- Email: [email protected]
Carbohydrate metabolism of the midgut of the silkworm Bombyx mori
ARCHIVES
OF
BIOCHEMISTRY
AND
Carbohydrate
80,
BIOPHYSICS
174-186
Metabolism of the Midgut Silkworm Bombyx mori Toshio Ito and Yasuhiro
From
(1959)
the Sericultural
Experiment Received
Station, July
of the
Horie
Suginami-h-u,
Tokyo,
Japan
7, 1958
The physiology of the insect gut, especially of the midgut, is of interest since it functions as the principal organ in digestion, absorption, and excretion. Although the role of the midgut in these areas has been clearly established (l-3), little is known of the biochemical processes concerning the intermediary metabolism of the insect’ midgut. In a previous paper (4) the mitochondria isolated from the silkworm midgut have been shown to possess a terminal oxidase system and to be capable of oxidizing tricarboxylic acid (TCA) cycle members. However, no publication exists on the carbohydrate metabolism of the insect midgut. Recent studies on carbohydrate met,abolixm in insects have demonstrated the occurrence of glycolysis (5-13)’ and the pentose cycle (12, 14-17). A review of this subject is available (18). These results show that the carbohydrate metabolism of insects is similar to that of higher animals. In the present paper the results of studies on the carbohydrate metabolism of silkworm midgut are presented, demonstrating glycolysis and the existence of the pentose cycle. MATERIALS
AND
METHODS
Fifth instar larvae of the silkworm, Bombyx mori, were used throughout the experiments. The larvae were opened along the mid-ventral line, and the midgut, freed from Malpighian tubes, tracheae, and fat body, was washed several times in cold water and homogenized in the cold. The homogenates were strained through several layers of gauze. Soluble fractions were obtained by centrifuging the homogenate at 19,000 X g for 20 min. A solution of 0.97; KCI-O.01 M ethylenediaminetetraacetic acid (EDTA), adjusted to pH 7.0, was used in place of water in preparing the homogenates for respiration experiments. This medium was also used for isolating mitochondria. The mitochondria were isolated in a manner similar to that employed in the preceding paper (4). All operations were conduct,ed at O-3’%. In respiration experiments, conventional procedures were followed with the Warburg apparatus. Experimental conditions are described in the results. 1 Also,
manuscript
in preparation
by T. 174
Ito
and
G. Fraenkel.
METABOLISM
OF
SILKWORM
GUT
175
Over-all glycolysis was measured anaerobically in Thunberg tubes, using glucose as the substrate. After 1 hr. incubation of the reaction mixture, aliquots were removed and analyzed for lactic acid by the method of Barker (19). For the determination of glucose, the method of Hagedorn and Jensen (20) was used following deproteinization with Ba(OH)* and ZnSOd . Phosphorylase activity was measured by determining the amount of inorganic phosphate freed from glucose l-phosphate (G-l-P) in the presence of added glycogen. To minimize hydrolysis of G-1-P by nonspecific alkaline phosphatase, which is of high activity in the midgut.2 KF was added at a high concentration to the reaction mixture. Phosphoglucomutase activity was assayed by measuring the rate of disappearance of G-l-P. Hexokinase activity was determined by measuring hexose disappearance after removal of hexose 6-phosphate. Glucose or fructose was used as the substrate. The disappearance of adenosine triphosphate (.4TP) was simultaneously measured. Phosphohexoisomerase activity was assayed by measuring the rateof disappearance of fructose 6-phosphate (F-6-P). a-Glycerophosphate (a-GP) dehydrogenase and lactic dehydrogenase activity was measured spectrophotometrically with the Beckman model DU spectrophotometer. LY-GP dehydrogenase activity was also measured manometrically with the Warburg respirometer and anaerobically by means of the Thunberg tube in the presence of triphenyltetrazolium chloride (TTZ). The experiments on the pentose cycle were conducted by measuring the formation of rihose and sedoheptulose from glucose g-phosphate (G-6-P)) of ribose from 6.phosphogluconate (6-P-G), and of hexose and sedoheptulose from ribose 5-phosphate (R-5-P)) in the presence of triphosphopyridine nucleotide (TPN), respectively. The rate of disappearance of R-5-P was also simultaneously assayed. Inorganic phosphate was determined by the method of Fiske and SubbaRow (21) and fructose hy that of Roe (22). Ribose was assayed by the Dische orcinol method (23) and total hexoses by the anthrone reaction described by Scott and Melvin (24). Sedoheptulose was determined according to the Dische (23) cysteine-H2SOI method [cf. 141. Sedoheptulose was provided by Dr. H. Lipke of the University of Illinois. Other phosphorylated intermediates, diphosphopyridine nucleotide (DPN), and cytochrome c were ohtained from Nutritional Biochemicals Co. TPN was extracted from sheep liver and purified by ion-exchange chromatography according to the prodecure of Horecker and Kornberg (25). Partially purified TPX was also used for pentose cycle experiments with satisfactory results. Reduced diphosphopyridine nucleotide (DPNH) solution was prepared by the procedure of Beisenherz et al. (26). RESULTS
Respiratory f?ubstratesfor Midgut Homogenates In B previous study (4) the silkworm midgut was shown to oxidize TCB cycle members. Before demonstrating the individual enzymes of the glycolytic scheme, an attempt was made to get information on the suh&rates which can be utilized for respiration by midgut homogennt,es. The substrates tested were t’he intermediates in carbohydrate metabolism, a Manuscript
in preparation
by T. Horie.
176
Respiratory
IT0
Substrates for Recombination
AND
HORIE
TABLE
I
Midgut Homogenates and the Effect on Oxidation of Mitochondria and Soluble Fraction
of
Each flask contained 50 pmoles phosphate buffer at pH 7.2; 4 Imoles MgCl! ; 0.1 pmole cytochrome c; 5 Imoles ATP, 100 pg. DPN; 5 rmoles nicotinamide; 15 Imoles substrate; 0.5 ml. of 20yo homogenate made in 0.9% KCI-O.01 M EDTA (Expt. l), or 9.1 mg. soluble fraction and 4.3 mg. mitochondria (Expt. 2). Total volume 2.0 ml.; temperature 30°C.; gas phase, air. &./hr./mg. Substrate
Expt.
1
Qo,
dry weight
T
Homogenate
Expt.
-
Soluble
fraction
-No substrate n-Glucose Glucose l-phosphate Glucose 6-phosphate Ribose 5-phosphate Fructose 6-phosphate Fructose 1,6-diphosphate 3-Phosphoglycerate a-Glycerophosphate Glycerol Acetate Pyruvate Lactate Succinate
6.0 7.9 13.2 15.9 8.4 12.0 9.4 9.4 14.5 7.3 9.9 5.9 5.5 10.9
2 Soluble fraction and mitochondria
0.7 0.3 0.3 0.2 0.3 0.4 0 2.3 0 -
-
13.9 18.8 19.8 14.7 14.6 13.8 13.0 22.7 15.7 -
-
glucose, and succinate, the latter for comparison with previous values (4). As shown in Table I, all the substrates tested, with the exception of pyruvate and lactate, were utilized as respiratory substrates. Further study is necessary on the oxidation of pyruvate since the cofactors for its oxidation may be limiting in this system. G-l-P, G-6-P, F-6-P, and cu-GP were oxidized with respiratory rates greater than those of succinate oxidation. Such high respiratory rates with phosphorylated intermediates were previously observed for the pea aphid (14) and the housefly (27). A locust muscle suspension also oxidized some phosphorylated intermediates (28). The soluble fraction scarcely oxidized the intermediates involved, but someoxidation occurred in the presence of wGP (Table I, Expt. 2). Further addition of washed mitochondria to the soluble fraction resulted in a conspicuous
enhancement
in oxygen
uptake,
indicating
that
the oxygen
uptake is evoked via terminal oxidases associated with mitochondria (4). Enhancement
of respiration
by the recombination
has also been shown in the housefly (27).
