Download A Comparative Study of the Distribution of Soluble

Clinical Science and Molecular Medicine (1974)46, 501-510.
A COMPARATIVE STUDY O F THE DISTRIBUTION
O F SOLUBLE A N D PARTICULATE GLYCYL-L-
LEUCINE HYDROLASE I N THE SMALL INTESTINE
M A N J U S R I DAS
AND
A. N. R A D H A K R I S H N A N
Wellcome Research Unit, Christian Medical College Hospital, Tamil Nadu, India
(Received 2 October 1973)
SUMMARY
1. A comparative study has been made of glycyl-L-leucinehydrolase activity in the
soluble and particulate fractions of intestinal mucosa from monkey, guinea-pig, rabbit
and rat. The specific activity of the soluble enzyme is very high in monkey and guineapig, and lower in rabbit and rat. The particulate enzymes from all the four species
show low specific activities and form l-lO% of the total activity.
2. The pH optima in all cases lie in the range 74-78.The K,,,values of the substrate
were similar for both soluble and particulate enzyme from monkey and guinea-pig, but
in the rabbit and rat the K,,, value with the particulate enzyme was higher than with
the soluble enzyme.
3. The particulate enzyme activity in all cases was the highest in the distal regions of
the intestine, whereas the soluble enzyme showed maximal activity in the proximal
and middle regions.
Key words : intestinal peptidases, dipeptides, rat, rabbit, monkey, guinea-pig.
Subcellular fractionation procedures have shown that intestinal dipeptidase activity is present
predominantly in the soluble fraction of the small intestinal mucosa (Peters, 1970; Robinson,
1963). The particulate fractions in general contain less than 10% of the total activity, and in the
guinea-pig (Peters, 1970) this activity is confined almost entirely to the brush border fraction.
It has been suggested that dipeptide hydrolases in the brush border and soluble fraction of small
intestine are distinct enzymes. This suggestionwas based on the differences in heat inactivation,
sensitivity to p-hydroxymercuribenzoate and electrophoretic mobilities between the soluble
and brush border enzymes (Fottrell, Keane & Harley, 1972; Kim, Birtwhistle & Kim, 1972).
In the present paper we report the differences in the distribution of the soluble and particulate
glycyl-L-leucinehydrolase activities along the length of the intestine of four species : monkey
(Macaca radiata), rabbit, guinea-pig and rat. Glycyl-L-leucine hydrolase was chosen for
Correspondence: Professor A. N. Radhakrisbnan, Wellcome Research Unit, Christian Medical College
Hospital, Vellore 632004,Tamil Nadu, India.
P
501
502
Manjusri Das and A . N. Rudhakrishnun
this study since it has been shown in the monkey to represent the major dipeptidase activity in
the soluble fraction, capable of hydrolysing an extremely wide range of dipeptides (Das &
Radhakrishnan, 1973).
MATERIALS A N D METHODS
Chemicals
Glycyl-L-leucine was obtained from Sigma Chemical Company, U.S.A. Tris, ultrapure, was
from Schwarz-Mann, U.S.A. Spectrophotometric grade ethanol was prepared from rectified
spirits, by shaking with powdered silver oxide and then distilling the decanted fluid, and this
gave a low absorbance at 210 nm. All other chemicals were of analytical grade.
Preparation of soluble and particulate fractions
After the animals were killed (under Nembutal anaesthesia for the monkeys and decapitation
for the other animals) the entire small intestine from pyloric to ileocaecal end was taken out.
The intestine was washed with 0-154mol/l KC1 and cut open longitudinally. Then it was cut
lengthwiseinto five equal segments (for intestines from rabbit, guinea-pig and rat). The monkey
intestines were cut into six equal segments. The mucosa was scraped from each segment with a
blunt knife and approximately 10% homogenates were prepared with 0.154 mol/l KC1 by
using a Teflon-glass homogenizer. The homogenate was filtered through nylon cloth (St
Martins Bolting Cloth 9N; Henry Simon Ltd, Cheshire) to remove unbroken cells. The filtrate
was then centrifuged at 105 000 g for 60 min (Beckman Spinco, model L, rotor type 50) to
obtain the soluble fraction. The pellet was washed twice by resuspending it in 0.154 mol/l KC1
and centrifuging at 105 000 g for 60 min. The washed pellet was called the particulate fraction.
