gastrin antisera

Regulatory Peptides, 6 (1983) 33-41

33

Elsevier

The reactivity of mononucleotides with cholecystokinin/gastrin antisera J e n s F. R e h f e l d University Department of Clinical Chemistry, Rigshospitalet, DK-2100 Copenhagen, Denmark

(Received28 January 1983; acceptedfor publication8 February1983)

Summary Dibutyryl cyclic GMP has been reported to interact with antisera specific for C-terminal tetrapeptide amide common for cholecystokinin (CCK) and gastrin. Moreover, cyclic nucleotides elute by gel chromatography in the same position as the free CCK/gastrin tetrapeptide. Therefore, we have examined the reactivity of 25 mononucleotides with eight CCK and gastrin antisera. The results show that the nucleotides all bind poorly to the antisera (nucleotide concentration required > 1 mM). Hence, endogenous cyclic nucleotides, which are present in biological extracts in pM to nM concentrations, do not interfere with immunochemical CCK or gastrin measurements. The antisera displayed highly individual patterns of reactivity without preferential binding of di- or monobutyryl cyclic nucleotides (AMP, GMP or IMP). Thus, the present results do not support the idea of structural resemblance between the C-terminus of CCK/gastrin peptides and butyryl derivatives of cyclic GMP. Enzymatic treatment of the antral tetrapeptide-like immunoreactivity showed that nucleotides do not contribute to this material, which appears exclusively peptidergic. cholecystokinin; cyclic nucleotides; gastrin; mononucleotides; radioimmunoassay

Introduction Cholecystokinin (CCK) and gastrin share the C-terminal tetrapeptide, Trp-MetAsp-Phe-NH2, which constitutes their active site. The tetrapeptide is well preserved during evolution [1], and immunoreactivity resembling the free tetrapeptide amide has now been found in brain and gastrointestinal tissue [2-4]. Recently, dibutyryl cyclic GMP was found capable of interfering with the receptor and antibody binding of CCK [5-11], perhaps due to structural resemblance between the nucleotide and the C-terminal tetrapeptide [5], or to interaction between the nucleotide and CCK itself [8]. 0167-0115/83/$03.00 © 1983 ElsevierSciencePublishersB.V.

34 Most gastrin and CCK antisera are specific for the common C-terminus [12]. Therefore, we have now studied the reactivity of various mononucleotides with CCK/gastrin antisera in order to decide whether nucleotides contribute to the endogenous tetrapeptide-like immunoreactivity.

Materials and Methods

Nucleotides All nucleotides examined are shown in Table II. They were obtained from Sigma Chemical Co. (St. Louis, MO, USA) or Boehringer (Mannheim, F.R.G.). Their antibody binding was measured in the concentrations 0.3, 1, 3, 10, 30, 100, 300/~M and 1, 3 and 10 mM. The concentrations were controlled by absorption at 260 nm. Peptides Highly purified porcine CCK-33 was generously donated by V. Mutt (Karolinska Institutet, Stockholm, Sweden). Synthetic, sulphated CCK-8 was generously donated by M. Ondetti (The Squibb Institute of Medical Research, Princeton, N J, U.S.A.). Synthetic, non-sulphated human gastrin-17, the C-terminal tetra- and pentapeptide amides were generous gifts from J.S. Morley (Peptide Research, I.C.I., Cheshire, U.K.). A n tisera Human gastrin-17 and porcine CCK-33 coupled by carbodiimide to bovine serum albumin served as antigen. The antisera were raised in white Danish rabbits as previously described [13,14]. Seven C-terminal directed gastrin/CCK-antisera (Nos. 2604, 2605 (1), 2609, 2716, 2717, 2720 and 4562 (14) and one antiserum specific for sequence 25-30 of CCK-33 (No. 4698) were selected for the present study. Characteristics of the antisera are given in Table I. Tracers Monoiodinated human gastrin-17 [15] and porcine CCK-33 [13] were used as tracers for the C-terminal and CCK-specific antisera, respectively. Enzymes Pronase ('for analytical purposes') from Boehringer Mannheim, F.R.G., achymotrypsin (type II), trypsin (type XI, DPCC treated) and phosphodiesterase (3',5'-cyclic nucleotide) from Sigma Chemical Co., St. Louis, MO, U.S.A., were incubated for 30-120 min in 0.05 M sodium phosphate buffer, pH 7.5 (1 mg enzyme/ml) at 37°C, 25°C and 37°C, respectively, with a sample of tetrapeptide-like immunoreactivity isolated from porcine antral tissue after gel filtration. The incubations were terminated by boiling for 30 rain. Radioimmunoanalysis The radioimmunoanalysis was performed in disposable glass tubes using 0.02 M