of these two
fractions,
METhBOLISM
OF
SILKWORM
177
GU1
Over-all Glycolysis The homogenat’e wns tested for its glycolytic xtivit~y. The results are present,ed in Table II. With t’he complete system, and conditions known as opt,imal for mammalian preparations, glucose disappeared w&h t,he product,ion of lactic acid. The glucose which disappeared under these condit,ions ww usually slightly in ewess of the la& acid (IA) produced. On withdrawal of fluoride, the Me of glucose disnppeur:mce and lact#ic acid formation scarcely enhanced. &., was approsimut,ely 12 with the complete syst,em, even in the absence of F-, which is higher than the value given by the cockroach muscle (8). In t,he silkworm midgut, the rat,io of lwtic acid formed to glucose utilized is approsimat,ely ten times that obtained wit’h adult male housefly homogenates (11). In the absence of F-, hesosr diphosphatc (HDI’), and pyruvatr, the rate of glucose disappearance dropped slightly, while la&* acid production dropped ronspicuously. However, tbe level of la&c acid production was not reduced to zero. This is different from the housefly, where no lactic acid was produced in the absence of those t’hree components (11). The amounts of lactate formed from glucose (Table II) or from various intermediates (see t,est below) are very large, which seems to be due at least t,o the fart, that Barker’s method for lact,ate is only moderately specific. T,4BLE Lactic
Acid
Fornlation
and
Glucose
II IJtiEization
by Midgut
Homogenate
Each Thunberg tube contained 100 pmoles phosphate buffer at pH 7.2; 14 pmoles MgCIZ ; 8 pmoles HDP; 10 pmoles pyruvate; 30 pmoles KF; 31.4 mg. homogenate (dry weight). Side arm contained 12 pmoles ATP; 0.4 mg. DPN; 50 &moles nicotinamide; 20 pmoles n-glucose. Total volume 4.4 ml.; temperature 37°C.; gas phase, Nz . Incubation period, 1 hr.
System pmoles/mg.
Homogenate Complete Minus KF Minus HDP, and KF Soluble fractionb Complete Rlitochondriac Complete
pyruvate,
a Control value at zero components. * Dry weight 24.7 mg. c Dry weight 28.6 mg.
time
being
dry wt.
pmoles/mg.
dry wt.
0.539 0.516 0.110
0.356 0.317 0.260
12.1 11.6 2.5
-
0.470
-
-
0.043
-
subtracted.
Control
tube
contained
all
the
178
ITO
AND
HORIE
Table II also shows that the rate of glucose utilization by the soluble fraction is ten times that of the mitochondria. This is in accord with the findings on enzyme localization in the housefly (11). Phosphorylase The glycogen content of the silkworm midgut has been shown to increase several fold shortly after glucose or fructose ingestion (29), which is suggestive of the presence of phosphorylase. The enzymic evidence for the presence of phosphorylase in the midgut is shown in Table III, by phosphorus liberation from G-l-P in the reaction mixture containing glycogen. In the absence of glycogen a considerable amount of phosphorus was still liberated. Nonspecific alkaline phosphatase might have accounted for the removal of G-l-P. The possibility also exists that the action of the other enzymes such as a specific hexose-1-phosphatase may be involved in such a phenomenon, since this enzyme has been demonstrated in silkworm blood (30). Such a high rate of phosphorus liberation does not occur in the silkworm fat body (31). Phosphoglucomutase A steady disappearance of G-l-P, measured as hydrolyzable P in N HCl at 100°C. for 10 min., was recognized (Table IV). During the course TABLE III Phosphorylase in Midgut Homogenate The incubation mixtures contained 25 pmoles Tris buffer at pH 7.2; 30 Imoles cysteine; 600 rmoles KF; 25 pmoles G-l-P; 22.0 mg. homogenate (dry weight). Total volume 4.0 ml.; temperature 37°C. Phosphorus
liberated,
pmoles
0 min.
20 min.
45 min.
80 min.
0 0
2.19 1.10
5.00 2.01
7.16 3.76
With glycogen (2%) Without glycogen
TABLE Phosphoglucomutase
IV
in Midgut
Homogenates
The incubation mixtures contained 25 rmoles Tris buffer at pH 7.2; 10 Fmoles MgClz ; 120 kmoles cysteine; 50 rmoles G-1-P; 5.25 mg. homogenate (dry weight). Total volume 4.2 ml.; temperature 37°C. G-l-P
Without KF With KF (40 rmoles)
decreased,
pm&s
0 min.
20 min.
40 min.
60 min.
0 0
4.79 1.47
10.65 2.20
15.55 4.79
METABOLISM
OF
SILKWORM
179
GUT
of the reaction, the amount of phosphorus compounds stable to hydrolysis in N HCl was increased, suggesting the formation of G-6-P from G-l-P. In the presence of KF, which is known to inhibit phosphoglucomutase (32)) the rate of G-l-P disappearance was decreased (Table IV). Phosphoglucomutsse activit,y has also been reported in the silkworm fat body (31). Hexokinase In insects, the presence of hexokinase activity has been demonstrated (11, 33). The occurrence of this enzyme was also reported in the locust (28). The previous report (29) showing an increase in glycogen after hexose ingestion by silkworm midgut suggest’s the presence of hexokinase in this tissue. Table V shows the ability of midgut homogenate to phosphorylate both glucose and fructose in the presence of ATP. In the absence of ATP, no utilization of hexoses occurred. The decrease in labile P, probable ATP, was almost proportional to the disappearance of hexoses. Kerly and Lenback (33) have considered that locust muscle contains a single, nonspecific hexokinase. Phosphohexoisomerase The rate of disappearance of F-6-P is shown in Table VI. The reaction proceeded quickly and reached equilibrium in 10 min. The equilibrium mixture contained F-6-P at a greater level than that shown in the housefly TABLE Hexokinase
V
in Midgut
Homogenates
The incubation mixtures contained 25 pmoles Tris buffer at pH 7.2; 20 pmoles MgClz ; 15pmoles ATP; either 17.4 mg. homogenate, 300rmoles BF, 4~moles glucose, total volume 3.1 ml.; or 33.3 mg. homogenate, 200 pmoles KF, 6 pmoles fructose, total volume 3.4 ml. Temperature 30°C. Hexose
Glucose Fructose
0 min.
10 min.
0 0
1.13
TABLE The incubation mixtures F-6-P; 41.0 mg. homogenate
0
2.23
20 min.
40 min.
60 min.
1.57
2.18 3.19
3.66
2.79
VI
in Midgut
Homogenates
contained 75 pmoles Tris buffer at pH 7.2; 24 kmoles (dry weight). Total volume 5.4 ml.; temperature 3O’C. F-6-P
2 min.
pm&s
2.00
Phosphohexoisomerase
0 min.
decreased,
decreased,
,umoles
4 min.
6 min.
10 min.
16 min.
30 min.
4.06
5.50
8.23
9.35
9.72
180
IT0
ASD
HORIE
(15). The formation of fructose in the presence of G-6-P was determined also with silkworm midgut homogenate, and t’he results confirmed t’he ahi1it.y of t.he homogenate to isomerize G-6-P to F-6-P. In the silkworm the presence of phosphohexoisomerase has been reported in the blood (31). A conversion of F-6-P to G-6-P is also known in t’he pea aphid (14). Formation
of Lactic Acid from Phosphorylated
Intermediutes
The ability of the homogenate to form lactic acid from some phosphorylated intermediates was measured. An increase in lactic acid formation occurred when R-5-P, HDP, or 3-phosphoglycerabe (3-PG) was used as substrate, but only very slightly with cr-GE’. The best yield was obtained with HDP, probably because DPNH formed in the course of HDP metnbolism acted in turn as a cofactor of lactic dehydrogenase. Pyruvate was as effective as HDP. Addition of 3-PG to HDP resulted in an increase in lactic acid production (from 0.110 to 0.150 pmole), while a maximum production was obtained when pyruvate, HDP, and 3-PG were present simultaneously (0.206 pmole). Lactic Dehydrogenase The fact that lactic acid is formed from glucose or glycolytic intermediates suggests the presence of lactic dehydrogenase. Figure 1 shows direct evidence of this enzyme activity by means of spectrophotometric
MINUTES FIG. 1. Oxidation of DPNH by midgut homogenate Each cuvette contained 200 rmoles phosphate buffer 10 rmoles pyruvate; 2.4 mg. homogenate (dry weight).
in the presence of pyruvate at pH 7.4; 0.12 rmole DPNH; Total volume 3.0 ml.
