Biochem. J. (1977) 166, 331-338 Printed in Great Britain
331
Characteristics and Postnatal Development of the Acid Lipase Activity ofthe Rat Small Intestine By PAUL M. COATES, SPENCER A. BROWN, JOCELYN JUMAWAN and OTAKAR KOLDOVSKY The Joseph Stokes Jr. Research Institute, Children's Hospital ofPhiladelphia, and Department of Pediatrics, University ofPennsylvania,Philadelphia, PA 19104, U.S.A. (Received 14 February 1977)
Acid lipase was identified in the rat small intestine by using esters of 4-methylumbelliferone as substrates. Maximum activity towards the oleate ester was found at pH4.0. In adult animals, the activity of acid lipase exhibited both latency and sedimentability, indicating a lysosomal localization. The activity of acid lipase was practically the same along the height of the villus, thus paralleling the distribution of acid f-galactosidase. In adult rats, the activity of acid lipase in proximal (jejunum) and middle (mid-jejunum) sections of the small intestine was practically the same and exceeded the activity in the distal (ileum) section by a factor of 2. In suckling rats, the activity of the enzyme in the mid-jejunum exceeded that in the jejunum and ileum by 2.5- and 1.5-fold respectively. During postnatal development, the acid lipase activity of the mid-jejunum showed a peak between days 10 and 15, at which time it exceeded the adult mid-jejunum activity by 5-6-fold. The activity of various lysosomal enzymes undersubstantial changes during early postnatal development in the small intestine of mammals (Koldovsky, 1969, 1972). Their activity is generally higher in newborns than in adults; during the suckling period, marked differences in the developmental pattern of the jejunum and ileum are observed. Their activity gradually decreases in the jejunum, but there is a transient increase in the ileum where the activity exceeds that of the jejunum some 5-10-fold in the suckling period (Koldovsky et al., 1972). Later, at the beginning of weaning, the activity in the ileum decreases abruptly, until the difference in activities between jejunum and ileum disappears. This developmental pattern has been described for acid glycosidases such as acid I-galactosidase(EC3.2.1 .23) (Koldovsky & Chytil, 1965), fi-glucuronidase (EC 3.2.1.31) (Koldovsky &Palmieri, 1971), N-acetyl-f8-Dglucosaminidase (EC 3.2.1.30) (Koldovsky & Palmieri, 1971) and a-galactosidase (EC 3.2.1.22) (Jumawan et al., 1972), but also for arylsulphatases (EC 3.1.6.1) (Heringova et al., 1968; Danovitch & Laster, 1969) and alkaline phosphatase (EC 3.1.3.1, found in the 22000g supernatant and not sedimenting with microvilli) (Pelichova et al., 1967). The high activity of those lysosomal hydrolases during the suckling period led us to comment on their possible role in the digestion of some milk components (Koldovsky, 1972). Since the earlier studies only dealt with a limited group of enzymes, we questioned whether other lysosomal enzymes possess similar characteristics. Acid lipase (EC 3.1.1.3) is a lysosomal enzyme that Vol. 166 goes
has been described in a number of tissues such as liver (Mahadevan & Tappel, 1968; Teng & Kaplan, 1974), brain (Eto & Suzuki, 1971), aorta (Takano et al., 1974) and leucocytes and fibroblasts (Patrick & Lake, 1973; Cortner et al., 1976). It was a logical candidate for such a study, since it is an enzyme with quite a different physiological function, i.e. it is responsible for the hydrolysis of triacylglycerols and of cholesteryl esters (Cortner et al., 1976; Sloan & Fredrickson, 1972). Multiple forms of acid lipase have been identified, and the genetically determined deficiency of one of these forms results in the inborn errors of metabolism Wolman's disease and cholesteryl ester-storage disease (Cortner et al., 1976). In these two disorders, acid lipase deficiency causes the intracellular accumulation of neutral lipids in most tissues (Patrick & Lake, 1973). We here give the first description of the presence of acid lipase in rat small intestine. Experiments are reported showing several of its biochemical characteristics and changes in its activity in various sections of the small intestine during early postnatal development of the rat. Experimental Materials 4-Methylumbelliferone, its oleate, elaidate, palmitate and nonanoate esters and its 8-D-galactopyranoside were obtained from Research Products International (Elk Grove Village, IL, U.S.A.). 4-Methylumbelliferyl myristate was from Isolab (Akron, OH,
332
P. M. COATES, S. A. BROWN, J. JUMAWAN AND 0.
U.S.A.), 4-methylumbelliferyl laurate and p-nitrophenyl ,B-galactoside were from Nutritional Biochemicals (Cleveland, OH, U.S.A.); sucrose, lactose, Triton X-100, sodium taurocholate, L-a-phosphatidylcholine and diethyl p-nitrophenyl phosphate (E600) were from Sigma Chemicals (St. Louis, MO, U.S.A.). Glucostat Special was from Worthington (Freehold, NJ, U.S.A.). Bovine serum albumin (fraction V) was from Schwarz-Mann (Orangeburg, NY, U.S.A.). p-Nitrophenyl N-acetyl-fl-D-glucosaminidewas from Calbiochem(LaJolla, CA, U.S.A.). All other chemicals were reagent-grade. Deionized water was used throughout. Animals Pregnant Charles River rats were obtained and gave birth in our animal house. On day 3 after birth, the size of the litter was decreased to eight or nine pups. The pups were kept with the mother until they were killed. Male rats from the same strain were used at the age of 2-3 months for some studies as adults. Preparation of intestine Fed rats were decapitated between 09.00 and 10.00 h and the small and large intestines were excised. In most experiments, the duodenum and large intestine were discarded and the small intestine was divided along its length into three segments (Koldovskf & Chytil, 1965): the jejunum, midjejunum and ileum. The segments were flushed with ice-cold 0.9 % (w/v) NaCl and then homogenized in deionized water (1 g wet wt. of tissue in 8 vol. ofwater) in a Potter-Elvehjem homogenizer by using a Teflon piston with a clearance of 0.15 mm at a speed of 500-600rev./min. The activities of acid lipase, acid ,B-galactosidase, sucrase (EC 3.2.1.26) and lactase (neutral, microvillar f-galactosidase, EC 3.2.1.23) are stable in frozen intestinal tissue and the tissue could be frozen at -40 to -70°C for up to 1 month before analysis. Isolation ofepithelial cells along the height ofthe villus Cells were isolated from different heights of the cillus-crypt units of adult rats (starved for 24h, but with access to water) by the method of Weiser (1973). The entire jejunoileum was used for these studies. Enzyme analyses were performed on freshly isolated cells.
