Clinica Chimica Acta, 195 (1990) 2740
27
Elsevier
CCA 04896
Interactions of pancreatic secretory trypsin inhibitor in small intestinal juice: its hydrolysis and protection by intraluminal factors Thomas C. Freeman, Rupert Davies and John Calam Gastroenterology Unit, Department of Medicine. Royal Postgraduate Medical School, London (UK) (Received Key words:
1 February
Trypsin
1990; revision received
inhibitor;
Trypsin;
Jejunum;
15 September
1990; accepted
5 October
1990)
Cholecystokinin
Summary
It has been proposed that modulation of cholecystokinin (CCK) release by proteinases, proteinase inhibitors and protein is mediated by a pancreatic secretory trypsin inhibitor (PSTI), also called monitor peptide, in the rat. When human [‘251]-PSTI was incubated with fasting small bowel juice or activated pancreatic juice > 88% of tracer eluted from gel chromatography in the characteristic position of hydrolysed PSTI. However, when the small bowel juice had been preincubated with soybean trypsin inhibitor 3 g/l, casein 5 g/l or lactalbumin 30 g/l, the hydrolysis of PST1 diminished so that 95%, 32%, and 33% respectively, now eluted in the characteristic position of free (i.e. intact and not bound to an enzyme) PSTI. When [‘251]-PSTI was incubated with pure trypsin, chymotrypsin, elastase or enterokinase > 95% of tracer eluted in the position of PSTI-enzyme complex. Incubation of PST1 with trypsin plus one other enzyme was required to produce hydrolysis. The degree of protection of PST1 from hydrolysis in duodenal juice produced by these substances correlates with their affects on CCK release. Our findings support the hypothesis that PSTIs mediate the modulation of CCK release by intraluminal proteinases, proteinase inhibitors and proteins. -
The peptide hormone cholecystokinin is released from the duodenum by intralumiAbbreviations; PSTI, Pancreatic
Secretory
Trypsin
Inhibitor;
BAPNA,
Na-Benzoyl-DL-Arginine-p-nitro-
anihde. Correspondence Medical School,
0009-8981/90/S
to: John Calam, Gastroenterology Unit, Department Du Cane Rd., London W12 ONN, UK.
03.50 0 1990 Elsevier Scientific Publishers
of Medicine,
B.V. (Biomedical
Division)
Royal
Postgraduate
28
nal protein [1, 21 and trypsin inhibitors [3, 41; release is suppressed by intraluminal trypsin in rats [5] and man [6, 71. These effects are believed to be involved in the pancreatic hypertrophy and neoplasia which occur in rats fed proteinase inhibitors [8], and in the suppression of pain, which occurs when patients with chronic pancreatitis are given enzyme supplements [9]. Pancreatic juice of the rat has recently been found to contain two pancreatic secretory trypsin inhibitors (PSTIs) [lo]. PST1 I, also called monitor peptide, strongly stimulates CCK release in the absence of trypsin [1 11. It has been postulated that the changes in CCK release produced by trypsin, proteinase inhibitors and proteins are mediated by the intraduodenal concentration of free (i.e. intact and not bound to enzymes) monitor peptide [12]. Thus trypsin might bind monitor peptide diminishing CCK release, whereas ingested trypsin inhibitors and protein might displace monitor peptide from trypsin or compete for its active site, resulting in free monitor peptide in the lumen and CCK release. Other mammals, including man, possess PSTIs [13-181 which are secreted in pancreatic juice and are homologous with rat PST1 I [lo]. Thus other PSTIs might play an analogous role to rat PST1 I in the control of CCK release. We therefore examined the interactions between human PST1 and human small bowel juice, in the presence or absence of proteins and proteinase inhibitors, as well as the interaction between PST1 and various purified pancreatic enzymes. We now report that the concentrations of free PST1 produced by different substances correlates with their effect on CCK release. This is consistent with the hypothesis that PSTI’s are involved in the modulation of CCK release. Materials All chemicals were purchased from BDH, Poole, Dorset, UK and enzymes and inhibitors were purchased from Sigma, Gillingham, Dorset, UK, unless otherwise stated. Methods Purljication of PSTI
PST1 was extracted from human pancreatic juice by a method based on that used by Iwai et al. [l l] to extract monitor peptide. Briefly, pancreatic juice not required for clinical investigation was collected from postoperative pancreatic drains and stored at - 20°C. The juice was pooled and mixed with an equal volume of 0.1 mol/l sodium citrate, and the pH adjusted to 2.5. Sodium chloride was then added to a final concentration of 1 mol/l, the mixture maintained at 80°C for 40 min, centrifuged at 3 500 x g for 45 min at 4°C and the supernatant concentrated on a C-18 Sep-Pak cartridge (Waters Associates, Milford, MA) equilibrated with 6.5 mmol/l trifluoroacetic acid in water. The cartridge was eluted with 80% acetonitrile in 6.5 mmol/l trifluoroacetic acid, and the eluent lyophilised. The eluent was reconstituted in 0.05 mol/l ammonium bicarbonate, pH 8.1, and applied to a 1.5 x 100 cm Sephadex G-50
29
superfine column (Pharmacia, Uppsala, Sweden) eluted with the same buffer. Fractions containing trypsin inhibitor activity (see below) were pooled, lyophilised and further purified by reverse phase high performance liquid chromatography on a 10 x 100 mm Dynamax C-8 column (12 pm, 300 A, Rainin, Woburn, MA), eluted with a gradient of 1630% acetonitrile in 13 mmol/l trifluoroacetic acid. Trypsin inhibitor activity eluted in a number of fractions as several poorly resolved peaks. The trypsin inhibitor activity was pooled and applied to a Mono S column (Pharmacia) equilibrated with ammonium acetate 0.1 mol/l, pH 3.5 and eluted with a gradient of ammonium acetate 0.1 mol/l, pH 3.5-4.5. When the three peaks of trypsin inhibitor that eluted from the Mono S column were rerun on a 4.6 x 250 mm Dynamax C-8 reverse phase high pressure liquid chromatography column (12 ,um, 150 A) eluted with a gradient from 22-30s acetonitrile in 13 mmol/l trifluoroacetic acid, peaks I and III emerged as single peaks whereas peak II separated into 2 peaks; II, and 112. The molecular masses of the four peaks, were determined using a ZAB-SE mass spectrometer (VG Instruments, Altrincham, Cheshire) and the amino-acid sequence of peak 1 was analysed of using a protein sequencer (Model 470, Applied Biosystems, Foster City, CA, USA). Iodination of PSTI
The predominant form of human PST1 (peak III) was radio-iodinated with i2jI by the chloramine T method [19]. Briefly, 10 pg of PST1 was dissolved in 50 ~1 of 0.4 mol/l phosphate buffer, pH 6.6, added to 0.5 mCi of Na*251(Amersham, Aylesbury, Bucks, UK) and mixed. The iodination reaction was started by adding 10 ~1 of 8.8 mmol/l chloramine T in 0.04 mol/l phosphate buffer, pH 6.6, and stopped 12 set later by adding 100 pl of 1.3 mmol/l sodium metabisulphate in 0.04 mol/l phosphate buffer. Purification of the [‘251]-PSTI was performed by reverse phase high performance liquid chromatography (Gilson, Luton, Beds., UK) using a C-18 column (0.46 x 30 cm, Techsil, 5 pm, HPLC Technology, Macclesfield, Cheshire, UK) previously equilibrated with 15% acetonitrile in 13 mmol/l trifluoroacetic acid. The column was eluted with a gradient of 15-45s acetonitrile in 13 mmol/l trifluoroacetic acid run over 30 min. Fractions containing [‘251]-PSTI were neutralised by the addition of an equal volume of 0.2 mol/l Tris, bovine serum albumin 10 g/l, divided into portions, and frozen at - 20°C until use. Typically the tracer had a specific activity of 43 ,&i/pg. Trypsin and trypsin inhibitor assays
