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Peptide hydrolases in holothurian intestinal mucosa
C,mp. Bimht,m Ph~.sh,L Vol. 6 0 C p p 169 to 170. 1981
0306-4492 g1,'030169-02502 00.'0 Copyright O 1981 Pcrtzamon Press Lid
Pr,nted in Great Britain. All rights reserved
PEPTIDE HYDROLASES IN HOLOTHURIAN INTESTINAL MUCOSA S. MCGETTIGAN1, M. CANNINGI, G. O'CuINN 1 and D. B. JOHNSON1 t Department of Biochemistry, University College, Galway and 'Department of Biochemistry, Regional Technical College, Galway, Ireland (Received 9 September 1980)
Almtract--l. Peptide hydrolases fro/n intestinal mucosa of H. forskali and N. raixta were studied by starch gel electrophoresis. 2. Dipeptides hydrolyzed were L-I.,cU-L-Lcuand L-Gly-L-Leu.L-Gly-L-Tyr and L-Leu-L-Tyrwere not hydrolyzed. 3. L-L,eu-L-Lcu-L-Leuwas hydrolyzed by extracts of both species. 4. The brush border fraction of H. forskali contained di- and tri- peptidase activity.
INTRODUCTION Although a number of studies have been made on enzymes of the digestive tracts of holothurians (see for example, Johnson et al., 1980) much more information is required to provide a complete understanding of the digestive process in this class of echinoderms. In thig communication we report observations on peptide hydrolase activities in the intestines of Holothuria forskali and Neopentadactyla mixta. MATERIALS AND METHODS N. mixta (()stergren, 1898), samples were collected at Kilkerrin Bay and H. (Pannignothuria)forskali (Delle Chiaje, 1828) at Carraroe and Beama, Co. Galway. N. mixta intestinal mucosal preparations were made by extruding the epithelium using pressure applied by forceps. In H. forskali an end-gut section was distinguished from the remainder, or fore-gut, by the colour of the lining mucosal cells. In this case the mucosal preparations from each section were made also by extrusion. Alternatively sections of H. forskali intestine were frozen and, when thawed, shaken in extracting buffer: this was sufficient to remove the mucosal layer. The buffers used for extractions were 0.15 M borate, pH8.4 or 0.1 M phosphate, pH7.0. Extraction was in a Potter-Elvejhem homogenizer. Extracts, were subjected to electrophoresis on horizontal blocks of starch gel as described by Dolly & Fottrell (1969). Peptidases were detected using L-amino and oxidase, horse radish peroxidase and potassium iodide (Donlon & Fottrell, 1972). RESULTS AND DISCUSSION The substrates used and those which elicited positive peptide hydrolase activities were shown in Table 1. It can be seen that the activity was specific
for C-terminal leucine residues. In Fig. 1 it is shown that these activities appeared in multiple forms in total homogenates of intestinal mucosa from the two holothurian species examined. When homogenates were fractionated an indication of subcellular distribution of the dipeptidase (L-Leu-L-Leu) activities was obtained. One fra~ionation scheme was as follows: centrifugation at 1000@ for 10min, with further centrifugation at 36,000 g for 30 min. In the case of N. mixta extracts the 36,000 g pellet and supernatant solution contained both a and fl bands, those in the supernatant solution staining much more rapidly and more strongly than those in the pellet. In the case of H.forskali samples the 36,000 0 pellet contained bands A and C, while band B appeared in the corresponding supematant solution. When that supernatant solution was centrifuged at 100,000g the major activity remained in the soluble fraction. A weaker activitiy associated with the microsomal pellet could be released by treatment with sodium deoxychelate (5% w/v in 0.1 M phosphate buffer, pH 7.0), and then it was found to migrate on electrophoresis as band B. A further fractionation procedure (Schmitz et al., 1973) provided a purified brush border preparation having 60% of the mucosal sucrase activity (Clifford & Johnson, unpublished observations). This fraction was found to stain for both L-Lcu-L-I.,cU and L-I-,cU-L-Lcu-L-Leu hydrolyzing activities: the location of these activities in relation to Fig. 1 was not established. The number of bands and their electrophoretic migration altered when extraction and electrophoresis conditions were altered. Figure 2 shows patterns obtained with mucosal preparations (36,000 0 supernatant) from both fore and end-gut of
