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Oligosaccharidases from Macracanthorhynchus hirudinaceus (Acanthocephala) from swine
Comp. Biochem. Physiol., 1968, Vol. 26, pp. 281 to 289. Pergamon Press. Printed in Great Britain
OLIGOSACCHARIDASES FROM
MACRACANTHORHYNCHUS HIRUDINACEUS ( A C A N T H O C E P H A L A ) F R O M SWINE* T. T. DUNAGAN and T. M. YAU Department of Physiology, Southern Illinois University, Carbondale, Illinois 62901, U.S.A.
(Received 28 December 1967) A b s t r a c t - - 1 . Enzymatic activities of cell-free extracts of Macracanthorhynchus
hirudinaceus indicates that maltose, turanose and trehalose are hydrolyzed but with maltose being broken down four times as fast as any other disaccharide tested. 2. M. hirudinaceus cell-free extracts readily hydrolyze dextrin but not: (1) the polysaccharide, inulin (2) the trisaccharides, raffmose and melizitose (3) disaccharides, sucrose, lactose and melibiose (4) methyl substituted aldohexoses, o~-D-methyl glucopyranoside and C~-D-methylmannoside. 3. One isozyme of maltase was observed using para-nitrophenyl-c~-D glucopyranoside in starch gel electrophoresis. 4. The pH optimum for maltase was 4.5 using 0"2 M Tris malate buffer, but with 0-2 M acetic acid-sodium acetate buffer, the pH optimum was 5"0. The optimum temperature in either case was 50°C. 5. Trehalase had a pH optimum of 5"8 and an optimum temperature of 45°C using 0.2 M phosphate buffer. INTRODUCTION THE UTILIZATION of exogenous carbohydrates by helminths has been most extensively studied for cestodes in which only a very limited n u m b e r of monosaccharides are metabolized to any extent and interestingly enough fructose is not in this group. Disaccharides such as maltose and trehalose have not been hydrolyzed except in Cittotaenia sp. However, Acanthocephala and the Nematoda are not nearly so restricted in their carbohydrate utilization. Laurie (1957) reported the fermentation of maltose by Moniliformis dubius and later (1959) found trehalose in the same species. M o r e recently, Fisher (1964) presented information indicating that Macracanthorhynchus hirudinaceus and M. dubius synthesized trehalose from exogenous sources. However, there is no published information on trehalase or maltase activity in any acanthocephalan and only scattered information on nematodes (Feist et al., 1965). It is the purpose of this study to provide information on the exogenous utilization of a variety of mono- and oligosaccharides by M. hirudinaceus and to partially characterize trehalase and maltase. * This study was supported in part by the Office of Research and Projects of Southern Illinois University and by Research Grant AI-05717 from the National Institutes of Health. 281
282
T. T. DUNAGAN AND T. M. YAu
T h e r e s u l t s p r e s e n t e d in this p a p e r a r e b a s e d e x c l u s i v e l y o n cell-free extracts. T h i s s h o u l d b e k e p t in m i n d w h e n c o n s i d e r i n g t h e r e s u l t s since t h e p r o p e r i n t e r p r e t a t i o n o f i n f o r m a t i o n o b t a i n e d f r o m c e l l - f r e e e x t r a c t s is n o t n e c e s s a r i l y selfe v i d e n t . I t is k n o w n , for e x a m p l e , t h a t t h e p H a c t i v i t y c u r v e for m a l t o s e f e r m e n t a t i o n b y y e a s t in vivo is s i g n i f i c a n t l y d i f f e r e n t f r o m t h a t o f t h e cell-free m a l t a s e . T h u s , o n e w o u l d n o t n e c e s s a r i l y e x p e c t M. hirudinaceus w i t h its " p r o b l e m s " o f m e m b r a n e t r a n s p o r t a t i o n etc. to h y d r o l y z e m a l t o s e at t h e s a m e rate in vivo as t h a t o f a c e l l - f r e e extract. MATERIALS AND METHODS
Cell-free preparation of Acanthocephala Acanthocephala were washed free from adhering debris with 0"1 M (pH 6"8) phosphate buffer and then homogenized in a precooled mortar. T h e homogenate was centrifuged at 20,000 g for 20 rain and the clear supernatant removed and used for enzyme analysis.
Preparation of glucose-free carbohydrate substrates Most commercial preparations of carbohydrates contain traces of D-glucose. Therefore, attempts were made to remove these traces by micro-organisms as well as by the technique of recrystallization for maltose. A loopful of yeast cells (from the Lindegren Carbondale Culture Collection) capable of hydrolyzing glucose but not the other substrates concerned was inoculated into 50 ml of 2% glucose nutrient broth. T h e cells were allowed to grow aerobically for 24 hr on a shaker at 30°C. After 24 hr these freshly grown cells were harvested by centrifugation and thoroughly washed twice with distilled water. The cell concentration was adjusted to 1 x 10 s cells/ml by the aid of a haemocytometer and 5"0 ml of this cell suspension added to 10 g of carbohydrate in 45 ml of distilled water. These yeast-sugar solutions were allowed to sit at 30°C for 4 hr. After the 4 hr, each solution was filtered through a sterilized Seitz filter to remove the yeast cells. T h e resulting s u g a r - approximately 50 ml of 20% sugar--was stored at 0°C for further use. In order to prevent bacterial contamination, 3 drops of toluene were added to each sugar stock solution.
