LACTATE DEHYDROGENASE ACTIVITY AND {SOZY MES
1N CERTMN STRNNS 0F STAPHYLOCOCCUS AUREUS

Thesis for the Game of M. S.
MICHIGAN STATE UNIVERSHY
ALAN E STOCKLAND
1967

 

 

 

”MAUI

LACTATB DEHIDROGENASE ACTIVITY AND ISOZYMES IN
own! ms or W m

bra-n3. Stockhnd

not.» dohydragmu (LDB) and 1.03 "can“ um
menu In strain: 29. 35. 6, 81. and #2!) of Staghgocgcun
m 0.100606 tro- ttn groups in tho mutational-Blur
an" ot phase propaguung “ruins. Cons I." cultivated
1:: Brain Hurt Inn-tan (Dirac) and» llama-0pm.!“ condi-
tion. and lama-Bod am tho and of the log pin-o. Quanta-
an luau-cum:- I». «ma! out on final. 0011- by nonpar-
138 the Optical donation for dilutions of wanna all
Mod «11.. The human and phouphoru contain no
uhmuutly «tanned for neural dilutions of nit-d «11-.
{DE activity III mum]. at the and of omen). growth
and In aunt-d ”automaton-0:210:11: by wanton of
pea unto-blu- «tannin (331') nuns Mouth-nu. admin.
«manna. (HAD) and platinum. nacho-unto (m3) .-
coupling Igniti-

Crud. man. cum" um propane both by an new
extraction technique (Vadohn. u .19 1961‘. Appl. Manual.
anon-1013) and by mutton. ma activity for than
mm pmttfll tron tho m1“. nnu- In dour-mod
u previously mum-d and all: by mounting cm at. or
“D reduction at 3110 um. Pro- both nun: method- tho rollov-
138 order of activity an: oburvodx 29 > 81 > 6 > M) 521).

Alan E. Stocklund

m3 unznu tra- flttm attains. "pro-mung the
nu plunge numbing group. of tho Int-mttml-Bldr
an". urn upmtod by ”171516. 501 cinnamon-nu.
Tho buds nor. «booted by incubation at tho 3.1- m c nom-
non containing mum hot.“ (60;). 1 .1; 0.2 n urn-am
butt-r pH 7.5, 22 :13 Kc» (0.06 a). 2 :13 m3. 1 as: m.
5 l3] and 2131'. It as. connottuly the 15 stain. Ion found
to pole." g mum of 5 “an“. no who: at "can"
pu- Itnhll varied from 2 to to Ian. 3 append In 13 of
tho 15 strain. and 1m fallow-d 11: “may by mbcrn 2
and 1.

MATE DEYDBOGENLSB ACTIVITY AND 180sz3 IN
mma arms or W m

by

Anna. amen-ma

A THESIS

sub-issue to
moms-n sut- Univ-nu:
1n.partlnl tnltlllnant or the roqntrununbn
for*tholdcgruo or

mama OP scxmcs
Dopcrblant or niorobaology and Publla noulth

1967

Ammmm

m author nan to than: D:- Mu L- San
Clo-unto to: his sum-t. sauna“. and 11an I133..-
uona duflng tho count of this may.

11

TaBLE CF CCHTENTS

Page
ACKR WLEDSEKZNT e e e e e e e e e e e e e e e a e e e e 11
LIST OF TABLES e e e e e e e e e e e e e e e e e e e e V
LIST CF FIGURES e e e e e e e e e e e e e e e e e e e e V1
IKTKCDUCTICRe e e e e e e e e e e e e e e e e e e e e e 1
HISTORICAL REVIEW e e e e e e e e e e e e e e e e e e e 3

Preliminary UOfk e e e e e e e e e e e e e e e e e

Improved Techniques to Assay Lactate
Dehydrosenaee. e e e e e e e e e e e e e e e e e h

nethode e: Extracting crude Lactate
Dehydrogenasoo e e e e e e e e e e e e e e e e e

Isozymee e e e e e e e e e e e e e e e e e e e e e
MATEEIALS A39 METHCDS e e e e e e e e e e e e e e e e e 10

Organisms. e e e e e e e e e e e . e . e e e e . e 10

Medium and Cultivation e e e e e e e e e e e e e e 10

Quantitative Methode to xeeaure Cell
Population e e e e e e e e e e e e e e e e a e e 10

Determination of Growth Phaee Shaving Maximal
Lactate Dehydragcnaee Activity e e e e e e e e e 11

Preparation of Crude Lactate Dchydrosenase e e l e 12
Acetone Powder. e e e e e e e e e e e e e e e 13
Soniontion. e e e e e a e e e e e e e e e e e 13

Also: Method. Employed for the Crude L93
3132303 Preparation. e e e e e e e e e e e e e e 1“

NAD Reduction e e e e e e e e e e e e e e e e in
NET Reduction e e e e e e e e e e e e e e e e 15

Calculation of Lactate Dehydrogenaee Activation

Energy e e e e e e e e e e e e e e e e e e e e e 1,

Separation of Lactate Dehydrogenaee Ieozyaee by
Aorylamide 6.1 ElectrOphoreeie e a e e e e e e e 15

ill

TABLE OF CCETENTS (continued)

w:
.4233
19

CA

Densitometcr Studies e e e e e e e e e e e e e e e

RESULTS e e e e e e e e e e e e eve e e e e e e e e e e 17

Quantitative Kcasnremcnta for Two Strainc of
fib aureue. e e e e e e e e e e e e e e e e e e e 17

Cell Enumeration by the Plate Count
Technique e e e e~e e e e e e e e~e e e e e 17

Total Cell flitrOgan and Cell Phosphorus l7

Absorbancy Spectrum for Lara Nitro—Blue
TOBIBZOIIQE (HBT)e e e e e eve e e e e e e e e e 22

Correlation of Grovth of fit aureue and Lactate
Dchydrogenaao Activity e e e e e e~e e e e e e e 22

Assay of Lactate Dehydrogcnaee Activity for Five
StalnIOfégurmlleeeeeeeeeeeeee 22

MlthOd II HAD Reduction. e e e e e e e e e e 22
Method III Reduction Of flBTe e e e e e e e e 22

Determination of Activation Energy and the
Cptimal Temperature for LDH Activity 0 e e . e e 23

Comparison of the LDH Ieczyme Pattern Among Fifteen
Straine ct‘gc.gg;ggg e e e e e e e e e e e e e e 23

DISCUSSIOHe e e e e e e e e e e e e e e e e etc 0 e e e 37
SUMMARY e e e e e e e e e e e e e e e e e e e e e ee e “h

urmmacman...........‘...“My... 1:6

iv

Table
1.

2e

3.

