The Further Purification of Motilin, a Gastric Motor Activity Stimulating Polypeptide from the Mucosa of the Small Intestine of Hogs

Department of Physiology andSurgcry, University of British Columbia, Varzcouver, British CoEunibia and The Clrernistry Department II, Karolinska Institute, Stocklzokrn, Sweden

Received November 5 , 1970

BROWN, J. C., MUTT, V., and DRYBURGH, J. R. 1971. The further purification of motilin, a gastric motor activity stitnulating polypeptide from the mucosa of the small intestine of hogs. Can. J . Physiol. Pharmacol. 49, 399-405.

A polypeptide has been isolated from the duodenal mucosa of hogs and has been named motilin. Motilin stimulates motor activity in both antral and fundic gland area pouches of the stomach of dogs. Pt will stimulate pepsin output, with no change in H' secretion, from fundic gland area pouches. Motilin differs from the other characterized gastrointestinal polypeptides both chemically and in its physiological actions. In particular, the presence of phenylalanine as N-terminal residue, the absence of histidine, and presence of large amounts of glutamic acid and glutamine distinguish it from cholecystokinin-pancreozymin and secretin.

BROWN, b. C., MUTT, V.. et DRYRURGII. J . W. 1971. The further purification of motilin, a gastric motor activity stinmulating polypeptide from the nmucosra of the small intestine of hogs. Can. J. Physiol. Pharnmacol. 49, 399-4135.

Wous avons is016 B partir de la muqueuse duodenale de porc. un polypeptide que nous avons nomrnk motiline. I1 stimufe la motilitC des poches de I'antre pylorique et du fundus de l'estornac de chien. I1 stirnule kgalernent la libkration de pepsine sans motifier la skcrbtion d'H+ B partir des aires ~Ccrbtrices du fundus. La motiline diffhre des autres polypeptides gastro-intestinaux dbjB connus par caractCristiques chimiques et ses actions physiologiques. En particulier. les caract2res qui le distinguent de la s@cr@tine et du CCK-PZ sont la prCsence de phbnylalanine csmme rksidu N-terminal, l'ahsence d'histidine et l'abondance de glutamine et d'acide glutanmique.

Inf roductis~l Materials and Methods

Brown et ab. (1966) demonstrated that al- kalinization of the duc~denum of the dog would induce powerful motor activity changes in trans- planted pouches of the fundus of the stomach. The nature of the animal preparation suggested that a blood-borne stimulatory material was involved. Brown (3967) was able to dem- onstrate the prescncc of this material iin crude duodenal extracts and Brc~wn and Parkes ( 196'7) were able to produce this material free fronn activities possessed by other gastrointes- tinal hormones. The original starting material, the preparation Pancreozymin (Boots Pure Drug Co., EngIand). used by Brown and Parkes ( 196'7) proved unsuitable for serious attempts at purification.

This report deals with the purification of a polypeptide froin duodenal extracts, which has potent stimuIatory activity for motor activity in fundic and antral pouches. The starting ma- terial was a side fraction produced during the purification of secretin by Mutt and Jorpes at

Carboxynmethylcellu~ose (CM 11 and 22) were products of Whatman Co., England. DEAE-Sephadex and other Sephadex products were obtained from T'harmaci,~, Sweden. Triethylan~insethyicell~~lose (TEAE-cellulose) was a product sf Serva. Heidel- berg, West Germany.

Fractions were tested for stimulatory activity in three conscious dogs. These animals had been pre- pared several months previsusly with pouches of the acid-secreting, fundic gland area of the stomach which wcre vagally and syln~pathetically denervated, and a vagally denervated antral pouch. Sympathetic de- nervation of the fundic pouches was achieved by stripping the nerves from the splenic artery and vein, removing the spleen, and severing all mesenteric con- nections to the pouch. Motor activity was recorded using Statham pressure transducers and pressure waves were monitored on a Gilson pen recorder. The pressure waves were analyzed for size of contractions, measured in mrn Hg, and duration of contractions measured in seconds. An index of motor activity was calculated as follows for all periods (Brown and Pederson 1970) : sum of (amplitude in mm Hg X duration in seconds

of each wave) time in minutes X 10

h e Karolins ka Institute, Stockholm, sweden. All periods were of 1 0-min duration and the infusions

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480 CANADIAN JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY. VOL. 49, 1971

of test fractions and standard were not commenced until at least three basal periods were obtained. An arbitrary unit has been established. It is the amount of stimulatory polypeptide present in 1.0 mg of Boots Pancreozymin which has been observed as being a reproducible preparation. For purposes of assay both fractisns and standard were injected intravenously over a period of 60 s. The standard was always given in a dose of 1.0 mg/kg, i.e. 1.0 motilin unit/kg.

