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The properties of trehalase from the mosquito-parasitizing water mold, Lagenidium sp
;OUHN.AL
OF INVERTEBRATE
The
Properties
PATHOLOGY
22,
of Trehalase Mold, THOMAS
Biochemistry
313-320 (1973)
Laboratory,
from the Mosquito-Parasitizing Lagenidium sp.l JR.~ AND A. DOMNAS"
MCINNIS,
Department
Chapel
Hill,
Received
Water
of Botany, North Corolinn Febrztury
University 2761/,
of North
Cn?olincl.
16, 1973
The soluble trehalase from the phycomycete Lagenidium sp., a parasite of many species of mosquitoes, was purified by acid titration, acetone precipitation, and Sephadex G-ZOO chromatography to give a i70-fold increase in specific activity over the crude extract,. The enzyme was specific for t~rehalose. A p-glucosidase was ropurified with the trehalase, but did not interfere with its characterization. Lagendium trehalase had a K, of 1.43 mM, and E. of 11.4 kc&mole, and a pH of optimum activity of 5.56.5, and a molecular weight of 72,000. It was denatured by 30 min incubation at temperatures above 5O”C, severely inhibited by heavy metals, and competitively inhibited by sucrose. No other reported inhibitors, including mannitol and ATP, were effective. Suggested physiological roles for the enzyme include the breakdown of stored trehalose in the mycelium and zoospores, and the digestion of hemolymph trehalose in infected mosquito larvae.
Trehalase (EC 3.2.1.28, cY,a’-glucoside 1-glucohydrolase) has been found in a wide variety of organisms and tissues including bacteria, fungi, lower and higher plants, insects, and mammals (Hey and Elbein, 1968; Ceccnrini, 1966; Hill and Sussman, 1963; Roberts and Tovey, 1969; Gussin and McCormack, 1970; Friedman, 1960; Dahlqvist, 1960). The enzyme has been extensively studied since its discovery by Bourquelot (1893) in fungi. According to certain investigators, t,he principal role of trehalase in fungi is to digest the storage carbohydrate trehalose, which constitutes *This work was supported in part by grants from the American Cancer Society, the University of North Carolina Research Council, the Society of Sigma Xi, and a United States Army Medical Research Development Grant DADA 17-72-C-2168. ’ Present address: Department of Botany, Clemson University, Clemson, South Carolina 29631. ‘Requests for reprints should be sent to A. Domnas.
up to 3% of the mycelial dry weight in some species (Cochrane, 1958). The enzyme has also been implicated in breaking the dormancy of Neurospora ascospores (Hill and Sussman, 1964) and in initiating basidiospore germinat,ion (Williams and Niederpruem, 1968). Ascomycetes and Basidiomycetes have been the favored source for trehalase isolation in the fungi, but this report, surprisingly, is the first investigation of trehalase from a water mold. Our reasons for studying this enzyme were 2-fold: (1) the fungus grows very well on trehalose in a defined medium indicating the presence of a trehalase, and, (2) the fungus is a virulent parasite of culicine and aedine mosquito larvae, and recently the host range specificity was widened to include members of the anopheline group (Giebel and Domnas, unpubl.) . Inasmuch as insects have a, potent trehalose-trehalase metabolism, we considered that a comparative study of the two systems in the host-parasite pair would
313 Copyright All rights
0 1973 by Academic Press, Inc. of reproduction in sny form reserved.
314
MCINNIS,
JR.
shed considerable light on the physiological events during disease development. It was ‘desirable therefore (1) to isolate and characterize the enzyme from Lager&&m sp. and to study its properties and (2) to isolate and study the trehalase from the larvae, particularly from the standpoint of inhibition and repression. This report describes the results of (1). MATERIALS
AND
METHODS
Culture of jun.gus. The original isolate of Lagenidium sp. was donated by C. J.
Umphlett of Clemson University and was maintained by monthly transfers on plates of peptone-yeast extract-glucose agar (Fisher Scientific, Inc.). Mycelium for enzyme extraction was grown in shake cultures of peptone-yeast extract-glucose broth (Fisher Scientific, 1nc.j in 250-ml Erlenmeyer flasks at 20%. Blended mycelium from the agar plates was used as inoculum. Isolation
and purification
of trehalase.
