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Immunochemical studies on big gastrin using NH2-terminal specific antisera
Regzdatory Peptides, 1 (Ire80)169-I86 © Elsevier/North-Holland Bio medical Press
l ~',_?
IMMUNOCHEMICAL STUDIES ON BIG GASTRIN USING NHz-TERMINAL SPECIIrC ANTISERA
G.J. DOCKRAY
Physiological Labor~to~'y° UniuersiO, o f l.,i~e,pao!, L i~,erpoot L69 3BX. l..!nHed .Y,itrg~L~m Accepted for l)ublieation tt August 1980
SUMMARY
Methods are described for obtainiog antisera specific for the NH,-terminal regions of human and porcine big gastrin (G341 that can bc use'~l in radioimmunoassays. Three antisem have been characterized in detail" one (L66~ ,aised to human 1 - t 5 (Tyrq-Pro~-Ser ~) G34 h~s atl antigenic determinant in tl~e 1 - 6 r e n a n o f human G34; a second (LI 07) raised to 1- 19 hG34 has an antigenic determinant in the 1 - I 2 region. Both these antisera react weakly with porcine G34. A third antiserum {L331 raised ,~o porcine G34 has an antigenic determinant in the 1 - 1 2 region of this peptide, and reacts weakly with human G34. In human antral extracts fractionated on Sephadex (;50. L66 and L1137 revealed a minor peak of immunoreactivity cerrespondir~g to G34, and a major peak correspor/ding to the NH2-r.erminal tryptic peptide of G34. Concentrations of the latter peptide were closely similar to those of GI7 (i.e. the COOH-terminal tryptic peptide of'G34), consistent with the idea that G34 is cleaved within G-cells by a trypsir~-!ike enzyn,~; to yield GI ?. Antiserum L33 revealed smal~ amour)is of immm~oreactivity ;n an'~ral extracts of dog and cat, but did not reveal significant immunoreactiv:~ty in rat an~rai extracts, in contrast, L66 reacted with rat antral extracts, bL, f not dog or cat. The sequences of G34 in these species are not known, but the results suggest significant differences compared witl; human ar~(l porci~e G34, and i~adicate a high degree of species-specificity with Nl-l~-lerminal G34 antiscra. gastrin; immunochemical studies; region-specific antibodies .
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Address correspondence to' G,.~ Dockrav, Physiological Laboratory., Uni~,,ersity a~ L~verpc 01, Brownlow Hill, P.O, Box i47, !.iverpon! L69 313X, U,K.
170 INTRODUCTION
The principal biologically active fames of the antral hormone gastrin are now estaNished to be peptides of i7 ',amino acid residues (GI 7, little gastrin), and 34 amino acid resi~lues (G34, big gastrin), both of which occur in sulphated and unsulphated forms (Fig+ !) [6,71. The COOH-terminal heptadecapeptide of G34 is virtua.r.ty identical with G17, and is liberated by trypt~y'~ieavage of G34, which suggests that G34 might be a biosynthetic precur,,Jar of GI 7 [ 7,81. In support of this view, equimolar amounts of G 17 and a / peptide with the chroma,ographic and immunochemical properties cf the NJ-12-terminal tryptic peptide of G34 (NTG34) are fo~.:nd in hog an tral mucosa, and have been localized to a single population of gastrin cells [3}. Preliminary experiments+ des.crJbed in more detail below, indicated that the antiserum used to detect porcine NTG34 reacted weakly with human G34. T'~e present series of experiments were fllerefore directed toward the production and cha,racterJzation of antiser~ spedfic for the NH~-terminus of h.'.tm~r, G34 tt~at might in the future be useful for clinical studies, tn additio+~, ';hose antisera were used in studies of antral extracts of some common laboratory mammals in order to provide a basis for future e~perimentaI studie~. MAT ERIA LS AND METHODS
Peptides
The fo!towing peptides, isolated from hog antrai mucosa or human gastrinoma:,, were generously made available by Professor R.A. Grzgory and Dr. H.J. Tracy" porcine sulphated big gastr/n (pG34s), porcine u~tsulphated big gastrin (l:G34ns), porcine sulphated little gastrin (pG17s), hamw~ unsutphaied big gastrin (hG34ns), and human unst~lphated little ge~.,trin (lag 17ns). The following synthetic fragments of hurnan or porcine big ga.~,~ns were prepared ant generously donated by the tal:e Professor G.W. Kerner and members of fie r;eptide synthesis group at tlac Department of Organic Chemistry, University of Liverpool: 1~6, 1-12 and 1~19 sequ,mces of both h~man and porcine C34, and analogues with substitutions at positions 4, 5, ~. 8, 9 (see Table i ar.d Fig. 1 for further details). n rise ra
Three methods were used to obtain at~Iizera spec:~fic for th.~ NH2-terminus of human or porcine G34. (1) Antisera to synthetic 1- 19 hG34 were raised !.~:,,immunization o" tab+ bits with 1-19 hG34 conjugat.qd to bovine seium album~n (BSA) by glutarai ~
t7t
dehyde. Synthetic 1 - 1 9 hG34 (500 nmol) was conjugated to BSA (50 nmol) by addition of 20/al of 5% glutaraldehyde to peptides and protein diluted in a total volume of 0.7 m! t 2~osphate buffer (0.25 M, pI! 7.4). The reaction was allowed to proceed at 22°C for 30 rain, and the reaction products sop,lrated by gel filtration on Sephadex GS0 (t x 30 cm). In a ty|,~cal re~¢lioti the yield was 30% based on the incorporation of a small amount of '~io labelled t - 1 9 hG34 into material eluting in the void volJ'me. Three rabi~i~Ls were immunized by multiple intradermal injections wi¢.: the conj~gate in doses equivalent to 50 nmol 1 - 1 9 hG34 emulsified in Freund's complete adjuvant. The rabbits were boosted at 6-wk intervals with Aoses equivalent to 30 nmot of hapten, and bleedings were taken after 6-113 days. Two of the 3 rabbits produced antisera after the first boost, and titres rose until the fourth boost and the~after were relatively constant, The highest titre antiserum (L107) was selected for further detaiTed study. (2) Antisera to 1 - ! 5 (TyrT-ProS-Ser9) hG34 were raised in rabbits irr~ munized with this analogue conjugated to BSA through the tyrosine residue i~ ~osition 7 using a triazine reaction [~. ]. These conjugates were prepared and generously donated by the late Professor G.W. Kenner and co-workers~ Fi~e rabMts were immunized with the conjugate in doses equivalent to 150 nmol of peptide and were boosted with similar doses using, the schedule described above. Four rabbits survived the immunization course, and 3 of these produced antisera; the highest titre antiserum (L66)was used in the studies described here. (3) Antisera to pG34 were raised in rabbits immunizedwilt, pG34s coniugated to BSA by carbodiimide. Natural pG34s (300 nmol) was conjugated ~o BSA (30 nmot) by addition of l-ethyl-3-(3-dimethylaminopropyl)carbodiimide-HCl (I mg) in a volume of 2,5 ml phosphate buffer (0~I M, pH 7.5). The reaction was allowed t~ proceed a,~ 4°C for 24 ~ and the conjugate was then dialysed against 4.5 litres distil.,'ed water at 4~C for 24 h. The ir~cot-poration of peptide into conjugate was 95% and 55% on two occasion.~, as def.-rmined by recoveI3, of a trace amount of ~31-1abelled pG34 added to the reaco tion mixture. Three rabbits were immunized with doses equivaler~t to 3C nmol pG34s and boosted with 25 nmel equivalent of peptide: ~s des,'ribed above. All 3 rabbits produced anfisera after the second boost, and titrea rose until the fomXh boost and then remained relatively constant. The ~ntiserum (L33) showing the highest ¢itre was used in the present expe,-hnents. Early bleedLqgs of this rabbit contained not only NH2-termi~al ~peci~ic antibodies. but also COOH-terminal specific an~bodies, and it was necessary to remove these by immunoaftSnity adsorption (see below).
:t72 Radiolabelled peptides Two types of ~2Sl-tabelled peptides were routinely used as tracer with NH2-terminal specific G34 antisera. (1) Nal;uml pG34 was labelled with t~Sl by the chloramine T metlmd [10] as foilows: 0.5 mCi tXSl was reacted with pG34ns (0.5 nmel) by addition o f chloramine T (20 nrnoI) iJ~ a total volume o f 50 tsl of phosphate buffer (0.25 M, pit 7.4). The reaction was stopped after 15 sec by addition o f an excess of sodium met~bisv"iphite (500 nmol), and monoiodo G34 separated from free t2*l and unincorporated peptide byion-exchange chromatography on DE cellulose (1 × 10 cat.) eluted wits a gradient from 0.05 to 0.5 M ammonium ,;arbenate using ~. 150 ml conical flask as mixing vessel. The iodinated G34 was presumably labeiled at the tyrosine in the COOH-terminaI re~ion, since trypsinization of the label yielded a single iodinated fragment with the chromatographk and immunochemical properties of the COOHterminal tryptic peptide of G34, i.e. G I7. (2) it proved possibte {o iodina~e the ~.-.12 and 1 ~ I 9 fragment~', of porcine and lxuman G34 by a modification of the chlora:nine T metl'.od that involved tongP,- reaction r:imes and greater concentrations of chlornmine T. Thus peptide t t . 0 nmol) was reacted with ~2Sl (0.5 mCi) by addition or chtor~mine T (350 nrngl) in a volume of 30/al o f phosphate buffer. The reaction was allowed to proceed for 60 set and was stopped by addition o f an exces~ o f sodium metabisulphite as before. There is no ; :rosine in the NH=termit~at region o f G34. and these fragments are presumably iodinated at their histidine residue in position 9. It is known tlaat histidine is relatively more d~'ficutt to iodin~*e than tyrosine I9], which probably accounts for the fact ~hat more extreme reaction conditions were needed. ~ e reaction products were separated by gel filtration on Sephadcx G50 superfine (I × 30 cm) etutod with ammoni.urn bicarbonate and ~,rewashed with ~SA. in~.'orporat~'on of iodine int~ peptides was estimated to l-,e 31% in 4 ct~c2secutive ic~dinations o f I - 1 2 pG34, which corresponds to a specific activity :)f C. 15 rnCi/ nmol. h~ a few experiw.ents a tyrosine-substi~uted analogue of 1 - 1 5 hG34 (i.e. Tyr~-Pr.'.)~-Ser9) was iodinated with ~sI by the cMoramine T method a.s described for pG34 and purified on Sepl:,adex G50 supe,fine (l " 25 cm) eluted with 0.05 M amn~o,fium bicarbonate. Preparatio¢~ o f "mmu~eaclsorba~ r Antisera raise6 to ~latural pG34 origina!Iy contained both C O O H - a n d Ntl2-terminal specific antibodies, and it proved necessary to remove the COOH-terminai reactive population in order to o!~r.~in anlisera of the desired spec.:ificity for ,'be present studies. Th~s was achieved hy immun©adsorp2on of tl~e COOH-termi~a~ reactive population to G i 7 imn-,obitized on Sepha-
t73
rose, Thus pGI 7s (2/~mol) was conjugated to 4 g AH Sepharose (Phr~rr~acia Fine Chemicals) using the metlaods recommended by the manufacturer. Antiserum L33 (1 ml) wao diluted I : i 0 with sodium barbltone buffer (0.02 M, pH 8.4) and gently rotated with 1.5 g Sepharose-GI7 conjugate for 2 h at 22°C. The antiserum was separated by centrifuging and re-treated with a second batch of Sepharose as before. The removal of COOlI-terminal reactive antibodies was monitored by binding of ~:'~t G17 (Fig. 2).
