Purification and properties of follicle-stimulating and luteinizing hormones from horse pituitary glands

Purification Luteinizing

I~eprrrlmcnzt

and

Properties

Hormones

of Zoology, Received

from

Cn,iversity January

of Follicle-Stimulating Horse

of Wisconsin,

14, 1970; acccptctl

Pituitary

Madison, i\pril

and Glands’

Ti*i.sconain

53706

9, 1970

.2 method is descrihctl for the purific*ation of follicle-stimulatit~g hormone (FSII) and luteinizing hormone (LH) from horse pituitary glands. The glands were exI racted twice in 40y0 ethanol , and the actjive protein was recovered hy acetone preripitation. Illactive protein was removed by dialysis of the acetone precipit,ate in dilute KaCl, precipitation with HP03 , gel filt,rat)ion on Scphadex G-100, and ionexchange chromatography on C?Il-Sephadex. FSH and LH nclivities were separated l)p preparative disc elrctrophoresis at pH 8.9. Additional pluification was accomplished by gel filtration of FSH on Sephadcx G-75 and LT-T on Sephadcx G-100. The FSII, pontainiug 90 units per mg of NISI-FSH-$1 and 0.16 llnits per mg of XIII-LII-Sl, was obtnilketl in a yield of 26 mg per kg of fresh tissue. It was homogene011s by disc electrophoresis at pII 8.9 and pI-I 7.5, Oncht,erlony double diffusion, immuIlocloctrophorrsis, and sedimentation eqllilibrium ultracentrifugation. After disc c,lcctrophorcsis at pH 4.3, t,wo zones wcrc apparent. but FSH activity was lost. The molecular weight of the horse FSH was 33,200 by ultraccnt,rifugation and 47,900 by gel filtration. An isoclect,ric point of pH 4.1 was determined by electrofocusing. The LH, containing 5.3 units per mg of NIII-LH-HI by the ovarian ascorbic aciddcplet.ion assay and 34 units per mg by t,hc ventral prostate-weight assay, showed lo:;, FSH activity. Several components were seen after Ollchterlony double diffusion and immunoclcci rophorrsis. Het,erogencit,y was apparent) by disc rlcctrophoresis, and bioassay of gel slices indicated a number of LII compolrellts and an FSH component with a different electrophoretic mobility than the major horse FSH fraction. Four LH caomponents with isoelectric points at p1-I 4.5, 5.9, 6.6, and 7.3 were shown b? c~lrc~trofoc~usirlg. r\n FSII component with an isocllectric point al pH 4.8 was also prclsrlltz. The molecular weight of t,he horse J,TT preparation was 41,500 by ldtracen1rifllg:r t ion and 63,800 by gel filtration.

Altllough Ilorse pituitnr\glands we krm~v~-~l to contain high levels of gonadotropins (l&I), tllev llave not been utilized :ts :L source for pukication of NH3 and TIH

as extensivelv as 11avc 41eep and human pituitaries. Highly purified preparations of sheep and human TSH and LH have been prepared (5-12) :md their physical and chemical propertics are being studied. Since purified horse FSH and LH- llave not been available, little is knows of their physicochemical charnctc~ristics. Biological studies of horse gonadotropins indiwte that they

1 This investigation was supported by Public llcalih Pcrvice Training Grant, 5.TOl-H1)0010404, from the Sational Institute of Child Health and Human l)f~vcloprnc~ni ; The Ford Foundation Grant, K-505; and 1hcb National Institlltes of IIeali h Research Grant. G1I 02154. 2Prcsent address: Hunting Laboratories, ?vIassachusetts General Ilospital, Boston, I*Iassarhusrtts 02114. 3’l’hc nbbreviat ions llsed arc: FYII, folliclestimnlat ing hormone; LIT, lutrinizing hormone;

PMSG, pregnant mare serum gonadotropin; HCG, human chorionic gonadotropin; VPW, ventral prostate weight; OAAD, ovarian ascorbic acid depletion; IU, intcruntional unit; RP, relative potK!rK!y.