Five-day-old mycelia were collected on filter paper using a Biichner funnel, washed with several volumes of chilled standard buffer (0.05 M phosphate, pH 6.0) and suspended in a minimum volume of buffer. The mycelia were first disrupted in a chilled Waring Blendor microcup, and the resulting mycelial fragments were further broken with glass beads in a Braun cell disruptor (VWB Scientific, Inc.). The suspension was centrifuged at 10,000 g for 10 min, and the supernatant was decanted and recentrifuged at 40,000 g for 10 min. The washed pellet from the first centrifugation showed significant trehalase activity, but no attempt was made to solubilize that enzyme. The supernatant from the second centrifugation was titrated with ice-cold 0.5 N HCI to a pH of 4.0, the solution was centrifuged, and the supernatant, which contained all the activity, was decanted. This dilute protein solution was concentrated in an Aminco Diaflo ultrafiltration apparatus with a PM-10 filter (American Instrument
AND
DOlMNAS
Co.) to approximately g0 the original volume. Ice-cold acetone was added dropwise to the concentrate with constant stirring until the acetone represented 70% of the total volume of the solution. This precipitated all trehalase activity, which was recovered by centrifugation. It was found that the acetone precipitation was much more quantitative when the extract was kept at an acid pH. The pellet was dissolved in a minimum volume of standard buffer, and the solution was dialyzed overnight against the buffer. The dialyzed supernatant was applied to a 2.5 by 50.0 cm. Sephadex G-200 column and the enzyme was eluted with the standard buffer. Active fractions were pooled and used for characterization tests. Protein was determined by the method of Lowry et al. (1951). Protein in the column effluent was monitored by measuring its absorbance at 280 nm. Assay and definition of units. The standard assay mixture consisted of 0.1 ml of enzyme, 1.9 ml of 0.05 M phosphate buffer, pH 6.0, and 2.0 ml of 0.2 M a,cu’-trehalose dihydrate (Sigma Chemical Co.). The assay mixtures along with appropriate blanks were incubated at 30°C for 30 min, and the reactions were stopped by placing the solutions in a boiling water bath. In order to determine the amount of glucose released by the reaction, 2.0 ml aliquots of the assay mixtures were mixed with 2.0 ml of Glucostat Special reagent (Worthington Biochemical Co.), prepared with 0.01 M phosphate buffer, pH 7.0. These mixtures were incubated for 10 min at 30°C and the reactions were terminated by the addition of 6 drops of 4 N HCI to each tube. The absorbance of each reaction mixture was measured at 420 nm, and the glucose concentration determined from a standard curve. One unit of activity was defined as the amount of enzyme which catalyzed the release of 1 pmole of glucose in 30 min at 30°C and at pH 6.0. Thin-layer chromatography. Samples of the assay mixture were chromatographed
on glass plates coated with silica gel G (Merck, Inc.). A butanol-ethanol-acetone-water (5:4:3:2 v/v) mixture was the developing solvent, and the products were detected by spraying the plates with an anisaldehyde-sulfuric acid reagent (50.0 ml of glacial acetic acid, 1.0 ml of concentrated sulfuric acid, and 0.5 ml of anisaldehyde), followed by heating the plates at 100% for 10 min. Disc gel chromatography. Acrylamide disc-gel (7.5’j;,) electrophoresis was performed in duplicate at 2% in a Tris-glytine buffer system. One tube was stained with Amido Schwarz solution and then washed with 7% HAC to remove excess stain (Davis, 1964). The second tube of the pair was sliced in 1/rc-inch segments and usually assayed for trehalase act.ivity. Molecular weight determination. Molecular weight was determined with a Sephadex G-200 column (Pharmacia K z5/1co) calibrated with Pharmacia standards (Granath and Kvist, 1967). RESULTS
Growth of Lagenidium and Trehalase Development Lagenidium sp. grew rapidly when grown on peptone-yeast extract-glucose broth, reaching a maximum weight after 5 days incubation (Fig. 1). The amount of trehalase extractable from the mycelium correspondingly reached a maximum after 5 days growth. This same pattern of enzyme development was observed with de-
COY ZIP,, ,‘“‘,r Q?’ c Prc,r,-
___-l_-l_~
Fraction Crude extract Acidified supernatant Acetone precipitate Sepadex C-ZOO fractions
OF SOLUBLE
Volume (ml) 803 805 17 24.6
. -iB.lc: ii.
FIG. 1. Growth curve of Lagenicktm sp. on peptone-yeast extract,-glucose broth and the corresponding trehalasc development.
fined media, with either glucose or trehalose as the carbon source. This suggest’edthat, the enzyme was constitutive, and &day-old mycelium was selected for enzyme isolation. Purification of Trehalase A summary of the purification procedure is shown in Table I. The pooled Sephadex fractions yielded a 170-fold increase in specific activity over the crude extract’. The elution profile of the Sephadex G-200 column (Fig. 2) shows that another cnzyme, p-glucosidase, was eluted along wit,h the trehalase, and that their peaks overlapped. Several other column chromatographic techniques were attempted in order to separate the t,wo enzymes, but none was found which improved the purity of trehalase. A DEAE-cellulose column, which has been successfully used in trehalase
TABLE PURIFICATION
31.-,
TREHALhSE
Lagenidium
1
TEEHALA~E
Total protein (wm) 2448 2053 68 2.8
FROM
LagenitEiwx
Total units”
Specific activity ( x IO-“)
Purification factor
83.2 83.2 56.4 16.2
3.40 4.05 83.0 579.0
1.2x 24.4x 170. ox
Recovery (5%)
100 100 68 12-28 19.5 --. 11One unit of activity is the amount of enzyme required to release 1 pmole of glucose in 30 min at 30°C. IX
316
MCINNIS,
JR.
FIG. 2. Elution of trehalase and @glucosidase from a column of Sephadex G-200. Protein measured by its absorbance at 280 nm. The column was 2.5 x 50.0 cm, and the eluting buffer was 0.05 M phosphate, pH 6.0.
purification by other researchers, either completely denatured the enzyme, or irreversibly bound it. /LGlucosidase was successfully eluted from this column by a number of developing methods, but no trace of trehalase was ever detected in the effluent. Activity
against Other Substrates
The pooled Sephadex fractions were tested for activity against several a-linked substrates (Table II), but none other than trehalose were hydrolyzed. Furthermore, the ,&glucosidase, when purified by DEAEcellulose chromatography, did not attack trehalose. These results demonstrated that the contaminating p-glucosidase would not interfere with the trehalase assay and characterization tests. TABLE SPECIFICITY
Substrate Trehalose Sucrose Lactose Maltose p-Nitrophenylcr-n-glucoside Cellobiose
2
OF TREHALASE
FROM
Bond type
Lagenidiuma Glucose released (Pd
a a 01 a
571 0 0 0
;
0 46gb
DStandard assay conditions described in text; 0.02 M substrates substituted for trehalose in the assay. b Activity due to contaminating &glucosidase.
AND
DOMNAS
0 FIG. 3. Temperature stability of trehalase. Standard assay mixtures minus trehalose were preincubated at various temperatures for 30 minutes. At the end of this time, 0.02 M trehalose was added, and the standard assay was performed as described in the text.