LIST OF TABLES

Pam.
comparieon of LDH activity of five etraine of
g. aux-cue by measuring the rate of NAD reduction
of com-II i‘ree extmcte prepared by conicationo e e 25

Com ariecn of LDH activity or five ctraine of s.
gfireuc by manning the rate of BAD reduction of
ca Iii-cc extracte prepared by the acetone powder
technique e e e e e e e e e e e e e e e e e e e e

 

Comparison of the number of LB?! ieozyaee from
cell-free extracts (prepared by conication) of

15 atraine or s. aurcua after acrylamide gel
electrophoreeie e53 Eeneitoneter ecanninge. e e e 36

LIST OF FIGURES
Figure Page

1. Comparison of colony count perwnl of
Staphylococcus aurgye (cultures of ctraine 6 and
3A in Brain Heart'ln?ueion) and Optical deneity
measured at 620 an. e e e e e e e e e e e e e e e 13

2. concariaon of colony count perwnl of
‘ Staphylococcus aureue (Strains 6 and 3A) and
Optical density measured at 620 an of oelle
harvested from Brain Heart Infusion and
reauapended in distilled water after washing‘
four tlmOBe e e e e e e e e e e e e e e e e e e e 19

3. Comparieon of colony count of gpachzlgccccgg
sarong (atroine 6 and 3A) and milligrams of cell

rcgeno e e e e e e e e e e e e e e e e e e e e 20

h. Comparison of colony count of gtanh lccgggug
ourcug (straine 6 and 3A) and milligrams of‘cell
phosyhorue. e e e e e e e e e e e e e e e e e e e 21

5. The absorption apcctrun or the indicator dye.
para.nitro-blue tCSIBZOIIQfl e e e e e e e e e e e 22

6. Lactate dehydrogenaee activity per fixed number
of colic in relation to growth phase or
ggaghylcccccus aurcuc (strain 6) cultured in
Brain"Heart Infusion under nicrcaerophilic
conditions fit 37 Ce e e e e e e e e e e e e e e e 23

7. Comparison of lactate dehydrosenaee activity
(using cell-tree extracts prepared by eonication)
or five etraine or §taphylococcue aureua by
measuring the rate of reduction of nitro-blue
tetrazoliun e e e e e e e e e e e e e e e e e e a 27

8. Arrheniue plot for lactate dehydrcgenaee activity
usingetrainefllandoorapmoeoceoo 29

9. Densitometer ecanninge of LDH iaozymee of
‘g. gyrcug (Group I or the International-Blair
aeriJET‘atter separation by acrylamide gel
electrochoreeie.and detection by war. e e e e e e 31

10. Denaitometer acanninge ct Lnfi ieozymoa or
g. aggggg (Group II of the International Blair
caries) after eeparation by acrylamide gel
electrcphcreaie and detection by NET. e e e . . . 32

vi

LIST OF FIGURES (continued)

Page

ll. Deneitometer 08811111113! of LDH ieozymee of
§e aurcue (Group III of the International-Blair
series) after separation by acrylanide gel
electrophoreaie and detection by NET e e e e e e e 33

12. Densitoncter coming: of LDH leozymee of
§. anrcue (Group IV and Mine. of the International-
Blair acriee) after separation by acrylamide gel
electrophorecie and detection by NET e e e e e e e 35

711

INTRODUCTION

Heat etudiee involving the function or enryaee in

W; m are concerned with thoee mien are
produced extracellular-lye Extensive work on extracellular

eneyaee or g. mg each ae coagulaee. lipaee. and pace-
phataae hae overemadoeed reeearch concerning the intraceln-
lular eczyaee.

Currently. lactate dehydrogenaee (ma) appeare to «mend
each attention: homer. reporte in thie area are largely
canned to clinical etndiee on tie-lee or the higher nit-ale.
including an. The concept of aaltiple aolecular tone of
enayeee (imynee) hae extended our enderetendina or theee
biologically active aeoroaolecalee and it ie euggeeted that
tbie action be applied to the etudy of bacterial ”annuity
and elaeaiticatioa.

lather and milieu (1961) deaonetrated electrophoretie
differencee in the ieceyae patterne or LOB in nor-eel and
dieeeeed tieazee of liver. heart. and earn- anon; the himer
.iaale. If electmhoretie technique for eeperation or
ieeeyaee are extended to certain wees-1.1 enryaee and air-
terencee in iecayae pattern- are found among certain etnine
at g. m a non and potentially valuable criterion for
elaenrieation eoald boom available. hecent work by
W and vans (1966) hae anon-tram m Ialie
dehydrosuaeee in W gall. deeignated a and a. the
latter being induced under aerobic conditiene men tor-inal

l

2
reapinticn ie the ecuroe or energy and the tricarbcxylie
acid. pawn: eervee in a cyclic annner. Xaplan and Cictti
(1961) denonetrated that aalic dehydrOgenaeee differ cam
eertain bacillue meciee. and they etate that individual
can variaticne aey bee-em in phylogenetic and. taxoncaie
eudiee. mie approach ie apported by the vcrk ct haunt
ad mlhaher (1965) in Ihieh repreeentativee e: 30 epeciee
of tits were claeaitied on the baeie ct ehether they poeeeeeed
l. 2. 3. or 5 aajcr ieozynee ct ma.

Obeerved variatiene in ieeeyae patterne cams pathogenic
and nun-pathome lay alee provide edae ineizht into the
inherent virulence et theee ermine. lvler (1965) ha
axon that ccegcleee peeitive etraine er etaphyleceeci have
greater m eetivity tin “patheguie etraine end xedaia.
et el. (1966) have noted greater eeccinie dehydroaaaee
aetivity m m pathOgenie etraiae. “a. thoee aeeeciated
aeet oft. vith eevere eepeie.

He therefore deter-med 1m activity and ma ieoayae
patterne tron crude extraate ei‘ certain phase-prepasating
attaine et g. m. vith the intention ci‘ correlating ma
iaeeyae petteine nth phase steeping. lie next “slit a
eerreependuoe to eoae ether unique feature that theee
etreiae poeeeeeed eepecially thoee ncet often connected vith
teetere contributing te the erganiae'e pamogeaicity. each
ae cocaine and penicillinue production (solo-on and can
cleaente. 3.963%

HISTORICAL REVIEW
Milli”! H021!

Early ctndicc of biological oxidation er dchydrosmatien
were per-forced by mcabers (1916) on rained acccle. Hie
aperiacntc. capleyina a epecially decisncd tube for anaero-
biceic, moved that intracellular acterial tron living cellc
eac capable of oxidising certain organic acidc in the chance
of 0118.1: eccc neonate to tolerate. hie mm tube
techaiece beccae the general acthcd for the ctcdy at dehy-
drogenation eiace it penittcd macrobiccic under weenie
auditiue of tine. White. and pfic

meetel .6 whethe- (192$) extended thie procedure te
aicroerscaiuc and deacnetrated that a rented and aerated
cccpeccicc of 3. 39;; could reduce aethylene blue vita varied
ntetratce alch ac lactate. Bach (1935) tectee 66 couponadc
ac hydrogen centre for ctaphylececcnc ad upleycd aethyine
hlae ac the {incl hydra. acceptor: econ; the .bctratec
teated. the coat active eere lactate. glance. at aeanoec.