Enzyme assay
For measurement of enzyme activity, the spectrophotometric method of Josefsson & Lindberg (1965a) was used with some modifications. The assay mixture contained glycyl-L-leucine
(2.5 pmol), Tris-HC1 buffer (5 pmol), pH 7-6 or 7-8 (depending on the animal species used),
and the enzyme, in a total volume of 0-05 ml. After incubation at 37°C for a time sufficient to
hydrolyse about 20% of the substrate (as determined in separate experiments), 0.01 ml of the
assay mixture was added to 2 ml of spectrophotometric grade ethanol. After centrifugation,the
readings were taken at 210 nm with a Carl Zeiss spectrophotometer (PMQ 11), and read against
a blank, in which the enzyme reaction was stopped at zero time by addition of ethanol. By these
modifications the concentration of glycyl-L-leucine employed in this procedure, unlike in the
original procedure of Josefsson & Lindberg (1965a) and in our earlier study (Das & Radhakrishnan, 1972), was maintained at saturating levels.
One unit of enzyme activity is defined as the amount required to catalyse the hydrolysis of
1 pmol of glycyl-L-leucine/min at 37°C at substrate concentration 50 mmol/l.
For K,,, determinations, a paper-chromatographic method of assay (Das & Radhakrishnan,
1973) was used with 0.1 mol/l Tris-HC1 buffer, pH 7.6 or 7.8, since the spectrophotometric
method of assay which depends on differences in absorbance was less reliable, especially at
lower levels of substrate.
Optimum pH of the reaction was determined separately with Tris-HC1 buffer (0.1 mol/l)
in the range pH 7-9 for each animal species for the pellet and supernatant fractions.
Intestinal dipeptidase
503
Protein estimation
Protein was estimated by the method of Lowry, Rosebrough, Farr & Randall (1951) with
crystalline bovine serum albumin used as standard.
RESULTS
p H optima
Fig. 1 shows the variation of enzyme activity with pH for soluble and particulate glycyl-Lleucine hydrolase activities from the four species. The pH optima for all the fractions are quite
similar and lie in the range 7.6-7-8. The pH optima values for the different fractions are given
in Table 1.
I
C
._
W
c
e
a
*
0
m
E
I
C
.-
E
-
E.=i
-
7
8
9
D
x
2,
-
Guinea- pig
0
L
-x
0
r
a,
._
40
0
W
T
30
_I
I
-
a
2
-
u
20
0 4
0-.
i'.
0.35
'*\
0.3
0
I
I
I
7
8
9
7
8
9
PH
FKG.
1. Variation of enzyme activity with pH. The soluble enzyme ( 0 )and the particulate enzyme
( 0 ) were assayed with 0.1 mol/l Tris-HC1 buffer, with glycyl-L-leucine (50 mmol/l) by the
spectrophotometric method.
504
Manjusri Das and A . N. Radhakrishnan
K, and V,,,,,. values
The K,,, and V,,,. values for the different fractions were determined by Lineweaver-Burk
plots of the rate data (Figs. 2-5). The values obtained by the plots are given in Table 1. One
I / [ G I Y C Y I -L- leucine] (rnrnol/O-'
FIG.2. K , values for glycyl-L-leucine (monkey). Lineweaver-Burk plots for glycyl-L-leucine
hydrolysis by the soluble ( 0 )and particulate ( 0 )enzymes from monkey intestine. Vis defined as
fimol of glycyl-L-leucine hydrolysed/min. The enzymes were assayed by the paper chromatographic method.