Enzymatic Properties
The observations made above indicated a high degree of specificity of the enzyme for trehalose. Thin layer chromatography of assay mixtures sampled at intervals of from 0 to 24 hr of incubation revealed glucose to be the only detectable product. This indicates that the reaction proceeds without the intervention of glucoside addition products. This is further evidence that p-glucosidase was not active in the assay system, since this enzyme forms glycoside addition products that would have been detectable if present. The pH of optimum activity of trehalase was in a broad range from 5.5 to 6.5. The enzyme was stable for at least 30 min at 30°C at all pHs between 4.0 and 7.0, but showed a rapid loss of activity outside of this range. The enzyme was stable at all temperatures below 45°C for at least 30 min, but was completely denatured in this time at 60°C (Fig. 3). The enzyme was stored frozen for several months without any loss of activity. The increase in the reaction rate with increasing temperature from loo to 40% was used to calculate the activation energy for the enzyme. An Arrhenius plot of this data (Fig. 4) gave a value of E, equal to 11.4 kcal/mole.
Lagenidium
.3 I i
TREHALASE
i.0 I
\
> 2.6 D 22.4
2.0
3.1
3.2
3.3 3.4 3.5 I/T (OK-‘) X lO-3
3.6
FIG. 4. Arhennius plot of the effect of increasing temperatures on trehalase activity. Standard assays were carried out at various temperatures between 10” and 40°C: and the activation energy was calculated from the slope of the line of the plotted data.
A value for the Michaelis constant (K,) of 1.43 m;M was calculated from a Lineweaver-Burk plot (Fig. 5). The effects of various substances added to the reaction mixture on the trehalase activity are shown in Table III. Heavy metals severely inhibited the reaction, but two other reported trehalase inhibitors, mannitol (Horikoshi and Ikeda, 1966) and ATP (Panek, 1969) had no effect. Sucrose significantly inhibited the reaction, and Lineweaver-Burk plots (Fig. 6) revealed the inhibition to be competitive. Figure 7 shows that the molecular weight of the purified enzyme as estimated by Sephadex G-200 was 72,000, based on the assumption that trehalase is a globular protein, like the standards. Results of the disc gel showed (Fig. 8) that the trehalase ran very close to the p-glucosidase, but was completely separated from other impurities. Only one band was found with trehalase activity.
FIG. 5. Linrwearer-Burk plot of relationship between substrate concentration and trehalase activity. Standard assays were carried out with various concentrations of t,rehalose substituted for the standard 0.02 M concentration.
TABLE EFFECTS
OF
VARIOUS
3 COMPOUNDS
ON
TREHALASE ACTIVITY"
-
Compounds
% of original activity remaining
added
~~~~ ATP 10 mM Sucrose5mM
100 100
x0
Mannose 5 mM Mannitol 5 nlM
100
Maltose 5 mM Glucose 5 mM CaCl, 0.5 mM HgCI, 0.5 mM Pb Acetate 0.5 mM CuCll 0.5 mM Dichloromecurobene rate 0.5
I00 I) 75 60 100
rn~
---_.
0 Compounds were added to the Standard Mixture in the concentrations indicated.
Assay
reported for four other trehalases, those from hybrid yeast (7.9 kcal/mole, Avigad et al. 1965) ; the silkworm (9.4 kcal/mole, Saito, 1960) ; and lily pollen (8.2 The properties of Lagenidium trehalase kcal/mole, Gussin and McCormack, 1970) were quite similar to those of other tse- and the tobacco hornworm larvae (17.66 halases examined in higher fungi. The pH kcal/mole, Dahlman, 1971). The E, for optimum and Michaelis constant were of Lagenidium trehalase was higher than the the same order of magnitude, and the resis- first three but less than that of the horntance to heat denaturation was similar to worm trehalase. In fact, the only major differences beall except for that reported in the heat-tolerant Neurospora trehalase (Hill and Suss- tween this trehalase and those reported for man, 1963). The activation energy has been other fungi were in its behavior on DEAE-
31s
MCINNIS,
JR.
.4ND
DOhfNAS
-6 -4 +2 FIG. 6. Effect of sucrose on trehalase activity. Lineweaver-Burk plots of the effect of various substrate concentrations on the reaction rate in the presence of sucrose. No sucrose (0); 0.05 M sucrose (0); 0.10 M sucrose (0). FIG. 8. Disc gel eleetrophoresis of purified treha!ase. Band 1 is the dye marker, bands 2, 3, 4 are inactive protein, band 5 is the trehalase, and band 6 is ,%glucosidase.
gested that this inhibition serves to prevent trehalose utilization until the sucrose supply is exhausted; whereupon the reserve 0.4 trehalose is utilized for energy until sucrose again becomes available. It appears un0.3 likely, however, that the same is true for Lagenidium. The fungus does not utilize sucrose for growth, and so the sugar would FIG. 7. Plot of the K., values of various moleculikely not exist in the fungus as a metabolar weight standards. The K,, values of Lagenidium lite. As this matter has not as yet been sp. trehalase and p-glucosidase are shown with examined, however, the presence of sucrose their corresponding apparent molecular weights. in Lagenidium cannot be ruled out at this time. Results recently obtained suggest that cellulose and its inhibition by sucrose. The pure cultures of Lagenidium sp. growing on trehalose are severely inhibited by increasloss of trehalase during DEAE-cellulose ing concentrations of sucrose. This might chromatography has been reported only once: Carnie and Porteous (1962) reported suggest the presence of a membrane-bound the same result in examining trehalase from trehalase, and we have repeatedly observed and measured an “insoluble” trehalase asrabbit intestine. The competitive inhibition of trehalase by sucrose has been reported sociated with the pellet obtained from fracfor trehalases from the honeybee and DYO- tion 1 (Table I). Such membrane-bound sophila by Huber and Lefebvre (1971) and trehaloses have been investigated by SackLefebvre and Huber (1970). It was sug- tor (1970) and Glasziou and Gayler
hXp??ZidiU??t
(1968), and the same may be true in Lagenidium. Nothing is known about sucrose levels in mosquito larvae, but sucrases are known to exist in insects (Huber and Lefebvre, 1971). Perhaps the most intriguing aspect of this enzyme is its role in mosquito parasitism. Mosquito larval hemolymph contain large amounts of trehalose (Wyatt and Kalf, 1957), and Lagenidiuwl can utilize exogenous trehalose as was shown in our pure culture studies. Once infection has occurred and the fungus has multiplied and spread, a competition could ensue between both organisms for the common sub&rate. The presence of a membrane-bound trehalase in the fungus would permit access to the substrate, and its consequent depletion would be very important contributory factor to the ultimate death of the larvae. Some insects possess a trehalase inhibitor (Friedman, 1961) in their hemolymph, and if it is present in the mosquito larvae, caunot aparently inhibit the fungal trehalase in situ but could perhaps in vivo. This and related aspects of trehalsse are being studied relative to the larvale trehalase. The role of t,he enzyme in the fungus is quite undefined other-than the obviois. It, may be necessary for utilization of st’ored trehalose in zoospores for energy in the case of swimming analogous to that of insect trehalose in flight energetic demands (Sacktor, 1970). &me data-have suggested that the storage of trehalose may be the necessary condition for zoospore morphogenesis.