Beth Fleieeh ad scent-om (192M imam“ the
mechani- of the dehydroscnaec reaction and head that
cyanide added te aneclc ticccc atoppcd the oxygen uptake bat
did act affect the rate ct aethylue blue redaction. Since
there eae ac etoichicaetrie rclcticaaxip tattle. the cynidc
and aethylue blae. the dye did not act by containing chea-
ioally with the cyanide. ‘i‘hie tact indicated that the dehy-
dresenaec qetca vac ecparate free that of the cytochroaec

and their mdccceo 3 '

t
Inproved Techniqnee to Aceay Lactate Dchydrosmaee

Lactate daydrogmaec (ma) activity nay be aeeeired by
lane-etric. epectrophotcnctrio. and coloriaetrio technique
(nachlae. et al- l960). rec epeotrophotoactric acccye have
been coed tor LDH preparatione. one which aeacnrcc the ’
appearance or HADH at 3M) cc during one usyaatic oxidation
ct lactate (Hcilandc. 1955). and another IIhioh ace-rec the
“mm of Him at 350 an daring the cnxyaic reduction ‘
er pyrnvate (Plea-con, et al.‘ 1956). the ecloriaetric
teemiqac (nachlac. et al- l9“) depende en the color chase .
tench ecccrc during reduction of certain oxidation-reduction
dyee aich ae the tetrazoliuc um. .

the uploynent ct acthylne the ea a final hydrogen
acceptor rcqciree anaerobic conditione bccucc it ic eccily
reezidiaed in elrc Forbee and cover (1951) am aeed
tetraaolim chloride cc the final hydrcg. acceptor to
replace aethyleae blue. Polleein; thic work. ccverel other
derivativec of tetracoliec site each vith differ-it cidc
group- can tected and town can core aiitablec manna.
eclte offered certain advantasec over acthylene bloc. aich
ae amplification of pnednre. nun-reoridatioe in air. and
creator eucdtivity to redaction. i‘hic incrccecd ecacitivity
appeared depuduit on certain electroacgative creepc attached
either te the 3-2 phuyl or he) phenyl rinse (ncchlae. et el-
l9“). m ample. iode-phcnyl tetraaolica chloride (m!)
and para nitro-hlne tetrczclina chloride (HM) were confidently

5
acre ccncitive hictcchenioal indicatcrc or aiccinic dehydro-

cuaec activity thm thedr enubetituted enclosec

the cfliciucy of hydra“ tractor daring in vitro
dehydrcgcncec etodicc hac been invcctisctedc term (1935)
toand cpccitic oe-factere (Wynn ecch ac nicotinnide
adenine dimleotidc. m) . indiepaieable couponcnt or we
dehydrogencce m. Dickene end nonrein (1938) met
daecnctrcted the value or phenarinc dcrivctivcc ac electron
earriere in a variety ct ddiydrcguaec queue. pneceeicc
come-circa vac found to be c. ccec effective electron
eerrier and to currently he cut eidely sued to: theee
wet.“

iroann end Phcrnakic (1962) ”re-ind that cynido We
theeelorchenseduring reductienetaneotetraeoli- ealtc
It aey prevat conpetitiea by the cytochroae oridaee eyctec
forthchydregei‘ionereleaeed inthe reaction. or itacyact
cc an immediate hydrogen acceptor and in turn tracetcr
the: to the tetraxoliee alto

the reportc e! invcctigctore vary on no optical pa and
mm to: a tetraeelina eeio to accept eieeticce during
the ccnvcrcicn of lactate to pyrnvatec uaohlaec ct cl. (1961)
etadied the ma reactiuc at 37 a no p! 7.0. thence Allen
(1961) H9101“ roee taperatcrc (ea. 25 c) and pH 7c!» ihic
reactiuhcd tebeoarricd eetintheearh cincelight
ecconpeecc the phenccine cetheeclfctcc

conditionc for optical encyce production alcc vary over
a wide range. Renoir-cute for acre growth or ctcphyleccccnc

s
are not cc ctringcnt cc thoce tor acrincl production of cone
ma canine and Moe (1962) moved that L38 in
extractc tron etaphylocoeoac grove at ca Vet no abcet t-
tiaee creator in preparation tron anaerobically cultivated
colic over the aerobic onecc

hethodc of wracting Crude LIX-I

Vcdcnra. ct ale (1965) have aplcycd coveral cethedc
of extracting intracellalar pretence tron bacterial cello
uoh ac acetone-peed». tolanc. euic treat-at. fleecing
and thaeins. cad art-tinge vein: theee acmede to obtain

out-m mum m- we mills: W
mm and W ism m. m mt ~1-
troataent ad manna fielded core protein in the cell-tree .
mnetetiieldid medium” however. melon-te-
'peeeer aothod tor all 3 ermine yielded proteaecc vith
the MM epeolfie activitye m the one! been met!
detaindicatod that eeaie treatentlad crindieeeorcthe
pretend eethede or did-tenet“: cello for he pnrpocc
or obtaining the aariael activity an intracellular
Pm ”In”.

leceyace

moneyace. ”hum. arokne—teorietiaealtiple
eclecalar tone. icee ee 'iucyaec'e for eruple. i‘ive
ieoeyaceetmarefeeadieteet dieletalaceclecad
eppclla and Meet (1962) have .hioeted thie ally-e to

7
various phyciochcmiccl cnclycicc “rhcy treated one icozyae
e! oryetalline rm tron hoof heart with 5 H mince-Hal
or with l! h area to dieeeciate vie ma aolecnle into four
inactive cnbnnite or equal molecular vcightc ihe tour nib-
Inite of beef m3 appeared to be very ciailar polypeptide»
ac analyzed ty 3 and c teraincl cainc and peptide patternc
(Winger-printing”) alter trypcia direction. but there cat-
anitccocld be ccparctcd intctvo clccceco<cnonn the
bane of charge. Aceorting thecc too kinda of ccbnnitc into
all poccihle greapc ct roar could theoretically yield five ‘
different aolecnlar {one of ma. all dietinmieied by charge
differonoo‘ec

nectrophorecic. calt fractionatica. cltracentrircgction.
Ibetrate Specificity. may. Weill etry. and
reaction kinetice repree-it the principal acthodc of coparatiaa
nltiple aoleccler force of aayacc (Harhcrt. 1965). Since
he electrophorctic acthcd sivoe good reultc aith a ainiaaa
"otter-t. it ieaect ccaaoaly eaploycdc PM etalc
£1960}. for ecaaple. otcerved at leaet tee urological” and
electrophoreticelly dietinct lactate dehydrogenaccc prccmt
iarabbit aileenand ecruc theyalcoi‘oehd thatuiclogoac
rabbit cal haaaa than have ciailar but not idaitical LDH
i-IIIO totternee Appella end Mort (1962) apieyed
electrophoretio acuiodc to detect chance in the 1m iccryne
pattern of pig heart daring development. Kaplen and Clotti
(1961) have boa able to dcaonatrate that neeborn rat heart
“Cull"! coma 1m rotten eliditly different from that

8

found in the adult organ and confirmed thie difference by
Manolosical etudieee

The phenomenon of clectrophcrotic reparation or iecxyace
hac proved to he of clinical value cince the icccync pattern
of certain cnzyaec ci‘tai are related to dicturbanccc or the
aetabolic oyctcne Lather and Skillcn (1961) have ehoen that
the ma icozyne pattern or the five handc tron human ecrua
departe tron the normal pattern after cardiac infarction and
iaundicec In a later publication. Lather (1965). pcctulatcd
that thie electrophorotic procedure will alco provide a
aeoi‘ul tool in differential diagnocic of acute pmcroatitic
and cancer. Fowler and Poarcon (1961:) have ctudied the
diamcctic and promo-tic aimiticanec of ecrua enxyacc
(eczc mm for auccclar dyctrcphy and other neurologic
dieeacecc In the cace of auccular dyatrOphy. coma enzyacc
(ecsc LDH) have been aeedi (l) to detect clinically healthy
heterozygouc carrierc or the dyctrophie may (2) to dicccvcr
preclinical caccc of nucoulcr dyctrophyi and (3) to aceict
in evaluating the natural couree or the diceacec Hill and
Levi (1951:) have reported com 11:13 elevaticne in neoplastic
diecacec Comparing noml adult ticeuc and naliaiant tumorc.
Latner and’acillon (1961) round a ehitt to the clover
aigrctins zonec in the ma icoryacc tron the diceaced ticaicec

