ninhydrin (Goldbarg~ 1959).
The soluble fraction was found to contain about 72% of the act-
ivity. The ribosomal fraction wa~ found to contain a~out 7%
of.the activity and the membr~ne fraction contained about 20%·
· ..: of the activity. The ribosomal fraction and the membrane fra-
tion were combined to form the particulate fraction and com-
par~d with aminopeptidase of the soltible fractiono The amino-
peptidase of the soluble fraction. was completely inactivated
by trypsiri and chymotrypsin, but the aminopeptidase 6f the
particulate fraction was completely resistant to these enzymese
. Patterson and co-workers (1963) showed that the enzymes which· ,.~.-- (7) J..\_,.;~.\ :':.1 ':!.\dO,) 10~::1x ...... _: ......
hydrolyze L-leucineamide were localized entirely in the soluble
fraction, whereas the enzymes which hydrolyze L-leucine-beta-
naphthylamide were distributed between the soluble and.the par-
tuculate fractions. When the soluble fraction was chromographed
on DEAE-cellulose, at least _three enzymes which hydrolyze the
·chromatographic substrate were separated from the enzymes which
cleave L-leucineamidee They assigned the name aryl aminopep-
tidase to the enzyme which hydrolyzes unsubstituted amides and
peptides that possess a free a-amino group _and are of the L-
configuration ..
Aminopeptidase activity has been found in rat· kidney,
liver, intestine, spleem, pancreas, and stomach which was in
hibited by EDTA, Ca++, Mn++, Co++, and Cu++, and wh:Lch had
a pH optium ·'of 7. 0 to 7 .. 4:. Human serum hydrolyzed L-alaine
beta-naphthylamide., Green ~ a·l (1955) found that Mg++ also
inhibited the reaction. Goldbarg ~ aJ.... (1959) ,·reported that
human serum, urine, bile, and tissues contained enzymes cap-
able of hydrolyzing amino-acyl-beta-naphthylamides. They con-
tributed this activity to leucine aminopeptidase although no
metal activator was required. Wolff and Resnik (1963) found
an animopeptidase capable of hydrolyzing L-leucine-beta-naph- ++ . ++ . ++ thylamide in the occular lens of cattle. Mtt , Co , and Mg
inhanced th~ .activity. . Chelating agents and heavy metal ions
inhibited the reaction. McDonald ~~(1964) showed that
puromycin did not inhibit leucine aminopeptidase' but 'that··
it did inhibit.the enzymes which hydrolyze amino acid beta-
naphthylamide. (8) z;~ -4 .~ •• ~:;l.~·~·-·· ~~-~!.J
,.'.
That the hydrolysis.of L:leucine-beta-naphthylami~e is
catalyzed soley by leucine amin~pept~dase has been questioned
for some time. Differences in reaction retes, rete specifit-
ies, and effect of inhibitors and activating metals have caused
~ .... ··a great deal of confusion$ Behal ~t ~ (1964) presented data
. ' ~ suggesting that. the. enzymes "t'IThich hydrolyze L-leucylglycine
· and L-leucine-beta-naphthylamide are different enzymes. It ·'... · ·has been shown that crystalline leucine aminopeptidase has
·-.activity on L-leucine-beta-naphthylamide substrafes, but that
this activity is much: less than that on L-leucylglycine~
Because of· these difference just dDscribed, it has been
.suggested that this activity on L-leucine-beta-naphthylamide
be called~arylamida~e. It is the presence of this arylamidase
in certain bacteria which will be the subject of this report$
' ,. ·~ .
. ·' :
(9) 1·.··· ;·
METHODS AND PROCEEDURES
The following bacteria are grown on appropiate agar at.
37° C:
·.·· 1~ Shigella flexneri (MCG A-1 Type i)
2. ·Shigella dysenteriae (MCG A-7)
3. Shigella sonei (MCG ·A-10)
4. Escherichia coli (ATCC # 1130)}
5. Escherichia coli ~MCG # E-2)
·· .. · .-. 6. Neisseria catarrhalis (ATCC # 11430)
7. Neisseria catarrhalis (MCG H-2) ·, . ~ ' ' I: ' 8~ Alcaligenes faecalis (ATCC # 8750)
9. Neisseria perfl~va (ATCC # 14799)
10. Staph1y1ococcus ·albus (MCG C-5)
11. Staph1ylococcus aureus (MCG C-13)·
12. Bacillus subtlis (MCG R-4)
13. Bacillus cereus (ATCC # 14579)
14. Bacillus megaterium (ATCC # 14581)
15. Sarcina lutea (ATCC # 38)
16. Micrococcus lysodeikticus (ATCC # 398)
·· 17. Streptococcus veridans (MCG V-14)
The surface of the agar is inoculated with a 24 hour broth
culture and allowed to incubate for 24 - 30 hours. After in-
· cubation the cells are harvested with saline at 4° C and washed e:t ··three times with 0.15 M NaCl at 4° c. The pell~ from the
last sedimentation is resuspended in distilled wzter, sonified~
and then centrifuged at a ghigh speed to prepare a cell free
extract. This extract is then assayed for arylamidase activitye (10) ; ·.~ . ~ _,.I !"j.r'• i,' 10 ~.it '1 A •'I(;~) ~~j~.t .;_~~~.:
I. Arylamidase Assay
A. Assay with Chromogenic Substrates
This method ·is based on the hydrolysis of amino
acyl-beta-naphtylamides (AA-BNA), as shown in Figure ls
According to Goldparg and Rutenburg (1959) this method
is sensitive enou~h to detect 0~02 u moles of beta
naphthylamine accurately and reproducibily.
1.. Substrates
Amino acid beta-naph~hylamides were used in
th.is study.. They are available commercially from
either :Hann Research La bora torie s, Inc.. or Cyclo <- Chemical Corporation~
2. Assay Procedure
The amount of .beta-naphthylamine (BNA) lib-
erated was determined by a modification'of the
procedure of Goldbarg ..
a. Preparation of incubation mixtures
Incubation mixtures had a volume of 1 .. 0 ml.
I. containing 0.34 micromoles of AA-BNA,
50 micromoles of phosphate buffer of the
desired pH, metal ion chlorides up to a
. }', maximum of 10 micromole, and enzyme sample.,
b. Determination of liberated BNA
After incubation for the d~sired interval
at 37° C., the reactibn was stopped by
-:i· the addition of 0.5 ml. of 2 .. ~5 M tri-
cloroacetic acid (TCA); any precipitated
(11) . ~
. '
. ' l'; ·,·... . . • . :8 .. · ·. N~C--CH-R A r y 1a rn ida s e· · '.; .' II ·. I >· . ··:, 0 NH 2- HC~
: L- amino acid p-naphthylamide· Hydrochloride /3-naph~hyl9m~r\~ .