AVIGAD, G., ZIV, O.! AND NEUFELD, E. 1965. Intracellular trehalase of a hybrid yeast. Biockem. 715-722.
BORQUELOT, E. 1893. Sur un ferment soluble nouveau dCboublant treh.alose en glucose. C. R. Acad. Sci., 116, 826828. CARNIE.
J.
A.,
AND
PORTEOUS,
CECCARINI, C. 1966. Trehalase from Diet yostalizrm dixoideum: Purification and properties. Srience, 151, 454-456. COCHR.~NE, 1’. W. 1958. “Physiology of the Fungi.”
Wiley, New York. I~.WLIM~X, D. 0. 1971. Purifi~xlion and propcrtit*r of trehalnse from tobacco hornworm 1arv:tc. J. Insect. DAHLQVIST,
Pkysiol.,
17, 1677-1687.
A. 1960. Characterization of hog int~‘ntin;\1 trt~halsse. Srfn Ckern. &and., 14. Q-16. P.kvrs, 13. .I. 1964. Disc-clectrophoresis. 11. Met ho:ls :111(1:tlllblicntion to hllman serum protc-in?. 11 r~?t. A’. 1.. Acnrl. Sci., 131, 404-427. FRItiumv~ 6. 1960. Tlrrt purification and tirs of trehnla$ls isolated from Pkormia Arrk. lli~~che)n. Biopkys., 87, 252-258.
pro1~‘r~eyP,cn.
IN. S. 1961. Inhibition of trehalase activity in the> hr\r~mol~mph of Pkormia rrgin~. A:rh.
~RIIWM
Rioclwm. Hioplr ys.. 93, 550-554. GL.~SZI~U, K. T., ANU G~TLER, K.
R. 1968. Sugar tranaIlori : OI~CWI~C~~CC of trchalase aciivit: in sugar cant. Planta, 85, 299-302. (;RANATIT, k-. 11.. .4SD ~r~s'r, 1% E. 1967. Molrcula~ wright distribut,ion analysis by gel chromatogrally
on Sqhdcx.
J. Chromntogr.,
28, 69-81,
GUSSIN, ,4. E. S., AND MCCOR~IACK, J. H. 1870. Trchalast~ and the enzymes of trehalose ltiosgnthesis in &lli~rn lon~ifEor~m. Phytochcmistry, 9, 1915-1920. HEY, ii. E., AND ELBEIN, il. D. 1968. Partial Furification and propcrfies of a trehalase from Streptomyces l~ygr~s~opicus. f. Bacler-iol., 96, 105-110. HILL, E. l’., AND SUS~MAN, A. S. 1963. Purification and I”.opertics of trehalase(s) from n’eurospora. Arch. Biochem. Biopkys., .180-m? HILL, E. P., ASD SUSSMAN, A. S. 1964.
ment of trehalase Xewosporrc.
J.
and invertase
Bacterial.,
88,
102,
Devclopactivitv in
1556-1566:
H~RIKO~~I, Ii., ANL, IKF,DA, Y. 1966. Trehrdas’ in conidia of Aspergillus orytne. J. Bacte&)l., 91,
1883-1887.
HUBER, R. E., AXD LEFEBVRE, Y. A. 1971. The purification and some properties of soluble trehalase and sucrase from Drosophila
REFERENCES
J., 97,
?ll!l
TREHALASE
J.
W.
1962.
The
solubilization, thermolability, chromatographic purificat.ion and intracellular distribution of some glycosidases of rabbit small intestine. Biochem. J.. 85, 62&629.
nwlanognater. C(cn. J. Biochem., LEFEHVRE, Y. A., AKD HCBER, R. E.
49, 1155-1164.
1970. Solubilization, puritiration and some properties of trehalase from honeybee (8pis meZlifexz). Arch. Biochem.
Biopkys.,
140, 514-518.
LOWRY, 0. H.,
ROSEBROUGH, N. J., FARR, A. I,., AXD RANDALL, R. J. 1951. Protein measuremrnl with the Folin phenol reagent.s. J. BioZ.
Cltem., PANEK, A.
193,
265-275.
J. 1969. Adenosine triphosphate inhibit.ion of yr:xjt f rc~hxlnse. .f. Rocfei-iol.. 99, 904-005.
320
MCINNIS,
JR.
R. M., AND TOVEY, Ii. D. ,1969. Trehalase activity in Selaginella martens& Arch. Bio-
ROBERTS,
them. SACETOR,
Biophys.,
133,
408-412.
B. 1970. Regulation of intermediary metabolism, with special reference to the control mechanisms in flight muscle. Advan. In-
sect Physiol. SAITO, J. 1960.
7, 267-347.
Trehalase
of the silkworm
Bombyr
AND
DOMNAS
mori: Purification and properties of the enzyme. J. Biochem. (Tokz/o), 48, 101-109. WILLIAMS, C. F., AND NIEDERPRUEM, D. 1968. Trehalase in schizophyllum commune. Arch. MikrobioE., 60, 377-383. WYATT, C. R., AND KALF, G. F. 1957. The chemistry of insect hemolymph. II. Trehalose and other carbohydrates. J. Gen. Physiol., 40, 833-847.