METABOLISM
OF
SILKWORM
181
GUT
measurements at 340 mp. The enzymic activit’y was dependent on the concentration of t.he substrate. Spectrophotometric evidence for this enzyme has been obtained with several insects (11, 13).i P’cntose Cycle The occurrence of the pentose cycle has been demonstrnt,ed in the pea aphid (15), the honey bee (l(i), t’he blowfly (12), and the housefly (15, 177, but no data are available on this cycle in insect midgut. A preliminary experiment showed the presence of this cycle in gut homogenates by the formation of sedoheptulose (increase in the optical densit,y at 510 rnp) and the simultaneous disappearance of glucose (decrease in the optical density at, 410 nip) in the reaction system containing G-6-P and TPN. The time course of formation of ribose and sedoheptulose was subsequently followed with the same reaction mixture (Table VII). The rate of ribose formation surpassed that of sedoheptulose formation. A conversion of 6-P-G to ribose by midgut homogenate was also observed, evidencing the presence of 6-P-G dehydrogenase nctivit,y (Table VII). Further evidence for pentose cycle activity was obtained by determining the formation of sedoheptulose and hexoses, when R-5-P was used as the substrate. Figure 2 shows the rapid formation of sedoheptulose and of hexoses with the swift disappearance of ribose. The recovery of carbon in this case was more than 100%. The formation of hexoses occurred as quickly as that of sedoheptulose in silkworm midgut, while in the pea aphid the accumulation of hexoses has been reported to be gradual (14). a-Glycerophosphate
Dehydrogenase
The presence of cr-GP dehydrogenase has been demonstrated in several species of insects (11, 13, 35, 36). Direct evidence for the presence of this TABLE Formation
of Ribose
and Sedoheptulose 6-Phosphogluconate
VII
from Glucose 6-Phosphate, by Midgut Homogenates
The incubation mixtures contained 100 pmoles Tris ; either 10 pmoles G-6-P, 33.6 mg. homogenate; MgClz homogenate. Total volume 10 ml.; temperature 30°C. Compound
buffer at pH 7.2; 100 rmoles or 10 pmoles 6-P-G, 35.8 mg.
0 min
5 min
15 min.
30 min.
I-
B.
From G-6-P Ribose Sedoheptulose From 6-P-G Ribose
from
i j
A.
and of Ribose
0 0 i
60 min.
120 min.
~____
O
0.245 -
0.30 0.197
0.416 0.25
:
0.55 0.372
-
182
IT0
AND
HORIE
20
40
60
MINUTES FIG. 2. Formation of hexoses and sedoheptulose from R-5-P by midgut homogen ate. The incubation mixtures contained 100 pmoles Tris buffer at pH 7.2; 100 pmoles MgClz ; 10 pmoles R-5-P; 32.2 mg. homogenate (dry weight). Total volume 10 ml.; temperature 30°C.
2o.oo, 0
2 MINUTES
3
4
FIG. 3. Reduction of DPN by midgut soluble fraction in the presence of wGP. Each cuvette contained 40 pmoles phosphate buffer at pH 8.2; 0.3 pmole DPN; 80 &moles wGP; 2.4 mg. soluble fraction (dry weight). Total volume 3.0 ml.
METABOLISM
OF
SILKWORM
183
GUT
enzyme in the silkworm midgut was obtained by measuring the rate of DPN reduction. As shown in Fig. 3, the soluble fraction of the midgut is able to reduce DPN in the presence of a-GP. However, the total change in optical density is rather low. Recent studies by Zehe and McShan (13) have shown the presence of particulate (r-G!? dehydrogenase in insect muscle which is not DPN specific. The mitochondria isolated from silkworm midgut were, therefore, tested in this respect. The result showed that, the addition of DPN resulted in a very slight increase in oxygen uptake (Table VIII). This seems to confirm the findings of Zebe and McShan. Antimycin A greatly reduced the rate of oxygen uptake. Midgut mitochondria also possessed DPN-linked wGP dehydrogenase, which was demon&rated in an anaerobic experiment by the use of TTZ (Table IX).
TABLE Oxidation
VIII
oj or-Glycerophosphate
by Midgut
Mitochondria
Each flask contained 100 I.tmoles phosphate buffer at pH 7.2; 0.1 pmole cytochrome c; 4 pmoles ATP; 100 pg. DPN; 10 pmoles Nmoles oc-glycerophosphate; 10.5 mg. mitochondria (dry weight). ml.; temperature 30°C.; gas phase, air. System
4 pmoles MgClz ; nicotinamide; 20 Total volume 2.0
Qok” pl.,‘hr./mg.
Complete Minus DPN Antimycin A (3 pg.) 0 Endogenous
oxygen
uptake
added was subtracted.
TABLE Reduction
dry wezgkt
47.3 43.5 19.0
IS
of Tetrazolium (TTZ) Salt by Midgut in the Presence of wGlycerophosphate
Mitochondria
Each Thunberg tube contained 200 pmoles phosphate buffer at pH 7.3; 10.0 mg. mitochondria (dry weight). Side arm contained 2 pmoles TTZ; 50 pmoles a-glycerophosphate; where necessary, 30 fig. DPN. Total volume 5.3 ml.; temperature 30°C. After 1 hr. incubation, reaction was stopped by adding 2 ml. of lOye trichloroacetic acid, and triphenylformazan was extracted with ethyl acetate. System
Optical
density
480 In/L
Complete Minus DPN Minus DPN
and LU-GP
0.234 0.125 0.055
184
IT0
AND
HORIE
DISCUSSION
From the present study, a glycolytic pathway was shown to operate in silkworm midgut tissues. Calculating the values in Table II, approximately 1.5 moles Iact,ic acid was formed with the disappearance of 1 mole glucose under the complete system. However, in the housefly the amount of lactic acid produced reaches barely one-tenth that of glucose phosphorylated, on the basis of the same weight of tdssue. As mentioned above, the QLA value is very high in the silkworm midgut, as compared with that in cockroach muscle. Thus the conclusion may be drawn of a very act’ive glycolysis in the midgut. The glycolytic pathway in the silkworm midgut seems t’o resemble the analogous system established in mammals, as shown by the accumulation of lactic acid from intermediates in the Embden-Meyerhof system and the demonstration of glycolytic enzymes. Though the presence of lactic dehydrogenase has been demonstrated in insects, it was recently pointed out that lactic dehydrogenase activity is relatively low in the muscles, and that insect flight muscles do not possess the typical glycolytic system (13). The dependence of insect cr-GP dehydrogenase on DPN has been confirmed (11, 13, 35, 36), and the occurrence of two types of cr-GP dehydrogenase has been reported in insects (13); one is contained in a soluble fraction and requires DPN, the other is associated with the particulates and is not DPN specific. LU-GP dehydrogenuse contained in the soluble fraction of the midgut is apparently DPN linked (Fig. 3), whereas that in the mitochondria seems to be only partly DPN specific (Tables VIII and IX). wGP was also used by the homogenate at a high rate as a respiratory substrate (Table I). Since LU-GP dehydrogenase in insect muscles is believed to supply large amounts of energy without accumulating intermediates (13), a-GP dehydrogenase of the midgut is considered to serve directly in respiration, either via the DPNH oxidation system or cytochrome system just as in the case of succinic dehydrogenase. There seems to be a possibility that respiration insensitive to antimycin A might occur via DPNH oxidation (Table VIII), since midgut mitochondria have been shown to oxidize DPNH (4). The present study also indicates that the pent,ose cycle occurs in the silkworm midgut. Whether this cycle might function as a major energy source has not been determined. It is hardly possible to evaluate quantitatively the extent, of the participation of this cycle in the carbohydrate metabolism of the midgut. The results only show that the rate of R-5-P oxidation is lower than that of G-l-P, G-6-P, F-6-P, or LU-GP. The question whether the hexose monophosphate pathway in the midgut serves to synthesize essential metabolites such as amino acid (37) or ket,o acid (38) must be investigated in the future.
