Multiple forms of dipeptidases in normal human intestinal mucosa and in mucosa from children with coeliac disease

CLINICA CHIMICA ACTA

555

MULTIPLE FORMS OF DIPEPTIDASES

IN NORMAL HUMAN INTESTINAL

MUCOSA AND IN MUCOSA FROM CHILDREN

J. 0. DOLLY

P.

F.

FOTTRELL

of~~ache~~~~~y, UlaBversity College, GaEway ~~~e~a~d~

Depadnent (Received

AND

WITH COELIAC DISEASE

August 22, 1969)

SUMMARY

Dipeptidases and tripeptidases from normal human intestinal mucosa and from mucosa of patients with coeliac disease were compared by starch gel electrophoresis. The number of major peptidase bands was identical in both types of mucosa, although the intensity of some peptidase bands was weaker in coeliac mucosa, especially when leucyl proline was the substrate.

INTRODUCTION

Due mainly to the development of a suitable assay procedure’ more information is now available on the levels of various dipeptidases in histologically normal human intestinal mucosa and in mucosa from patients with coeliac disease (gluten-induced enteropathy). Thus, Lindberg and col1.a found that patients with gluten-induced enteropathy had relatively low levels of intestinal dipeptidases but a general increase in activities was detected after the patients were treated with a gluten-free diet. We recently detected multiple molecular forms of peptidases in normal human intestinal mucosa3 and the present study was undertaken to investigate whether all these multiple peptidases were present in the mucosa of patients with coeliac disease. MATERIALS

AND METHODS

Histologically-normal mucosa was obtained by peroral biopsy from eight children with subacute diarrhoea but with normal faecal fat excretion and lactose tolerance curves. Biopsies were also taken from ten children with coeliac disease who were on diets containing gluten immediately before biopsies were taken. Biopsies were taken from the first loop of the jejunum with a Crosby-Kugler capsule under X-ray control and graded histologically as described by McNicholl and Egan*. Mucosa was extracted for 3 min at less than 5” in a micro Potter-Elvehjem homogeniser with 40 ,ul of cold 0.1 IV NaCl per mg of tissue. The homogenate was then centrifuged at IZOOOg for 30 min at z” and the clear supernatant used for electrophoresis. Pieces of Whatman 3 MM paper saturated with IO ~1 of extract from either coeliac or normal Clin. Chim. Ada, 26 (1969) 555-558

DOLLY, FOTTRELL

556

mucosa were inserted side by side in horizontal starch gels and subjected to electrophoresis at 4” (ref. 5). After electrophoresis, gels were sliced longitudinally and a number of procedures were used to locate peptidases on the cut surfaces of the gels. The method described by Lewis and Harris6 in which a chromogen is oxidised in the presence of a peptide, L-amino acid oxidase and peroxidase was very satisfactory but had two disadvantages, (I) the oxidised chromogen diffused in the gels and (2) commercially available L-amino acid oxidase (from Crotalzcs adamalzteus) was inactive with II naturallyoccurring amino acids including glutamic, proline, alanine and aspartic and was weakly active with three othersB. Methods based on the formation of an insoluble formazan dye were therefore developed to detect free glutamic, aspartic and alanine. For example, to detect release of free glutamic acid from peptides containing this amino acid, the cut surfaces of the gels were covered after electrophoresis and incubated at 37” with an agar overlay prepared immediately beforehand by mixing 30 ml of z”/o agar (Difco) with a solution of the following: 25 ml 0.2 M phosphate buffer pH 7.5; 40 mg peptide; 0.1 ml glutamate dehydrogenase (Sigma, Type II) ; 60 mg NAD; 1.5 mg phenazine methosulphate (Sigma) ; 15 mg nitroblue tetrazolium (Sigma); 0.5 ml 60 mM MgCl,; 3 ml 0.2 n/r NaCl. Similar procedures were used to detect the release of aspartate or alanine from peptides except that 40 mg of z-oxoglutarate and either aspartate aminotransferase or alanine aminotransferase were included in the agar overlay. RESULTS AND DISCUSSION

The hydrolysis of 31 dipeptides and 5 tripeptides has been studied using the aforementioned methods with extracts of intestinal mucosa from various mammalian species including human; these results will be presented elsewhere. Peptidase zymograms were very reproducible when freshly prepared extracts of the same piece of tissue were compared and when different extraction procedures were used, such as the inclusion of sulphydryl reagents (e.g. dithioerythritol) or neuraminidase in the extracting medium ‘9*. Zymograms of peptidases from children with histologically normal mucosa and from mucosa of children with coeliac disease are shown in Fig. I. The relative intensities of some peptidase bands were reduced in zymograms of coeliac mucosa especially when leucyl proline was the substrate (Fig. I). However, as shown in Table I for glutamyl tyrosine, the number and relative mobilities of peptidase bands were practically identical in normal and coeliac mu-