0.06 0.10 100 0.005 0.005 < 10 -7

Reactivity with: CCK-33 (s) CCK-8 (s) G-17 (ns) G/CCK-5 G/CCK-4 Nucleotides 12.5 8.3 100 0.0 ! 0.006 < 10 - 7

150 0.83 0.98

2605

11.6 16.7 100 1.82 0.56 < 10-7

140 0.63 0.94

2609

3.3 13.6 100 0.60 0.01 < 10-7

320 4.20 0.96

2716

a~dity, heterogeneityandspecificity)

10.0 40.0 100 4.00 3.00 < 10-7

500 0.33 0.83

2717

12.0 10.8 100 0.02 < 10-7

400 2.30 0.46

2720

1.50 1.15 100 0.15 0.01 < 10-7

3800 4.80 0.90

4562

100 100 < 10 - 3 < 10- 3 < I0 - 3 < 10-7

12 0.12 i .02

4698

125I-gastrin-17 [15] was used. b Effective equilibrium constant according to Ekins and N e w m a n [22]. c Index of heterogeneity according to Sips [21].

a Antiserum dilution at which 50% of 2 fmol tracer is b o u n d at equilibrium. ~251-CCK-33 [2] was used as tracer with antiserum 4698. With the other antisera

125 0.97 0.99

2604

Antiserum No.

Titer × 103 a K ° f f X l012 b ac

Parameter

CharacteristicsofCCK/g~trin-antisera(titer,

TABLEI

36 veronal buffer, pH 8.4, containing 0.1% bovine serum albumin as diluent. The incubation mixture consisted of 2.0 ml antiserum diluted as indicated in Table I, 250 /xl tracer corresponding to approximately 2 fmol peptide and 150/~1 nucleotide or peptide solution. The solutions were mixed at room temperature. After two days of incubation at 4°C antibody bound and 'free' tracer were separated by addition of Amberlite resin (gastrin tracer) or protein-coated charcoal (CCK-tracer) and centrifuged as previously described [13,16]. Supernatant and pellet were counted in automatic gamma scintillation counters (Selektronik, HOrsholm, Denmark; and LKB-Wallac, Sweden).

Isolation of endogenous tetrapeptide-fike immunoreactivity Porcine antral mucosa was frozen 20-30 min post mortem in a local abbattoir. The mucosa was cut into pieces weighing a few mg in the frozen state and immersed in boiling water (pH 6.6) for 20 min (10 m l / g tissue). The boiled water extract was applied to a calibrated Sephadex G-50 superfine column (2000 × 25 mm) and eluted with 0.25 M N H 4 H C O , pH 8.2, at a flow rate of 20 m l / h . Fractions of 3 ml were collected and assayed using antiserum 2609, which recognizes the free tetrapeptide amide (Fig. 1 and Table I). The fractions eluted as the tetrapeptide were pooled and divided in portions incubated with chymotrypsin, phosphodiesterase, pronase or trypsin as described above.