OF INVERTEBRATE
The
Properties
PATHOLOGY
22,
of Trehalase Mold, THOMAS
Biochemistry
313-320 (1973)
Laboratory,
from the Mosquito-Parasitizing Lagenidium sp.l JR.~ AND A. DOMNAS"
MCINNIS,
Department
Chapel
Hill,
Received
Water
of Botany, North Corolinn Febrztury
University 2761/,
of North
Cn?olincl.
16, 1973
The soluble trehalase from the phycomycete Lagenidium sp., a parasite of many species of mosquitoes, was purified by acid titration, acetone precipitation, and Sephadex G-ZOO chromatography to give a i70-fold increase in specific activity over the crude extract,. The enzyme was specific for t~rehalose. A p-glucosidase was ropurified with the trehalase, but did not interfere with its characterization. Lagendium trehalase had a K, of 1.43 mM, and E. of 11.4 kc&mole, and a pH of optimum activity of 5.56.5, and a molecular weight of 72,000. It was denatured by 30 min incubation at temperatures above 5O”C, severely inhibited by heavy metals, and competitively inhibited by sucrose. No other reported inhibitors, including mannitol and ATP, were effective. Suggested physiological roles for the enzyme include the breakdown of stored trehalose in the mycelium and zoospores, and the digestion of hemolymph trehalose in infected mosquito larvae.
Trehalase (EC 3.2.1.28, cY,a’-glucoside 1-glucohydrolase) has been found in a wide variety of organisms and tissues including bacteria, fungi, lower and higher plants, insects, and mammals (Hey and Elbein, 1968; Ceccnrini, 1966; Hill and Sussman, 1963; Roberts and Tovey, 1969; Gussin and McCormack, 1970; Friedman, 1960; Dahlqvist, 1960). The enzyme has been extensively studied since its discovery by Bourquelot (1893) in fungi. According to certain investigators, t,he principal role of trehalase in fungi is to digest the storage carbohydrate trehalose, which constitutes *This work was supported in part by grants from the American Cancer Society, the University of North Carolina Research Council, the Society of Sigma Xi, and a United States Army Medical Research Development Grant DADA 17-72-C-2168. ’ Present address: Department of Botany, Clemson University, Clemson, South Carolina 29631. ‘Requests for reprints should be sent to A. Domnas.
up to 3% of the mycelial dry weight in some species (Cochrane, 1958). The enzyme has also been implicated in breaking the dormancy of Neurospora ascospores (Hill and Sussman, 1964) and in initiating basidiospore germinat,ion (Williams and Niederpruem, 1968). Ascomycetes and Basidiomycetes have been the favored source for trehalase isolation in the fungi, but this report, surprisingly, is the first investigation of trehalase from a water mold. Our reasons for studying this enzyme were 2-fold: (1) the fungus grows very well on trehalose in a defined medium indicating the presence of a trehalase, and, (2) the fungus is a virulent parasite of culicine and aedine mosquito larvae, and recently the host range specificity was widened to include members of the anopheline group (Giebel and Domnas, unpubl.) . Inasmuch as insects have a, potent trehalose-trehalase metabolism, we considered that a comparative study of the two systems in the host-parasite pair would
313 Copyright All rights
0 1973 by Academic Press, Inc. of reproduction in sny form reserved.
314
MCINNIS,
JR.
shed considerable light on the physiological events during disease development. It was ‘desirable therefore (1) to isolate and characterize the enzyme from Lager&&m sp. and to study its properties and (2) to isolate and study the trehalase from the larvae, particularly from the standpoint of inhibition and repression. This report describes the results of (1). MATERIALS
AND
METHODS
Culture of jun.gus. The original isolate of Lagenidium sp. was donated by C. J.
Umphlett of Clemson University and was maintained by monthly transfers on plates of peptone-yeast extract-glucose agar (Fisher Scientific, Inc.). Mycelium for enzyme extraction was grown in shake cultures of peptone-yeast extract-glucose broth (Fisher Scientific, 1nc.j in 250-ml Erlenmeyer flasks at 20%. Blended mycelium from the agar plates was used as inoculum. Isolation
and purification
of trehalase.
Five-day-old mycelia were collected on filter paper using a Biichner funnel, washed with several volumes of chilled standard buffer (0.05 M phosphate, pH 6.0) and suspended in a minimum volume of buffer. The mycelia were first disrupted in a chilled Waring Blendor microcup, and the resulting mycelial fragments were further broken with glass beads in a Braun cell disruptor (VWB Scientific, Inc.). The suspension was centrifuged at 10,000 g for 10 min, and the supernatant was decanted and recentrifuged at 40,000 g for 10 min. The washed pellet from the first centrifugation showed significant trehalase activity, but no attempt was made to solubilize that enzyme. The supernatant from the second centrifugation was titrated with ice-cold 0.5 N HCI to a pH of 4.0, the solution was centrifuged, and the supernatant, which contained all the activity, was decanted. This dilute protein solution was concentrated in an Aminco Diaflo ultrafiltration apparatus with a PM-10 filter (American Instrument
AND
DOlMNAS
Co.) to approximately g0 the original volume. Ice-cold acetone was added dropwise to the concentrate with constant stirring until the acetone represented 70% of the total volume of the solution. This precipitated all trehalase activity, which was recovered by centrifugation. It was found that the acetone precipitation was much more quantitative when the extract was kept at an acid pH. The pellet was dissolved in a minimum volume of standard buffer, and the solution was dialyzed overnight against the buffer. The dialyzed supernatant was applied to a 2.5 by 50.0 cm. Sephadex G-200 column and the enzyme was eluted with the standard buffer. Active fractions were pooled and used for characterization tests. Protein was determined by the method of Lowry et al. (1951). Protein in the column effluent was monitored by measuring its absorbance at 280 nm. Assay and definition of units. The standard assay mixture consisted of 0.1 ml of enzyme, 1.9 ml of 0.05 M phosphate buffer, pH 6.0, and 2.0 ml of 0.2 M a,cu’-trehalose dihydrate (Sigma Chemical Co.). The assay mixtures along with appropriate blanks were incubated at 30°C for 30 min, and the reactions were stopped by placing the solutions in a boiling water bath. In order to determine the amount of glucose released by the reaction, 2.0 ml aliquots of the assay mixtures were mixed with 2.0 ml of Glucostat Special reagent (Worthington Biochemical Co.), prepared with 0.01 M phosphate buffer, pH 7.0. These mixtures were incubated for 10 min at 30°C and the reactions were terminated by the addition of 6 drops of 4 N HCI to each tube. The absorbance of each reaction mixture was measured at 420 nm, and the glucose concentration determined from a standard curve. One unit of activity was defined as the amount of enzyme which catalyzed the release of 1 pmole of glucose in 30 min at 30°C and at pH 6.0. Thin-layer chromatography. Samples of the assay mixture were chromatographed