Acid hydrolyses were performed with 6.0 M HCI in an atmosphere of argon at 118 "C for 20-22 h. The HCl was removed in a high vacuum. No correc- tion has been made for incomplete hydrolysis. Amino acid composition was determined qualitatively by a two-dimensional paper chromatographic technique (Wedfield 1953 ) . and quantitatively according to Spackman et al. (1958) using a Biochrona BC200 amino acid analyzer. Tryptophan was tested for quantitatively by the method of Gaitonde and Dovey (1970). N-terminal amino acid determinations were performed by the phenylisothiocyanate method of Edrnan (1950a,b) and the dansyl method (DNS) of Gray and Hartley (1 963 ) .

Results Preparation of Starting Materiak

The starting n~aterial for this study was a side-fraction produced during the purification of the gastrointestinal hormone secretin on carboxymethylcellulose, essentially by the method described by Mutt ( 1 9 5 9 ~ ) . This pro- cedure has been slightly modified and 0.0125 M phosphate buffer, pH 8.0, was used to dc- velop the column. Figure 1 shows the chro- inatogrann produced when 12.0 g of partially purified secretin were fractioned on a 14 >< 15 crn column of carboxymethyIcellulose. Partially purified secretin was obtained by extraction of crude secretin into methanol, precipitating with ether, and reprecipitating from water by satura- tion with NaCl (Mutt 1959h).

Fractions 1-5 were pooled, adsorbed onto alginic acid, and eluted with 0.2 M HCl, and Cl- was exchanged for acetate by the method described by Brown et al. (1970) . This ma- terial was found to contain 35-50 motilin units/mg, and is referred to as the starting material. The average yield at this stage was 1.5-2.0 g. Refractionation on CM-cellulose

In a typical experiment 355 mg of starting material were dissolved in 15 rnl of 0.02 M NPT,HCB3/CQ2 to a final pH of 6.5. The start- ing buffer had been exposed to C 0 2 until the pH fell to 6.5. It was maintained at this level by holding in an atmosphere of C 0 2 . The solu-

w . w

4 12 86 Fraction Number

FIG. 1. Chromatogram of 12.0 g of crude secretin on a column 14 cna X 15 em sf carboxymethylcellulose (CM 11). Fractions were of 1 liter. Fractions 1-5, the shaded area, were pooled and IyophiIized for further pamrification.

tion was applied to a column (2.5 x 10 cm) of CM-cellulose ( Whatman CM 2 2 ) which had been previously equilibrated with (6.02 M NH.l- HCO:{, pH 6.5, and developed with the same buffer. This was followed by 0.2 M ~ g H C B 3 buffer, pH 8.0. The eluate was collected in 6.0-n11 fractions at a flow rate of 360 inl/h.

The absorbance of the fractions was mea- sured at 280 mb~, through a l .0 cln light path. The results are shown in Fig. 2. Fractions 12- 40 inclusive were pooled and liyophilized. This procedure yiclded 105.5 mg of dried material, which is referred to as M I . (an assay, M 1 was found to contain approximately 2000 units/

Fractionation on Sephadex G-25 In a typical experiment 37 mg of fraction

M 1 were dissolved in 3.0 ml of 0.2 M acetic acid, applied to a column of Sephadex G-25 fine (0.9 x 98 cm), and cluted with 0.2 M acetic acid. The eluate was collected in 4.0-1111

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BROWN ET AL.: PURIFICATION OF MOTILIN

Fraction Number

FIG. 2. Refsactionation of 355 mg of fraction 1 from CM 11. Fractions of 6.0 rnl at a flow rate of 360 ml/h were collected. Tubes 12-40, the shaded area, were pooled and lyophilized. The fraction is referred to as fraction M 1.

5 10 15 20 2 5

Fraction Number

FIG. 3. Chromatogram of 37 rng of fraction M 1 on Sephadex G-25 fine. Elution was with 0.2 M acetic acid, and fractions of 4.0 ml were collected at a flow rate of 20 rnl/h. Tubes 14-18, the shaded area, were pooled and lyophilized. This fraction is referred to as fraction hl 2.