Table 1. Peptidase activity in H. forskali and N. mixta intestines Speoes N. mixta H. forskaliFore-gut End-gut
Substrate L-Leu-L-Leu-L-Leu +
L-Leu-L-Leu +
L-Gly-L-Leu
+ Not tested
+ +
+
m
+
m
+
169
L-Leu-L-Tyr --
L-Gly-L-Tyr _
170
S. McGETTIGANet al. z
0
®
L - Lau - L -~U
A
H. forskoli
a
B
S
N. m i x t o
D
L-LOu-L-- L I ~ - L - LlltJ
H. forskoli
0 000
0
N. mixto
Fig. 1. Zymograms of peptide hydrolases in whole homogenates of N. mixta and H. Jbrskali intestinal mucosa: homogenized in 0.1 M phosphate buffer, 0.1 M. electrophoresed at 150 V for 15 hr and stained in the presence of L-Leu-L-Leu or L-Leu-t-Leu-L-Leu. Z
®
0
A
O0 O 0 0 0
8
0
C D
0 0
Fig. 2. Zymograms of peptide hydrolases in H. fi~rskali intestinal mucosa (36.000@ supernatant solution): homogenized in 0.15 M borate buffer, pH 8.4: A, B: fore-gut: C. D: end-gut. A. C: L-Leu-L-Leu: B, D: L-Gly-L-Leu.
H. forskali following homogenizing for 15min in 0.15 M borate buffer, pH 8.4. Figure 3 shows patterns obtained with the same supernatant fraction of N. mixta: extraction and staining were as in Fig. 2, and electrophoresis was at 100V for 17hr or 150V for 16hr. In both species there was some electrophoretic correspondence between L-Leu-L-Leu and L-GIy-L-Leu hydrolyzing activities. Likewise there was some correspondence between di- and tri-peptidase bands (bands B and fl, Fig. 1). The patterns shown in Fig. l suggest that the peptidases of the two species are quite distinct. Purification and characterization studies would be required to establish the nature of such differences. Peptide hydrolases are widespread in nature and in intestinal mucosa these enzymes are thought to play a role in the terminal stages of protein digestion. Such enzymes occur also in other tissues where they are considered to function in the metabolism and turnover of endogenous protein. Our results do not clearly indicate which of these two roles the hydrolases we have demonstrated play in holothurian intestines. However, it is clear that in H. forskali there are more forms of peptide hydrolase activity in the well differentiated mucosal surface of the fore-gut region than in the end-gut region. This observation indicates a role for some of these enzymes in the digestion of peptides of exogenous origin. This is supported by our observation that some di- and tri-peptidase activity was localized in the brush border fraction. Acknowledgement--The authors thank the National Board for Science and Technology for financial assistance. REFERENCES
Z
®
OW
[] []
0
O0 00
Fig. 3. Zymograms of peptide hydrolases of N. mixta intestinal mucosa {36,000 g supernatant solution): homogenized in 0.15M borate buffer, pH 8.4. A,B: 100V for 12hr; C: 150V for 16hr; A: L-Gly-L-Leu stain: B. C: L-Leu-L-Leu stain.
DOLLY J. O. & FOTTRELL P. F. (1969) Multiple forms of dipeptidases in normal human intestinal mucosa and in mucosa from children with coeliac disease. Clinica chim. Acta 26. 555-558. DONLON J. & FOTrgELL P. F. (1972) Studies on substrate specificities and subcellular location of multiple forms of peptide hydrolases in guinea-pig intestinal mucosa. Comp. Biochem. Physiol. 4lB, 181-193. JOHSSON D. B.. RUSHEB.. GLENN B., CANNINGM. & SMITH T. (1980) In Echinoderms--Present and Past (Proceedings of a symposium held in Brussels 3-8 September. 1979) (Edited by JANGOt;X M. & BALKEMAA. A.) pp. 313-317. Rotterdam. SCHMITZ J.. PREISER H.. MAESTRACCI D.. GHOSH B. K.. CERDA J. J. & CRANE R. K. (1973) Purification of the human intestinal brush border membrane. Bioehim. biophys. Acta 323. 98-112.