Latency and sedimentability These studies were performed by modification of the methods of Galand & Forstner (1974) and of Peters et al. (1975). Erythrocytes from mid-jejunum of fed adult rats were isolated by the method of Weiser
KOLDOVSKY?
(1973). The first three washes of the mucosa (which contained approx. 50% of the total protein) were pooled and the pooled washes were homogenized in 15ml of 0.3M-sucrose/1 mM-EDTA/22mM-ethanol, pH7.4 (Peters et al., 1975) or 10ml of0.28 M-mannitol (Galand & Forstner, 1974) in a Dounce homogenizer with ten strokes of pestle A. The homogenate was centrifuged for 10min at 800gat 4°C in a Sorvall RC-5 centrifuge equipped with rotor SS-34. The resulting post-nuclear supernatant was then divided into two portions: one was centrifuged immediately at 40000g for 10min at 4°C; to the other was added 0.1 vol. of 1.0% (w/v) Triton X-100. The mixture was kept at 4°C for 30min and then centrifuged at 40000g for 10min at 4°C. Pellets from both samples were resuspended in sucrose/EDTA/ethanol or in mannitol to the original volume of post-nuclear supernatant. Enzyme activities were measured as described below on supernatants and pellets and the percentage sedimentable activity was calculated as: Activity in pellet x 100 Activity in pellet and supernatant Latency was determined in the post-nuclear supernatant by measuring the total activity [substrate + sucrose/EDTA/ethanol or mannitol + 0.1 % (w/v) Triton X-100] and free activity (substrate + sucrose/ EDTA/ethanol or mannitol alone) at pH5.5 to avoid disruption of the lysosomes that occurs at pH4.0, and in the presence of 10,uM-diethyl p-nitrophenyl phosphate to avoid interference by neutral nonspecific esterase (see the Discussion section). The percentage latent activity was calculated as: Total activity-free activity x 100 Total activity Enzyme assays The activity of acid lipase was determined as described by Cortner et al. (1976). Briefly, 4methylumbelliferyl oleate was present at a final concentration of 50,gm, with 8011M-L-a-phosphatidylcholine, 300,pM-sodium taurocholate and 175mMsodium acetate buffer, pH4.0, in a final volume of 2.Oml. The reaction was monitored at 37°C with an Aminco-Bowman spectrophotofluorimeter with excitation and emission wavelengths of 325 and 455nm respectively. The assay with other esters of 4-methylumbelliferone was carried out in a similar fashion. When assays were performed in the presence of
diethyl p-nitrophenyl phosphate, samples were preincubated for 10min at 37°C in 0.2M-sodium acetate buffer containing 10,lM-inhibitor, then added to substrate containing the inhibitor at the same concentration. Acid /-galactosidase activity was measured at pH 3.5 by usingp-nitrophenyl fl-D-galactopyranoside 1977
RAT SMALL-INTESTINAL ACID LIPASE
(Koldovsky et al., 1972) or 4-methylumbelliferyl fl-Dgalactopyranoside by the method of Kolodny & Mumford (1976). N-Acetyl-ft-D-glucosaminidase was measured at pH4.0 by the method of Koldovsky et al. (1972). Sucrase and lactase were determined by the methods ofDahlqvist (1964) with slight modifications (Koldovsky et al., 1972, 1975). All assays were performed under conditions of linear activity with time and the amount of enzyme. Protein was measured by the method of Lowry et al. (1951) with bovine serum albumin (fraction V) as the standard. Significance of the difference of means was evaluated by using the Student's t test. Results In initial experiments, two basic properties of acid lipase were studied, namely the activity towards 4-methylumbelliferyl esters of different chain length, and the dependence of activity on pH. In these studies, jejunum, mid-jejunum and ileum from young adult (2-3 month) and suckling (12-15 day) rats were used. The results of experiments using different esters are summarized in Table 1. At pH4.0, the nonanoate ester was hydrolysed most rapidly, then the oleate. The other esters, including elaidate, which is the transisomer of oleate, were generally hydrolysed at considerably slower rates than was oleate. The nonanoate and oleate esters were used in further studies. Fig. 1 shows that the mid-jejunum from both adult and suckling rats hydrolysed 4-methylumbelliferyl oleate maximally at pH4.0. Further, preparations from suckling rats showed considerable activity below pH4.0; this activity was not observed in adult tissue. Hydrolysis of 4-methylumbelliferyl nonanoate in preparations from suckling rats showed a broad range of pH optimum, with specific activities lower
333 than those in preparations from adults. In adult rats, activity increased with pH, to a maximum at pH6.0. Above pH 6.0, the adult tissues show considerable activity not observed in the tissues of suckling rats. The activity in other sections of the small intestine, the jejunum and ileum, showed essentially the same pH-dependency as did that in the mid-jejunum. However, at the pH optimum for hydrolysis of the oleate ester, the mid-jejunum showed higher activity of acid lipase than did either the jejunum or ileum. Further, substantial differences were observed between the activities of different intestinal segments of suckling and adult rats. Therefore we considered the presence of an activating or inhibiting substance. 'Mixing' experiments were performed, where the activity measured in the assay of two mixed samples was compared with the expected activity calculated as the mean of activities determined in assay of the individual samples. All pair combinations were tested from the jejunum, mid-jejunum and ileum of both suckling and adult rats. In two separate experiments, the observed values for acid lipase activity were not found to differ from theoretical values by more than 10 %. The next series of experiments were designed to characterize the acid lipase activity.