In other clinical applieaticnc. Kelly and Groirt (1961)
have demonstrated variatione in LDH activity during growth of
viruc in the embrymting chicken egg. The activiticc or the
fluidc tron infected eggc at the 72nd hour. or later, acre

9

approximately 19 times higher than thcee tron non-infected
eggcc Based on there data. they developed a qualitative
teat to detect infection by influenza viruec

Other applicaticnc or immune variatione have how
etudiedc Kuplan and Cictti (1961) have mom that the oxalic
dehydrOgcnneee in bacteria display concidornble diversiticne-
tion in their activity with certain cc-tactcrc ac NAD and
MADP. 8115110qu ieozynce or closely related apecicc generally
pocceea quite similar propertieec In contract. the cello
dehydrcgcnacec of the hecilluc group no a whole had remark-
able dittorenccc in their activitye 'I‘hccc properticc might
eventually provide valuable insight into phylogenetic and
taxonomic studiocc Shem (i965) hac etctcd that electrophore—
tic ctudioc on icocynec may have application in geneticc and
in the analytic of enzyme relationaiipc. and predicted that
the accunulnting date on electrophoretic varictionc will
becoae significant in understanding the molecular bneie for

variation.

EATER IALB AND HE‘I‘HCD 3
Organi an e

Fifteen phage prepagating etraine of W
mg belonging to the International-Blair eeriee (Blair
and Carr. 1960) were eelected for theee etudiee. Group 1-—
P. e. 29e52A/‘79. 80. 52. Group nap. a. 3A. 33. 3C. 55.
Group Illa-P. 3. 6o ‘37. 5’3. 770 Group Iva-Po 8e #21). Group
hiecellaneoueuP. 8. 81. 83MB.

Medium and cultivation

stock culturee were grown on Nutrient Agar (Dirco)
elante and refrigerated at h (3. Gene for assay md prepara-
tion of LDH were cultivated in Bill under nioroaerOphilio
cmditione since preliminary etudiee had shown cello grown
under reduced oxygen teneion had eignitieantly greater mu
activity. Thie obeervation ie eupported by Colline and
Laeoellee (1962) who found greater LDH activity in etaphylococci
groin anaerobically. Hicroaerophilie conditione were obtained
by filling Earlermeyer flame with the medium to the neck and
cloeing it with a plastic stopper. 'lheoe conditione would
exiet during the lag phase and exponential growth) however,
an anaerobic state could be armed when the etationary phase

ie reached.
Quantitative Measuremente of Cell Populatione

Straine 6 and 3A 1were cultivated in Brain Heart Inmeicn
lo

11
(3111) under aicroaerophilie conditione at 37 c and eanplee
were removed at inter-vale of one hour. The optical denaity
or each ample wae neamred in 13 r 100 n tubee upon moval
and the viable cell number determined by the plate comt
aethod. For a eecond quantitative aeaeument. 500 al
volume or celle were cultivated under nicroaerophilic condi-
tione and removed at 8 houre by centrifugation at 10,000 r G
for 15 ninutee. The cello were waited 1! timer and renepended
in distilled water. Optical deneity and viable cell number
were correlated by raking plate eounte of appropriate cell
dilution»

The nitrogen and pheephorae content or etraine 6 and 3a
were analyzed according to the aethode mggeeted by Unbreit
(1957b more were modificatione of the nierokjeldehl and
neeelerization techniquee for nitrogen. and the Filte-
aat‘oarow method for total phoephorue.

Determination of Growth Phaee mowing harinal LDH Activity

cultures of g. mg in five hundred al or an: eere
incubated at 3? c under aicroaerophilic condition“ Seaplee
were removed at irregular intervale during a 32 hour period
and refrigerated. at a convnient tine after thie period.
all unplee were adjueted to an optical dueity of 0.222 at
620 an with freeh BHI. one-a1 portioae or each adiueted
enple were incubated 30 ninutee at 37 c with 3 ll of an ma
reactim nixture prepared according to the aethod of Allen
(1961). The final concentratione of reactante in thie LDH

12
preparation were: 0.025 M trie (hydroxyaethyl) minonethane
buffer pH 7“; 0.1 h lactic acid: 0.001 M nicotinanide adenine
dinuclectide (HAD); 0.5 lug/bl nitrc-blue tetrazolitm (1131‘):
0.005 M potassium cyanide; and 0.02 ng/nl phenazine metnomlfate
(PMS). Although RAD wae included in thie m3 reaction mixture,
ite ouieeion may be Justified for thie experiment since whole
oelle were uplcyed. After incubation, 3 ul of chlorofom
were added to each mph and the contente nixed thoroughly.
The mplee were allowed to etand at rote temperature lo
ainutee. agitated once acre. and after eeparation of the two
layere, the top aqueoue layer wae treneferred to 504:1 plaetie
centrifuge tubee by a Paeteur pipette. ihie portion no then
centrifuged at 20,000 r a in a Screen angle centrifuge (aodel
33-1) for 10 ninutu to remove the ineoluble debrie. After
centrifugation. the mpermtant fluid wae trand’erred to teat
tubee and 3 a]. of water added to each before ueamring abeorb-
eney at 625 an in 13 r 100 an epeetrophotoaeter tuhee. A
blank wee prepared in a ciailar neuter except that 1 al of
fret: BIII replaced 1 ml of cell euepeneion.

Preparatim of Crude LDR

Both the acetone dry powder procedure of Vadehra. et a1.
(1965) and sonic disintegration (ESE 100 watt Ultraeonic
Di eintegrator) were employed to obtain an LDH preparation.

13

Acetgge 293225

For the acetone extraction technique. §. w (strain
6) wae cultivated in 1 liter of m1 at 37 c under micro-
aerophilic conditions for lo hours. after which the calla
were removed by centrifugation at 10.000 x d for l5 ninutee
in a sorvall (model RC—Z) refrigerated centrifuge. The celle
were then washed II tines with ham). portions of distilled
water and romspendcd in ideal of cold distilled water.
Thie mrpensicn was added dropwiee to a 500-nl Earlemeyer
flack containing 200 ml of cold acetone (-20 c). it the
cells were added. the mixture wae ewirled continuouely on a
magnetic stirring apparatus. The treated cell mapcneion wan
next filtered through a Buohner funnel using Ho. 1 Uhatnan
filter paper and the cellular aaee removed to a deeeicator
at 8 c for at least 21: hours. The remlting powder wae then
ground in a mortar using gloat beade (125-177 u). no al
cold 0.05 M trie-HCl buffer (pH 7.5) were added and the
viecoue mopeneion etirred thoroughly at 15 minute intervale
for one hour. The debrie wae moved by centrifucation at
50,000 r G for to minutes at h c. The expernatant fluid
containing the crude Lou extract wae etored at b c for future

W
Celle were prepared for eonication by cultivating a
liter of g. cums in ml for eight houre. me celle were

centrifuged at 10,000 r G for 15 ainutee. washed four tiaee

10
with 00 m1 portion. of dietilled water and rem-pended in
two ml 0.05 h trio-Hcl buffer (pH 7.5). The tube containing
this mopencion wee immersed in on ice-eelt mixture and the
cello subjected to conic disintegration (i.e. 30 occonde
eonication was alternated with 30 eeoonde or cooling for a
total period of 20 I111). The conic dieintegration method
required 10 minotee at 7-9 aicrone to obtain 80 to 90% disrup-
tion or the cells. The debrie wee finally moved by
centrifugation at 20.000 x 0 for 30 minutee and the supernatant
fluid stored at h C for future LEE aooayee