Trypsin and trypsin inhibitor assays were performed on column eluates using NaBenzoyl-Dt_-Arginine-p-nitroanilide (BAPNA) as substrate by a modification of the procedure reported by Erlanger et al. [20]. Briefly, 40 mg of BAPNA was dissolved in 1 ml of dimethyl sulphoxide (DMSO) and diluted with 100 ml of assay buffer, Tris buffer 0.05 mol/l, pH 8.2, containing 0.02 mol/l CaC12, previously warmed to 37°C. For the measurement of trypsin, 100 ,ul of column eluate was made up to 2 ml with
30
distilled water and the mixture maintained at 37°C for 5 min. The reaction was started by the addition of 2.5 ml of prewarmed BAPNA solution to each tube. After 10 min the reaction was terminated by adding 0.5 ml of 5.2 mol/l acetic acid and the absorbance of the tubes was read on a UV/visible spectrophotometer (PU 8720 Phillips, Cambridge, UK) at 410 nm. One trypsin unit (TU) was defined as an increase of 0.001 absorbance units at 410 nm per 5 ml of the reaction mixture. Trypsin inhibitor activity was measured similarly. 100 ~1 of column eluate was made up to 1 ml with distilled water and incubated with 1 ml of bovine trypsin solution (20 mg/l in 1 mmol/l HCl) for 5 min at 37°C. The samples were then treated as described for the trypsin assay. Trypsin inhibitor activity was expressed as the number of trypsin units inhibited compared to control tubes, under these conditions. Collection of small bowel juices
Small bowel fluid was collected from 2 patients with jejunostomies and two normal volunteers undergoing duodenal aspiration for another study. The juice was divided into 500 ~1 fractions, immediately frozen on solid carbon dioxide and stored at - 20°C. Pancreatic juice and bile were collected from a post-operative drains. The patients had undergone surgery for periampullary tumours. Pancreatic juice was stored at -20°C and the bile at 4°C. Pancreatic juice was activated by a modification of the method described by Rinderknecht et al. [21], by incubation for 1 h at 37°C with enterokinase, 0.05 mIU/l of juice. Incubations of PSTI and chromatographic analysis
This study employed size exclusion chromatography as used by Eddeland et al. to examine interactions of PST1 in plasma [22]. Chromatography was performed on a column (1 x 100 cm) packed with Sephadex G-75 (Pharmacia) eluted with 50 mmol/l Tris, 2 mmol/l CaCl2, 150 mmol/l NaCl buffer, pH 7.6. Samples of juice and solutions of enzymes, 300 ~1, were incubated with approximately 20 ~1 (10 000 c.p.m.) of [‘251]-PSTI, for 40 min at 37°C. A mixture of blue dextran and phenol red in 30 ~1 column buffer was then added, and the mixture rapidly applied to the column. In experiments involving the incubation of the [‘251]-PSTI with more than one enzyme, an equal volume of each enzyme solution (1 g/l) was used so that the total incubation volume was 300 ~1. The purified enzymes were bovine trypsin (EC 3.4.21.4) chymotrypsin (EC 3.4.21.1) and carboxypeptidase A (EC 3.4.17.1) and porcine elastase (EC 3.4.21.36) enterokinse (EC 3.4.21.9) and carboxypeptidase B (EC 3.4.17.2). The following samples and mixtures were applied to the column following the 40min incubation time described above. i. The column was calibrated by running [‘251]-PSTI (in 300 ,~l of column buffer) and trypsin (1 g/l in column buffer) separately and after incubation together. ii. Small bowel juice (4 samples, 300 ~1) with [‘251]-PSTI.
31
iii. Activated pancreatic juice with [1251]-PSTIwith and without an equal volume of bile. Also, bile only with [iZ51]-PSTL iv. Small bowel juice (100 ~1) incubated with soybean trypsin inhibitor (300 fig in 200 ~1 of column buffer) for 40 min at 37°C and then incubated with [‘z51]-PSTI. v. Same bowel juice (100 ~1) with column buffer (200 ~1) and [‘251]-PSTI, incubated alone and with casein or lactalbumin at concentrations in the incubation mixture of l-15 g/l juice and 10-30 g/l juice, respectively. vi. Purified enzymes (1 g/l in column buffer) with [1251]-PSTI. vii. The [1251]-PSTItracer was subjected to chromatography at regular intervals to ensure that its elution position had not changed with time. The column was eluted at about 20 ml/h and fractions were collected every 3 min and counted on a gamma counter (LKB, 1260 Multigamma II, Turka, Finland). The V, and Vt were located by absorbance at 560 nm. The position of elution of the peaks of radioactivity is expressed as a percentage of the time between elution of blue dextran and phenol red. Where more than one peak of radioactivity emerged, the radioactivity in each peak was divided by the total eluted radioactivity. Results The purification procedure for human PST1 lead to the separation of PST1 into 4 forms after the final reverse phase chromatography step. The molecular masses these forms of PST1 as determined by mass spectrometry were 16242.5, IIi:6241.8, I11:6241.6 and IIL6242.5, compared with the predicted molecular mass for protonated PST1 of 6242.1. Amino-acid sequence analysis of peak I showed the N-terminal tridecapeptide sequence of peak I was equal to that of human PSTI. When [‘251]-PSTTwas applied to a Sephadex G-75 column before and after incubation with purified enzymes or small bowel juice with or without various intraluminal factors, the radioactivity eluted as follows. i. Calibration of the column showed free [‘251]-PSTI to elute at 64.3 4 1.569, M= 6, (mean + SE), trypsin eluted at 30.0 f 2.0%, n = 3, and after the incubation of trypsin with [1251]-PSTI, the trypsin-PST1 complex eluted at 26.15 1.4%, n=4 (Fig. 1). In some column runs tracer eluted after free PSTI, usually in a discrete peak between X0-85%, indicating hydrolysis of PSTI. Occasionally radioactivity eluted after 85Sr; (see below). ii. After the incubation of [‘251]-PSTI with samples of small bowel juice >0.88 of tracer eluted in the position of hydrolysed PST1 (2 samples) (Fig. 2a), or slightly later (93% and 100%). After incubation of [‘251]-PSTI with one of the samples of jejunal juice 0.11 of tracer eluted in the position of PSTI-enzyme complex. iii. After the incubation of [1251]-PSTI with activated human pancreatic juice all the tracer eluted from the column in the hydrolysed position (Fig. 2b). Addition of bile to the incubation mixture did not change the elution position of the tracer. After incubation of [1251]-PSTI with bile all of the tracer eluted from the column in the position of free PSTI. iv. Pre-incubation of small bowel juice with an excess of soybean trypsin inhibitor
(A)
CC)
=‘
,” 600-
f
d ,:
dOO20001, 0
I, 20
I. 40
I, 60
I 80
100
% ELUTION
Fig. I. Elution profile of (A) [iZ51]-PSTI,(B) trypsin and (C) [Y]-PST1 after incubation with trypsin, from a Sephadex G-75 column (1 x 100 cm), equilibrated and eluted with 50 mmol/l Tris, 2 mmol/l CaC12, 150 mmol/l NaCl buffer pH 7.6. The elution position of the [1251]-PSTI(A&C) was measured by counting the radioactivity (counts per minute/fraction) and the elution of trypsin (C) by measuring the trypsin activity (TU) in the column eluate.