45

differ from gonadotropins of other species in their action on the reproductive organs of test animals (13-21). It is of interest to compare the chemical structures of the horse gonadotropins with those of other species in light of their differing biological action. In this communication methods are described for the preparation of highly purified horse pituitary FSH and LH along with the results of several physicochemical determinations. MATERIALS

ANI~)

RlETHOI)S

autopsy was performed on the morning of Day 29. The rats were weighed on J)ay 25 and Day 29 at autopsy. Ovaries, uteri, and adrenals of thr females, and testes, ventral prostates, seminal vesicles, and adrenals of males were taken and weighed on a Met tier analytical balance. T’entrul prostate weight u.ssay.s. LH was determined quantitatively by the ventral prostate weight, (\-PW) assay (28). Ilypophysectomized male rats were treated with I,11 as described in the preceding section, and ventral prostate weights were recorded. The relative potencp of the unknown solutions in terms of NIII-LH-Sl was calculated by the parallel-line method (23).

Bioassay Procedures

Plofein Determination

Intacl rat assn!/s. Rolitine assa?;s were based on the increase in weight of I he sex and accessory glands of intact immattlre female and male rats. Twenty-one-da)--old 50. to GO-g rats (Holteman, Co., Madison, Wis.) were given nine injections (0.5 ml each in 0.9C;; saline) twice a day for 4.5 d ays a,nd sacrificed on the morning of Day 6. Increase in ovarian weight was taken as an indication of FSH activity ant1 increase in ventral prostate and seminal vesicle weights as an indication of LH activity. Augmenlaliorb cl.s.s~cy.s. Quantitative deterrnimtion of FSH activity was done by the augment ittion assay method (22) using 50 IU of IICG. Three dose lcvcls (follr animals per dose) were given in addition to the IICG controls. The data were analyzed by the parallel-lint method (23) and expressed in terms of the NIH-FS:Il-Sl st.and:trd. One milligram of the standard was considered to have 1 unit of FSH activity.

Protein was estimated by the biuret method (29) and by the method of Lowry e/ ~2. (30) Itsing bovine serum albumin as st,andard.

Omrian

ascorbic

ucitl-depletion

(OA Al))

test.

LH activity was qllantitat,ed by the OAAI) test (24). The method was modified so that hot h ovaries were t,akcn 4 hr aftrr tail vein injections of I,TT. Ascorbic acid was detcrrnined by the method of Schaffert and Kingsley (25). The data were analyzed by the parallel-line method (23) adjllsting ascorbic acid content to the mean ovarian weight for the entire assay using covarinnce analysis (2ti, 27). The data were rxpresscd in terms of the NIHLH-Sl standard, where 1 rng of standard was considered to contain 1 unit of LII aclivity. Hypophysectottlirctl rat assa~,.s. The final preparations of FSH and LH were tested by assay in hypophysec’t omized rats. I101 tzmau rats (50-60 g) were hypophysectornized on I)ay 22 by Hormone Assay Laboratories (Chicago) and shipped on the same day. The rats \I-erc kept 3 days before starting the injections on the morning of I)ay 25. They were given rat chow-, cerelosc, oranges, and water. Injections were give11 twice daily for 4 d:>ys and

Polyacrylnmide gel electrophoresis at pIi 8.9 was performed according to the method of Ornstein and Davis (31, 32). I~lcct,rol)horrsis at ~114.3 was according to the proccdllre of Rcisfeld c/ al. (33), and at pH 7.5 according Lo Williams and lleisfrld (34).

Antibodies to fraction llPCI\IFS, wit.h hoi h FSH and I,11 activities, were prepared by immunizing New Zealand White rabbits. Injections of antigens emldsified in complete Frrrmd’s adj uvant were given s\tbcutaneously once rach week for 6 weeks. ;Intiserum was collected 5 days after the last injection. Experirnr~~ts were performed using the antiserum and, also, antiserum adsorbed wit,h normal horse serum. Acqur gel dolcblc t/Q”~r.sion. The homogeneity of the purified FdII and LH fract.ions was investigated 1)~ the double-diffusion nlcthod of Ouchterlony (35), using l.25yG xgar gels in 0.9% saline containing O.OlC,& sodium azide as preservative. Antigens were diffused against I he antisera for 7 days and wet-stained with light green stain. Ztrl,tl/rnoclectrophoresiR. The method of Schedegger (4G) was followed. Microscope slides were coated with 1.5 mm of 0.87; agar dissolved in 0.05 ionic strength sodium b:trbit:~l, pH X.1. Sample wells 3 nmi in diameter wore filled with antigen (90 rg protein/ml) alld elec*trophorcsis was performed for 2.5 hr at 80 \- and 8 mA The crater trough was then filled with antiserum against MPC~lFS and diffrlsion allowed IO proceed for 7 days.