METABOLISM
OF
SILKWORM
GUT
185
In insects as in higher forms, most glycolytic enzymes have been shown not, to be associated with the mitochondrin, and the activities of hexose monophosphate shunt are located in t’he soluble fraction. Similarly, t,hese activities in the silkworm midgut. do not seem t’o be associated with the mitochondria, with the exception of c&P dehydrogennse. Association of the glyrolyt,ic enzymes with the mitochondrin hss been reported in the case of wGP dehydrogenase (13,33.5,36) and triosephosphate isomerase (II). The situation of t’he midgut in silkworm physiology seems t,o be cornplicated, since this tissue not only fun&ions in digestion proper, but also sholvs very high artivit,y in carbohydrate (4) and phosphorus (39) metubolism. It, is thus concluded t’hat the midgut is capable of metaholizing carbohydrates as an energy source for it’s own respiration and for synthesis of essential metubolit,es. Such a high metabolic activity seems to account for the muscular as well as the srcret’ory nature of t,his organ. On t’he other hand, the midgut is also capable of t,rnnsferring metabolites through its wall into the blood wit,hout thorough breakdown, as evidenced hy t,hr fact, that hlood glucose increases markedly several hours after glucose ingestion (39). Silkworm blood contains G-6-P (40, 11) and phosphohexoisomerase, but neither phosphoglucomutase nor phosphorylase (30). Thus a functional differentiation exists between the midgut and the blood. It is desirable to st’udy the relation of these two organs in the met,abolism of carbohydrates in insects in the fut’ure. SUMMARY
1. Intermediates of the glycolytic pathway were satisfactorily utilized as respiratory subst#rat’es for midgut homogenates of the silkworm. No oxidation occurred by the soluhle fraction alone. 2. A glycolyt,ic pathway was shown to operat’e, since glucose and phosphorylated intermediat’es were used for lactic acid formation. 3. Phosphorylnse, phosphoglucomutase, hexokinase, phosphohexoisomerase, lactic dehydrogenase, and cY-glycerophosphate dehydrogennse activities were determined. 4. The data showed t’hat possibly three pathways exist for the oxidation of CX-GP: DPX-specific dehydrogenase in the soluble fraction and dehydrogenases associat#ed with the mitochondria; one of them is DPr\J specific, the other nonspecific. 5. The presence of the pentose cycle was recognized in t,he midgut. REFEREKC’ES 1. DAY, M. F., ANI) WATERHOUSE, D. F., ed.). pp. 311-30. John Wiley & Sons, WATERHOUSE, D. F., Ann. Rev. Enlontol. WATERHOUSE, 1~. F., ANU DAY, &I. F., in pp. 331-49. John Wiley R- Sons. Inc.,
zn “Insect Physiology” (K. I>. Roeder, Inc., Xew York, 1953. 2, 1 (1957). “Insect Physiology” (K. D. Roeder, ed.), New York, 1953.
186 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
26.
27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37.
38. 39. 40. 41.
IT0
AND
HORIE
ITO, T., HORIE, Y., AND ISHIEAWA, S., J. Insect PhysioZ. 2 (1958). In press. GILMOUR, D., Biol. BUZZ. 79, 297 (1940). GIL~MOUR, D., Biol. Bull. 80, 45 (1941). GILMOUR, D., J. Cellular Comp. Physiol. 18, 93 (1941). BARRON, E. S. G., AND TAHMISIAN, T. N., J. Cellular Comp. Physiol. 33,57 (1948). HUMPHREY, G. F., J. Cellular Comp. Physiol. 34, 323 (1949). HUMPHREY, G. F., AND SIGGINS, L., Australian J. ExptZ. BioZ. Med. Sci. 27, 353 (1949). CHEFURKA, W., Enzymologia 17, 73 (1954). MCGINNIS, A. J., CHELDBLIN, V. H., AND NEWBURGH, R. W., Arch. Biochem. Biophys. 63, 427 (1956). ZEBE, E. C., AND MCSHAN, W. H., J. Gen. Physiol. 40,779 (1957). NEWBURGH, R. W., AND CHELDELIN, V. H., J. BioZ. Chem. 214, 37 (1955). CHEFUREA, W., Biochim. et Biophys. Acta 17, 295 (1955). HOSKINS, D. D., CHELDELIN, V. H., AND NEWBURGH, R. W., J. Gen. Physiol. 39, 705 (1956). CHEFURBA, W., Enzymologia 18, 209 (1957). ROCKSTEIN, M., Ann. Rev. Entomol. 2, 19 (1957). BARKER, S. B., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. III, p. 241. Academic Press, New York, 1957. HAGEDORN, H. C., AND JENSEN, B. N., Biochem. 2. 136, 46 (1923). FISKE, C. H., AND SUBBAROW, Y., J. BioZ. Chem. 66, 375 (1925). ROE, J. H., J. BioZ. Chem., 107, 15 (1934). DISCHE, Z., J. BioZ. Chem., 204, 983 (1953). SCOTT, T. A., AND MELVIN, E. H., Anal. Chem. 26, 1656 (1953). HORECKER, B. L., AND KORNBERG, A., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. III, p. 879. Academic Press, New York, 1957. BEISENHERZ, G., BUTCHER, T., AND GARBADE, K.-H., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. I, p. 391. Academic Press, New York, 1955. SACKTOR, B., J. Biophys. Biochem. Cytol. 1, 29 (1955). REES, K. R., Biochem. J. 68, 196 (1954). HORIE, Y., AND TANAKA, M., J. Sericult. Sci. Japan 26,40 (1957). FAULKNER, P., Biochem. J. 60, 590 (1955). SHIGEMATSU, H., J. Sericult. Sci. Japan 26, 115 (1956). NAJJAR, V. A., in “Phosphorus Metabolism” (W. D. McElroy and B. Glass, eds.), Vol. I, p. 500. Johns Hopkins Press, Baltimore, 1951. KERLY, M., AND LEABACK, D. H., Biochem. J. 67, 245 (1957). FAULKNER, I’., Biochem. J. 64, 436 (1956). SACKTOR, B., AND COCHRAN, D. G., Biochim. et Biophys. Acta 26, 649 (1957). SACKTOR, B., AND COCHRAN, D. G., Biochim. et Biophys. Acta 26, 200 (1957). WEISSBACH, A., AND HORECKER, B. L., in “,4 Symposium on Amino Acid Metabolism” (W. D. McElroy and B. Glass, eds.), p. 1048. Johns Hopkins Press, Baltimore, 1955. TOLBERT, N. E., AND COHAN, M. S., J. BioZ. Chem. 204, 649 (1953). ITO, T., HORIE, Y., AND TANAKA, M., Proc. 10th Intern. Congr. Entomol. 2, 283 (1958). WYATT, G. R., LOUGHHEED, T. C., AND WYATT, S. S., J. Gen. Physiol. 39, 853 (1956). WYATT, G. R., AND KALF, G. F., J. Gen. Physiol. 40,833 (1957).