Dependency ofactivity on concentration ofhomogenate This was studied by using homogenates of all three small intestinal parts from adult and suckling rats. With 4-methylumbelliferyl oleate, the reaction was linear in all tissues in the range 3.5-5,ug of protein/ assay; in the ileum of suckling rats, a wider range, up to lOO,g of protein/assay, was observed. With 4-methylumbelliferyl nonanoate, the activity was linear- in all tissues studied from 3.5-804ug of protein/ assay; in addition, the reactions using jejunum of adult and ileum of suckling rats were both linear up to 120,ug of protein/assay.
Table 1. Hydrolysis of 4-methylumbelliferyl esters by homogenates of different segments of the small intestine of suckling and adult rats All esters were prepared at a final concentration of 50M and assays were at pH4.0. The results of determinations by using esters other than oleate are expressed relative to the activity towards the oleate ester (=1.0). Activities towards the oleate ester were expressed as nmol/min per mg of protein. Sucklings were 12-15 days old; adults were 2-3 months old. These are results of a typical experiment reproduced three times with identical results. N.T., Not tested. Adult Suckling Substrate Oleate (18:1 cis) Elaidate (18:1 trans) Palmitate (16:0) Myristate (14:0) Laurate (12:0) Nonanoate (9: 0) Vol. 166
Jejunum
Mid-jejunum
29.7 0.7 0.4 0.7 1.0 2.0
43.8 0.7 0.5 0.6 N.T. 1.9
Ileum 34.7 0.5 0.4 0.5 0.6 1.8
Jejunum 25.5 0.6 0.7 0.9 1.0
3.3
Mid-jejunum 16.0 0.6 0.6 0.8 N.T. 2.9
Ileum 10.9 0.6 0.6
0.9 0.9 2.7
334
P. M. COATES, S. A. BROWN, J. JUMAWAN AND 0.
-
100 - (a)
._.
80
_
60
_
KOLDOVSKVt
(b)
*4-
co
*E0 E \o oo
40 _
20
0
_
L
2
3
4
5
6
7
8
1
3
4
5
6
7
8
9
pH Fig. 1. Dependency of acid lipase activity on pH Homogenates of mid-jejunum from (a) 2-month-old adult and (b) 12-14-day-old suckling rats were assayed in 0.2Msodium acetate buffers from pH 3.0 to 5.5, 0.2M-citric acid/sodium phosphate buffers from pH2.5 to 7.0 and 0.2Msodium phosphate buffers from pH6.5 to 8.0. Substrates used were 4-methylumbelliferyl oleate (0) and nonanoate (0). Results are expressed as percentage of the maximum activity for each substrate. These are the results of a typical experiment; two other experiments gave similar results.
Linearity of enzyme reaction with time ofincubation Although most assays using esters of 4-methylumbelliferone were carried out for 0.5-2.0min as a result of the great sensitivity of this technique, dilute samples (approx. 5,ug/assay) showed linear increase in activity with time up to 30min.
Dependency of activity on substrate concentration Homogenates ofjejunum and ileum from adult and suckling rats were assayed at pH4.0 with concentrations of 4-methylumbelliferyl oleate and nonanoate of 1-1OO4uM. The concentrations of all other reactants (L-a-phosphatidylcholine, sodium taurocholate, sodium acetate buffer) were kept constant. Protein concentration ranged from 5 to I0O,g of protein/assay. Saturation was achieved at 50AuM for both substrates tested using all tissue samples. Apparent Vmax. values found in two independent experiments were within the range observed in the developmental studies (see Fig. 4), in which these substrates were used at a final concentration of 50AM. Apparent Km values for 4methylumbelliferyl oleate ranged from 5.5 to 10.5,UM and for nonanoate from 5.5 to 9.8AM, with no systematic differences in these values among the tissue samples tested in two independent experiments. The apparent Km values for the oleate ester are similar to those found using human lymphocytes, fibroblasts,
(5-15.uM; P. M. Coates & J. A. Cortner, unpublished work). A final concentration of 50AM was used for both substrates in all further studies. aorta and liver
Effect of diethyl p-nitrophenylphosphate on activity Diethyl p-nitrophenyl phosphate is known to inhibit some lipases and esterases (Hayase & Tappel, 1970). Since the previously described pH-dependency experiment (Fig. 1) indicated the possibility of more than one enzyme activity hydrolysing the substrates used, we tested the effect of the inhibitor on these tissues with both esters as substrates. The hydrolysis of the oleate ester at pH4.0 was resistant to inhibition (mean residual activity± 1 S.D. = 105 ±20 %). Identical results were obtained for all three parts ofthe small intestine from suckling and adult rats. With the nonanoate ester at pH4.0, residual activity was 80±10%, reflecting some inhibition. In parallel studies at pH6.0, residual activities were 42±12% for 4-methylumbelliferyl oleate hydrolysis and 17±10% for 4-methylumbelliferyl nonanoate hydrolysis. These results suggest the existence of at least two enzyme activities. One has a pH optimum of 4.0, with activity towards both oleate and nonanoate esters, and resistance to inhibition by diethyl p-nitrophenyl phosphate. The other has a pH optimum 1977
335
RAT SMALL-INTESTINAL ACID LIPASE
Table 2. Sedimentability of acid hydrolases in iso-osmotic postnuclear supernatant from homogenates of adult rat mid-jejunum enterocytes, in the presence and absence of0.1% Triton X-100 Results are expressed as percentage of sedimentable activity (see the Experimental section), and are shown as mean +S.E.M. for the number of independent experiments shown in parentheses. Acid lipase, 4-methylumbelliferyl ester Oleate (5) 60.6+4.9 32.8+6.2 37.8+8.1
-Triton X-100 +Triton X-100 Difference
Nonanoate (5)
57.2±4.6 13.6+4.7 43.6+2.0
of 6.0, is active towards the nonanoate ester (but not the oleate ester), and is sensitive to inhibition. This latter activity will not be discussed further.