IIORSI~:

PITUITARY

FSTl

ANI)

LI I

17

05.

0 : 40 TUBE

NUMBER

FIG. 1. &l

filtration of 1.66 g of MI’S (sample ~0150 ml) on a 6 x 90.cm column of Sephadex C-100 using 0.05 nf phosphate, pH 7.2, as eluant. Fractions of 12 ml were collected at 20-min intervals. Tubes containing biological activity were pooled to give fraction MPC; as indicated. controlled by gravity. The pH 8.9 buffer system of Ornstein and Davis (31, 32) was used as modified in information received from Canalco, Inc. (Rockville, Md.). The separator gel consisted of 1 ml of 1O70 gel layered under 7 ml of 5% gel solution. The use of a 1O70 gel underlayer (42) reduced the amount of swelling at the elution slit so that. smoother flow of buffer across the bottom of the gel was acconplished, and blockage of flow during the later stages of electrophoresis was avoided. Twenty-two milligrams of the active fraction from CM-Sephadex (MPCMF3) were layered lmdrr the upper electrode buffer in 3 ml of 20cz stIcrose and a starting current, of 6 mA was maintained for 1 hr and 10 min until all protein was 1 mm into the separator gel. The current was then inrreased to 20 m,4 for 2.5 hr at which time it, was increased to 28 mA for the remainder of the experiment. 1*;111tion buffer flow was maintained at 170 ml/hr rlntil the FSH was eluted, at which time it, was decreased to 90 ml/hr. Fract,ions were collected at, 3.min intervals (Fig. 3). Rechromatography on Sephadex G-75 (PSH) or G-100 (LH). The FSH fraction from preparative disc electrophoresis (PDFSFI) was extensively dialyzed in0.025% ammonium acetate, lyophilized, taken up in 2 ml of 0.05 M phosphate in 0.1 M NaCl, pH 7.0, and chromatographed on a 2 X Wcm column of Sephadex (i-75 eqllilibrated with the above buffer (Fig. 4). A flow rate of 30 ml/hr was maintained.

FSH,

LH

\

J

MPCMFI

0

1 ’ 0

MPCMFE

MPCMF3.

I

I

40

80 TUBE

I

t

120

NUMBER

FIG. 2. IoIl-exchange chromatography of MPG (450 mg protein) on a 2 X 29.cm column of CM Sephades C-50. The starting buffer was 0.02 31 phosphate, pH 6.0. This was followed by a gradient, to pH 8.0 at I and a stepwise increase to 0.15 M phosphate in 0.1 M SaCl, pH 8.0 at II. Fractions of 7 ml were collected at, Urnin intervals. FSH and LH activities were recovered in the stippled area.

The LII fraction (PI)LII) was concentrated similarly to the PI)FSH and chromatographed on a 1.4 X l&cm column of Sephadex (i-100 in 0.05 M phosphate in 0.1 1~ NaCl, pH 7.0, at a flow rate of 15 ml/hr (Fig. 5).

.r :

i i

_-‘!,VENTRAL ! , PROSTATES i-4 I

2 N :

15-

100 80 g I60 5; % 40

I

2

3 HOURS

4

!il

FIG. 3. I’reparativc disc electrophorcsis of RH’CRIFS (22 mg prokin) at. pH 8.9 on a Carudco PI)-2/320 column. The starting current of 6 rnh was increased to 20 rnA at I and 28 rnA at II. Thr~ initial flow- rate of 170 ml/hr was decreased to 90 ml/hr at, Il. Fractions were collwted at :I-min intervals.

5 : 0

20

0 TUBE

NUMBER

FIG. 5. (;el

filtration of 1’l)LIl (G.8 mg pr,,kin) on :L 1.1 X 1%cm colrun~l of Sephadex G-100. The elrmnt was 0.05 11 phos~~h:~~c~ iti 0.1 M SaCI, pII 7.0. Frnct iolls of :3 ml w(‘rc wllrctcd at 12.nlitl intervals.