Test for carbohydrate hydrolytic activity A quantitative manometric method was devised for hydrolytic activity of cell-free extract from Acanthocephala using yeast cultures capable of using only glucose, mannose and fructose and which are not capable of hydrolyzing di- or oligosaccharides. By employing a yeast culture of the above genotype, (Lindegren Carbondale Culture Collection) hexoses liberated by enzymatic hydrolysis of the ceU-free extract were determined qualitatively in the form of carbon dioxide liberated by the micro-organism. T h e following sugars were chosen as representative of the different classes of carbohydrates; turanose (Tur), maltose (MAD, sucrose (Suc), a-D-methyl-glucopyranoside (aMG), melezitose (Mez), trehalose (Tre), lactose (Lac), melibiose (Mel), raffinose (Raf), ~-D-methyl-mannoside (aMM), inulin (Inu) and dextrin (Dex).
Maltase: cell-free preparation by yeast A maltose fermenting yeast culture from the Lindegren Carbondale Culture Collection was inoculated into 50 ml of maltose nutrient broth containing: 5 g, yeast extract; 3"5 g, peptone; 2 g, ammonium sulfate; 2 g, potassium phosphate; 1 g, magnesium sulfate; 20 g, maltose monhydrate; and this was brought to 1000 ml with distilled water. T h e yeast cells were incubated in this medium at 30°C on a shaker for 36 hr. After this time, the cells were harvested by centrifugation; washed twice with distilled water and twice with p H 6"8 phosphate buffer. T h e 10 ml of cell paste obtained was then homogenized for
OLIGOSACCHARIDASES F R O M M A C R A C A N T H O R I I Y N C H U S
283
HIRUDINACEUS
20 min with a rotary pestle at 1000 rev/min. After homogenization, 16 ral of cold phosphate buffer (pH 6.8) was added and the resulting preparation centrifuged at 0°C at 25,000 g for 35 min. The clear supernate was stored under refrigeration for further use. An a-glucosidase that hydrolyzes not only maltose, but sucrose, ~-methyl-glucoside and turanose was also prepared using another yeast culture from the same collection.
Maltose purification Maltose was purified by the technique of Wolfrom & Thompson (1962). Twice reerystallized material was quite satisfactory and gave a negative test for glucose.
Quantitative determinations of carbohydrate hydrolytic activity Assays were conducted using a glucose oxidase coupled enzyme system of Worthington Biochemical Co. in a Beckman DB Spectrophotometer. The assay mixture contained 2"8 ml of 0.1 M phosphate buffer (pH 6"8), 0"1 ml cell-free extract (4.752 mg protein/m1), and 0-1 ml of substrate (e.g. maltose--200"0 mg/ml). This mixture was incubated for 25 min at 30°C after which 5"0 ml of glucostat and 0.1 ml substrate were added and the reaction continued for an additional 5 min. At this time, the entire reaction was adjusted to pH 1"0 with 6 N HC1. It should be noted that the second addition of substrate does not significantly interfere with the results because of the inhibitory nature of Tris buffer in the glucostat reagent. However, this does provide a check in case the substrate is not entirely free from glucose. RESULTS M a n o m e t r i c m e a s u r e m e n t s of carbon dioxide production (Table 1) in the yeast experiment indicate that M. hirudinaceus extracts contain enzymes capable of hydrolyzing maltose, dextrin, trehalose and turanose in decreasing order of magnitude. T h e r e is also an indication of activity on melezitose and lactose b u t this activity is sufficiently low that it could be attributed to causes other t h a n the presence of a specific e n z y m e in the parasite extract. T h e results obtained with glucostat (Fig. 1) were parallel with those obtained with the micro-organism. I t is obvious f r o m this histogram that the maltase activity is very high c o m p a r e d to that of the substrates tested. TABLE I~VoLUME
p H 4.5 p H 6-8 p H 8.6
OF CARBON DIOXIDE IN m l PRODUCED BY 1 x l0 s CELLS AFTER INCUBATION AT 3 0 ° C IN A REACTION MIXTURE ~
Tur
Mal
Suc
0.5 0.2 0.I
9.0 8"I 7"3
0 0 0
MG
Mez
Tre
Lac
Mel
0 0 0
0.2 0 0.I
0.4 0.7 0.7
0"I 0.1 0
0 0 0
12 hr
OF
Raf a M M
Inu Dex
0.1 0 0
0"I 0 0
0 0 0
4.9 2.4 3.0
* The reaction mixture contained 1.0 nd cell-free extract, 2.2 ml 0"2 M phosphate buffer, and 0"8 ml of carbohydrate. The final volume of 4"0 ml contained 4 per cent carbohydrate. A n u m b e r of different molecular f o r m s of maltases have been observed in other animals (five different forms reported f r o m h u m a n intestine, three different swine intestinal maltases, two different molecular forms in Neurospora). However, only
284
T. T. DUNAGANAND T. M. YAU
one form appeared in M. hirudinaceus. This molecular form was repeatedly observed in starch gel electrophoresis using para-nitro-phenyl-~-D-glucopyranoside. Maltase may also have been responsible for the hydrolysis of turanose (Table 1) due to overlapping substrate specificity.
450
40/)_ "o
g
350 _
o
~
300
-
250
-
200
-
150 _ 100 _ 50.
0
~
FIG. 1. Histogram showing activity of cell-free extract of M . hirudinaceus on reaction mixture containing 20 mg of the appropriate carbohydrate substrate. Incubation continued 25 min at 3°C prior to glucose assay. Assay mixture contained 2.8 ml of 0.2 M phosphate buffer (pH 6.8), 0"1 ml cell-free extract (4.752 mg protein/m1), and 0"1 ml of substrate (200"0 mg/ml).