Lucy Hethode mplcyed for the ma Extract Preparation

no W __t

The tiret procedure employed for assaying LDH activity
wae e elimtly modified method of Hielmde described in
agthgdg H! W (Colowick and chlan. 1955). The rate
of RAD reduction woe neamred in B-el curettee at 30 c and
300 an in a Becman DU epectrOphotometer (model 2000). Each
test woe carried out no follower to the curette there wae
added 1.87 mi trio-Hal buffer (0.05 H. pH 7.5). 0.21 :1 KC?!
(0.06 h, pH 7.5). 0.15 ml HAD (0.02 H). and 0.02 I1 enzyme
preparation (2 mg protein/ml). The nirtnre wae allowed to
equilibrate before adding 0.75 :1 lactate (0.5 H 01. he form).
The optical density wee neeeured at 30 second intervale for
3 minutes.

15

E? reduction

a.“

 

The second procedure for measuring activity depended
upon the color formation of an cridetiOn-reéuction dye,
para nitronblue tetrccoliun. To a cuvette there was added
0.20 ml of the LEI! preparation (1.25 mg: protein per ml) end
3 ml of the LIE reaction mixture (held at 37 C). The contente
were nixed rapidly by drawing and expelling the total mixture
3 times. The optical dentity at 625 mu was immediately let
to 0 and measured at 60 occond intervale in the Bockman DU
spectrOphotoneter to determine the rate for the reduction of

the para nitro—blue tetrazolium.
calculation of LDH Activation Exam

The activation energy (Es) was determined by the Eda
reduction method (previously described) which was carried
out at 20, 25, 30. 35. 00, 105, and 50 c. The reciprocal of
absolute temperature woe plotted against Ln. LDH activity
and Ea‘wet calculated from the elepe of the line.

separation of the ma Ieozynee by Aerylaaide
Gel Electmphorocie

The electrophoretic method'ueed wae that deecribed by
Davie (1960). To each gel there woe added 0.02 ml of 12.5 mg
protein/ml of the LOB preparation. 'After polymerization of
the gels. electrOphoreaie was carried out at 5 c to minimize
poeeible dcnaturation of the enzyme by ohmic heating and a

y 16
a current of 0 ma was ueed per tube. When the tracer dye
1 al 0.0011 (bran phenol blue) wae within S m of the bottom
of the tube (ca. 1 hour), the current waa turned off and the
gele were placed in ice-cold water until they could be removed
conveniently from the tubes. The gele were then innereed in
the ma reaction aixture (Allen, 1961:) and incubated at room
temperature in the dark for 30 ainutee. After development
of the bands. the gels were moved and placed in 7% acetic
acid to prevent further diffusion of the band a.

Deneitoueter Studies

The ma patterne of the 15 etraine were analyzed by a
aicrodcnoitcneter (Canalco, aodel P) equipped with a record-
ins chart. The meter wee eat to full ecale with an optical
denaity range of 2. The gain wae act to 5 and a chart speed
of 3 iuchee per uinute wee employed for the recorder.

RESULTS

Quantitative Meaeurenente for Two Straine of g. aureug

Qell Enumereti 93 b: the Plate count zechnigue
A linear relaticnmip between cell papulation and

optical deceit: or 2 different etreine of g. 3% ie
reported in Piguree l and 2. In Fig.1 aeemrenente were
nade on whole cultures. and in Figure 2 the remlte tor
weaned celle that were reeuepended in dietilled water are
flown. Optical deneit: incremente of 0.1 were obtained with
1.23 x 108 waned cella/nl whereaa 0.69 r 108 unwaehed celle
were required to aleo give 0.1 optical deneity increment»
In both caeee the neaeuremnte reflected the degree or cell
diaper-ion which waa alnoet twice ae much in the caee of the
weaned cello.

0 el tro and el o ho

cell nitrogen and phosphorue (Piguree 3 and N) were
compared to cell deneity eeing waehed preparatione or etraine
6 and 3A. In both caeee a linear relationahip wee obeerved
although the linee did not paea through the origin. Err-ore
umallr inherent within papulation aeaeureaente nay account
for thia alight dimlaceaent. The nitrogen content wae
5.910 r 10.11 nag/cell whereaa the phcephorue yield wae
1.09 x 10"“ wan.

l7

 

18

 

4+ 0/
O STRAIN 6
A STRAIN 3A
3,.
E ,A
x O
S
u: 2 >
do
2
a
Z
r:
8.1»
/A
A0
y
0
00 Of? 0: Ofé

o. D. (620mp)

Comparison of colony count per m1 of
Staphylococcus aureus (cultures of strains
6 and 3A in Brain Heart Infusion) and
Optical density measured at 620 mu.

(CELL NUMBER/Mi.)x 10‘”
u a u o

no

19

o STRAIN 6
e STRAIN 3A

 

L A L

o 0.1 0.2 0.0 0.4 0.5
O.D.(620mu)

Figure 2. Comparison of colony count per ml of
Staphylococcus aureus (strains 6 and 3A)
and OpticaI"d§nsIEy measured at 620 mu
of cells harvested from Brain Heart
Infusion and resuspended in distilled
water after washing four times.

 

CEll NUMBER x 108

00

b.

(a)

 

20

0 STRAIN 6

 

A STRAIN 3A A/
/0/
7A
A
Q,/
/“’
f0
.03 02 .013
mg.CE|.L N

Figure 3.

Comparison of colony count of
Staphllococcus aureus (strains 6 and 3A)
and milligrams of cell nitrogen. The
washed cells were digested in pyrex tubes
with 5N sulfuric acid (containing 150 mg
of COpper selenite per liter) at 180 C for
12 hours and the amount of nitrogen then
determined by nesslerization.

can NUMBER x 103

U!

‘

(J

N

 

21

 

Figure 4.

A
O STRAIN 6
u A STRAIN 3A
L
0
ll
6 C)
‘ o
/ A
* 8
.002 6.004 .006
mg.CELL P

Comparison of colony count of Staphylococcus
aurues (strains 6 and 3A) and milligrams of“
cell phosphorus. The washed cells were
digested in pyrex tubes containing lON
sulfuric acid at 150 C for 1 hour and the
amount of cell phosphorus determined by the
Fiske-Subbarow method.

22
Absorbancy Spectrua tor Para Nitrc-Blus Tetrazoliun (NET)

The absorption spectrum of reduced NET was measured
(fig. 5) using oxidised NET as the blank. Absorbanoy wee
neasured spectmphotomstrically (Banach and Leah. model
Spectronic 20) between 375 an and 725 an at intervals of
25 nu. hazinal absorption occurred in the region or 625 mu.

correlation of Growth of g. pursue and mu Activity

ma activity was neasured in standardised senplu (Pig.
6) taken at irregular intervals from a non-shake culture
(strain 6) during growth. The highest LDH activity per fixed
number of cells (adjusted to optical density 0.222) was
observed to reach a neximun near the end or the exponential
phase. i.e. at 7 hours or at an optical density or 0.75 for
the original culture.