No NO ..::· . . 2 ... HCI HN-CH -CH -NH -2HCI 2 2 ~ \. '·
~HN-CH 2-C H2;-NH2-"2HCI
N; N I ·Cl- ~ ~N=N. ~ ~-
Azo-dye ..
FIGURE 1 : REACTION. FOR ~RYLAMIDASE A.SSAY .. CFro.m<.Goldbarg and Rutenburg .1958)
(12)
:.·
.:>·' I;,.J -·· ~-··""·1 L :.,.,_:_ __ ·•·--·~....J- .. ~.:..-=---~ ..... -~ ·.. ~ - ~~ . \.).•. JyCJ 1'· :(~~io~)-) 'J.\.:1,),) Otl.:JX I· '---~·"' .-t.-9!5!}_/ t:
Figure 1. Schemat~c diagram of the reactions that take
take place in the arylamidase assay as des-
cribed by Goldbarg and Rutenburg.
,'•
·~ .,. ~- ·. ,·.: /
protein was removed by centrifigation*
"Zero time" tubes were prepared exactly
as sample tubes; however, TCA was added
immediately after'tbe addition of enzyme
and the tubes were not incubated. Next~
,. 2 ., 1.0 ml. of 1.45 x 10- M sodium nitrate
:,, was added, the tubes liTere mixed liThll, :... .-
and allowed to stand for three minutes~
To this solution was added 1.0 ml. of
1. 8 -2 · '1:. 3 · x 10'· · M ammonium sulfama te, the
tubes were shaken and allowed to stand
for two minutes. At this time, 2 ml.
of ethanolic N-(1-naphthyl)-ethylenedamine
dihydrochloride was added with vigorous
shaking. After standing for thirty min~
utes to allow the color to develop, the
tubes were read at 580 millimicrons on a.
Bausch and Lomb Spectronic 20 Model
Spectrophotometer against either water
or a "zero time" blank. One unit of
AP activity liTas defined as the amount of
enzyme that hydrolyzes one millimicromole
of substrate per hour under the conditions
of the experiment~:
BNA standards were prepared in a
1 S imilar manner.. The curve of hydrolysis
is shown in Figure 2. (13) ""-Jf· t:..:~"\ . - " : J-·, 11;~; [ ':f~.~ l~~:J':~~J '!,O~J. ~--
I'" \ ... ~ ·".(
Figure 2. Standard curve for determination of liberated
beta-naphthylamine. r .
. '· ··~
...... '· .,~
.. •j
: ~ ' ,....,
·.... ·:.I .5
...
j:.' .
J . · I. 0 > I- (/')- . ·z LJ..J 0 .·· --l ·. ;-, <( . . -~ . u· o·.5. 1--. ·a. 0..
~ . <: - ·i-
.:;:: ...
.:I 25 50' 75 ... ·._.JQO .. . . .· ...... , . . MILUMICROMOLEi OF 13-NAPHTHYLAMfNf: ; .·...... ·._ ~· .· ·. Fl GURE -~:.STANDARD CURVE FOR DETERMINATION. OF ·f)-NAPHTHYLAM I NE> H.YDR.OLYSIS .· .· ·. 0 0 • . • ' ·,· • ' • '',. • . • '. • . I • ••• • • • • • ' ,' • ( 14) .' . . '·:,- .• ·. • "• ' •. ,· . '\ . • • • • • • •
. •
. ' ~- '; . . "' .·. ,2'£ bi-: )..·.;·1(.~1~{;. OU3X\' -~-._....·-" ·~~}tlil{
.... ' .. - .,· .... ··.
3. Reagents for BNA Assay··
a~ Substrate Solutions
All are 1~37 x 10-JM and of the L configuration
alanyl-B:NA
leucyl-BNA
Nethionyl-BNA
_a-aspartyl-BNA
arginyl-BNA
g;lycyl-BNA
phenylalanyl-BNA
isoleucyl-BNA.
,·, .. valyl~BNA
seryl-BNA
B-aspartyl-BNA
theonyl.:..BNA
histidyl-BNA
prolyl-BNA
cys~inyl-BNA
b. Phosphate buffers
The desired pH was obtained by mixing
appropriate volumes of the following stock
solutions: 0.2 M K HP0 , 34~4 gm® of K HP0 g 2 4 2 4 34~4 gm. of K HP0 per liter of water~ 2 4 and 0 .. 2 M KH Po , .. 2 7. 2 gm. of KH Po q per 2 4 2 liter of water.
c. Buffered substrate
Equal volumes of substrate and buffer (15) / .. ' ' ,ill ...... ··-'···'··--~--- ...... ~ ...... " .. ____ _ ~.l ••••• •.... -), ;,j():) ~; -~~·=,~J- •t~:{~~
are mixed.
d~ Trichloroacetic acid
2a5 M,· 400 gm. of TCA per liter. of solution0
ee Sodium nitrite
1.4s·x 10 -2M, 1 gm. of sodium nitrite
per liter of waters
f. Ammonium sulfamate
. ~.J'· 8 x 10 -2H, 5 gm .. of ammonium sulfamate
per liter of water.
g. N-{1-naphthyl)-ethenediamine dihydro
chloridee 1.9 X 10-3M, 0~5 gme per liter
of' water.
h.. Standard BNA solution
1.93 x 10-3M, 50 mg. BNA per liter of :··
water.
i.. Netal ions
Stock solutions of OelO for the following
metal ion chlorides were prepared:
CoC1 -6H 0 2 2 MnC1 -4H 0 2 2 ZnC1 -6H 0 2 2 :tv!gC1 0 2-6H2 CaCl 2
B. PROTEIN DETERHINATIO~
Protein concentration was determined by measuring
the absorbance at 280 mu.
C. CHROMATORAPHIC FRACTIONATION OF CELL FREE EXTRACTS (16) ·-----·· ···--·--·-·. -·-----~------~.-.-';·: ..~ ...... /-~~~J .. _. ____ . \AdOOi: ~.~eJ
A modification of the method of Peterson (1938)
as desc,ribed by Behal ~ al (1962) was used to carry.
out chromat~graphic fractiona±ion of the cell free ex-
tracts on DEAE-cellulose colQmns with gradient elution~
Column dimensions ,.,ere 1 x 40 em., in which 'vas packeq.