0306-4492 g1,'030169-02502 00.'0 Copyright O 1981 Pcrtzamon Press Lid
Pr,nted in Great Britain. All rights reserved
PEPTIDE HYDROLASES IN HOLOTHURIAN INTESTINAL MUCOSA S. MCGETTIGAN1, M. CANNINGI, G. O'CuINN 1 and D. B. JOHNSON1 t Department of Biochemistry, University College, Galway and 'Department of Biochemistry, Regional Technical College, Galway, Ireland (Received 9 September 1980)
Almtract--l. Peptide hydrolases fro/n intestinal mucosa of H. forskali and N. raixta were studied by starch gel electrophoresis. 2. Dipeptides hydrolyzed were L-I.,cU-L-Lcuand L-Gly-L-Leu.L-Gly-L-Tyr and L-Leu-L-Tyrwere not hydrolyzed. 3. L-L,eu-L-Lcu-L-Leuwas hydrolyzed by extracts of both species. 4. The brush border fraction of H. forskali contained di- and tri- peptidase activity.
INTRODUCTION Although a number of studies have been made on enzymes of the digestive tracts of holothurians (see for example, Johnson et al., 1980) much more information is required to provide a complete understanding of the digestive process in this class of echinoderms. In thig communication we report observations on peptide hydrolase activities in the intestines of Holothuria forskali and Neopentadactyla mixta. MATERIALS AND METHODS N. mixta (()stergren, 1898), samples were collected at Kilkerrin Bay and H. (Pannignothuria)forskali (Delle Chiaje, 1828) at Carraroe and Beama, Co. Galway. N. mixta intestinal mucosal preparations were made by extruding the epithelium using pressure applied by forceps. In H. forskali an end-gut section was distinguished from the remainder, or fore-gut, by the colour of the lining mucosal cells. In this case the mucosal preparations from each section were made also by extrusion. Alternatively sections of H. forskali intestine were frozen and, when thawed, shaken in extracting buffer: this was sufficient to remove the mucosal layer. The buffers used for extractions were 0.15 M borate, pH8.4 or 0.1 M phosphate, pH7.0. Extraction was in a Potter-Elvejhem homogenizer. Extracts, were subjected to electrophoresis on horizontal blocks of starch gel as described by Dolly & Fottrell (1969). Peptidases were detected using L-amino and oxidase, horse radish peroxidase and potassium iodide (Donlon & Fottrell, 1972). RESULTS AND DISCUSSION The substrates used and those which elicited positive peptide hydrolase activities were shown in Table 1. It can be seen that the activity was specific
for C-terminal leucine residues. In Fig. 1 it is shown that these activities appeared in multiple forms in total homogenates of intestinal mucosa from the two holothurian species examined. When homogenates were fractionated an indication of subcellular distribution of the dipeptidase (L-Leu-L-Leu) activities was obtained. One fra~ionation scheme was as follows: centrifugation at 1000@ for 10min, with further centrifugation at 36,000 g for 30 min. In the case of N. mixta extracts the 36,000 g pellet and supernatant solution contained both a and fl bands, those in the supernatant solution staining much more rapidly and more strongly than those in the pellet. In the case of H.forskali samples the 36,000 0 pellet contained bands A and C, while band B appeared in the corresponding supematant solution. When that supernatant solution was centrifuged at 100,000g the major activity remained in the soluble fraction. A weaker activitiy associated with the microsomal pellet could be released by treatment with sodium deoxychelate (5% w/v in 0.1 M phosphate buffer, pH 7.0), and then it was found to migrate on electrophoresis as band B. A further fractionation procedure (Schmitz et al., 1973) provided a purified brush border preparation having 60% of the mucosal sucrase activity (Clifford & Johnson, unpublished observations). This fraction was found to stain for both L-Lcu-L-I.,cU and L-I-,cU-L-Lcu-L-Leu hydrolyzing activities: the location of these activities in relation to Fig. 1 was not established. The number of bands and their electrophoretic migration altered when extraction and electrophoresis conditions were altered. Figure 2 shows patterns obtained with mucosal preparations (36,000 0 supernatant) from both fore and end-gut of