Assay of LDH Activity for Five strains or g. m

nomad 1: W
In the first method LDH activity of 5 strains of g. aureus

(Tables 2 and 3) was measured using the NAB reduction proce-
dure. ma preparations from both soniceted cells and from
the acetone dry powder eethcd were shown to have the following
order of LDH activity based on units activity per cell: 29 >
81 > 6 > 31 > 13213.

hsthod II: Reduction of 382
The second technique used to compare LDH activity

(Fig. 7) unpleyed only the extracts from soniceted cells.

(17

(lb

.0
Ln

$3
n.

OPTICAL DENSITY
p

.0
a)

’ \

. (3.0.0009

 

69%

O

\

O

’0

f
[0/

 

400 600 80'6—
WAVE I.E NGTH (mp)

Figure 5. The absorption spectrum of the indicator dye,
para nitrc-blue tetrazolium.

24

.0 mmowmg mcow

LDH (0.0. or REDUCED Nsreczsmp)

D-OCOO O

e in M

”:3

m

smmmmmmao a no as o msmcH paccm mama
mo omega 29309 c
pdmccc Hmcfip o no Ha\mHHco 0H N me.

.5

MM .0 caswdm

 

maacc ho H0983: woman Hog m «bdpcm cmozcwcacm cc cpmpo
fancier:
0 Nu 0% o.— m. o . 0—0.
.
llAVslllllllllllllllllllllll AV. ”WW
/ 4
. d \ no.
_ \
a o ._.
c >523 :mu \
Q hmz cmunowm
a;
L m.
IIO 0......0 .
4 .0—
m.r

 

 

 

(”mom can} Auswaa 1133

25

 

 

2:3 .84 03.3 :3 a 8.... 3 n3
cam.m on." oem.am use” a n~.e NH 4n
oom.a~ as.“ . coo.mwa ”do” w ou.c HHH c
03.5 ms." 23.8.“ :3 u more .SE .8
85.3 wed Songs 33 u 86 a e~

3.4.8. I 4!: I 4 I I

sedan usKwndeo nacho
3:33 .8326 segue—5 gonna no sense: scene Sauce
Tease“: new 1

 

.souasouece hp ceueueac nauseous
83.32.. e33 .88 on can us 8332... a: co 32 .5
neaaa-ses:ap .eaocc mod no use an» as cede-ea use no
ummuwmummmMMme nonsense hasnouueus :« ceaseauucc. a
8 a 3m no .593. n no suffice :3 as dispensed 2— eased.

26

 

neo.n as.c oc~.ma «How u on.w an awn
nan.n ne.o ooe.on «god a as.» an «n
nue.n ea.o coo.mn "so” u mo.e Ham v
on~.: n«.~ coo.me «and w oo.w .oeaa um
nao.n en.” oo~.nm "dog a no.» a an
Auaommua mmwmwwsc. .: r. 1‘ 4:.
adeo asneo mane
asapauoq cacaoenm hoax-ease. «antenna no sen-ea omega casuam

 

9:5! eel

auapacaa and

Sass—nose acumen success on» an condoned nauseous

   

eat—Hoe evoke Icky on can an .8336: 042 no cash on»
Baeaenauedhc Seen» as cede-ls 35E
sum 0 wean—vane rescues: ca covet—3:3 cuckoo
can he nuance n no hugged m5 no dosage—co ow ages

27

1.0» STRAIN

 

 

 

C) 25? C]
’5: A 81 O
E t 6 A
.n 0 3A
‘3 E] 420
V O
>- A ‘
.—
" e
‘2 0A
u: -
o 0.5 A ‘
5 a ..
ES 23. 4A. ‘. E]
o D A .D
A ‘ .D
e
OA‘ 500
. 56
a . J .
8 10 20
MINUTES

Figure 7. Comparison of lactate dehydr0genase activity
(using cell-free extracts prepared by sonica-
tion) of five strains of Staphylococcus
aureus by measuring the rate of reduction

of para nitro-blue tetrazolium.

23
The rate of reduction of NET was measured with a Beclonan DU
speotmphotometer (Model 2&00). The strains tested gave the
same order of LDH activity as that observed in the NAB
reducticn‘method.

Determination of Activation Energy

t va ion or and the 0 tinal em a ture or LDH Act$zi§z

Two LDH preparations from sonicated cells of strains 6
and 81 were compared for Lbs activity at temperatures ranging
from 20 C to 50 c (Fig. 8). The optimal temperature for LDH
activity occurred at #5 c for both strains.

The Arrhenius plot (Fig. 8) to determine activation
energy shows a discontinuity of the slope above #5 c. This
is apparently due to myms denaturation. Using the equation
slope - 2:;3 the activation energy (3‘) was’caleulated to be
3.260 Keel.

Comparison of the LDH Ieozyme Pattern Among Fifteen
Strains of‘g. 523333

The isozyae pattern for 15 phage prepagatmng strains of
g. m representing groups I. 11. III. IV. and sun. of
the International-Blair series (Blair and Carr. 1960) were
coepared. The number of isozymes obtained for each strain
was as follows: two for strains 29. 52, SZAIB, 3A. 30. 55.
and BSA/B: three for strains 80. 33. 6. R7, 77. and h2D|
and four bands for strains 81 and 5h. Densitometer tracings
tor'thsse strains are shown in Figures‘9, 10. ll. and 12.

lN. ACTIVITY

6.3

6.0

5.7

 

29

 

/6 A STRAIN 8)
o A O STRAIN 6
' O
\A
o\
A
~ A
3] 32 313 32 3d:
1/7 x 104
Figure 8. Arrhenius plot for lactate dehydrogenase activity

using strains 81 and 6 of Staphylococcus aureus.
The rates were measured by NAD reduction at

3&0 mu, using a temperature range from 20 C to
50 C.

30
The bands were designated according to the ratio of their
relative migrations to the tracer dye. brom phenol blue.
This ratio is analogous to the hf value used in paper
chromatography. The overall range of ratios was .50 to .80
and isozymss were arbitrarily divided into 5 components, each
differing by an increment of .06 and designated as follows:
.50 to .56-isozyne 5: .56 to .62-isozyme h; .62 to .68.-
isosyae 33 .68 to .7b-oiaozyme 2) .7t to .80-misery“ l.

The vertical distance of the densitometer tracings from
the recording chart was reduced by b and the horizontal
length was left unaltered. Zero on the abscissa represents
the position of the slowest moving band of that particular
ample.

Figure 9 represents densitometer tracing of mu ieozynes
for group I of the International-Blair series. Strains 29 and
80 both have isozyaes l and 2. whereas isozyme 3 was noted
in both 80 and 52A/79. Isozyue h was observed in both 52 and
52”?” however, strain 52 was mown also to possess isosyae 5.

Figure 10 represents densitometer tracings of LDH
isosyees from group II. All 1: strains have isosyaes 2 and
3. whereas only 33 possesses isozyme 1.

Figure ll represents densitometsr tracings for group III.
strains 6. 5b. and possibly 77 have ieozyaee l and 1L. whereas
all 0 strains have an isozyms 3 in common. Only strain 51» and

t7 possess an iacryms 5.

ABSORBANCY

 

31

 

 

 

 

 

 

 

 

Figure 9.

PS 29 PS 80
2
2
‘ 1
3
3
PS 52 W PS 52A/79
4.
4‘0 60 0 210 4‘0 6‘0
MILLIMETERS

Densitometer scannings of LDH isczymes of
Staphylococcus aureus (Group I of the
International-Blair series) after separation
by acrylamide gel electrOphoresis and

detection by NBT.