Table 2 presents a comparison of information on optimum pH and temperature for maltase and trehalase which have been obtained from a number of organisms. It is interesting to note that data on M. hiru&'naeeus and the intestine in which it is found are very different. This observation further supports the notion that the same enzymes from parasite and host are different molecules. Glucosidases are normally divided into three classes according to the bonds broken (~-1,4; ~-1,6; both). Since maltase and trehalase are both e~-oligoglucosidases, it was originally considered that the maltase activity was the result of a single enzyme capable of hydrolyzing both ~-1,4 and ~-1,6 glucosidic bonds. However, when the information in Fig. 2 is examined, it is clear that the hydrolysis of these substrates is the result of separate enzymes. Using 0.2 M Tris malate buffer, (tertiary amines are competitive inhibitors of maltases) the pH optimum for maltose was 4.5; with 0.2 M acetic acid-sodium acetate buffer, the pH optimum was 5.0. A fractionation
OLIGOSACCHARIDASES F R O M M A C R A C A N T H O R H Y N C H U S H1RUDINACEUS
285
of the crude homogenate with a m m o n i u m sulfate in which the precipitate f r o m the 20-30 per cent fraction contained m o s t of the e n z y m e activity produced no significant shift in the o p t i m u m t e m p e r a t u r e b u t did lower the o p t i m u m p H slightly. Trehalase was not examined in partially purified fractions and for some reason we were unable to find trehalase in all samples examined. However, the majority of parasites examined had this enzyme. Using 0-1 M phosphate buffer, trehalase had a p H o p t i m u m of 5.8 and an o p t i m u m t e m p e r a t u r e of 45°C. TABLE 2 - - A
C O M P A R I S O N OF pH O P T I M U M OF MALTASE A N D TREHALASE FROM M . hirudinaceus W I T H T H A T F R O M T H E I N T E S T I N E OF I T S H O S T A N D O T H E R C O M M O N SOURCES*
Enzyme
Optimum pH
Optimum temp. °C
Maltase Maltase Maltase Maltase Maltase Trehalase Trehalase Trehalase Trehalase Trehalase
5"8 6"0 5.2-7"2* 6.5-7.5 4.5-5"0 5"6 6.5 5'7 6"0 5"8
50-76* 37 -37 50 45 37 40-50 37 45
Organism
Reference
Human intestine Human intestine mucosa Swine intestine Swine intestine mucosa M. hirudinaceus Phormia regina Melolontha
Bakers' yeast Swine intestine M . hirudinaceus
Auricchio et al. (1965) Gottschalk (1950) Gottschalk (1950) Dahlqvist (1960) This report Friedman (1960) Courtois et al. (1962) Panek & Souza (1964) Dahlqvist (1960) This report
* Where there is a wide range of pH or temperature for a given enzyme it may indicate a circumstance where multiple forms of enzymes are present. 10 o
9--
K 8
/
x
-
~6_ E 5-
~
._E x
3 2-
'~
1
°~
0
-
I 1
I 2
I I 3 4
I 5
I I 6 7 pH
I 8
I I I I 9 10 11 12
FIG. 2. Enzymatic activity of maltase and trehalase in cell-free extracts of Buffers used were glycine hydrochloride--glyclne (pH 2-3.6); acetic acid-sodium acetate (pH 3"6-5-8); potassium dihydrogen phosphate-disodium hydrogen phosphate (pH 5.8-7.4); Tris hydrochloride-Tris (pH 7"4-9"5). All determinations on maltase ( e . . . . e) and trehalase (© ©) were run at 50°C
M . hirudinaceus expressed as a function of pH.
T. T. DUNAGANAND T. M. YAU
286
Table 3 presents information obtained from inactivation studies conducted with p H 5.8 phosphate buffer (M/15) at 4°C using 8- and 24-hr incubations. It is clear from this information that the degree of inactivation is not a function of time. Notice that the percentage of activity of the various samples at 8 hr is similar to that of 24 hr. However, during the initial stage the degree of inactivation may be proportional to the time of incubation. Likewise, it is also clear that the disaccharidases in M. hirudinaceus are more stable with respect to urea inactivation 10 _
~ e .
"~•~. 7
~~
!1 ~
\',
3 I
0 0
I 1
I 2
I 3
I 4
I l
I 6
I 7
! 8
I, 9
Temperature .. C ° x 10
FIG. 3.--Enzymatic activity of maltase and trehalase in cell-free extracts of M. hirudinaceus expressed as a function of temperature. Assay of maltase conducted at pH 5"0 using 0"2 M acetic acid-sodium acetate buffer and at pH 5.8 for trehalase using same type buffer: 0 - - - 0 = maltase cativity; © ©--trehalase activity. TABLE
3 - - I N A C T I V A T I O N STUDIES CONDUCTED W I T H pH 5 " 8 PHOSPHATE BDI~IqlR AT 4 o c USING 8 - AND 2 4 - h r INCUBATIONS. PERCENTAGE ACTIVITY AS COMPARED W I T H THE CONTROL SAMPLE
Test No urea (control) 1"0 M urea 2.0 M urea 3"0 M urea 4"0 M urea 5"0 M urea
Enzyme
8-hr incubation
24-hr incubation
Maltase Trehalase Maltase Trehalase Maltase Trehalase Maltase Trehalase Maltase Trehalase Maltase Trehalase
100 100 104 108 95 95 77 37 71 30 63 30
100 100 106 109 95 87 78 37 70 35 59 37
(M/15)
OLIGOSACCHARIDASES FROM M A C R A C A N T H O R H Y N C H U S HIRUDINACEUS
287
than the disaccharidases of certain micro-organisms. For example, a 2.5 M concentration of urea inactivates more than 50 per cent of the initial a-glucosidase activity in yeast within 2 hr of incubation at 4°C. TABLE 4---MALTASE ACTIVITY EXPRESSED PER m g
PROTEIN FOR FEMALE M .
hirudinaceus
Maltase activity/rag protein Section Anterior Middle Posterior
Maximum
Minimum
Mean
2329 2287 1620
1065 1056 AAA.
