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Isolation of a polydisperse high-molecular-weight glycogen from rat liver
ANALYTICAL
BIOCHEMISTRY
111,
Isolation
137-145 (1981)
of a Polydisperse High-Molecular-Weight Glycogen from Rat Liver’ DORB
M. HAVERSTICK
AND ALVIN
H.
GOLD
Department of Pharmacology, St. Louis University School of Medicine, St. Louis, Missouri 63104 Received September 15, 1980 A new method is described for the isolation of glycogen from rat liver using centrifugation, gentle heating, and gel chromatography. The prepared polysaccharide was judged by both sucrose density gradient centrifugation and the absorbance spectrum of an I,-glycogen complex to be highly branched, polydisperse, and of an unusually high molecular weight upon comparison to other glycogens. Using adult fasted rats, this glycogen was shown to be better than high-molecular-weight cold water-ethanol extracted glycogen for the binding of glycogen metabolizing enzymes. Further, the addition of 0.5% (w/v) of the glycogen to a crude liver extract from newborn rats facilitated the isolation of an almost 700-fold purified glycogen synthase with 40% recovery. It is suggested that this glycogen could also be used to study the role of enzyme binding in the regulation of carbohydrate metabolism.
phorylase binding to endogenous glycogen. The enzyme-glycogen complex can be separated from other components of liver homogenates by high-speed centrifugation and glycogen can be degraded with endogenous phosphorylase upon dialysis of the enzyme-glycogen complex against a phosphate-containing buffer. Phosphate ion is subsequently removed by a second dialysis and synthase and phosphorylase can be separated by ion-exchange chromatography on DEAE-cellulose, to be used for studies as such (6) or subjected to further purification (8). This method works well with normal, fed rats where the glycogen content is within the range of 3-5% of liver wet weight. With adrenalectomized fasted rats (9), alloxan diabetic rats (10,l l), as well as newborn rats (12), glycogen contents are usually less than 1% of liver wet weight. Differential centrifugation does not uniformly result in the synthase remaining in a solubilized form for (NH&SO, precipitation (7) and usually a portion of the enzyme
Recently, several methods have been developed for isolation of glycogen synthase (l-7) each have different degrees of difficulty and success regarding purity and yield. These procedures include (NH&SO, or ethanol precipitation of solubilized synthase followed by ion-exchange chromatography, affinity chromatography, calcium phosphate gel chromatography, or disulfide interchange as a means to separate the synthase from glycogen before further isolation techniques are used. In general, these methods seem harsh in that severe manipulative procedures are required for preparation of the enzyme essentially free of contaminating proteins and glycogen. A more gentle technique has been developed (8) and successfully used in this laboratory (6), for enzyme isolation from liver. Briefly, the method utilizes the advantageous property of synthase and phos’ Supported by NIH grants HD-07788 and AM-21 149 as well as by grants from the Greater St. Louis Diabetic Childrens Welfare Association and the American Diabetes Association. 137
0003-2697/81/030137-09$02.00/O Copyright All rights
0 1981 by Academic Press, Inc. of reproduction in any form reserved.
138
HAVERSTICK
sediments with the microsomal protein fraction from which extraction is difficult. The addition of exogenous high-molecular-weight glycogen (13) to bind solubilized synthase (14) prior to further isolation has not been extensively utilized. In some preparations where synthase is bound to liver glycogen, removal of the polysaccharide requires the addition of amylase (45). Also, preparation of high-molecularweight glycogen by the method of Bueding and Orrell (13) is difficult and the yield is variable. The present report describes a simple method for the preparation of a highly branched, polydisperse, high-molecularweight glycogen as judged by sucrose density gradient centrifugation and the absorption spectrum of an I,-glycogen complex. The glycogen can be used to isolate synthase and phosphorylase from liver extracts of animals when endogenous glycogen is low. The method for enzyme isolation, described in this report, does not depend upon the addition of exogenous amylase for hydrolytic removal of the added polysaccharide. METHODS
AND MATERIALS
Preparation of polydisperse, high-molecular-weight glycogen. Glycogen pellets
were prepared from an 8000g supernatant from the homogenate of livers of normal, fed Sprague-Dawley rats (male, 250-300 g) by centrifugation at 50,OOOg (60 min). The preparation of the glycogen pellets is the same as that used for starting material for isolation of glycogen synthase described previously (6,7). Briefly, the method consists of blending livers in 3 vol of ice-cold 0.25 M sucrose containing 0.1 M glycylglycine, pH 7.4, 0.1 M KF and 0.02 M 2mercaptoethanol (Buffer A). An 8000g (10 min) supernatant was prepared from the homogenate and packed glycogen pellets were obtained by centrifugation of this supernatant at 50,OOOg (60 min). After re-
AND
GOLD
moval of the supernatant and microsomal fraction overlaying the glycogen, the pellets were washed by recentrifugation at 50,OOOg (60 min) following resuspension of the glycogen pellets in 0.1 M glycylglycine, pH 7.4, containing 0.1 M KF, 0.02 M 2mercaptoethanol and 20% (v/v) glycerol (Buffer B). The pellets contained about 60-70% of synthase and phosphorylase activities, assayed by standard procedures described previously (6,7) as well as 60% of glycogen as measured by the anthrone method (15), both values determined relative to the 8000g supernatant. The pellets were stored at -80°C and could be used for either the isolation of synthase (6-8), or preparation of polydisperse, high-molecular-weight glycogen described below. This high-molecular-weight glycogen, herein termed glycogen Type-X, was prepared by suspending the 50,OOOg pellets in 10 ml of buffer (10 mM EDTA, pH 7.4), per gram of frozen pellet. The solution was heated to 60°C and maintained at this temperature for 15 min to assure inactivation of enzymes and denaturation of proteins complexed to the glycogen. After the heat step, the solution was cooled and centrifuged at 15,OOOg (10 min) to remove denatured proteins. About 17-20% of the glycogen was lost by centrifugation. To remove ions, peptides and other byproducts of the heat denaturation step, the supernatant from the 15,OOOg centrifugation was passed through a Sephadex G-25 column (30 x 4.4 cm) equilibrated with 10 mM EDTA, pH 7.4, at room temperature. The glycogen, identified as a white solution, was collected as a single fraction and concentrated into pellets by centrifugation at 75,000g (60 (min). The pellets were stored at -80°C and could be used as such upon suspension. For weight determinations of the amount of glycogen used in the experiments to be described, the pellets were freeze-dried and stored at 4°C until use. The final storage form of the glycogen yielded no noticeable differences in properties of the glycogen