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40% CANADIAN JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY. VOE. 49, 1971

fractions at a flow rate of 20 ml/h. The absor- pooled and lyophilized. This procedure yielded bance of the fractions was measured at 288 n n , ~ 7.0 mg of highly active material which is re- through a 1.0 cm light path. The chromatogam ferred to as M 2. This material assayed at 4000 is shown in Fig. 3. Fractions 14-1 8 were units/mg.

FrCI~ti0I-i Number

FIG. 4. Chromatogram of 10.4 rng fraction M 2 on TEAE-cellulose. The eluate was col- lected in 2.0 rnl fractions at a flow rate of 60 ml/h. Tubes 1-5, the shaded area, were pooled and lyophilized. This fraction is referred to as fraction &I 3.

t 1.0 unit /kg

Sample Number

FIG. 5. Graph summarizing the effect s f 1.0 unit/ kg of motilin fraction kf 2 or M 3 on H' secretion from fundic pouches. The graph shows the mean of 12 observations in three animals -Cs.e. of the mean.

Q 0 1 2 3 4 5

S ~ r n g l e Number

FIG. 6. Graph summarizing the effect of the injec- tion of 1.0 unit/kg sf rnotilin M 2 or M 3 on pepsin secretion from fundic pouches. The graph shows the mean of seven observations in three animals t s .e . of the mean.

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BROWN ET AL.: PURIFICATION OF MOTILIN 403

Fractionation on TEA E-celkuiose In a typical experiment 10.4 mg of fraction

M 2 were dissolved in 1 .O ml sf 0.01 M NH3 and 1.0 ml of 0.01 WH4H6103. The pH of this solution was 7.2, and it was adjusted to pH 8.1 by the addition of 0.01 M M,. The solu- tion was applied to a 0.9 x 5.0 cm column of TEAE-cellulose and eluted first with 0.01 M M4H6Io3 buffer at pH 7.8. This was followed by 0.2 M NH4PTC03 buffer at pH 8.0. The eluate was collected in 2.0-ml fractions at a flow rate of 60 ml/h. The absorbance of the fractions was measured at 280 mp through a 1.0 cm light path. The chromatogram is shown in Fig. 4. Fractions 1-5 were pooled and lyophilized. This procedure yielded 5.7 mg of highly active material. This material is referred to as M 3 and on assay was found tb contain 8000 - 10 000 units/mg. Amino Acid Analyses and N-terminal Residue

Determinations Amino acid analyses have revealed the ab-

5 -

0 o i 2 3 4 5

Sample Number

FIG. 7. Graph summarking the effest of the injec- tion of 1.0 unit/kg of mti l in M 2 or M 3 on the index of motor activity in fundic pouches. The graph shows the mean of 13 observations *see. of the mean.

sence of histidine, tryptophan, and cystine. A predominance of glutamic acid or glutamine residues was also observed. The N-terminal amino acid was shown to be phenylalanine. Dansylation also confirmed the presence of tyrosine and lysine in the molecule.

Gastric Elgects of Motikin (M 2 and M 3) The results are summarized in Figs. 5-8.

Three animals were used in the investigation of each parameter, and each animal was used at least twice. All graphs represent the mean see.

Figure 5 shows the effect of the intravenous injection of motilin, 1.0 unit/kg, on H + output from fundic pouches. No significant increase in W+ secretion was obtained. However, this dose of the polypeptide produced a significant increase in pepsin output from the fundic pouches which continued into the second period after injection (Fig. 6). The large in- creases in the indices of fundic (Fig. 7) and antral (Fig. 8) motor activity were seen to

0 0 1 2 3 4 5

Sample Number FIG. 8. Graph summarizing the effect of the injec-

tion of 1.8 unit/kg of motilk fraction M 1 or M 2 on the index of motor activity in antral pouches. The graph shows the mean of nine observations in three animals -es.e. of the mean.

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404 CANADIAN JOURNAL OF PHYSIOLOGY AND PHARMACOLOGY. VOL. 49, 8978

Fundc Pouch

A - - L - ~ L - 0 I60 80 Antml Pouch

Q A h .