3 gm. of DEAE-cellulose, 0~9 meq~ per gram. The coluw~s
were eluted with a phosphate buffer gradient-sodium
chloride system® Initial sodium chloride ~olarity,
phosphate molarity, and pH were 0.00, 0.005, and 8o6
respectively and final concentrations were OQl, 0~4~
and 3~5 respectively. Elution was carried out ·at 4°
C at a flow rate of 0.3-0$4 ml. per minute. Fraction
volumes '\vere 6 .. 0 ml. (Figure 3): D. STARCH GEL ELECTROPHORESIS
Horizontal starch gel electrophoresis is carried
out according to the method of Smithies (1955)~. Tris-
borate buffer at a concentration. of 0.05 M and a'pH
8.6 is used to prepare the starch ge~. The ·chambers
are filled with sodium borate at a concentration of
0.,01 M at a pH ·of 8.·6. · Electrophoresis:>is carried out
with 200 volts and 5 milliamperes current. ·At the end
of 16~hours the current is removed and the starch is
sliced and stained for enzyme activity. The substrate
L-leucine BNA at a concentration:of 0.027 M is added
and the st~rch strip is incubated for.·one hour with
the substrate solution. is removed and the starch gel
is washed with distilled water. After washing, the
( 17). . . ~ ' -~ . -
' '·.· . . . · . ..
.:.._ -..
_. ',,
. - ~- .:
. ._ .. _·,-
I.- 125 mI ErIe n m ey·e r F I ask 2.-125 ml Erlenmeyer Flask 3.--250ml. Erlenmeyer Flask. •a.--Column Dimensions I. 0 x 6 0 c m~ ·: ..·
• .·. ':"j .. ·. Packlng·=- 1.0 x 40.cm. with . DEA E-ce II til os·e I~ 13xl00cm;·tes.t · · U · · · tube in Fracticin collecto.t· · ·
·-;· '· --~,,··;
.• .. , Figure 3 : · ELUTION . SYSTEM .. FOR CHROMATOGRAPHIC< ;-·-.. PROCEDURES . ' .. ··_. -. ~ .-· ~~- ·; ..
. _:.'
·_·,· ..
. .'. - ' . _._·. '• :_·.·,·;'. 1 ' ' • • "' " . . ·:-· . ' . .~· ::/ '.· ·:- .:_ .·_ ., . , - . ·: .·_. .· .··
. ·' -~ ~- . . - - ..i .~d(J ...)' :L9~";J~_'
. } . -.... •
.I
Figure 3. Diagram of the elution system used for DEAE-
cellulose chromatography •
. ! ), J ..... l.-:~~--~-".. ·.· ·.·F . '·~:.;~10 t' Oi:J::::lX•: 1l ____c~:li;~l ;...·-' ·-·· _;.;;.J
fast blue B dye is added. The dye will indicate the
enzyme activity by staining released BNA with a red
color at the site of enzyme activity.
E. SUBSTRATE SPECIFICITY
The rate nf hydrolysis of synthetic amino acid
B-naphthylamines are determinedo Partially purified
fractions from DEAE-cellulose columns are ·incubated
from 30-60 minutes with different amino acid B-naph-
thylamines. The arylamidase assay is then carried
out as described above •
. F. .l-f.ETAL ION REQUIREMENTS
The metal ion dependence of the arylamidase activity
was determined by dialyzing the partially purified enzyme
fractions against 0.01 M ethylenediaminetetracetate
(EDTA) overnight. The same fractions, after treatement
with.EDTA, were incubated with the substrate leucine
BNA. To this same incubation mixture Mn++, Co++, Mg++~ ++ ++ Ca++ zn , Cu_ , and H o as a control were added~ ' 2 After a time interval of from 30-60 minutes the ary~
!amidase assay is carried out.
G. INHIBITORS
Enzyme inhibitors such as puromycin and EDTA
(0.1 ml.) are added to an incubation mixture of partially
purified arylamidase fractions from DEAE cellulose colunms
(0.4 ml.) and the amino acid B-na,phthylamine substrate
(0.5 ml.). After a time interval_ of from 30-60--minutes,
the arylamidase assay is carried out. (19) •·-- ·: ~j. - ~ L,:.. -l~Ejw·
. RESULTS.
It was found that N~ catarrhalis was the richest bacterial
source of an enzyme hydrolyzing the substrate leucine-BNA"
~rylamidase was piesent in all the.Gram negative organisms tested~
~10 Gram positive organisms, Sarcina lutea and MG lysodeikticus,
also had appreciable arylamidase activitye There is consider-
able variation in the amount of this enzyme activity in each
of these bacteria. (Table I).
I. ELECTROPHORISIS
Cell free extracts of each of the organisms that had aryl-
amidase activity were electrophoresed on a starch gel. Since
the electrpohoretic mobility of arylamidase from N. catarrhalis
had already been established in our laboratory, the extract
trom this organism ~as used in each case. as a reference~ Aryl-
amidase from each organism appeared as a single component
(Figure IV). Each arylamidase component migrated approximately
11 em., except for Sh. sonnei. In order to determine whether
the enzyme from Sh. sonnei did in fact have a mobility different
from that of N. catarrhalis, equal amounts of the extract of
both N. catarrhalis and Sh. sonnei were mixed and electrophor-
_esed along with anothe·r sample of only!!· catarrhalis .. It was
assumed that if these were different enzymes, two components'
would result from this mixture (Figure V). Of all the extracts
that were studied by starch gel electrophoresis, only one dev-
· ient was found. Sarcina lutea had t1~o components, one·_.,which
migrated the· same distance as· N. catarrhalis and one which
migrated approximately 5.5 em~ (Figure VIII)
(20) .i;£j \ J. r.i(j·~;'ti , ~~x:~J
The cell free extracts of each of these bacteria were
assayed for arylamidase activity as previously described~
TABLE I
, . . ·Arylamida se ac tivi ty on the substrate L-leuc ine BNA from
cell free extracts of several organisms
UNITS OF ORGANIS~i ACTIVITY* per.mg. of protein
Neisseria catarrhalis 100
Sarcina lutea 51
Shigella dysenteriae 42
Escherichia coli 37 Proteus vulgaris 24
.Micrococcus lysodeikiticus 20
Alcaligenes faecalis '17
-Shigella sonnei 14
Shigella flexneri 13
Staphlylococcus aureus 0.
Staphlylococcus albus 0
Bacillus subtillus 0
Bacillus cereus 0
Bacillus 'megatherium 0
Streptococcus veridans 0
*An arbitrary value of 100 is assigned to the organism
having the highest level of enzyme activity per mg. protein
(21) : .... : ~; ,t
,,~ ' ' '' -·· .. I',:.