Sedimentability and latency studies The results of sedimentability experiments are summarized in Table 2. The percentage sedimentable
Acid 8I-galactosidase (5) N-Acetyl-f8-D-glucosaminidase (4) 62.0+6.8 64.3+6.8 21.3+5.8 28.6+7.3 33.4+9.1 43.0+ 1.0
activity of acid lipase in iso-osmotic medium was similar to that observed for acid 8-galactosidase and N-acetyl-fl-D-glucosaminidase. There was no difference between using sucrose/EDTA/ethanol and mannitol as the iso-osmotic medium; the result of both types of experiments were therefore pooled. In one experiment, the pellets were homogenized with 20 strokes of pestle A, and the results were identical with those obtained with 10 strokes. When 4-methylumbelliferyl nonanoate was used as substrate, there was less sedimentable activity (57 %) than was seen with the oleate ester as substrate (70 %), but the difference was not significant (P>0.25). In all cases, the addition of 0.1 % (w/v) Triton X-100 released significant activity from the pellet into the supernatant.
Determination of latency by the method of Peters et al. (1975) revealed that acid lipase activity measured '!
0
OX
0
20
40 %
60
80
100
of total protein
Fig. 2. Enzyme activities along the height of the villus The protein contents of all fractions were totalled and the percentage of protein recovered in each fraction was calculated. The activity of each fraction is expressed as the percentage of maximum activity. (a) *, Acid lipase measured with 4-methylumbelliferyl oleate; o, acid lipase measured with 4-methylumbelliferyl nonanoate; A, acid 8-galactosidase; (b) El, lactase; *, sucrase. These are the results of a typical experiment reproduced four times with the same results.
Vol. 166
with 4-methylumbelliferyl oleate as substrate at pH5.5, in the presence of lOpM-diethylp-nitrophenyl phosphate, was 58 % latent (56 %, 60 %). When measured with 4-methylumbelliferyl nonanoate, however, Triton X-100 apparently inhibited 50% of the enzyme activity. Even in the presence of 10pCMdiethyl p-nitrophenyl phosphate, which should inhibit non-specific neutral esterase activity, no latency of this residual activity could be demonstrated. Thus, after establishing the characteristics of acid lipase activity, we studied three biological properties of the enzymes: their distributions along the height of the villus and along the length of the small intestine, and finally, changes in acid lipase activity during postnatal development. Distribution of acid lipase activity along the height of the villus-crypt units This was determined by using homogenates of cells isolated from different heights of the villi of the intestinal mucosa of adult rats. Fig. 2 shows that the activity of acid lipase was practically the same along the height of the villus and follows that of acid ,-galactosidase (lysosomal). Sucrase and lactase showed distributions typical of brush-border enzymes,
P. M. COATES, S. A. BROWN, J. JUMAWAN AND 0. KOLDOVSKY
336
20
40
12
is
30
9
O
20
6-
5
10
3-
0
3
2
I
D
4
5
6
C
0
D I
2
3
4
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C
0
D I
2
3
4
5
6
C
Fraction Fig. 3. Distribution ofacid lipase and acid l-galactosidase activities along the length of the rat small intestine (a) Acid lipase measured with 4-methylumbelliferyl oleate; (b) acid lipase measured with 4-methylumbelliferyl nonanoate; (c) acid f-galactosidase, in suckling (e) and adult (0) rats. Fractions are designated D (duodenum), 1-6 (small intestine) and C (colon). These are the results of a typical experiment reproduced three times with the same pattern. (a)
50 > 40 .6C)
O 30
>
20
w
101
0
5
10
15 20 25 30
0
5
10
15 20 25 30
0
5
lo 15
20
25
30
Age in days Fig. 4. Postnatal changes of acid lipase and acid .l-galactosidase in the rat small intestine Jejunum (0), mid-jejunum (A) and ileum (A). (a) Acid lipase measured with 4-methylumbelliferyl oleate; (b) acid lipase measured with 4-methylumbelliferyl nonanoate; (c) acid /i-galactosidase. Symbols denote mean values for four to ten animals. Bars indicate 1 S.E.M. above or below the mean; S.E.M. not given if smaller than the symbol used.
decreasing in activity from the villus tip to the crypt. When acid lipase assays were carried out in the presence of 10,lM-diethyl p-nitrophenyl phosphate, there was no change in the distribution of activity; this inhibitor simply decreased the rate of hydrolysis of 4-methylumbelliferyl nonanoate by 20-25 %. Distribution of acid lipase activity along the length of the small intestine
This was studied by dividing the tissue from adult and suckling rats into six parts of equal length
(Koldovsky et al., 1972). In addition, homogenates of duodenum and large intestine were assayed. Fig. 3 shows that in suckling rats, acid lipase measured with either 4-methylumbelliferyl ester as substrate, is predominantly located in the mid-jejunum. This is in contrast with the pattern observed for acid I8-galactosidase, which is primarily located in the ileum of suckling rats. In the adult, the peak ofactivity for hydrolysis of both esters is shifted to the jejunum. The activity towards 4-methylumbelliferyl oleate in the post-jejunal sections was always higher in the 1977
337
RAT SMALL-INTESTINAL ACID LIPASE
suckling than in the adult rats. The activity towards the nonanoate ester in the jejunum was three times as it~gfmn the adult as in the suckling rat, but in the postjejunal sections the activity was similar in both adults and sucklings. Developmental changes in acid lipase activity The activity of acid lipase measured with the oleate ester did not differ along the length of the small intestine of newborn rats. After birth, however, considerable differences were observed between different sections of the intestine (Fig. 4). In the jejunum, this activity did not change between days 5 and 25; later it decreased. In the mid-jejunum, the activity increased from day 0 to a peak at 12-14 days, when it reached a value 2-3 times that in the jejunum. The activity then decreased until 20 days, when it was comparable with that in thejejunum. In the ileum, there was a more moderate increase in activity with age, but it paralleled that seen in the mid-jejunum. The activity towards the nonanoate ester was also highest in the mid-jejunum between 12 and 14 days; the jejunum showed a moderate peak of activity between 5 and 10 days: later, no change in activity was seen. The ileal activity exhibited a peak between day S and 15 and then gradually decreased until days 25 to 30, when values practically equal to those found in the newborn were observed. In the 25- and 30-dayold rats, there was no significant difference (P>0.25) between the activity of mid-jejunum and jejunum, whereas these activities were significantly higher than those found in the ileum (P