Antiserum specificity Two approaches were used to establish the specificity of G34 antisera. Firstly, the binding of a range of dilutions of antiserum to several different tabeHed peptides was sr.udied. Se~.ondly, using optimal radioimmunoass,ly conditions the relative molar poter'.cies of G34 fragments and analogues were compared for inlfibition of binding of label to antiserum. In routine assays I - ! 2 hG34 was used as a standard with antisera L66 and L107, and 1--12 pG34 was used with L33. Natural hG34 and pG34 were also used as standards where appropriate, tt was found that intact hG34 was slightly more potent than its NH2-terminal tryptie peptide with L66 and El07. The NH2terminal tryptic peptide was, however, equipotent with the synthetic 1--I2 hG34. When necessary, samples, including intact natural G34, were tr)psinized by incubation with I0 gg of TPCK-trypsin (Worthington Biochemica!s) in I.C mt ammonium bicarbonate for 60 rain at 22-'C. The reaction was stopped by briefly heating to boiling point. Radio mtmunoassay systems Antisera were used routinely at dilutions which bound 50'/~ of a tr~tce amount (2000 cpm) of label. Incubations were carried out at 4~C for ¢8 13 i~,l a volume of 2.0 ml of 0.02 M sodium barbitone pH 8~4. containing 0.2% sodium azide and i% (~/v) commercial BSA soluzinr~ (Bo~umin. Orthodi,~t~nostics). Antibody-bound label was ~eparated from free label by addition ~f charcoal (Nodte, BDH) and centrifagaticm a, 2000 x g for lO rnin a~ 4°( '. lt~ assays using pG34 label, 0.2 ml of a suspension rontaining charcoal (!00 tug/ ml), de×tran (I0 mg/ml) and plasma (100/al/ml) was added: in ~say~ u qinLz 1-12 or 1 - 1 9 label, 100 #1 of a suspension of charcoal (i00 mg/ml) and dextran (10 mg/ml) was added. Both antibody-bound label and flee l.~be~ were routinely counted. Control tubes in which antibody was omitted were included for all samples to assess non-specific binding - which was iess titan 8% of total counts in aU studies described here. COOH-tenni~at specific assa~s Samples were routinely assayed with a COOH-terminal specific a~tiserum
!74 (1296) that reacts with G34 about 60% compared with G17, and has previously been described in detail [21. Tissue ex" gracts
Fresh human antra! mueosa was obtained from patients undergoing aatrectomy for recurrent peptic ulcer (n -.--4). Antral mucosa and pituitary glands were obtained from hogs killed at a local abattoir. Ttae pars nervosa was zeparated from pars distatis and the two separately extracted. Antral mucosa of dogs and cats w e r e obtained from Nembutal-anaesthetized anlreals, and antral mucosa of rats obtained from animals killed by a blow on the head~ In all cases tissues were frozen on dry ice as soon as possible and stored at -70~C prior to extraction. The tissues were weighed, thinly sliced, and while still frozen were quickly transferred to boiling water for extract~o~l. The tissues were then boiled for 2 rain (0.! g/mI), homogenized, extracted at 20°C for 30 rain, and centrifuged (2000 × g, 10 rain). Repeated extraction of the pellet in~!~.ted that over 90% of recoverable G17, G34 and NTG34 was obtaine~ in the initial extraction. Pilot ~tudies also indicated that th.~ yield o~" 0oth G17 and NTG34 obtained in boiling water extracts (pH approx. 6.5) was considerably higher than in acetic acid. Tis~.:ae e×~racts were fractionated by gel filtration on columns of Sephadex G~0 saperfine (1 × I00 cm) eluted with 0.02 M ~odium barbitone (pH 8.4). ~SA ~;nd ~2si were added to each sample before elution to provide markers for the void volume, estimated from absorption at 280 ;ira (=0%), and salt region (=I00%). The columns were also calibrated in separate runs with (334, G 17 and the fra;,,ments obtained by trypsinization of G34. RESULTS Antisen~m specificity
tn initial studies the specificity of the antisera was analyzed en the basis of b~rJding properties with various labelled peptides. The two antisera (L66 and L107) raised to fragments of hG34 bound ~2~[-labeIted NH~-terminal 1 - t 2 sequence of hG34, but not surprisingly failed to bind G17, iJ:dicating specificTty for the NHa-terminal region of G34 (Table I). T~-_ese two antisera fa{ied to bind labelled t - t 2 pG34, suggesting specificity for humaa~ versus porcine big ga~trin~. Antiserum L33 raised to pG34 was ini~ially found to bind G17, G34 and NH2-terminal fragments of G34, suggesting the wesence of bo~h COOH- and NIf~.-terminal specif~:.: araribody populations. However, treatment with G ~,7-conjugated Sepharose removed the COOH-termhaz! reactive antibodies, so that the treated antiserum no longer bound G I7 label, but still bound iodinated G34 and its 1-12 NP~-termi~iat fragment (Fig. 2),
t,'5 TABLE t
Bii~ding properties of three G34 antlsera with various labelled peptjdes Anti-
Antigen
Label
sor~lm
pG34
hG 17
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L33 *
pG34
1,66
1-15 <1 : 100 (Tyr 7-Pro8Ser9) hG34 1-t9 hG34
L107
l : 3000
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1-t2 pG34 .
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<1 : t00
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~
1---t 2 hG~t4 . . . . . . . . . . . . . . . . . . . . . . . . .
t :2500
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1-15 (Ty r "~4'rn ~. .
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t00 < t ; 1(~fJ
<1 : I 0 0 < I :
t00
i :
4000
<1 :t00 < I :
t00
t : t0000
1 : 704)CI
-~
The dilution of ax:tJ.serum binding 50% of the tracer ( 2 0 0 0 cpm) is shown. * After remm, al of C O C H . t e r m i n a l antibodies by imrm, noadsorption (see t~,'xt).
although with slightly Iower titre. Antiserum L33 failed to bind ! --12 hG34. indicating specificity for porcine compared with human big gastrir:- (Table i). Detailed studies of speci[icity were based on competitive binding exped~ ments in which fragments and analogues of the NH2-temlinaI sequence of G34 were cempared for their capacity to inhibit binding of" label to a~tiserum, The antisera raised to hG34 bound 1.-12 hG34, ! .... 19 hG34 and intact hG34 with approximately similar potency (Table II). With L66 the immunochemical potency of 1--6 hG34 was not markedly different from tI~;~ of t - 1 2 , indicating that the antigenic determinant [or L66 lie; wifllin the NH2-terminal hex.apepfide. Moreover, with this antiserum the porcine t 6 fragment was significantly less active than its h~man counte.rpart; since th,_~ge peptides differ only in the substitution of Pro for Leu at position 4 i~ is clear that this residue is an important part o f the antigenic de~ermina~l f(;~ L66. la keeping with this observation, it was found that other peptides w~tfl substitutions in positions 4 or 5 had marked|y reduced immunochemi~::~ r etency with L66, bat substitutions in positions 7. 8 a~d 9 had little ,.)r rL(,' influence on immunoreactivity. In contrast, with antiserum L~0?, the NIt~terminal 1~6 fragment had low immunoreactivity compared with 1-. t2. Moreover, substitutions in posifiens 4, 5, 7, 8 and 9 ail caused a :;harply dkmini~i~ed immunoreactive potency. Together the results indicat<.- ti~,~t L~07 is sgecific for at least the 4 - 9 sequence of hG34. With antiserum L33 the t ~ 1 2 and i - 1 9 fragments of pG34 had ~milar immunoreactivity to intact pG34~ but the t-.-6 fragment was markedly less active. A~ important par~ of the antigenic determinant for L33 was th-?reg>re
176 TABLE II lmmunoci~emical potencies of NH2~terminal fragments and analogues o f G34 with three antisera * Peptide
Antiselx~m ~L66
Lt07
L33
lluman G34 fragme,tts }-12 hG34 hG34n~ NTG 34 I ~19 hG34 I - 6 hG34 i -- ~3 (TyrT-Pro~-Ser 9) t --, i 2 (HisT-ProS-Pr99) i ~ 12 (Ser%Pro~-Ifi,~ 9) 1 --I 2 (Fro%SerB-His 9) t - 12. (His °.-ProS-Set 9)
1.00 2.t 0,90 1.4 0.54 0,7 1.0O 0,55 0,66 0.8 2
1.00 2.4 1.10 3,2 0.0005 0.003 0.051 0.11 0.003
0.0003 0,02 0.017 0.000 002 0.03 0.000 012 0.000 005 0.000 004 0.000 009
Poeci,~e G ~4 _fragments t - i 2 pG 34 pG34 NTG34 i - 1 9 p(;34 1-6pG34 t - t 2 (HisT-ProS-Ser 9) 1 ~ ! 2 (HL~%ProS-Pro 9)
0,1300 35 0,001 4 0.000 3 0.000 3 0.000 52 0.000 7
0,000 0.00][ 0.002 0.000 0,000 0.000
Ottmees 1 - t 2 (Gln4.ProS-His~'-ProS-Set 9) | --I 2 (Gln4-LcuS-His'LProB-Ser 9)
0.000 0.000
0.000 3 0.000 3
2 3
3 8 6 5 3 3
1.00 2.4 1.15 1,8 0.000 0~000 0.00t
02 34 4
0.t3.)0 001 0.0'O0 00 ]
* Immunochemical potencies are expressed relative to 1 - 12 }zG34 m the ca.-e of L66 and L I 0 7 , and relative to I ~ 1 2 p G 3 4 in the case of L33. Potencies were calculated on the basis of the ratio of concentrations of 1 - 1 2 G34 and tes~ peptide needed to mhibR bindhlg of la~c] t~ a.utiserurn by 50%. See Fig. 1 for sequences of [he a~thentic p,_:ptides.
localized to the 7 - 1 2 region. I-Iowevcr, since human [ - 1 2 was significantly less active than porcine t - i 2 G34, and these peptides differ only at position 4, this residue was also an important p,~rt of the antigenic determkL'~t for L33. Analogues with substitutions at posit~ons 4.5, 7, 8 and 9 atl had significantly lower immunoreactivity compared wi~h !:he native sequence of !~ 12 pG34, again confirming the importance of the 4~- 9 region. The resuAs of the specificity swdies are summarized in Figure i.
