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Distribution. of Arylamidase in Some .Selected Bacteria

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Distribution. of Arylamidase in Some .Selected Bacteria }·' '. •, ,·' ,·· - ;. ~ .' - ' ',.- . :··,· 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~
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