1785 1701 927
Data from thirty-two worms representing eight different hosts. Each worm was cut into three equal sections and the appropriate section of four different worms pooled for each sample. Data shows maximum, minimum and mean of the activity. 0"2 M acetic acid buffer pH 5"2. Table 4 shows the variation in the amount of maltase present in female worms and also shows that the distribution of activity favors the anterior part of the worm but only slightly over the middle portion. The posterior one-third however, has decidedly less activity. DISCUSSION The question as to whether disaccharidases are found exclusively or predominantly intracellular is important to the overall understanding of parasite requirements. Dahlqvist (1960) and Dahlqvist & Thomson (1963) working on rats observed that trehalose was not absorbed unless it was simultaneously hydrolyzed. Therefore intraceUular hydrolysis was the rate limiting factor for trehalose absorption. Apparently the majority of disaccharides are absorbed unhydrolyzed following which they are hydrolyzed within the cells. Dahlqvist & Thomson (1963) suggested that the disaccharide absorption mechanism consisted of three steps, namely: (1) entry of disaccharide into the mucosal cells, (2) hydrolysis, and (3) exit of the monosaccharides into the portal blood in the lower intestine. Of this group, the third step is the rate limiting step for the absorption of maltose in the rat but the availability of trehalase seems to be the deciding factor in trehalose absorption. If one considers the location of maltase, which according to Dahlqvist (1960) is predominantly found in the posterior part of the swine intestine, one would expect those parasites located in the duodenum to possess an active ~-ghicosidase activity toward maltose. Moreover one might expect the parasite to possess minimal amounts of trehalase since it is in an area of the intestine where there is high swine trehalase activity. However, if the work of Dahlqvist & Thomson (1963) on the rat is also true for swine, the disaccharidases would not be available for
288
T. T. DUNAGANANDT. M. YAU
acanthocephalan use since they are found in the mucosal cells rather than secreted into the intestinal juices. More recently Johnson (1967) has presented electron microscope evidence using hamster intestine which he believes supports the idea that disaccharidases "are contained in the glycocalyx.., external to the plasma membrane of the microvillus". Regardless of the outcome of these views, most agree that maltase is not "secreted" into the lumen of the intestine but remains a part of the cell. This would support the accepted thesis that these parasites have their own complement of enzymes independent of their host and are capable of hydrolyzing complex carbohydrates. Although the actual location of disaccharidases in the intestine varies among species, trehalase has been reported highest in the anterior intestine of the majority of animals examined. As is true with these parasites, the hydrolysis of maltose and trehalose in other organisms is, as a rule, due to the action of distinct enzymes. It is also interesting to note that other tests in this laboratory indicate that a-methyl glucoside is not hydrolyzed by maltase extracted from M . hirudinaceus. In fact, rather than acting as a substrate, which is the case for maltase obtained from other sources, s-methyl glucoside behaves as a competitive inhibitor. Trehalose is of widespread occurrence in the lower orders of the plant kingdom where it is often important as a reserve carbohydrate. The function of trehalose in yeast has been reported as an energy source for cell division in addition to helping regulate osmotic pressure. Likewise, the presence of trehalose in insects is supposed to be that of an energy source for flight. Apparently the presence of this sugar is not unique but is rather widespread even among the parasites. REFERENCES AURICCHIO S., SEMRNZAG. & RUBINO A. (1965) Multiplicity of human intestinal disaccharidase---II. Characterization of the individual maltases. Biochim. biophys. Acta 96, 498-507. COURTOIS J. E., Pm~K F. & ZANOtrZIM. A. K. (1962) Oxidases of the cockchafer. Ct. R. Soc. Biol. 156, 565-566. DAHLQVXSr A. (1960) Characterization of three different hog intestinal maltases. Acta chem. scand. 14, 1-8. DnI-mOVlST A. & BORGSTROMB. (1961) Digestion and absorption of disaccharides in man. Biochem.J. 81, 411-418. DAHLQVISTA. & THOMSOND. L. (1963) The digestion and absorption of sucrose by the intact rat. J. Physiol. 167, 193-209. FEIST C. F., READC. P. & FISHERF. M., JR. (1965) Trehalose syntheses and hydrolysis in .4scaris suum. J. Parasit. 51 (1), 76-78. FISHERF. M. (1964) Syntheses of trehalose in Acanthocephala. J. Parasit. 50 (6), 803-804. FRIEDMAN S. (1960) The purification and properties of trehalase isolated from Phormia regina Meig. Archs Biochem. Biophys. 87, 252-258. Gox'rSCHALKA. (1950) Ct-D Glucosidases. In The Enzymes (Edited by SUMNERJ. B. & MYRa),CK K.), Vol. 1, pt. 1. Academic Press, New York. JOHNSON C. F. (1967) Disaccharidase: Localization in hamster intestine brush borders. Science 155, 1670-1672. LAtraI~. J. S. (1959) Aerobic metabolism of Moniliformis dubius (Acanthocephala). Expl Parasit. 8, 188-197.
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YIVCHUS HIRUDIN.4CEUS
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LAURIE J. S. (1957) The in vitro fermentation of carbohydrates by two species of cestodes and one species of Acanthocephala. Excpl Parasit. 6, 24-5-260. PANEK A. & SOUZA V. O. (1964) Purification and properties of bakers' yeast trehalase..7. Biol. Chem. 239 (6), 1671-1673. WOLFROM M. L. ~ THOMPSON A. (1962) In Methods in Carbohydrate Chemistry (Edited by WHISTLER R. L. & WOLFROM M. L.), Vol. I, p. 334. Academic Press, New York.