177
TRYPSIN T
~r
PG34: .GL~PL~L~O~L.Y_-.L._Eu_-. . _G_._L_N _-_6Ly-PRO- PRO-HI s-LEu.-VAL-ALA-AsP-LEu-AL),-LYS-LYs-GL~-$t.YL33 HG34: 'GLP~LE~Gt~Y~PR~GL~m~LY~FR~-P~-Hi~LF~"VAL~ALA~sP~PR~ER~LY~;~LY~5U~G~Y -
L66. ........
. . .
--
= - - - - - ----L 1 0 7
20
PG311:
54
30
- PRr3-TRP-MET-(GLu)5-ALA-TyR-GLY-IRP-MET-AsF-FHE-N_ ~ .
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1296
HG5,:
- PRO-TRP-LEo-(GLu)5-ALA-TYR-GL~'-TRP"-M~i-As;--PHE;~-H2"
Fig. 1. Amino acid sequence of porcine 0 3 4 (pG34) a~>~dhuman G34 (hG~,i), showing antigenic determinants for 1296, L33, L66 and L107, For details about artdserum specificity see text and Tables I and II. Note that these sequences differ from tiaos~ erigina]ly described [8] ir~ the 7 - 9 ;region ~4].
~L~}PG34
~I HG17
L~ Z
DtLUTtON OF ANTg_,~:RUM Fig. 2. Binding of 12sI-labelled G34 aI~d G 17 by antiserum L33 before and aflEer ir.im~r~osorp~ion w,th G~7 con3t-'.gate4 I'o Sephamm to remove COOH-termii~at spetiii: an~ibodies,
17g
Antrat extracts." man and pig
Extracts of human arttral mucosa inhibited binding of label to L66 (Fig. 3) and LI07 (not shown) in paraltet with 1--12 hG34. Similarly extracts of porcine anlral mucosa produced a graded inhibition of binding of label to L33 that was paral]el to 1- 12 pG34 (Fig, 4). These results establish the validity of assays with these antisera for the estimation of NH2-termina! immunoreactivity in antral extracts. in human antral extracts the concentrations of immunoreaetive material measured with L66 (21.4 _+ 1.7 nmoI/g, n---4; 1-12 hG34 standard)were comp~rabEe to those measured with the COOH-lerminal assay using antiserum 1296 (17.9 +_.2.1 nmol/g; G17 standard). Similar results have been obtained previously for hog antral mucosa using antisera L33 and 1296. Fractionation of' hog antra! exeracts or.~ Sephadex G50 revealed a major peak of im~nunoreactivity with L33 that eluted just before the main peak read by 1296 (Fig. 5), Both antisera revealed a minor peak of immunoreactivity eluting in the position of G34. ~l~e m~:jor peak read by L33 emerged in a simi/ar position ~o the NH~-terminal reactive fragment produced by tryptic cleavage oi G34, while that read by 1296 corresponded to G17 (Fig. 5). Closely
Ab L66 100"
130• [&
roB0. ...I
\ Z
z
F'EPT~DE COi~C~?¢tTRAT~C,~-,! [ p moles/I
ib
~J~,q~l/~_. c~I"I~IACTS Ing oeiuiuvdent/ml ] Fig. 3. CompetRive binding curves for antiserum L66 and 1 - 12 hG34 label with a l~un~.an anttal ¢xb'act (concentration expressed it., ng equivalents ,~: ~.nrral muct, sa), hG34, t ~, 12 nG34 and I~-12 pG34. Binding is expressed as a percentage t:~f IL:_ initial ratio of b¢}u a to free label in the absel~ee of competii~g peptides.
179
Ab L 33 lot~
80
O
Extract Z t~
¢t,u 20i1 .
.
,
1"o' PEPTIOE CONCENTRATION [ p m o ~ s / I ] ANTRAL EXTRACTS [ ng eq~J~valent/ ml]
Fig. 4. Competitive binding curves for antiserum L33 and 1-]2 p(;34 label with a hog ontral extract, pG34, I ~ 12 pG34 and 1- 12 hG34. See t:~g. 3 far further details.
similar results were obtained for human antral mucosa fractionated by gel filtration and assayed with antisera L66, L107 and 1296 (Fig. 6). Tlms L66 and L I 0 7 revealed a major peak of activity eluting tn the position of tt~c NH2~terminal tryptic peptidc, and !296 revealed G I 7-like activity. All 3 an~isera revealed a minor peak eluting in the position of G34, but interestingly the concentrations of this material measured by. L66 were significar~qy higher than those with L107 and 1296. Further work i~ needed to establis~ the significance of this observation, tn the past Rehfetd [ 12] has described a small peak of immunoreactive gastrin (designated component l) that eiutes just before G34 on gel filtration, and reacts with COO[-J-~erminal sp.ccif~c antisera. In the present study no s~gnificant arnou~t~ of componer~t [ ',yore found in antral extracts. However, a partially purified preparation of compr,o nent [ (generously donated by Dr, H.J. Tracy), which had been obtained from a human.gastrinoma and was essentially free of G34 and ()17o was found to react with both L66 and L ! 0 7 . The concentration of this component measured with L66 and L107 were comparable to those measured with 1 296, when G34 was ased as a radioimmunoassay standard. A n t r a l extracts." d(.g, cat and rat In antral extracts o f dog and cat, antiservm 1296 revealed concentrations
180
I
15"
'~"i
pG34,TRYI~INIZED
10'
5"
z
0
!
J
t~
HOG" ANTRAL MUCOSA
,
~
10"
A,L PERCENTEUJTIONVOLUMEF~OTEINTO'~I Fig. 5, Fractione~2ion o n S e p h a d e x GS0 o f an extract o f hog antrat m u e o s a tic- .vet panel), and a ~n~!pt:~ (~f p ( ; 3 4 treated with trypsin ( u p p e r panel). C o J u m n eluate~ w,.'re assayed with antisera L't3 !o . . . . . -o) and 1296 (o- . . . . ~1. Elut~ion p o s i t i o n s of G 3 4 and G I 7 are indicated b y arrows .~t t'.l~ top.
of immunoreactivity that were c(~mparable to those in man and pig (,tog, mean 6.5 nmol/g, n = 2; cat, 17.4 nmo!/g, n - 2). In contrast, antiserum L33 revealed a b o u t 20 times lower concentrafio~~ (dog, 0.34 nmot/g; cat, ~ ~" nmo//g), and L66 ~nd L107 fai!ed to demonstral~.: measurable amount,, ~f immunoreactivity. Fractionatior~, o f dlese extracts on S~:phadex G 5 0 revealer patterns of immunoreactive c o m p o n e n t s similar to those iv. pig .rod man, i.e.
18;I NTG34
30
L i07
L~
kl.l
30 ~ .J
L66
0 O Z 20>.-
.> loI,,-
~:0
020"
~
'
~
~
O 20 4.0 60 80 tOO P£RCENT F..LUTION VOLUME PROTEIN TO '2'~1
I~ig. 6. Fractton~tion on Bephadex G 5 0 o f an extract o f human ant.r~i m u c o ~ . Cctumn L107 (Zop panel), In s e p a r a t e r u n s t h e c o l u m n s were emlibrated w i t h G 3 4 a n d its t y p t i c peptides -~ N'I G 3 4 and G 17 - t h e elution p o s i t i o n s o f w h i c h are i~dicated b y a n o w s at t h e top.
eiuates were azsayed with ~296 (lower p~nel), L66 (middle panel) and
a mii~or peak corresponding to G34, aim major peaks corresponding to G I / and NTG34 (Fig. 7). The concentrations of G34qike activity measured with L33 were approximately 5% those estimated with 1296. When the peak corresponding to G34 was pooled, lyophi!ized and digested with trypsin° two peaks of activity were get, crated that had the gel filtration and immunochemical properties of G17 and NTG34 (Fig. 8), but again concentrations measured with L33 were about 5% those with 1 296. in rat antrai extracts 1296 measured 4.2 _+ 0.9 nmoltg (n = 4) compared with 4.7 _+ 1.3 nmoI/g with L66.~ and < 0 , t nmoi/g w~th LI07 and L33. On Sephadex gel filtration the rat extracts were re~olved into a minor peak of G344ike activity that reacted with both L66 and 1296, and major pt.aks of activity corresponding to G] 7 (1 296) and an NH~-terminal fragment of G34 (L66) (resutts not shown),
182
~G34
~GI/ -70
?, / | i
t
.' ~q
;
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i
-50
-40 1'5-
L33
~.,,..,+
i
~ :1296
I-
.j J
O"
-~0 <
__.. 2-0-
e e
t
R~ -40
-30 ~-0-20 05. FaERCEN7 EL.UTION MOi.tJME PROTEIN 3"O'~-3 Fig. 7. F r a c t i o n a t i o n z~ ,g~l,t~adex G 5 0 of e x t r a c t s of dog antrai m u c o s a ( b o t t o m pa;~el) and cat antzai mucosa ( t o p p~nel). F o r details see ~:igs. 5 and 6. Note that the scal,, fo~ assays with L33 (left side) differs fro~t: t.ha! for 1 2 9 6 (i-ight side).
N o tt-an I r a / t & s u es
Antiserum L33 did not reveal significant amoun.~ of immunoreactiv~v (<0.0t nmot/g) in extracts of hog gastric fundus, duodc~ra,.iejunum~ ileum or colon. Rehfeld previously reported that in b,~h adenob, yp~,~physis and neurohypophysis of the pig p~:~.~itaz-y there were small amounts of g~:~stfin
34 ¢17 r4000
~7 ,,,x. i t00+ ~4~-iti
I .: ~e
t;!
j,,x7
,-
li-~,.l£~-t
"~r~ ,0
~.
