Plant Physiol. (1974) 54, 414-415
Short Communication
Starch in Fungi
III. ISOLATION AND PROPERTIES OF AN AMYLOSE-PRECIPITATING FACTOR FROM LENTINELLUS
URSINUS FRUIT BODIES'
Received for publication January 8, 1974 and in revised form April 22, 1974
DEREK A. MCCRACKEN
Department of Biological Scien2ces, Illiniois State University, Normal, Illinois 61761
at 20,000g for 5 min at 0 C. The 60 and 90% ammonium sulfate fractions were obtained in the same way.
A factor which precipitates amylose has been isolated from
Each of the ammonium sulfate pellets were redissolved in a
Lentinellus ursinus (Fr.) Kuhner fruit bodies. This factor volume of 0.01 M tris-HCl buffer (pH 7.0) equal to the amount
could be a protein or a polypeptide. Glucose, maltose, and of crude extract used. The 30% fraction containing the APF
amylopectin do not affect the binding of amylose. Amylose bind- activity was re-fractionated to obtain the 0 to 10%, 10 to 20%,
ing is unaffected by temperature (4 to 40 C) or pH (6 to 8.5). and 20 to 30% ammonium sulfate pellets.
In separate experiments the sensitivity of APF to pH
changes was determined by adjusting the pH of APF solutions
to either 4.2 or 9.5. The pellets obtained after centrifugation at
20,000g for 5 min were re-dissolved in the original of volume
of 0.01 M tris-HCI buffer (pH 7.0) and the pH of the supernatants was adjusted to 7.0.
Certain fungi have recently been shown to produce a unique
DEAE-cellulose was used as an adsorbent with the APF
starch which is composed of short chain amylose molecules sample prepared by re-fractionation with ammonium sulfate.
that are deposited in the hyphal wall (3, 8, 10). This amylose The DEAE-cellulose was first placed in 0.01 M tris-HC1 buffer
is synthesized in the cytoplasm (9) which contains a starch (pH 7.0), then this swollen material was added to the APFphosphorylase (EC 2.4.1.1) (unpublished results). ADPglu- containing fraction (1.0 g of dry DEAE-cellulose/10 ml of
cose glucosyltransferase (EC 2.4.1.11) has not been demon- extract). The mixture was stirred for 1 min, then centrifuged
strated in these fruit bodies but glycogen-producing fungi have for 5 min at 0 C at 20,000g. The supernatant fluid was saved
been shown to contain this enzyme (1). Since both starch- for APF activity determination while the pellet was washed
phosphorylase and ADPglucose glucosyltransferase possess the successively with 10 ml each of 1.0 M NaCl (in 0.01 M triscapability of producing relatively long chain amylose molecules HCI buffer, pH 7.0), 1.0 M amino-methyl-propanol buffer (pH
(2, 4) the production of only short chain molecules by these 8.5), H20, and 0.5 M citrate buffer (pH 6.0).
fungi, both naturally and after incubation with glucose 1-phosFractionation with cold acetone resulted in the loss of 70%
phate (9), was unexpected. Then, in experiments to measure of the APF activity and so was not used in the purification of
Lentinellus amylase activity by digestion of amylose, I found APF. The final isolation procedure consisted of obtaining the
that the fungal extract caused the amylose to precipitate. The 0 to 30% ammonium sulfate fraction of the original extract,
present report describes the isolation and some of the prop- re-fractionating this to obtain the 0 to 10% ammonium sulfate
erties of this amylose-precipitating factor.
fraction, subsequently adjusting the pH of this fraction to 9.5,
and saving the supernatant fluid after centrifugation. The pH
MATERIALS AND METHODS
of this final fraction was returned to 7.0 prior to its use.
No attempt was made to determine specific activity because
Lentinellus ursinus (Fr.) Kiihner fruit bodies were collected it is not
known whether the amylose-precipitating factor is a
locally and stored at 4 C until needed.
protein or not. Instead the isolation proceeded on the basis of
Isolation of APF2. All of the steps in the isolation procedure the % yield of APF units.
were carried out at room temperature unless otherwise indiMeasurement of APF Activity. The reaction mixture concated. Fruit bodies were blended in distilled water (2 ml H,O/g tained 0.5 ml of the solution
to be tested plus 0.5 ml of 0.2%
fresh weight) and then filtered through two thicknesses of fine amylose (average chain length 300 glucose units; Nutritional
cloth. The filtrate was centrifuged at 20,000g for 10 min at 0 C Biochemical Corporation) solution (pH 7.0). After 5 min the
and the resulting supernatant fluid was the crude extract. This precipitate was centrifuged in a clinical centrifuge and 0.5 ml
extract was brought to 30% saturation with ammonium sulfate of the supernatant fluid was added to 0.5 ml of iodine solution
and the precipitated material was collected by centrifugation (0.2% I2 2.0% KI) in a 50-ml volumetric flask containing 15
to 20 ml of H20. The resulting solution was made to volume
1 This research was supported by grants from the Illinois Acad- and the absorbance measured at 660 nm. The absorbances of
emy of Science and Illinois State University Faculty Research test solutions were compared to that of an amylose standard
prepared in the same way except that water replaced the test
Grant Program.
2 Abbreviation: APF: amylose-precipitating factor.
solution. If the test solution precipitated 100% of the amylose
414
ABSTRACT
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Plant Physiol. Vol.
