Role of pancreatic L-asparagine in homeostasis of L-asparagine HARRY
A. MILMAN,
MILMAN, YOUNG. ostasis
DAVID
of Toxicology,
Laboratory
HARRY A., DAVID
Role of pancreatic L-usparagine.
A. COONEY,
National
synthetase
M.
in home-
of Am. J. Physiol. 236(6): E746-E753, 1979 or Am. J. Physiol.: Endocrinol. Metab. Gastrointest. Physiol. 5(6): E746-E753, 1979.-L-Asparagine synthetase from mouse pancreas was found to be associated principally with the exocrine pancreas and to be dependent on the age of the animal, but not on gender, diet, or the presence of tumor under the conditions examined. The function of the pancreatic enzyme appears to be to supply L-asparagine for the synthesis of pancreatic proteins. This function is suggested by the high specific activity of L-asparagine in pancreatic proteins after intravenous treatment of BDFr mice with L-[U-‘4C]aspartate. The pancreas is also able to function as a storage depot for L-asparagine under conditions in which the concentration of the amino acid in the blood is in excess. Unlike the liver, the pancreas is unable to add L-asparagine to the circulation when the concentration of the amide is below normal limits, leukemia 5178Y; exocrine circulation
pancreas;
ALTHOUGH
GOOD
THERE
IS
diet; age; protein
EXPERIMENTAL
AND
Cancer Institute,
A. COONEY, AND DAVID
L asparugine
synthetase
synthesis;
EVIDENCE
that the liver plays an important role in regulating the concentration of L-asparagine in the blood (30), various features of this homeostatic control await further clarification. Thus, although the enzymatic equipment for the control of increased L-asparagine in plasma is present in rodent liver in the form of L-asparaginase (L-asparagine amidohydrolase; EC 3.5.1.1) active under physiological conditions, the mechanism by which low levels of Lasparagine in plasma are elevated by hepatic control has yet to be elucidated. Orthodox L-asparagine synthetase (L-glutamine hydrolyzing; EC 6.3.5.4), the enzyme catalyzing the amidation of L-aspartate, would be the most obvious of such a mechanism. Yet, a recent survey of the specific activity of the biosynthetic enzyme in the livers of several mammalian species revealed that only the liver of the guinea pig was equipped to synthesize L-asparagine at a prominent rate (19). In this species L-asparaginase in plasma effectively eliminates L-asparagine (13). It would be expected that depletion would induce the biosynthesis of the amide and therefore may be regarded as an exceptional case, not representative of the controls that operate in subjects with normal concentrations of L-asparagine in plasma. In the same survey (19), it was observed that mammalian pancreas, in contradistinction to mammalian liver, consistently contained L-asparagine synthetase at a specific activity higher than in any of the
DAVID Bethesda,
M. YOUNG Maryland
20205
other organs examined. On the other hand, L-asparaginase was present at a very low specific activity in the pancreas. These findings suggest that the pancreas and the liver may act in concert to maintain the homeostasis of Lasparagine in plasma. These studies were designed to characterize the behavior of L-asparagine synthetase in the pancreas of the mouse under conditions of growth, development, and nutritional variations in order to gain some insight into the responsiveness of the enzyme to changes in its amino acid environment. Furthermore, the ability of the pancreas of the dog to regulate the concentration of L-asparagine in plasma under conditions in which the concentration of the amino acid is unduly elevated or reduced also was investigated. MATERIALS
AND
METHODS
Enzymes. L-Asparaginase (EC 3.5.1.1) from Escherichia coli (340 IU/mg protein) was purified at the Merck Institute for Therapeutic Research, West Point, PA, and provided by the Drug Research and Development Branch of the National Cancer Institute. L-Aspartate-4decarboxylase from Alcaligenes faecalis (EC 4.1.1.12) was purified by the method of Tate and Meister (27). The enzyme exhibited a specific activity, with z-aspartate as substrate, of 77 IU/mg protein and was stored as previously described (8). z-Glutamate decarboxylase from E. c&i (EC 4.1.1.15,50 IU/mg protein) was purified by the method of Shukuya and Schwert (24). Malate dehydrogenase (EC 1.1.1-37; 720 IU/mg protein) and Lglutamate-oxaloacetate transaminase (EC 2.6.1.1; 190 IU/mg protein) were purchased from Boehringer Mannheim Corp., New York, NY. Pronase was obtained from Calbiochem, Los Angeles, CA. Subtilisin was purchased from NOVO, Copenhagen. Collagenase was a product of Worthington Biochemical Corp., Freehold, NJ. Radiochemicals. L-[4-14C]aspartate (sp radioact 12.9 &i/pmol), L-[UJ4C]aspartate (sp radioact 231 pCi/ pmol), and L-[U-14C]asparagine (sp radioact 185 @Zi/ pmol) were the products of Amersham Corp., Silver Spring, MD. Purity (99%) of these chemicals was assessed as previously described (19), Chemicals and drugs. L-Asparagine was purchased from Schwartz/Mann Biological Research, Rockville, MD, and stored in a desiccator over calcium chloride until used. Reduced diphosphopyridine nucleotide (NADH) was obtained from Sigma Chemical Co, St.
I3746
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PANCREATIC
L-ASPARAGINE
SYNTHETASE
Louis, MO. L-Glutamine and Z-oxoglutarate were purchased from Calbiochem. Streptozotocin, actinomycin D, cycloheximide, and emetine were obtained through the Drug Research and Development Branch of the National Cancer Institute. All other chemicals used were of reagent grade. Measurement of L -asparagine synthetase activity. Tissue samples were homogenized at 4OC in 9 vol (wt/vol) of 0.1 M Tris-HCI buffer (pH 7.6) containing I mM dithiothreitol and U,5 mM disodium EDTA. The homogenates were transferred to Eppendorf 1,500~~1 disposable plastic centrifuge vessels (Brinkman Instrument Co., Silver Spring, MD) and centrifuged in the Eppendorf Zentrifuge at 12,000 g for 6 min. L-Asparagine synthetase activity in the 12,000 g supernatants was assessed by a radiometric technique the full details of which have been described previously (19) Measurement of L -asparuginase activity. L-Asparaginase activity was measured radiometrically (9) with a sensitivity of 0.00001 IU/ml. Measurement of L -asparagine. L-Asparagine was measured in the 12,000 g heated (100°C) dog plasma either by a radiometric technique (8), by an enzymatic spectrophotometric method (6) in which as little as 5 nmol of amide can be estimated, or by a radiometric, gasometric procedure (7), Estimation of protein. Estimation of protein in the 12,000 g (6 min) supernatant of organ or tumor homogenates was conducted according to the method of Lowry et al. (17) using bovine serum albumin (Armour Pharmaceutical Co., Chicago, IL) as the standard. Measurement of arteriovenous (A- V) difference of Lasparagine acruss pancreas of anesthetized dug, Beagle dogs, weighing lo-13 kg, were starved for 16 h and then anesthetized with intravenous sodium pentobarbital. A midline abdominal incision was made extending from the xiphoid process to 7 cm anterior to the symphysis pubica. The pancreas was exposed and a cannula inserted into the craneo-pancreaticoduodenal vein, slightly distal to its junction with the portal vein. A three-way stopcock was placed on the exposed end of the cannula, and a small amount of heparinized saline was injected into the cannula to prevent clotting. The cannula was secured in place with monofilament nylon sutures. The femoral artery and vein were isolated and cannulated, and a three-way stopcock was inserted in the same manner. Blood samples (2 ml) were collected from the femoral artery and craneo-pancreaticoduodenal vein periodically and transferred to l&ml Falcon tubes (Falcon, Oxhard, CA) containing two drops of heparin. The tubes were centrifuged at 2,500 g for 10 min, and the clear supernatant was transferred to polycarbonate tubes, which were immediately frozen on dry ice. The frozen tubes were transferred to a boiling water bath and boiled for 5 min after which the caps of the tubes were retightened. The tubes were boiled for an additional 15 min and then centrifuged at 105,000 g for 20 min. The clear supernatant was assayed radiometrically for L-asparagine. When L-asparaginase activity was to be measured in dog plasma, separate blood samples (I ml) were taken and transferred to Eppendorf 1,500~~1 centrifuge tubes containing 2 drops of heparin. The tubes were immedil
E747 ately centrifuged at 12,000 g for 5 s in the Eppendorf Zentrifuge and the supernatant transferred to fresh Eppendorf vessels, which were immediately frozen. L-ASparaginase activity was measured in 5-~1 aliquots of the plasma as described. RESULTS
Localization of L -asparagine synthetase activity in mouse pancreas. Of the seven mouse organs examined (Fig. l), the specific activity of L-asparagine synthetase was found to be highest in pancreas. This activity was distributed evenly throughout the organ (27.69 & 2.98 nmol/mg protein per hour in the gastroduodenal portion; 33.60 t 2.48 nmol/mg protein per hour in the splenic portion). Mouse testis also exhibited considerable activity (approximately 200 nmol/g h) in confirmation of previous reports (14, 19). Noteworthy was the finding of low or negligible L-asparagine synthetase activity in mouse liver, even in the presence of 0.2 M (NH&SO+ a compound that inhibits mouse hepatic L-asparaginase activity by greater than 95%. The presence of L-asparagine synthetase in high activities in the pancreas of the mouse led us to speculate that this activity might be originating in p-cells. Islets of Langerhans therefore were isolated (IO), and the activity of L-asparagine synthetase was measured in clusters of 30 -t 5 islets. Only 0.02 nmol of L-asparagine was synthesized per hour per 30 islets, an apparent L-asparagine synthetase activity representing 0.26% of the total activity measured in mouse pancreas.’ Furthermore, the activity of L-asparagine synthetase (47.47 t 7.37 nmol/mg protein per hour) in the pancreata of BALB/c mice treated intravenously with streptozotocin, (200 mg/kg) did not differ significantly from that measured in the pancreata of mice treated with 0.005 M sodium citrate, pH 4.0 (vehicle) (40.07 + 6.16 nmol/mg protein per hour), although glucosurea and atrophic islets were observed in drug-treated animals. Moreover, normal mice maintained on a diet supplemented with 5% D-glucose for 14 days in order to stimulate insulin synthesis and release (Table 1) had activities of L-asparagine synthetase in their pancreata comparable to those measured in the pancreata of control mice. Effect of age, gender, genetic variation, diet, and presence of tumor on L-asparugine synthetase activity in mouse pancreas. L-Asparagine synthetase activity in mouse pancreas increases with the age of the animal until day 20 and maintains a constant level at maturity (data not shown). The specific activity of L-asparagine synthetase in mouse pancreas was independent of the gender of the subject; mean t SE synthetase activity in the pancreata of five adult, male BALB/c mice (36.26 t 5.77 nmol/mg protein per hour) was equivalent to that measured in the pancreata of five adult, female BALB/c mice (31.31 t 1.79 nmol/mg protein per hour). l
’ Inasmuch as mouse pancreas contains approximately 600 islets, the L-asparagine synthetase activity in the endocrine pancreas, therefore is equivalent to approximately 0.4 nmol/h. Because the total L-asparagine synthetase activity in mouse pancreas is approximately 150 nmol/h, the contribution by the endocrine pancreas is aDDroximatelv 0.26%.