Gel fiitratiffn 0s the n&aphosphafe superWhen fraction _\Il’S was chromatographed WI Sephades G-100 in 0.05 RI phosphate, pH 7.2, the biological activities appeared in the secondpeak of the chromatogram (Fig. 1) and were designated fraction VOLUME ml !\llX. This resulted in an X-fold increase in FIG. 4. (kl filtration of PIIFSH (8.8 mg prospecific activity and a, protein yield of 500 tein) on a 2 x 87.cm colrmu1 of Sephadex G-75. The mgjkg fresh tissue. I\II’G was 13 times as LL~U~II( was 0.05 M phosphate in 0.1 11 NnCl, pH 7.0. active as NH-FSH-Sl and equal in potency Fractions of (i ml were collected at. 12.rnin interto NH-LH-Sl (Table I). vals. FSlI activity was elllted in the? stippled area. C:l/-Sephatlex chro~)lafography. A 4 to L fold purification was achieved by ion-exl:I’SUI,TS ANI) 1)ISCUSSION change chromatography on CXI-Sephudcx Pw~fication l+ocedu~~es C-50 (Fig. ‘2). The active fraction (;LIPCI\II’A\~~ outline of the methods used in purifica3) contained 54 units/mg of FSH and 5.S tion of FSH and LH from horse pituitaries units;mg of LH, with :t protein Jield of 90 is given in Diagram 1. Protein content and mgjkg fresh t,issue. This represent,ed 533“: quantitative assaysof the fractions obtained of the IJSH activity originally present in the are slwwl in Table I. ethanol extract and 59% of the LH. Extraction of glantls. Augmentation assays Plel)alatioe tbisc electTophoresis. Horse piof the 40 ‘3 ethanol extract (EE) indicated tuitary FSH and LH were separated by disc a pot’ency of 0.49 units/mg of FSH and elect~rophorcsisat pH S.9 (Fig. 3). The first OAAD assays indicated 0.047 units/mg of major peak nft~cr t’he .dye front contained T,H (Table I). The average protein recovery follicle-stimulating :lctwty and wts desig\\-:LsK-3.7g/kg fresh glands. nated l,DIBH. The trailing edge of this peak HPOa precipitation. The addition of HPO, consisted of :I fraction with both FSH and to a. 2 % solution of El3 in 0.025 M phosphate, LH nctivit’ics, and this was followed by elupH 7.3, resulted in precipitation of inactive tion of the lutcinizing liormont~ which I\-:ts protein at pH 3.2. A s-fold purification was designated as I’DLH. obtained. The relative potenq- of the me& PDF’SH contained 77 unit’s/mg of I’SH phosphate supernate (I\II’S) was 1.7 units/ and 1.5 units/mg of I,H, while I’DLH conmg of ICSH and 0.1s units of LH (Table I) tained 5.5 units/mg TJH and 9 units/mg of 14th :I protein yield of 4.7 g,/kg fresh tissue. FSH. nate.

50 Extraction

of

ground

glands

in

40 percent

Shake I Residue

3.5

Supernate

ethanol

hours,

1

centrifuge

(Sl)

Shake 3 hours in 40 percent ethanol, centrifuge Rl

Supernate Add

______( ombined 5 volumes

of

Sclpernates acetone I precipitate

Acetone Dialyze,

ceritrif,Jge

I

I EE 1

R2

Add HP03

to

(Sl+S2)

pH 4.2,

cei-ltrif'llge

YIP'S

MP'F

Concentrate on ultrafilter, G-100 chromatography MPG Concentrate on Lltrafilter, desalt on G-25, CM-Sephadex chromatography

I MPCMFI Preparative

Sephadex G75Fl

G75F2 (FSH)

1

MPCMF3

MPCMF2

G-75

disc I PDFSH-LH (save)

G75F3

Rechrormatoqraphy on G-75 (PDFSH) or G-100 (PDLH). Aft,er separation of I;SH and LH by preparative disc clectrophoresis, final purification was achieved by chromntography of thr~ I’DFSH on Sephadcs G-75 (Icig. 4) and the l’Dl,H on Seph;~dex G-100 (Fig. 5). The fin:~l I:SH fraction obtained by (i-7.5 gel filtration (G751+?2) was 90 timw as active as the SIH-E’SH-Sl standnrd and it COILtained 0.16 units/mg of TJH wlu.x~ ass:~yrtl by the OA4D method, and 1.3 unit,s/mg of LH by the VPW method (Table I). A yield of 26 mg protein per kg fresh glands was obtained, representing a recovery of 26 ‘:; of thtl &ivity of the original extr:lct. The :tc