331
Characteristics and Postnatal Development of the Acid Lipase Activity ofthe Rat Small Intestine By PAUL M. COATES, SPENCER A. BROWN, JOCELYN JUMAWAN and OTAKAR KOLDOVSKY The Joseph Stokes Jr. Research Institute, Children's Hospital ofPhiladelphia, and Department of Pediatrics, University ofPennsylvania,Philadelphia, PA 19104, U.S.A. (Received 14 February 1977)
Acid lipase was identified in the rat small intestine by using esters of 4-methylumbelliferone as substrates. Maximum activity towards the oleate ester was found at pH4.0. In adult animals, the activity of acid lipase exhibited both latency and sedimentability, indicating a lysosomal localization. The activity of acid lipase was practically the same along the height of the villus, thus paralleling the distribution of acid f-galactosidase. In adult rats, the activity of acid lipase in proximal (jejunum) and middle (mid-jejunum) sections of the small intestine was practically the same and exceeded the activity in the distal (ileum) section by a factor of 2. In suckling rats, the activity of the enzyme in the mid-jejunum exceeded that in the jejunum and ileum by 2.5- and 1.5-fold respectively. During postnatal development, the acid lipase activity of the mid-jejunum showed a peak between days 10 and 15, at which time it exceeded the adult mid-jejunum activity by 5-6-fold. The activity of various lysosomal enzymes undersubstantial changes during early postnatal development in the small intestine of mammals (Koldovsky, 1969, 1972). Their activity is generally higher in newborns than in adults; during the suckling period, marked differences in the developmental pattern of the jejunum and ileum are observed. Their activity gradually decreases in the jejunum, but there is a transient increase in the ileum where the activity exceeds that of the jejunum some 5-10-fold in the suckling period (Koldovsky et al., 1972). Later, at the beginning of weaning, the activity in the ileum decreases abruptly, until the difference in activities between jejunum and ileum disappears. This developmental pattern has been described for acid glycosidases such as acid I-galactosidase(EC3.2.1 .23) (Koldovsky & Chytil, 1965), fi-glucuronidase (EC 3.2.1.31) (Koldovsky &Palmieri, 1971), N-acetyl-f8-Dglucosaminidase (EC 3.2.1.30) (Koldovsky & Palmieri, 1971) and a-galactosidase (EC 3.2.1.22) (Jumawan et al., 1972), but also for arylsulphatases (EC 3.1.6.1) (Heringova et al., 1968; Danovitch & Laster, 1969) and alkaline phosphatase (EC 3.1.3.1, found in the 22000g supernatant and not sedimenting with microvilli) (Pelichova et al., 1967). The high activity of those lysosomal hydrolases during the suckling period led us to comment on their possible role in the digestion of some milk components (Koldovsky, 1972). Since the earlier studies only dealt with a limited group of enzymes, we questioned whether other lysosomal enzymes possess similar characteristics. Acid lipase (EC 3.1.1.3) is a lysosomal enzyme that Vol. 166 goes
has been described in a number of tissues such as liver (Mahadevan & Tappel, 1968; Teng & Kaplan, 1974), brain (Eto & Suzuki, 1971), aorta (Takano et al., 1974) and leucocytes and fibroblasts (Patrick & Lake, 1973; Cortner et al., 1976). It was a logical candidate for such a study, since it is an enzyme with quite a different physiological function, i.e. it is responsible for the hydrolysis of triacylglycerols and of cholesteryl esters (Cortner et al., 1976; Sloan & Fredrickson, 1972). Multiple forms of acid lipase have been identified, and the genetically determined deficiency of one of these forms results in the inborn errors of metabolism Wolman's disease and cholesteryl ester-storage disease (Cortner et al., 1976). In these two disorders, acid lipase deficiency causes the intracellular accumulation of neutral lipids in most tissues (Patrick & Lake, 1973). We here give the first description of the presence of acid lipase in rat small intestine. Experiments are reported showing several of its biochemical characteristics and changes in its activity in various sections of the small intestine during early postnatal development of the rat. Experimental Materials 4-Methylumbelliferone, its oleate, elaidate, palmitate and nonanoate esters and its 8-D-galactopyranoside were obtained from Research Products International (Elk Grove Village, IL, U.S.A.). 4-Methylumbelliferyl myristate was from Isolab (Akron, OH,
332
P. M. COATES, S. A. BROWN, J. JUMAWAN AND 0.
U.S.A.), 4-methylumbelliferyl laurate and p-nitrophenyl ,B-galactoside were from Nutritional Biochemicals (Cleveland, OH, U.S.A.); sucrose, lactose, Triton X-100, sodium taurocholate, L-a-phosphatidylcholine and diethyl p-nitrophenyl phosphate (E600) were from Sigma Chemicals (St. Louis, MO, U.S.A.). Glucostat Special was from Worthington (Freehold, NJ, U.S.A.). Bovine serum albumin (fraction V) was from Schwarz-Mann (Orangeburg, NY, U.S.A.). p-Nitrophenyl N-acetyl-fl-D-glucosaminidewas from Calbiochem(LaJolla, CA, U.S.A.). All other chemicals were reagent-grade. Deionized water was used throughout. Animals Pregnant Charles River rats were obtained and gave birth in our animal house. On day 3 after birth, the size of the litter was decreased to eight or nine pups. The pups were kept with the mother until they were killed. Male rats from the same strain were used at the age of 2-3 months for some studies as adults. Preparation of intestine Fed rats were decapitated between 09.00 and 10.00 h and the small and large intestines were excised. In most experiments, the duodenum and large intestine were discarded and the small intestine was divided along its length into three segments (Koldovskf & Chytil, 1965): the jejunum, midjejunum and ileum. The segments were flushed with ice-cold 0.9 % (w/v) NaCl and then homogenized in deionized water (1 g wet wt. of tissue in 8 vol. ofwater) in a Potter-Elvehjem homogenizer by using a Teflon piston with a clearance of 0.15 mm at a speed of 500-600rev./min. The activities of acid lipase, acid ,B-galactosidase, sucrase (EC 3.2.1.26) and lactase (neutral, microvillar f-galactosidase, EC 3.2.1.23) are stable in frozen intestinal tissue and the tissue could be frozen at -40 to -70°C for up to 1 month before analysis. Isolation ofepithelial cells along the height ofthe villus Cells were isolated from different heights of the cillus-crypt units of adult rats (starved for 24h, but with access to water) by the method of Weiser (1973). The entire jejunoileum was used for these studies. Enzyme analyses were performed on freshly isolated cells.