before incubation with [1251]-PSTI resulted in >0.95 of the tracer eluting from the column in the position of free PST1 (Fig. 2~). v. After the incubation of [1251]-PSTI in jejunostomy fluid Cjejunostomy 1, Table I) diluted 1:2 in column buffer, 0.25 of the tracer eluted in the position of enzymePST1 complex and 0.75 at 88%, with no peak of free PST1 (Fig. 3a). When casein (l-l 5 g/l juice) was added to the incubation mixture the elution position of the tracer altered. With increasing casein concentration (Fig. 3~): Tracer eluting in the free position increased to a maximum, then decreased with casein > 6 g/l juice. Tracer eluting in the position of PSTI-enzyme complex increased linearly and the amount of tracer eluting in the position of hydrolysed PST1 was greatly decreased, even with low concentrations of casein. Lactalbumin caused similar changes to the elution pattern of
33
TRYPSIN-PST1 COMPLEX
iooo-
(A)
FREE
PST1
600‘
,i/
\ 0
20
40
6'0
60
160
40
60
60
100
60
60
100
= (8)
e
12oo-
E
1000-
z s !
&loo600400-
E
200-
0
20
600 -
CC)
500' 400-
om.,,,,,.,., 0 20
40 %
Fig. 2. Elution
profile of [1251]-PSTI from a Sephadex
with 50 mmol/l Tris, 2 mmol/l with (A) duodenal 37°C with soybean
G-75 column
(1 x 100 cm), equilibrated
CaCl*, 150 mmol/l NaCl buffer pH 7.6, after incubation
juice, (B) pancreatic trypsin
ELUTION
inhibitor.
the radioactivity
juice and (C) duodenal The elution
position
juice after preincubation
of the [rZ51]-PST1 was measured
(counts per minute/fraction)
in the column
and eluted
for 40 min at 37°C for 40 min at by counting
eluate.
the tracer but considerably higher concentrations of lactalbumin were required (Fig. 3b). For example 30 g/l juice of lactalbumin had to be added to produce similar affects to 5 g/l juice of casein. vi. After the incubation of [‘251]-PSTI with either chymotrypsin, elastase or enterokinase, > 0.95 of the tracer eluted in a similar position to the PSTI-trypsin complex, However, when [ ‘251]-PSTI was incubated with trypsin and one of the other three enzymes, only about 0.35 of the tracer now eluted in the position of complex, the remainder eluting in the other two positions. The amount tracer eluting in the hydrolysed position increasing from 0.19 with enterokinase through 0.34 with chymotrypsin to 0.49 with elastase. Incubation with chymotrypsin, elastase and enterokinase in the absence of trypsin, resulted in 0.90 complex and only 0.10 hydrolysed (Fig.
34 TABLE 1
INCUBATIONMIXTURE
ENZYME-PST COMPLEX
FREE PST1
HMROLSED PST1
SMALLBOWELJUICES
1
Jejurlostomy Jejunostomy Normal Normal
2 1 2
1 .o 1.0 1.0
Pancreatic Juice Bile Pancreatic Juice + Bile
1.0 1
0.05
Jejunostomy (100 ul +, 200 ul Buffer) Jejunostomy + 1 mg Lactalbumin Jejunostomy + 2 mg Lactalbumin Jejunostomy + 3 mg Lactalbumin
5.25 0.27 0.38 0.35
+ 5.1 mg Casein + 5.5 mg Casein + 1.5 mg Casein + 1.5 mg Casein
.o 1.0
Jejunostomy + SBTI
Jejoonstomy Jejunostomy Jejunostomy Jejunostomy
0.89
0.11
5.27 0.38
0.54 0.72
0.95
0.30 0.33
0.75 0.73 0.32 0.32
0.19 0.32 0.30 0.17
0.54 0.30 0.16 0.1 I
ENZYMES
Trypsin Chymotrypsin Elastase Enterokinase
5.92 1.0 1.0 1.5
Trypsin and Chymotrypsin Trypsin and Elastase Trypsin and Enterokinase
0.32 0.35 0.36
zhymotrypsin. Etastase, Enterokinase
0.90
Trypsin, Chymotrypsin, Elastase, Enterokinase
0.12
5.34
0.54
Trypsin, Chymot~ps~n, Etastase, Enterokinase, Carboxypeptidase A & 8
0.24
0.30
0.46
0.08
0.34 O.tS 0.44
0.34 0.49 0.19 0.10
46
ECUnON
X ELUTION
Fig. 3. Elution profile of [i*SI]-PSTI from a Sephadex G-75 column (1 x 100 cm), equilibrated and eluted with 50 mmol/l Tris, 2 mmol/l CaCl?, 150 mmol/l NaCl buffer pH 7.6, after incubation for 40 min at 37°C with (A) duodenal juice (100 ~1f200 /cl column buffer), (B) duodenal juice plus lactalbumin (1) 10 g/l, (2) 20 g/l (3) 30 g/l of juice and (C) duodenal juice pius casein (I) I g/l (2) 5 g/1 (3) 10 g/1 (4) IS g/l of juice. The elution position of the [125i]-PSTIwas measured by counting the radioactivity (counts per minute/fraction) in the column eluate.
4a). With these 3 enzymes plus trypsin only 0.12 now eluted as complex with 0.34 free and 0.54 hydrolysed (Fig. 4b). The addition of carboxypeptidases A and B to this mixture caused no apparent change (Fig. 4~). Discussion In this study we have shown for the first time that PST1 is cleaved into smaller fragments by fasting small bowel juice due to the combined action of pancreatic serine proteinases. The presence of trypsin in the incubation mixture was essential for this hydrolysis. Hydrolysis was inhibited completely by soybean trypsin inhibitor,
TAYPSIN-PST1 COMPLEX
FREE
PST1
(A)
600 500 400 300 200 100 0-a.
0
8. 20
0
20
I., 40
60
, 80
/ 100
40
60
80
100
(C)
%
Fig. 4. Elution
profile of [r251]-PST1 from a Sephadex
with 50 mmol/l
Tris, 2 mmol/l
with (A) chymotrypsin, and (C) trypsin,
CaClr,
elastase
chymotrypsin,
of the [‘*51]-PST1 was measured
ELUTION
G-75 column
(1 x 100 cm), equilibrated
150 mmol/l NaCl buffer pH 7.6, after incubation
and enterokinase, elastase,
(B) trypsin,
enterokinase
by counting
chymotrypsin,
and carboxypeptidase
the radioactivity eluate.