OF
BIOCHEMISTRY
AND
Carbohydrate
80,
BIOPHYSICS
174-186
Metabolism of the Midgut Silkworm Bombyx mori Toshio Ito and Yasuhiro
From
(1959)
the Sericultural
Experiment Received
Station, July
of the
Horie
Suginami-h-u,
Tokyo,
Japan
7, 1958
The physiology of the insect gut, especially of the midgut, is of interest since it functions as the principal organ in digestion, absorption, and excretion. Although the role of the midgut in these areas has been clearly established (l-3), little is known of the biochemical processes concerning the intermediary metabolism of the insect’ midgut. In a previous paper (4) the mitochondria isolated from the silkworm midgut have been shown to possess a terminal oxidase system and to be capable of oxidizing tricarboxylic acid (TCA) cycle members. However, no publication exists on the carbohydrate metabolism of the insect midgut. Recent studies on carbohydrate met,abolixm in insects have demonstrated the occurrence of glycolysis (5-13)’ and the pentose cycle (12, 14-17). A review of this subject is available (18). These results show that the carbohydrate metabolism of insects is similar to that of higher animals. In the present paper the results of studies on the carbohydrate metabolism of silkworm midgut are presented, demonstrating glycolysis and the existence of the pentose cycle. MATERIALS
AND
METHODS
Fifth instar larvae of the silkworm, Bombyx mori, were used throughout the experiments. The larvae were opened along the mid-ventral line, and the midgut, freed from Malpighian tubes, tracheae, and fat body, was washed several times in cold water and homogenized in the cold. The homogenates were strained through several layers of gauze. Soluble fractions were obtained by centrifuging the homogenate at 19,000 X g for 20 min. A solution of 0.97; KCI-O.01 M ethylenediaminetetraacetic acid (EDTA), adjusted to pH 7.0, was used in place of water in preparing the homogenates for respiration experiments. This medium was also used for isolating mitochondria. The mitochondria were isolated in a manner similar to that employed in the preceding paper (4). All operations were conduct,ed at O-3’%. In respiration experiments, conventional procedures were followed with the Warburg apparatus. Experimental conditions are described in the results. 1 Also,
manuscript
in preparation
by T. 174
Ito
and
G. Fraenkel.
METABOLISM
OF
SILKWORM
GUT
175
Over-all glycolysis was measured anaerobically in Thunberg tubes, using glucose as the substrate. After 1 hr. incubation of the reaction mixture, aliquots were removed and analyzed for lactic acid by the method of Barker (19). For the determination of glucose, the method of Hagedorn and Jensen (20) was used following deproteinization with Ba(OH)* and ZnSOd . Phosphorylase activity was measured by determining the amount of inorganic phosphate freed from glucose l-phosphate (G-l-P) in the presence of added glycogen. To minimize hydrolysis of G-1-P by nonspecific alkaline phosphatase, which is of high activity in the midgut.2 KF was added at a high concentration to the reaction mixture. Phosphoglucomutase activity was assayed by measuring the rate of disappearance of G-l-P. Hexokinase activity was determined by measuring hexose disappearance after removal of hexose 6-phosphate. Glucose or fructose was used as the substrate. The disappearance of adenosine triphosphate (.4TP) was simultaneously measured. Phosphohexoisomerase activity was assayed by measuring the rateof disappearance of fructose 6-phosphate (F-6-P). a-Glycerophosphate (a-GP) dehydrogenase and lactic dehydrogenase activity was measured spectrophotometrically with the Beckman model DU spectrophotometer. LY-GP dehydrogenase activity was also measured manometrically with the Warburg respirometer and anaerobically by means of the Thunberg tube in the presence of triphenyltetrazolium chloride (TTZ). The experiments on the pentose cycle were conducted by measuring the formation of rihose and sedoheptulose from glucose g-phosphate (G-6-P)) of ribose from 6.phosphogluconate (6-P-G), and of hexose and sedoheptulose from ribose 5-phosphate (R-5-P)) in the presence of triphosphopyridine nucleotide (TPN), respectively. The rate of disappearance of R-5-P was also simultaneously assayed. Inorganic phosphate was determined by the method of Fiske and SubbaRow (21) and fructose hy that of Roe (22). Ribose was assayed by the Dische orcinol method (23) and total hexoses by the anthrone reaction described by Scott and Melvin (24). Sedoheptulose was determined according to the Dische (23) cysteine-H2SOI method [cf. 141. Sedoheptulose was provided by Dr. H. Lipke of the University of Illinois. Other phosphorylated intermediates, diphosphopyridine nucleotide (DPN), and cytochrome c were ohtained from Nutritional Biochemicals Co. TPN was extracted from sheep liver and purified by ion-exchange chromatography according to the prodecure of Horecker and Kornberg (25). Partially purified TPX was also used for pentose cycle experiments with satisfactory results. Reduced diphosphopyridine nucleotide (DPNH) solution was prepared by the procedure of Beisenherz et al. (26). RESULTS
Respiratory f?ubstratesfor Midgut Homogenates In B previous study (4) the silkworm midgut was shown to oxidize TCB cycle members. Before demonstrating the individual enzymes of the glycolytic scheme, an attempt was made to get information on the suh&rates which can be utilized for respiration by midgut homogennt,es. The substrates tested were t’he intermediates in carbohydrate metabolism, a Manuscript
in preparation
by T. Horie.
176
Respiratory
IT0
Substrates for Recombination
AND
HORIE
TABLE
I
Midgut Homogenates and the Effect on Oxidation of Mitochondria and Soluble Fraction
of
Each flask contained 50 pmoles phosphate buffer at pH 7.2; 4 Imoles MgCl! ; 0.1 pmole cytochrome c; 5 Imoles ATP, 100 pg. DPN; 5 rmoles nicotinamide; 15 Imoles substrate; 0.5 ml. of 20yo homogenate made in 0.9% KCI-O.01 M EDTA (Expt. l), or 9.1 mg. soluble fraction and 4.3 mg. mitochondria (Expt. 2). Total volume 2.0 ml.; temperature 30°C.; gas phase, air. &./hr./mg. Substrate
Expt.
1
Qo,
dry weight
T
Homogenate
Expt.
-
Soluble
fraction
-No substrate n-Glucose Glucose l-phosphate Glucose 6-phosphate Ribose 5-phosphate Fructose 6-phosphate Fructose 1,6-diphosphate 3-Phosphoglycerate a-Glycerophosphate Glycerol Acetate Pyruvate Lactate Succinate
6.0 7.9 13.2 15.9 8.4 12.0 9.4 9.4 14.5 7.3 9.9 5.9 5.5 10.9
2 Soluble fraction and mitochondria
0.7 0.3 0.3 0.2 0.3 0.4 0 2.3 0 -
-
13.9 18.8 19.8 14.7 14.6 13.8 13.0 22.7 15.7 -
-
glucose, and succinate, the latter for comparison with previous values (4). As shown in Table I, all the substrates tested, with the exception of pyruvate and lactate, were utilized as respiratory substrates. Further study is necessary on the oxidation of pyruvate since the cofactors for its oxidation may be limiting in this system. G-l-P, G-6-P, F-6-P, and cu-GP were oxidized with respiratory rates greater than those of succinate oxidation. Such high respiratory rates with phosphorylated intermediates were previously observed for the pea aphid (14) and the housefly (27). A locust muscle suspension also oxidized some phosphorylated intermediates (28). The soluble fraction scarcely oxidized the intermediates involved, but someoxidation occurred in the presence of wGP (Table I, Expt. 2). Further addition of washed mitochondria to the soluble fraction resulted in a conspicuous
enhancement
in oxygen
uptake,
indicating
that
the oxygen
uptake is evoked via terminal oxidases associated with mitochondria (4). Enhancement
of respiration
by the recombination
has also been shown in the housefly (27).