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MILMAN,
Spleen
8roin
Testis
Pancreas
MOUSE
Kidney
Lung
Liver
ORGANS
1. L-Asparagine synthetase activity mouse. L-Asparagine synthetase was measured of organ homogenates of mouse as described. of 5 animals. FIG.
in principal organs of in 12,ooO g supernatants Values are means & SE
1. Effect of diet m activity of L -asparagine synthetase
TABLE
Expt
Diet
NO
Treatment
L-Asparagine Synthesized (nmol/mg of protein/h t SE) Pancreas
2
2
27.1 -+ 3.1
L-Asparagine-free L- Asparagine-free L-Asparagine-free L-Asparagine-free L-asparagine, bwwt) z-Asparagine-free L-asparagine, M/wt) L-Asparagine-free L-asparagine,
Tumor
L5178Y/AS L5178Y/AR
27.3 zk 2.0 29.2 f: 1.4
2.0 k 0.4 52.6 I!I 3.1
19.9 * 3*7
+ 2% +
L5178Y/AS
23.7 t 5.2
+
L5178Y/AR
21.4 k 2.5
1*4 -+ 0.2
2% 42.8 t 6.1
2Y0
bwwt)
3
D-Glucose, 5% + Purina chow Water + Purina chow
31.5 AI 4*7 25.7 k 1.9
Values are means t SE of duplicate determinations on samples from 5 animals. Normal male I3DFI mice were housed in metabolism cages and maintained either on Purina chow or starvation for 54 h (expt I); or 5% D-glucose and Purina chow or Purina chow alone for 14 days feqt 4). BDFI mice, injected with 107 cells of L5178Y/AS or L5178Y/ AR subcutaneously, were maintained on a diet free of L-asparagine (Nutritional Biochemicals Carp,, Cleveland, OH) or supplemented with 2’% (wt/wt) L-asparagine for the first 10 days of tumor growth (expts 2 and 3). All mice were killed by cervical dislocation at the times specified, and their pancreata or pancreata and tumors were removed, homogenized, and assayed for L-asparagine synthetase activity as described.
Although the activity of L-asparagine synthetase in the pancreas of 20 strains of adult mice examined ranged from 23,91 t 3.30 nmol/mg protein per hour in ADKzFl mice to 72.12 -t 1.96 nmol/mg protein per hour in ZWB/ F1 mice (data not shown), this variation cannot be totally
COONEY,
AND
YOUNG
attributed to genetic factors because age and prior conditioning of the test animals were not strictly controlled. The influence of diet on mouse pancreatic L-asparagine synthetase activity can be appreciated from the results in Table 1. In mice starved for as long as 54 h, the activity of pancreatic L-asparagine synthetase (34.0 t 4.7 nmol/ mg protein per hour) was comparable to that measured in mice fed Purina chow laboratory mouse feed (37.3 k 2.9 nmol/mg protein per hour). (All mice were housed in metabolism cages to avoid possible ingestion of feces). This constancy should be contrasted with the observation that mice maintained on a starvation diet lost an average of 6.0 g, whereas control mice gained a mean of 0.8 g. Mice maintained for 10 days on a diet free of L-asparagine (Nutritional Biochemicals Corp., Cleveland, OH) (Table 1) had pancreatic L-asparagine synthetase activities comparable to those found in the pancreas of mice fed a diet supplemented with 2% (wt/wt) L-asparagine. Our studies with mice bearing L5178Y (Table 1) indicate that low activity of L-asparagine synthetase is detectable even in the sensitive form of the tumor (L5178/ AS) (19). On day 10 of tumor growth (after implantation of 107 cells subcutaneously), when tumor diameter was approximately 1.4 cm, the presence of either L-asparaginase-sensitive or -resistant forms of the leukemia 5178Y had no effect on the activity of L-asparagine synthetase in the pancreas of tumor-bearing mice. Function of L -Asparagine Synthetase of Mouse Pancreas Effect of pancreatic L -asparagine synthetase on protein synthesis. Cassano and Hansson (4) observed that the pancreas takes up radioactive L-glutamine to the greatest extent of all the organs studied and that this amide apparently is used for protein synthesis within that organ. It appeared likely that L-asparagine, the next lower homologue of L-glutamine, would be used in an analogous manner, It can be seen (Figs. 2 and 3) that pancreas invariably takes up and incorporates L-[U-‘4C]asparagine and L-[U14C]aspartate into its proteins to the greatest extent of the ten organs studied; (sp act of L-asparagine, 27,000.0 pCi/pmol; sp act of L-aspartate, lJ60.2 pCi/pmol). Small intestine had the next highest specific activity of these amino acids in its proteins (Figs. 2 and 3); (L-asparagine, 11,653.a pCi/pmol; L-aspartate, lJU4.3 pCi&mol). The rate of incorporation of these amino acids into lung proteins, however, was lower than that seen in liver; (7,500.O pCi/pmol for L-asparagine; 676.6 pCi/pmol for L-aspartate). Although kidney was able to assimilate Lasparagine to a prominent degree (sp act, 7,367.5 pCi/ pmol), the specific activity of L-aspartate in its proteins was low (220.0 pCi/pmol). Of the ten organs studied, brain apparently utilized L-asparagine and L-aspartate to the lowest extent. When L-[UJ4C]aspartate was the injectate (Fig. 4) and the specific activity of L-asparagine was measured in the proteins of several organs 20 min after injection, proteins of pancreas contained L-asparagine at the highest specific activity (1,493.4 pCi/pmol). This value reflects both syn-
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PANCREATIC
L-ASPARAGINE
E749
SYNTHETASE
T
F-l
Kidney
Pancreas
Lung
Testis
Thymus
FIG. 2. hCorporatiOn of L-[U-14C]asparagine into proteins of various organs of mouse. Ten BALB/c mice were starved for 16 h and then given iv injections of 10 &i (0.2 ml) of L-[W4C]asparagine or 0.2 ml of saline. All mice were killed 20 min later by cervical dislocation, and organs listed in figure were removed and digested as described (7). Labeled (7) and unlabeled (5) L-asparaginyl residues were measured and speaccordingly. cific activities calculated Values are means t SE of duplicate determinations on samples from 5 animals.
LlLL
Muscle
MOUSE ORGANS
I t
Kidney
P8ncreas
Lung
Brain
Testis
Thymus
Muscle
Heart
Small Intestine
FIG. 3. Incorporation of L-[W4C]aspartate into proteins of various organs of mouse. Ten BALB/c mice were starved for I6 h and then given iv injection of 10 @i (0.2 ml) of L-[U-14C]aspartate or 0.2 ml of saline, All mice were killed 20 min later by cervical dislocation, and organs listed in figure were removed and digested as described (7). Labeled (7) and unlabeled (5) L-aspartyl residues were measured and specific activities calculated accordingly. Values are means & SE of duplicate determinations on samples from 5 animals.