54, 1974
415
AMYLOSE-PRECIPITATING FACTOR
then the determination was repeated using diluted test solution. One unit of APF activity is the amount required to precipitate 10% of the amylose.
Properties of APF. The specificity of APF was determined
by using amylopectin (Calbiochem) instead of amylose in the
APF assay. Glucose, maltose, and amylopectin were tested as
possible competitive inhibitors of APF activity. In addition to
amylose and APF the reaction mixture contained 0.5 ml of
either 0.2% amylopectin solution, 0.5 M maltose, or 4.0 M
glucose.
Activity determinations were run on APF preparations at
pH 6.0, 7.0, and 8.5.
The effect of temperature on APF activity was investigated
by incubating reaction mixtures at various temperatures.
RESULTS AND DISCUSSION
Table I contains the results of the various isolation procedures. DEAE-cellulose adsorbed 100% of the APF and none of
the elution procedures were successful. Adsorption of APF to
DEAE-cellulose is due to the charged groups, not to the polyglucan nature of cellulose, since APF does not bind to plain
cellulose. Amylose-precipitating substances isolated from rice
grains were identified as fatty acids (7). However, the APF from
Lentinellus has a combination of properties usually associated
with proteins or polypeptides, not fatty acids: (a) thermal
lability (APF is destroyed above 50 C); (b) precipitation at pH
4.2; (c) precipitation by ammonium sulfate and acetone; (d)
adsorption to DEAE-cellulose. Thus APF would be similar to
concanavalin A which also reacts with carbohydrates, albeit
with differing specificity (5).
Properties of APF. Unlike concanavalin A the amylose-precipitating factor from Lentinellus precipitates amylose not
amylopectin. Furthermore, the polysaccharide-binding sites of
APF differ from those of concanavalin A because APF is not
inhibited by glucose or maltose (6). Since glucose and maltose
do not compete with amylose for APF it is possible that
amylose binding is related to the helical structure of the
amylose molecule. The lack of binding by amylopectin could be
due either to the outer chains being too short or to steric interference among the branches.
APF is a very stable substance whose activity is the same
from 4 C to 40 C and from pH 6 to pH 8.5. Solutions of APF
have been kept for a week at 4 C and they still retain considerable activity. Even after 24 hr at room temperature APF
solutions retained better than 78% of their original activity.
Freeze drying, on the other hand, resulted in as much as 40
to 50% loss of activity.
Physiological Role of APF. As I indicated above, fungal
starch is unique in that it is a wall component composed of
only short chain amylose molecules. In an earlier paper Dodd
and I (3) suggested that the physiological role of fungal
amylose may be intimately related to its being a short chain
Table I. Relative Effectiveniess of Various Procedures Used to
Isolate APF
Fraction
Yield of APF Units'
Crude extract
30% ammonium sulfate
60%Wc ammonium sulfate
90% ammonium sulfate
pH 4.2 pellet2
pH 4.2 supernatant3
pH 9.5 pellet2
pH 9.5 supernatant'
10%c, ammonium sulfate4
20% ammonium sulfate4
30% ammonium sulfate4
100
85
5
3
100
0
0
64
85
0
1
1 APF unit: the amount required to precipitate 10%c of the
amylose.
2 Pellet redissolved in 0.01 M tris (pH 7.0).
3 pH adjusted to 7.0.
4These fractions were obtained by refractionating the original
30%/ ammonium sulfate fraction.
molecule. The presence of an amylose-precipitating factor may
serve to prevent the amylose-synthesizing enzymes from producing amylose molecules too long for their physiological role.
LITERATURE CITED
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2. BAILEY, J. M. AND W. J. WHELAN. 1961. Physical properties of starch. I. Relationship between iodine stain and chain length. J. Biol. Chem. 236: 969973.
3. DODD, J. L. AND D. A. MICCRACKEN. 1972. Starch in fungi. Its molecular structure in three genera and an hypothesis concerning its physiological role.
Mycologia 64: 1341-1343.
4. Doi, A. 1967. Enlargement of amylopectin by ADP-d-glucose: a-1,4-glucan
a-4 glucosyltransferase of spinach. Biochim. Biophys. Acta 146: 603-605.
5. GOLDSTEIN, I. J., C. E. HOLLERMAN, AND J. M. MERRICK. 1965. Protein-carbohydrate interaction. I. The interaction of polysaccharides with concanavalin A. Biochim. Biophys. Acta 97: 68-76.
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A with polysaccharides. Biochemistry 4: 876-883.
7. HAMADA, I. 1961. The mechanisms of starch accumulation in rice plants. VI.
Identification of amylose-precipitating substances contained in rice grains.
Chem. Abstr. 61: 4714 g (1964).
8. MCCRACKEN, D. A. AND J. L. DODD. 1971. Molecular structure of starch-type
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174: 419.
9. MCCRACKEN', D. A., M. J. NADAXAVKAREN, AND J. L. DODD. 1973. Starch in
fungi. II. Induction of amyloidity in members of diverse genera. Amer. J.
Bot. 60: 940-943.
10. MEEUSE, B. J. D. AND D. M. HALL. 1973. Studies on the cell wall starch of
Hericium. Ann. N.Y. Acad. Sci. 210: 39-45.
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