ABSORBANCY

 

 

 

 

 

 

 

 

 

 

 

32 g
I
I 3 PS 3A PS 38
. 3
2
\ 1
PS 3: 3 PS 55
3 A
2 2
20 4b 67) o {o if 60
MILLIMETERS
Figure 10. Densitometer scannings of LDH isozymes of

Staphylococcus aureus (Group II of the
International-Blair series) after separation

by acrylamide gel electrOphcresis and detection

ABSORBANCY

33

 

 

 

 

 

4.
1 ’\
3 .
PS 6 PS 54
3
5 1
4 /\
3
I
2
I) PS 47 PS 77
2
1

 

 

 

‘

 

 

 

afiéE22-gf”//\J . k
0 20 410 60 0 2‘0 40 $0
'MILLIMETERS

Figure 11. Densitometer scannings of LDH isozymes of
Staphylococcus aureus (Group III of the
International-Blair'series) after separation

by acrylamide gel electrcphoresis and detection

3“

Figure 12 represent. denutoneter tracing: for group Iv
and the miscellaneous group. Strain 1:21) 1- the only
representative or group IV and 1t hee leozymee 2. 3. end 5.
Strains 83MB and 81. representing the niecellaneoue group.
both possess leozymee 3 and h, whereae 81 else he: isozyme-
1 and 2e

Table 3 represente a composite of LDH leozymee for an
15 strains. Ieozme 3 was most often encountered among the
etrelna tested, followed in decreeeing frequency by isozymes
2. 1, h, and 5e

ABSORBANCY

 

35

 

 

 

 

 

 

 

 

 

ii
P511201
2
[iii
3 /
PS 83A/B PS 81
3 2
.4
4.
2‘0 ‘0 640 o 270 410 6‘0
MILLIMETERS
Figure 12. Densitometer scannings of LDH isozymes of

Staphylococcus aureus (Group IV and Misc.
of the International—Blair series) after

separation by acrylamide gel electrOphoresis
and detection by NBT.‘

36

Table 3. Comparison of the number of use isozymes from
cell-tree extracts (prepared by eonication) of
15 strains of finphylonoccug aureus after
acrylamide gel electrcphoreeie ana densitometer

 

 

 

scannings.
strain Tghageu I enzyme _
Group m h 5

29 I + + W
52 I + 4-
52M? I I + 4»
8311/3 m so. + 4-
3A II 4- +
38 II + e-
55 n + +
BB II + + +
80 I + + +
77 III 4- + +

6 III 4- + +
“7 ' III + + +
#21) IV 4- +
81 Misc. + + + +
515 III + + + +

'bxscussxcu

our preliminary studies concerning LEE in §. m have
indicated that the production of thie enzyme ie enhanced by
cultivating the etaphylococci under aicroaerophilic condi-
tione. Thie finding in supported by colline and Laecellee
(1962) who found that mapeneione of anaerobically cultivated
§. m in nutrient broth gave approximately 10 times the
ma activity ae did aerobically grown cello. certain media
aleo appear to favor production of me enzyme» e.g.. we
found greater LDH activity in preparatione cultivated in mil
over those grown in trypticaee ac: broth.

Viable colony oounte and optical density were compared
aeing named and unnamed celle. The apparent cell count
for nested celle nae alaoet twice ae high ae that for unnamed
eelle at equivalent optical deneitiee. thue indicating a
higher degree of clump diaper-ion in the fomer case.
staphylococcal unclunped uepenaione are eepecially difficult
to prepare (Kinda and Peter-on. 1963) becauee of their nuccue
nature and Elek (1959) etetee that a, warm in a
eyathetic amino-acid medium producee large quantitiee of
aucoid material. we cultivated etaphylococci in a medium
(ml) containing a high amount of protein material and a
large quantity of nucoid material aae produced. heduced oxygen
tension may also increaee the aucoid by-prodeucte. Russell
(1950) etatee that if the degree of aeration ie reduced,
aicroorganime will begin to excrete a variety of energy-rich

37

38

compounds instead of the fully-oxidized carbon dioxide. The
excreted products of respiration by heterotmphic organic"
growing under limiting conditions consists of two distinct
types of conpound as complex carbohydrate gums typically
produced by many groups of bacteria under aerobic conditions,
and a variety of simple soluble compound such as certain
aliphatic acids. aldehydes. ketones. alcohols. and simple
dibasio and hydroxy-soide.

Binds and Peterson (1963) found that the addition of
0.5% Tween 80 greatly enhanced the dispersion of staphylococcal
cells and enabled them to obtain reproducible viable counts.
viewing with distilled water also enhanced dispersion of
the cells by reaoving much of this nucoid material.

Linear relationmips of both nitrogen and phosphorus to
cell number sore observed; however. in both instances the
line did not pass through the origin. Errors usually inherent
in pepulaticn neaairenents (e.g. dead cells) may account for
this displacement. The nitrogen content was calculated to
be 5.911 r 10"“ ng per cell shereas the phosphorus yield was
1.09 r 10"n mg per cell. Bennett and Williams (1957) calcula-
ted nitrogen and phosphorus values for hicrococgug W
var. 9.9.5933 cultivated on beef extract agar (solid medium)
and obtained 0.50 x 10"“ mg nitrogen per cell and 0.20 x 10"11
ng phosphorus per cell. The differences note our values may
be due in part to the growth medium and cathode employed for
these studies. Secondly. the tins of harvesting the cells
may partially account for these variations since certain
organic materials containing phosphorus or nitrogen nay

39
accmulate over a longer cultivation time.

Since little infomtion was available on bacterial LDH.
it see necessary to adapt and modify certain procedures used
in histochmical studies (Wises, et al. 1962). Tetrazoliua
salts are comonly used in the clinical studies of LDH
isozymes (Lather and Skillen. 1961) because of their high
sensitivity to reduction and their resistance to reoxidation.
There are currently available amorous indicator dyes to
detect reducing mamas. but 531' and Zap-iodOphenyl-B-p-
nitrOphenyl-S-phmyl tetrazoliun chloride (INT) appear to be
the most sensitive tetrazoliun indicators (Hachlas, et al.
1960) because of certain electronegative grape on the 14-2
or No3 phenyl rings. of ten indicator dyes tested by us. NET
and INT gave the strongest response to staphylococcal ma.
since N31? was routinely used by us under modified conditions
from those found in the literature. it was necessary to run
an absorbmxcy spectrum for this tetrasoliun salt. haxinal
absorbency for the purple fomazan produced from N3]: upon
reduction was observed in the region of 625 nu.

Maximal LDK activity by g. m occurred at the end
of exponential growth. This was 7 hours (optical density
0.75) for strain 6 cultivated in BE]: as a non-make culture
and using a 10% inoculum. Hershey and Bronfenbrenner (1938)
have sheen that the maximal rate of oxygen uptake per cell
for g. 33;; occurs at the beginning of exponential growth.
since our organises were grown under nicroaerOphilic conditions.
increasing If?! activity may be expected to occur during the

no
decline in oxygen tension by the end of the log phase. The
apparent decrease in L133 activity per cell during the stationary
phase would also be expected since there is an increasing num-
ber of dying cells in the medium at this phase. A130 1311
shich had leaked into the aediuu during autolyuis or old cells
would have been disproportionately diluted during the prepare-
tion of the samples.