PERCENT ELATION VOLUME !'~OTEtN TO "miq
Fig. 8, lFr&ctio.~ation ori Sephad~z GS0 of pooled concvsl.trate of the (i34 ~'a~:~ion~ ot czf antral extracts previousiy run on Sephadex (bottom pallel) an(t the .~me con;:er,.rzHe after dige,~tion with trypsin (top panel). For further det~il~ see Figx, 5, 6 nnd 7,
G34 ~
JGt?
w ~ 800t ,
3 z _I
0
j 600j i
~00~
Z
~>
'
0 20 .~0 60 .80 ~j PER(SENT ELUTIO"J VOLUME PROTEIN fQ ~-~'#
Fig. 9, Fractionation on $ephade× GS.0 of an extract of hog posterior pitl~i,taty, F'~ details see Fig. 5.
t84
[ 13] In the present study, extracts of pig pars nervosa contained 30.6 +i l . 3 pmol/g immunoreactive gastrin measured witt] 1296, and 34-~ 12 pmol/g (n = 4) with L33. Extracts of adenohypophysis contained less than 2 pmoI[g with 1296, and were not studied further with L33, On Sephadex G50 gel filtration the pars nervosa activity eluted as about equal amounts of G34 and GI 7 (I 29671 and NTG34 ~L33) (Fig. 9). DISCUSSION [n the present study radioimmurmassays have been developed using anti-
sera reacting at the NHz-terminus of human G34, These assays have been used to identify and estimate molecuIes witll the properties of G34 and its N]-l:-~erminaI tryptic peptide in antral extracts of mar~ and other mammals. The present studies confirm and extend those previously obtained in pig [3 ]. The Ni-l~-.:erminat specific antiserum (L33) used in previous studies has been shown here to react weakly with human G34, and is therefore of limited use. "lwo approacfles were used to o~tain antisera specific for the NH2-terminu.~ of hG24. In both cases rabbits were immunized with NHa-terminal frag,,ner~ts cou;~led to carrier proteins iz~ such a way as to maximize *~e chances of o~.tain;-ng NHz-terminal reactive antisera. In one case conjugation was achieved '~y triazine coup}ing through an analogue substituted with tyrosine at position 7, and in the other case by glutaraldehyde coupling through *.he epsilon a~nino groups of lysines at position 16 and 17. The a,,.tisera obtained reacted better with human {ban porcine 1--12 G34, suggest.rag specificity for a region iz:cluding posi{~on 4. The antigenic determinant of one antiserum was iocalize~-{ to the Nl-12-ter~ninal hexspeptide (L66), and that of the other inch~ded at least the 4 ....9 region. Interpretation of previous immun,;,:hemical studies on big gastrin has been complicated by an error in the originally reported sequence. Thus synthetic peptides with tile original sequence (parclue: His~-Pr@-Prog: human: His'-ProS-Ser~) had iGw immuno~be:,qk:al ~,otency wi,h antiserum L33 compared with natural peptides. In th.. preser~? study ]t was ,it~own that antiserum L66 witll specificity for the 1 .-6 sequence of hG34 did not di.~,criminate beiween fragments of G34 with the original a~d the revised s.equerlce, whereas antiserum LI07, wlaic]~ has an an{agenjc determinant extending into the 7~9 region, clearly distinguished bet~,.'een peptides with the original seq~ef~ce, which had low potency, and those wiih the revised sequent.e, which were fully active compared with natural hG34. Broadly similar laalterns of immunoreactJve forms of gastriri were seen when L66 and LI07 were used to assay human antral extracts after gel filtralion. In both cases a major peak of immunoreacli'eity elated in the position of NT(;34. The concentrations of NFIa-termina] fragment were. similar
t85
to those of GI 7 -- wilich represents the COOH-terminal try. ptic peptide of G34. These results therefore a~'a'ee with, and extend, those atready obtained in pig, and are consistent with the cleavage of G34 within gastrin cells by z trypsin-like enzyme, Recently, ~'anaihara et ai. [141 have reported similm' findings using several NH~-terminal specific G34 antisera. In one respect their results differ from the present findings, in that on Sephadex G50 they id,elltiffed large amounts o f NH~-terminal immunoreactivity eluting after GI;'. whereas in the present study there is a major peak etuting betbre G17. "l'h~~. reason~ for these differences remain to be established. Antiserum L66 wo~Id have been capable of demonstrating NHT-terminal fragments as small as the I - 6 sequence, but no evidence of significant amounts o f sttch fragment,., were found in the human tissue extracts. The amounts of G34-1ike material measured by L66 were slightly, buz consistently, higher than those measured wiZh 1296 and L107, The significance of this is unclear, but it is of interest that in hog antral mucosa there it; evidence of a form of G34 that reacts with NH~-terminal but not COOHtet~nina~ specific antisera [51, and may perhaps be a COOH-terrni~al extend~:d gastrin. Conceivably there is a form of G34 extended at either COOH-terminus or NH2-terminus which reacts better with L66 than other anfisera. In this context it is significant that. in studying hog g astrb~ rnRNA, Noyes et at. [ 11 ] have obtained evidence for a precursor o f al least 140 residues, which is likely to contain G34 in its mid-region Thus. in ' ~ conversion o f precursor to G34, both COOH- and NH~-terminal extended forms of G34 might be fovned. The different patterns of immunoreactive material measured bv L33, L66 and L 107 in rat, dog and cat coutd be explained by species differences in the sequence of the NH~-t,=rminal regions o f G34. Because COOH-terminal regions are important in determining the biological a~tMty of these peptid ~.s, one wo~dd anticipate that these reNons might be conserved, whereas NH~terminal regions are relatively unimportant and are not conserved. The present study opens the way for more deZailed ar~atysis of the .forn.s of gastfin in human blood and tissues. The currently avai.~able NH~-termin~l specitqc ar._tisera have relatively tow affinity, and are unsuitable for ,Jire~ as;ay of circulating ga~trin~ However, there is no reason wl~y high-affinity antisera might not be obtained in the fut:tre. Alternatively. one might concentrate the immunoreactive forms in blood prior to assay_ Using the latter approach we have recently obtained evidence for the existence of tow concentrations of NH2-terminal fragments o f G34 in the peripheral circular.ion of man after feeding (Pauwels and Dockray, unpubl, studies). Further sttadies of these forms should offer insigh~ into the mechanisms of synthesis and secretion of gaxtrin in health and disease.
156 ACKN(3'WI.EDGEMENTS
TMs work was supported by a g~'ant from the MRC. Skilled technical assistance was pcovided by Carol Higgin~, Elaine Clarke al~d Gavin Laing: Mrs. Eva McElroy kindly typed the mat~uscfipt. Professors R.A. Gregory and G.W. Kenner and Dr. H.J. Tracy generou~ly provided peptide samples. R I.~.l'~E R ENCES I Agarwal, K.L., Grudzinska, S., Ken~,~er, G.W., Rogers, N.H., Sheppard, R.C. and Mc L GuJgan, J.C., lmmunochemicat differentiation between gastrin and feinted peptide hormones through a hover conjugation of peptides to proteins, Experientia, ] 7 (197 ! ) 5~-5~5, 2 D~ckray, G.J. ~ d Walsh, J.|-l., Amino terminaI gas~rin fragment in serum of Zoliing~.r --EItisor~ syndrome patients, G~stroenterro~ogy, 68 (1975) 2 2 0 - 2 3 0 . 3 Dockray, R.J., VaiHant, C. and Hopkins, C.R., Biosyrtthetic relationships of big and little g~t~-~na, N~itu~e (London), 274 (1978) 770-.-772. 4 Dockr.~y, G.J., Gregory, R.A~, Kenner, G.W., Moore, S,, Rama~:handren, K.L. and 'damage, R., The use of immunochemicat methods in strtictural studies ~n hu~;~an big gus~rin. In A. MiyosIli (Ed.), Gut Peptides, Kodanstla, Tokyo, ! 97~), pp. 3 8 - 4 0 . 5 Gregory', R.A., Some asb",:ets of the struc[ure of g~stril~ and gastric-like ft~rms and fragments in gut and brain. In J.F. Rehfdd aud E. Amdrup (Eds.), G~strins and the Vagus, Academic Press, London, 1979, pp. 4 7 - 5 5 . 6 GregorT, R.A. and Tracy, t1.J., The constitution a~ad prope_~r~s of two gastrins extracted from hog ar~tJ-al mu~ '~sa, Gut, 5 (1964) 103 - 117. 7 Gregory, R.A. and Tracy, H J., !sol~ioa of two 'big ga~t~ins' from Zollir,ger-Ellison turnout ti~:~ue, Lancet, 2 (19-'2) 797-799. 8 Gregory, R.A. and Tr~c?, H.j., The chemistry of the g~.strins: So,he rece~, advznces. ;n J,C, Thompson (Ed°), Gastrointestinal Hormones, University ~)f t e x a s Press, Austin, Te:~as, 1975, pp. I3-24. 9 Hughes, W.L., The chemistry of iodination, N.Y. Acad. $c~., (t o5 7) 3- i 8. 10 Hunter, W.M. and Greenwood, E.C., Preparation of iodine ~ 1abetted growth hormone c~f high ~pecific activity, Nature (London); ! 94 ( ~962) 495 -496. i t N oyes~ ~.~.i., Mevm'ech, M., Stein, R. and Agarwa~. K.L., Detection :rod partial ~equence a~:~.~;~s of gastrin mRNA By using an oligodeonynuc]eotMe pr.~be, Prec. Nnt. Acad. Sci. USA, 76 fl979) 1770-.I774. 12 Rt~hfeld, J.F., Three components of gastrin in human serum. Gel filtration studies o.~ the molecular size of ~mmur~ereactive seru:~ ga.~trin, Biochim, Biophys A e t a 285 (1972) 364-372. ! 3 Rehfeld, J.F., Localisation of gastritis ,~ ne~ro- and adenohypophysis, Nature (~..ondon), 271 (]978) 771-773. 14 Yanzilmra, M., Yanaihara, C., Ozaki, J., Mochizuki~ "F., Shimizu, F., Mihara, S., NagM. K., Sato, H. and Sakagarni, M., Human big gas~rin N-~cr;i',i'aal fragment immunorc~..* tivity. In A. Miyoshi (Ed.), Gut Peptides, Kodansha, Tokyo, ~970, pp. 16-33.
l ~',_?