on glass plates coated with silica gel G (Merck, Inc.). A butanol-ethanol-acetone-water (5:4:3:2 v/v) mixture was the developing solvent, and the products were detected by spraying the plates with an anisaldehyde-sulfuric acid reagent (50.0 ml of glacial acetic acid, 1.0 ml of concentrated sulfuric acid, and 0.5 ml of anisaldehyde), followed by heating the plates at 100% for 10 min. Disc gel chromatography. Acrylamide disc-gel (7.5’j;,) electrophoresis was performed in duplicate at 2% in a Tris-glytine buffer system. One tube was stained with Amido Schwarz solution and then washed with 7% HAC to remove excess stain (Davis, 1964). The second tube of the pair was sliced in 1/rc-inch segments and usually assayed for trehalase act.ivity. Molecular weight determination. Molecular weight was determined with a Sephadex G-200 column (Pharmacia K z5/1co) calibrated with Pharmacia standards (Granath and Kvist, 1967). RESULTS
Growth of Lagenidium and Trehalase Development Lagenidium sp. grew rapidly when grown on peptone-yeast extract-glucose broth, reaching a maximum weight after 5 days incubation (Fig. 1). The amount of trehalase extractable from the mycelium correspondingly reached a maximum after 5 days growth. This same pattern of enzyme development was observed with de-
COY ZIP,, ,‘“‘,r Q?’ c Prc,r,-
___-l_-l_~
Fraction Crude extract Acidified supernatant Acetone precipitate Sepadex C-ZOO fractions
OF SOLUBLE
Volume (ml) 803 805 17 24.6
. -iB.lc: ii.
FIG. 1. Growth curve of Lagenicktm sp. on peptone-yeast extract,-glucose broth and the corresponding trehalasc development.
fined media, with either glucose or trehalose as the carbon source. This suggest’edthat, the enzyme was constitutive, and &day-old mycelium was selected for enzyme isolation. Purification of Trehalase A summary of the purification procedure is shown in Table I. The pooled Sephadex fractions yielded a 170-fold increase in specific activity over the crude extract’. The elution profile of the Sephadex G-200 column (Fig. 2) shows that another cnzyme, p-glucosidase, was eluted along wit,h the trehalase, and that their peaks overlapped. Several other column chromatographic techniques were attempted in order to separate the t,wo enzymes, but none was found which improved the purity of trehalase. A DEAE-cellulose column, which has been successfully used in trehalase
TABLE PURIFICATION
31.-,
TREHALhSE
Lagenidium
1
TEEHALA~E
Total protein (wm) 2448 2053 68 2.8
FROM
LagenitEiwx
Total units”
Specific activity ( x IO-“)
Purification factor
83.2 83.2 56.4 16.2
3.40 4.05 83.0 579.0
1.2x 24.4x 170. ox
Recovery (5%)
100 100 68 12-28 19.5 --. 11One unit of activity is the amount of enzyme required to release 1 pmole of glucose in 30 min at 30°C. IX
316
MCINNIS,
JR.
FIG. 2. Elution of trehalase and @glucosidase from a column of Sephadex G-200. Protein measured by its absorbance at 280 nm. The column was 2.5 x 50.0 cm, and the eluting buffer was 0.05 M phosphate, pH 6.0.
purification by other researchers, either completely denatured the enzyme, or irreversibly bound it. /LGlucosidase was successfully eluted from this column by a number of developing methods, but no trace of trehalase was ever detected in the effluent. Activity
against Other Substrates
The pooled Sephadex fractions were tested for activity against several a-linked substrates (Table II), but none other than trehalose were hydrolyzed. Furthermore, the ,&glucosidase, when purified by DEAEcellulose chromatography, did not attack trehalose. These results demonstrated that the contaminating p-glucosidase would not interfere with the trehalase assay and characterization tests. TABLE SPECIFICITY
Substrate Trehalose Sucrose Lactose Maltose p-Nitrophenylcr-n-glucoside Cellobiose
2
OF TREHALASE
FROM
Bond type
Lagenidiuma Glucose released (Pd
a a 01 a
571 0 0 0
;
0 46gb
DStandard assay conditions described in text; 0.02 M substrates substituted for trehalose in the assay. b Activity due to contaminating &glucosidase.
AND
DOMNAS
0 FIG. 3. Temperature stability of trehalase. Standard assay mixtures minus trehalose were preincubated at various temperatures for 30 minutes. At the end of this time, 0.02 M trehalose was added, and the standard assay was performed as described in the text.
Enzymatic Properties
The observations made above indicated a high degree of specificity of the enzyme for trehalose. Thin layer chromatography of assay mixtures sampled at intervals of from 0 to 24 hr of incubation revealed glucose to be the only detectable product. This indicates that the reaction proceeds without the intervention of glucoside addition products. This is further evidence that p-glucosidase was not active in the assay system, since this enzyme forms glycoside addition products that would have been detectable if present. The pH of optimum activity of trehalase was in a broad range from 5.5 to 6.5. The enzyme was stable for at least 30 min at 30°C at all pHs between 4.0 and 7.0, but showed a rapid loss of activity outside of this range. The enzyme was stable at all temperatures below 45°C for at least 30 min, but was completely denatured in this time at 60°C (Fig. 3). The enzyme was stored frozen for several months without any loss of activity. The increase in the reaction rate with increasing temperature from loo to 40% was used to calculate the activation energy for the enzyme. An Arrhenius plot of this data (Fig. 4) gave a value of E, equal to 11.4 kcal/mole.