(counts
and eluted
for 40 min at 37°C
elastase
and enterokinase,
A&B. The elution
per minute/fraction)
position
in the column
partially by casein and weakly by lactalbumin. These results are consistent with the proposed role of PST1 in the modulation of CCK release by proteinases, their inhibitors and proteins. This study was prompted by the observation that rat PST1 I, which has 52% homology with human PST1 [lo], unlike exogenous proteinase inhibitors, stimulates CCK release in the absence of trypsin [l 11. This led to the suggestion that intraluminal proteinases suppress CCK release by binding or hydrolysing PST1 and that intraluminal proteinase inhibitors and proteins displace enzyme bound PST1 or protect it from hydrolysis [12]. Moreover, Fushiki [23] also demonstrated that the intraduodenal ad-
37
ministration anti-PST1 I antibodies diminished protein stimulated CCK release in the rat. Thus the interactions of PST1 with intraluminal enzymes are central to the understanding of the mechanisms of CCK release. When PST1 was incubated in the presence of fasting small bowel juice or pancreatic juice the majority of tracer eluted between 80 and loo%, due to the hydrolysis of PST1 by pancreatic enzymes. Preincubation of the small bowel juice with trypsin inhibitor, a potent stimulant of CCK release in both rats [3] and man [4], resulted in the PST1 eluting in free position. Soybeans contain two trypsin inhibitors; Kunitz and Bowman-Birk, with dissociation constants, &, for bovine trypsin of 3.1 x lo-‘* and 2.9 x lob7 respectively [24]. Human PST1 has a Ki for bovine trypsin [25] of 3.8 x IO-*. Thus displacement of human PST1 from human trypsin by soybean trypsin inhibitors is consistent with published kinetic data. In addition, our findings indicate that soybean trypsin inhibitors have sufficient affinity with the other human serine proteinases to displace human PSTI. This study has demonstrated that casein is better at preventing the hydrolysis of human PST1 than is lactalbumin. Liddle et al. [2] showed that casein inhibited the tryptic hydrolysis of an alternative substrate to a greater extent than lactalbumin. Fushiki et al. 1231showed that trypsin released more trichloroacetic acid-soluble peptides from casein than from lactalbumin. Thus it appears that trypsin has a greater affinity for casein than for lactalbumin, consistent with our findings. Interestingly Liddle [2] and Fushiki [23] both found casein to be a more potent stimulant of CCK release than la&albumin in the rat. The results of the present study therefore support the idea that PSTIs mediate CCK release by proteins. The aim of the studies with pure enzymes was to determine which enzyme or enzymes cause the marked hydrolysis of PST1 in small bowel juice. No individual serine proteinase hydrolysed PST1 but all four; trypsin, chymotrypsin, elastase and enterokinase, formed PSTI-enzyme complexes. The binding of PST1 to elastase and enterokinase was unexpected because neither enzyme is inhibited by PSTI 1131.This might reflect non-inhibitory interactions or traces of trypsin present in the commercial enzyme preparations. Combinations of trypsin with one other serine proteinase caused partial hydrolysis. The enzyme pair with most effect was trypsin plus elastase, but a mixture of the four enzymes had most effect. The addition of carboxypeptidase did not have any demonstrable effect on hydrolysis. When PST1 was incubated with chymotrypsin, elastase and enterokinase in the absence of trypsin, very little hydrolysis was observed (10%). Thus trypsin appears to render PST1 susceptible to digestion by the other enzymes. Trypsin is known to cleave the Arg’*-Ilei9 bond at the reactive site of PST1 [26]. The product of this reaction retains trypsin inhibitor activity, but prolonged incubation of the trypsin-PST1 complex leads to return of tryptic activity, an effect known as temporary inhibition 1271.No combination of purified enzymes produced as complete hydrolysis of PST1 as whole small bowel juice. This may reflect differences between human enzymes and the purified animal enzymes used in this part of the study. The nature of interactions of PST1 with the different pancreatic enzymes is also relevant to its protective role within the pancreas. Trypsin is unique amongst pan-
3x
creatic proteinases in having the ability to activate the zymogens of all the other pancreatic proteinases. Inhibition of trypsin is therefore central to protection and PST1 has its greatest inhibitory effect on this enzyme. However, the results of the present study show that PST1 would be rapidly destroyed within the gland if multiple enzymes were activated, as occurs in acute pancreatitis. Obstruction to the flow of juice out of the gland causes pancreatitis. PST1 may fail to protect the pancreas under conditions of stasis because its inhibitory effect is temporary [27]. Interest in the survival of PST1 in small intestinal juice has been increased by the finding that PSTIs have effects other than the inhibition of trypsin. Human PST1 has growth factor activity [28-301. The atrophy of the small intestine which occurs during fasting [3 1] might be due to increased hydrolysis of PST1 and the structurally related proteins such as EGF, in the absence of other enzyme substrates. The results of this study are consistent with the proposed role of PST1 in CCK release. Further studies are required to determine whether PST1 effects the growth of the gut, and the CCK-releasing activities of PSTIs in other species including man. Acknowledgement We are grateful to the Wellcome Trust for financial support, and to Linda Poulter and Janice Young of ICI Pharmaceuticals for Mass Spectrometry and Sequence Analysis of PSTI. References 1 Liddle trypsin 2 Liddle
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N, Nagata
Nature
H, Schwarz Hoppe-Seyler’s
F, Minchiotti secretory
amino acid sequence activity.
W, Fritz
aus schafpankreas.
E, Bortolotti
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I9 Hunter
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J Biol Chem 1969;244:26462657.
N, Cohen
Archs Biochem
H, Renner
W. The preparation Biophys
IG, Curmack
and properties
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1961;95:271-278,
C. Activation
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7313699372. 22 Eddland
A, Ohlsson
with trypsin 23 Fushiki
T, Kajiura
in rat pancreatic trypsin
K. Studies on the pancreatic H, Fukuoka
enzyme
and monitor
24 Hanlon
peptide.
protease
25 Matsuda
K, Ogawa
inhibitor
R, Laskowski
27 Laskowski 28 Freeman
factors
of the inhibition
in plasma and its complex
trypsin
WL, Sakagami
from hepatoma
of rat and bovine
mediator
with dietary
inhibition carcinoma
trypsins
and some properties
urine. Enzyme
protein,
by naturally
(Kazal). of trypsin.
J. Pancreatic
of a low mole-
1985;34: 129-139.
and kinetics of the reactive
inhibitor
B, Calam J, Woodburn rat pancreatic
for an intraluminal
response
1986;84B:53-57.
T, Ishida M, Mori T. Purification
MJ. Thermodynamics
M, Wu FC. Temporary
of AR4-2J
of the pancreatic
from acute pancreatitis
secretory
TC, Curry
29 McKeehan
inhibitor
1989; 119: 622-627.
Comp Res Physiol
M, Kitahara
in bovine pancreatic
growth
Reconstitution
J Nutr
inhibitors.
cular weight trypsin 26 Sealock
trypsin
S-I, Kido K, Semba T, Iwai K. Evidence
secretion:
MH, Liener IE. A kinetic analysis
occurring
secretory
in vivo and in vitro. Stand J Clin Lab Invest 1978;38:5077515.