·· ... :
'' :· ... :· •r.' -·,1 _·,>·' ,. ',!
Figti~~ 4.· St~rch gel electrophoresis of cell f~ee ext~~ct~~ . ' . - ' ·· N~ ·.-~a.tarrhalis, .§!i· ·f'lexneri., 2h,~ · dysenteriae~ ·and·
. ' - .. Sh. sonnei ·showing-the migration of.·arylanii.dase~ . . iondition~- previo~sly;des6ribed •.
/ ' ... . :'. ·: - . ' . -~-' . /''
. I·.:. '·:.··.--
. ;' ., ··' ·. ,·_,
.· __ ,_
~--t • ' ' Figure 5•· Starch gel electrophoresis of c~ll free ex~~acts~rif
. on;ly N. catarrhal is and . a mixture of equal par·ts of··: : __ .·. N. catarrhalis .and §ll.. ·. sonnei showing the ·migrationt..i:_:·
.of arylamidase under:conditions·previously described
·.. :....
,.._:t· ... . ··' ,... ~ .... :,,-· .\·
' : ~ . ..;' ·-•-,'
'.:.·_.-,·.· .<.
·.-.:.. - • ! .. ) ......
' -·~ . ~ .. . .
' -~' ... -·.::· . .'. . ~ --
• •• 1 ,·
• '', ',I • ·~::, ~:· ._.,, '·.·.. _·t·' :...... -·._., ..-
. ' ', ~ .
,·
. :."· l'' .. \;_ ·,_ ELECTROPHORESIS OF ARYLAMIDASE
Starch gel electrophoresis of cell free extracts Of· 1. Neisseria catarrhalis -· 2. Shigella flexneri. _3. Shigella ~iae. 4. Shigella sonnei ~~--~~~------~~~~ Neisseria Shigella Shigella Shigella catarrhalis flexneri dysenteriae sonnei
0 ~---r--.,.-----,~r----...... ,r---:~"---- ORIGIN
2 : t 3 Distance 4 in em 5
6
7
8
9
10
11
Figure 4 ELECTROPHORESIS OF ARYLAMIDASE
Starch gel electrophoresis of cell free extracts of: l. Neisseria ,atarrhalis (50mA)
2. Neisseria catarrhal is (25m A) +Shigella sonnei (25mA) ·I Neisseria Neisseria I cata rrha I is cata r rha I is + Shigella I. Sonnei 0 ORIGIN
l -
2 ~ i 3 + :Distance 4 in I. em 5 - 6 - 7 -
8
10 - -·:.-'
. . -~. '
•':- ··'·· . . ' ~ . ' ';;F
.,·
,· _ .. ,
. ' ·... - Figure _6~ starch--gel electrophoresis of ary,lamida~e. from ·cell:~·.,
free extracts of·li·· cat~~rhalis, _!~. J?erflava, .'r ·.
• .,l migra t·ion of this enzyme under conditions previously-<:
des~ribed. ,·· · .._ ,· ,\,:. ., .
.··-!_·
. ., .· . ·-~- _;. -, .. ··.· .. :
·· ..
Figure 7~ Staich gel ele~t~oph6resis of cell free extracts-of
_li. catarrhalis, Proteus vulgaris, Aerobacter sp .. ,_
-..... ~ and .!• coli the _mi·gr~ tion ·of ary~amida se_-_:-· -··-· :.·:· under conditions p~e_yiously described~--· ·: :·.
. ' . ~- ··-·.
,\·: -·
. ~- ' ..
. ~·. -.r.
,·
-~ ' . • ,'1
.. ··.
·. : .... :.
)-. ._, .. ·.. ----·· ELECTROPHORESIS OF ARYLAMIDASE
Starch gel electrophoresis of cell free extracts of: l. Neisseria catarrhalis 2. Neisseria perflava 3. MTCrOCOccUSTYSQdeikticus 4. Alcaligenes faecalis
Neisseria Neisseria Micrococcus Alcaligenes catarrhal is perflava lysodeikticus faecal is
0 ORIGIN
2 i 3 - Distance 4 in em 5 6 l 7 8 .
9
!l 10 .~ .. ,.,,., .. ,,., ~ 11 ·····""·"·'•·· ------~------:------#·------·---;--:------... ·~------~------~---- Figure_6·
r ELECTROPHORESIS OF ARYLAMIDASE
Ir Starch gel electrophoresis of cell free extracts of: I 1. Neisseria cata rrha I is 2. Proteus vulgaris 3. Aerobacter sp. I 4. Escherichia coli MCG I
Neisseria Proteus Aerobacter Escherichia coli MCG catarrhal is vulgaris sp. coli
0 ORIGIN
1
2 I l t 3 + Distance 4 in em 5 ! 6 7
8 .
I 9 I 10
. I. 11 l:"]:.ji
/_----~------·· ·.·- --~.:..::._ ___ ---, ------~ ·------_....,------~-~~---.--.--·- Figuer 7· . ~ 23) t:lii ~', •' .. ; :~_.:.._: ___ ,__ .. iJ h.--- .. -· ... - __ .:_ __ . -- ____ .. _._ .
I' I l( j ~ j !' ;:.:.j~)~; r 'Oi.ClX~ (OtJ::lX i. - ..--·""' ·--..--..H...-~
Figure 8. Starch gel electropho~esis of cell free extracts ·of
~· catarrhalis, ~· £211 (ATCC), Alcaligenes faecalis,
and Sarcina lutea showing the migration of aryl-
amidase under conditions previously described. ELECTROPHORESIS OF ARYLAM IDASE
Starch gel electrophoresis of cell free extracts of: l. Neisseria catarrhalis ATCC 2. Escherichia coli ATCC-- 3. Alcaligenes faecalis 4. ~arcina lutea
Neisseria Escherichia Alcaligenes Sarcina cata rrha I is coli faecal is lutea
0 -----...-...----,.------,,.-.,ro-o---- OR I GIN 1 . 2 ~ t 3 + .. 4 Distance in 5• em 6
7 8
9
- -~------·------:;------______.. _ ----.------· --·------.-·---,-~------·- :-- ~- -~------Figure 8.
(2-4) • -. ,•, i:• \~~~~~: -· -... ---~·
II~ DEAE-CELLULOSE CHRO}fATOGRAPHY
The extracts of those bacteria with arylamidase activity
were fractionated by DEAE-cellulose chromatographye It was ...
found that in the extracts of all those bacteria, there was
one distinct component that appeared from fraction 40-63
(Figures IX-XV) .. ·. The amount of activity recovered from each
of these bacteria paralled the data found on TABLE I. In ex-
·tracts of Sarcina IH:t~e~, however, two distinct conponents
were found. These two components h~ve arbitrarily been inde-
tified in the order of elution, and are defined as· follows:
a~arylamidaie, activity in fractions 30-37 and B-arylamidase,
activity in fractions 50-59. Less a-arylamidase than B~aryl-
·amidase was recovered.