Lagenidium
.3 I i
TREHALASE
i.0 I
\
> 2.6 D 22.4
2.0
3.1
3.2
3.3 3.4 3.5 I/T (OK-‘) X lO-3
3.6
FIG. 4. Arhennius plot of the effect of increasing temperatures on trehalase activity. Standard assays were carried out at various temperatures between 10” and 40°C: and the activation energy was calculated from the slope of the line of the plotted data.
A value for the Michaelis constant (K,) of 1.43 m;M was calculated from a Lineweaver-Burk plot (Fig. 5). The effects of various substances added to the reaction mixture on the trehalase activity are shown in Table III. Heavy metals severely inhibited the reaction, but two other reported trehalase inhibitors, mannitol (Horikoshi and Ikeda, 1966) and ATP (Panek, 1969) had no effect. Sucrose significantly inhibited the reaction, and Lineweaver-Burk plots (Fig. 6) revealed the inhibition to be competitive. Figure 7 shows that the molecular weight of the purified enzyme as estimated by Sephadex G-200 was 72,000, based on the assumption that trehalase is a globular protein, like the standards. Results of the disc gel showed (Fig. 8) that the trehalase ran very close to the p-glucosidase, but was completely separated from other impurities. Only one band was found with trehalase activity.
FIG. 5. Linrwearer-Burk plot of relationship between substrate concentration and trehalase activity. Standard assays were carried out with various concentrations of t,rehalose substituted for the standard 0.02 M concentration.
TABLE EFFECTS
OF
VARIOUS
3 COMPOUNDS
ON
TREHALASE ACTIVITY"
-
Compounds
% of original activity remaining
added
~~~~ ATP 10 mM Sucrose5mM
100 100
x0
Mannose 5 mM Mannitol 5 nlM
100
Maltose 5 mM Glucose 5 mM CaCl, 0.5 mM HgCI, 0.5 mM Pb Acetate 0.5 mM CuCll 0.5 mM Dichloromecurobene rate 0.5
I00 I) 75 60 100
rn~
---_.
0 Compounds were added to the Standard Mixture in the concentrations indicated.
Assay
reported for four other trehalases, those from hybrid yeast (7.9 kcal/mole, Avigad et al. 1965) ; the silkworm (9.4 kcal/mole, Saito, 1960) ; and lily pollen (8.2 The properties of Lagenidium trehalase kcal/mole, Gussin and McCormack, 1970) were quite similar to those of other tse- and the tobacco hornworm larvae (17.66 halases examined in higher fungi. The pH kcal/mole, Dahlman, 1971). The E, for optimum and Michaelis constant were of Lagenidium trehalase was higher than the the same order of magnitude, and the resis- first three but less than that of the horntance to heat denaturation was similar to worm trehalase. In fact, the only major differences beall except for that reported in the heat-tolerant Neurospora trehalase (Hill and Suss- tween this trehalase and those reported for man, 1963). The activation energy has been other fungi were in its behavior on DEAE-
31s
MCINNIS,
JR.
.4ND
DOhfNAS
-6 -4 +2 FIG. 6. Effect of sucrose on trehalase activity. Lineweaver-Burk plots of the effect of various substrate concentrations on the reaction rate in the presence of sucrose. No sucrose (0); 0.05 M sucrose (0); 0.10 M sucrose (0). FIG. 8. Disc gel eleetrophoresis of purified treha!ase. Band 1 is the dye marker, bands 2, 3, 4 are inactive protein, band 5 is the trehalase, and band 6 is ,%glucosidase.
gested that this inhibition serves to prevent trehalose utilization until the sucrose supply is exhausted; whereupon the reserve 0.4 trehalose is utilized for energy until sucrose again becomes available. It appears un0.3 likely, however, that the same is true for Lagenidium. The fungus does not utilize sucrose for growth, and so the sugar would FIG. 7. Plot of the K., values of various moleculikely not exist in the fungus as a metabolar weight standards. The K,, values of Lagenidium lite. As this matter has not as yet been sp. trehalase and p-glucosidase are shown with examined, however, the presence of sucrose their corresponding apparent molecular weights. in Lagenidium cannot be ruled out at this time. Results recently obtained suggest that cellulose and its inhibition by sucrose. The pure cultures of Lagenidium sp. growing on trehalose are severely inhibited by increasloss of trehalase during DEAE-cellulose ing concentrations of sucrose. This might chromatography has been reported only once: Carnie and Porteous (1962) reported suggest the presence of a membrane-bound the same result in examining trehalase from trehalase, and we have repeatedly observed and measured an “insoluble” trehalase asrabbit intestine. The competitive inhibition of trehalase by sucrose has been reported sociated with the pellet obtained from fracfor trehalases from the honeybee and DYO- tion 1 (Table I). Such membrane-bound sophila by Huber and Lefebvre (1971) and trehaloses have been investigated by SackLefebvre and Huber (1970). It was sug- tor (1970) and Glasziou and Gayler
hXp??ZidiU??t
(1968), and the same may be true in Lagenidium. Nothing is known about sucrose levels in mosquito larvae, but sucrases are known to exist in insects (Huber and Lefebvre, 1971). Perhaps the most intriguing aspect of this enzyme is its role in mosquito parasitism. Mosquito larval hemolymph contain large amounts of trehalose (Wyatt and Kalf, 1957), and Lagenidiuwl can utilize exogenous trehalose as was shown in our pure culture studies. Once infection has occurred and the fungus has multiplied and spread, a competition could ensue between both organisms for the common sub&rate. The presence of a membrane-bound trehalase in the fungus would permit access to the substrate, and its consequent depletion would be very important contributory factor to the ultimate death of the larvae. Some insects possess a trehalase inhibitor (Friedman, 1961) in their hemolymph, and if it is present in the mosquito larvae, caunot aparently inhibit the fungal trehalase in situ but could perhaps in vivo. This and related aspects of trehalsse are being studied relative to the larvale trehalase. The role of t,he enzyme in the fungus is quite undefined other-than the obviois. It, may be necessary for utilization of st’ored trehalose in zoospores for energy in the case of swimming analogous to that of insect trehalose in flight energetic demands (Sacktor, 1970). &me data-have suggested that the storage of trehalose may be the necessary condition for zoospore morphogenesis.