of these two
fractions,
METhBOLISM
OF
SILKWORM
177
GU1
Over-all Glycolysis The homogenat’e wns tested for its glycolytic xtivit~y. The results are present,ed in Table II. With t’he complete system, and conditions known as opt,imal for mammalian preparations, glucose disappeared w&h t,he product,ion of lactic acid. The glucose which disappeared under these condit,ions ww usually slightly in ewess of the la& acid (IA) produced. On withdrawal of fluoride, the Me of glucose disnppeur:mce and lact#ic acid formation scarcely enhanced. &., was approsimut,ely 12 with the complete syst,em, even in the absence of F-, which is higher than the value given by the cockroach muscle (8). In t,he silkworm midgut, the rat,io of lwtic acid formed to glucose utilized is approsimat,ely ten times that obtained wit’h adult male housefly homogenates (11). In the absence of F-, hesosr diphosphatc (HDI’), and pyruvatr, the rate of glucose disappearance dropped slightly, while la&* acid production dropped ronspicuously. However, tbe level of la&c acid production was not reduced to zero. This is different from the housefly, where no lactic acid was produced in the absence of those t’hree components (11). The amounts of lactate formed from glucose (Table II) or from various intermediates (see t,est below) are very large, which seems to be due at least t,o the fart, that Barker’s method for lact,ate is only moderately specific. T,4BLE Lactic
Acid
Fornlation
and
Glucose
II IJtiEization
by Midgut
Homogenate
Each Thunberg tube contained 100 pmoles phosphate buffer at pH 7.2; 14 pmoles MgCIZ ; 8 pmoles HDP; 10 pmoles pyruvate; 30 pmoles KF; 31.4 mg. homogenate (dry weight). Side arm contained 12 pmoles ATP; 0.4 mg. DPN; 50 &moles nicotinamide; 20 pmoles n-glucose. Total volume 4.4 ml.; temperature 37°C.; gas phase, Nz . Incubation period, 1 hr.
System pmoles/mg.
Homogenate Complete Minus KF Minus HDP, and KF Soluble fractionb Complete Rlitochondriac Complete
pyruvate,
a Control value at zero components. * Dry weight 24.7 mg. c Dry weight 28.6 mg.
time
being
dry wt.
pmoles/mg.
dry wt.
0.539 0.516 0.110
0.356 0.317 0.260
12.1 11.6 2.5
-
0.470
-
-
0.043
-
subtracted.
Control
tube
contained
all
the
178
ITO
AND
HORIE
Table II also shows that the rate of glucose utilization by the soluble fraction is ten times that of the mitochondria. This is in accord with the findings on enzyme localization in the housefly (11). Phosphorylase The glycogen content of the silkworm midgut has been shown to increase several fold shortly after glucose or fructose ingestion (29), which is suggestive of the presence of phosphorylase. The enzymic evidence for the presence of phosphorylase in the midgut is shown in Table III, by phosphorus liberation from G-l-P in the reaction mixture containing glycogen. In the absence of glycogen a considerable amount of phosphorus was still liberated. Nonspecific alkaline phosphatase might have accounted for the removal of G-l-P. The possibility also exists that the action of the other enzymes such as a specific hexose-1-phosphatase may be involved in such a phenomenon, since this enzyme has been demonstrated in silkworm blood (30). Such a high rate of phosphorus liberation does not occur in the silkworm fat body (31). Phosphoglucomutase A steady disappearance of G-l-P, measured as hydrolyzable P in N HCl at 100°C. for 10 min., was recognized (Table IV). During the course TABLE III Phosphorylase in Midgut Homogenate The incubation mixtures contained 25 pmoles Tris buffer at pH 7.2; 30 Imoles cysteine; 600 rmoles KF; 25 pmoles G-l-P; 22.0 mg. homogenate (dry weight). Total volume 4.0 ml.; temperature 37°C. Phosphorus
liberated,
pmoles
0 min.
20 min.
45 min.
80 min.
0 0
2.19 1.10
5.00 2.01
7.16 3.76
With glycogen (2%) Without glycogen
TABLE Phosphoglucomutase
IV
in Midgut
Homogenates
The incubation mixtures contained 25 rmoles Tris buffer at pH 7.2; 10 Fmoles MgClz ; 120 kmoles cysteine; 50 rmoles G-1-P; 5.25 mg. homogenate (dry weight). Total volume 4.2 ml.; temperature 37°C. G-l-P
Without KF With KF (40 rmoles)
decreased,
pm&s
0 min.
20 min.
40 min.
60 min.
0 0
4.79 1.47
10.65 2.20
15.55 4.79
METABOLISM
OF
SILKWORM
179
GUT
of the reaction, the amount of phosphorus compounds stable to hydrolysis in N HCl was increased, suggesting the formation of G-6-P from G-l-P. In the presence of KF, which is known to inhibit phosphoglucomutase (32)) the rate of G-l-P disappearance was decreased (Table IV). Phosphoglucomutsse activit,y has also been reported in the silkworm fat body (31). Hexokinase In insects, the presence of hexokinase activity has been demonstrated (11, 33). The occurrence of this enzyme was also reported in the locust (28). The previous report (29) showing an increase in glycogen after hexose ingestion by silkworm midgut suggest’s the presence of hexokinase in this tissue. Table V shows the ability of midgut homogenate to phosphorylate both glucose and fructose in the presence of ATP. In the absence of ATP, no utilization of hexoses occurred. The decrease in labile P, probable ATP, was almost proportional to the disappearance of hexoses. Kerly and Lenback (33) have considered that locust muscle contains a single, nonspecific hexokinase. Phosphohexoisomerase The rate of disappearance of F-6-P is shown in Table VI. The reaction proceeded quickly and reached equilibrium in 10 min. The equilibrium mixture contained F-6-P at a greater level than that shown in the housefly TABLE Hexokinase
V
in Midgut
Homogenates
The incubation mixtures contained 25 pmoles Tris buffer at pH 7.2; 20 pmoles MgClz ; 15pmoles ATP; either 17.4 mg. homogenate, 300rmoles BF, 4~moles glucose, total volume 3.1 ml.; or 33.3 mg. homogenate, 200 pmoles KF, 6 pmoles fructose, total volume 3.4 ml. Temperature 30°C. Hexose
Glucose Fructose
0 min.
10 min.
0 0
1.13
TABLE The incubation mixtures F-6-P; 41.0 mg. homogenate
0
2.23
20 min.
40 min.
60 min.
1.57
2.18 3.19
3.66
2.79
VI
in Midgut
Homogenates
contained 75 pmoles Tris buffer at pH 7.2; 24 kmoles (dry weight). Total volume 5.4 ml.; temperature 3O’C. F-6-P
2 min.
pm&s
2.00
Phosphohexoisomerase
0 min.
decreased,
decreased,
,umoles
4 min.
6 min.
10 min.
16 min.
30 min.
4.06
5.50
8.23
9.35
9.72
180
IT0
ASD
HORIE
(15). The formation of fructose in the presence of G-6-P was determined also with silkworm midgut homogenate, and t’he results confirmed t’he ahi1it.y of t.he homogenate to isomerize G-6-P to F-6-P. In the silkworm the presence of phosphohexoisomerase has been reported in the blood (31). A conversion of F-6-P to G-6-P is also known in t’he pea aphid (14). Formation
of Lactic Acid from Phosphorylated
Intermediutes
The ability of the homogenate to form lactic acid from some phosphorylated intermediates was measured. An increase in lactic acid formation occurred when R-5-P, HDP, or 3-phosphoglycerabe (3-PG) was used as substrate, but only very slightly with cr-GE’. The best yield was obtained with HDP, probably because DPNH formed in the course of HDP metnbolism acted in turn as a cofactor of lactic dehydrogenase. Pyruvate was as effective as HDP. Addition of 3-PG to HDP resulted in an increase in lactic acid production (from 0.110 to 0.150 pmole), while a maximum production was obtained when pyruvate, HDP, and 3-PG were present simultaneously (0.206 pmole). Lactic Dehydrogenase The fact that lactic acid is formed from glucose or glycolytic intermediates suggests the presence of lactic dehydrogenase. Figure 1 shows direct evidence of this enzyme activity by means of spectrophotometric
MINUTES FIG. 1. Oxidation of DPNH by midgut homogenate Each cuvette contained 200 rmoles phosphate buffer 10 rmoles pyruvate; 2.4 mg. homogenate (dry weight).
in the presence of pyruvate at pH 7.4; 0.12 rmole DPNH; Total volume 3.0 ml.