Results

Binding of nucleotides In concentrations from 300 nM to 300/~M neither of the 25 nucleotides could displace the tracer from either the CCK-specific antiserum or the seven C-terminal gastrin/CCK-antisera (Table I). Subsequently, only the three most used C-terminal antisera in our laboratory were incubated with the nucleotides in 1-10 mM concentrations. As shown in Table II only 10 mM 8-bromo-cGMP could displace 125I-gastrin-17 beyond the detection limit using antiserum 2604. Neither di- nor monobutyryl cGMP, cAMP and clMP had significant effects on antiserum 2604 which although C-terminal directed is highly gastrin-specific and only binds CCK-peptides and the free C-terminal tetra- and pentapeptide poorly (Table I). A different pattern appeared with antiserum 2609, which reagts well with CCKpeptides and the free C-terminal tetra- and pentapeptides (Table I). Both di- and monobutyryl c G M P as well as monosuccinyl cGMP could displace the tracer. Also GDP, G T P and 8-bromo-GMP were bound significantly. Above all, however, 8-bromo-cAMP was bound. Finally, monobutyryl-cAMP and clMP, AMP, ATP and ITP were also bound, although to a lesser extent. Antiserum 4562 has with respect to binding of CCK-peptides and the free C-terminal tetra- and pentapeptides a position between Ab. 2604 and Ab. 2609 (Table I). But with respect to nucleotide binding antiserum 4562 is not between the two other antisera (Table II). In 10 mM concentrations 19 of the 25 nucleotides

inosine inosine inosine inosine inosine

adenosine adenosine adenosine adenosine adenosine adenosine adenosine adenosine adenosine adenosine

guanoslne guanosme guanosme guanosme guanosme guanoslne guanosme guanosme guanosme guanosme

3',5'-monophosphate 3',5'-monophosphate 5'-monophosphate 5'-diphosphate 5'-triphosphate

3',5'-monophos ~hate 3',5'-monophos ~hate 3',5'-monophos ~hate 3',5'-monophos )hate Y,5'-monophos 3hate 3',5'-monophos ~hate 3'-monophos ~hate 5'-monophos ~hate 5'-diphosphate 5'-triphosphate

3',5'-monophosphate 3'-monophosphate 5'-monophosphate 5'-diphosphate 5'-triphosphate

3',5'-monophosphate

3',5'-monophosphate 3',5'-monophosphate 3',5'-monophosphate 3',5'-monophosphate

0 0 0 0 0

0 0 0 0 0 0 0 0 0 0 0 0 0 0 0

0 3 0 0 1 0 0 0 0 1

0 0 0 3 1

2 7

1 1

0 0 0 0 0

4 0 0

0 0 0

8 0 0 0 1

5 8 0 1 4 2 0 0 3 7

3 0 0 5 5

5 10

7 5 5

1 0 0 0 5

2 3 0 0 10 0 0 1 0 0

1 l 1 1 7

4 0

7 3 2

0 0 0 0 5

0 5 0 0 22 0 2 0 0 1

2 0 0 5 8

5 0

10 10 4

3

1

10

1

3

Ab. 2609 a

Ab. 2604 a

m M nucleotide

a Detection limit using ab. 2609 is 6 pmol and using abs. 2604 and 4562 9 pmol synthetic h u m a n gastrin-17/l.

O 2-monobutyryl

N 6, O 2-dibutyryl O 2-monobutyryl N6-monobutyryl O Z-monosuccinyl 8-bromo

N 2, O 2-dibutyryl O 2-monobutyryl N 2-monobu tyryl O 2-monosuccinyl 8-bromo

Nucleotide

9 0 0 4 11

3 7 0 0 64 1 7 4 4 18

4 0 3 10 15

11 8

15 13 10

10

4 0 0 0 9

8 0 0 2 2 4 0 0 0 9

3 6 7 6 9

4 2

3 7 7

1

6 3

5 8 9

8 2 0 2 10

7 7 5 5 5 3 4 0 5 9

2 6 10 10 15

3

Ab. 4562 a

23 10 8 I1 17

17 14 11 14 15 4 11 4 17 25

9 13 18 24 26

7 5

10 15 18

10

Ability of 25 nucleotides to displace 125I-gastrin-17 from C-terminal gastrin/CCK-antisera. The displacement potency is expressed as pmol synthetic h u m a n gastrin-17 per liter