AVIGAD, G., ZIV, O.! AND NEUFELD, E. 1965. Intracellular trehalase of a hybrid yeast. Biockem. 715-722.
BORQUELOT, E. 1893. Sur un ferment soluble nouveau dCboublant treh.alose en glucose. C. R. Acad. Sci., 116, 826828. CARNIE.
J.
A.,
AND
PORTEOUS,
CECCARINI, C. 1966. Trehalase from Diet yostalizrm dixoideum: Purification and properties. Srience, 151, 454-456. COCHR.~NE, 1’. W. 1958. “Physiology of the Fungi.”
Wiley, New York. I~.WLIM~X, D. 0. 1971. Purifi~xlion and propcrtit*r of trehalnse from tobacco hornworm 1arv:tc. J. Insect. DAHLQVIST,
Pkysiol.,
17, 1677-1687.
A. 1960. Characterization of hog int~‘ntin;\1 trt~halsse. Srfn Ckern. &and., 14. Q-16. P.kvrs, 13. .I. 1964. Disc-clectrophoresis. 11. Met ho:ls :111(1:tlllblicntion to hllman serum protc-in?. 11 r~?t. A’. 1.. Acnrl. Sci., 131, 404-427. FRItiumv~ 6. 1960. Tlrrt purification and tirs of trehnla$ls isolated from Pkormia Arrk. lli~~che)n. Biopkys., 87, 252-258.
pro1~‘r~eyP,cn.
IN. S. 1961. Inhibition of trehalase activity in the> hr\r~mol~mph of Pkormia rrgin~. A:rh.
~RIIWM
Rioclwm. Hioplr ys.. 93, 550-554. GL.~SZI~U, K. T., ANU G~TLER, K.
R. 1968. Sugar tranaIlori : OI~CWI~C~~CC of trchalase aciivit: in sugar cant. Planta, 85, 299-302. (;RANATIT, k-. 11.. .4SD ~r~s'r, 1% E. 1967. Molrcula~ wright distribut,ion analysis by gel chromatogrally
on Sqhdcx.
J. Chromntogr.,
28, 69-81,
GUSSIN, ,4. E. S., AND MCCOR~IACK, J. H. 1870. Trchalast~ and the enzymes of trehalose ltiosgnthesis in &lli~rn lon~ifEor~m. Phytochcmistry, 9, 1915-1920. HEY, ii. E., AND ELBEIN, il. D. 1968. Partial Furification and propcrfies of a trehalase from Streptomyces l~ygr~s~opicus. f. Bacler-iol., 96, 105-110. HILL, E. l’., AND SUS~MAN, A. S. 1963. Purification and I”.opertics of trehalase(s) from n’eurospora. Arch. Biochem. Biopkys., .180-m? HILL, E. P., ASD SUSSMAN, A. S. 1964.
ment of trehalase Xewosporrc.
J.
and invertase
Bacterial.,
88,
102,
Devclopactivitv in
1556-1566:
H~RIKO~~I, Ii., ANL, IKF,DA, Y. 1966. Trehrdas’ in conidia of Aspergillus orytne. J. Bacte&)l., 91,
1883-1887.
HUBER, R. E., AXD LEFEBVRE, Y. A. 1971. The purification and some properties of soluble trehalase and sucrase from Drosophila
REFERENCES
J., 97,
?ll!l
TREHALASE
J.
W.
1962.
The
solubilization, thermolability, chromatographic purificat.ion and intracellular distribution of some glycosidases of rabbit small intestine. Biochem. J.. 85, 62&629.
nwlanognater. C(cn. J. Biochem., LEFEHVRE, Y. A., AKD HCBER, R. E.
49, 1155-1164.
1970. Solubilization, puritiration and some properties of trehalase from honeybee (8pis meZlifexz). Arch. Biochem.
Biopkys.,
140, 514-518.
LOWRY, 0. H.,
ROSEBROUGH, N. J., FARR, A. I,., AXD RANDALL, R. J. 1951. Protein measuremrnl with the Folin phenol reagent.s. J. BioZ.
Cltem., PANEK, A.
193,
265-275.
J. 1969. Adenosine triphosphate inhibit.ion of yr:xjt f rc~hxlnse. .f. Rocfei-iol.. 99, 904-005.
320
MCINNIS,
JR.
R. M., AND TOVEY, Ii. D. ,1969. Trehalase activity in Selaginella martens& Arch. Bio-
ROBERTS,
them. SACETOR,
Biophys.,
133,
408-412.
B. 1970. Regulation of intermediary metabolism, with special reference to the control mechanisms in flight muscle. Advan. In-
sect Physiol. SAITO, J. 1960.
7, 267-347.
Trehalase
of the silkworm
Bombyr
AND
DOMNAS
mori: Purification and properties of the enzyme. J. Biochem. (Tokz/o), 48, 101-109. WILLIAMS, C. F., AND NIEDERPRUEM, D. 1968. Trehalase in schizophyllum commune. Arch. MikrobioE., 60, 377-383. WYATT, C. R., AND KALF, G. F. 1957. The chemistry of insect hemolymph. II. Trehalose and other carbohydrates. J. Gen. Physiol., 40, 833-847.