III. PROPERTIES OF THE ENZYMES
The ~rylamidase activity·in the extract of these organ-
isms on a number of amino-acid-B-naphthylamines was determin-
ed~ ·An arbitrary value of 100 was assigned to the substrate
giving maximum activity. These values '~ere determined for aryl-
amidase activity in the most active fractions from a DEAE-
cellulose chromatography which resulted in a 300 fold purifi~
cation of the enzyme$ In(~·each case alanine-BNA was hydro-
lyzed more rapidly than leucine-BNA (TABLE II).
A study was made of the effect of inhibitors on partially
purified arylamidase obtained from the most active fractions
resulting from DEAE-cellulose chromatography of the cell free
extracts of each of these organisms. The arylamidase assay
was carried out as previously described. An arbitrary value
(25) ...... j_ e;...,.;_~·---'-· ~... ' . \ :1.\,l(.l(j/ t~~:'~i
Figure 9 .. Column chromatographic profile of arylamidase activity
from the cell free extract of N. catarrhalis.
Figure 10. Column chromatographic profile of arylamidase activity
from the cell free extrarit 6f Sh~ flexneri. '.·· ·.,_, ·. ,'•,, -·· .. :: .·.· .
•• - ,.· • >1. COLUMN CHROMATOGRAPHIC PROFILE OF ARYLAMIDASE FROM NEISSERIA CATARRHALIS .
2 . 0 >- r-- ' > l (f) z >I w I 5 0
't: ...J <{ u I .0 •'' -:~ ..... a.. -- 0 0 5
0
.J ••
COLUMN CHROMATOGRAPHIC PROFILE 'OF ARYLAMIDASE ACTIVITY OF SHIGELLA FLEXNERI
2.0 :·" j- > t- ~ 1.5 ··o.,~w
_,_.} ..
.i : ...J 1.0 -:;·:.···_·;_ .. - ·.~ ' 0 ...... ,.;:;;;;;.._~~;;,;,.;;;;;:~_..;;;.~;....,..--....-....;._-...... -----.-~;;;;;...,...;;;;....,;;;;..;;;;...--r \ 10 20 30 40 50 60 70 ·-' 80 FRACTION NUMBER -.-PT;------:r ---:.----: -~------~~ '' '; ;~- ~~.--.-:~--~-:.--~---~- ._.-.- ---··.- -. - -.--:;----:---:--~--:---:- .. -.--___ -:~--r---~-3- . ':, :: ' .. y ·:.:· ·.. .· .. ..-: . '.:· - ~ • '' 1_·, . itlii~~j-'.:t;o, ·.. ·· x·, '', ._ .. ' ' •: '':._. ~:""• -,' ' -, - ;. ,.,. ', ' ' ,':.' ,•' ;, 1.:...-~------::, '< ... ·. __ ,- '\'' j .~' l Figure 11. Column· chroma to graphic profile of arylamidase from cell free extracts o.f ·sh. dysenteriae. Figure 12. Column chromatographic profile of arylamidase from the cell free extract of Sh. sonnei. . !_··. COLUMN CHROMATOGRAPHIC PROFILE OF ARYLAMIDASE ACTIVITY OF SHIGELLA DYSENTERIAE >- ·.I- I. 5 ··. -~ ·-(/) z L&J 0 1.0 _J <( 0 I- Q. 0 0.5 10 20 30 40 50 60 70 80 FRACTION NUMBER -- . . - .. -- .. ·- ... - . - .....,. ·-·. ·,- ...---. . --~. ---,---,------·------~------~------~- ---- Figure 11 COLUMN CHROMATOGRAPHI.C ARYLAMIDASE ACTIVITY OF PROFILE OF. SHIGELLA SONNEI > t- en z LLJ Q .....J 30 40 50 60 70 80 ---....._.____,_~ -·--- -_-...... _,- ~-.....______·Figure ·12 ____ .. ( 27) L:.--, '1 ~~~f:~i~ ...... _T ,,- '• Column chroma to graphic profile of arylamidas-e from the cell free extract of Proteus vulgaris. Figure 14. Column chromatographic profile of arylamidase from the cell free extract of E. ~· 1:·· '-.:·:·. - '. ..._ ~ J • ...- . ~ ) - :·"' .." .·. '· . -· . ' : .' ~ . . ·,, '• j' : \..... - ... ,. ...~ •' _1~ • ;_.: ••• -•• ~ .\ .. -: . ·~ .· ; . -~ ~ ' .. - . ~ . ··~ ' ~~~~~~~~~-~--~-~~~~-~'---~--~--~~--~~~-·~-~--·~-~-~~~~~~--~ COLUMN CHROMATOGRAPHIC PROFILE OF ARYLAMIDASE ~ 2 .O ACTIVITY OF PROTEUS VULGARIS . j g_ ] 1.5 >..... CJ) z LLJc I .0 . -~- -:._.' ' ...J <( (.) h: 0.5 0 _. ·.:·. ' 10 20 30 40 50 60 70 80 FRACTION NUMBER . \ ·' .. . -' ,.-. ·. . . -' ...... -' . - . ' ·' . : '· ~- ' .' : . .. . ; _. ' . '· .·. ·... .. , . '· ~.:.:__:-·.'~,··-~· _.:_:__~····;.._...,·. -~- ~~·..;.;.;...·, ~·...,...,-·..:.;;.;·<:...--:.····._,_·-.-,;· _.:.;,;_,;::; '-...;.,:_-·,·:,...... ;..' ____,~-·_..::,_,>·r~-·--~·-·-=-·~~::~-:·~,;,.;..<·:.,...::...·__ '.,..-_,~-c.__;,·.=""""-~·::.:.,.,·.:_,:..._··~:....,.;.....:__:;_,·_,:..;..:-··--:.:...... :...:...... ;_...:....:.'·-· -::..:_---'-'-it . - .. I'l COLUMN CHROMATOGRAPHIC PROFILE OF 1 : ·-f ARYLAMIDASE FROM ESCHERICHIA. COLI ·.,__ ·. 1 .· - ·l 2.0 i . I .. ~z -1 I.LJ I .5 ·.--1 0 . . .·'1 .-.--.- :.: .. ··l ...J <( .. ·.. . 'I u I .0 ...... '. ~--·_>. ·, :.·1 a.. ·. -, .. - -~~ '.• -j 0 . : '.·:'·I 0.5 .. -.- : .I ·. . j 10 30 40 50 60 70 80 .··I -.·I FRACTION NUMBER ' . ' ... •:: 'l ". ~----:_---~--~---:--:-~-·. :~.-.--;----::-::.-:"'·-;:--.:-- . ··:.·-;- :.. -.~.. -~.- -~--:;-~·:····-:-·:-=;·'·,~-- :·-:·r:-- r~_-:-·7~-~----:.~-::;~-- -:,.~ ·:· ~---~- -: - '- ·_·-. -- .. : : .•=.\ : :F~g:ure. 7_4 _- . - . ... __ :, ·/ .·· ____ ·-· ' ..- _(2_8) •. 