electrophoresis PDLH Sep,hadex

G-10;l!;,dHF2

GlOOLHFl (La

tivit>T of G7.?1;2 (l:SH) in h\-pophysectomizcd female and mule rats is shown in Table II and VPW assays are given in Table III. In previous reports on the purification of horse pituitary ITSH, the highest wlntivc potency attained n-as 7 units/mg (43, 44). The LH content of thtw prcp:uxt,iorls w:w not quantitated. The final LH fraction (GlOOLHl~‘2) obtained by G-100 gel filtration uxs X3 times :IS active as NH-LH-Sl whcrl assayed b) t,hr OAAD method (Table I) and XZ.C, tirnes the LH standard by the VI’W assay (Tablt III). It contained 9.1 units/mg of 1;SH, which represents about 10”; FSH on :L \vt:igllt basis. The Aion of G1001jHl~2 in

ttOl:SI~:

ls.i 1.7 0.5 0.00 0.0:39

0 03 1 0. ox 0 Olli

t’t’t’ITt’~.~t:\r’

PSI1

ANI)

101) x7 it 5:1 :i:< :I “Ii 2

.-I1

t.tt

0. o-ii (o.o:3M. 08) A’, 0. 18 O.!lX 3.7X 1 .5-1 5.5 0.1 Ii 5 1‘3 1 .:3 :x3.(\

(0. 12-o. 30) -:i (0.5%1.56-2 (3.35 9.57)-t (0.7% 2.90-2 (3.0 10.0-2 (0.00 0. “(i ) -:< (3 1 I). 1 ) -3 (0.7’ 2 M-2 (ll;.~;klll

1-R

TABI,E VPW

Ass.ius

FSH

.4ssiry

Fraction

G100LIIF2

Arithmetic average G75F2 (FSII)

(P!?)

IT I (:75F2

FRACTION

nose

number

NIII-LH-Sll

Arithmetic

OF

Jndex of Inerision

AND

LIT

individual slope

GlOOI,ITF2

RI’ units/ma (955

SIII-LIT-S1 CL)

Index discr$yx$

of

OAAD)”

1 2 1 2 2

3.5, 4.5, 1.0, 0.25, 1.0,

18” 18 4 1 1

0.2F2 0.221 0.262 0.221 0.221

1.82 9.30 7.89 li.(ici 15.36

1 2

4.0, 4.0.

16 16

0.2G2 0.221

8.27 9.26

average

a OAAD averages from Table I. * Four animals were used at each

FMCU~N

0.81 40.9 25.1 34.8 33.6 1.36 1.21 1.30

(17.3-210) (14.8-46.2) (17.7-97.1) (l(i.(i-118) (0.72-2.92) (0.73-2.21) (0.72-2.56)

7.7 -1.7 ti G fi .3 8.5 7.8 8.1

dose level

+

I

II

Ill

V

FIG. 6. Polyacrylamide gel electjrophoresis of purified horse gonadotropins. I: FSII fraction G75F2 (100 pg) at pH 8.9 in a 7.5% acrylamide gel; II: FSH fraction G75F2 (75 pg) at pH 7.5 ill a 7.59; acrylamide gel;1 III: LH fract,iolr GlOOLHF2 (11Opg) at pH 4.3 in a 55, acrylamide gel; IV: LH fraction G100LIIF2 (100 rg) at pII 8.9 in a 7.55; acrplamide gel; V: FSH fraction (+75F2 (100 rg) at pH 4.3 in a 57, acrylarnide gel.

6) although bioassay revealed thrl loss of FSH activity. NH activity was not detected when elcctrophorrsis at pH 4.3 was conducted with the electrodes reversed, indicating the hormone had not been migrating

in the opposite direction. It has been reported (46, 47) that several enzymes are inactivated during electrophorcsis at pH 4.X because of oxidation by the ammonium persulfatc catalyst. To determine whether