HIGH-MOLECULAR-WEIGHT
and, for convenience, the freeze-dried form was generally used. Other glycogen forms used. In order to produce a glycogen similar to the glycogen acceptor for glucose used for the assay of synthase and phosphorylase activities (6,7,11), an alkali digest of Type-X was prepared. For this preparation, Type-X glycogen was heated at 100°C in 20% KOH (final concentration) for 30 min. The glycogen was precipitated from solution with 66% ethanol (final concentration) and centrifuged at 2200 rpm (10 min) in a Sorvall GLC-1. After resuspending the glycogen pellet in water and reprecipitation with 66% ethanol, the glycogen was collected by a second centrifugation and freeze-dried before use. The KOH-ethanol treatment is essentially that used for extracting glycogen from tissue for quantitative determination by the anthrone method of assay (15). The glycogen prepared by this method is termed KOH-rat.’ A high-molecular-weight glycogen was isolated from rat liver using the cold waterethanol extraction procedure described by Bueding and Orrell (13). This glycogen is termed high-molecular-weight rat (HighM W-rat). Glycogen Type III from rabbit liver (Sigma Chemical Co., St. Louis, MO.) is the standard glycogen used for assay of synthase and phosphorylase (6,7,11). The glycogen is prepared from liver by hot KOH digestion of tissue followed by ethanol precipitation as described above. This glycogen will be termed KOH-rabbit. Glycogen Type V-S, also purchased from Sigma Chemical Company, is a high-molecular-weight glycogen obtained from rab2 Abbreviations used: KOH-rat, glycogen prepared from Type-X glycogen by hot KOH digestion and ethanol precipitation; KOH-rabbit, glycogen prepared from whole liver by the same KOH-ethanol treatment; GE-complex, glycogen-enzyme complex; HighMW-rabbit and High-MW-rat, high-molecular-weight glycogens obtained from rabbit and rat respectively, prepared by the method of Beuding and Orrell (13).
GLYCOGEN
ISOLATION
139
bit liver prepared by the method of Bueding and Orrell (13). This glycogen is termed high-molecular-weight rabbit (High-MWrabbit). To determine if the heat denaturation of the proteins associated with the 50,000c~ glycogen pellets altered the glycogen, the glycogen-enzyme (GE) complex, herein termed GE-complex, was used as well in the characterizations to be described. The GEcomplex was prepared from the 50,OOOg glycogen pellet, described above, by suspending the preparation in 10 ml buffer (10 mM EDTA, pH 7.4), per gram of frozen pellet. Although enzymes were inactive, protein remained associated with glycogen. Analytical techniques used. Sucrose density gradient centrifugation was done to compare the sedimentation characteristics of the various glycogens. Five-milliliter tubes were prepared with linear 5-40% aqueous sucrose gradients as described by Martin and Ames (16). The glycogens, 0.1 ml of 5 mg/ml in water, were layered onto the gradient tubes and centrifugation was done at 12,500 rpm for 105 min in a Beckman SW 50.1 rotor. Three hundred-microliter fractions were collected dropwise by piercing the bottom of each gradient tube. The fractions were assayed for glycogen by the method described by Krisman (17) whereby 1.0 ml of the KI-I,-CaCl, reagent is added to each fraction, mixed, and the absorbance is determined at 460 nm. The KI-I,-CaCl, solution is 10% KI and 1% I, in saturated CaCl,. Determination of the absorbance spectra of iodine complexes of the various glycogens was done essentially as described by Krisman (17). Two hundred and fifty micrograms of each glycogen, as determined by the anthrone assay, was mixed with 3.0 ml of the KI-I,-CaCl, solution and the absorbance was scanned from 600 to 375 nm using a GCA/McPherson 700 series uv-visible recording spectrophotometer. The reference cuvette contained only the KI-I,-CaCI, solution.
140
HAVERSTICK
Proteins were assayed by the methods of Bradford (18) or Lowry (19). Glycogen was quantitatively determined by the method of Krisman (17) or the anthrone method of Hassid and Abraham (15). Glycogen synthase and phosphorylase were assayed by the filter paper method of Thomas et al. (20) as described previously (6,7,11). Briefly, [i4C]glucose is transferred from UDP-[14C]glucose to glycogen acceptor for synthase determinations whereas for phosphorylase, [14C]glucose is transferred from [14C]glucose-l-P to glycogen. All reagents used were of the best quality available and were purchased from either Fisher Scientific, Springfield, New Jersey or Sigma Chemical Company, UDP-[14C]glucose was purchased from New England Nuclear, Boston, Massachusetts, and [‘“Clglucose-l-P from Amersham, Arlington Heights, Illinois. RESULTS
Table 1 shows a typical isolation of Type X glycogen. For convenience of comparison, yield is expressed relative to the 50,OOOg pellets used as starting material. Since these pellets contain about 60% of the glycogen in the 8OOOg supernatant from which they were prepared (6), the final yield of Type-X glycogen is roughly 50% of the 8000g supernatant. These values were determined by the anthrone method for glycogen TABLE PREPARATION
OF GLYCOGEN
Volume step 50,ooog Pellet 15,coog Supernatant Sephadex G-25 &late
1
(ml)
Roteinb
TYPE-X”
GlyCOgell~
(mgiml) (mdml)
Percentage yield (Glycogen)
47 42
0.50 0.08
4.6 4.3
loo 83
64
nil”
3.0
83
0 This represents an average preparation 300-g rats. b Determined by the Bradford method (18). CDetermined by the anthrone method (1%. d Not measurable by either the Bradford protein determination.