Liver
MOUSE ORGANS
thesis and incorporation of the amide. Proteins of small intestine had the next highest rate of L-asparagine synthesis and incorporation (386.8 pCi/pmol) ,- Stuprisingly, femoral muscle, the specific activity of L-asparagine was 245.5 pCi/pmol. Direct measurement of L-asparagine synthetase in this tissue, however, showed low levels of activity (0.15 -t 0.07 nmol/mg protein per hour). The high specific activity of L-asparagine in muscle may be a reflection of the low concentrations of free, unl .abeled Lasparagine in that tissue (3). Brain, testis, lung, and liver had low levels of L-asparagine in their proteins after administration of L-[U-14C]aspartate. In order to confirm that the high specific activity of L-asparagine in pancreatic proteins after treatment with @J-14C]aspartate was due to the synthesis of the amide in that organ, mice were treated with cycloheximide (100 mg/kg), emetine (32 mg/kg), or actinomycin D (ZOOmg/ kg) in order to inhibit protein synthesis or with an
equivalent volume of saline (Table 2, expt 1) and the Lasparagine synthetase activity and concentration of Lasparagine were measured in the pancreas 1.5 h after treatment. As can be seen (Table 2), these agents did not affect the activity of the enzyme significantly under these conditions; however, the concentration of L-asparagine in the tissue was elevated by approximately 200% of control. Lower doses of actinomycin D (250 or 400 pg/kg; Table 2, expt 2) did inhibit L-asparagine synthetase in pancreas by 55-75% of control; however, this effect was observed 48 h after treatment. Effect of pancreatic L -asparugine synthetase on circulating levels of L-asparugine. Woods and Handschumacher (30) reported that rat liver plays a regulatory role in maintaining the concentration of L-asparagine in the plasma at a steady state, presumably through a balance between its L-asparaginase and L-asparagine synthetase activities. The pancreas, by virtue of its high
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E750
MILMAN,
Pancreas
Lung
Brain
Testis
Thymus
Muscle
Heart
Small Intestine
AND
YOUNG
FIG. 4. Synthesis of L-[U-14C]asparagine and its incorporation into proteins of various organs of mouse. Ten BALB/ c mice were starved for 16 h and then given iv injection of 10 &i (0,2 ml) of L[U-14C]aspartate or 0.2 ml of saline. All mice were killed 20 min later by cervical dislocation, and organs listed in figure were removed and digested as described (7). Labeled (5) and unlabeled (7) L-asparaginyl residues were measured and specific activities calculated accordingly. Values are means & SE of duplicate determinations on samples from 5 animals.
dl Kidney
COONEY,
Liver
MOUSE ORGANS 2. Effect of inhibitors on activity of mouse pancreatic L -asparagine synthetase
TABLE
Time of Sacrifice, h Posttreatment
Treatment
Expt 1 Saline,
0.2 ml
Mean L-Asparagine Synthkized, nmol/mg Protein h
1.5
25.94 4 11.97
1.5
27.16
t
1.5
25,57
t- 13.47
1.5
20,87
-+ 5.89
48 48
44.93 18.99
t 3.13 X!I 3.70"
48
Il.16
t
Cycloheximide,
10.50
100w/kg Emetine, 32 mg/ kg Actinomycin D, 20 w/k- -
m
Mean Free L-ASparagine, nmol/mg Protein and nmol/ g wet wt
3.43 383.6 6.91 700.0 8.30 799.6 5.52 576.0
-t AI t f. k
0.86 47.4 1.45" 32,6* 2.41* k 97.5* t 0.58* AI 65,6*
Expt2 Saline, 0.2 ml Actinomycin D, 250~dkg
Actinomycin
In a second experiment, the pancreatic ducts of beagle dogs were ligated, and the exocrine pancreas was allowed to degenerate (2). Sham-operated animals served as controls. Measurements of L-asparagine were conducted on heated (100°C) plasma samples obtained before and after such ligation (8). Amylase and lipase activities in the serum (Harleco, Philadelphia, PA) were used as a measure of the degeneration of the exocrine pancreas (25). The levels of L-asparagine in plasma remained essentially unchanged throughout the experiment (data not shown). Amylase and lipase activities, however, rose sharply, peaking at day 5, and then declined rapidly as the pancress degenerated (data not shown). Clinical observations of test animals included marked diarrhea and steatorrhea. L-Asparagine synthetase activity in the pancreas of the dogs killed 20 days after pancreatic duct ligation was 84 nmol/(g h) compared to 1,538 nmol/(g h) in the pancreas of normal beagle dogs (19). In a separate study, the splenic portion of the pancreas of mice was surgically removed and the concentration of L-asparagine in plasma was monitored (data not shown). No significant differences in the concentration of L-asparagine in the serum were seen in these partially pancreatectomized animals versus their sham-operated counterparts, suggesting that even in the mouse, the normal concentration of L-asparagine in the blood is partly independent of pancreatic function. We next investigated the competence of pancreas to regulate L-asparagine under conditions in which the concentration of the amide in the“blood is increased. This could be envisioned as occurring postprandially, in the presence of increased L-asparagine in the diet or in disease states in which protein breakdown is occurring. In the first of a series of experiments, beagle dogs were anesthetized with sodium pentobarbital and, after a baseline level of L-asparagine had been established, L-asparagine in normal saline was infused into the femoral vein at the rate of 25 pmol/min (0.5 ml/min) for 123 min, then increased to 50 pmol/min (1.0 ml/min) for an additional 90 min. In a typical experiment shown in Fig. 5, it can be seen l
D
1.06"
400/%/kg Expt 1. Values
are means t SE of duplicate determinations on samples from 5 animals. Twenty adult, male GP Swiss mice were starved for 16 h and then divided into 4 equal groups. Each group of mice was treated with the appropriate drug or saline by intravenous injection, All mice were killed 1.5 h later by cervical dislocation, and their pancreata were removed, homogenized, and assayed for L-asparagine synthetase activity and L-asparagine as described. Expt 2. Values are means k SE of duplicate determinations on samples from 10 animals. Thirty adult, male GP Swiss mice were divided into 3 equal groups. Each group of mice was treated with an injection of actinomycin D (250 or 400 pg/kg ip) or with an equivalent volume of saline. The mice were killed (48 h after treatment) by cervical dislocation, and their pancreata were removed, homogenized, and assayed for L-asparagine synthetase activity and L-asparagine, * P c 0.05 compared to the saline control animals.
content of L-asparagine synthetase, also would appear to be equipped to regulate the homeostasis of L-asparagine. To test this possibility, the concentration of L-asparagine in the arterial and venous blood across the pancreas of the anesthetized dog was monitored (data not shown). No significant or consistent negative A-V difference of L-asparagine across the pancreas was observed in six experiments.
l
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PANCREATIC
L-ASPARAGINE I
E751
SYNTHETASE
1
I
1
1
T
1
case in the blood. The measurement of a high specific activity of L-asparagine synthetase in mammalian pancreas (19) suggested that the organ might have an additional role, namely, the regulation of the concentration of L-asparagine in the blood, in a manner similar to the liver as proposed by Woods and Handschumacher (30). Our present studies, however, led us to conclude that this is not the case. Whereas the liver is able to add Lasparagine to the circulation under conditions in which the blood perfusing it is low in the amino acid (3), the pancreas does not appear to do so (Fig. 6) This finding is further supported by the observation that the activity of L-asparagine synthetase from mouse pancreas is unaffected by changes in diet (Table l), This result should be contrasted with the work of Patterson and Orr (ZU), who have shown that the activity of L-asparagine synthetase from rat liver was increased six- to eightfold after the rats were maintained on an L-asparagine-free diet for 5 days. Both liver and pancreas, however, are able to withdraw L-asparagine from the blood when the concentration of the amide is above normal limits. In the case of the liver, the amino acid is metabolized to L-aspartic acid, which then enters the citric acid cycle (3); in the case of the pancreas, some of the L-asparagine taken up by the organ is utilized for protein synthesis (Fig. 2); however, most of the excess circulating amino acid enters the pancreatic free pool of the amide, which expands or contracts as the need arises. This result is suggested by the observation that a lower concentration of L-asparagine is seen in the craneo-pancreaticoduodenal venous blood of the dog as opposed to the arterial blood during infusion of the amino acid (Fig. 5), signifying expansion of the pancreatic free pool of L-asparagine, whereas a greater concentration of L-asparagine is seen in the craneo-pancreaticoduodenal venous blood over that in the arterial blood after termination of the infusion of the amino acid (Fig. 5), suggesting contraction of the free pool of the amide. The role of pancreatic L-asparagine synthetase in the homeostatis of L-asparagine appears to be confined primarily to meeting the intrinsic amino acid requirement of important pancreatic proteins (Fig. 4). The importance of L-asparagine in exocrine pancreatic proteins can be appreciated from an inspection of the primary structures of several digestive enzymes produced by this organ, Trypsin and chymotrypsin each contain 14 L-asparaginyl residues; pancreatic ribonuclease contains 10 L-asparaginyl residues. Moreover, Putnam and Neurath (21) reported that the NH&erminal amino acid of carboxypeptidase A is L-asparagine. Finally, in the activation of chymotrypsin from the inactive proenzyme, chymotrypsinogen, an L-threonine+asparagine dipeptide is released (29). Attempts to link L-asparagine synthetase with the endocrine pancreas were fruitless. L-Asparagine synthetase activity in the islets of Langerhans of the muuse was low (approximately 0.26% of the total activity found in the pancreas), and treatment of mice with streptozotocin, a potent P-cell cytotoxic agent (22), did not alter Lasparagine synthetase levels in the pancreata of experimental animals although diabetes was produced by this treatment. Moreover, mice maintained on a glucose-enriched diet for 2 wk in order to stimulate insulin synthesis l
A
0 TIME
c (minutes1
FIG. 5. Effect of pancreas on concentration of L-asparagine in plasma during course of L-asparagine infusion. A beagle dog was anesthetized with pentobarbital after starvation for 16 h. Then its pancreas was exposed, and femoral artery, femoral vein, and craneo-pancreaticoduodenal vein were cannulated. Samples (2 ml) of blood were withdrawn into heparinized tubes at time periods shown in figure for measurement of L-asparagine. At 35 min (A), 25 pmol of L-asparagine in normal saline were infused per minute into femoral vein at 0.5 ml/ min. At 158 min (B), infusion rate was increased to 1 ml/min (50 pmol/ min). At 248 min (C), infusion was discontinued. l , Concentration of L-asparagine in craneo-pancreaticoduodenal venous blood; 0, concentration of L-asparagine in the femoral arterial blood. Values are means of duplicate determinations. At no time did individual values vary from means by more than 5%
that pancreas vigorously takes up L-asparagine from the circulation du ring L-asparagine infusion; that is to say, there is a substantial positive A-V difference of L-asparagine across the pancreas at all time periods examined during the infusion of the amide. This phenomenon also was seen in two 0 ther identical experiments. At the termination of the infusion, L-asparagine app arently leaves the pancreas at a higher rate than it enters it (i.e., a negative A-V difference is observed). We next examined the effect of pancreas on circulating levels of L-asparagine when the concentration of the amino acid is significantly below normal limits. In order to test this hypothesis, 6 IU of agouti L-asparaginase was infused into the femoral vein of the anesthetized dog. This concentration of enzyme was sufficient to deplete L-asparagine from the blood almost totally (Fig. 6). Termination of amidohydrolysis was accomplished by infusion of 10 ml of serum of a rabbit immunized to agouti L-asparaginase. It can be seen (Fig. 6) that L-asparagine in the serum was restored at the same rate in the craneopancreaticoduodenal venous blood as in the arterial supply; that is, no significant A-V difference of L-asparagine across the pancreas could be detected. DISCUSSION
Pancreas has long been considered to be an organ primarily involved in the synthesis of enzymes responsible for digestion and in the production of insulin and glucagon for the regulation of the concentration of glu-
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MILMAN,
60
COONEY,
AND
YOUNG
FIG. 6. Effect of pancreas on concentration of L-asparagine in plasma after infusion of L-asparaginase. A beagle dog was starved for 16 h and then anesthetized with pentobarbital. Its pancreas was exposed, and femoral artery, femoral vein, and craneo-pancreaticoduodenal vein were cannulated. Samples (2 ml) of blood were withdrawn into heparinized tubes at time periods indicated in figure for measurements of L-asparagine and/ or L-asparaginase. At 36 min (A), I ml (6 IU) of agouti L-asparaginase was infused into femoral vein. l , concentration of L-asparagine in craneo-pancreaticoduodenal venous blood; 0, concentration of L-asparagine in femoral arterial blood; A, L-asparaginase activity in femoral arterial blood. Values are means of duplicate determinations. At no time did individual values vary from means by more than 5%.