Fro. 7. Disc elcctrophorcsis of E’S11 frwtion (+75F2 (top) :111ti LFI fraction (;lOOLIIF2 (bottom) at pH 8.9 in 10cjO polyncry1:tmide gels. One gel of c~;tch sample m-as stained with amide black 10B and :L second sliced into :&mm thick sections and assayed in intact irnmatr~rc male and female rats. G75B2 was applied in a sample of 310~18 protein per gel, and GlOOLHF2 wns applied in A sample of ‘240 pg protein per gel

this w~,s causing the loss of I;SH activity, polymerization of t’hc gels w’;1s cat:+zed by riboflavin in p1:u.x of pcrsulfate. 1SH :ctivity w;w :Ignin lost n-hen run :it pH 4.3 in these gels. This evidence indiwtes t’hat horse pituitwy I’SH is inwtiwltrd by disc electrophoresis :Lt pH 1.3. The appc:wance of two mnjor zones at this pH m:ry suggest thnt the I:SH is sep:watrd into imtctive subu&s, similar t,o the form:ltion of inactive subunits by T,H :lt, low pH (4%53). Tl~r: FSH V-U not inxtiwrted by clectropllowsis :lt pH S.9 (Fig. 7) :md tile xtivit) coincided with t,lic single zone (R, OX) obt:ked. The I,H frxction (GlOOLHE’%) :ippe:ired :w :L broad zone :lfter clectrophoresis :rt pH s.!) (Fig. 6). A number of b:rnds could be seen throughout the zone indicating several components were present. This w:is cow firmed by ttlt~ctrol)lloresis :It) pH 3.3 (E’ig. 6), aft,er \~h& I,H wtivity could be dctectrd :lt’

the :tnode twtl of t#hr gel XH indiwtctl b\ slicing :tnd :ws:~y in intact immature Ink. Analyt,ical disc clectroplioresis of GlOOLHl’Z :tt pH S.9 resulted in :I diffuar b:mcl of protc4n (l’ig. 6). Wllen gels run concurrcritl~ were shcrd ink) X-mm thick s&ions :md :ISwyed in intnct, rats, three active compontwts could be dctwttd (Fig. 7). These consisted of :L Itxding LH Ix:& (R, O.%), and 13H and LH frxtion (R, 0.24), and :l tkling T,H Ix& (R, 0.19). These three component,s migrnkd more slowly th:m the IJSH of G75E’Z shown directly :ibove in l:ig. 7. Thus, it, is noteworthy that the I%H :&vity \\-hich :tppears in GlOOI,HE’2 is distinctl? different from th:it of Gi.51’2 in terms of electrophorctic migr:ttion (R, 0.24 vs h’, 0.X). rlqar ye1 double tli’usion. The puritit~d horse ICSH fmct,ion formed onl>, :L singl(l prrcipit~iti line :Ig:kist :wtiserum t,o fr:lct’iotl A~II’C~;\II~‘.‘: (Fig. S). The I,H fmction fortncd sewr:Ll Iwcipititl lines (Fig. S), 01i~~of which \v:~s continuous with the I’SH frwt,ion G7.51?2. This nwv be th(l rwult of cont:~min:kion of tbc LH kction \vit#ll NH. However, it \V:IS shovel b\ :tn:dytic:ll disc clectrophoresis, :w previously discussed, th:Lt the P’SH :lssoci:tted with frnction GlOOLHI~‘2 has :L different electrophorctic mobilit8J- (R, 0.24) t,h:m thnt of (;75FF’:! (R, 0.3s). Electrofocusing cxprriments, discuswd in the following sect8ion, show tlr:\t t,hr I:SH of GlOOT,Hl~:! h:ts :I higher isoelectric point (pH 4.8) th:m tht> I;SH of (:7:iIc’,, (pH 4.1). It also has :t Ilighcr molcculw \vcight as shown by wdimcntntion equilibrium ultracentrifug:klon :md GlOO gel fikration. In the light, of t,hese findings, the continuity of t,he prccipitin lines m:l,v b(b due to :L nonspecific reaction. I,lullul~oelectl-opl/olesis. The I’SH frxtion (:75I? :rIso cxhibitcd :L single component :tfter immunortlectrophoresis (Fig. S). IIImunoelect~ropborrsis of t,lle I,H frxction G lOOLHl~2 resultrd in rcsolut,ion of t’wo precipitin lines (F’ig. S). When comp:lwd \\-it11 the I:SH fmction (;75E‘Z, thr: :mtigtw of GlOOLHI9 did not migrate M f:w tr)w:trd the :modC, confirming results of :malytic:~l disc clrctrophorwis :md isoclcctric focusing. Ble~t~oSoczl,~jn!l. Tlrt> isoelrctric point of

