Some aspects of human placental enzymology

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SOITE A$FL'CTs

OF

^HIJIIATI

PIACENTAL ENZYIJOI,OGY E=E=t3:=;zE=:+----.3 2.a=:

A TI{EStrS SI,,EMITTED FOR TI{E DEffiEE OF DOCTO! OF PHXTO6OPHT

OF TI{E UNIVER$ITY OF AI'CKI.AIIID.

Jtr$tE, 1973"

I

^agrce

to this thesis

^belng consultedL and,/or photocoplqd

fo1 the

^purpose

of

icsaanch

or nnlvate

stud,i nr.ovlded

that

due acknodedgment 13 ^{mlde whe}

of

researth

or private

study pnovlded that-due edgrrcnt

fq

gla{e wherc

appr6p.iiiie ind'thai'rrU perirlisfon is

obtalned

before

any

materlal ls

publ ished

, .- - -? /s

c.,-u{.. sl{al&

4

.FQFItrSW nTq jgn€L_r1E--ltnsJE.fH Lcs J.l"{a GTTNAFCOLOGY

Slgned.

GONTENTS

Page SUMilIARV

Chaener

l,'- .

n"lJL-o,UJQ TISI]I

A.

^TI{E INACTIVATIOI\I CIF NEURO}IYPOPHYSEAL HORMCFIES.

l-

^{Ttre Hormones}

(a)

oxytocln

(n)

rUasopressins

(c)

TS.sEup

ulte

of; inactlva;#on

(e) ldolecular slte of inactlvation (i) (il)

ltit)

(ro)

2e

DeEradation by Ll-vee TiEsue

S.

Degtadatlon bY KldneY Tlssue,

!.

Degnadatlo.n

ht Uterine

Tiesue

5.

Degradation

by

Placental

Tissue

^(a)

I

3

Amlnopeptidaie

acti.on I0

C-terminsl ll,

Disulph'lde

bond 1l

Tlssue amLnopeptldase

Il

t2

I.4.

l5

The

structule of the

^hurnan

placanta ,

¹⁷

(U)

Deginadation

of

^neurohYPo-

phiseal,

hormones

⁷³

6.

Degradatlon

by

hegnanoy Serstn O-tiytoci'naoe

(a)

General ^c,ctt-ment

(b-)

Mati.rods

of aosay (l) BioloEical

(tt)

chentcat

(c) turif ication

(al

Properttes

k) Orlsins

(f

)

^srrriction

25 26 26

,w

29 33 36

l

B.

^Af,KALINE

(,a)

(b)

(cl (c)

(e)

*{OSPI{ATASE

IN

^PREqIANCY.

General conrnent Methods ^rof, a:gsay

furlfi"catf,on

Propertles

(antlbocly productton)

Hlstological localization

(ii)

40

,42

43

44 45

54 54 54 5?

57 G. GLINIICAI AFPITICATICF{ OF 0XYTCICINASE ^A}tD

DEf,ERI{INATIOI{€

[,U'CE$T.AI ALI$tINE P}.I05F}TATASE

D.

AIIT OF THtrS ^iiJORK

Chapter

2,

lilETllODS AND hIATERIALS-

A.

GEN,ERAL METHOD6,

1,.

Instrumen,tation

2.

Electrophoresis Oxytoolnase

Placental

alkalin,e

phosphatase

Acfylamide

gels in thln

^aheets

Acrilarnlde

gels In

^eolumnE

Horizontal

starc'h ^gels

3.

Inuounological Techniques (a)

(u)

(a), (n)

(")'

(*)

(n)

46 47 48

50

51

52 53 53

Gel

dlffusion

Mieroiununoel e.ctro ^{hore sI s}

4.

Fhoqplorous

Estinatlon

5.

P,otaas{um ,Estlmatlon

6. Proteln Estlnatien

B.

L.

OXYTOCINASE

Assay Proceciures

(a) !.cysttne-dlip-nitroanilide

^{as subetrate}

(i)

maoual _Frasedure

(lt),ar,r"tomated

Pro-cedure

(U) S-benzyl:!-cy,steirie'g-nitroanill'de;

^{as substrate}

(r)

rnanual _Pro'cedure

(fi)

autornated Procedure

fur'i

fication

Antl-,oxYtoci.nas€ ^c-otrumns

(')

Antioxytoci'lll,ds€ ^bor,rnd

to

^septrarose

(i) Prroaratlon

^{of, calurnn}

(fi)

ahsorPti'on

of

antigen

(iri) elution of

antigen

(iv) effect of

neuramlnidlse on elutl'on

tr

Enzacryl

Glutaraldehyde

DEAE cellulo-se

CystXne aminopeptidase

ectivtty cf

piaeen'b'nl

sxtracts

(a)

Pre.oaratisn

of

honogerrate and rel'ilova1

of

oxytoclnase

(b)

^pH optl.mwn

(c) effect

^of, neuraminidase

(U) effect of

^len,gth

of gestatlon

^{and toxaemia}

(e) effeqt of

^EDTA

(f ) effect of

su,bstra'to

(lix)

78 78 79 80 80 80 b9 60

62 64 6B

72

2.