Biochemistry
site peptide-bond
hydrolysis
1973; 12:3 139-3 146.
J Biol Chem 1953;204:797-805. secretory
trypsin
inhibitor
(PSTI) stimulates
cells. Gut 1989;30(5):A752.
Y, Hoshi H, McKeehan
cells are tumour-associated
KA. Two apparent proteinase
human
inhibitors.
endothelial
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J Biol Chem 1986;261:53788
5383. 30 Ogawa
M, Tsushima
pancreatic 31 Feldman intestinal
secretory
T, Ohba Y, et al. Stimulation trypsin
EJ, Dowling adaptation
inhibitor.
Res Commun
RH, NcNaughton
of DNA synthesis Chem Pathol
in human
Pharmacol
fibroblasts
J, Peters TJ. Effect of oral versus intravenous
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1985;50: 1555158. nutrition
1976;70:712-719.
on
27
Elsevier
CCA 04896
Interactions of pancreatic secretory trypsin inhibitor in small intestinal juice: its hydrolysis and protection by intraluminal factors Thomas C. Freeman, Rupert Davies and John Calam Gastroenterology Unit, Department of Medicine. Royal Postgraduate Medical School, London (UK) (Received Key words:
1 February
Trypsin
1990; revision received
inhibitor;
Trypsin;
Jejunum;
15 September
1990; accepted
5 October
1990)
Cholecystokinin
Summary
It has been proposed that modulation of cholecystokinin (CCK) release by proteinases, proteinase inhibitors and protein is mediated by a pancreatic secretory trypsin inhibitor (PSTI), also called monitor peptide, in the rat. When human [‘251]-PSTI was incubated with fasting small bowel juice or activated pancreatic juice > 88% of tracer eluted from gel chromatography in the characteristic position of hydrolysed PSTI. However, when the small bowel juice had been preincubated with soybean trypsin inhibitor 3 g/l, casein 5 g/l or lactalbumin 30 g/l, the hydrolysis of PST1 diminished so that 95%, 32%, and 33% respectively, now eluted in the characteristic position of free (i.e. intact and not bound to an enzyme) PSTI. When [‘251]-PSTI was incubated with pure trypsin, chymotrypsin, elastase or enterokinase > 95% of tracer eluted in the position of PSTI-enzyme complex. Incubation of PST1 with trypsin plus one other enzyme was required to produce hydrolysis. The degree of protection of PST1 from hydrolysis in duodenal juice produced by these substances correlates with their affects on CCK release. Our findings support the hypothesis that PSTIs mediate the modulation of CCK release by intraluminal proteinases, proteinase inhibitors and proteins. -
The peptide hormone cholecystokinin is released from the duodenum by intralumiAbbreviations; PSTI, Pancreatic
Secretory
Trypsin
Inhibitor;
BAPNA,
Na-Benzoyl-DL-Arginine-p-nitro-
anihde. Correspondence Medical School,
0009-8981/90/S
to: John Calam, Gastroenterology Unit, Department Du Cane Rd., London W12 ONN, UK.
03.50 0 1990 Elsevier Scientific Publishers
of Medicine,
B.V. (Biomedical
Division)
Royal
Postgraduate
28
nal protein [1, 21 and trypsin inhibitors [3, 41; release is suppressed by intraluminal trypsin in rats [5] and man [6, 71. These effects are believed to be involved in the pancreatic hypertrophy and neoplasia which occur in rats fed proteinase inhibitors [8], and in the suppression of pain, which occurs when patients with chronic pancreatitis are given enzyme supplements [9]. Pancreatic juice of the rat has recently been found to contain two pancreatic secretory trypsin inhibitors (PSTIs) [lo]. PST1 I, also called monitor peptide, strongly stimulates CCK release in the absence of trypsin [1 11. It has been postulated that the changes in CCK release produced by trypsin, proteinase inhibitors and proteins are mediated by the intraduodenal concentration of free (i.e. intact and not bound to enzymes) monitor peptide [12]. Thus trypsin might bind monitor peptide diminishing CCK release, whereas ingested trypsin inhibitors and protein might displace monitor peptide from trypsin or compete for its active site, resulting in free monitor peptide in the lumen and CCK release. Other mammals, including man, possess PSTIs [13-181 which are secreted in pancreatic juice and are homologous with rat PST1 I [lo]. Thus other PSTIs might play an analogous role to rat PST1 I in the control of CCK release. We therefore examined the interactions between human PST1 and human small bowel juice, in the presence or absence of proteins and proteinase inhibitors, as well as the interaction between PST1 and various purified pancreatic enzymes. We now report that the concentrations of free PST1 produced by different substances correlates with their effect on CCK release. This is consistent with the hypothesis that PSTI’s are involved in the modulation of CCK release. Materials All chemicals were purchased from BDH, Poole, Dorset, UK and enzymes and inhibitors were purchased from Sigma, Gillingham, Dorset, UK, unless otherwise stated. Methods Purljication of PSTI
PST1 was extracted from human pancreatic juice by a method based on that used by Iwai et al. [l l] to extract monitor peptide. Briefly, pancreatic juice not required for clinical investigation was collected from postoperative pancreatic drains and stored at - 20°C. The juice was pooled and mixed with an equal volume of 0.1 mol/l sodium citrate, and the pH adjusted to 2.5. Sodium chloride was then added to a final concentration of 1 mol/l, the mixture maintained at 80°C for 40 min, centrifuged at 3 500 x g for 45 min at 4°C and the supernatant concentrated on a C-18 Sep-Pak cartridge (Waters Associates, Milford, MA) equilibrated with 6.5 mmol/l trifluoroacetic acid in water. The cartridge was eluted with 80% acetonitrile in 6.5 mmol/l trifluoroacetic acid, and the eluent lyophilised. The eluent was reconstituted in 0.05 mol/l ammonium bicarbonate, pH 8.1, and applied to a 1.5 x 100 cm Sephadex G-50
29
superfine column (Pharmacia, Uppsala, Sweden) eluted with the same buffer. Fractions containing trypsin inhibitor activity (see below) were pooled, lyophilised and further purified by reverse phase high performance liquid chromatography on a 10 x 100 mm Dynamax C-8 column (12 pm, 300 A, Rainin, Woburn, MA), eluted with a gradient of 1630% acetonitrile in 13 mmol/l trifluoroacetic acid. Trypsin inhibitor activity eluted in a number of fractions as several poorly resolved peaks. The trypsin inhibitor activity was pooled and applied to a Mono S column (Pharmacia) equilibrated with ammonium acetate 0.1 mol/l, pH 3.5 and eluted with a gradient of ammonium acetate 0.1 mol/l, pH 3.5-4.5. When the three peaks of trypsin inhibitor that eluted from the Mono S column were rerun on a 4.6 x 250 mm Dynamax C-8 reverse phase high pressure liquid chromatography column (12 ,um, 150 A) eluted with a gradient from 22-30s acetonitrile in 13 mmol/l trifluoroacetic acid, peaks I and III emerged as single peaks whereas peak II separated into 2 peaks; II, and 112. The molecular masses of the four peaks, were determined using a ZAB-SE mass spectrometer (VG Instruments, Altrincham, Cheshire) and the amino-acid sequence of peak 1 was analysed of using a protein sequencer (Model 470, Applied Biosystems, Foster City, CA, USA). Iodination of PSTI