IO
OLIGOSACCHARIDASES FROM
MACRACANTHORHYNCHUS HIRUDINACEUS ( A C A N T H O C E P H A L A ) F R O M SWINE* T. T. DUNAGAN and T. M. YAU Department of Physiology, Southern Illinois University, Carbondale, Illinois 62901, U.S.A.
(Received 28 December 1967) A b s t r a c t - - 1 . Enzymatic activities of cell-free extracts of Macracanthorhynchus
hirudinaceus indicates that maltose, turanose and trehalose are hydrolyzed but with maltose being broken down four times as fast as any other disaccharide tested. 2. M. hirudinaceus cell-free extracts readily hydrolyze dextrin but not: (1) the polysaccharide, inulin (2) the trisaccharides, raffmose and melizitose (3) disaccharides, sucrose, lactose and melibiose (4) methyl substituted aldohexoses, o~-D-methyl glucopyranoside and C~-D-methylmannoside. 3. One isozyme of maltase was observed using para-nitrophenyl-c~-D glucopyranoside in starch gel electrophoresis. 4. The pH optimum for maltase was 4.5 using 0"2 M Tris malate buffer, but with 0-2 M acetic acid-sodium acetate buffer, the pH optimum was 5"0. The optimum temperature in either case was 50°C. 5. Trehalase had a pH optimum of 5"8 and an optimum temperature of 45°C using 0.2 M phosphate buffer. INTRODUCTION THE UTILIZATION of exogenous carbohydrates by helminths has been most extensively studied for cestodes in which only a very limited n u m b e r of monosaccharides are metabolized to any extent and interestingly enough fructose is not in this group. Disaccharides such as maltose and trehalose have not been hydrolyzed except in Cittotaenia sp. However, Acanthocephala and the Nematoda are not nearly so restricted in their carbohydrate utilization. Laurie (1957) reported the fermentation of maltose by Moniliformis dubius and later (1959) found trehalose in the same species. M o r e recently, Fisher (1964) presented information indicating that Macracanthorhynchus hirudinaceus and M. dubius synthesized trehalose from exogenous sources. However, there is no published information on trehalase or maltase activity in any acanthocephalan and only scattered information on nematodes (Feist et al., 1965). It is the purpose of this study to provide information on the exogenous utilization of a variety of mono- and oligosaccharides by M. hirudinaceus and to partially characterize trehalase and maltase. * This study was supported in part by the Office of Research and Projects of Southern Illinois University and by Research Grant AI-05717 from the National Institutes of Health. 281
282
T. T. DUNAGAN AND T. M. YAu
T h e r e s u l t s p r e s e n t e d in this p a p e r a r e b a s e d e x c l u s i v e l y o n cell-free extracts. T h i s s h o u l d b e k e p t in m i n d w h e n c o n s i d e r i n g t h e r e s u l t s since t h e p r o p e r i n t e r p r e t a t i o n o f i n f o r m a t i o n o b t a i n e d f r o m c e l l - f r e e e x t r a c t s is n o t n e c e s s a r i l y selfe v i d e n t . I t is k n o w n , for e x a m p l e , t h a t t h e p H a c t i v i t y c u r v e for m a l t o s e f e r m e n t a t i o n b y y e a s t in vivo is s i g n i f i c a n t l y d i f f e r e n t f r o m t h a t o f t h e cell-free m a l t a s e . T h u s , o n e w o u l d n o t n e c e s s a r i l y e x p e c t M. hirudinaceus w i t h its " p r o b l e m s " o f m e m b r a n e t r a n s p o r t a t i o n etc. to h y d r o l y z e m a l t o s e at t h e s a m e rate in vivo as t h a t o f a c e l l - f r e e extract. MATERIALS AND METHODS
Cell-free preparation of Acanthocephala Acanthocephala were washed free from adhering debris with 0"1 M (pH 6"8) phosphate buffer and then homogenized in a precooled mortar. T h e homogenate was centrifuged at 20,000 g for 20 rain and the clear supernatant removed and used for enzyme analysis.
Preparation of glucose-free carbohydrate substrates Most commercial preparations of carbohydrates contain traces of D-glucose. Therefore, attempts were made to remove these traces by micro-organisms as well as by the technique of recrystallization for maltose. A loopful of yeast cells (from the Lindegren Carbondale Culture Collection) capable of hydrolyzing glucose but not the other substrates concerned was inoculated into 50 ml of 2% glucose nutrient broth. T h e cells were allowed to grow aerobically for 24 hr on a shaker at 30°C. After 24 hr these freshly grown cells were harvested by centrifugation and thoroughly washed twice with distilled water. The cell concentration was adjusted to 1 x 10 s cells/ml by the aid of a haemocytometer and 5"0 ml of this cell suspension added to 10 g of carbohydrate in 45 ml of distilled water. These yeast-sugar solutions were allowed to sit at 30°C for 4 hr. After the 4 hr, each solution was filtered through a sterilized Seitz filter to remove the yeast cells. T h e resulting s u g a r - approximately 50 ml of 20% sugar--was stored at 0°C for further use. In order to prevent bacterial contamination, 3 drops of toluene were added to each sugar stock solution.
Test for carbohydrate hydrolytic activity A quantitative manometric method was devised for hydrolytic activity of cell-free extract from Acanthocephala using yeast cultures capable of using only glucose, mannose and fructose and which are not capable of hydrolyzing di- or oligosaccharides. By employing a yeast culture of the above genotype, (Lindegren Carbondale Culture Collection) hexoses liberated by enzymatic hydrolysis of the ceU-free extract were determined qualitatively in the form of carbon dioxide liberated by the micro-organism. T h e following sugars were chosen as representative of the different classes of carbohydrates; turanose (Tur), maltose (MAD, sucrose (Suc), a-D-methyl-glucopyranoside (aMG), melezitose (Mez), trehalose (Tre), lactose (Lac), melibiose (Mel), raffinose (Raf), ~-D-methyl-mannoside (aMM), inulin (Inu) and dextrin (Dex).