9.

(u)

(")

(d)

v4 v4 74

(1")

.C.

GLUTATTIIQI(E OXTTOGIN TF{"1NSH}0iiOG!NA5E

l.

^{Assay trroeedute}

2. furlflqatlon

3.

Floperti.es

of

Gl.utathlon€-oxytocin Tian'shydro; ena$e

(") stability

(U)

Inhtbtt.tron and

activators

(c) Dlstrldrtion

(a) Specificlty

Antibody Prep:aration and

Rrificatlon

DEGRADATTS.I OF OXYJOCIN tsY ^PLACENTAT TII$IOGEI"IATES

Degradatlo,o o,f

labelled

axyt..ctn

Chy.motryptLc

actlvity of placental

homoganates

(a)

^{Ebrzyrne Assay}

(U) Inhlbltion of

^eneytre

activlty

(c) Effect of oxytocln

on chymotryptlc

activlty (a)

Freparatlpn o-f

rat tlssue

homogenaies

8l

82

85 85 85 85 86

8?

89

9I

92 92 D.

t,

2,

9E 93 95 96 t00

Er

Ir

ALKATINE PI{OSP}{ATASE

Assay Prioce&rre

(a)

Marnral. method

(b)

Automated nre'thod f;or

,(c)

Autornated rnethod

for

Puri,f lcati,on

Antibody' productlon

total,

al,.kaltne PhosPhatase

placental alkaline

phosPhatase 2:.

3;

(vj

F.

I.

2.

LOCALIZATION oF ENZYT{IES X}l PLAONTAI TISSUE

Histo'l,ogloatr

(alkallne

phoephatase) Iircnune'flu,ot e Fcent kotefu'r ^Te acing,

(al

Preparatlon

of labelled

antibqdy

(u) keporation of tissue

seetlonE

(e) Fluorescent staXnLng

(i) Dlrect

Tachnique

(il)

Ind;i,rect Technique

(A)

Exarntnatlon

of

stai-ned se:tt'ons

$u ee't.Lllil arr ^Loeal l,uatlon

(a)

Ultrae entrlfugatl.on

(f)

Glucose-6-phosphataso

(ti)

Glucose-6-phosphate dehl'drog€nase

(ifi)

Cytochrome oxida',e

(fv)

Succlnate dehYdlogenase

(v)

Alkal!.ne phosphatase

(vi)

Oxytocin,ase

(vif )

^C-o transhydrogrenase

(vfi i)

CarboxyanrLdopeptldase

. (rx)

chyurotry,psln

(*).. (3n) t'yrusine

release

(x1)

A.rtibody

effects

(U)' Ultracentrlfugatloo in a

Eucrase gradtent

(f}

Proteln

(ft)

Fhosphollpid

(fti)

Ribonucleic acld

ENZtIT'1E LE\IEIS

IN

^PREG{A}ICY

gxytocinaEe _and .Fl.acental

Alkatine

,Phesphatase as an trndex

of

F!.acental Ftmction

Inaetivation of

SEtum Enzymes withr Antlsera

tc2

it02

r05 I05

1q7 107

109 110

trlr

Itr

112

1I3

rts

113

tl3

IJ.3

rt3

113

II3

Itr?

t17

118

120

t20

G.

I.

2.

(vi)

Chapter

3'

^RESdll-TS.

A,.

OtrTocINASE

l.

^Assay Fr,ocedu,u'es

(a) !-eystine-dt-g-nitro,anillde

as substrate

(i)

manurtr mathori

(if ;

autoilatad nrethad

(n) S-,banzyt-!-cysteine-g-nl.troaniltde

as substrate

(i)

'uran'rral rrrethod

(ii)

autonate{i method

?. hrXf,tcation

(a) furification by fractionation

and

gel filtration (U) Hmific,ation by

atrttenzyrne chronnatography

(i)

adsorptlon on antien,/y$e ^coJ.urnn

(lt)

elu' 1qn ftorn anticnzyme ^coitunn

(lfi1 ,effect of

neuramir:irisse on

elution

4,a

Gyetlns ,tminopeptldase

Activity of

Pltrcental llomogenates'

t22

L26

LzE

[36

13A

(a) (u)

(c)

(d) (e)

pH optimuun

Neurarni nida,se e f ^f e';t

Cl.lnical

state

Inhi.bltion by

^EDIA

$$strate

139 140 1.4s

r46

I46

L47 148

I48

152 B.

X,.