FIG. 8. Imm~u~ological studies of pkwificd horse pit rlitary golladotropirls. I: Agar gel tloublc difflwioll followed by wet staining with light green. The c-etlter well was filled with rabbil, antiserrm~ against fraction RIPCMF3 (colltaillillg FSII slld LIT :tc.tivitic~s). alld lhe peripheral wells cwlltainrd (1) LIT fraction (:lOOLHF2, (2) FSH fraction (:75B2. (3) MPC,\lF3, and (4) (;lOOLHF2. II: Tnlln~uloelr~~ropl~oresis of FSII fraction G75F2 at A a11t1 fraction MPCMFS cwntaining both FSIT atrtl I,H activities at H. The trollgh was filled with alltiserrlm lo RIPCMF3. Ill : Imrnluloclr~tropl~oresis of J,ll fractiolr (+lOOLHF2 at C and fraction MPCMFS wlltaining both PSI1 :111d I,ll activities at I<. The trough was filled with alitiserrim to MPC1lF3

I

0

5

IO

15

20

25

30

0 TUBE

5

IO

15

20

215

NUMBER

FIG. 9. l.Xect refocusing of FSII fraction (;75F2 alld LH frac.tiull (;lOOLTIF2 il) a 1’; strlt~tioll of pH :J-10 ampholytes. All LKB 8101 clectrofocrlsing colr~mn was r~setl. The cnthcdc, at the top, cwiisisled of 2’ ; (v/v) ethylclrcdiaminc and thr allode, at the bet tom, WIIsisted of 0.2 ml IbPOa , 14 ml IT,0 and 12 g sucrcw. A potential of 310 C: was applied for 30 hr, the rlurent having stabilized at 0.9 1~4 after a total of 20 hr. The FSH sample cow tained 1.8 mg protein alIt the I,11 sample cotltailled 2.0 mg proteitl. hliql~ots from each tube were assaycd at a dose level of 5-g c~qllivalrnts of fwsh tissrw ill intact immnt llrr malt and female rats.

22

1 ’ .2 201 u c -

5.8 56

r2

FIG. 10. IIigh-speed sedimrntution eyuilibrirun wntrifugation of FSH fraction G75F2 in 0.1 ionic strength pho!jphate. pli 5.0. The sample was run :\I 39,977 rpm :rt 1”.

I+SH fraction G75F2 n-as located at pH 4.1 by the electrofocusing technique. Only a single peak of FSH activity was evident by assay in intact rats (Icig. 9). No previous reports on the isoelectric point of horse pituitary l:SH are available. The isoelc&ric point of human FSH has been reported at pH 4.25 (54) and at about pH 4.5 for most other species (55). The horse pituitary LH fract’ion GlOOLHI;L’ exhibited four peaks of activity in the nmpholyte gradient. One LH component, became isoelectric at pH 4.5-4.8 and contained the majority of the FSH activity that appeared in GlOOLHE’2 (Fig. 9). It can be seen that this I’SH actlivity is located at a higher pH (43) t,h:rn hhe F’SH of G751(:! (4.1) and npparent~ly is a different entity. A second I,H component, appeared at pH 5.9, a third at pH 6.6, and a fourth at pH 7.3. Low amounts of FSH activity were present in these fractions also. The above results were confirmed by electrofocusing in polyacrylnmide gels according to a modification of the techniqw of Wrigley (56). There are no rc1)nrt.s available on the isoelectric point of horse pituitary I,H, but, the isoelectric point’ of ovine LH has been reported at pH 7.3-7.7 (-57, 5~). The existence of LH as several disclY~t(~ c,l(,rt,l,ol)horrti(: components has re-