1 • ' . . ' ' . ./ ~ .. - : . . Figure 15. Column chromatographic profile of arylamidase from the cell free extract of Alcali~enes faecalis. Figure 16. Column chromatographic profile of arylamidase from the cell free extract of Sarcina lutea...... '• ·-',·,,: .... ' ' ,. ' • - ~·- ' • • 1', ' ' • .. ·r__:..:;~-==-=~---..--:..-~~- ~~~--~....:..._:_._~ COLUMN CHROMATOGRAPHIC PROFILE OF ARYLAMIDASE I ACTIVITY OF ALCALIGENES FAECALIS. I 2-.0 I > t- en z w 0 ...J I .0 · <( 0 ..... a.. 0 0.5 10 20 30 40 50 60 70 80 -1 [______FRACTION NUMBER . , .. ~ ··--:;-;-:,:-·:-=- --~~-.--.. --.-.- ~---·- -:-· :·...-.·---~~-~- -.~; .. -.-..!. .. - • i~~.;--;· -----~------;""------··:--=------... ---:.--·------. --·----~ - -· · · · ··- ·.. ···.: :_·_--· .··Fig~r~ :-l'~J'-~·:·"<·. ·. :,· · -. · -~-· •,, .··. ' J - •. -.. • '. ·.' • ' • . • • - ..• •• - ~~ ~~ .' : • ' · .. ' _' : . '·:_,:· / ' ,' . ' ' ' ·' ' .'-. .- . ·' . ' ,,:.'' . . . ' . .. :• :· :; . ·. ' '. ·.: _· •. . ' ·, ,·,_; .. • • • : , : :', \ • ' • -' • ' ·'"' ' • " ;.· '. r, ,/.' • • •• ·:~. r' ·•' '' ,,., • .'; ';- • ~· - ' \ ·.-, '.::. • •.,..- '• I ,I .. ' , ' '•· .,,._, ': .. •, .: ' ; . ! ·. : :. : •. ' - ...... •' ·.:-~ '. ' ' ':. '. : :-; ·. .. ·:. . . • ' !" - .- ~' : · .. ... - -- . '. ' ' COLUM'N. CHROMATOGRAPH·, c:-P-ROFILE' OF ' : ~ ' , I ARYLAMIDASE ACT~IJ.V~OF SARCINA LUT.~ · 2.0 1->- 1.·5 en z LIJ c__. .5 FRACTION NUMBER _ ---~--- ~~-· --~-. ---~------~., -:F--l.--."g·.. --,-u·:.e·~-_.--:-1- _6_,--.~-.-:.":::-~-:--:--.~"--=--.· -- ···-. ·- . -~.;~---~~ --.. ·:-'_-.·-·. · .. ----:--~,------'-~---.--- ... ' ' ...... ,. .. ' _.'' ' ' : ·.. . ' :.' : ( 2 9 ) ' - . -. .. .. •. .. .,...... :- - -~ ..:·. . .. : ; - . ·' . ; ' • ~-· •• J • ._ • ' ' - ':. ,_·:. ·.:.· -·~'·;'"-~~. . . .: .. ·~...:- ..... ~--·-' . ~ ...... ~ •.. -.;.. _~·------r~< . -L .:id \ ),,-1(,~) f. 'OcDX\ ... •• • ~~J.' TABLE II Arylamidase. activity of cell free extracts of several· ~a.lCJilRirSD•~ --- A~4INO ACID Nq) ShOll Sh.a. Sh. E.o RESIDUE -ca tarrhalis dysenteriae flex:neri sonnei coli alanine 100 100 100 100 100 leucine 28 39 34 41 31 methionine 27 27 30 .32 20 a-a sparti·c acid 26 22 2l1 -20 14 :· arginine 14 14 12 12 8 glycine 13 12 10 9 6 phenylalanine 1 1 1 0 2 isoleucine 0 0 0 0 0 ·valine 0 0 0 0 0 serine 0 0 0 0 0 B-aspartic acid 0 0 0 0 o. threonine 0 0 0 0 0 histidine 0 0 0 0 0 proline 0 0 0 0 0 cystine 0 0 0 0 0 (30-a) ---1 TABLE -· +: \. :organisms on s·ev'eral -amino ·acid .beta-naphthylamines ·Pr.oteu~ . Alcaligene-s a-enzyme· _Sarcin~ B-·enzyme . Sar.c ina vulga~is: faecalis lu.tea lute a ·,'' 100 100 100 · .. 100 20 'J~: i6' 24 30 - '~. ' • -~ f - . - ·.· ~ ' .:.. ." . 14 12 -..· .~ . .' . · ...... _io 9 8 10 ;- 9 4. "• 5 4· ._, ' ' , .. 1 1 '2 -·.0. ·.·. 0 0 0 0 ·o .·.;.; ,_. .1-. o.· ···:_; .0· __ .. · 0 0 o":- •. --: ·- ·.' - ...... 0 ;. 0 ·o ' -,: 0 0 .() 0 .. 0 o· '' 0 ·.; 0 . 0 ·:-' o . ·-··-.o .. ·. ··:.- :. ~.1 ' _t.;; . . - L--'-'-'-'--~•i :._ .. :.. ;·_'~ ·j )..,,j(JIJi 'tO~I::':lX/ - ~---.:-.:.j..::.~-- t~~~~~~- of 100 was assigned to the substance giving_maximum activity on the substrate leucine-BNA~ It was found that in each case EDTA at a concentration of 0~01 M had no effect on arylamidase activity. EDTA at a concentration of 0~10 M, however~ decreas- ed the activity of_arylamidase about one third ... Puromycin at a concentration of 0.001 M decreased-the activity of this en~- yme about 50% (TABLE III). The ef£ect of metal ions on-the activity of arylamidase was investigated using partially purified fractions obtained of each of these organisms. The arylamidase assay was carried out as previously described using leucine-BNA as the substrate. An arbitrary value of 100 was as~igned to the sub- s_tance giving maximum activity.· The designation H o was tised 2 to indicate that water displaced the substance being tested~c It 1,ras found in all cases that Co++ enhanced the activity of this enzyme. Mn ++ , c a ++ , and Zn++ , h owever, decreased the act- ivity of arylamidase while Cu++ completely inhibited activity·. (TABLE IV). Attempts were made to define differences between the two arylamidase components isolated from the cell free extracts of Sarcina lutea. Studies were carried out on pH optimas with partially purified fractions of each component obtained from DEAE-cellulose chromatography. The pH optima of the a-enzyme ;. was found ·to be 7. 8 't'rhile the pH optima of the B-enzyme was M~chaelis constant studies·.~·on ·the a-enzyme from Sar_s.f~a.. lutea· showed the Km = 9 x l0-5 using alanine--BNA as the sub- ...- ...... ~--...... (31) . l .• :.;;- ··--j_··~c!·l:,_.. ,~--...