t 100 E3
TIME
(minutes)
and release did not have elevated L-asparagine synthetase activity in their pancreata at the termination of the study when compared to that measured in the pancreata of control mice (Table 1). It is possible, therefore, that the hypoinsulinemia that occurs in some patients after treatment with L-asparaginase may be due to the extremely low levels of L-asparagine synthetase in the islets of Langerhans. The possibility that L-asparagine is synthesized in the exocrine pancreas and subsequently is transported to the p-cells for insulin synthesis has not been investigated. Such a possibility is made likely by the work of MalaisseLagae and co-workers (18). These investigators observed that the exocrine tissue has almost no direct arterial supply; furthermore, the exocrine and endocrine cells may exert a mutual and direct control of one another’s secretory activity. Another feature of pancreatic organization is the presence around the islets of acini, which are composed of large secretory cells packed with zymogen granules. The same investigators indicate that such an anatomical segregation coincides with a functional heterogeneity of the exocrine tissue. It can be concluded, therefore, that the activity of Lasparagine synthetase is primarily associated with the exocrine pancreas and is modulated by factors such as the age of the animal, but not by gender, diet, or the
presence of tumor under the conditions examined. Moreover, the function of the enzyme is to supply L-asparagine for the synthesis of important pancreatic proteins. Furthermore, whereas the pancreas appears to be able to reduce the concentration of L-asparagine in plasma during an increased amino acid load, it is unable to add Lasparagine to the circulation when the concentration of the amide is below normal levels. Further studies attempting to correlate the activity of L-asparagine synthetase with pancreatic function are presently being investigated. The authors wish to thank Dr. H. Olson and Mrs. V. King for their assistance in the performance of the partial pancreatectomy in mice, and Mason Research Institute, Worcester, MA for conducting the pancreatic duct ligation experiments in beagle dogs. The excellent typing of the manuscript by Ms. Joyce Winters is also greatly appreciated. Present address of H. A. Milman: Toxicology Branch, Carcinogenesis Testing Program, National Cancer Institute, Bethesda, MD 20205. Present address of D. M. Young: Dept. of Veterinary Science, Veterinary Research Laboratory, Montana State Univ., Bozeman, MT 59715. This work is taken in part from a dissertation by H. A. Milman that was presented to the Dept. of Pharmacology, Graduate School of Arts and Sciences, George Washington Univ., Washington, DC, in partial fulfillment of the requirements for the PhD. Send reprint requests to: Dr. Harry A. Milman, National Cancer Institute, 7910 Woodmont Avenue, Room 3C25, Bethesda, MD 20205. Received
30 June
1978; accepted
in final
form
9 February
1979.
REFERENCES 1. ARFIN, S. M. Asparagine synthesis in the chick embryo liver, Biochim. Biophys. Acta 136: 233-244,1967. 2. BANTING, F. G., AND C. H, BEST. The internal secretion of the pancreas. Lab. CZin. Med. 7: 465-480, 1922. 3. BERGSTROM, J., P. FURST, L. 0, NOREE, AND E. VINNARS. Intracellular free amino acid concentration in human muscle tissue. J. Appl. Physiol. 36: 693-697, 1974. 4. CASSANO, G. B., AND E. HANSSON, Uptake of [‘4C]glutamine in the tissues of the mouse studied by whole-body autoradiography. J. Neurochem. 12: 851-855, 1965,
5. COONEY, D. A., R, L, CAPIZZI, AND R. E. HANDSCHUMACHER. Evaluation of L-asparagine metabolism in animals and man. Cancer Res. 30: 929-935, 1970. 6. COONEY, D. A., R, DAVIS, AND G. VANATTA. A spectrophotometric method for the simultaneous measurement of L-glutamine and Lasparagine in biological materials. And. Biochem. 40: 312-326, 1971. 7. COONEY, D. A., E. R. HOMAN, T. CAMERON, AND U. SCHAEPPI. The measurement of 4-[14C]-L-asparagine in body fluids and tissues: methodology and application. J. Lab. Ch. Med. 81: 455-466, 1973,
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PANCREATIC
L-ASPARAGINE
8. COONEY,
D. A., AND pi. A. MILMAN. A radiometric technique for measuring L-asparagine in picomole quantities. Biochem. J. 129: 953-960, 1972. 9. COONEY, D. A., H. A. MILMAN, AND A. M. GUARINO. Investigations of the biosynthesis of L-asparagine in the skate Raja oceLZafa and dogfish Squab acanthias. Mt. Desert Island Biol. Lab. BUZZ. 13: 27-28,1973.
COONEY, D. A., K A. MILMAN, AND E. R. HOMAN. A radiometric technique for measuring nicotinamide adenine dinucleotide in femtomole quantities. Methodology and application. Anal Biochem. 63: 528-542, 1975. 11, GAILANI, S., A. NUSSBAUM, T. OHNUMA, AND A. FREEMAN. Diabetes in patients treated with asparaginase, CZin. PharmacoZ. Therap. 12: 487-490, 1971. 12. GOLDBERG, A. I., D, A. COONEY, J. P. GLYNN, E. R. HOMAN, M. R. GASTON, AND H. A. MILMAN. The effects of immunization to Lasparaginase on antitumor and enzymatic activity. Cancer Res, 33: 10.
256-261, 13.
14.
15.
16. 17.
18.
19.
E753
SYNTHETASE
1973.
HOLCENBERG, 3. S. Guinea pig asparagine synthetase, Biochim. Biophys, Acta 185: 228-238, 1969. HOROWITZ, B., B. K. MADRAS, A. MEISTER, L, J, OLD, E. A. BOYSE, AND E. STOCKERT. Asparagine synthetase activity of mouse leukemias. Science 160: 533-535, 1968. HUANG, Y. Z., AND W. E. KNOX. Glutamine-dependent asparagine synthetase in fetal, adult and neoplastic rat tissues. Enzyme 19: 314-328, 1975, KHAN, A., M. ADACHI, AND J. M. HILL. Diabetogenic effect of Lasparaginase. CZin. Endocrinol. Metab. 29: 1373-1376, 1969. LOWRY, 0, H., N. J, ROSENBROUGH, A, L. FARR, AND R. J. RANDALL. Protein measurement with the Folin phenol reagent. J. BioZ. Chem, 193: 265-275, 1951. MALAISSE-LAGAE, F., M. RAVAZZOLA, P. ROBBERECHT, A. VANDERMEERS, W. J, MALAISSE, AND L. ORCI. Exocrine pancreas: evidence for topographic partition of secretory function. Science 190: 795-797, 1975. MILMAN, H. A., AND D. A. COONEY. The distribution of L-aspara-
gine synthetase in the principal organs of several mammalian and avian species. Biochem. J. 142: 27-35, 1974. 20. PATTERSON, JR., M. K., AND E. R. ORR, Degeneration, tumor, dietary, and L-asparaginase effects on asparagine biosynthesis in rat liver. Cancer Res, 29: 1179-1183, 1969. 21, PUTNAM, F. W., AND H. NEURATH. Chemical and enzymatic properties of crystalline carboxypeptidase. J. BioZ. Chem. 166: 603-619, 1946. 22. SCHEIN, P. S., D, A. COONEY, M. G. MCMENAMIN, AND T. ANDERSON. Streptozotocin diabetes-further studies on the mechanism of depression of nicotinamide adenine dinucleotide concentrations in mouse pancreatic islets and liver. Biochem, Pharmacol. 22: 26252631,1973. 23. 24. 25.