Latency and sedimentability These studies were performed by modification of the methods of Galand & Forstner (1974) and of Peters et al. (1975). Erythrocytes from mid-jejunum of fed adult rats were isolated by the method of Weiser
KOLDOVSKY?
(1973). The first three washes of the mucosa (which contained approx. 50% of the total protein) were pooled and the pooled washes were homogenized in 15ml of 0.3M-sucrose/1 mM-EDTA/22mM-ethanol, pH7.4 (Peters et al., 1975) or 10ml of0.28 M-mannitol (Galand & Forstner, 1974) in a Dounce homogenizer with ten strokes of pestle A. The homogenate was centrifuged for 10min at 800gat 4°C in a Sorvall RC-5 centrifuge equipped with rotor SS-34. The resulting post-nuclear supernatant was then divided into two portions: one was centrifuged immediately at 40000g for 10min at 4°C; to the other was added 0.1 vol. of 1.0% (w/v) Triton X-100. The mixture was kept at 4°C for 30min and then centrifuged at 40000g for 10min at 4°C. Pellets from both samples were resuspended in sucrose/EDTA/ethanol or in mannitol to the original volume of post-nuclear supernatant. Enzyme activities were measured as described below on supernatants and pellets and the percentage sedimentable activity was calculated as: Activity in pellet x 100 Activity in pellet and supernatant Latency was determined in the post-nuclear supernatant by measuring the total activity [substrate + sucrose/EDTA/ethanol or mannitol + 0.1 % (w/v) Triton X-100] and free activity (substrate + sucrose/ EDTA/ethanol or mannitol alone) at pH5.5 to avoid disruption of the lysosomes that occurs at pH4.0, and in the presence of 10,uM-diethyl p-nitrophenyl phosphate to avoid interference by neutral nonspecific esterase (see the Discussion section). The percentage latent activity was calculated as: Total activity-free activity x 100 Total activity Enzyme assays The activity of acid lipase was determined as described by Cortner et al. (1976). Briefly, 4methylumbelliferyl oleate was present at a final concentration of 50,gm, with 8011M-L-a-phosphatidylcholine, 300,pM-sodium taurocholate and 175mMsodium acetate buffer, pH4.0, in a final volume of 2.Oml. The reaction was monitored at 37°C with an Aminco-Bowman spectrophotofluorimeter with excitation and emission wavelengths of 325 and 455nm respectively. The assay with other esters of 4-methylumbelliferone was carried out in a similar fashion. When assays were performed in the presence of
diethyl p-nitrophenyl phosphate, samples were preincubated for 10min at 37°C in 0.2M-sodium acetate buffer containing 10,lM-inhibitor, then added to substrate containing the inhibitor at the same concentration. Acid /-galactosidase activity was measured at pH 3.5 by usingp-nitrophenyl fl-D-galactopyranoside 1977
RAT SMALL-INTESTINAL ACID LIPASE
(Koldovsky et al., 1972) or 4-methylumbelliferyl fl-Dgalactopyranoside by the method of Kolodny & Mumford (1976). N-Acetyl-ft-D-glucosaminidase was measured at pH4.0 by the method of Koldovsky et al. (1972). Sucrase and lactase were determined by the methods ofDahlqvist (1964) with slight modifications (Koldovsky et al., 1972, 1975). All assays were performed under conditions of linear activity with time and the amount of enzyme. Protein was measured by the method of Lowry et al. (1951) with bovine serum albumin (fraction V) as the standard. Significance of the difference of means was evaluated by using the Student's t test. Results In initial experiments, two basic properties of acid lipase were studied, namely the activity towards 4-methylumbelliferyl esters of different chain length, and the dependence of activity on pH. In these studies, jejunum, mid-jejunum and ileum from young adult (2-3 month) and suckling (12-15 day) rats were used. The results of experiments using different esters are summarized in Table 1. At pH4.0, the nonanoate ester was hydrolysed most rapidly, then the oleate. The other esters, including elaidate, which is the transisomer of oleate, were generally hydrolysed at considerably slower rates than was oleate. The nonanoate and oleate esters were used in further studies. Fig. 1 shows that the mid-jejunum from both adult and suckling rats hydrolysed 4-methylumbelliferyl oleate maximally at pH4.0. Further, preparations from suckling rats showed considerable activity below pH4.0; this activity was not observed in adult tissue. Hydrolysis of 4-methylumbelliferyl nonanoate in preparations from suckling rats showed a broad range of pH optimum, with specific activities lower
333 than those in preparations from adults. In adult rats, activity increased with pH, to a maximum at pH6.0. Above pH 6.0, the adult tissues show considerable activity not observed in the tissues of suckling rats. The activity in other sections of the small intestine, the jejunum and ileum, showed essentially the same pH-dependency as did that in the mid-jejunum. However, at the pH optimum for hydrolysis of the oleate ester, the mid-jejunum showed higher activity of acid lipase than did either the jejunum or ileum. Further, substantial differences were observed between the activities of different intestinal segments of suckling and adult rats. Therefore we considered the presence of an activating or inhibiting substance. 'Mixing' experiments were performed, where the activity measured in the assay of two mixed samples was compared with the expected activity calculated as the mean of activities determined in assay of the individual samples. All pair combinations were tested from the jejunum, mid-jejunum and ileum of both suckling and adult rats. In two separate experiments, the observed values for acid lipase activity were not found to differ from theoretical values by more than 10 %. The next series of experiments were designed to characterize the acid lipase activity.