I il /kg Boots P Z - Fw& Pouch

Antral Pouch

10 Minute P e r ~ d

FIG. 9. Record showing motor activity pressure waves in both antral and fundic pouches to injections of the standard, 1 unit E 1 mg/kg Boots Pancreozymin (PZ) and 2 ,ug s f motilin zz 1 unit/kg. The records are continuous. (Redrawn from an actual recording.)

commence within 2 min of the injection of the justification in applying an identifiable name to polypeptide. Increases in both frequency and the polypeptide. height sf contractions of the pressure waves The name 66motilin" has beell chosen be- were observed (Fig. 9) . cause of the initial observation that the poly-

peptide will stimulate motor activity in fundic Discussi~n pouches. which is the most specific action of

The physiological and the chemical data ob- tained to date from studies with this polypep- tide reveal it to be quite distinct from the known identified gastrointestinal hormones ex- tracted from the mucosa of the upper small intestine of the hog. In particulal-, the identi- fication of phenylalanine as the N-terminal amino acid, the presence of glutamine and glutamic acid in large amounts, and the ab- sence of histidine distinguish it from secretin and cholecystokinin-pancreozymin. The stim- u l a to r~ effect on pepsin secretion from fundic pouches is shared with secretin but secretin does not stimulate motor activity in fundic pouches. The action of the polypeptide in stimulating antral motor activity is similar to that seen with gastrin and cholecystokinin- pancreozymin, but this polypeptide has no pancreatic effects and will not stimulate W+

ihis polypeptide. Under similar conditions, neither cholecystokinin-pancreozy~nin nor gas- trin do so.

It is possible that motilin is the polypeptide released when the duodenum becomes alkalin- ized (Brown et al. 1966). However, support for this hypothesis must await more intensive comparative studies and the identification of motilin in blood and tissues. Brown (1967) and Brown and Parkes (1967) described the separation of a material with fundic pouch motor-stimulating activity from a crude com- mercial extract of the upper small intestine mucosa. Physiologically, motilin has the same actions as the crude preparation of Brown and Parkes ( 1967).

Structural work on the molecule and a more complete study on physiological actions have been commenced.

iecretion from fundic pouches. Because of We wish to thank Mr. K. G. He,e for the these demonstrable differences from other gas- illustrations. trointestinal hormones we fcel that there is Supported by grant MA 1972 from the Canadian

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BROWN ET AL.: PURIFICATION OF MOTILIN 485

Medical Research Council to J. C. Brown and by grants to Emeritus Professor E. Jorpes and Professor V. Mutt from U.S. Public Health Service, National Hnstitute of Wealth (No. AM 06410-07), the Squibb Institute for Medical Research, New Brunswick, N.J., U.S.A., the Swedish Cancer Society, and the Swedish Medical Research Council.

BROWN, J. C.: JOHNSON, L. P., and MAGEE, D. F. 1966. Effect of duodenal alkalinization on gastric motility. Gastroenterology, 50, 333-339.

BROWN, J. C. 1967. The presence of a gastric motor stimulating property in duodenal extracts. Gas- troenterology, 52, 225-229.

BROWN, J. C., and PARKES, C. 0. 1967. The separa- tion sf frrndic pouch motor activity stimulatory and inhibitory fractions from pancreozymin. Gastroenterology, 53, 73 1-736.

BROWN, b, C., and PEDEKSON, R. A. 1970. A multi- parameter study on the action of preparations containing chslecystokinin-pancreozymin. Scand. J. Gastruent. 5, 537-541.

BROWN, J. C., R~UTT, V., and PEDERSON, R. A. 1970. Further purification of a polypeptide demonstrat- ing enterogastrone activity. J. Physiol. (Lond.) , 209, 57-64.

EDMAN, P. 1950a. Preparation of phenyl thiohydan- toins from some natural amino acids. Acta Chem. Scand. 4, 277-282.

1950h. Methods for determination of the amino acid sequence in peptides. Acta Chem. Scand. 4, 283-293.

GAITONDE, M. K., and DOVEY, T. 1970. A rapid and direct method for the quantitative determination of tryptophan in the intact protein. Biochem. J. 117, 907-91 1.

GRAY, W, R., and HAR'TLEY, B. S. 1963. A fluorescent end-group reagent for proteins and polypeptides. Biochem. J. 89, 59P.

MUTT, V . 1959a. Chromatography of secretin on carboxymethyl cellulose. Acta Chem. Scand. 13, 1247-1248.

1959h. Preparation of highly purified secretin. Arkiv. Kemi. 15, 69-74.

WEDFIELD, R. R. 1953. Two-dimensional paper chro- matographic systems with high resolving power for amino acids. Bioehem. Biophys. Acta, 18, 344-345.

SPACKMAN, D. M., STEIN, W. H., and MOORE, S. 1958. Automatic recording apparatus for use in the chromatography of amino acids. Anal. Chem. 30, 1 190-1206.

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