using livers
or Lowry
from four
methods
for
AND GOLD
FIG. 1. Sucrose density gradient centrifugation of isolated glycogens. Linear 5 to 40% gradient tubes were prepared with aqueous sucrose solutions and loaded with 0.5 mg of the various glycogens. Centrifugation was at 12,500 rpm for 105 min in an SW 50.1 rotor. Fractions (300 ~1) were collected dropwise by piercing the bottom of each tube and glycogen was identified by the addition of 1.0 ml of Krisman’s KI-I,-CaCl, solution (17). The absorbance of each fraction, determined at 460 nm, is expressed relative to the peak fraction (100%). Top to bottom of the tube reads left to right. (A) Type-X (B) KOH-rabbit (C) GE-complex (D) KOH-rat (E) High-MW-rabbit (F) High-MW-rat.
analysis. It should be recognized that glycolipid and glycoprotein components of the SOOOg supernatant contain anthrone-positive material which would be removed during the isolation of the Type-X glycogen. This would indicate artificially low yields of glycogen in the final preparation. The sedimentation characteristics resulting from sucrose density gradient centrifugation of the glycogen preparations are presented in Fig. 1. It can be seen that Type-X and GE-complex glycogens (Figs. 1A and C, respectively) sediment as polydisperse, high-molecular-weight compounds relative to either the KOH-rabbit or the KOH-rat glycogens (Figs. 1B and D, respectively) which have been established to be low-molecular-weight polysaccharides (9,13,21). It is also noted that there is some low-molecular-weight material associated with the Type-X and GE-complex glycogens
HIGH-MOLECULAR-WEIGHT
GLYCOGEN
ISOLATION
141
which does not enter the sucrose density gradient. The nature of this material is not known. The KOH-rat glycogen (Fig. 1D) was more polydisperse than KOH-rabbit (Fig. 1B) as shown by the slightly greater amount of high-molecular-weight material sedimenting in the tube. The High-MWrabbit (Fig. 1E) and High-MW-rat (Fig. 1F) glycogens show sedimentation patterns which are similar in that both appear polydisperse and show a peak of material which sediments slower than the major peak of Type-X or GE-complex glycogens but which sediments faster than the major component of KOH-rat and KOH-rabbit glycogens. These results suggest that the cold 0.25 water-ethanol extraction procedure of Bueding and Orrell (13) results in a species of glycogen with a molecular weight less WAYELENGlH lnml than the Type-X glycogen but greater than the KOH-rabbit and KOH-rat glycogens; FIG. 2. Absorbance spectra of the I,-glycogen complexes. Each type of glycogen (250 pg) was mixed with however, the cold water-ethanol prepara3.0 ml of the KI-I,-CaCI, solution of Krisman (17). tion is less polydisperse than the Type-X The absorbance was scanned from 600 to 375 nm glycogen . against a reference cuvette containing only the Comparative absorption spectra of the I2 Krisman reagent. (A) upper: GE-complex, lower: Type complexes of the various glycogens are X (B) upper: KOH-rabbit, lower: KOH-rat (C) upper: presented in Fig. 2. Krisman (17) has High-MW-rabbit, lower: High-MW-rat. shown the wavelength of maximum absorbance (A,,3 of the I,-glycogen complexes pectin-I, complexes which are nonbranched is shifted to longer wavelengths upon or less branched than glycogen, respecdecreased branching of glycogen. The ab- tively. The A,,, of cold water-ethanol sorption spectra were done in the present glycogen of Bueding and Orrell (13) is also study to determine the degree of branching at a shorter wavelength suggesting a highly of Type-X glycogen relative to the other branched polymer (Fig. 2C, upper, Highpreparations. The extent of branching might MW-rabbit; lower, High-MW-rat). This glyreflect the capability of the Type-X material cogen has been shown to readily bind to bind glycogen-metabolizing enzymes in soluble synthase (14). the liver extracts. In order to facilitate the isolation of It can be seen in Fig. 2 that the A,,, glycogen synthase from liver of newborn of Type-X (Fig. 2A, lower curve) and GE- rats, wherein glycogen contents are low complex (Fig. 2A, upper curve) is more (12), Type-X glycogen was added to an clearly defined and at a shorter wavelength 8000g (10 min) supernatant extract of liver than the A,,, of KOH-rabbit (Fig. 2B, homogenized in 5 vol (v/w) of Buffer A. upper curve) suggesting the high-molecularThe results of the experiment are shown weight glycogens are highly branched. in Table 2 where varying amounts of Type-X These results agree with observations by glycogen are added to the SOOOg extract, Krisman (17) wherein the A,,, of glycogen the preparation centrifuged (75,OOOg, 60 is less than the A,,, of amylose or amylomin), and the synthase activity determined
142
HAVERSTICK
AND GOLD
TABLE ISOLATION
GLYCOGEN
OF LIVER
Percentage glycogen”
2
SYNTHASEFROM
Control
NEWBORN
0.25
RATS
0.50
1.0
Step
Yield
Purity*
Yield
Purity
Yield
Purity
Yield
Purity
8,OOOg supernatantr 75,OOOg supernatant 75,OOOg microsomal layer 75,OOOg pellet
100 5 25 38
0.1 1.2 9
100 11 29 46
0.2 1.1 35
100 23 70
0.1 0.6 23
100 13 16 50
0.1 0.06 28
U Percentage glycogen indicates the final concentration (w/v) of glycogen Type-X added to an SOOOgsupernatant prepared from a homogenate of livers of 24- to 48-h-old rats (homogenized in 5 vol of 0.1 M glycylglycine, pH 7.4, 0.25 M sucrose, 0.1 M KF, and 0.02 M 2-mercaptoethanol, Buffer A). c Purity represents the ratio of the specific activity (units synthaseimg protein) to the specific activity of the SOOOgsupernatant. c The 8OOOg supernatant was centrifuged at 75,OOOg for 60 min and three fractions were isolated: supernatant: soft microsomai layer (resuspended in 50% of the 8OOOgsupernatant volume in 0.1 M glycylglycine, pH 7.4, 20% (v/v) glycerol, 0.1 M KF, and 0.02 M 2-mercaptoethanol, Buffer B): firmly packed glycogen pellet (resuspended in the same manner as the microsomal layer).