T A B L E II

38

25 Z 20 tn

15

Vo

0-3~

/GilTH 1 v~

o

,,<, ,fl(C C O

10

z

05

i

~

i

02

J

i

04

J

06

EFFLUENT

i

08

VOLUME

(11 Fig. 1. Gel chromatography of a boiling water extract of porcine antral mucosa (10 m l / g mucosa). 5 ml extract were apphed to a Sephadex G-50 superfine column (25 ×2000 mm) and eluted at 4°C with 0.25 M N H 4 H C O 3 at a flow rate of 20 m l / h . The column was previously calibrated with 125I-albumin (V0) , porcine gastrin-34 (G-34), gastrin- 17 (G- 17), 22 NaCl (Vt ), the C-terminal tetrapeptide amide common to CCK and gastrin (G-4) and cyclic nucleotides (cGMP and cAMP). Fractions of 3 ml were collected and assayed using antiserum 2609 (for characteristics, see Table l), monoiodinated gastrin-17 (22) and synthetic human gastrin-17 as standard. The fractions eluted as G-4 and cyclic nucleotides (between the broken lines) were pooled, lyophilized and reconstituted in sodium phosphate buffer, which again were divided in five equally large portions for incubation with enzymes (Fig. 2).

displaced the tracer significantly from antiserum 4562, while one nucleotide affected antiserum 2604 and 13 antiserum 2609. In contrast to the other antisera, the 8-bromo derivatives had no particular effect on antiserum 4562 (Table II). z n,-" F(./) i

02

0A

0Z ~ Ig Control

Trypsin

Chymotrypsin

Pronase

Phosphodiesterase

ENZYMES

Fig. 2. Immunoreactivity of the tetrapeptide-like material (Fig. 1, fractions between broken lines) after incubation with various enzymes. The immunoreactivity was measured by radioimmunoassay using antiserum 2609 (see Table I), monoiodinated synthetic human gastrin-17 (22) and synthetic human gastrin-17 as standard.

39

Effect of enzymes on the endogenous tetrapeptide-like immunoreactivity While pronase and chymotrypsin entirely removed the tetrapeptide-like immunoreactivity in the antral extracts (Fig. 1), phosphodiesterase and trypsin had no effect (Fig. 2).