Flc:. 11. l
cently been demonstr:ated in t#he human (59) and sheep (60). The above results indicate horse LH is similar in this respect. rllolecular weight defernu?Lations. Srdimcntation equilibrium studies of the horn 1;SH (fraction G75W) indicated an :~pparent weight, average molecular n-eight of 33,200, :wsuming a partial specific volume CC) of 0.72 ml!‘g. This is similar to the molecular weights obtained for FSH of otther -pecies. Sheep I’SH was sho\vrl t,o have :I molcrular weight of 31,000 -32,000 (6, 61, 62) :m(l human 1;SH :t molecular weight of 31,000 (s-2). Evidence that human FSH forms MI aggregating system has been obtained by ,s;edimentntion equilibrium studies ((3) :md gel filtmtion (64). An aggregnhirig syhteni for ovine I’SH was also shown (7). Yo e\-itlence of aggregation it-as shown in the plewit study of horse pituitary FXH :IS incliwt~d by the straight line plot, of In c v,\ 6 (Fig. 10). GlOO gel filtrnt’ion studies ot’ fraction G732 indiwte a molecular lveight of 47,900 for 11orsc pituitary NH (Fig. 11’1. The higher value obtained by gel filt~ration for the ICSH is probabl!- a reflection of tile csrbohydratc content. Whitnker (38) folmd that, glycoproteins exhibit, an :tbnormally high molecular weight by this method. Ovamucoid, which had a molecular w-eight of 27,000 by ultracentrifugatior~ studies, shopped

56

BRASELTON

AND

a molecular weight of 45,000 by gel filtration. R,eichert and Jiang (63) used the gelfiltration technique to obtain a molecular weight of 45,000 for LH of several species. These were shown to be %,OOO-33,000 by ultracentrifugation. Human FSH appeared to have a molecular weight of 32,4SO by gel filtration (52) whereas a molecular weight of 31,000 was indicated by ult8racentrifug:ttion (54). PA\ISG, another glycoprotein, was reported to have a molecular weight of Z&O00 as shown by ultracentrifugation (66) but exhibibed a molecular weight of GS,OOO when examined by gel filkation (67). The apparent weight average molecular weight of the horse LH (fraction GlOOLHE’2) was found to be 44,500 by sedimentation equilibrium ultracentrifugation. This molecular Jveight is much greater than that reported for LH of other species. Molecular weight est’imates in t,lie range of %,OOO34,000 have been given for bovine, ovine, porcine, and human LH (57, 65, E-70). The lack of curvature in the plot of In c vs 1.’ (Fig. 12) indicates that the preparation of horse pituitary LH is homogeneous in terms of molecular weight and is not undergoing aggregation. The molecular weight of GlOOLHE’2 was 72 70 t 68

66,ir 64 t 6 2c ” c

60-

5.6-

5.2-

12. High-speed sedimentation equilibrium centrifugation of LH fraction G100LHF2 in 0.1 ionic strength phosphate, pH 5.0. The sample was run at 27,993 rpm at 4”. FIG.

M&HAN

also estimatr~d by gel filtration on Sephadex G-100 (Fig. 11). A molecular weight of 63,800 was indicated by this procedure. The much higher estimate by gel filtration is again probably due t’o the carbohydrate content of the molecule. Reichert and Jinng (65) reported that horse pituitary LH with a relutive potency of 0.9 units/mg was eluted from Sephadex G-100 at the void volume, which would indicate a molecular weight greater Ohan 150,000. The discrepant)between this and the 63,800 found in the present study is unexplained, although the buffer systems used may have played a part. Reichert and ,Jiang (65) did not examine their horse pituitary LH by ultracentrifugation. Recent studies of a number of glycoproteins, reviewed by Gottschalk (71), have revealed that heterogeneity exists among the carbohydrate groups within a single species of glycoprotein molecule. This may be a result of the enzymatic, nonribosomal type of biosynthesiss or of enzymatic degradation (71). This characteristic heterogeneity of carbohydrate moieties in glycoproteins most likely accounts for the appearance of the purified FSH and LH as broad zones after analytical disc electrophoresis, ngar gel diffusion, and immunoelectrophoresis. In conclusion, a, procedure has been described for the preparation of highly purified horse pituitary I”SH and LH. The FSH fraction is suitable for chemical characterization, and studies in this regard are in progress. The LH fraction is shown to consist of several species of LH and an FSH species with different physicochemical properties than the major horse pituitary FSH fraction. ACKNOWLEI)GMENTS The technical assistance of Mr. H. .J. Grimek, Mr. K. Yano, and Mr. J. 1). Hodge is gratefully acknowledged. The authors thank Mr. S. B. Bangalore for t,he immunological studies, Mr. L. Lut,ter for the sedimentation equilibrium, and iLIr. Louis Nuti for the dry-weight determinations. REFERENCES 1. HELLTIAUY, A. A., l’roc. Sot. Erp. Bid. 30, 641 (1933). 2. HILL, Ii.. T., J. I’hqsiol. 83, 137 (1935).

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