in the resulting supernatant, microsomal, and glycogen pellet fractions. It can be seen that increasing the concentration of Type-X glycogen results in an increase in the amount of synthase sedimenting in the glycogen pellet when compared to the preparation to which no Type-X glycogen was added. It is noted that there is some glycogen in the control preparation which sediments as a pellet containing about 38% of the synthase in the 80008 supernatant. However, this pellet contains more protein as reflected by the specific enzyme activity (units/mg protein). In this preparation it is about one-fourth the specific activities of the preparations to which Type-X glycogen was added. The results show also that the addition of 0.5% (w/v) of Type-X glycogen is optimal in terms of the yield of synthase obtained in the pellet although enzyme purity is somewhat less than with the other preparations. The glycogen pellet obtained as outlined in Table 2 can be taken through room temperature phosphorolysis in the presence of phosphate buffer to remove glycogen, followed by DEAE-cellulose (DE-52, Whatman) ion-exchange chromatography for sep-
aration of synthase and phosphorylase (6). The isolation of synthase in this experiment is shown in Table 3. For this experiment, the glycogen pellet of the 0.5% (w/v) addition of Type-X glycogen (Table 2) was centrifuged a second time at 75,OOOg (60 min). The glycogen pellet from the second centrifugation was then suspended in modified Buffer B (Buffer B at pH 6.9 containing 0.05 M KH,PO,) and dialyzed overnight at room temperature against this buffer. The phosphate was removed by a second dialysis, in the cold, against Buffer B. Comparative anthrone analysis shows that about 90% of the glycogen was removed by this method. This solution (cleared solution, Table 3) was then adsorbed to a DE-52 column (7 x 2 cm) equilibrated with Buffer B. After the “breakthrough” fraction (as monitored by 280-nm absorbance) washed from the column, phosphorylase was eluted with Buffer B supplemented with 0.1 M NaCl and synthase eluted in Buffer B containing 0.2 M NaCl. Sodium chloride was removed from the peak synthase fractions by dialysis against Buffer B; these fractions represent the “column eluate” of Table 3. It can be seen that synthase isolated by this
HIGH-MOLECULAR-WEIGHT
GLYCOGEN
TABLE ISOLATION
OF SYNTHASE
FROM
LIVERS
143
ISOLATION
3
OF NEWBORN
ANIMALS
USING
TYPE-X
GLYCOCEN
Fraction
Protein (total mg)
Units (synthase)
S.A.”
Purity” fold
Yield (%)
8OOOgsupernatant First 75,OOOg pellet Second 75,OOOgpellet Cleared solution” Column eluate
1075.0’ 35.0 12.8 4.8 0.6
115 80 55 75 46
0.11 2.3 4.3 15.6 76.7
1.0 21 39 142 697
100 70 48 65 40
’ Specific activity, defined as units synthaseimg protein. B Purity is defined as in Table 2. V Equivalent to 19 g (wet wt) of liver from pooled liters of 24- to 4%h-old rats. ” Cleared solution refers to the second 75,OOOg pellet after endogenous phosphorolysis the text.
procedure is 700-fold purified relative to the 80008 extract and is about eight times more pure than when a DE-52 ion-exchange separation is used to isolate the synthase from an (NH&SO, (45% saturation) fraction as described previously by this laboratory (7). Table 3 is representative of the majority of preparations by this method; however, on two occasions enzyme purifications of 900-fold relative to the 8000g supernatant have been obtained. Fasting of adult rats for 16-24 h depletes liver glycogen to less than 1% of tissue wet weight resulting in the partial solubilization of glycogen synthase and phosphorylase. The 8000g supernatant from the fasted rats was used as synthase and phosphorylase source material to compare the efficiency of enzyme binding to Type-X and High-MWrabbit glycogens, the latter of which binds solubilized synthase quite readily (14). For this experiment, 1% (w/v) of the glycogen was added to the supernatant, the solution was mixed and centrifuged at 75,OOOg (60 mitt) to separate supernatant, microsomal layer, and glycogen pellet. A control was identically treated except that exogenous glycogen was not added to the 8000g supernatant. The choice of 1% (w/v) glycogen is based on a comparative study with adult liver preparations at varying concentrations of glycogen as done with the liver extracts
as described in
of young rats shown in Table 2 (data for the adult is not shown). The glycogen pellets were centrifuged a second time (75,OOOg; 60 min) after suspension in Buffer B. The washed glycogen pellets were resuspended in the phosphate-containing pH 6.9 buffer, as described for Table 3, for the endogenous phosphorolysis of the added glycogens. Phosphate was removed by a second dialysis against Buffer B and each fraction of the entire preparation was assayed for protein and glycogen contents as well as for synthase and phosphorylase activities. Table 4 shows the units of synthase and phosporylase activity per milligram of protein and the percentage recovery of the enzymes relative to the 8000g starting material for each fraction of the control and glycogen-supplemented preparations. The results show that after the first 75,000g centrifugation, the control fraction had the majority of synthase and phosphorylase activities in the supernatant whereas there was no synthase and very little phosphorylase in the supernatant of either the High-MW-rabbit or Type-X glycogen-supplemented extracts. With the Type-X glycogen, most of the synthase and phosphorylase recovered (65% of synthase, 59% of phosphorylase) sediments with the glycogen pellet. However, with the High-MW-rabbit glycogen-treated preparation, the synthase is equally distributed
144
HAVERSTICK
AND
TABLE ISOLATION
OF SYNTHASE
AND
4
PHOSPHORYLASE
FROM
Control Phosphorylase Yield
8,OoOg 75,000~ 75,OOOg 75,OOOg Second Cleared
Supernatant Supernatant Microsomal layer Pellet 75.OOOg pellet solution
” Definition
for S.A.,
S.A.”