Table 1. Peptidase activity in H. forskali and N. mixta intestines Speoes N. mixta H. forskaliFore-gut End-gut
Substrate L-Leu-L-Leu-L-Leu +
L-Leu-L-Leu +
L-Gly-L-Leu
+ Not tested
+ +
+
m
+
m
+
169
L-Leu-L-Tyr --
L-Gly-L-Tyr _
170
S. McGETTIGANet al. z
0
®
L - Lau - L -~U
A
H. forskoli
a
B
S
N. m i x t o
D
L-LOu-L-- L I ~ - L - LlltJ
H. forskoli
0 000
0
N. mixto
Fig. 1. Zymograms of peptide hydrolases in whole homogenates of N. mixta and H. Jbrskali intestinal mucosa: homogenized in 0.1 M phosphate buffer, 0.1 M. electrophoresed at 150 V for 15 hr and stained in the presence of L-Leu-L-Leu or L-Leu-t-Leu-L-Leu. Z
®
0
A
O0 O 0 0 0
8
0
C D
0 0
Fig. 2. Zymograms of peptide hydrolases in H. fi~rskali intestinal mucosa (36.000@ supernatant solution): homogenized in 0.15 M borate buffer, pH 8.4: A, B: fore-gut: C. D: end-gut. A. C: L-Leu-L-Leu: B, D: L-Gly-L-Leu.
H. forskali following homogenizing for 15min in 0.15 M borate buffer, pH 8.4. Figure 3 shows patterns obtained with the same supernatant fraction of N. mixta: extraction and staining were as in Fig. 2, and electrophoresis was at 100V for 17hr or 150V for 16hr. In both species there was some electrophoretic correspondence between L-Leu-L-Leu and L-GIy-L-Leu hydrolyzing activities. Likewise there was some correspondence between di- and tri-peptidase bands (bands B and fl, Fig. 1). The patterns shown in Fig. l suggest that the peptidases of the two species are quite distinct. Purification and characterization studies would be required to establish the nature of such differences. Peptide hydrolases are widespread in nature and in intestinal mucosa these enzymes are thought to play a role in the terminal stages of protein digestion. Such enzymes occur also in other tissues where they are considered to function in the metabolism and turnover of endogenous protein. Our results do not clearly indicate which of these two roles the hydrolases we have demonstrated play in holothurian intestines. However, it is clear that in H. forskali there are more forms of peptide hydrolase activity in the well differentiated mucosal surface of the fore-gut region than in the end-gut region. This observation indicates a role for some of these enzymes in the digestion of peptides of exogenous origin. This is supported by our observation that some di- and tri-peptidase activity was localized in the brush border fraction. Acknowledgement--The authors thank the National Board for Science and Technology for financial assistance. REFERENCES
Z
®
OW
[] []
0
O0 00
Fig. 3. Zymograms of peptide hydrolases of N. mixta intestinal mucosa {36,000 g supernatant solution): homogenized in 0.15M borate buffer, pH 8.4. A,B: 100V for 12hr; C: 150V for 16hr; A: L-Gly-L-Leu stain: B. C: L-Leu-L-Leu stain.
DOLLY J. O. & FOTTRELL P. F. (1969) Multiple forms of dipeptidases in normal human intestinal mucosa and in mucosa from children with coeliac disease. Clinica chim. Acta 26. 555-558. DONLON J. & FOTrgELL P. F. (1972) Studies on substrate specificities and subcellular location of multiple forms of peptide hydrolases in guinea-pig intestinal mucosa. Comp. Biochem. Physiol. 4lB, 181-193. JOHSSON D. B.. RUSHEB.. GLENN B., CANNINGM. & SMITH T. (1980) In Echinoderms--Present and Past (Proceedings of a symposium held in Brussels 3-8 September. 1979) (Edited by JANGOt;X M. & BALKEMAA. A.) pp. 313-317. Rotterdam. SCHMITZ J.. PREISER H.. MAESTRACCI D.. GHOSH B. K.. CERDA J. J. & CRANE R. K. (1973) Purification of the human intestinal brush border membrane. Bioehim. biophys. Acta 323. 98-112.