26.
27, 28.
29. 30.
31,
SHAW, M. T., C, C. BARNES, F. J. F. MADDEN, AND K. D. BAGSHAW. L-Asparaginase and pancreatitis. Lancet 11: 721, 1970. SHWKUYA, R., AND G, W. SCHWERT. Glutamic acid decarboxylase. J. Biol, Chem, 235: 1649-1652, 1960. SONG, H., N. W. TIETZ, AND C. TAN. Usefulness of serum lipase, esterase, and amylase estimation in the diagnosis of pancreatitisa comparison. Chin. Chem. 16: 264-268, 1970. TAN, C. L., S. P, CHIANG, AND K, P. WEE. Acute haemorrhagic pancreatitis following L-asparaginase therapy in acute lymphoblastic leukemic-a case report. Singapore Med. J. 15: 278-282, 1974. TATE, S. S., AND A. MEISTER. Studies on the sulfhydryl groups af L-aspartate+decarboxylase. Biochemistry 7: 3240-3247, 1968. WEETMAN, R. M.,- AND R. L. BAEHNER. Latent onset of clinical pancreatitis in children receiving L-asparaginase therapy. Cancer 34: 780-785, 1974. WHITE, A., P. HANDLER, AND E. L. SMITH. Principles of Biochemistry. New York: McGraw, 1968, p. 252. WOODS, J. S., AND R, E. HANDSCHUMACHER, Hepatic homeostasis of plasma L-asparagine. Am. J. PhysioZ. 221: 1785-1790, 1971. YIP, C, C., AND M. L. MOWLE. Structure and function of insulin: preparation and biological activity of guinea pig des-B-asp3’), desA-asn”-insulin. Can, J. Biochem. 54: 866-871, 1976.
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A. MILMAN,
MILMAN, YOUNG. ostasis
DAVID
of Toxicology,
Laboratory
HARRY A., DAVID
Role of pancreatic L-usparagine.
A. COONEY,
National
synthetase
M.
in home-
of Am. J. Physiol. 236(6): E746-E753, 1979 or Am. J. Physiol.: Endocrinol. Metab. Gastrointest. Physiol. 5(6): E746-E753, 1979.-L-Asparagine synthetase from mouse pancreas was found to be associated principally with the exocrine pancreas and to be dependent on the age of the animal, but not on gender, diet, or the presence of tumor under the conditions examined. The function of the pancreatic enzyme appears to be to supply L-asparagine for the synthesis of pancreatic proteins. This function is suggested by the high specific activity of L-asparagine in pancreatic proteins after intravenous treatment of BDFr mice with L-[U-‘4C]aspartate. The pancreas is also able to function as a storage depot for L-asparagine under conditions in which the concentration of the amino acid in the blood is in excess. Unlike the liver, the pancreas is unable to add L-asparagine to the circulation when the concentration of the amide is below normal limits, leukemia 5178Y; exocrine circulation
pancreas;
ALTHOUGH
GOOD
THERE
IS
diet; age; protein
EXPERIMENTAL
AND
Cancer Institute,
A. COONEY, AND DAVID
L asparugine
synthetase
synthesis;
EVIDENCE
that the liver plays an important role in regulating the concentration of L-asparagine in the blood (30), various features of this homeostatic control await further clarification. Thus, although the enzymatic equipment for the control of increased L-asparagine in plasma is present in rodent liver in the form of L-asparaginase (L-asparagine amidohydrolase; EC 3.5.1.1) active under physiological conditions, the mechanism by which low levels of Lasparagine in plasma are elevated by hepatic control has yet to be elucidated. Orthodox L-asparagine synthetase (L-glutamine hydrolyzing; EC 6.3.5.4), the enzyme catalyzing the amidation of L-aspartate, would be the most obvious of such a mechanism. Yet, a recent survey of the specific activity of the biosynthetic enzyme in the livers of several mammalian species revealed that only the liver of the guinea pig was equipped to synthesize L-asparagine at a prominent rate (19). In this species L-asparaginase in plasma effectively eliminates L-asparagine (13). It would be expected that depletion would induce the biosynthesis of the amide and therefore may be regarded as an exceptional case, not representative of the controls that operate in subjects with normal concentrations of L-asparagine in plasma. In the same survey (19), it was observed that mammalian pancreas, in contradistinction to mammalian liver, consistently contained L-asparagine synthetase at a specific activity higher than in any of the
DAVID Bethesda,
M. YOUNG Maryland
20205
other organs examined. On the other hand, L-asparaginase was present at a very low specific activity in the pancreas. These findings suggest that the pancreas and the liver may act in concert to maintain the homeostasis of Lasparagine in plasma. These studies were designed to characterize the behavior of L-asparagine synthetase in the pancreas of the mouse under conditions of growth, development, and nutritional variations in order to gain some insight into the responsiveness of the enzyme to changes in its amino acid environment. Furthermore, the ability of the pancreas of the dog to regulate the concentration of L-asparagine in plasma under conditions in which the concentration of the amino acid is unduly elevated or reduced also was investigated. MATERIALS
AND
METHODS
Enzymes. L-Asparaginase (EC 3.5.1.1) from Escherichia coli (340 IU/mg protein) was purified at the Merck Institute for Therapeutic Research, West Point, PA, and provided by the Drug Research and Development Branch of the National Cancer Institute. L-Aspartate-4decarboxylase from Alcaligenes faecalis (EC 4.1.1.12) was purified by the method of Tate and Meister (27). The enzyme exhibited a specific activity, with z-aspartate as substrate, of 77 IU/mg protein and was stored as previously described (8). z-Glutamate decarboxylase from E. c&i (EC 4.1.1.15,50 IU/mg protein) was purified by the method of Shukuya and Schwert (24). Malate dehydrogenase (EC 1.1.1-37; 720 IU/mg protein) and Lglutamate-oxaloacetate transaminase (EC 2.6.1.1; 190 IU/mg protein) were purchased from Boehringer Mannheim Corp., New York, NY. Pronase was obtained from Calbiochem, Los Angeles, CA. Subtilisin was purchased from NOVO, Copenhagen. Collagenase was a product of Worthington Biochemical Corp., Freehold, NJ. Radiochemicals. L-[4-14C]aspartate (sp radioact 12.9 &i/pmol), L-[UJ4C]aspartate (sp radioact 231 pCi/ pmol), and L-[U-14C]asparagine (sp radioact 185 @Zi/ pmol) were the products of Amersham Corp., Silver Spring, MD. Purity (99%) of these chemicals was assessed as previously described (19), Chemicals and drugs. L-Asparagine was purchased from Schwartz/Mann Biological Research, Rockville, MD, and stored in a desiccator over calcium chloride until used. Reduced diphosphopyridine nucleotide (NADH) was obtained from Sigma Chemical Co, St.
I3746
Downloaded from www.physiology.org/journal/ajpendo by ${individualUser.givenNames} ${individualUser.surname} (163.015.154.053) on October 31, 2018. Copyright © 1979 American Physiological Society. All rights reserved.