Dependency ofactivity on concentration ofhomogenate This was studied by using homogenates of all three small intestinal parts from adult and suckling rats. With 4-methylumbelliferyl oleate, the reaction was linear in all tissues in the range 3.5-5,ug of protein/ assay; in the ileum of suckling rats, a wider range, up to lOO,g of protein/assay, was observed. With 4-methylumbelliferyl nonanoate, the activity was linear- in all tissues studied from 3.5-804ug of protein/ assay; in addition, the reactions using jejunum of adult and ileum of suckling rats were both linear up to 120,ug of protein/assay.
Table 1. Hydrolysis of 4-methylumbelliferyl esters by homogenates of different segments of the small intestine of suckling and adult rats All esters were prepared at a final concentration of 50M and assays were at pH4.0. The results of determinations by using esters other than oleate are expressed relative to the activity towards the oleate ester (=1.0). Activities towards the oleate ester were expressed as nmol/min per mg of protein. Sucklings were 12-15 days old; adults were 2-3 months old. These are results of a typical experiment reproduced three times with identical results. N.T., Not tested. Adult Suckling Substrate Oleate (18:1 cis) Elaidate (18:1 trans) Palmitate (16:0) Myristate (14:0) Laurate (12:0) Nonanoate (9: 0) Vol. 166
Jejunum
Mid-jejunum
29.7 0.7 0.4 0.7 1.0 2.0
43.8 0.7 0.5 0.6 N.T. 1.9
Ileum 34.7 0.5 0.4 0.5 0.6 1.8
Jejunum 25.5 0.6 0.7 0.9 1.0
3.3
Mid-jejunum 16.0 0.6 0.6 0.8 N.T. 2.9
Ileum 10.9 0.6 0.6
0.9 0.9 2.7
334
P. M. COATES, S. A. BROWN, J. JUMAWAN AND 0.
-
100 - (a)
._.
80
_
60
_
KOLDOVSKVt
(b)
*4-
co
*E0 E \o oo
40 _
20
0
_
L
2
3
4
5
6
7
8
1
3
4
5
6
7
8
9
pH Fig. 1. Dependency of acid lipase activity on pH Homogenates of mid-jejunum from (a) 2-month-old adult and (b) 12-14-day-old suckling rats were assayed in 0.2Msodium acetate buffers from pH 3.0 to 5.5, 0.2M-citric acid/sodium phosphate buffers from pH2.5 to 7.0 and 0.2Msodium phosphate buffers from pH6.5 to 8.0. Substrates used were 4-methylumbelliferyl oleate (0) and nonanoate (0). Results are expressed as percentage of the maximum activity for each substrate. These are the results of a typical experiment; two other experiments gave similar results.
Linearity of enzyme reaction with time ofincubation Although most assays using esters of 4-methylumbelliferone were carried out for 0.5-2.0min as a result of the great sensitivity of this technique, dilute samples (approx. 5,ug/assay) showed linear increase in activity with time up to 30min.
Dependency of activity on substrate concentration Homogenates ofjejunum and ileum from adult and suckling rats were assayed at pH4.0 with concentrations of 4-methylumbelliferyl oleate and nonanoate of 1-1OO4uM. The concentrations of all other reactants (L-a-phosphatidylcholine, sodium taurocholate, sodium acetate buffer) were kept constant. Protein concentration ranged from 5 to I0O,g of protein/assay. Saturation was achieved at 50AuM for both substrates tested using all tissue samples. Apparent Vmax. values found in two independent experiments were within the range observed in the developmental studies (see Fig. 4), in which these substrates were used at a final concentration of 50AM. Apparent Km values for 4methylumbelliferyl oleate ranged from 5.5 to 10.5,UM and for nonanoate from 5.5 to 9.8AM, with no systematic differences in these values among the tissue samples tested in two independent experiments. The apparent Km values for the oleate ester are similar to those found using human lymphocytes, fibroblasts,
(5-15.uM; P. M. Coates & J. A. Cortner, unpublished work). A final concentration of 50AM was used for both substrates in all further studies. aorta and liver
Effect of diethyl p-nitrophenylphosphate on activity Diethyl p-nitrophenyl phosphate is known to inhibit some lipases and esterases (Hayase & Tappel, 1970). Since the previously described pH-dependency experiment (Fig. 1) indicated the possibility of more than one enzyme activity hydrolysing the substrates used, we tested the effect of the inhibitor on these tissues with both esters as substrates. The hydrolysis of the oleate ester at pH4.0 was resistant to inhibition (mean residual activity± 1 S.D. = 105 ±20 %). Identical results were obtained for all three parts ofthe small intestine from suckling and adult rats. With the nonanoate ester at pH4.0, residual activity was 80±10%, reflecting some inhibition. In parallel studies at pH6.0, residual activities were 42±12% for 4-methylumbelliferyl oleate hydrolysis and 17±10% for 4-methylumbelliferyl nonanoate hydrolysis. These results suggest the existence of at least two enzyme activities. One has a pH optimum of 4.0, with activity towards both oleate and nonanoate esters, and resistance to inhibition by diethyl p-nitrophenyl phosphate. The other has a pH optimum 1977
335
RAT SMALL-INTESTINAL ACID LIPASE
Table 2. Sedimentability of acid hydrolases in iso-osmotic postnuclear supernatant from homogenates of adult rat mid-jejunum enterocytes, in the presence and absence of0.1% Triton X-100 Results are expressed as percentage of sedimentable activity (see the Experimental section), and are shown as mean +S.E.M. for the number of independent experiments shown in parentheses. Acid lipase, 4-methylumbelliferyl ester Oleate (5) 60.6+4.9 32.8+6.2 37.8+8.1
-Triton X-100 +Triton X-100 Difference
Nonanoate (5)
57.2±4.6 13.6+4.7 43.6+2.0
of 6.0, is active towards the nonanoate ester (but not the oleate ester), and is sensitive to inhibition. This latter activity will not be discussed further.