IMMUNOCHEMICAL STUDIES ON BIG GASTRIN USING NHz-TERMINAL SPECIIrC ANTISERA
G.J. DOCKRAY
Physiological Labor~to~'y° UniuersiO, o f l.,i~e,pao!, L i~,erpoot L69 3BX. l..!nHed .Y,itrg~L~m Accepted for l)ublieation tt August 1980
SUMMARY
Methods are described for obtainiog antisera specific for the NH,-terminal regions of human and porcine big gastrin (G341 that can bc use'~l in radioimmunoassays. Three antisem have been characterized in detail" one (L66~ ,aised to human 1 - t 5 (Tyrq-Pro~-Ser ~) G34 h~s atl antigenic determinant in tl~e 1 - 6 r e n a n o f human G34; a second (LI 07) raised to 1- 19 hG34 has an antigenic determinant in the 1 - I 2 region. Both these antisera react weakly with porcine G34. A third antiserum {L331 raised ,~o porcine G34 has an antigenic determinant in the 1 - 1 2 region of this peptide, and reacts weakly with human G34. In human antral extracts fractionated on Sephadex (;50. L66 and L1137 revealed a minor peak of immunoreactivity cerrespondir~g to G34, and a major peak correspor/ding to the NH2-r.erminal tryptic peptide of G34. Concentrations of the latter peptide were closely similar to those of GI7 (i.e. the COOH-terminal tryptic peptide of'G34), consistent with the idea that G34 is cleaved within G-cells by a trypsir~-!ike enzyn,~; to yield GI ?. Antiserum L33 revealed smal~ amour)is of immm~oreactivity ;n an'~ral extracts of dog and cat, but did not reveal significant immunoreactiv:~ty in rat an~rai extracts, in contrast, L66 reacted with rat antral extracts, bL, f not dog or cat. The sequences of G34 in these species are not known, but the results suggest significant differences compared witl; human ar~(l porci~e G34, and i~adicate a high degree of species-specificity with Nl-l~-lerminal G34 antiscra. gastrin; immunochemical studies; region-specific antibodies .
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Address correspondence to' G,.~ Dockrav, Physiological Laboratory., Uni~,,ersity a~ L~verpc 01, Brownlow Hill, P.O, Box i47, !.iverpon! L69 313X, U,K.
170 INTRODUCTION
The principal biologically active fames of the antral hormone gastrin are now estaNished to be peptides of i7 ',amino acid residues (GI 7, little gastrin), and 34 amino acid resi~lues (G34, big gastrin), both of which occur in sulphated and unsulphated forms (Fig+ !) [6,71. The COOH-terminal heptadecapeptide of G34 is virtua.r.ty identical with G17, and is liberated by trypt~y'~ieavage of G34, which suggests that G34 might be a biosynthetic precur,,Jar of GI 7 [ 7,81. In support of this view, equimolar amounts of G 17 and a / peptide with the chroma,ographic and immunochemical properties cf the NJ-12-terminal tryptic peptide of G34 (NTG34) are fo~.:nd in hog an tral mucosa, and have been localized to a single population of gastrin cells [3}. Preliminary experiments+ des.crJbed in more detail below, indicated that the antiserum used to detect porcine NTG34 reacted weakly with human G34. T'~e present series of experiments were fllerefore directed toward the production and cha,racterJzation of antiser~ spedfic for the NH~-terminus of h.'.tm~r, G34 tt~at might in the future be useful for clinical studies, tn additio+~, ';hose antisera were used in studies of antral extracts of some common laboratory mammals in order to provide a basis for future e~perimentaI studie~. MAT ERIA LS AND METHODS
Peptides
The fo!towing peptides, isolated from hog antrai mucosa or human gastrinoma:,, were generously made available by Professor R.A. Grzgory and Dr. H.J. Tracy" porcine sulphated big gastr/n (pG34s), porcine u~tsulphated big gastrin (l:G34ns), porcine sulphated little gastrin (pG17s), hamw~ unsutphaied big gastrin (hG34ns), and human unst~lphated little ge~.,trin (lag 17ns). The following synthetic fragments of hurnan or porcine big ga.~,~ns were prepared ant generously donated by the tal:e Professor G.W. Kerner and members of fie r;eptide synthesis group at tlac Department of Organic Chemistry, University of Liverpool: 1~6, 1-12 and 1~19 sequ,mces of both h~man and porcine C34, and analogues with substitutions at positions 4, 5, ~. 8, 9 (see Table i ar.d Fig. 1 for further details). n rise ra
Three methods were used to obtain at~Iizera spec:~fic for th.~ NH2-terminus of human or porcine G34. (1) Antisera to synthetic 1- 19 hG34 were raised !.~:,,immunization o" tab+ bits with 1-19 hG34 conjugat.qd to bovine seium album~n (BSA) by glutarai ~
t7t
dehyde. Synthetic 1 - 1 9 hG34 (500 nmol) was conjugated to BSA (50 nmol) by addition of 20/al of 5% glutaraldehyde to peptides and protein diluted in a total volume of 0.7 m! t 2~osphate buffer (0.25 M, pI! 7.4). The reaction was allowed to proceed at 22°C for 30 rain, and the reaction products sop,lrated by gel filtration on Sephadex GS0 (t x 30 cm). In a ty|,~cal re~¢lioti the yield was 30% based on the incorporation of a small amount of '~io labelled t - 1 9 hG34 into material eluting in the void volJ'me. Three rabi~i~Ls were immunized by multiple intradermal injections wi¢.: the conj~gate in doses equivalent to 50 nmol 1 - 1 9 hG34 emulsified in Freund's complete adjuvant. The rabbits were boosted at 6-wk intervals with Aoses equivalent to 30 nmot of hapten, and bleedings were taken after 6-113 days. Two of the 3 rabbits produced antisera after the first boost, and titres rose until the fourth boost and the~after were relatively constant, The highest titre antiserum (L107) was selected for further detaiTed study. (2) Antisera to 1 - ! 5 (TyrT-ProS-Ser9) hG34 were raised in rabbits irr~ munized with this analogue conjugated to BSA through the tyrosine residue i~ ~osition 7 using a triazine reaction [~. ]. These conjugates were prepared and generously donated by the late Professor G.W. Kenner and co-workers~ Fi~e rabMts were immunized with the conjugate in doses equivalent to 150 nmol of peptide and were boosted with similar doses using, the schedule described above. Four rabbits survived the immunization course, and 3 of these produced antisera; the highest titre antiserum (L66)was used in the studies described here. (3) Antisera to pG34 were raised in rabbits immunizedwilt, pG34s coniugated to BSA by carbodiimide. Natural pG34s (300 nmol) was conjugated ~o BSA (30 nmot) by addition of l-ethyl-3-(3-dimethylaminopropyl)carbodiimide-HCl (I mg) in a volume of 2,5 ml phosphate buffer (0~I M, pH 7.5). The reaction was allowed t~ proceed a,~ 4°C for 24 ~ and the conjugate was then dialysed against 4.5 litres distil.,'ed water at 4~C for 24 h. The ir~cot-poration of peptide into conjugate was 95% and 55% on two occasion.~, as def.-rmined by recoveI3, of a trace amount of ~31-1abelled pG34 added to the reaco tion mixture. Three rabbits were immunized with doses equivaler~t to 3C nmol pG34s and boosted with 25 nmel equivalent of peptide: ~s des,'ribed above. All 3 rabbits produced anfisera after the second boost, and titrea rose until the fomXh boost and then remained relatively constant. The ~ntiserum (L33) showing the highest ¢itre was used in the present expe,-hnents. Early bleedLqgs of this rabbit contained not only NH2-termi~al ~peci~ic antibodies. but also COOH-terminal specific an~bodies, and it was necessary to remove these by immunoaftSnity adsorption (see below).
:t72 Radiolabelled peptides Two types of ~2Sl-tabelled peptides were routinely used as tracer with NH2-terminal specific G34 antisera. (1) Nal;uml pG34 was labelled with t~Sl by the chloramine T metlmd [10] as foilows: 0.5 mCi tXSl was reacted with pG34ns (0.5 nmel) by addition o f chloramine T (20 nrnoI) iJ~ a total volume o f 50 tsl of phosphate buffer (0.25 M, pit 7.4). The reaction was stopped after 15 sec by addition o f an excess of sodium met~bisv"iphite (500 nmol), and monoiodo G34 separated from free t2*l and unincorporated peptide byion-exchange chromatography on DE cellulose (1 × 10 cat.) eluted wits a gradient from 0.05 to 0.5 M ammonium ,;arbenate using ~. 150 ml conical flask as mixing vessel. The iodinated G34 was presumably labeiled at the tyrosine in the COOH-terminaI re~ion, since trypsinization of the label yielded a single iodinated fragment with the chromatographk and immunochemical properties of the COOHterminal tryptic peptide of G34, i.e. G I7. (2) it proved possibte {o iodina~e the ~.-.12 and 1 ~ I 9 fragment~', of porcine and lxuman G34 by a modification of the chlora:nine T metl'.od that involved tongP,- reaction r:imes and greater concentrations of chlornmine T. Thus peptide t t . 0 nmol) was reacted with ~2Sl (0.5 mCi) by addition or chtor~mine T (350 nrngl) in a volume of 30/al o f phosphate buffer. The reaction was allowed to proceed for 60 set and was stopped by addition o f an exces~ o f sodium metabisulphite as before. There is no ; :rosine in the NH=termit~at region o f G34. and these fragments are presumably iodinated at their histidine residue in position 9. It is known tlaat histidine is relatively more d~'ficutt to iodin~*e than tyrosine I9], which probably accounts for the fact ~hat more extreme reaction conditions were needed. ~ e reaction products were separated by gel filtration on Sephadcx G50 superfine (I × 30 cm) etutod with ammoni.urn bicarbonate and ~,rewashed with ~SA. in~.'orporat~'on of iodine int~ peptides was estimated to l-,e 31% in 4 ct~c2secutive ic~dinations o f I - 1 2 pG34, which corresponds to a specific activity :)f C. 15 rnCi/ nmol. h~ a few experiw.ents a tyrosine-substi~uted analogue of 1 - 1 5 hG34 (i.e. Tyr~-Pr.'.)~-Ser9) was iodinated with ~sI by the cMoramine T method a.s described for pG34 and purified on Sepl:,adex G50 supe,fine (l " 25 cm) eluted with 0.05 M amn~o,fium bicarbonate. Preparatio¢~ o f "mmu~eaclsorba~ r Antisera raise6 to ~latural pG34 origina!Iy contained both C O O H - a n d Ntl2-terminal specific antibodies, and it proved necessary to remove the COOH-terminai reactive population in order to o!~r.~in anlisera of the desired spec.:ificity for ,'be present studies. Th~s was achieved hy immun©adsorp2on of tl~e COOH-termi~a~ reactive population to G i 7 imn-,obitized on Sepha-
t73
rose, Thus pGI 7s (2/~mol) was conjugated to 4 g AH Sepharose (Phr~rr~acia Fine Chemicals) using the metlaods recommended by the manufacturer. Antiserum L33 (1 ml) wao diluted I : i 0 with sodium barbltone buffer (0.02 M, pH 8.4) and gently rotated with 1.5 g Sepharose-GI7 conjugate for 2 h at 22°C. The antiserum was separated by centrifuging and re-treated with a second batch of Sepharose as before. The removal of COOlI-terminal reactive antibodies was monitored by binding of ~:'~t G17 (Fig. 2).