(%)
S.A.
0.20 0.14 0.08 0.06
loo 50 8 4
5.0 5.0 1.0 1.0
-
-
and cleared
solution
LIVERS
High
Synthase
Fraction
GOLD
100 61 5 I -
DISCUSSION
This investigation describes a method for preparing a high-molecular-weight polydisperse glycogen from rat liver. The procedure is relatively simple and mild in that
RATS Type-X
Phosphorylase
Yield
Phosphorylase
Synthase Yield
S.A.
(%I
S.A.
(%)
S.A.
0.2 0.0 0.4 3.3 19.0 4.8
100 0 56 46 23 10
5.2 0.7 5.7 23.3 182.0 86.0
100 10 34 16 9 8
0.2 0.0 0.3 2.0 9.1 6.8
are the same as for Table
between microsomal and glycogen pellet fractions and the majority of total phosphorylase recovered (57%) is in the microsomes. Of significance as well is the observation that there is a threefold greater recovery of synthase and phosphorylase in the cleared solution of the Type-X glycogen preparation than of the High-MW-rabbit glycogen-treated preparation even though both glycogens were initially at the same concentration. These results suggest the polydisperse nature of the Type-X glycogen affords protection for synthase and phosphorylase activities. In addition, the Type-X glycogen possibly contains components which more readily bind these enzymes than does the cold water-ethanol extracted High-MW-rabbit glycogen. Analyses before and after phoshorolysis show that both glycogen-supplemented preparations lost about 96% of anthrone-positive material suggesting that the glycogens are equally efficient substrates for phosphorylase and other degradative enzymes which might be present.
ADULT
MW-rabbit
Synthase
Yield (%)
OF FASTED
Yield (%) loo 0 33 60 40 30
Yield S.A.
cm
4.2 0.2 4.0 25.0 105.0 146.0
100 3 25 40 22 27
3.
the glycogen pellets obtained by high-speed centrifugation and complexed with synthase and phosphorylase can be used for starting material to isolate either enzymes or the polysaccharide (3,4,6,8). The method of glycogen preparation is mild in that a heat step in the absence of strong alkali appears sufficient to denature proteins of the enzymeglycogen complex. In addition, ethanol precipitation of glycogen is avoided as is the use of other organic solvents, as described by Bueding and Orrell (13). The polydisperse nature of the Type-X glycogen provides a structure to which synthase can bind for extraction from tissue homogenates which have low endogenous glycogen. Enzyme isolation procedures developed for normal animals can then be followed (6-8). The binding of phosphorylase as well as synthase to the high-molecular-weight glycogen allows for endogenous phosphorolysis of the added glycogen to remove the polysaccharide prior to enzyme isolation and does not necessitate the addition of amylase for hydrolysis of the polysaccharide (4,5), or removal of glycogen by chemical dissociation (3). The Type-X glycogen seems to protect both synthase and phosphorylase as there is no loss of either activity during the phosphorolysis at room temperature. This Type-X glycogen should prove suitable for isolation of synthase and phosphorylase
HIGH-MOLECULAR-WEIGHT
from livers of glycogenic hormone-deficient animals where glycogen levels are low (e.g., adrenalectomized, diabetic). It is interesting to note as well that the concentration of Type-X glycogen added to the 8OOOg supernatant is, when calculated on the amount added per gram liver wet weight, within the physiological range of 3-6%. Since High-MW-rabbit glycogen was added at the same concentration as Type-X, the greater fraction of synthase and phosphorylase bound to Type-X glycogen suggests that the Type-X material contains components that bind these enzymes more efficiently than the High-MW-rabbit glycogen. This observation suggests that Type-X glycogen might be used in further studies to describe the nature of the interaction between enzymes of glycogen metabolism and the polysaccharide (14).
GLYCOGEN
145
ISOLATION
5. Jett, M. F., and Soderling, Chem. 254,6739-6745.
T. R. (1979)
J. Biol.
6. Gold, A. H., Dickemper, D., and Haverstick, D. M. (1979) Mol. Cell. Biochem. 25, 47-59. 7. Haverstick, D. M., and Gold, A. H. (1980)1. Viol.