PANCREATIC
L-ASPARAGINE
SYNTHETASE
Louis, MO. L-Glutamine and Z-oxoglutarate were purchased from Calbiochem. Streptozotocin, actinomycin D, cycloheximide, and emetine were obtained through the Drug Research and Development Branch of the National Cancer Institute. All other chemicals used were of reagent grade. Measurement of L -asparagine synthetase activity. Tissue samples were homogenized at 4OC in 9 vol (wt/vol) of 0.1 M Tris-HCI buffer (pH 7.6) containing I mM dithiothreitol and U,5 mM disodium EDTA. The homogenates were transferred to Eppendorf 1,500~~1 disposable plastic centrifuge vessels (Brinkman Instrument Co., Silver Spring, MD) and centrifuged in the Eppendorf Zentrifuge at 12,000 g for 6 min. L-Asparagine synthetase activity in the 12,000 g supernatants was assessed by a radiometric technique the full details of which have been described previously (19) Measurement of L -asparuginase activity. L-Asparaginase activity was measured radiometrically (9) with a sensitivity of 0.00001 IU/ml. Measurement of L -asparagine. L-Asparagine was measured in the 12,000 g heated (100°C) dog plasma either by a radiometric technique (8), by an enzymatic spectrophotometric method (6) in which as little as 5 nmol of amide can be estimated, or by a radiometric, gasometric procedure (7), Estimation of protein. Estimation of protein in the 12,000 g (6 min) supernatant of organ or tumor homogenates was conducted according to the method of Lowry et al. (17) using bovine serum albumin (Armour Pharmaceutical Co., Chicago, IL) as the standard. Measurement of arteriovenous (A- V) difference of Lasparagine acruss pancreas of anesthetized dug, Beagle dogs, weighing lo-13 kg, were starved for 16 h and then anesthetized with intravenous sodium pentobarbital. A midline abdominal incision was made extending from the xiphoid process to 7 cm anterior to the symphysis pubica. The pancreas was exposed and a cannula inserted into the craneo-pancreaticoduodenal vein, slightly distal to its junction with the portal vein. A three-way stopcock was placed on the exposed end of the cannula, and a small amount of heparinized saline was injected into the cannula to prevent clotting. The cannula was secured in place with monofilament nylon sutures. The femoral artery and vein were isolated and cannulated, and a three-way stopcock was inserted in the same manner. Blood samples (2 ml) were collected from the femoral artery and craneo-pancreaticoduodenal vein periodically and transferred to l&ml Falcon tubes (Falcon, Oxhard, CA) containing two drops of heparin. The tubes were centrifuged at 2,500 g for 10 min, and the clear supernatant was transferred to polycarbonate tubes, which were immediately frozen on dry ice. The frozen tubes were transferred to a boiling water bath and boiled for 5 min after which the caps of the tubes were retightened. The tubes were boiled for an additional 15 min and then centrifuged at 105,000 g for 20 min. The clear supernatant was assayed radiometrically for L-asparagine. When L-asparaginase activity was to be measured in dog plasma, separate blood samples (I ml) were taken and transferred to Eppendorf 1,500~~1 centrifuge tubes containing 2 drops of heparin. The tubes were immedil
E747 ately centrifuged at 12,000 g for 5 s in the Eppendorf Zentrifuge and the supernatant transferred to fresh Eppendorf vessels, which were immediately frozen. L-ASparaginase activity was measured in 5-~1 aliquots of the plasma as described. RESULTS
Localization of L -asparagine synthetase activity in mouse pancreas. Of the seven mouse organs examined (Fig. l), the specific activity of L-asparagine synthetase was found to be highest in pancreas. This activity was distributed evenly throughout the organ (27.69 & 2.98 nmol/mg protein per hour in the gastroduodenal portion; 33.60 t 2.48 nmol/mg protein per hour in the splenic portion). Mouse testis also exhibited considerable activity (approximately 200 nmol/g h) in confirmation of previous reports (14, 19). Noteworthy was the finding of low or negligible L-asparagine synthetase activity in mouse liver, even in the presence of 0.2 M (NH&SO+ a compound that inhibits mouse hepatic L-asparaginase activity by greater than 95%. The presence of L-asparagine synthetase in high activities in the pancreas of the mouse led us to speculate that this activity might be originating in p-cells. Islets of Langerhans therefore were isolated (IO), and the activity of L-asparagine synthetase was measured in clusters of 30 -t 5 islets. Only 0.02 nmol of L-asparagine was synthesized per hour per 30 islets, an apparent L-asparagine synthetase activity representing 0.26% of the total activity measured in mouse pancreas.’ Furthermore, the activity of L-asparagine synthetase (47.47 t 7.37 nmol/mg protein per hour) in the pancreata of BALB/c mice treated intravenously with streptozotocin, (200 mg/kg) did not differ significantly from that measured in the pancreata of mice treated with 0.005 M sodium citrate, pH 4.0 (vehicle) (40.07 + 6.16 nmol/mg protein per hour), although glucosurea and atrophic islets were observed in drug-treated animals. Moreover, normal mice maintained on a diet supplemented with 5% D-glucose for 14 days in order to stimulate insulin synthesis and release (Table 1) had activities of L-asparagine synthetase in their pancreata comparable to those measured in the pancreata of control mice. Effect of age, gender, genetic variation, diet, and presence of tumor on L-asparugine synthetase activity in mouse pancreas. L-Asparagine synthetase activity in mouse pancreas increases with the age of the animal until day 20 and maintains a constant level at maturity (data not shown). The specific activity of L-asparagine synthetase in mouse pancreas was independent of the gender of the subject; mean t SE synthetase activity in the pancreata of five adult, male BALB/c mice (36.26 t 5.77 nmol/mg protein per hour) was equivalent to that measured in the pancreata of five adult, female BALB/c mice (31.31 t 1.79 nmol/mg protein per hour). l
’ Inasmuch as mouse pancreas contains approximately 600 islets, the L-asparagine synthetase activity in the endocrine pancreas, therefore is equivalent to approximately 0.4 nmol/h. Because the total L-asparagine synthetase activity in mouse pancreas is approximately 150 nmol/h, the contribution by the endocrine pancreas is aDDroximatelv 0.26%.
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MILMAN,
Spleen
8roin
Testis
Pancreas
MOUSE
Kidney
Lung
Liver
ORGANS
1. L-Asparagine synthetase activity mouse. L-Asparagine synthetase was measured of organ homogenates of mouse as described. of 5 animals. FIG.
in principal organs of in 12,ooO g supernatants Values are means & SE
1. Effect of diet m activity of L -asparagine synthetase
TABLE
Expt
Diet
NO
Treatment
L-Asparagine Synthesized (nmol/mg of protein/h t SE) Pancreas
2
2
27.1 -+ 3.1
L-Asparagine-free L- Asparagine-free L-Asparagine-free L-Asparagine-free L-asparagine, bwwt) z-Asparagine-free L-asparagine, M/wt) L-Asparagine-free L-asparagine,
Tumor
L5178Y/AS L5178Y/AR
27.3 zk 2.0 29.2 f: 1.4
2.0 k 0.4 52.6 I!I 3.1
19.9 * 3*7
+ 2% +
L5178Y/AS
23.7 t 5.2
+
L5178Y/AR
21.4 k 2.5
1*4 -+ 0.2
2% 42.8 t 6.1
2Y0
bwwt)
3
D-Glucose, 5% + Purina chow Water + Purina chow
31.5 AI 4*7 25.7 k 1.9
Values are means t SE of duplicate determinations on samples from 5 animals. Normal male I3DFI mice were housed in metabolism cages and maintained either on Purina chow or starvation for 54 h (expt I); or 5% D-glucose and Purina chow or Purina chow alone for 14 days feqt 4). BDFI mice, injected with 107 cells of L5178Y/AS or L5178Y/ AR subcutaneously, were maintained on a diet free of L-asparagine (Nutritional Biochemicals Carp,, Cleveland, OH) or supplemented with 2’% (wt/wt) L-asparagine for the first 10 days of tumor growth (expts 2 and 3). All mice were killed by cervical dislocation at the times specified, and their pancreata or pancreata and tumors were removed, homogenized, and assayed for L-asparagine synthetase activity as described.
Although the activity of L-asparagine synthetase in the pancreas of 20 strains of adult mice examined ranged from 23,91 t 3.30 nmol/mg protein per hour in ADKzFl mice to 72.12 -t 1.96 nmol/mg protein per hour in ZWB/ F1 mice (data not shown), this variation cannot be totally
COONEY,
AND
YOUNG
attributed to genetic factors because age and prior conditioning of the test animals were not strictly controlled. The influence of diet on mouse pancreatic L-asparagine synthetase activity can be appreciated from the results in Table 1. In mice starved for as long as 54 h, the activity of pancreatic L-asparagine synthetase (34.0 t 4.7 nmol/ mg protein per hour) was comparable to that measured in mice fed Purina chow laboratory mouse feed (37.3 k 2.9 nmol/mg protein per hour). (All mice were housed in metabolism cages to avoid possible ingestion of feces). This constancy should be contrasted with the observation that mice maintained on a starvation diet lost an average of 6.0 g, whereas control mice gained a mean of 0.8 g. Mice maintained for 10 days on a diet free of L-asparagine (Nutritional Biochemicals Corp., Cleveland, OH) (Table 1) had pancreatic L-asparagine synthetase activities comparable to those found in the pancreas of mice fed a diet supplemented with 2% (wt/wt) L-asparagine. Our studies with mice bearing L5178Y (Table 1) indicate that low activity of L-asparagine synthetase is detectable even in the sensitive form of the tumor (L5178/ AS) (19). On day 10 of tumor growth (after implantation of 107 cells subcutaneously), when tumor diameter was approximately 1.4 cm, the presence of either L-asparaginase-sensitive or -resistant forms of the leukemia 5178Y had no effect on the activity of L-asparagine synthetase in the pancreas of tumor-bearing mice. Function of L -Asparagine Synthetase of Mouse Pancreas Effect of pancreatic L -asparagine synthetase on protein synthesis. Cassano and Hansson (4) observed that the pancreas takes up radioactive L-glutamine to the greatest extent of all the organs studied and that this amide apparently is used for protein synthesis within that organ. It appeared likely that L-asparagine, the next lower homologue of L-glutamine, would be used in an analogous manner, It can be seen (Figs. 2 and 3) that pancreas invariably takes up and incorporates L-[U-‘4C]asparagine and L-[U14C]aspartate into its proteins to the greatest extent of the ten organs studied; (sp act of L-asparagine, 27,000.0 pCi/pmol; sp act of L-aspartate, lJ60.2 pCi/pmol). Small intestine had the next highest specific activity of these amino acids in its proteins (Figs. 2 and 3); (L-asparagine, 11,653.a pCi/pmol; L-aspartate, lJU4.3 pCi&mol). The rate of incorporation of these amino acids into lung proteins, however, was lower than that seen in liver; (7,500.O pCi/pmol for L-asparagine; 676.6 pCi/pmol for L-aspartate). Although kidney was able to assimilate Lasparagine to a prominent degree (sp act, 7,367.5 pCi/ pmol), the specific activity of L-aspartate in its proteins was low (220.0 pCi/pmol). Of the ten organs studied, brain apparently utilized L-asparagine and L-aspartate to the lowest extent. When L-[UJ4C]aspartate was the injectate (Fig. 4) and the specific activity of L-asparagine was measured in the proteins of several organs 20 min after injection, proteins of pancreas contained L-asparagine at the highest specific activity (1,493.4 pCi/pmol). This value reflects both syn-
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PANCREATIC
L-ASPARAGINE
E749
SYNTHETASE
T
F-l
Kidney
Pancreas
Lung
Testis
Thymus
FIG. 2. hCorporatiOn of L-[U-14C]asparagine into proteins of various organs of mouse. Ten BALB/c mice were starved for 16 h and then given iv injections of 10 &i (0.2 ml) of L-[W4C]asparagine or 0.2 ml of saline. All mice were killed 20 min later by cervical dislocation, and organs listed in figure were removed and digested as described (7). Labeled (7) and unlabeled (5) L-asparaginyl residues were measured and speaccordingly. cific activities calculated Values are means t SE of duplicate determinations on samples from 5 animals.