METABOLISM
OF
SILKWORM
181
GUT
measurements at 340 mp. The enzymic activit’y was dependent on the concentration of t.he substrate. Spectrophotometric evidence for this enzyme has been obtained with several insects (11, 13).i P’cntose Cycle The occurrence of the pentose cycle has been demonstrnt,ed in the pea aphid (15), the honey bee (l(i), t’he blowfly (12), and the housefly (15, 177, but no data are available on this cycle in insect midgut. A preliminary experiment showed the presence of this cycle in gut homogenates by the formation of sedoheptulose (increase in the optical densit,y at 510 rnp) and the simultaneous disappearance of glucose (decrease in the optical density at, 410 nip) in the reaction system containing G-6-P and TPN. The time course of formation of ribose and sedoheptulose was subsequently followed with the same reaction mixture (Table VII). The rate of ribose formation surpassed that of sedoheptulose formation. A conversion of 6-P-G to ribose by midgut homogenate was also observed, evidencing the presence of 6-P-G dehydrogenase nctivit,y (Table VII). Further evidence for pentose cycle activity was obtained by determining the formation of sedoheptulose and hexoses, when R-5-P was used as the substrate. Figure 2 shows the rapid formation of sedoheptulose and of hexoses with the swift disappearance of ribose. The recovery of carbon in this case was more than 100%. The formation of hexoses occurred as quickly as that of sedoheptulose in silkworm midgut, while in the pea aphid the accumulation of hexoses has been reported to be gradual (14). a-Glycerophosphate
Dehydrogenase
The presence of cr-GP dehydrogenase has been demonstrated in several species of insects (11, 13, 35, 36). Direct evidence for the presence of this TABLE Formation
of Ribose
and Sedoheptulose 6-Phosphogluconate
VII
from Glucose 6-Phosphate, by Midgut Homogenates
The incubation mixtures contained 100 pmoles Tris ; either 10 pmoles G-6-P, 33.6 mg. homogenate; MgClz homogenate. Total volume 10 ml.; temperature 30°C. Compound
buffer at pH 7.2; 100 rmoles or 10 pmoles 6-P-G, 35.8 mg.
0 min
5 min
15 min.
30 min.
I-
B.
From G-6-P Ribose Sedoheptulose From 6-P-G Ribose
from
i j
A.
and of Ribose
0 0 i
60 min.
120 min.
~____
O
0.245 -
0.30 0.197
0.416 0.25
:
0.55 0.372
-
182
IT0
AND
HORIE
20
40
60
MINUTES FIG. 2. Formation of hexoses and sedoheptulose from R-5-P by midgut homogen ate. The incubation mixtures contained 100 pmoles Tris buffer at pH 7.2; 100 pmoles MgClz ; 10 pmoles R-5-P; 32.2 mg. homogenate (dry weight). Total volume 10 ml.; temperature 30°C.
2o.oo, 0
2 MINUTES
3
4
FIG. 3. Reduction of DPN by midgut soluble fraction in the presence of wGP. Each cuvette contained 40 pmoles phosphate buffer at pH 8.2; 0.3 pmole DPN; 80 &moles wGP; 2.4 mg. soluble fraction (dry weight). Total volume 3.0 ml.
METABOLISM
OF
SILKWORM
183
GUT
enzyme in the silkworm midgut was obtained by measuring the rate of DPN reduction. As shown in Fig. 3, the soluble fraction of the midgut is able to reduce DPN in the presence of a-GP. However, the total change in optical density is rather low. Recent studies by Zehe and McShan (13) have shown the presence of particulate (r-G!? dehydrogenase in insect muscle which is not DPN specific. The mitochondria isolated from silkworm midgut were, therefore, tested in this respect. The result showed that, the addition of DPN resulted in a very slight increase in oxygen uptake (Table VIII). This seems to confirm the findings of Zebe and McShan. Antimycin A greatly reduced the rate of oxygen uptake. Midgut mitochondria also possessed DPN-linked wGP dehydrogenase, which was demon&rated in an anaerobic experiment by the use of TTZ (Table IX).
TABLE Oxidation
VIII
oj or-Glycerophosphate
by Midgut
Mitochondria
Each flask contained 100 I.tmoles phosphate buffer at pH 7.2; 0.1 pmole cytochrome c; 4 pmoles ATP; 100 pg. DPN; 10 pmoles Nmoles oc-glycerophosphate; 10.5 mg. mitochondria (dry weight). ml.; temperature 30°C.; gas phase, air. System
4 pmoles MgClz ; nicotinamide; 20 Total volume 2.0
Qok” pl.,‘hr./mg.
Complete Minus DPN Antimycin A (3 pg.) 0 Endogenous
oxygen
uptake
added was subtracted.
TABLE Reduction
dry wezgkt
47.3 43.5 19.0
IS
of Tetrazolium (TTZ) Salt by Midgut in the Presence of wGlycerophosphate
Mitochondria
Each Thunberg tube contained 200 pmoles phosphate buffer at pH 7.3; 10.0 mg. mitochondria (dry weight). Side arm contained 2 pmoles TTZ; 50 pmoles a-glycerophosphate; where necessary, 30 fig. DPN. Total volume 5.3 ml.; temperature 30°C. After 1 hr. incubation, reaction was stopped by adding 2 ml. of lOye trichloroacetic acid, and triphenylformazan was extracted with ethyl acetate. System
Optical
density
480 In/L
Complete Minus DPN Minus DPN
and LU-GP
0.234 0.125 0.055
184
IT0
AND
HORIE
DISCUSSION
From the present study, a glycolytic pathway was shown to operate in silkworm midgut tissues. Calculating the values in Table II, approximately 1.5 moles Iact,ic acid was formed with the disappearance of 1 mole glucose under the complete system. However, in the housefly the amount of lactic acid produced reaches barely one-tenth that of glucose phosphorylated, on the basis of the same weight of tdssue. As mentioned above, the QLA value is very high in the silkworm midgut, as compared with that in cockroach muscle. Thus the conclusion may be drawn of a very act’ive glycolysis in the midgut. The glycolytic pathway in the silkworm midgut seems t’o resemble the analogous system established in mammals, as shown by the accumulation of lactic acid from intermediates in the Embden-Meyerhof system and the demonstration of glycolytic enzymes. Though the presence of lactic dehydrogenase has been demonstrated in insects, it was recently pointed out that lactic dehydrogenase activity is relatively low in the muscles, and that insect flight muscles do not possess the typical glycolytic system (13). The dependence of insect cr-GP dehydrogenase on DPN has been confirmed (11, 13, 35, 36), and the occurrence of two types of cr-GP dehydrogenase has been reported in insects (13); one is contained in a soluble fraction and requires DPN, the other is associated with the particulates and is not DPN specific. LU-GP dehydrogenuse contained in the soluble fraction of the midgut is apparently DPN linked (Fig. 3), whereas that in the mitochondria seems to be only partly DPN specific (Tables VIII and IX). wGP was also used by the homogenate at a high rate as a respiratory substrate (Table I). Since LU-GP dehydrogenase in insect muscles is believed to supply large amounts of energy without accumulating intermediates (13), a-GP dehydrogenase of the midgut is considered to serve directly in respiration, either via the DPNH oxidation system or cytochrome system just as in the case of succinic dehydrogenase. There seems to be a possibility that respiration insensitive to antimycin A might occur via DPNH oxidation (Table VIII), since midgut mitochondria have been shown to oxidize DPNH (4). The present study also indicates that the pent,ose cycle occurs in the silkworm midgut. Whether this cycle might function as a major energy source has not been determined. It is hardly possible to evaluate quantitatively the extent, of the participation of this cycle in the carbohydrate metabolism of the midgut. The results only show that the rate of R-5-P oxidation is lower than that of G-l-P, G-6-P, F-6-P, or LU-GP. The question whether the hexose monophosphate pathway in the midgut serves to synthesize essential metabolites such as amino acid (37) or ket,o acid (38) must be investigated in the future.