Chem. 255, 1351- 1357. 8. Lin, D. C., and Segal. H. L. (1973)5. Biol. Chem. 248, 7007-7011. 9. Mersmann, H. J., and Segal, H. L. (1969) J. Biol. Chem. 244, 1701-1704. 10. Gold, A. H. (1970) J. Biol. Chem. 245, 903-906. 11. Haverstick, D. M., Dickemper, D., and Gold, A. H. (1979) Biochem. Biophys. Res. Commun. 87, 177- 183. 12. Gold. A. H., and Haverstick, D. M. (1977) Arch. Biochem. Biophys. 184, 441-452. 13. Bueding, E., and Orrell, S. A. (1964)5. Biol. Chem. 239, 4018-4020. 14. Ernest, M. J., and Kim, K.-H. (1974) J. Biol. Chem. 249, 5011-5018. 15. Hassid, W. Z.. and Abraham, S. (1957)in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 3, pp. 34-50, Academic Press, New York. 16. Martin, R. G., and Ames, B. N. (1961) J. Biol.
Chem. 236, 1372- 1379.
REFERENCES 1. Sanada, Y., and Segal, H. L. (1971) Biochem. Biophys. Res. Commun. 45, 1159-1168. 2. Hussein, A. I., and Mendicino, J. (1973) J. Bio/. Chem. 248,685-696. 3. McVerry, P. H., and Kim, K.-H. (1974) Biochemistry 13,3505-3511. 4. Killilea, S. D., and Whelan, W. J. (1976) Biochemistry 15, 1349- 1356.
17. Krisman, C. R. (1962) Anal. B&hem. 4, 17-23. 18. Bradford, M. M. (1976) Anal. Biochem. 72, 248254. 19. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. 20. Thomas, J. A., Schlender, K. K., and Larner, J. (1968) Anal. Biochem. 25, 486-499. 21. Orrell, S. A., and Bueding, E. (1964)J. Biol. Chem. 239, 4021-4026.
BIOCHEMISTRY
111,
Isolation
137-145 (1981)
of a Polydisperse High-Molecular-Weight Glycogen from Rat Liver’ DORB
M. HAVERSTICK
AND ALVIN
H.
GOLD
Department of Pharmacology, St. Louis University School of Medicine, St. Louis, Missouri 63104 Received September 15, 1980 A new method is described for the isolation of glycogen from rat liver using centrifugation, gentle heating, and gel chromatography. The prepared polysaccharide was judged by both sucrose density gradient centrifugation and the absorbance spectrum of an I,-glycogen complex to be highly branched, polydisperse, and of an unusually high molecular weight upon comparison to other glycogens. Using adult fasted rats, this glycogen was shown to be better than high-molecular-weight cold water-ethanol extracted glycogen for the binding of glycogen metabolizing enzymes. Further, the addition of 0.5% (w/v) of the glycogen to a crude liver extract from newborn rats facilitated the isolation of an almost 700-fold purified glycogen synthase with 40% recovery. It is suggested that this glycogen could also be used to study the role of enzyme binding in the regulation of carbohydrate metabolism.
phorylase binding to endogenous glycogen. The enzyme-glycogen complex can be separated from other components of liver homogenates by high-speed centrifugation and glycogen can be degraded with endogenous phosphorylase upon dialysis of the enzyme-glycogen complex against a phosphate-containing buffer. Phosphate ion is subsequently removed by a second dialysis and synthase and phosphorylase can be separated by ion-exchange chromatography on DEAE-cellulose, to be used for studies as such (6) or subjected to further purification (8). This method works well with normal, fed rats where the glycogen content is within the range of 3-5% of liver wet weight. With adrenalectomized fasted rats (9), alloxan diabetic rats (10,l l), as well as newborn rats (12), glycogen contents are usually less than 1% of liver wet weight. Differential centrifugation does not uniformly result in the synthase remaining in a solubilized form for (NH&SO, precipitation (7) and usually a portion of the enzyme
Recently, several methods have been developed for isolation of glycogen synthase (l-7) each have different degrees of difficulty and success regarding purity and yield. These procedures include (NH&SO, or ethanol precipitation of solubilized synthase followed by ion-exchange chromatography, affinity chromatography, calcium phosphate gel chromatography, or disulfide interchange as a means to separate the synthase from glycogen before further isolation techniques are used. In general, these methods seem harsh in that severe manipulative procedures are required for preparation of the enzyme essentially free of contaminating proteins and glycogen. A more gentle technique has been developed (8) and successfully used in this laboratory (6), for enzyme isolation from liver. Briefly, the method utilizes the advantageous property of synthase and phos’ Supported by NIH grants HD-07788 and AM-21 149 as well as by grants from the Greater St. Louis Diabetic Childrens Welfare Association and the American Diabetes Association. 137
0003-2697/81/030137-09$02.00/O Copyright All rights
0 1981 by Academic Press, Inc. of reproduction in any form reserved.