I /[GIYCYI-L-
leucind ( r n r n o l /l ) - '
FIG.3. K,,, values for glycyl-L-leucine (guinea-pig). For details see the legend to Fig. 2.
Intestinal dipeptidase
505
I / CGlycyl-~-leucinel (mrnol/l)-'
FIG. 4. K,,,
values for glycyl-L-leucine (rabbit). For details see the legend to Fig. 2.
-02
0
0.2
0.4
0.6
0.8
1-0
I / L G l y c y l - ~ - leucinel (rnrnol/l)-'
FIG.5. K, values for glycyl-L-leucine(rat). For details see the legend to Fig. 2.
Manjusri Das and A . N . Radhakrishnan
506
TABLE
1. p H optimum, Km,Vmax.and activity of the soluble andparticulate glycy-L-leucine hydrolase
K,,,
and Vmax.values are the means of closely similar duplicate determinations. Total activity in the whole
intestine was calculated from the data in Figs. 6(a) and 6(b). The values given in parentheses refer to measurements in individual animals
Animal species
(length of
intestine, cm)
Enzyme
fraction
Monkey
(112, 126, 136)
Soluble
7.8
2.2
Particulate
7-8
3.2
Soluble
7.8
2.1
Particulate
7.8
13.4
Soluble
7.6
4.7
Particulate
7.6
6.2
0.39
Soluble
7.6
3-0
2-6
Particulate
7-6
8.0
0-29
Rabbit
(220,235)
Guinea-pig
(130, 135)
Rat
K,
pH
I-')
optimum (-01
Vmax.(')
39
0.5
14
1.0
39
(50955)
Specific enzyme
x Total enzyme
activity in the intestine activity (unitslmg of
(units)
protein)
76.4
(60.3, 76, 92.5)
0.73
(0.47, 0.8, 0.92)
40
(34, 39, 46)
0.53
(0.39, 0.57, 0.63)
18-7
(17, 20.3)
0.93
(0.83, 1.0)
14
( 1 5 , ~
0.92
(0.78, 1.06)
13-1
(12.8, 13.4)
0.098
(0*088,0*109)
38
(33,42)
0.39
(0-38.0.4)
0.15
(0*141,0*157)
0.014
(0.013, 0.015)
2.3
(2.2, 2.4)
0.28
(0*26,0-29)
(') V,,,,,. is defined as pmol of glycyl-L-leucine hydrolysed min-' mg of protein-' at saturating substrate
concentrations.
general feature about the K, values shown in the table is that, within the same species, the
soluble enzyme tends to have a lower K,,, than that of the particulate enzyme. Differences between
the supernatant and pellet enzymes are particularly noticeable with the preparations from rabbit
and rat. Monkey and guinea-pig show smaller differences between the K, values of particulate
and soluble enzymes.
SpeciJic and total enzyme activities
Total supernatant enzyme activity is quite high in monkey, rabbit and guinea-pig but
comparatively much lower in the rat (Table 1). For total pellet activity, the order of decrease is:
rabbit > monkey > guinea-pig > rat. The pellet enzyme activity as a percentage of total
activity was the highest in rat (lo%), followed by rabbit ( 5 7 3 , monkey (1%) and guinea-pig
(0*8%), showing that, in general, the pellet peptidase forms a very small component.
The soluble enzymes from monkey and guinea-pig have similar and high specific activities
and are quite different from those of rabbit and rat soluble enzymes (Table 1). The soluble
enzyme from the rat intestine has an atypically low specific activity. The specific activities of all
the particulate enzymes were between 1 and 10% of that of the soluble enzymes.
507
Intestinal dipeptiduse
.--.--.,
2ooooPx
4000
‘0
5000
200-P
i-
‘40
l 20
o t
u
L
L
L
.