METABOLISM
OF
SILKWORM
GUT
185
In insects as in higher forms, most glycolytic enzymes have been shown not, to be associated with the mitochondrin, and the activities of hexose monophosphate shunt are located in t’he soluble fraction. Similarly, t,hese activities in the silkworm midgut. do not seem t’o be associated with the mitochondria, with the exception of c&P dehydrogennse. Association of the glyrolyt,ic enzymes with the mitochondrin hss been reported in the case of wGP dehydrogenase (13,33.5,36) and triosephosphate isomerase (II). The situation of t’he midgut in silkworm physiology seems t,o be cornplicated, since this tissue not only fun&ions in digestion proper, but also sholvs very high artivit,y in carbohydrate (4) and phosphorus (39) metubolism. It, is thus concluded t’hat the midgut is capable of metaholizing carbohydrates as an energy source for it’s own respiration and for synthesis of essential metubolit,es. Such a high metabolic activity seems to account for the muscular as well as the srcret’ory nature of t,his organ. On t’he other hand, the midgut is also capable of t,rnnsferring metabolites through its wall into the blood wit,hout thorough breakdown, as evidenced hy t,hr fact, that hlood glucose increases markedly several hours after glucose ingestion (39). Silkworm blood contains G-6-P (40, 11) and phosphohexoisomerase, but neither phosphoglucomutase nor phosphorylase (30). Thus a functional differentiation exists between the midgut and the blood. It is desirable to st’udy the relation of these two organs in the met,abolism of carbohydrates in insects in the fut’ure. SUMMARY
1. Intermediates of the glycolytic pathway were satisfactorily utilized as respiratory subst#rat’es for midgut homogenates of the silkworm. No oxidation occurred by the soluhle fraction alone. 2. A glycolyt,ic pathway was shown to operat’e, since glucose and phosphorylated intermediat’es were used for lactic acid formation. 3. Phosphorylnse, phosphoglucomutase, hexokinase, phosphohexoisomerase, lactic dehydrogenase, and cY-glycerophosphate dehydrogennse activities were determined. 4. The data showed t’hat possibly three pathways exist for the oxidation of CX-GP: DPX-specific dehydrogenase in the soluble fraction and dehydrogenases associat#ed with the mitochondria; one of them is DPr\J specific, the other nonspecific. 5. The presence of the pentose cycle was recognized in t,he midgut. REFEREKC’ES 1. DAY, M. F., ANI) WATERHOUSE, D. F., ed.). pp. 311-30. John Wiley & Sons, WATERHOUSE, D. F., Ann. Rev. Enlontol. WATERHOUSE, 1~. F., ANU DAY, &I. F., in pp. 331-49. John Wiley R- Sons. Inc.,
zn “Insect Physiology” (K. I>. Roeder, Inc., Xew York, 1953. 2, 1 (1957). “Insect Physiology” (K. D. Roeder, ed.), New York, 1953.
186 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
26.
27. 28. 29. 30. 31. 32. 33. 34. 35. 36. 37.
38. 39. 40. 41.
IT0
AND
HORIE
ITO, T., HORIE, Y., AND ISHIEAWA, S., J. Insect PhysioZ. 2 (1958). In press. GILMOUR, D., Biol. BUZZ. 79, 297 (1940). GIL~MOUR, D., Biol. Bull. 80, 45 (1941). GILMOUR, D., J. Cellular Comp. Physiol. 18, 93 (1941). BARRON, E. S. G., AND TAHMISIAN, T. N., J. Cellular Comp. Physiol. 33,57 (1948). HUMPHREY, G. F., J. Cellular Comp. Physiol. 34, 323 (1949). HUMPHREY, G. F., AND SIGGINS, L., Australian J. ExptZ. BioZ. Med. Sci. 27, 353 (1949). CHEFURKA, W., Enzymologia 17, 73 (1954). MCGINNIS, A. J., CHELDBLIN, V. H., AND NEWBURGH, R. W., Arch. Biochem. Biophys. 63, 427 (1956). ZEBE, E. C., AND MCSHAN, W. H., J. Gen. Physiol. 40,779 (1957). NEWBURGH, R. W., AND CHELDELIN, V. H., J. BioZ. Chem. 214, 37 (1955). CHEFUREA, W., Biochim. et Biophys. Acta 17, 295 (1955). HOSKINS, D. D., CHELDELIN, V. H., AND NEWBURGH, R. W., J. Gen. Physiol. 39, 705 (1956). CHEFURBA, W., Enzymologia 18, 209 (1957). ROCKSTEIN, M., Ann. Rev. Entomol. 2, 19 (1957). BARKER, S. B., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. III, p. 241. Academic Press, New York, 1957. HAGEDORN, H. C., AND JENSEN, B. N., Biochem. 2. 136, 46 (1923). FISKE, C. H., AND SUBBAROW, Y., J. BioZ. Chem. 66, 375 (1925). ROE, J. H., J. BioZ. Chem., 107, 15 (1934). DISCHE, Z., J. BioZ. Chem., 204, 983 (1953). SCOTT, T. A., AND MELVIN, E. H., Anal. Chem. 26, 1656 (1953). HORECKER, B. L., AND KORNBERG, A., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. III, p. 879. Academic Press, New York, 1957. BEISENHERZ, G., BUTCHER, T., AND GARBADE, K.-H., in “Methods in Enzymology” (S. P. Colowick and N. 0. Kaplan, eds.), Vol. I, p. 391. Academic Press, New York, 1955. SACKTOR, B., J. Biophys. Biochem. Cytol. 1, 29 (1955). REES, K. R., Biochem. J. 68, 196 (1954). HORIE, Y., AND TANAKA, M., J. Sericult. Sci. Japan 26,40 (1957). FAULKNER, P., Biochem. J. 60, 590 (1955). SHIGEMATSU, H., J. Sericult. Sci. Japan 26, 115 (1956). NAJJAR, V. A., in “Phosphorus Metabolism” (W. D. McElroy and B. Glass, eds.), Vol. I, p. 500. Johns Hopkins Press, Baltimore, 1951. KERLY, M., AND LEABACK, D. H., Biochem. J. 67, 245 (1957). FAULKNER, I’., Biochem. J. 64, 436 (1956). SACKTOR, B., AND COCHRAN, D. G., Biochim. et Biophys. Acta 26, 649 (1957). SACKTOR, B., AND COCHRAN, D. G., Biochim. et Biophys. Acta 26, 200 (1957). WEISSBACH, A., AND HORECKER, B. L., in “,4 Symposium on Amino Acid Metabolism” (W. D. McElroy and B. Glass, eds.), p. 1048. Johns Hopkins Press, Baltimore, 1955. TOLBERT, N. E., AND COHAN, M. S., J. BioZ. Chem. 204, 649 (1953). ITO, T., HORIE, Y., AND TANAKA, M., Proc. 10th Intern. Congr. Entomol. 2, 283 (1958). WYATT, G. R., LOUGHHEED, T. C., AND WYATT, S. S., J. Gen. Physiol. 39, 853 (1956). WYATT, G. R., AND KALF, G. F., J. Gen. Physiol. 40,833 (1957).