Maltase: cell-free preparation by yeast A maltose fermenting yeast culture from the Lindegren Carbondale Culture Collection was inoculated into 50 ml of maltose nutrient broth containing: 5 g, yeast extract; 3"5 g, peptone; 2 g, ammonium sulfate; 2 g, potassium phosphate; 1 g, magnesium sulfate; 20 g, maltose monhydrate; and this was brought to 1000 ml with distilled water. T h e yeast cells were incubated in this medium at 30°C on a shaker for 36 hr. After this time, the cells were harvested by centrifugation; washed twice with distilled water and twice with p H 6"8 phosphate buffer. T h e 10 ml of cell paste obtained was then homogenized for
OLIGOSACCHARIDASES F R O M M A C R A C A N T H O R I I Y N C H U S
283
HIRUDINACEUS
20 min with a rotary pestle at 1000 rev/min. After homogenization, 16 ral of cold phosphate buffer (pH 6.8) was added and the resulting preparation centrifuged at 0°C at 25,000 g for 35 min. The clear supernate was stored under refrigeration for further use. An a-glucosidase that hydrolyzes not only maltose, but sucrose, ~-methyl-glucoside and turanose was also prepared using another yeast culture from the same collection.
Maltose purification Maltose was purified by the technique of Wolfrom & Thompson (1962). Twice reerystallized material was quite satisfactory and gave a negative test for glucose.
Quantitative determinations of carbohydrate hydrolytic activity Assays were conducted using a glucose oxidase coupled enzyme system of Worthington Biochemical Co. in a Beckman DB Spectrophotometer. The assay mixture contained 2"8 ml of 0.1 M phosphate buffer (pH 6"8), 0"1 ml cell-free extract (4.752 mg protein/m1), and 0-1 ml of substrate (e.g. maltose--200"0 mg/ml). This mixture was incubated for 25 min at 30°C after which 5"0 ml of glucostat and 0.1 ml substrate were added and the reaction continued for an additional 5 min. At this time, the entire reaction was adjusted to pH 1"0 with 6 N HC1. It should be noted that the second addition of substrate does not significantly interfere with the results because of the inhibitory nature of Tris buffer in the glucostat reagent. However, this does provide a check in case the substrate is not entirely free from glucose. RESULTS M a n o m e t r i c m e a s u r e m e n t s of carbon dioxide production (Table 1) in the yeast experiment indicate that M. hirudinaceus extracts contain enzymes capable of hydrolyzing maltose, dextrin, trehalose and turanose in decreasing order of magnitude. T h e r e is also an indication of activity on melezitose and lactose b u t this activity is sufficiently low that it could be attributed to causes other t h a n the presence of a specific e n z y m e in the parasite extract. T h e results obtained with glucostat (Fig. 1) were parallel with those obtained with the micro-organism. I t is obvious f r o m this histogram that the maltase activity is very high c o m p a r e d to that of the substrates tested. TABLE I~VoLUME
p H 4.5 p H 6-8 p H 8.6
OF CARBON DIOXIDE IN m l PRODUCED BY 1 x l0 s CELLS AFTER INCUBATION AT 3 0 ° C IN A REACTION MIXTURE ~
Tur
Mal
Suc
0.5 0.2 0.I
9.0 8"I 7"3
0 0 0
MG
Mez
Tre
Lac
Mel
0 0 0
0.2 0 0.I
0.4 0.7 0.7
0"I 0.1 0
0 0 0
12 hr
OF
Raf a M M
Inu Dex
0.1 0 0
0"I 0 0
0 0 0
4.9 2.4 3.0
* The reaction mixture contained 1.0 nd cell-free extract, 2.2 ml 0"2 M phosphate buffer, and 0"8 ml of carbohydrate. The final volume of 4"0 ml contained 4 per cent carbohydrate. A n u m b e r of different molecular f o r m s of maltases have been observed in other animals (five different forms reported f r o m h u m a n intestine, three different swine intestinal maltases, two different molecular forms in Neurospora). However, only
284
T. T. DUNAGANAND T. M. YAU
one form appeared in M. hirudinaceus. This molecular form was repeatedly observed in starch gel electrophoresis using para-nitro-phenyl-~-D-glucopyranoside. Maltase may also have been responsible for the hydrolysis of turanose (Table 1) due to overlapping substrate specificity.
450
40/)_ "o
g
350 _
o
~
300
-
250
-
200
-
150 _ 100 _ 50.
0
~
FIG. 1. Histogram showing activity of cell-free extract of M . hirudinaceus on reaction mixture containing 20 mg of the appropriate carbohydrate substrate. Incubation continued 25 min at 3°C prior to glucose assay. Assay mixture contained 2.8 ml of 0.2 M phosphate buffer (pH 6.8), 0"1 ml cell-free extract (4.752 mg protein/m1), and 0"1 ml of substrate (200"0 mg/ml).