Antiserum specificity Two approaches were used to establish the specificity of G34 antisera. Firstly, the binding of a range of dilutions of antiserum to several different tabeHed peptides was sr.udied. Se~.ondly, using optimal radioimmunoass,ly conditions the relative molar poter'.cies of G34 fragments and analogues were compared for inlfibition of binding of label to antiserum. In routine assays I - ! 2 hG34 was used as a standard with antisera L66 and L107, and 1--12 pG34 was used with L33. Natural hG34 and pG34 were also used as standards where appropriate, tt was found that intact hG34 was slightly more potent than its NH2-terminal tryptie peptide with L66 and El07. The NH2terminal tryptic peptide was, however, equipotent with the synthetic 1--I2 hG34. When necessary, samples, including intact natural G34, were tr)psinized by incubation with I0 gg of TPCK-trypsin (Worthington Biochemica!s) in I.C mt ammonium bicarbonate for 60 rain at 22-'C. The reaction was stopped by briefly heating to boiling point. Radio mtmunoassay systems Antisera were used routinely at dilutions which bound 50'/~ of a tr~tce amount (2000 cpm) of label. Incubations were carried out at 4~C for ¢8 13 i~,l a volume of 2.0 ml of 0.02 M sodium barbitone pH 8~4. containing 0.2% sodium azide and i% (~/v) commercial BSA soluzinr~ (Bo~umin. Orthodi,~t~nostics). Antibody-bound label was ~eparated from free label by addition ~f charcoal (Nodte, BDH) and centrifagaticm a, 2000 x g for lO rnin a~ 4°( '. lt~ assays using pG34 label, 0.2 ml of a suspension rontaining charcoal (!00 tug/ ml), de×tran (I0 mg/ml) and plasma (100/al/ml) was added: in ~say~ u qinLz 1-12 or 1 - 1 9 label, 100 #1 of a suspension of charcoal (i00 mg/ml) and dextran (10 mg/ml) was added. Both antibody-bound label and flee l.~be~ were routinely counted. Control tubes in which antibody was omitted were included for all samples to assess non-specific binding - which was iess titan 8% of total counts in aU studies described here. COOH-tenni~at specific assa~s Samples were routinely assayed with a COOH-terminal specific a~tiserum
!74 (1296) that reacts with G34 about 60% compared with G17, and has previously been described in detail [21. Tissue ex" gracts
Fresh human antra! mueosa was obtained from patients undergoing aatrectomy for recurrent peptic ulcer (n -.--4). Antral mucosa and pituitary glands were obtained from hogs killed at a local abattoir. Ttae pars nervosa was zeparated from pars distatis and the two separately extracted. Antral mucosa of dogs and cats w e r e obtained from Nembutal-anaesthetized anlreals, and antral mucosa of rats obtained from animals killed by a blow on the head~ In all cases tissues were frozen on dry ice as soon as possible and stored at -70~C prior to extraction. The tissues were weighed, thinly sliced, and while still frozen were quickly transferred to boiling water for extract~o~l. The tissues were then boiled for 2 rain (0.! g/mI), homogenized, extracted at 20°C for 30 rain, and centrifuged (2000 × g, 10 rain). Repeated extraction of the pellet in~!~.ted that over 90% of recoverable G17, G34 and NTG34 was obtaine~ in the initial extraction. Pilot ~tudies also indicated that th.~ yield o~" 0oth G17 and NTG34 obtained in boiling water extracts (pH approx. 6.5) was considerably higher than in acetic acid. Tis~.:ae e×~racts were fractionated by gel filtration on columns of Sephadex G~0 saperfine (1 × I00 cm) eluted with 0.02 M ~odium barbitone (pH 8.4). ~SA ~;nd ~2si were added to each sample before elution to provide markers for the void volume, estimated from absorption at 280 ;ira (=0%), and salt region (=I00%). The columns were also calibrated in separate runs with (334, G 17 and the fra;,,ments obtained by trypsinization of G34. RESULTS Antisen~m specificity
tn initial studies the specificity of the antisera was analyzed en the basis of b~rJding properties with various labelled peptides. The two antisera (L66 and L107) raised to fragments of hG34 bound ~2~[-labeIted NH~-terminal 1 - t 2 sequence of hG34, but not surprisingly failed to bind G17, iJ:dicating specificTty for the NHa-terminal region of G34 (Table I). T~-_ese two antisera fa{ied to bind labelled t - t 2 pG34, suggesting specificity for humaa~ versus porcine big ga~trin~. Antiserum L33 raised to pG34 was ini~ially found to bind G17, G34 and NH2-terminal fragments of G34, suggesting the wesence of bo~h COOH- and NIf~.-terminal specif~:.: araribody populations. However, treatment with G ~,7-conjugated Sepharose removed the COOH-termhaz! reactive antibodies, so that the treated antiserum no longer bound G I7 label, but still bound iodinated G34 and its 1-12 NP~-termi~iat fragment (Fig. 2),
t,'5 TABLE t
Bii~ding properties of three G34 antlsera with various labelled peptjdes Anti-
Antigen
Label
sor~lm
pG34
hG 17
.
L33 *
pG34
1,66
1-15 <1 : 100 (Tyr 7-Pro8Ser9) hG34 1-t9 hG34
L107
l : 3000
.
.
.
.
.
1-t2 pG34 .
.
.
.
<1 : t00
.
.
.
.
.
.
.
.
~
1---t 2 hG~t4 . . . . . . . . . . . . . . . . . . . . . . . . .
t :2500
,
.
1-15 (Ty r "~4'rn ~. .
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
t00 < t ; 1(~fJ
<1 : I 0 0 < I :
t00
i :
4000
<1 :t00 < I :
t00
t : t0000
1 : 704)CI
-~
The dilution of ax:tJ.serum binding 50% of the tracer ( 2 0 0 0 cpm) is shown. * After remm, al of C O C H . t e r m i n a l antibodies by imrm, noadsorption (see t~,'xt).
although with slightly Iower titre. Antiserum L33 failed to bind ! --12 hG34. indicating specificity for porcine compared with human big gastrir:- (Table i). Detailed studies of speci[icity were based on competitive binding exped~ ments in which fragments and analogues of the NH2-temlinaI sequence of G34 were cempared for their capacity to inhibit binding of" label to a~tiserum, The antisera raised to hG34 bound 1.-12 hG34, ! .... 19 hG34 and intact hG34 with approximately similar potency (Table II). With L66 the immunochemical potency of 1--6 hG34 was not markedly different from tI~;~ of t - 1 2 , indicating that the antigenic determinant [or L66 lie; wifllin the NH2-terminal hex.apepfide. Moreover, with this antiserum the porcine t 6 fragment was significantly less active than its h~man counte.rpart; since th,_~ge peptides differ only in the substitution of Pro for Leu at position 4 i~ is clear that this residue is an important part o f the antigenic de~ermina~l f(;~ L66. la keeping with this observation, it was found that other peptides w~tfl substitutions in positions 4 or 5 had marked|y reduced immunochemi~::~ r etency with L66, bat substitutions in positions 7. 8 a~d 9 had little ,.)r rL(,' influence on immunoreactivity. In contrast, with antiserum L~0?, the NIt~terminal 1~6 fragment had low immunoreactivity compared with 1-. t2. Moreover, substitutions in posifiens 4, 5, 7, 8 and 9 ail caused a :;harply dkmini~i~ed immunoreactive potency. Together the results indicat<.- ti~,~t L~07 is sgecific for at least the 4 - 9 sequence of hG34. With antiserum L33 the t ~ 1 2 and i - 1 9 fragments of pG34 had ~milar immunoreactivity to intact pG34~ but the t-.-6 fragment was markedly less active. A~ important par~ of the antigenic determinant for L33 was th-?reg>re
176 TABLE II lmmunoci~emical potencies of NH2~terminal fragments and analogues o f G34 with three antisera * Peptide
Antiselx~m ~L66
Lt07
L33
lluman G34 fragme,tts }-12 hG34 hG34n~ NTG 34 I ~19 hG34 I - 6 hG34 i -- ~3 (TyrT-Pro~-Ser 9) t --, i 2 (HisT-ProS-Pr99) i ~ 12 (Ser%Pro~-Ifi,~ 9) 1 --I 2 (Fro%SerB-His 9) t - 12. (His °.-ProS-Set 9)
1.00 2.t 0,90 1.4 0.54 0,7 1.0O 0,55 0,66 0.8 2
1.00 2.4 1.10 3,2 0.0005 0.003 0.051 0.11 0.003
0.0003 0,02 0.017 0.000 002 0.03 0.000 012 0.000 005 0.000 004 0.000 009
Poeci,~e G ~4 _fragments t - i 2 pG 34 pG34 NTG34 i - 1 9 p(;34 1-6pG34 t - t 2 (HisT-ProS-Ser 9) 1 ~ ! 2 (HL~%ProS-Pro 9)
0,1300 35 0,001 4 0.000 3 0.000 3 0.000 52 0.000 7
0,000 0.00][ 0.002 0.000 0,000 0.000
Ottmees 1 - t 2 (Gln4.ProS-His~'-ProS-Set 9) | --I 2 (Gln4-LcuS-His'LProB-Ser 9)
0.000 0.000
0.000 3 0.000 3
2 3
3 8 6 5 3 3
1.00 2.4 1.15 1,8 0.000 0~000 0.00t
02 34 4
0.t3.)0 001 0.0'O0 00 ]
* Immunochemical potencies are expressed relative to 1 - 12 }zG34 m the ca.-e of L66 and L I 0 7 , and relative to I ~ 1 2 p G 3 4 in the case of L33. Potencies were calculated on the basis of the ratio of concentrations of 1 - 1 2 G34 and tes~ peptide needed to mhibR bindhlg of la~c] t~ a.utiserurn by 50%. See Fig. 1 for sequences of [he a~thentic p,_:ptides.
localized to the 7 - 1 2 region. I-Iowevcr, since human [ - 1 2 was significantly less active than porcine t - i 2 G34, and these peptides differ only at position 4, this residue was also an important p,~rt of the antigenic determkL'~t for L33. Analogues with substitutions at posit~ons 4.5, 7, 8 and 9 atl had significantly lower immunoreactivity compared wi~h !:he native sequence of !~ 12 pG34, again confirming the importance of the 4~- 9 region. The resuAs of the specificity swdies are summarized in Figure i.