Analytic or cell free extracts required a suitable method I
or disrupting the cells to release the intracellular was
however. this presented the problem of possible denaturation ~
of the ammo. Veda-ire. at al. (1965) reported that of several I {
cell dimzpting methods an acetone extraction technique ave
the least denaturation of intracellular bacterial proteases
even though less total protein was. recovered. Althngh the
acetone extraction procedure gave us satisfactory rewlte. we
found higher apeciflc m3 activity and less apparent denatmra-
tlon using a sonication procedure.

The determination of NA!) reduction rates for sonicated
and acetone powder preparations clearly demonstrated the high
activity of strains 81. 6. and 29 as compared to strains 3A
and 13217. The mom relative order of LDH activity for these
otraina was confirmed by the RR? colorimetric method. hedzie.
et a1. (1966) has stated that strains isolated from patients
with staphylococcal infections demonstrated higher activity
of certain commas (mg. mccinic dehydrogenaso) than strains
isolated from healthy carriers. Ivlcr (1965) has found that
the enzyme levels of IJJH in coagulase positive strains were

oonoi.do:m’:-._.~1y higher than in coagulaoc negative strains: smd

hi
Baird-Parker (1965) reported thet etrein 81 end 80 ere
frequently eeeocieted with eevere eepeie in hoepitele end
that etrnin 6 ie often inpliceted in food poieoning one".
coegnleee and penicillineee production contribute to the
pethogenicity of the etephylocccci end etrcine 29. 81. end
6 ere extrunely high producer-e of either or both or theee
tie enzymes .( Solomon and San Clmente. 1963). our dete n
man for strain 6 indicete thet the mine). LDH ectivity ie
etteincd precieely et the end or log growth; therefore. in
order to ccnpare urinal ectivity or eech etrein. neeedreuente
were uniformly made from emplee collected et the end or leg
pheee for each etrein.

. The optimal tapereture for ma eetivity or cell-tree
extrecte (etrnine 6 end 81) tree #5 c and the ectiretion
energy celculeted tron en Arrhenine plot wee 3.260 Keel.
me plot mowed e diecontinnity or the elope et #5 c thne
indicating denetnreticn er the enzyme ebove thie taper-eta“.
airer'e work (19153) hee mom the activation energy of g. 32;;
L08 ee 19.1300 Keel. end the inactivation tenpereture ee 1&5 c.
inere ere eererel pceeible mean for thie eix-i‘old difference
in eetivetion energiee of bacterial L011. Sizer (19103) employed
nixed whole celle of g. 22;; and eddednc additional cofaetore
to decrease the ectivetion energy. Instead we need cell-tree
extrecte or g. m end added contectcre m3 end HAD. Since
whole celle will not ellow imediete interaction of the
intracellular LDH with the eubetrete. the rate of enzyme
eetivity may alco. in pert. be e neeeure of cell well end

1:2
nenbrane permeability. Another possibility for the discrep-
ancy in activation energiee ie in the method of ite neelire-
nent. whereaa we neamred the rate of BAD reduction. Sizer
neeeured the tine required for 75% reduction of methylene
blue.

Good resolution 0! most LDH ieozyaee was obtained by
ueing 0.25 to 1.0 mg protein per sample gel. Resolution nae
inpaired if more than 1 as of protein nae need per eanple. L.“
On the other hand some bende would be nieeed it the mount of L}
protein nae leee than 0.25 as per emple. Better recolution

 

could be eeauned with purification. An occaeimal band may
appear in the control eemple. inie phenomenon nay be due
to endogenoue activity and appeere to be eliminated by incuba-
ting the acrylamide gale no longer than 30 ninutee.

We attempted uneueceeemlly to correlate LDH activity
with LDH ieozyae patterns: however. there did appear to be
e poeeible correlation between ma ieozynee in certain etraine
with annually high coagulaee and penicillinaae production.
For emple. in Group 1 or the Intemtionel-Bleir caries.
only etrain 80 had more than two USE icozynee. Thie etrain
eee the highest coagulaee and penicillineee producer of thie
group. Straine 5': end poeeibly 77 are the only repreeentativee
teeted in Group III that have more than 3 LEE ieozymee. These
two etraine are very high penicillinaoe producere, and in
addition etrain 510 ie also highly coagulaae active. In Group
hiecellaneoue only etrain 81 had more than two {DH isozymes.
Thie etrain ie only a fair coagulaee producer; however.

“3
penicillinase activity is extremely high.

Isozyae 3 appeared nest frequently (37% of the strains
tested) and may possibly represent a demon LDH ieozyne for
the genus: however. more strains, including coagulase negative
strains, will have to be examined to support this conclusion.
Seven of the fifteen strains possessed 2 LEE isozymes, six
had 3 £353 isozymes. and only two strains (518 and 81) had 1:
ieozymcs. None of the etraine tested possessed all 5 LB}: >14
isozymes. Although there appeared to be no definite correla-
tion of LIX-I isozyme patterns and the phage grouping among
the few strains studied there clearly are differences in LB?!
activity and 1.5:! isozyme patterns among the phage propagating
strains of :5. gm. The distribution and amount of the
isozyme may well form the basis for a new classification of
the staphylococci; however. a much broader analysis is
mandatory to support this preposition. This objective could
be accompliahcd by the following: (1) the analysie of a
greater number of strains: (2) the testing of cell-free
extracts from various phasee of growth: (3) the use of
moral different media and the effect of certain growth
inhibitors upon on ma isozymes patterns; and (h) the testing
of possible additive effects of ms isozymes by mixing

preparations from two or more strains.

SUI THAT? I

Selected strains of g. 232223 representing the 5 groups
of the International-Blair series were used for studies of
lactate dehydmgenaso (LDH). Cells mployed for assays
were cultivated in Bill at 37 C.

maximal L133 activity for whole cells appeared near the
end of exponential growth. This peak (0.9. 0.75) was reached
at 7 hours for strain 6 using a 10% inoculun.

Cell free extracts were prepared by an acetone ponder
method (Vadohra, et al. 1965) and by sonication. The sonica-

 

tion technique gave higher specific activity and less appar-
ent denaturaticn of the LDH enzyme.

LDH activity was determined by measuring the rate of
HAD reduction (Hollands. 1955) and nor reduction (Allen.
1961). In both assays. the relative order of LDH activity
for 5 strains of §. 51533 was: 29> 81>6 >3A>h2D.

The optimal temperature for LEI! activity in crude cell-
free extracts was 1:5 c and the activation energy. calculated
from an Arrhenius plot. was 3.260 Kcal.

The ma isozyne patterns for 15 strains of g. w
were determined by acrylamide gel electrophoresis (Davis.
1961:) and band development by HM (Allen. 1961). Five
isozymes were arbitrarily designated according to their
relative migrations to the tracer dye. bran phenol blue.

The slowest band was designated isozyme 5 and the fastest,
isozyme 1. Isczyme l was found in 7 strains, isozyme 2
M»

‘35
in 7 strains, isozyme 3 in 13 atrains, iaozyme It in 7
strains. and isozyme 5 in '16 strains. neat strains had 2 or
3 isozymes; however. none or these strains was fctmd to have
all 5 isozymes. Ioozyme 3 ethic}: appeared in 87;“: of these
strainc may possibly constitute a common L351 Home for the

genus.

 

1.

2.

3.

h.

5.

6.

7.

8.

9.

10.

11.

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qun..rx—.zrnnv~”
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' 1

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