LlLL
Muscle
MOUSE ORGANS
I t
Kidney
P8ncreas
Lung
Brain
Testis
Thymus
Muscle
Heart
Small Intestine
FIG. 3. Incorporation of L-[W4C]aspartate into proteins of various organs of mouse. Ten BALB/c mice were starved for I6 h and then given iv injection of 10 @i (0.2 ml) of L-[U-14C]aspartate or 0.2 ml of saline, All mice were killed 20 min later by cervical dislocation, and organs listed in figure were removed and digested as described (7). Labeled (7) and unlabeled (5) L-aspartyl residues were measured and specific activities calculated accordingly. Values are means & SE of duplicate determinations on samples from 5 animals.
Liver
MOUSE ORGANS
thesis and incorporation of the amide. Proteins of small intestine had the next highest rate of L-asparagine synthesis and incorporation (386.8 pCi/pmol) ,- Stuprisingly, femoral muscle, the specific activity of L-asparagine was 245.5 pCi/pmol. Direct measurement of L-asparagine synthetase in this tissue, however, showed low levels of activity (0.15 -t 0.07 nmol/mg protein per hour). The high specific activity of L-asparagine in muscle may be a reflection of the low concentrations of free, unl .abeled Lasparagine in that tissue (3). Brain, testis, lung, and liver had low levels of L-asparagine in their proteins after administration of L-[U-14C]aspartate. In order to confirm that the high specific activity of L-asparagine in pancreatic proteins after treatment with @J-14C]aspartate was due to the synthesis of the amide in that organ, mice were treated with cycloheximide (100 mg/kg), emetine (32 mg/kg), or actinomycin D (ZOOmg/ kg) in order to inhibit protein synthesis or with an
equivalent volume of saline (Table 2, expt 1) and the Lasparagine synthetase activity and concentration of Lasparagine were measured in the pancreas 1.5 h after treatment. As can be seen (Table 2), these agents did not affect the activity of the enzyme significantly under these conditions; however, the concentration of L-asparagine in the tissue was elevated by approximately 200% of control. Lower doses of actinomycin D (250 or 400 pg/kg; Table 2, expt 2) did inhibit L-asparagine synthetase in pancreas by 55-75% of control; however, this effect was observed 48 h after treatment. Effect of pancreatic L -asparugine synthetase on circulating levels of L-asparugine. Woods and Handschumacher (30) reported that rat liver plays a regulatory role in maintaining the concentration of L-asparagine in the plasma at a steady state, presumably through a balance between its L-asparaginase and L-asparagine synthetase activities. The pancreas, by virtue of its high
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E750
MILMAN,
Pancreas
Lung
Brain
Testis
Thymus
Muscle
Heart
Small Intestine
AND
YOUNG
FIG. 4. Synthesis of L-[U-14C]asparagine and its incorporation into proteins of various organs of mouse. Ten BALB/ c mice were starved for 16 h and then given iv injection of 10 &i (0,2 ml) of L[U-14C]aspartate or 0.2 ml of saline. All mice were killed 20 min later by cervical dislocation, and organs listed in figure were removed and digested as described (7). Labeled (5) and unlabeled (7) L-asparaginyl residues were measured and specific activities calculated accordingly. Values are means & SE of duplicate determinations on samples from 5 animals.
dl Kidney
COONEY,
Liver
MOUSE ORGANS 2. Effect of inhibitors on activity of mouse pancreatic L -asparagine synthetase
TABLE
Time of Sacrifice, h Posttreatment
Treatment
Expt 1 Saline,
0.2 ml
Mean L-Asparagine Synthkized, nmol/mg Protein h
1.5
25.94 4 11.97
1.5
27.16
t
1.5
25,57
t- 13.47
1.5
20,87
-+ 5.89
48 48
44.93 18.99
t 3.13 X!I 3.70"
48
Il.16
t
Cycloheximide,
10.50
100w/kg Emetine, 32 mg/ kg Actinomycin D, 20 w/k- -
m
Mean Free L-ASparagine, nmol/mg Protein and nmol/ g wet wt
3.43 383.6 6.91 700.0 8.30 799.6 5.52 576.0
-t AI t f. k
0.86 47.4 1.45" 32,6* 2.41* k 97.5* t 0.58* AI 65,6*
Expt2 Saline, 0.2 ml Actinomycin D, 250~dkg
Actinomycin
In a second experiment, the pancreatic ducts of beagle dogs were ligated, and the exocrine pancreas was allowed to degenerate (2). Sham-operated animals served as controls. Measurements of L-asparagine were conducted on heated (100°C) plasma samples obtained before and after such ligation (8). Amylase and lipase activities in the serum (Harleco, Philadelphia, PA) were used as a measure of the degeneration of the exocrine pancreas (25). The levels of L-asparagine in plasma remained essentially unchanged throughout the experiment (data not shown). Amylase and lipase activities, however, rose sharply, peaking at day 5, and then declined rapidly as the pancress degenerated (data not shown). Clinical observations of test animals included marked diarrhea and steatorrhea. L-Asparagine synthetase activity in the pancreas of the dogs killed 20 days after pancreatic duct ligation was 84 nmol/(g h) compared to 1,538 nmol/(g h) in the pancreas of normal beagle dogs (19). In a separate study, the splenic portion of the pancreas of mice was surgically removed and the concentration of L-asparagine in plasma was monitored (data not shown). No significant differences in the concentration of L-asparagine in the serum were seen in these partially pancreatectomized animals versus their sham-operated counterparts, suggesting that even in the mouse, the normal concentration of L-asparagine in the blood is partly independent of pancreatic function. We next investigated the competence of pancreas to regulate L-asparagine under conditions in which the concentration of the amide in the“blood is increased. This could be envisioned as occurring postprandially, in the presence of increased L-asparagine in the diet or in disease states in which protein breakdown is occurring. In the first of a series of experiments, beagle dogs were anesthetized with sodium pentobarbital and, after a baseline level of L-asparagine had been established, L-asparagine in normal saline was infused into the femoral vein at the rate of 25 pmol/min (0.5 ml/min) for 123 min, then increased to 50 pmol/min (1.0 ml/min) for an additional 90 min. In a typical experiment shown in Fig. 5, it can be seen l
D
1.06"
400/%/kg Expt 1. Values
are means t SE of duplicate determinations on samples from 5 animals. Twenty adult, male GP Swiss mice were starved for 16 h and then divided into 4 equal groups. Each group of mice was treated with the appropriate drug or saline by intravenous injection, All mice were killed 1.5 h later by cervical dislocation, and their pancreata were removed, homogenized, and assayed for L-asparagine synthetase activity and L-asparagine as described. Expt 2. Values are means k SE of duplicate determinations on samples from 10 animals. Thirty adult, male GP Swiss mice were divided into 3 equal groups. Each group of mice was treated with an injection of actinomycin D (250 or 400 pg/kg ip) or with an equivalent volume of saline. The mice were killed (48 h after treatment) by cervical dislocation, and their pancreata were removed, homogenized, and assayed for L-asparagine synthetase activity and L-asparagine, * P c 0.05 compared to the saline control animals.
content of L-asparagine synthetase, also would appear to be equipped to regulate the homeostasis of L-asparagine. To test this possibility, the concentration of L-asparagine in the arterial and venous blood across the pancreas of the anesthetized dog was monitored (data not shown). No significant or consistent negative A-V difference of L-asparagine across the pancreas was observed in six experiments.