~~ b~ . I"' CIIJ'-) i' . ,O.ti3XJ: ·. :~ ~-~j~~~ ------..._:., ·'. TABLE III Effect of inhibitors on arylamidase activi-ty of the enzyme from several organisms ·------EDTA EDTA PUROMYCIN ORGANISM H 0 {0.01 M) (0~10 M) (0.001 M) 2 N .. catarrha1is 100 100 72 55 Sh. flexneri 100 ioo 64 41 Sh. dysenteriae 100 100 68 Sh. sonnei 100 100 61 42 Proteus vulgaris 100 100 60 40 E. coli 100 100 64 Alcaligenes faecal is 100 100 38 a-enzyme Sarcina 100 100 lutea B-enzyme Sarcina 100 100 70 50. lutea ( J2) -~~ ·. ~·· ., ...... ,__ ~) .. :l(;~f. '-~~:3J TABLE IV Effect of metal ions 011 arylamidase from several organisms. ++ ++ ++ ++ ++ ORGANISM Mn++ Mg Co Ca Zn Cu H 0 2 N .. catarrhal is 100 93 100 87 50. 30 ' 98 Sh. flexneri 100 94 100 83 45 41 95 Sh .. dysenteriae 100 92 100 Bo 44 33 91 Sh. sonnei 100 96 100' 86 52 38 96 Proteus vulgaris 100 91 100 85. 42 34 97 E. coli 42 59 100 89 47 0. 59 Alcaligenes faecalis 100 92 100 81 53 35 90 a-enzyme Sarcina 100 95 100 85 54 36 95 lute a B-enzyme Sarcina 100 90 100 81 59 29 94 lutea (33} ,I ~. ' - l..:.~<.. ·" ~~} ~.-~·~\JO~) :ii2~:a,~, Figure 17. Michaelis constant comparison between activity of\ the a-enzyme of Sarcina~ on the· substrates leucine-BNA and alanine-BNA. Figure 18. Michaelis constant sutdy sho·w·ing the non-competitive inhibition by puromycin of arylamidase from B-enzyme of Sarcina lutea. ;·_,: .·· .. : . .' '..... "':.- ~ , . ' ~ .'~ .. ; .. , ~ ~·: ~- : - ~ ';:• .. ... ·: "".:• '1,' ... -~- . ·~.:. .. • -~.: • •• ~·f :-·:<:~-~·~·' .·-~ ..- ,• ... } .- /;.~\( ~;. . ... '. ~-- . :· .· -~ ' . ·i·.'. a -enzyme of Sarcina lute a 800 ,· :· .. : '. :. .. 600- 400- ,· 20 40 60 80 j_ X 10 3 [S] -~.~ .. ~-~-:--- ,_ : ·,'...... ~·!;_ ·~ ,_- ·<-. ·-_.- ~ -~ :-~] ·. ; .. -.....: it·r;, ·~ ,':;,: .· -•• i'1;'r!;~,~~~~S·t7i ii? "· ··: .. ·,:.j - .·, l ~-enzyme Sarcina lutea L -ala-BNA =Substrate . ;.,- ... ',· . ':_.,. v .. -4 Km=2.5xl0 5 10 15 20 25 30 35 40 I X 103 [5] ·::-·.-- ---,-- -.·.. ·, . ,. ~ .'·.-~ _.- - . . ·~ .' ' : ..· .. ·: . ·• -_· .. .-·. -- - ... -· . '. ·_ •. ~ . ;, ... ·· ·... ···'~lt3~~······t,,?. . . ~ - r- • • ~':. (' ,, ! ' . • --~\- •><>·_:~--~· :' . :_.· .. --·,.:,_·. '.,.·_ ,• strate. · For the B-enzyme from this bacte-ria using alanine as . . . . '. . . ' the substrate the Km had a v.alue of 2$5. x 10•5 . (FIGURE·~.:- x\tr~- XIX)~· Michaeli~ constant studies on the a-enzyme from: Sarcina ., J .....,... .·,·I . ' . .'~ 'a value· of 5 (FIGURE .. XIX)., Further inhibition ·inve·s~ t'··. tigation_s with puromycin using alanine· BNA as the subs.trate ·showed that· puromycin. non-completi t.ively. irihibi ted both·· the·····' .; a-arylamida·se· and the '!3-arylami~ase· ·from Sarcina lutea.,: . :-• :·.·· .. . .. ~ ,;-· ;.• ·,", .· ...... ·' ·•• 1' .... · ) .· . ·' ·, -...· .; '',: :_--· .,, ., .. .... -· '•,:. :,· .. . .-· '',l', . : -~ (35) • 'y' '•,'• . '.. .·.·: .;' ., (:,; _;.,:.! ;'),·r.J(JU( .o.~;:~x~r Figure 19. Michaelis constant study showing the non-competitive .inhibition of arylamidase from the a-enzyme of Sarcina lutea caused by puromycin. '.>I,'' .....'' ', ,': .. ' ,._·r ., _: ,_.··. ·.- ,· '. -'.-'. .· .. ' ' -~· .,_ ... : . . ' ' .. •·, I d -enzyme of Sarcina lutea I I 800 l I v 20 40 60 80 J... X 103 s -I - - ~------..-·- -- .. , ,....__.. - ..,.,. .--~- ----;--~~---- . . t. . ------• ; •• - .-: -~-·----:·-. ------.::~ ·------;--.------· 1 --~-- -- -·-·· ---~~:·:-· ...... ; - .. -~-~---: -·· --- ' . ~. ; . -;.. . . ' . . ' ~ . · .... _' ':'. :_ _(36} ...',.' ;,. "', DISCUSSION - . d.- ••• ' ;·. The purpose .of this investigation was to study the pro- . . . . f perties and occurrence of arylamidase in bacteria.. Upon stu- .. . . . . ' dying cell free extracts obtained. from approximately equal num- ;hers of both Gram negative and Gram positive organisms·,. i:t was •• ! : ·found that this enzyme occurred principally. in Gram negative. · ". "·extracts of or,t~y two of the Gram positive organisms tested~ , . Sarcina lutea and M. lXSQdeikticus. '• ,1_:· ·Arylamidaseshydrolyze amino-acid-B-naphthylamides which ,.are not naturally occurring compounds. The question arose then as. to the· funci-ton of this arylamidase. Some insight· into its function might be ·_gained by _studying its ·properties and com paring its action with that of enzymes with known· function on structurally related substrates. In general, bacterial arylamidases seem to be a group of similar enzymes. From the data obtained in this study,- there~:. did not appear to be many.different types of bacterial arylam- · idase·s .. · There was no re-lationship between the presence of . ~ ·, apprecia.ble amounts of arylamidase and the natural habitat of ~the bacteria studied. The use of starch gel electr9phoresis to obtain high-re- solution separation of proteins has been established by Smithies (1959). One oj. the most important recent advances in the chro matographic purification and characterization of proteins ha$~;;,: .. been the introduction of cellulose-based ion-exchangers by Sobe~ ·artd Peter~on (1954) •. The ~ccessibility of- the exchange groups .. . . ~· (37) . "1,-' •••• ~·{~ifu:.