Discussion

The present study has shown that the reactivity of nucleotides with C-terminal specific CCK/gastrin antisera is so poor, that endogenous cyclic nucleotides [17] do not interfere with radioimmunoassay measurements of C-terminal CCK/gastrin fragments. Moreover, the pattern of reactivity with the individual C-terminal antisera varied considerably. There was no preferential binding of butyryl cyclic GMPs, which have a unique binding to CCK receptors [5-7,9-11] or interference with CCK [8]. Thus, the present study does not support the idea of structural resemblance between butyryl cyclic CMPs and the C-terminal CCK/gastrin sequence. The results support, however, the concept of pronounced residue-specificity for each antiserum [18]. The study here was initiated because Robberecht et al. [11] reported that a C-terminal directed gastrin antiserum could bind cyclic nucleotides - - especially dibutyryl cGMP, and because cyclic nucleotides by gel chromatography elute in a position similar to the free C-terminal tetrapeptide of CCK and gastrin (Fig. 1). Hence, it became possible that nucleotides contributed to or perhaps constituted the tetrapeptide-like immunoreactivity in antral, intestinal and cerebral extracts [2-4]. Taken together the results exclude this possibility: First, the antisera employed were the same used for discovery and characterization of the tetrapeptide-like material [2-4]. Second, the antibody-binding of the nucleotides was so poor (107-fold lower than the free tetrapeptide and 109-fold lower than gastrin-17) that the antisera would not be able to measure endogenous cyclic nucleotides, which occur in pmol/g concentrations in tissue [17]. Third, the antisera displayed no parallelism between tetra- or pentapeptide crossreactivity (Table I) and nucleotide reactivity (Table II). Finally, the removal of the tetrapeptide-like-immunoreactivityby chymotrypsin and pronase (Fig. 2) shows that the tetrapeptide-like material is peptidergic. The lack of effect of trypsin agrees with the absence of arginyl and lysyl in the C-terminal CCK/gastrin sequence. The nucleotide concentrations used in this study (up to 10 mM) are the same as in previous CCK receptor [5-7,9-11] and antibody studies [5,8,11]. Such concentrations approach the order of magnitude of sodium chloride concentrations necessary to interfere with gastrin radioimmunoassays [16,19]. Hence, it is possible that the nucleotide interference is an unspecific salt effect, for which the antisera have slightly different sensitivities. In two of the three previous studies using C-terminal specific antisera [5,11] cGMPs had the most pronounced nucleotide effect, and the third study [8] included only dibutyryl cGMP itself. These reports, however, used only one C-terminal antiserum, which may explain the difference from the present results. Thus, a significant factor is not only the sequence-, but also the residue-

40 specificity. A s r e p o r t e d e l s e w h e r e [18], C - t e r m i n a l C C K / g a s t r i n a n t i s e r a , a l t h o u g h r a i s e d a g a i n s t the s a m e a n t i g e n a n d r e a c t i n g w i t h t h e s a m e s e q u e n c e , r e a c t h i g h l y i n d i v i d u a l l y w i t h e a c h residue.

Acknowledgement T h e skilfull t e c h n i c a l a n d s e c r e t a r i a l a s s i s t a n c e o f B o d i l Basse a n d L i n d a M y g i l is g r a t e f u l l y a c k n o w l e d g e d . T h e s t u d y w a s s u p p o r t e d b y g r a n t s f r o m the D a n i s h M R C a n d the N O V O F o u n d a t i o n .

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41 15 Stadil, F. and Rehfeld, J.F., Preparation of 125I-synthetic human gastrin-I for radioimmunoanalysis, Scand. J. Clin. Lab. Invest., 30 (1972) 361-369. 16 Stadil, F. and Rehfeld, J.F., Determination of gastrin in serum. An evaluation of the reliability of a gastrin radioimmunoassay, Scand. J. Gastroenterol., 8 (1973) 101-113. 17 Levine, R.A., Role of cyclic nucleotides in gastrointestinal diseases. In L. Volicer (Ed.) Clinical Aspects of Cyclic Nucleotides, Spectrum Publ. Inc., New York, 1977, pp. 229-261. 18 Rehfeld, J.F. and Morley, J.S., Residue-specific radioimmunoanalysis: A novel analytical tool, J. Biochem. Biophys. Methods, 7 (1983) 161-170. 19 Schwartz, T.W., Sakso, P. and Rehfeld, J.F., Immunochemical studies on gastrin in the urine, Clin. Chim. Acta, 89 0978) 381-386. 20 Sips, R., On the structure of a catalysts surface, J. Chem. Phys. 16 (1949) 490-501. 21 Ekins, R. and Newman, B., Theoretical aspects of saturation analysis, Acta Endocrinol., suppl. 147 (1970) 11-36. 22 Rehfeld, J.F., A unique high-titer antiserum to gastrin, Scand. J. Clin. Lab. Invest., 41 (1981) 723-727. 23 Andersen, B.N., de Magistris, L. and Rehfeld, J.F., Radioimmunochemical quantitation of sulfated and non-sulfated gastrins in biological fluids, Clin. Chim. Acta, 127 0983) 29-39.