l
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PANCREATIC
L-ASPARAGINE I
E751
SYNTHETASE
1
I
1
1
T
1
case in the blood. The measurement of a high specific activity of L-asparagine synthetase in mammalian pancreas (19) suggested that the organ might have an additional role, namely, the regulation of the concentration of L-asparagine in the blood, in a manner similar to the liver as proposed by Woods and Handschumacher (30). Our present studies, however, led us to conclude that this is not the case. Whereas the liver is able to add Lasparagine to the circulation under conditions in which the blood perfusing it is low in the amino acid (3), the pancreas does not appear to do so (Fig. 6) This finding is further supported by the observation that the activity of L-asparagine synthetase from mouse pancreas is unaffected by changes in diet (Table l), This result should be contrasted with the work of Patterson and Orr (ZU), who have shown that the activity of L-asparagine synthetase from rat liver was increased six- to eightfold after the rats were maintained on an L-asparagine-free diet for 5 days. Both liver and pancreas, however, are able to withdraw L-asparagine from the blood when the concentration of the amide is above normal limits. In the case of the liver, the amino acid is metabolized to L-aspartic acid, which then enters the citric acid cycle (3); in the case of the pancreas, some of the L-asparagine taken up by the organ is utilized for protein synthesis (Fig. 2); however, most of the excess circulating amino acid enters the pancreatic free pool of the amide, which expands or contracts as the need arises. This result is suggested by the observation that a lower concentration of L-asparagine is seen in the craneo-pancreaticoduodenal venous blood of the dog as opposed to the arterial blood during infusion of the amino acid (Fig. 5), signifying expansion of the pancreatic free pool of L-asparagine, whereas a greater concentration of L-asparagine is seen in the craneo-pancreaticoduodenal venous blood over that in the arterial blood after termination of the infusion of the amino acid (Fig. 5), suggesting contraction of the free pool of the amide. The role of pancreatic L-asparagine synthetase in the homeostatis of L-asparagine appears to be confined primarily to meeting the intrinsic amino acid requirement of important pancreatic proteins (Fig. 4). The importance of L-asparagine in exocrine pancreatic proteins can be appreciated from an inspection of the primary structures of several digestive enzymes produced by this organ, Trypsin and chymotrypsin each contain 14 L-asparaginyl residues; pancreatic ribonuclease contains 10 L-asparaginyl residues. Moreover, Putnam and Neurath (21) reported that the NH&erminal amino acid of carboxypeptidase A is L-asparagine. Finally, in the activation of chymotrypsin from the inactive proenzyme, chymotrypsinogen, an L-threonine+asparagine dipeptide is released (29). Attempts to link L-asparagine synthetase with the endocrine pancreas were fruitless. L-Asparagine synthetase activity in the islets of Langerhans of the muuse was low (approximately 0.26% of the total activity found in the pancreas), and treatment of mice with streptozotocin, a potent P-cell cytotoxic agent (22), did not alter Lasparagine synthetase levels in the pancreata of experimental animals although diabetes was produced by this treatment. Moreover, mice maintained on a glucose-enriched diet for 2 wk in order to stimulate insulin synthesis l
A
0 TIME
c (minutes1
FIG. 5. Effect of pancreas on concentration of L-asparagine in plasma during course of L-asparagine infusion. A beagle dog was anesthetized with pentobarbital after starvation for 16 h. Then its pancreas was exposed, and femoral artery, femoral vein, and craneo-pancreaticoduodenal vein were cannulated. Samples (2 ml) of blood were withdrawn into heparinized tubes at time periods shown in figure for measurement of L-asparagine. At 35 min (A), 25 pmol of L-asparagine in normal saline were infused per minute into femoral vein at 0.5 ml/ min. At 158 min (B), infusion rate was increased to 1 ml/min (50 pmol/ min). At 248 min (C), infusion was discontinued. l , Concentration of L-asparagine in craneo-pancreaticoduodenal venous blood; 0, concentration of L-asparagine in the femoral arterial blood. Values are means of duplicate determinations. At no time did individual values vary from means by more than 5%
that pancreas vigorously takes up L-asparagine from the circulation du ring L-asparagine infusion; that is to say, there is a substantial positive A-V difference of L-asparagine across the pancreas at all time periods examined during the infusion of the amide. This phenomenon also was seen in two 0 ther identical experiments. At the termination of the infusion, L-asparagine app arently leaves the pancreas at a higher rate than it enters it (i.e., a negative A-V difference is observed). We next examined the effect of pancreas on circulating levels of L-asparagine when the concentration of the amino acid is significantly below normal limits. In order to test this hypothesis, 6 IU of agouti L-asparaginase was infused into the femoral vein of the anesthetized dog. This concentration of enzyme was sufficient to deplete L-asparagine from the blood almost totally (Fig. 6). Termination of amidohydrolysis was accomplished by infusion of 10 ml of serum of a rabbit immunized to agouti L-asparaginase. It can be seen (Fig. 6) that L-asparagine in the serum was restored at the same rate in the craneopancreaticoduodenal venous blood as in the arterial supply; that is, no significant A-V difference of L-asparagine across the pancreas could be detected. DISCUSSION
Pancreas has long been considered to be an organ primarily involved in the synthesis of enzymes responsible for digestion and in the production of insulin and glucagon for the regulation of the concentration of glu-
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MILMAN,
60
COONEY,
AND
YOUNG
FIG. 6. Effect of pancreas on concentration of L-asparagine in plasma after infusion of L-asparaginase. A beagle dog was starved for 16 h and then anesthetized with pentobarbital. Its pancreas was exposed, and femoral artery, femoral vein, and craneo-pancreaticoduodenal vein were cannulated. Samples (2 ml) of blood were withdrawn into heparinized tubes at time periods indicated in figure for measurements of L-asparagine and/ or L-asparaginase. At 36 min (A), I ml (6 IU) of agouti L-asparaginase was infused into femoral vein. l , concentration of L-asparagine in craneo-pancreaticoduodenal venous blood; 0, concentration of L-asparagine in femoral arterial blood; A, L-asparaginase activity in femoral arterial blood. Values are means of duplicate determinations. At no time did individual values vary from means by more than 5%.
t 100 E3
TIME
(minutes)
and release did not have elevated L-asparagine synthetase activity in their pancreata at the termination of the study when compared to that measured in the pancreata of control mice (Table 1). It is possible, therefore, that the hypoinsulinemia that occurs in some patients after treatment with L-asparaginase may be due to the extremely low levels of L-asparagine synthetase in the islets of Langerhans. The possibility that L-asparagine is synthesized in the exocrine pancreas and subsequently is transported to the p-cells for insulin synthesis has not been investigated. Such a possibility is made likely by the work of MalaisseLagae and co-workers (18). These investigators observed that the exocrine tissue has almost no direct arterial supply; furthermore, the exocrine and endocrine cells may exert a mutual and direct control of one another’s secretory activity. Another feature of pancreatic organization is the presence around the islets of acini, which are composed of large secretory cells packed with zymogen granules. The same investigators indicate that such an anatomical segregation coincides with a functional heterogeneity of the exocrine tissue. It can be concluded, therefore, that the activity of Lasparagine synthetase is primarily associated with the exocrine pancreas and is modulated by factors such as the age of the animal, but not by gender, diet, or the
presence of tumor under the conditions examined. Moreover, the function of the enzyme is to supply L-asparagine for the synthesis of important pancreatic proteins. Furthermore, whereas the pancreas appears to be able to reduce the concentration of L-asparagine in plasma during an increased amino acid load, it is unable to add Lasparagine to the circulation when the concentration of the amide is below normal levels. Further studies attempting to correlate the activity of L-asparagine synthetase with pancreatic function are presently being investigated. The authors wish to thank Dr. H. Olson and Mrs. V. King for their assistance in the performance of the partial pancreatectomy in mice, and Mason Research Institute, Worcester, MA for conducting the pancreatic duct ligation experiments in beagle dogs. The excellent typing of the manuscript by Ms. Joyce Winters is also greatly appreciated. Present address of H. A. Milman: Toxicology Branch, Carcinogenesis Testing Program, National Cancer Institute, Bethesda, MD 20205. Present address of D. M. Young: Dept. of Veterinary Science, Veterinary Research Laboratory, Montana State Univ., Bozeman, MT 59715. This work is taken in part from a dissertation by H. A. Milman that was presented to the Dept. of Pharmacology, Graduate School of Arts and Sciences, George Washington Univ., Washington, DC, in partial fulfillment of the requirements for the PhD. Send reprint requests to: Dr. Harry A. Milman, National Cancer Institute, 7910 Woodmont Avenue, Room 3C25, Bethesda, MD 20205. Received
30 June
1978; accepted
in final
form
9 February
1979.
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PANCREATIC
L-ASPARAGINE
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SHAW, M. T., C, C. BARNES, F. J. F. MADDEN, AND K. D. BAGSHAW. L-Asparaginase and pancreatitis. Lancet 11: 721, 1970. SHWKUYA, R., AND G, W. SCHWERT. Glutamic acid decarboxylase. J. Biol, Chem, 235: 1649-1652, 1960. SONG, H., N. W. TIETZ, AND C. TAN. Usefulness of serum lipase, esterase, and amylase estimation in the diagnosis of pancreatitisa comparison. Chin. Chem. 16: 264-268, 1970. TAN, C. L., S. P, CHIANG, AND K, P. WEE. Acute haemorrhagic pancreatitis following L-asparaginase therapy in acute lymphoblastic leukemic-a case report. Singapore Med. J. 15: 278-282, 1974. TATE, S. S., AND A. MEISTER. Studies on the sulfhydryl groups af L-aspartate+decarboxylase. Biochemistry 7: 3240-3247, 1968. WEETMAN, R. M.,- AND R. L. BAEHNER. Latent onset of clinical pancreatitis in children receiving L-asparaginase therapy. Cancer 34: 780-785, 1974. WHITE, A., P. HANDLER, AND E. L. SMITH. Principles of Biochemistry. New York: McGraw, 1968, p. 252. WOODS, J. S., AND R, E. HANDSCHUMACHER, Hepatic homeostasis of plasma L-asparagine. Am. J. PhysioZ. 221: 1785-1790, 1971. YIP, C, C., AND M. L. MOWLE. Structure and function of insulin: preparation and biological activity of guinea pig des-B-asp3’), desA-asn”-insulin. Can, J. Biochem. 54: 866-871, 1976.
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