~·~'." _·:antibiotic assays with _penicillin ~ensi tive Staphylococcus following· incubation of penicillin with arylamidase~ it was ~oncluded_th~t penicillin a~idase and~ arylamidase are distint- ively different. Other possibilities included arylsulfatase, arylesterase, or phosphatase actionc Again subsequent data obtained in our . ~ ' .. laboratory have shown no relationship between their function and the function rif bacterial a~ylamidase. Sensiti~ity to puromycin has been used as a means to differ- entiatirig leucine aminopeptidase from arylamidase. Low concen- tra tions of puromycin significantly irihihi t · arylamidase; this·::· ·is not ·true of aminopeptidase. Puromycin was found. to be a competitive inhibitor of N., catarrhalis arylamidase, while pur- _omycin non ... competitively inhibited Sarcina lutea arlyamidase .. While all of these possible roles of bacterial arylamidase offer interesting, speculation, at the present no implication can be made between arylamidase function and protein biosyn- ·thesis., With the use of differential centrifugation, it was found' in our laboratory that the N., catarrhalis enzyme always appear ed in the soluble fraction. These results are consistant with .those reported on an enzyme found in E .. coli that:'is· active on leucine-BNA (Bolton, 1966)~ Since b~cterial wall peptides of Gram.positive organisms ·contain D-alanine, thej_lack of measurable amounts of arylamidase in cell free extracts of Gram positive bacteria could result (40). from the incomplete brealc dowTl of their cell walls during son- ication. If the enzyme ~ ti.Y.£,. lvere held near the cell membrane~.- absence of arylamidase from Gram positive organisms c~uld pas- sibly be an artifact of incomplete cell disruption. Although D-alanine BNA is not available at this time, arylamidase would=· ·not be expected to attack D-amino-acid residues. Branching ori the B-carbon atom results in decreased ~ffinity ·between the· substrate and the arylamidase enzymic active site., Dipeptides or amino-acid BNA ~~ose L-N-terminal residue has an . ' . \ . alkyl· ·group with branching on .the B-carbon atom are hydrolyzed at relatively low rates. "For example, valylglycine, isoleucyl :glycine, valine BNA~ .and isoleucine BNA were hydrolyzed very ·slowly, whereas leucylglycine, norleucylglycine, norvalylgly-: - ... cine, leucine BNA, norl.eucine BNA, phenylalanine BNA~ alanine BNA, and ·to ~orne· extent phenylalanylglycine·, are hydrolyzed relatively.repidly •.. Though it is clear that arylamidase and leucine aminopep . tidase are indeed different enzymes, at pr~sent no conclusions c~n be reached as to the function of this enzymeD (41) 1~"':-~~""-'l -.. c-~-··:- ~:~, "' . : ' [~:;,;? ~~~ -;~~-~~~ - ... ·; . :~ :' '.'·· .. ; .: :-,~··.:.~·-· .. . ~ .· ... •,:.' '.. ) .· .. . . . Nachlas; M. M .. , .T.· P .. G-oldstein; and A. M. Seligman, i962~ · ··· .. . s·t'udy of the chromogenic· substrates for the. assay.· :of: ... ·:· .. ·. aminopept-idase ·activ:;i.ty-.. _, Arch. Biochem .. · and Phys. ;· 9i:. -- 22).. . - '. ,I -·:,L ,· .. :patterson,,. E1.izabeth .K_ .. · ~ Shu~:Hs-i S~ith; E·~' L.. ' ·w. J e Polgiase , .. 'i949: Further . stU:~i·e s of ,_., . ·L,AP· specif'ici ty. J ~ Biol .. · Chern.,~ ~: 255 .,. - . '·. -.Sntith, E. t.·~- and H~ D~· Spackman;-· 1955 .. : LAP vs·•. 'ac·ti~~tion, _.:_, ... -~pe~ifici~y;:an~ mech~nism of acti6n. J.· Biol. _Chem~,: 212:271 •. " . S~ithi~s.~o., 19~5. Z6ne electrophoresis· in starch g~ls; group variations in the serum proteins of normal human adults • . '. ·.-. ~~-, ' .. Biochem. ,'· 61:629-641. , _ · ... -. ·:wolf ~rtd:Resnik, 1963. Aminopeptidase of ~he occula~·i~ns~ · Bioche~ica ~nd Biophysic-a., ll= 588-612_ .. .,_ ,_...... • ...... I ·, ~ ., . . ·.t···.. -·- -.-· ...... ·-" ·_, __ _ ·,:- ·.-. : . -· -:~.- ··.-. ,. ';._- . ' ', .... ·~··· . ~ .· - . -· ',. '··, .. -' -' .· i7 -. . (..45) .. - ... 1 - .:., ··.. ' ··. ·_._ . • t (' . ~. ~ .~ . . · ··'' .- .; . ... . ' '. -· ~- ; ._ .. ,_·7 l ,·.