177
TRYPSIN T
~r
PG34: .GL~PL~L~O~L.Y_-.L._Eu_-. . _G_._L_N _-_6Ly-PRO- PRO-HI s-LEu.-VAL-ALA-AsP-LEu-AL),-LYS-LYs-GL~-$t.YL33 HG34: 'GLP~LE~Gt~Y~PR~GL~m~LY~FR~-P~-Hi~LF~"VAL~ALA~sP~PR~ER~LY~;~LY~5U~G~Y -
L66. ........
. . .
--
= - - - - - ----L 1 0 7
20
PG311:
54
30
- PRr3-TRP-MET-(GLu)5-ALA-TyR-GLY-IRP-MET-AsF-FHE-N_ ~ .
.
.
.
.
.
.
.
1296
HG5,:
- PRO-TRP-LEo-(GLu)5-ALA-TYR-GL~'-TRP"-M~i-As;--PHE;~-H2"
Fig. 1. Amino acid sequence of porcine 0 3 4 (pG34) a~>~dhuman G34 (hG~,i), showing antigenic determinants for 1296, L33, L66 and L107, For details about artdserum specificity see text and Tables I and II. Note that these sequences differ from tiaos~ erigina]ly described [8] ir~ the 7 - 9 ;region ~4].
~L~}PG34
~I HG17
L~ Z
DtLUTtON OF ANTg_,~:RUM Fig. 2. Binding of 12sI-labelled G34 aI~d G 17 by antiserum L33 before and aflEer ir.im~r~osorp~ion w,th G~7 con3t-'.gate4 I'o Sephamm to remove COOH-termii~at spetiii: an~ibodies,
17g
Antrat extracts." man and pig
Extracts of human arttral mucosa inhibited binding of label to L66 (Fig. 3) and LI07 (not shown) in paraltet with 1--12 hG34. Similarly extracts of porcine anlral mucosa produced a graded inhibition of binding of label to L33 that was paral]el to 1- 12 pG34 (Fig, 4). These results establish the validity of assays with these antisera for the estimation of NH2-termina! immunoreactivity in antral extracts. in human antral extracts the concentrations of immunoreaetive material measured with L66 (21.4 _+ 1.7 nmoI/g, n---4; 1-12 hG34 standard)were comp~rabEe to those measured with the COOH-lerminal assay using antiserum 1296 (17.9 +_.2.1 nmol/g; G17 standard). Similar results have been obtained previously for hog antral mucosa using antisera L33 and 1296. Fractionation of' hog antra! exeracts or.~ Sephadex G50 revealed a major peak of im~nunoreactivity with L33 that eluted just before the main peak read by 1296 (Fig. 5), Both antisera revealed a minor peak of immunoreactivity eluting in the position of G34. ~l~e m~:jor peak read by L33 emerged in a simi/ar position ~o the NH~-terminal reactive fragment produced by tryptic cleavage oi G34, while that read by 1296 corresponded to G17 (Fig. 5). Closely
Ab L66 100"
130• [&
roB0. ...I
\ Z
z
F'EPT~DE COi~C~?¢tTRAT~C,~-,! [ p moles/I
ib
~J~,q~l/~_. c~I"I~IACTS Ing oeiuiuvdent/ml ] Fig. 3. CompetRive binding curves for antiserum L66 and 1 - 12 hG34 label with a l~un~.an anttal ¢xb'act (concentration expressed it., ng equivalents ,~: ~.nrral muct, sa), hG34, t ~, 12 nG34 and I~-12 pG34. Binding is expressed as a percentage t:~f IL:_ initial ratio of b¢}u a to free label in the absel~ee of competii~g peptides.
179
Ab L 33 lot~
80
O
Extract Z t~
¢t,u 20i1 .
.
,
1"o' PEPTIOE CONCENTRATION [ p m o ~ s / I ] ANTRAL EXTRACTS [ ng eq~J~valent/ ml]
Fig. 4. Competitive binding curves for antiserum L33 and 1-]2 p(;34 label with a hog ontral extract, pG34, I ~ 12 pG34 and 1- 12 hG34. See t:~g. 3 far further details.
similar results were obtained for human antral mucosa fractionated by gel filtration and assayed with antisera L66, L107 and 1296 (Fig. 6). Tlms L66 and L I 0 7 revealed a major peak of activity eluting tn the position of tt~c NH2~terminal tryptic peptidc, and !296 revealed G I 7-like activity. All 3 an~isera revealed a minor peak eluting in the position of G34, but interestingly the concentrations of this material measured by. L66 were significar~qy higher than those with L107 and 1296. Further work i~ needed to establis~ the significance of this observation, tn the past Rehfetd [ 12] has described a small peak of immunoreactive gastrin (designated component l) that eiutes just before G34 on gel filtration, and reacts with COO[-J-~erminal sp.ccif~c antisera. In the present study no s~gnificant arnou~t~ of componer~t [ ',yore found in antral extracts. However, a partially purified preparation of compr,o nent [ (generously donated by Dr, H.J. Tracy), which had been obtained from a human.gastrinoma and was essentially free of G34 and ()17o was found to react with both L66 and L ! 0 7 . The concentration of this component measured with L66 and L107 were comparable to those measured with 1 296, when G34 was ased as a radioimmunoassay standard. A n t r a l extracts." d(.g, cat and rat In antral extracts o f dog and cat, antiservm 1296 revealed concentrations
180
I
15"
'~"i
pG34,TRYI~INIZED
10'
5"
z
0
!
J
t~
HOG" ANTRAL MUCOSA
,
~
10"
A,L PERCENTEUJTIONVOLUMEF~OTEINTO'~I Fig. 5, Fractione~2ion o n S e p h a d e x GS0 o f an extract o f hog antrat m u e o s a tic- .vet panel), and a ~n~!pt:~ (~f p ( ; 3 4 treated with trypsin ( u p p e r panel). C o J u m n eluate~ w,.'re assayed with antisera L't3 !o . . . . . -o) and 1296 (o- . . . . ~1. Elut~ion p o s i t i o n s of G 3 4 and G I 7 are indicated b y arrows .~t t'.l~ top.
of immunoreactivity that were c(~mparable to those in man and pig (,tog, mean 6.5 nmol/g, n = 2; cat, 17.4 nmo!/g, n - 2). In contrast, antiserum L33 revealed a b o u t 20 times lower concentrafio~~ (dog, 0.34 nmot/g; cat, ~ ~" nmo//g), and L66 ~nd L107 fai!ed to demonstral~.: measurable amount,, ~f immunoreactivity. Fractionatior~, o f dlese extracts on S~:phadex G 5 0 revealer patterns of immunoreactive c o m p o n e n t s similar to those iv. pig .rod man, i.e.
18;I NTG34
30
L i07
L~
kl.l
30 ~ .J
L66
0 O Z 20>.-
.> loI,,-
~:0
020"
~
'
~
~
O 20 4.0 60 80 tOO P£RCENT F..LUTION VOLUME PROTEIN TO '2'~1
I~ig. 6. Fractton~tion on Bephadex G 5 0 o f an extract o f human ant.r~i m u c o ~ . Cctumn L107 (Zop panel), In s e p a r a t e r u n s t h e c o l u m n s were emlibrated w i t h G 3 4 a n d its t y p t i c peptides -~ N'I G 3 4 and G 17 - t h e elution p o s i t i o n s o f w h i c h are i~dicated b y a n o w s at t h e top.
eiuates were azsayed with ~296 (lower p~nel), L66 (middle panel) and
a mii~or peak corresponding to G34, aim major peaks corresponding to G I / and NTG34 (Fig. 7). The concentrations of G34qike activity measured with L33 were approximately 5% those estimated with 1296. When the peak corresponding to G34 was pooled, lyophi!ized and digested with trypsin° two peaks of activity were get, crated that had the gel filtration and immunochemical properties of G17 and NTG34 (Fig. 8), but again concentrations measured with L33 were about 5% those with 1 296. in rat antrai extracts 1296 measured 4.2 _+ 0.9 nmoltg (n = 4) compared with 4.7 _+ 1.3 nmoI/g with L66.~ and < 0 , t nmoi/g w~th LI07 and L33. On Sephadex gel filtration the rat extracts were re~olved into a minor peak of G344ike activity that reacted with both L66 and 1296, and major pt.aks of activity corresponding to G] 7 (1 296) and an NH~-terminal fragment of G34 (L66) (resutts not shown),
182
~G34
~GI/ -70
?, / | i
t
.' ~q
;
CttY
i
-50
-40 1'5-
L33
~.,,..,+
i
~ :1296
I-
.j J
O"
-~0 <
__.. 2-0-
e e
t
R~ -40
-30 ~-0-20 05. FaERCEN7 EL.UTION MOi.tJME PROTEIN 3"O'~-3 Fig. 7. F r a c t i o n a t i o n z~ ,g~l,t~adex G 5 0 of e x t r a c t s of dog antrai m u c o s a ( b o t t o m pa;~el) and cat antzai mucosa ( t o p p~nel). F o r details see ~:igs. 5 and 6. Note that the scal,, fo~ assays with L33 (left side) differs fro~t: t.ha! for 1 2 9 6 (i-ight side).
N o tt-an I r a / t & s u es
Antiserum L33 did not reveal significant amoun.~ of immunoreactiv~v (<0.0t nmot/g) in extracts of hog gastric fundus, duodc~ra,.iejunum~ ileum or colon. Rehfeld previously reported that in b,~h adenob, yp~,~physis and neurohypophysis of the pig p~:~.~itaz-y there were small amounts of g~:~stfin
34 ¢17 r4000
~7 ,,,x. i t00+ ~4~-iti
I .: ~e
t;!
j,,x7
,-
li-~,.l£~-t
"~r~ ,0
~.