L
d
1
2
3
4
5
1
2
Segment number
3
4
5
t
1
2
3
4
5
Segment number
FIG.6. Variation of solubleand particulate glycyl-L-leucine hydrolase activitiesalong the length of
the intestine: (a) in monkey and guinea-pig; (b) in rabbit and rat. The segments are numbered
starting from the pyloric end. The soIuble enzyme ( 0 , total activity; 0 , specific activity) and the
particulate enzyme (A, total activity; A, specific activity) were assayed as described in the text.
The results given are mean values from two animals in separate experiments (in (a), three with
monkey).
Variation of soluble and particulate enzyme activities along the length of the intestine
Figs. 6(a) and 6(b) show the variation of total and specific activities of the soluble and
particulate preparations from different intestinal segments of monkey, rabbit, guinea-pig and
rat. The general feature observed is that, in all the four species tested, the pellet enzyme showed
a higher activity in the distal regions of the intestine whereas the soluble enzyme shows a peak
of activity either in the proximal or in the middle region of the intestine. This is true when either
total or specific enzyme activity is considered.
Stability of the enzymes
The enzymes from monkey, rabbit and guinea-pig are stable at W C for at least 4 days.
The soluble enzyme from rat is extremely labile, losing about half of its activity in 24 h. The
enzyme in the pellet fraction in all cases was stable at 04°C for at least 4 days.
508
Manjusri Das and A . N . Radhakrishnan
DISCUSSION
Glycyl-L-leucine hydrolase activity is localized mainly in the cytosol fraction, as shown by the
ratio of supernatant enzyme to particulate enzyme activity in the four species. The ratio of
particulate to supernatant activity is very low in monkey and guinea-pig compared with that in
the rabbit and rat. Possibly in order to compensate for the low proportion of pellet activities in
monkey and guinea-pig, the K, values for glycyl-L-leucine with these enzymes are lower than
with the particulate enzyme from the other two species.
Multiple forms of dipeptidaseswith overlapping substrate specificitieshave been shown to be
present in the small intestine of various animal species, including human (Dolly, Dillon, Duffy
& Fottrell, 1971; Fottrell et al., 1972; Kim et al., 1972). However, in the present study there
was apparently only one kinetic system for glycyl-L-leucine hydrolysis, since the LineweaverBurk plots were single straight lines. Also, in the purification procedure of the ‘master’ dipeptidase from monkey small intestine, there was no observable heterogeneity of glycyl-Lleucine hydrolase activity and single peaks of enzyme activity were obtained even in the high
resolution fractionation steps, DEAE-Sephadex and hydroxyapatite column chromatography
(Das & Radhakrishnan, 1973). The multiple forms of dipeptidases observed by earlier workers
may be due to reasons other than molecular heterogeneity. A highly active enzyme such as
glycyl-L-leucinehydrolase may, by protein association, give rise to heterogeneity or the system
employed may result in enzyme association, giving polymeric active forms of the enzyme. Also,
it should be noted that exopeptidases like leucine aminopeptidase act on dipeptides.
The variation of dipeptidase activity along the length of the intestine in the rat has been
studied earlier by Robinson & Shaw (1960), and in the pig by Josefsson & Lindberg (1965b). In
these reports the activities measured were probably those of the supernatant enzymes, since
mucosal extracts were used for assays. In the pig, glycyl-L-leucine hydrolase activity per mg
of nitrogen was the highest in the middle region of the intestine. In the rat intestine, the ileum
showed the highest rate of glycyl-L-leucine hydrolase activity per mg of protein. Our studies
suggest that in the intestines of the four species tested, the peak of total enzyme activity (soluble
+particulate) is in the proximal or the middle region of the intestine.