The predominant form of human PST1 (peak III) was radio-iodinated with i2jI by the chloramine T method [19]. Briefly, 10 pg of PST1 was dissolved in 50 ~1 of 0.4 mol/l phosphate buffer, pH 6.6, added to 0.5 mCi of Na*251(Amersham, Aylesbury, Bucks, UK) and mixed. The iodination reaction was started by adding 10 ~1 of 8.8 mmol/l chloramine T in 0.04 mol/l phosphate buffer, pH 6.6, and stopped 12 set later by adding 100 pl of 1.3 mmol/l sodium metabisulphate in 0.04 mol/l phosphate buffer. Purification of the [‘251]-PSTI was performed by reverse phase high performance liquid chromatography (Gilson, Luton, Beds., UK) using a C-18 column (0.46 x 30 cm, Techsil, 5 pm, HPLC Technology, Macclesfield, Cheshire, UK) previously equilibrated with 15% acetonitrile in 13 mmol/l trifluoroacetic acid. The column was eluted with a gradient of 15-45s acetonitrile in 13 mmol/l trifluoroacetic acid run over 30 min. Fractions containing [‘251]-PSTI were neutralised by the addition of an equal volume of 0.2 mol/l Tris, bovine serum albumin 10 g/l, divided into portions, and frozen at - 20°C until use. Typically the tracer had a specific activity of 43 ,&i/pg. Trypsin and trypsin inhibitor assays
Trypsin and trypsin inhibitor assays were performed on column eluates using NaBenzoyl-Dt_-Arginine-p-nitroanilide (BAPNA) as substrate by a modification of the procedure reported by Erlanger et al. [20]. Briefly, 40 mg of BAPNA was dissolved in 1 ml of dimethyl sulphoxide (DMSO) and diluted with 100 ml of assay buffer, Tris buffer 0.05 mol/l, pH 8.2, containing 0.02 mol/l CaC12, previously warmed to 37°C. For the measurement of trypsin, 100 ,ul of column eluate was made up to 2 ml with
30
distilled water and the mixture maintained at 37°C for 5 min. The reaction was started by the addition of 2.5 ml of prewarmed BAPNA solution to each tube. After 10 min the reaction was terminated by adding 0.5 ml of 5.2 mol/l acetic acid and the absorbance of the tubes was read on a UV/visible spectrophotometer (PU 8720 Phillips, Cambridge, UK) at 410 nm. One trypsin unit (TU) was defined as an increase of 0.001 absorbance units at 410 nm per 5 ml of the reaction mixture. Trypsin inhibitor activity was measured similarly. 100 ~1 of column eluate was made up to 1 ml with distilled water and incubated with 1 ml of bovine trypsin solution (20 mg/l in 1 mmol/l HCl) for 5 min at 37°C. The samples were then treated as described for the trypsin assay. Trypsin inhibitor activity was expressed as the number of trypsin units inhibited compared to control tubes, under these conditions. Collection of small bowel juices
Small bowel fluid was collected from 2 patients with jejunostomies and two normal volunteers undergoing duodenal aspiration for another study. The juice was divided into 500 ~1 fractions, immediately frozen on solid carbon dioxide and stored at - 20°C. Pancreatic juice and bile were collected from a post-operative drains. The patients had undergone surgery for periampullary tumours. Pancreatic juice was stored at -20°C and the bile at 4°C. Pancreatic juice was activated by a modification of the method described by Rinderknecht et al. [21], by incubation for 1 h at 37°C with enterokinase, 0.05 mIU/l of juice. Incubations of PSTI and chromatographic analysis
This study employed size exclusion chromatography as used by Eddeland et al. to examine interactions of PST1 in plasma [22]. Chromatography was performed on a column (1 x 100 cm) packed with Sephadex G-75 (Pharmacia) eluted with 50 mmol/l Tris, 2 mmol/l CaCl2, 150 mmol/l NaCl buffer, pH 7.6. Samples of juice and solutions of enzymes, 300 ~1, were incubated with approximately 20 ~1 (10 000 c.p.m.) of [‘251]-PSTI, for 40 min at 37°C. A mixture of blue dextran and phenol red in 30 ~1 column buffer was then added, and the mixture rapidly applied to the column. In experiments involving the incubation of the [‘251]-PSTI with more than one enzyme, an equal volume of each enzyme solution (1 g/l) was used so that the total incubation volume was 300 ~1. The purified enzymes were bovine trypsin (EC 3.4.21.4) chymotrypsin (EC 3.4.21.1) and carboxypeptidase A (EC 3.4.17.1) and porcine elastase (EC 3.4.21.36) enterokinse (EC 3.4.21.9) and carboxypeptidase B (EC 3.4.17.2). The following samples and mixtures were applied to the column following the 40min incubation time described above. i. The column was calibrated by running [‘251]-PSTI (in 300 ,~l of column buffer) and trypsin (1 g/l in column buffer) separately and after incubation together. ii. Small bowel juice (4 samples, 300 ~1) with [‘251]-PSTI.
31
iii. Activated pancreatic juice with [1251]-PSTIwith and without an equal volume of bile. Also, bile only with [iZ51]-PSTL iv. Small bowel juice (100 ~1) incubated with soybean trypsin inhibitor (300 fig in 200 ~1 of column buffer) for 40 min at 37°C and then incubated with [‘z51]-PSTI. v. Same bowel juice (100 ~1) with column buffer (200 ~1) and [‘251]-PSTI, incubated alone and with casein or lactalbumin at concentrations in the incubation mixture of l-15 g/l juice and 10-30 g/l juice, respectively. vi. Purified enzymes (1 g/l in column buffer) with [1251]-PSTI. vii. The [1251]-PSTItracer was subjected to chromatography at regular intervals to ensure that its elution position had not changed with time. The column was eluted at about 20 ml/h and fractions were collected every 3 min and counted on a gamma counter (LKB, 1260 Multigamma II, Turka, Finland). The V, and Vt were located by absorbance at 560 nm. The position of elution of the peaks of radioactivity is expressed as a percentage of the time between elution of blue dextran and phenol red. Where more than one peak of radioactivity emerged, the radioactivity in each peak was divided by the total eluted radioactivity. Results The purification procedure for human PST1 lead to the separation of PST1 into 4 forms after the final reverse phase chromatography step. The molecular masses these forms of PST1 as determined by mass spectrometry were 16242.5, IIi:6241.8, I11:6241.6 and IIL6242.5, compared with the predicted molecular mass for protonated PST1 of 6242.1. Amino-acid sequence analysis of peak I showed the N-terminal tridecapeptide sequence of peak I was equal to that of human PSTI. When [‘251]-PSTTwas applied to a Sephadex G-75 column before and after incubation with purified enzymes or small bowel juice with or without various intraluminal factors, the radioactivity eluted as follows. i. Calibration of the column showed free [‘251]-PSTI to elute at 64.3 4 1.569, M= 6, (mean + SE), trypsin eluted at 30.0 f 2.0%, n = 3, and after the incubation of trypsin with [1251]-PSTI, the trypsin-PST1 complex eluted at 26.15 1.4%, n=4 (Fig. 1). In some column runs tracer eluted after free PSTI, usually in a discrete peak between X0-85%, indicating hydrolysis of PSTI. Occasionally radioactivity eluted after 85Sr; (see below). ii. After the incubation of [‘251]-PSTI with samples of small bowel juice >0.88 of tracer eluted in the position of hydrolysed PST1 (2 samples) (Fig. 2a), or slightly later (93% and 100%). After incubation of [‘251]-PSTI with one of the samples of jejunal juice 0.11 of tracer eluted in the position of PSTI-enzyme complex. iii. After the incubation of [1251]-PSTI with activated human pancreatic juice all the tracer eluted from the column in the hydrolysed position (Fig. 2b). Addition of bile to the incubation mixture did not change the elution position of the tracer. After incubation of [1251]-PSTI with bile all of the tracer eluted from the column in the position of free PSTI. iv. Pre-incubation of small bowel juice with an excess of soybean trypsin inhibitor
(A)
CC)
=‘
,” 600-
f
d ,:
dOO20001, 0
I, 20
I. 40
I, 60
I 80
100
% ELUTION
Fig. I. Elution profile of (A) [iZ51]-PSTI,(B) trypsin and (C) [Y]-PST1 after incubation with trypsin, from a Sephadex G-75 column (1 x 100 cm), equilibrated and eluted with 50 mmol/l Tris, 2 mmol/l CaC12, 150 mmol/l NaCl buffer pH 7.6. The elution position of the [1251]-PSTI(A&C) was measured by counting the radioactivity (counts per minute/fraction) and the elution of trypsin (C) by measuring the trypsin activity (TU) in the column eluate.