PERCENT ELATION VOLUME !'~OTEtN TO "miq
Fig. 8, lFr&ctio.~ation ori Sephad~z GS0 of pooled concvsl.trate of the (i34 ~'a~:~ion~ ot czf antral extracts previousiy run on Sephadex (bottom pallel) an(t the .~me con;:er,.rzHe after dige,~tion with trypsin (top panel). For further det~il~ see Figx, 5, 6 nnd 7,
G34 ~
JGt?
w ~ 800t ,
3 z _I
0
j 600j i
~00~
Z
~>
'
0 20 .~0 60 .80 ~j PER(SENT ELUTIO"J VOLUME PROTEIN fQ ~-~'#
Fig. 9, Fractionation on $ephade× GS.0 of an extract of hog posterior pitl~i,taty, F'~ details see Fig. 5.
t84
[ 13] In the present study, extracts of pig pars nervosa contained 30.6 +i l . 3 pmol/g immunoreactive gastrin measured witt] 1296, and 34-~ 12 pmol/g (n = 4) with L33. Extracts of adenohypophysis contained less than 2 pmoI[g with 1296, and were not studied further with L33, On Sephadex G50 gel filtration the pars nervosa activity eluted as about equal amounts of G34 and GI 7 (I 29671 and NTG34 ~L33) (Fig. 9). DISCUSSION [n the present study radioimmurmassays have been developed using anti-
sera reacting at the NHz-terminus of human G34, These assays have been used to identify and estimate molecuIes witll the properties of G34 and its N]-l:-~erminaI tryptic peptide in antral extracts of mar~ and other mammals. The present studies confirm and extend those previously obtained in pig [3 ]. The Ni-l~-.:erminat specific antiserum (L33) used in previous studies has been shown here to react weakly with human G34, and is therefore of limited use. "lwo approacfles were used to o~tain antisera specific for the NH2-terminu.~ of hG24. In both cases rabbits were immunized with NHa-terminal frag,,ner~ts cou;~led to carrier proteins iz~ such a way as to maximize *~e chances of o~.tain;-ng NHz-terminal reactive antisera. In one case conjugation was achieved '~y triazine coup}ing through an analogue substituted with tyrosine at position 7, and in the other case by glutaraldehyde coupling through *.he epsilon a~nino groups of lysines at position 16 and 17. The a,,.tisera obtained reacted better with human {ban porcine 1--12 G34, suggest.rag specificity for a region iz:cluding posi{~on 4. The antigenic determinant of one antiserum was iocalize~-{ to the Nl-12-ter~ninal hexspeptide (L66), and that of the other inch~ded at least the 4 ....9 region. Interpretation of previous immun,;,:hemical studies on big gastrin has been complicated by an error in the originally reported sequence. Thus synthetic peptides with tile original sequence (parclue: His~-Pr@-Prog: human: His'-ProS-Ser~) had iGw immuno~be:,qk:al ~,otency wi,h antiserum L33 compared with natural peptides. In th.. preser~? study ]t was ,it~own that antiserum L66 witll specificity for the 1 .-6 sequence of hG34 did not di.~,criminate beiween fragments of G34 with the original a~d the revised s.equerlce, whereas antiserum LI07, wlaic]~ has an an{agenjc determinant extending into the 7~9 region, clearly distinguished bet~,.'een peptides with the original seq~ef~ce, which had low potency, and those wiih the revised sequent.e, which were fully active compared with natural hG34. Broadly similar laalterns of immunoreactJve forms of gastriri were seen when L66 and LI07 were used to assay human antral extracts after gel filtralion. In both cases a major peak of immunoreacli'eity elated in the position of NT(;34. The concentrations of NFIa-termina] fragment were. similar
t85
to those of GI 7 -- wilich represents the COOH-terminal try. ptic peptide of G34. These results therefore a~'a'ee with, and extend, those atready obtained in pig, and are consistent with the cleavage of G34 within gastrin cells by z trypsin-like enzyme, Recently, ~'anaihara et ai. [141 have reported similm' findings using several NH~-terminal specific G34 antisera. In one respect their results differ from the present findings, in that on Sephadex G50 they id,elltiffed large amounts o f NH~-terminal immunoreactivity eluting after GI;'. whereas in the present study there is a major peak etuting betbre G17. "l'h~~. reason~ for these differences remain to be established. Antiserum L66 wo~Id have been capable of demonstrating NHT-terminal fragments as small as the I - 6 sequence, but no evidence of significant amounts o f sttch fragment,., were found in the human tissue extracts. The amounts of G34-1ike material measured by L66 were slightly, buz consistently, higher than those measured wiZh 1296 and L107, The significance of this is unclear, but it is of interest that in hog antral mucosa there it; evidence of a form of G34 that reacts with NH~-terminal but not COOHtet~nina~ specific antisera [51, and may perhaps be a COOH-terrni~al extend~:d gastrin. Conceivably there is a form of G34 extended at either COOH-terminus or NH2-terminus which reacts better with L66 than other anfisera. In this context it is significant that. in studying hog g astrb~ rnRNA, Noyes et at. [ 11 ] have obtained evidence for a precursor o f al least 140 residues, which is likely to contain G34 in its mid-region Thus. in ' ~ conversion o f precursor to G34, both COOH- and NH~-terminal extended forms of G34 might be fovned. The different patterns of immunoreactive material measured bv L33, L66 and L 107 in rat, dog and cat coutd be explained by species differences in the sequence of the NH~-t,=rminal regions o f G34. Because COOH-terminal regions are important in determining the biological a~tMty of these peptid ~.s, one wo~dd anticipate that these reNons might be conserved, whereas NH~terminal regions are relatively unimportant and are not conserved. The present study opens the way for more deZailed ar~atysis of the .forn.s of gastfin in human blood and tissues. The currently avai.~able NH~-termin~l specitqc ar._tisera have relatively tow affinity, and are unsuitable for ,Jire~ as;ay of circulating ga~trin~ However, there is no reason wl~y high-affinity antisera might not be obtained in the fut:tre. Alternatively. one might concentrate the immunoreactive forms in blood prior to assay_ Using the latter approach we have recently obtained evidence for the existence of tow concentrations of NH2-terminal fragments o f G34 in the peripheral circular.ion of man after feeding (Pauwels and Dockray, unpubl, studies). Further sttadies of these forms should offer insigh~ into the mechanisms of synthesis and secretion of gaxtrin in health and disease.
156 ACKN(3'WI.EDGEMENTS
TMs work was supported by a g~'ant from the MRC. Skilled technical assistance was pcovided by Carol Higgin~, Elaine Clarke al~d Gavin Laing: Mrs. Eva McElroy kindly typed the mat~uscfipt. Professors R.A. Gregory and G.W. Kenner and Dr. H.J. Tracy generou~ly provided peptide samples. R I.~.l'~E R ENCES I Agarwal, K.L., Grudzinska, S., Ken~,~er, G.W., Rogers, N.H., Sheppard, R.C. and Mc L GuJgan, J.C., lmmunochemicat differentiation between gastrin and feinted peptide hormones through a hover conjugation of peptides to proteins, Experientia, ] 7 (197 ! ) 5~-5~5, 2 D~ckray, G.J. ~ d Walsh, J.|-l., Amino terminaI gas~rin fragment in serum of Zoliing~.r --EItisor~ syndrome patients, G~stroenterro~ogy, 68 (1975) 2 2 0 - 2 3 0 . 3 Dockray, R.J., VaiHant, C. and Hopkins, C.R., Biosyrtthetic relationships of big and little g~t~-~na, N~itu~e (London), 274 (1978) 770-.-772. 4 Dockr.~y, G.J., Gregory, R.A~, Kenner, G.W., Moore, S,, Rama~:handren, K.L. and 'damage, R., The use of immunochemicat methods in strtictural studies ~n hu~;~an big gus~rin. In A. MiyosIli (Ed.), Gut Peptides, Kodanstla, Tokyo, ! 97~), pp. 3 8 - 4 0 . 5 Gregory', R.A., Some asb",:ets of the struc[ure of g~stril~ and gastric-like ft~rms and fragments in gut and brain. In J.F. Rehfdd aud E. Amdrup (Eds.), G~strins and the Vagus, Academic Press, London, 1979, pp. 4 7 - 5 5 . 6 GregorT, R.A. and Tracy, t1.J., The constitution a~ad prope_~r~s of two gastrins extracted from hog ar~tJ-al mu~ '~sa, Gut, 5 (1964) 103 - 117. 7 Gregory, R.A. and Tracy, H J., !sol~ioa of two 'big ga~t~ins' from Zollir,ger-Ellison turnout ti~:~ue, Lancet, 2 (19-'2) 797-799. 8 Gregory, R.A. and Tr~c?, H.j., The chemistry of the g~.strins: So,he rece~, advznces. ;n J,C, Thompson (Ed°), Gastrointestinal Hormones, University ~)f t e x a s Press, Austin, Te:~as, 1975, pp. I3-24. 9 Hughes, W.L., The chemistry of iodination, N.Y. Acad. $c~., (t o5 7) 3- i 8. 10 Hunter, W.M. and Greenwood, E.C., Preparation of iodine ~ 1abetted growth hormone c~f high ~pecific activity, Nature (London); ! 94 ( ~962) 495 -496. i t N oyes~ ~.~.i., Mevm'ech, M., Stein, R. and Agarwa~. K.L., Detection :rod partial ~equence a~:~.~;~s of gastrin mRNA By using an oligodeonynuc]eotMe pr.~be, Prec. Nnt. Acad. Sci. USA, 76 fl979) 1770-.I774. 12 Rt~hfeld, J.F., Three components of gastrin in human serum. Gel filtration studies o.~ the molecular size of ~mmur~ereactive seru:~ ga.~trin, Biochim, Biophys A e t a 285 (1972) 364-372. ! 3 Rehfeld, J.F., Localisation of gastritis ,~ ne~ro- and adenohypophysis, Nature (~..ondon), 271 (]978) 771-773. 14 Yanzilmra, M., Yanaihara, C., Ozaki, J., Mochizuki~ "F., Shimizu, F., Mihara, S., NagM. K., Sato, H. and Sakagarni, M., Human big gas~rin N-~cr;i',i'aal fragment immunorc~..* tivity. In A. Miyoshi (Ed.), Gut Peptides, Kodansha, Tokyo, ~970, pp. 16-33.