The typical difference in the sites of maximal activity for soluble and particulate enzyme has
been observed in all the four species tested and it might be a general phenomenon. These
results give some indication of the possible physiological roles of these enzymes in relation to
dipeptide transport. Peptide transport may represent a second major mode of transport of the
terminal products of protein digestion, as suggested by Matthews (1971). Dipeptide transport
is independent of amino acid transport, as shown by studies in cystinuria (Hellier, Perrett,
Holdsworth & Thirumalai, 1971) and in Hartnup disease (Asatoor, Cheng, Edwards, Lant,
Matthews, Milne, Navab & Richards, 1970). The sites for maximal transport for peptides and
amino acids are probably different. Matthews, Crampton & Lis (1971) showed that the site
for L-methionyl-L-methioninetransport is in the jejunal region of the rat intestine, in contrast
to that for L-methionine, which is in the distal ileum. Although free amino acids are better
absorbed in the lower part of the small intestine, absorption of protein occurs mainly in the
upper part (Matthews & Laster, 1965). These results indicate that peptide transport occurs at a
site more proximal than the site for amino acid transport. Very little is known about the exact
mechanism of peptide transport. Preliminary studies on the specificity of the dipeptide-uptake
system in monkey intestine has shown that the uptake of glycyl-L-leucine is unaffected in the
Intestinal dipeptidase
509
presence of amino acids, but is competitively inhibited by a number of dipeptides, including
peptides containing imino acids like glycyl-L-proline and L-prolylglycine, which are hydrolysed by separate enzymes, suggesting that the initial entry process is probably independent
of the brush border dipeptidase action (Manjusri Das & A. N. Radhakrishnan, unpublished
observations). The intracellular dipeptidases may not be directly involved in dipeptide transport, but may facilitate peptide transport indirectly by creating a steep concentration gradient
between the lumen and inside the cell (see Das & Radhakrishnan, 1973, for further discussion).
Thus, for the uptake of dipeptides, there could be a special entry mechanism independent of
dipeptidase action and another mechanism involving the action of the highly active intracellular dipeptidase, and these two mechanisms may operate additively. It has been shown that,
in the rat, the site for maximal absorptive capacity for the dipeptide L-methionyl-L-methionine
is in the jejunal region (Crampton, Lis & Matthews, 1973), which is also the site of the highest
soluble dipeptidase activity. Thus the sites of maximal activity of the cytoplasmic glycyl-Lleucine hydrolase and of the dipeptide transport system are possibly in the same region, i.e.
the proximal or middle regions of the intestine, suggesting a close relationship between the two
systems. Though the present work relates to a single dipeptidase activity, it may represent a
general phenomenon, since glycyl-L-leucine hydrolase activity in monkey intestine has been
shown to represent a major dipeptidase activity capable of hydrolysing most of the theoretically
possible dipeptides (Das & Radhakrishnan, 1973).
The particulate dipeptidase, as measured in this study, presumably reflects the brush border
enzyme since, at least in the guinea-pig, the particulate dipeptidase activity is confined almost
entirely to the brush border (Peters, 1970). On the basis of this assumption, the brush border
enzyme may be involved, especially in the terminal regions of the intestine in another mode of
dipeptide absorption, i.e. surface hydrolysis coupled to the uptake of free amino acids, a
mechanism suggested by Ugolev (1972), Heizer, Kerley & Isselbacher (1972) and Fujita,
Parsons & Wojnarowska (1972).
Thus, in protein digestion, the mixture of small peptides and amino acids produced in the
lumen by the action of pancreatic enzymes and by intestinal enzymes in the desquamated cells is
rapidly absorbed in the upper portion of the intestine, probably by the concerted operation of
three transport systems, namely, amino acid transport, peptide transport involving membrane
hydrolysis coupled to amino acid transport and, thirdly, a separate system for transport of
intact peptides followed by intracellular hydrolysis to yield amino acids inside the cell.
The observed efficient and rapid absorption of proteins in the intestine compared with the
slow rate of hydrolysis in vitro of protein to amino acids by pancreatic proteases (Fisher,
1954; Crane & Neuberger, 1960) might be explained on the basis of the concerted action of the
three mechanisms mentioned above.
ACKNOWLEDGMENTS
The authors are grateful to Professor S. J. Baker for his keen interest. The project was supported
by the Council of Scientific and Industrial Research, New Delhi, and by PL-480 funds under
contract no. 01 329 01.
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