Table 2 presents a comparison of information on optimum pH and temperature for maltase and trehalase which have been obtained from a number of organisms. It is interesting to note that data on M. hiru&'naeeus and the intestine in which it is found are very different. This observation further supports the notion that the same enzymes from parasite and host are different molecules. Glucosidases are normally divided into three classes according to the bonds broken (~-1,4; ~-1,6; both). Since maltase and trehalase are both e~-oligoglucosidases, it was originally considered that the maltase activity was the result of a single enzyme capable of hydrolyzing both ~-1,4 and ~-1,6 glucosidic bonds. However, when the information in Fig. 2 is examined, it is clear that the hydrolysis of these substrates is the result of separate enzymes. Using 0.2 M Tris malate buffer, (tertiary amines are competitive inhibitors of maltases) the pH optimum for maltose was 4.5; with 0.2 M acetic acid-sodium acetate buffer, the pH optimum was 5.0. A fractionation
OLIGOSACCHARIDASES F R O M M A C R A C A N T H O R H Y N C H U S H1RUDINACEUS
285
of the crude homogenate with a m m o n i u m sulfate in which the precipitate f r o m the 20-30 per cent fraction contained m o s t of the e n z y m e activity produced no significant shift in the o p t i m u m t e m p e r a t u r e b u t did lower the o p t i m u m p H slightly. Trehalase was not examined in partially purified fractions and for some reason we were unable to find trehalase in all samples examined. However, the majority of parasites examined had this enzyme. Using 0-1 M phosphate buffer, trehalase had a p H o p t i m u m of 5.8 and an o p t i m u m t e m p e r a t u r e of 45°C. TABLE 2 - - A
C O M P A R I S O N OF pH O P T I M U M OF MALTASE A N D TREHALASE FROM M . hirudinaceus W I T H T H A T F R O M T H E I N T E S T I N E OF I T S H O S T A N D O T H E R C O M M O N SOURCES*
Enzyme
Optimum pH
Optimum temp. °C
Maltase Maltase Maltase Maltase Maltase Trehalase Trehalase Trehalase Trehalase Trehalase
5"8 6"0 5.2-7"2* 6.5-7.5 4.5-5"0 5"6 6.5 5'7 6"0 5"8
50-76* 37 -37 50 45 37 40-50 37 45
Organism
Reference
Human intestine Human intestine mucosa Swine intestine Swine intestine mucosa M. hirudinaceus Phormia regina Melolontha
Bakers' yeast Swine intestine M . hirudinaceus
Auricchio et al. (1965) Gottschalk (1950) Gottschalk (1950) Dahlqvist (1960) This report Friedman (1960) Courtois et al. (1962) Panek & Souza (1964) Dahlqvist (1960) This report
* Where there is a wide range of pH or temperature for a given enzyme it may indicate a circumstance where multiple forms of enzymes are present. 10 o
9--
K 8
/
x
-
~6_ E 5-
~
._E x
3 2-
'~
1
°~
0
-
I 1
I 2
I I 3 4
I 5
I I 6 7 pH
I 8
I I I I 9 10 11 12
FIG. 2. Enzymatic activity of maltase and trehalase in cell-free extracts of Buffers used were glycine hydrochloride--glyclne (pH 2-3.6); acetic acid-sodium acetate (pH 3"6-5-8); potassium dihydrogen phosphate-disodium hydrogen phosphate (pH 5.8-7.4); Tris hydrochloride-Tris (pH 7"4-9"5). All determinations on maltase ( e . . . . e) and trehalase (© ©) were run at 50°C
M . hirudinaceus expressed as a function of pH.
T. T. DUNAGANAND T. M. YAU
286
Table 3 presents information obtained from inactivation studies conducted with p H 5.8 phosphate buffer (M/15) at 4°C using 8- and 24-hr incubations. It is clear from this information that the degree of inactivation is not a function of time. Notice that the percentage of activity of the various samples at 8 hr is similar to that of 24 hr. However, during the initial stage the degree of inactivation may be proportional to the time of incubation. Likewise, it is also clear that the disaccharidases in M. hirudinaceus are more stable with respect to urea inactivation 10 _
~ e .
"~•~. 7
~~
!1 ~
\',
3 I
0 0
I 1
I 2
I 3
I 4
I l
I 6
I 7
! 8
I, 9
Temperature .. C ° x 10
FIG. 3.--Enzymatic activity of maltase and trehalase in cell-free extracts of M. hirudinaceus expressed as a function of temperature. Assay of maltase conducted at pH 5"0 using 0"2 M acetic acid-sodium acetate buffer and at pH 5.8 for trehalase using same type buffer: 0 - - - 0 = maltase cativity; © ©--trehalase activity. TABLE
3 - - I N A C T I V A T I O N STUDIES CONDUCTED W I T H pH 5 " 8 PHOSPHATE BDI~IqlR AT 4 o c USING 8 - AND 2 4 - h r INCUBATIONS. PERCENTAGE ACTIVITY AS COMPARED W I T H THE CONTROL SAMPLE
Test No urea (control) 1"0 M urea 2.0 M urea 3"0 M urea 4"0 M urea 5"0 M urea
Enzyme
8-hr incubation
24-hr incubation
Maltase Trehalase Maltase Trehalase Maltase Trehalase Maltase Trehalase Maltase Trehalase Maltase Trehalase
100 100 104 108 95 95 77 37 71 30 63 30
100 100 106 109 95 87 78 37 70 35 59 37
(M/15)
OLIGOSACCHARIDASES FROM M A C R A C A N T H O R H Y N C H U S HIRUDINACEUS
287
than the disaccharidases of certain micro-organisms. For example, a 2.5 M concentration of urea inactivates more than 50 per cent of the initial a-glucosidase activity in yeast within 2 hr of incubation at 4°C. TABLE 4---MALTASE ACTIVITY EXPRESSED PER m g
PROTEIN FOR FEMALE M .
hirudinaceus
Maltase activity/rag protein Section Anterior Middle Posterior
Maximum
Minimum
Mean
2329 2287 1620
1065 1056 AAA.