before incubation with [1251]-PSTI resulted in >0.95 of the tracer eluting from the column in the position of free PST1 (Fig. 2~). v. After the incubation of [1251]-PSTI in jejunostomy fluid Cjejunostomy 1, Table I) diluted 1:2 in column buffer, 0.25 of the tracer eluted in the position of enzymePST1 complex and 0.75 at 88%, with no peak of free PST1 (Fig. 3a). When casein (l-l 5 g/l juice) was added to the incubation mixture the elution position of the tracer altered. With increasing casein concentration (Fig. 3~): Tracer eluting in the free position increased to a maximum, then decreased with casein > 6 g/l juice. Tracer eluting in the position of PSTI-enzyme complex increased linearly and the amount of tracer eluting in the position of hydrolysed PST1 was greatly decreased, even with low concentrations of casein. Lactalbumin caused similar changes to the elution pattern of
33
TRYPSIN-PST1 COMPLEX
iooo-
(A)
FREE
PST1
600‘
,i/
\ 0
20
40
6'0
60
160
40
60
60
100
60
60
100
= (8)
e
12oo-
E
1000-
z s !
&loo600400-
E
200-
0
20
600 -
CC)
500' 400-
om.,,,,,.,., 0 20
40 %
Fig. 2. Elution
profile of [1251]-PSTI from a Sephadex
with 50 mmol/l Tris, 2 mmol/l with (A) duodenal 37°C with soybean
G-75 column
(1 x 100 cm), equilibrated
CaCl*, 150 mmol/l NaCl buffer pH 7.6, after incubation
juice, (B) pancreatic trypsin
ELUTION
inhibitor.
the radioactivity
juice and (C) duodenal The elution
position
juice after preincubation
of the [rZ51]-PST1 was measured
(counts per minute/fraction)
in the column
and eluted
for 40 min at 37°C for 40 min at by counting
eluate.
the tracer but considerably higher concentrations of lactalbumin were required (Fig. 3b). For example 30 g/l juice of lactalbumin had to be added to produce similar affects to 5 g/l juice of casein. vi. After the incubation of [‘251]-PSTI with either chymotrypsin, elastase or enterokinase, > 0.95 of the tracer eluted in a similar position to the PSTI-trypsin complex, However, when [ ‘251]-PSTI was incubated with trypsin and one of the other three enzymes, only about 0.35 of the tracer now eluted in the position of complex, the remainder eluting in the other two positions. The amount tracer eluting in the hydrolysed position increasing from 0.19 with enterokinase through 0.34 with chymotrypsin to 0.49 with elastase. Incubation with chymotrypsin, elastase and enterokinase in the absence of trypsin, resulted in 0.90 complex and only 0.10 hydrolysed (Fig.
34 TABLE 1
INCUBATIONMIXTURE
ENZYME-PST COMPLEX
FREE PST1
HMROLSED PST1
SMALLBOWELJUICES
1
Jejurlostomy Jejunostomy Normal Normal
2 1 2
1 .o 1.0 1.0
Pancreatic Juice Bile Pancreatic Juice + Bile
1.0 1
0.05
Jejunostomy (100 ul +, 200 ul Buffer) Jejunostomy + 1 mg Lactalbumin Jejunostomy + 2 mg Lactalbumin Jejunostomy + 3 mg Lactalbumin
5.25 0.27 0.38 0.35
+ 5.1 mg Casein + 5.5 mg Casein + 1.5 mg Casein + 1.5 mg Casein
.o 1.0
Jejunostomy + SBTI
Jejoonstomy Jejunostomy Jejunostomy Jejunostomy
0.89
0.11
5.27 0.38
0.54 0.72
0.95
0.30 0.33
0.75 0.73 0.32 0.32
0.19 0.32 0.30 0.17
0.54 0.30 0.16 0.1 I
ENZYMES
Trypsin Chymotrypsin Elastase Enterokinase
5.92 1.0 1.0 1.5
Trypsin and Chymotrypsin Trypsin and Elastase Trypsin and Enterokinase
0.32 0.35 0.36
zhymotrypsin. Etastase, Enterokinase
0.90
Trypsin, Chymotrypsin, Elastase, Enterokinase
0.12
5.34
0.54
Trypsin, Chymot~ps~n, Etastase, Enterokinase, Carboxypeptidase A & 8
0.24
0.30
0.46
0.08
0.34 O.tS 0.44
0.34 0.49 0.19 0.10
46
ECUnON
X ELUTION
Fig. 3. Elution profile of [i*SI]-PSTI from a Sephadex G-75 column (1 x 100 cm), equilibrated and eluted with 50 mmol/l Tris, 2 mmol/l CaCl?, 150 mmol/l NaCl buffer pH 7.6, after incubation for 40 min at 37°C with (A) duodenal juice (100 ~1f200 /cl column buffer), (B) duodenal juice plus lactalbumin (1) 10 g/l, (2) 20 g/l (3) 30 g/l of juice and (C) duodenal juice pius casein (I) I g/l (2) 5 g/1 (3) 10 g/1 (4) IS g/l of juice. The elution position of the [125i]-PSTIwas measured by counting the radioactivity (counts per minute/fraction) in the column eluate.
4a). With these 3 enzymes plus trypsin only 0.12 now eluted as complex with 0.34 free and 0.54 hydrolysed (Fig. 4b). The addition of carboxypeptidases A and B to this mixture caused no apparent change (Fig. 4~). Discussion In this study we have shown for the first time that PST1 is cleaved into smaller fragments by fasting small bowel juice due to the combined action of pancreatic serine proteinases. The presence of trypsin in the incubation mixture was essential for this hydrolysis. Hydrolysis was inhibited completely by soybean trypsin inhibitor,
TAYPSIN-PST1 COMPLEX
FREE
PST1
(A)
600 500 400 300 200 100 0-a.