138
HAVERSTICK
sediments with the microsomal protein fraction from which extraction is difficult. The addition of exogenous high-molecular-weight glycogen (13) to bind solubilized synthase (14) prior to further isolation has not been extensively utilized. In some preparations where synthase is bound to liver glycogen, removal of the polysaccharide requires the addition of amylase (45). Also, preparation of high-molecularweight glycogen by the method of Bueding and Orrell (13) is difficult and the yield is variable. The present report describes a simple method for the preparation of a highly branched, polydisperse, high-molecularweight glycogen as judged by sucrose density gradient centrifugation and the absorption spectrum of an I,-glycogen complex. The glycogen can be used to isolate synthase and phosphorylase from liver extracts of animals when endogenous glycogen is low. The method for enzyme isolation, described in this report, does not depend upon the addition of exogenous amylase for hydrolytic removal of the added polysaccharide. METHODS
AND MATERIALS
Preparation of polydisperse, high-molecular-weight glycogen. Glycogen pellets
were prepared from an 8000g supernatant from the homogenate of livers of normal, fed Sprague-Dawley rats (male, 250-300 g) by centrifugation at 50,OOOg (60 min). The preparation of the glycogen pellets is the same as that used for starting material for isolation of glycogen synthase described previously (6,7). Briefly, the method consists of blending livers in 3 vol of ice-cold 0.25 M sucrose containing 0.1 M glycylglycine, pH 7.4, 0.1 M KF and 0.02 M 2mercaptoethanol (Buffer A). An 8000g (10 min) supernatant was prepared from the homogenate and packed glycogen pellets were obtained by centrifugation of this supernatant at 50,OOOg (60 min). After re-
AND
GOLD
moval of the supernatant and microsomal fraction overlaying the glycogen, the pellets were washed by recentrifugation at 50,OOOg (60 min) following resuspension of the glycogen pellets in 0.1 M glycylglycine, pH 7.4, containing 0.1 M KF, 0.02 M 2mercaptoethanol and 20% (v/v) glycerol (Buffer B). The pellets contained about 60-70% of synthase and phosphorylase activities, assayed by standard procedures described previously (6,7) as well as 60% of glycogen as measured by the anthrone method (15), both values determined relative to the 8000g supernatant. The pellets were stored at -80°C and could be used for either the isolation of synthase (6-8), or preparation of polydisperse, high-molecular-weight glycogen described below. This high-molecular-weight glycogen, herein termed glycogen Type-X, was prepared by suspending the 50,OOOg pellets in 10 ml of buffer (10 mM EDTA, pH 7.4), per gram of frozen pellet. The solution was heated to 60°C and maintained at this temperature for 15 min to assure inactivation of enzymes and denaturation of proteins complexed to the glycogen. After the heat step, the solution was cooled and centrifuged at 15,OOOg (10 min) to remove denatured proteins. About 17-20% of the glycogen was lost by centrifugation. To remove ions, peptides and other byproducts of the heat denaturation step, the supernatant from the 15,OOOg centrifugation was passed through a Sephadex G-25 column (30 x 4.4 cm) equilibrated with 10 mM EDTA, pH 7.4, at room temperature. The glycogen, identified as a white solution, was collected as a single fraction and concentrated into pellets by centrifugation at 75,000g (60 (min). The pellets were stored at -80°C and could be used as such upon suspension. For weight determinations of the amount of glycogen used in the experiments to be described, the pellets were freeze-dried and stored at 4°C until use. The final storage form of the glycogen yielded no noticeable differences in properties of the glycogen
HIGH-MOLECULAR-WEIGHT
and, for convenience, the freeze-dried form was generally used. Other glycogen forms used. In order to produce a glycogen similar to the glycogen acceptor for glucose used for the assay of synthase and phosphorylase activities (6,7,11), an alkali digest of Type-X was prepared. For this preparation, Type-X glycogen was heated at 100°C in 20% KOH (final concentration) for 30 min. The glycogen was precipitated from solution with 66% ethanol (final concentration) and centrifuged at 2200 rpm (10 min) in a Sorvall GLC-1. After resuspending the glycogen pellet in water and reprecipitation with 66% ethanol, the glycogen was collected by a second centrifugation and freeze-dried before use. The KOH-ethanol treatment is essentially that used for extracting glycogen from tissue for quantitative determination by the anthrone method of assay (15). The glycogen prepared by this method is termed KOH-rat.’ A high-molecular-weight glycogen was isolated from rat liver using the cold waterethanol extraction procedure described by Bueding and Orrell (13). This glycogen is termed high-molecular-weight rat (HighM W-rat). Glycogen Type III from rabbit liver (Sigma Chemical Co., St. Louis, MO.) is the standard glycogen used for assay of synthase and phosphorylase (6,7,11). The glycogen is prepared from liver by hot KOH digestion of tissue followed by ethanol precipitation as described above. This glycogen will be termed KOH-rabbit. Glycogen Type V-S, also purchased from Sigma Chemical Company, is a high-molecular-weight glycogen obtained from rab2 Abbreviations used: KOH-rat, glycogen prepared from Type-X glycogen by hot KOH digestion and ethanol precipitation; KOH-rabbit, glycogen prepared from whole liver by the same KOH-ethanol treatment; GE-complex, glycogen-enzyme complex; HighMW-rabbit and High-MW-rat, high-molecular-weight glycogens obtained from rabbit and rat respectively, prepared by the method of Beuding and Orrell (13).
GLYCOGEN
ISOLATION
139
bit liver prepared by the method of Bueding and Orrell (13). This glycogen is termed high-molecular-weight rabbit (High-MWrabbit). To determine if the heat denaturation of the proteins associated with the 50,000c~ glycogen pellets altered the glycogen, the glycogen-enzyme (GE) complex, herein termed GE-complex, was used as well in the characterizations to be described. The GEcomplex was prepared from the 50,OOOg glycogen pellet, described above, by suspending the preparation in 10 ml buffer (10 mM EDTA, pH 7.4), per gram of frozen pellet. Although enzymes were inactive, protein remained associated with glycogen. Analytical techniques used. Sucrose density gradient centrifugation was done to compare the sedimentation characteristics of the various glycogens. Five-milliliter tubes were prepared with linear 5-40% aqueous sucrose gradients as described by Martin and Ames (16). The glycogens, 0.1 ml of 5 mg/ml in water, were layered onto the gradient tubes and centrifugation was done at 12,500 rpm for 105 min in a Beckman SW 50.1 rotor. Three hundred-microliter fractions were collected dropwise by piercing the bottom of each gradient tube. The fractions were assayed for glycogen by the method described by Krisman (17) whereby 1.0 ml of the KI-I,-CaCl, reagent is added to each fraction, mixed, and the absorbance is determined at 460 nm. The KI-I,-CaCl, solution is 10% KI and 1% I, in saturated CaCl,. Determination of the absorbance spectra of iodine complexes of the various glycogens was done essentially as described by Krisman (17). Two hundred and fifty micrograms of each glycogen, as determined by the anthrone assay, was mixed with 3.0 ml of the KI-I,-CaCl, solution and the absorbance was scanned from 600 to 375 nm using a GCA/McPherson 700 series uv-visible recording spectrophotometer. The reference cuvette contained only the KI-I,-CaCI, solution.