GLI'TATH IONE ^CIXYTOCIN TRAN SIIpROGENASE

Assay Procedure

Vallat[on of

assay ^parameters

furl,fieatlon

¹⁵⁵

\v]1/

e Properti.es

(a) stabillty

(u,J Inhib,ttors

and

qctivators

(c) Distribution

(d) speclficity

Antihody Freparatlon anej

furlficaticn

DEGTADATICIII OF OXYTOCII{

Degrad.atton

of Iabelled

oxytocin

(3tr) ty"usin€ s*ytooin (rac)

gtybi,namlde ox'ytoc n

2.

C,hymo,tryptie

actlrrity

PLACENTAI, AI,KALINE PF.IOSFIIATASE

Puri.fication

and Antibody Chafacterizatio'n

LOCALIZATION OF EI{ZY,IJIES'

IN

PLACENTAI ^TXSSUE

l{lstologlcal

Loc,alizat,l on

,AIkaIine Fhosphatase

Fluorescent

Protein

Tracing

(a) Alka'tino

.7 Phosphatase

(b)

Oxyt,ocinase

(c)

Glutat,hione

oxytocln

tiranshydrogenase

Subcellular

Locatr izattron

(a)

Ultracentrt,fugation

(U)

^'Sucr'ose gnadient centrif;ugiation

F.,

^ENZYIJIE LEVELS IN. FNES[A}..IC:Y

1.

Oxytoclnase as an i,ndex

of

pl,acental _functtron

2n

^AlkaLi:ne phosphatase as an lndex

of ptacenta!

functlon

3. Inactivation of

serun eneyraes

with anti-sefa

(a)

orytocinase

('U)

_alkal-ine phosphatasg n

1.

trLI

Ir,

(a) (u)

lsB

158 159 160 163

L72

LW 166 L67 169

2.

L77

l82

r.88

188 193

r93 r99

201 204

(virt)

c, hp,gter

a. .

^. DI9$ilFSICt't

A.

^ENZYIU,ES DEffiADING ^OXYTOOTI'I

I,

Fregna:ncy Serun 0qrtoclnase Assay Procedursrs

RrrIfication

^Methcds

Enzyme ProBerrtl,eo

2, ^Fl aqental'Gl

utathlone

^0xytoe5.n Tlanshydrogena sc

(a) (b)

(")

(a)

(,b1

(.)

2CI5

na

224

229 229 230 231 235 237 .Assay Procedufcs

R.rnt f ic at

ion

^{Mer thod,s}

Enzyrne Propertiee

3.

^Placental Oarhoxyaurldopeptid,ase

Actlvlty 4.

Compartson

of

^Ertzy*es

Attacking

^Oxytocin

E.

^{PTACENTAL AIKALNIE} FIIOST+I,ATASE

(a)

Assay Frocedune

(U) Rrtfi.cation

^Method

LOOALTZATXON OF TISSUE :EII*TEY|II|ES

Techrtl,ques

of i.dentifytng

^En?ymes

ln

^tissues

iC.' l.

244 24Q

?4L

?.44

248 254

291

n2

233 253

ta)

(n) (o)

Histochemical

Immunof luor'escant Subcellular

2.

3,.

4.

Localizatlon of

Oxytoeinas€

Locallzatlon of Alkaltne

Fhosphatase

toc allz ati.on

of

glut athione-o)crttoci'n

tr

^an ahydroge,nase

D'

li

2r

CL]NtrCAt APPLICATION5 ^{CIF ENZY||IE} As.says

foa

pLacentall enzytles

ASTIVITIES

Funct,ion

of

O.xytosinase

'in

^preEnancy

(i

x)

Char..r'1. t.'t

:>.

^{Cal :i'l}

A.

I]',IACTIVATiCII OI CXYT(.)CiII

1'

C;<';'t'oci lase

2' Clul:athione-c;ylocin

t.ca.rsi,yCrog,:n.r:.c

3.

Car],o;<yani:rj;,rpt,il6sg

257

lJ'/

259 260

2r,-C

tvL zQt

elt,7

26,1

TISJUE

I.

2.

3.

LCCALIZATIC':'] OF

Placc-'n'uaI

all:al

Oxy t,cci n ase

E|'!ZYIIES

I"i

I)LhCi:|ll',:.L

iie

phcs;lratase

Gl tr t, a th i one- ^{o.x y',} c c i

n

tl' ^{a n r ) : yC.i' o gi ;',} a - ⁺

ChaPU:-i--

r;t-

Al-'l'.rlli"

t!l:.

l.

A!;irreviations

2.

A;!:,:rr:inrLedgeni::'rts

3.

i\;t;l

ications arising frcn

^{i;)i j :, ivoi'f:}

nFFrS rf,rnr^