0
8. 20
0
20
I., 40
60
, 80
/ 100
40
60
80
100
(C)
%
Fig. 4. Elution
profile of [r251]-PST1 from a Sephadex
with 50 mmol/l
Tris, 2 mmol/l
with (A) chymotrypsin, and (C) trypsin,
CaClr,
elastase
chymotrypsin,
of the [‘*51]-PST1 was measured
ELUTION
G-75 column
(1 x 100 cm), equilibrated
150 mmol/l NaCl buffer pH 7.6, after incubation
and enterokinase, elastase,
(B) trypsin,
enterokinase
by counting
chymotrypsin,
and carboxypeptidase
the radioactivity eluate.
(counts
and eluted
for 40 min at 37°C
elastase
and enterokinase,
A&B. The elution
per minute/fraction)
position
in the column
partially by casein and weakly by lactalbumin. These results are consistent with the proposed role of PST1 in the modulation of CCK release by proteinases, their inhibitors and proteins. This study was prompted by the observation that rat PST1 I, which has 52% homology with human PST1 [lo], unlike exogenous proteinase inhibitors, stimulates CCK release in the absence of trypsin [l 11. This led to the suggestion that intraluminal proteinases suppress CCK release by binding or hydrolysing PST1 and that intraluminal proteinase inhibitors and proteins displace enzyme bound PST1 or protect it from hydrolysis [12]. Moreover, Fushiki [23] also demonstrated that the intraduodenal ad-
37
ministration anti-PST1 I antibodies diminished protein stimulated CCK release in the rat. Thus the interactions of PST1 with intraluminal enzymes are central to the understanding of the mechanisms of CCK release. When PST1 was incubated in the presence of fasting small bowel juice or pancreatic juice the majority of tracer eluted between 80 and loo%, due to the hydrolysis of PST1 by pancreatic enzymes. Preincubation of the small bowel juice with trypsin inhibitor, a potent stimulant of CCK release in both rats [3] and man [4], resulted in the PST1 eluting in free position. Soybeans contain two trypsin inhibitors; Kunitz and Bowman-Birk, with dissociation constants, &, for bovine trypsin of 3.1 x lo-‘* and 2.9 x lob7 respectively [24]. Human PST1 has a Ki for bovine trypsin [25] of 3.8 x IO-*. Thus displacement of human PST1 from human trypsin by soybean trypsin inhibitors is consistent with published kinetic data. In addition, our findings indicate that soybean trypsin inhibitors have sufficient affinity with the other human serine proteinases to displace human PSTI. This study has demonstrated that casein is better at preventing the hydrolysis of human PST1 than is lactalbumin. Liddle et al. [2] showed that casein inhibited the tryptic hydrolysis of an alternative substrate to a greater extent than lactalbumin. Fushiki et al. 1231showed that trypsin released more trichloroacetic acid-soluble peptides from casein than from lactalbumin. Thus it appears that trypsin has a greater affinity for casein than for lactalbumin, consistent with our findings. Interestingly Liddle [2] and Fushiki [23] both found casein to be a more potent stimulant of CCK release than la&albumin in the rat. The results of the present study therefore support the idea that PSTIs mediate CCK release by proteins. The aim of the studies with pure enzymes was to determine which enzyme or enzymes cause the marked hydrolysis of PST1 in small bowel juice. No individual serine proteinase hydrolysed PST1 but all four; trypsin, chymotrypsin, elastase and enterokinase, formed PSTI-enzyme complexes. The binding of PST1 to elastase and enterokinase was unexpected because neither enzyme is inhibited by PSTI 1131.This might reflect non-inhibitory interactions or traces of trypsin present in the commercial enzyme preparations. Combinations of trypsin with one other serine proteinase caused partial hydrolysis. The enzyme pair with most effect was trypsin plus elastase, but a mixture of the four enzymes had most effect. The addition of carboxypeptidase did not have any demonstrable effect on hydrolysis. When PST1 was incubated with chymotrypsin, elastase and enterokinase in the absence of trypsin, very little hydrolysis was observed (10%). Thus trypsin appears to render PST1 susceptible to digestion by the other enzymes. Trypsin is known to cleave the Arg’*-Ilei9 bond at the reactive site of PST1 [26]. The product of this reaction retains trypsin inhibitor activity, but prolonged incubation of the trypsin-PST1 complex leads to return of tryptic activity, an effect known as temporary inhibition 1271.No combination of purified enzymes produced as complete hydrolysis of PST1 as whole small bowel juice. This may reflect differences between human enzymes and the purified animal enzymes used in this part of the study. The nature of interactions of PST1 with the different pancreatic enzymes is also relevant to its protective role within the pancreas. Trypsin is unique amongst pan-
3x
creatic proteinases in having the ability to activate the zymogens of all the other pancreatic proteinases. Inhibition of trypsin is therefore central to protection and PST1 has its greatest inhibitory effect on this enzyme. However, the results of the present study show that PST1 would be rapidly destroyed within the gland if multiple enzymes were activated, as occurs in acute pancreatitis. Obstruction to the flow of juice out of the gland causes pancreatitis. PST1 may fail to protect the pancreas under conditions of stasis because its inhibitory effect is temporary [27]. Interest in the survival of PST1 in small intestinal juice has been increased by the finding that PSTIs have effects other than the inhibition of trypsin. Human PST1 has growth factor activity [28-301. The atrophy of the small intestine which occurs during fasting [3 1] might be due to increased hydrolysis of PST1 and the structurally related proteins such as EGF, in the absence of other enzyme substrates. The results of this study are consistent with the proposed role of PST1 in CCK release. Further studies are required to determine whether PST1 effects the growth of the gut, and the CCK-releasing activities of PSTIs in other species including man. Acknowledgement We are grateful to the Wellcome Trust for financial support, and to Linda Poulter and Janice Young of ICI Pharmaceuticals for Mass Spectrometry and Sequence Analysis of PSTI. References 1 Liddle trypsin 2 Liddle
RA, Goldfine inhibitor, RA. Green
fats stimulate
GM, Conrad
J Physiol
4 Peikin SR, Springer
sin inhibitor-induced
in rats: Effects of food,
JA. Proteins
but not amino
acids, carbohydrates,
or
1986;251:G24336248.
cholecystokinin
after oral trypsin
inhibitor
meas-
1981;319:325-343.
CJ, Dockray
GJ, Calam
RL. Feedback
C, Louie DS, Tatum
J. Oral administration
in man. Gastroenterology regulation
hypersecretion
of cholecystokinin
cholecystokinin
in the rat. Am J Physiol
release of cholecystokinin
5 Green GM, Lyman
7 Owyang
CK, Williams
secretion
of plasma
1984;87:542-549,
RG. The release of rat intestinal
ured by bioassay.
6 Owyang
JA. Bioassay
Gastroenterology
cholecystokinin
3 Brand SJ, Morgan
2 stimulates
ID, Williams
and alcohol.
inhibitor
potatoe
enzyme secretion
as a mechanism
for tryp-
in rats. Proc Sot Exp Biol Med 1972;140:612.
D. Feedback
release by trypsin.
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