Sedimentability and latency studies The results of sedimentability experiments are summarized in Table 2. The percentage sedimentable
Acid 8I-galactosidase (5) N-Acetyl-f8-D-glucosaminidase (4) 62.0+6.8 64.3+6.8 21.3+5.8 28.6+7.3 33.4+9.1 43.0+ 1.0
activity of acid lipase in iso-osmotic medium was similar to that observed for acid 8-galactosidase and N-acetyl-fl-D-glucosaminidase. There was no difference between using sucrose/EDTA/ethanol and mannitol as the iso-osmotic medium; the result of both types of experiments were therefore pooled. In one experiment, the pellets were homogenized with 20 strokes of pestle A, and the results were identical with those obtained with 10 strokes. When 4-methylumbelliferyl nonanoate was used as substrate, there was less sedimentable activity (57 %) than was seen with the oleate ester as substrate (70 %), but the difference was not significant (P>0.25). In all cases, the addition of 0.1 % (w/v) Triton X-100 released significant activity from the pellet into the supernatant.
Determination of latency by the method of Peters et al. (1975) revealed that acid lipase activity measured '!
0
OX
0
20
40 %
60
80
100
of total protein
Fig. 2. Enzyme activities along the height of the villus The protein contents of all fractions were totalled and the percentage of protein recovered in each fraction was calculated. The activity of each fraction is expressed as the percentage of maximum activity. (a) *, Acid lipase measured with 4-methylumbelliferyl oleate; o, acid lipase measured with 4-methylumbelliferyl nonanoate; A, acid 8-galactosidase; (b) El, lactase; *, sucrase. These are the results of a typical experiment reproduced four times with the same results.
Vol. 166
with 4-methylumbelliferyl oleate as substrate at pH5.5, in the presence of lOpM-diethylp-nitrophenyl phosphate, was 58 % latent (56 %, 60 %). When measured with 4-methylumbelliferyl nonanoate, however, Triton X-100 apparently inhibited 50% of the enzyme activity. Even in the presence of 10pCMdiethyl p-nitrophenyl phosphate, which should inhibit non-specific neutral esterase activity, no latency of this residual activity could be demonstrated. Thus, after establishing the characteristics of acid lipase activity, we studied three biological properties of the enzymes: their distributions along the height of the villus and along the length of the small intestine, and finally, changes in acid lipase activity during postnatal development. Distribution of acid lipase activity along the height of the villus-crypt units This was determined by using homogenates of cells isolated from different heights of the villi of the intestinal mucosa of adult rats. Fig. 2 shows that the activity of acid lipase was practically the same along the height of the villus and follows that of acid ,-galactosidase (lysosomal). Sucrase and lactase showed distributions typical of brush-border enzymes,
P. M. COATES, S. A. BROWN, J. JUMAWAN AND 0. KOLDOVSKY
336
20
40
12
is
30
9
O
20
6-
5
10
3-
0
3
2
I
D
4
5
6
C
0
D I
2
3
4
5
6
C
0
D I
2
3
4
5
6
C
Fraction Fig. 3. Distribution ofacid lipase and acid l-galactosidase activities along the length of the rat small intestine (a) Acid lipase measured with 4-methylumbelliferyl oleate; (b) acid lipase measured with 4-methylumbelliferyl nonanoate; (c) acid f-galactosidase, in suckling (e) and adult (0) rats. Fractions are designated D (duodenum), 1-6 (small intestine) and C (colon). These are the results of a typical experiment reproduced three times with the same pattern. (a)
50 > 40 .6C)
O 30
>
20
w
101
0
5
10
15 20 25 30
0
5
10
15 20 25 30
0
5
lo 15
20
25
30
Age in days Fig. 4. Postnatal changes of acid lipase and acid .l-galactosidase in the rat small intestine Jejunum (0), mid-jejunum (A) and ileum (A). (a) Acid lipase measured with 4-methylumbelliferyl oleate; (b) acid lipase measured with 4-methylumbelliferyl nonanoate; (c) acid /i-galactosidase. Symbols denote mean values for four to ten animals. Bars indicate 1 S.E.M. above or below the mean; S.E.M. not given if smaller than the symbol used.
decreasing in activity from the villus tip to the crypt. When acid lipase assays were carried out in the presence of 10,lM-diethyl p-nitrophenyl phosphate, there was no change in the distribution of activity; this inhibitor simply decreased the rate of hydrolysis of 4-methylumbelliferyl nonanoate by 20-25 %. Distribution of acid lipase activity along the length of the small intestine
This was studied by dividing the tissue from adult and suckling rats into six parts of equal length
(Koldovsky et al., 1972). In addition, homogenates of duodenum and large intestine were assayed. Fig. 3 shows that in suckling rats, acid lipase measured with either 4-methylumbelliferyl ester as substrate, is predominantly located in the mid-jejunum. This is in contrast with the pattern observed for acid I8-galactosidase, which is primarily located in the ileum of suckling rats. In the adult, the peak ofactivity for hydrolysis of both esters is shifted to the jejunum. The activity towards 4-methylumbelliferyl oleate in the post-jejunal sections was always higher in the 1977
337
RAT SMALL-INTESTINAL ACID LIPASE
suckling than in the adult rats. The activity towards the nonanoate ester in the jejunum was three times as it~gfmn the adult as in the suckling rat, but in the postjejunal sections the activity was similar in both adults and sucklings. Developmental changes in acid lipase activity The activity of acid lipase measured with the oleate ester did not differ along the length of the small intestine of newborn rats. After birth, however, considerable differences were observed between different sections of the intestine (Fig. 4). In the jejunum, this activity did not change between days 5 and 25; later it decreased. In the mid-jejunum, the activity increased from day 0 to a peak at 12-14 days, when it reached a value 2-3 times that in the jejunum. The activity then decreased until 20 days, when it was comparable with that in thejejunum. In the ileum, there was a more moderate increase in activity with age, but it paralleled that seen in the mid-jejunum. The activity towards the nonanoate ester was also highest in the mid-jejunum between 12 and 14 days; the jejunum showed a moderate peak of activity between 5 and 10 days: later, no change in activity was seen. The ileal activity exhibited a peak between day S and 15 and then gradually decreased until days 25 to 30, when values practically equal to those found in the newborn were observed. In the 25- and 30-dayold rats, there was no significant difference (P>0.25) between the activity of mid-jejunum and jejunum, whereas these activities were significantly higher than those found in the ileum (P