1785 1701 927
Data from thirty-two worms representing eight different hosts. Each worm was cut into three equal sections and the appropriate section of four different worms pooled for each sample. Data shows maximum, minimum and mean of the activity. 0"2 M acetic acid buffer pH 5"2. Table 4 shows the variation in the amount of maltase present in female worms and also shows that the distribution of activity favors the anterior part of the worm but only slightly over the middle portion. The posterior one-third however, has decidedly less activity. DISCUSSION The question as to whether disaccharidases are found exclusively or predominantly intracellular is important to the overall understanding of parasite requirements. Dahlqvist (1960) and Dahlqvist & Thomson (1963) working on rats observed that trehalose was not absorbed unless it was simultaneously hydrolyzed. Therefore intraceUular hydrolysis was the rate limiting factor for trehalose absorption. Apparently the majority of disaccharides are absorbed unhydrolyzed following which they are hydrolyzed within the cells. Dahlqvist & Thomson (1963) suggested that the disaccharide absorption mechanism consisted of three steps, namely: (1) entry of disaccharide into the mucosal cells, (2) hydrolysis, and (3) exit of the monosaccharides into the portal blood in the lower intestine. Of this group, the third step is the rate limiting step for the absorption of maltose in the rat but the availability of trehalase seems to be the deciding factor in trehalose absorption. If one considers the location of maltase, which according to Dahlqvist (1960) is predominantly found in the posterior part of the swine intestine, one would expect those parasites located in the duodenum to possess an active ~-ghicosidase activity toward maltose. Moreover one might expect the parasite to possess minimal amounts of trehalase since it is in an area of the intestine where there is high swine trehalase activity. However, if the work of Dahlqvist & Thomson (1963) on the rat is also true for swine, the disaccharidases would not be available for
288
T. T. DUNAGANANDT. M. YAU
acanthocephalan use since they are found in the mucosal cells rather than secreted into the intestinal juices. More recently Johnson (1967) has presented electron microscope evidence using hamster intestine which he believes supports the idea that disaccharidases "are contained in the glycocalyx.., external to the plasma membrane of the microvillus". Regardless of the outcome of these views, most agree that maltase is not "secreted" into the lumen of the intestine but remains a part of the cell. This would support the accepted thesis that these parasites have their own complement of enzymes independent of their host and are capable of hydrolyzing complex carbohydrates. Although the actual location of disaccharidases in the intestine varies among species, trehalase has been reported highest in the anterior intestine of the majority of animals examined. As is true with these parasites, the hydrolysis of maltose and trehalose in other organisms is, as a rule, due to the action of distinct enzymes. It is also interesting to note that other tests in this laboratory indicate that a-methyl glucoside is not hydrolyzed by maltase extracted from M . hirudinaceus. In fact, rather than acting as a substrate, which is the case for maltase obtained from other sources, s-methyl glucoside behaves as a competitive inhibitor. Trehalose is of widespread occurrence in the lower orders of the plant kingdom where it is often important as a reserve carbohydrate. The function of trehalose in yeast has been reported as an energy source for cell division in addition to helping regulate osmotic pressure. Likewise, the presence of trehalose in insects is supposed to be that of an energy source for flight. Apparently the presence of this sugar is not unique but is rather widespread even among the parasites. REFERENCES AURICCHIO S., SEMRNZAG. & RUBINO A. (1965) Multiplicity of human intestinal disaccharidase---II. Characterization of the individual maltases. Biochim. biophys. Acta 96, 498-507. COURTOIS J. E., Pm~K F. & ZANOtrZIM. A. K. (1962) Oxidases of the cockchafer. Ct. R. Soc. Biol. 156, 565-566. DAHLQVXSr A. (1960) Characterization of three different hog intestinal maltases. Acta chem. scand. 14, 1-8. DnI-mOVlST A. & BORGSTROMB. (1961) Digestion and absorption of disaccharides in man. Biochem.J. 81, 411-418. DAHLQVISTA. & THOMSOND. L. (1963) The digestion and absorption of sucrose by the intact rat. J. Physiol. 167, 193-209. FEIST C. F., READC. P. & FISHERF. M., JR. (1965) Trehalose syntheses and hydrolysis in .4scaris suum. J. Parasit. 51 (1), 76-78. FISHERF. M. (1964) Syntheses of trehalose in Acanthocephala. J. Parasit. 50 (6), 803-804. FRIEDMAN S. (1960) The purification and properties of trehalase isolated from Phormia regina Meig. Archs Biochem. Biophys. 87, 252-258. Gox'rSCHALKA. (1950) Ct-D Glucosidases. In The Enzymes (Edited by SUMNERJ. B. & MYRa),CK K.), Vol. 1, pt. 1. Academic Press, New York. JOHNSON C. F. (1967) Disaccharidase: Localization in hamster intestine brush borders. Science 155, 1670-1672. LAtraI~. J. S. (1959) Aerobic metabolism of Moniliformis dubius (Acanthocephala). Expl Parasit. 8, 188-197.
OLIGOSACCHARIDASES FROM MACRACAIVTHORH
YIVCHUS HIRUDIN.4CEUS
289
LAURIE J. S. (1957) The in vitro fermentation of carbohydrates by two species of cestodes and one species of Acanthocephala. Excpl Parasit. 6, 24-5-260. PANEK A. & SOUZA V. O. (1964) Purification and properties of bakers' yeast trehalase..7. Biol. Chem. 239 (6), 1671-1673. WOLFROM M. L. ~ THOMPSON A. (1962) In Methods in Carbohydrate Chemistry (Edited by WHISTLER R. L. & WOLFROM M. L.), Vol. I, p. 334. Academic Press, New York.
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