+ N 71

Leu-Pro

Glu

Fig. I. Peptidase zymograms from normal (N) and coeliac (C) intestinal mucosa. Extracts were included side by side in the starch gel which was subsequently stained with a solution containing the appropriate substrate. Relative intensity of peptidase bands is indicated by shading. C&z. Claim. Acta, 26 (1969) 555-558

INTESTINAL TABLE: RELATIVE

DIPEPTIDASES

f ELECTROPHORETIC

GLUTAMYC -~-._

MOBILITIES

OF HUMAN

INTESTINAL

PEPTIDASES

WYUROLYSING

TYROSINE

Patied

A.C. M.M. B.McD.

Histology

Relative vmbilities

of major

(Grade)

pefitidase bands*+ ~--. b a

c

0.24

0.p

I.0

0.24

0.72

1.0

0.23

0.71 0.70 0.73

I.0 1.0 I.0

N N N XII III III III

P.&I&. R-L.

0.24 o.z=j 0.26 0.23

0.74

E.0

0.72

1.0

--

Grade III = flat mucosal surface. * Grade N = normal; ** The mobility of the most rapidly m&Ming band was given an arbitrary mobilities of other bands expressed as a fraction of this.

TABLE NUMBER COELIAC

value of 1.0 and

II AND

RELATIVE

INTENSITIES

OF

PEPTXDASE

BANDS

IN

ZYMOGRAMS

FROM

NORMAL

AND

MUC#SA*

OvevaB r-elat’ivaintmsity

No. of mltajorpeptida.w bands* -.-__- * Coeliac -_-_

iVoem.8

coetircc

z fal

+i++

i++

Leu-Pro Pro-Leu

++++

+

ClU-J’al

3 (2) 3 (1)

++++ ++++ ++++

+ + + + -t + + -t +

++++

SfS

Substvate

Normal GIu-Tyr

y-Glu-Leu

3 (21

z ;:i 3 (2) 3 (2)

y-Glu-&Vaph Gly-Glu

:: I:;

Glu-Tyr-Glu

; 1:; 3 (a)

-k--t++

+++

4 (2)

4 (2)

+-t-t+

+++

_“_ _“_.^_ “._ * All coeliac patients were Grade III (flat mucosal surface).

?f fiq%dasebaads

* * Figures in parentheses indicate the number of different biopsies tested.

cosa. This similarity between peptidases from coeliac and normal mucosa is also evident in Table II where eight different peptides are compared. These results showed that peptidase zymograms from coeliac and normal mucosa were very similar aIthough the relative intensities of some peptidase bands were different. While further studies with coeliac patients on gluten-free diets are warranted, it would appear from the results of others2 that the decreased intensities of peptidase bands on zymograms of coeliac mucosa are probably attributable to secondary phenomena resulting from a morphologically abnormal mucosa. ACKNOWLEDGEMENTS

We are grateful to Professor B. ~c~icholl and Dr. B. Mitchell, Department of Paediatrics, for taking and grading biopsies and for valuable advice on several occasions. The assistance of Xiss Ann Dillon with some experiments is gratefully acknowledged. We also thank the Medical Research Council of Ireland for a grant. Cl&.

Chim. Acta, 26 (rg6g) 555-558

5.58

DOLLY, FOTTRELL

REFERENCES I L. JOSRFSSON AND T. LINDBERG, Biochim. Biophys. Ada. 105 (1965) 149. 2 T. LINDBERG, A. NORDBN AND L. JOSEFSSON, Stand. J.Gastroenterol., 3 (1968) 3 J. 0. DOLLY AND P. F. FOTTRELL, Rio&em. J., III (1968) 30. 4 B. MCNICHOLL AND B. EGAN, C&z. Pediat,, 7 (1968) 544. 5 0. SMITHIES, Biochem. J., 61 (1955) 629. 6 W. H. I?. LEWIS AND H. HARRIS, Nature, 215 (1967) 351. 7 H. I;.HEIDRICH, Hoppe Seylcrs 2. Physiol. Chem., 349 (1968) 873. 8 J. C. ROBINSON AND J. E. PIERCE, ATatu~@,204 (1964) 472. Clin.Chim. Acta, 26 (1969) 555-558

177.