140
HAVERSTICK
Proteins were assayed by the methods of Bradford (18) or Lowry (19). Glycogen was quantitatively determined by the method of Krisman (17) or the anthrone method of Hassid and Abraham (15). Glycogen synthase and phosphorylase were assayed by the filter paper method of Thomas et al. (20) as described previously (6,7,11). Briefly, [i4C]glucose is transferred from UDP-[14C]glucose to glycogen acceptor for synthase determinations whereas for phosphorylase, [14C]glucose is transferred from [14C]glucose-l-P to glycogen. All reagents used were of the best quality available and were purchased from either Fisher Scientific, Springfield, New Jersey or Sigma Chemical Company, UDP-[14C]glucose was purchased from New England Nuclear, Boston, Massachusetts, and [‘“Clglucose-l-P from Amersham, Arlington Heights, Illinois. RESULTS
Table 1 shows a typical isolation of Type X glycogen. For convenience of comparison, yield is expressed relative to the 50,OOOg pellets used as starting material. Since these pellets contain about 60% of the glycogen in the 8OOOg supernatant from which they were prepared (6), the final yield of Type-X glycogen is roughly 50% of the 8000g supernatant. These values were determined by the anthrone method for glycogen TABLE PREPARATION
OF GLYCOGEN
Volume step 50,ooog Pellet 15,coog Supernatant Sephadex G-25 &late
1
(ml)
Roteinb
TYPE-X”
GlyCOgell~
(mgiml) (mdml)
Percentage yield (Glycogen)
47 42
0.50 0.08
4.6 4.3
loo 83
64
nil”
3.0
83
0 This represents an average preparation 300-g rats. b Determined by the Bradford method (18). CDetermined by the anthrone method (1%. d Not measurable by either the Bradford protein determination.
using livers
or Lowry
from four
methods
for
AND GOLD
FIG. 1. Sucrose density gradient centrifugation of isolated glycogens. Linear 5 to 40% gradient tubes were prepared with aqueous sucrose solutions and loaded with 0.5 mg of the various glycogens. Centrifugation was at 12,500 rpm for 105 min in an SW 50.1 rotor. Fractions (300 ~1) were collected dropwise by piercing the bottom of each tube and glycogen was identified by the addition of 1.0 ml of Krisman’s KI-I,-CaCl, solution (17). The absorbance of each fraction, determined at 460 nm, is expressed relative to the peak fraction (100%). Top to bottom of the tube reads left to right. (A) Type-X (B) KOH-rabbit (C) GE-complex (D) KOH-rat (E) High-MW-rabbit (F) High-MW-rat.
analysis. It should be recognized that glycolipid and glycoprotein components of the SOOOg supernatant contain anthrone-positive material which would be removed during the isolation of the Type-X glycogen. This would indicate artificially low yields of glycogen in the final preparation. The sedimentation characteristics resulting from sucrose density gradient centrifugation of the glycogen preparations are presented in Fig. 1. It can be seen that Type-X and GE-complex glycogens (Figs. 1A and C, respectively) sediment as polydisperse, high-molecular-weight compounds relative to either the KOH-rabbit or the KOH-rat glycogens (Figs. 1B and D, respectively) which have been established to be low-molecular-weight polysaccharides (9,13,21). It is also noted that there is some low-molecular-weight material associated with the Type-X and GE-complex glycogens
HIGH-MOLECULAR-WEIGHT
GLYCOGEN
ISOLATION
141
which does not enter the sucrose density gradient. The nature of this material is not known. The KOH-rat glycogen (Fig. 1D) was more polydisperse than KOH-rabbit (Fig. 1B) as shown by the slightly greater amount of high-molecular-weight material sedimenting in the tube. The High-MWrabbit (Fig. 1E) and High-MW-rat (Fig. 1F) glycogens show sedimentation patterns which are similar in that both appear polydisperse and show a peak of material which sediments slower than the major peak of Type-X or GE-complex glycogens but which sediments faster than the major component of KOH-rat and KOH-rabbit glycogens. These results suggest that the cold 0.25 water-ethanol extraction procedure of Bueding and Orrell (13) results in a species of glycogen with a molecular weight less WAYELENGlH lnml than the Type-X glycogen but greater than the KOH-rabbit and KOH-rat glycogens; FIG. 2. Absorbance spectra of the I,-glycogen complexes. Each type of glycogen (250 pg) was mixed with however, the cold water-ethanol prepara3.0 ml of the KI-I,-CaCI, solution of Krisman (17). tion is less polydisperse than the Type-X The absorbance was scanned from 600 to 375 nm glycogen . against a reference cuvette containing only the Comparative absorption spectra of the I2 Krisman reagent. (A) upper: GE-complex, lower: Type complexes of the various glycogens are X (B) upper: KOH-rabbit, lower: KOH-rat (C) upper: presented in Fig. 2. Krisman (17) has High-MW-rabbit, lower: High-MW-rat. shown the wavelength of maximum absorbance (A,,3 of the I,-glycogen complexes pectin-I, complexes which are nonbranched is shifted to longer wavelengths upon or less branched than glycogen, respecdecreased branching of glycogen. The ab- tively. The A,,, of cold water-ethanol sorption spectra were done in the present glycogen of Bueding and Orrell (13) is also study to determine the degree of branching at a shorter wavelength suggesting a highly of Type-X glycogen relative to the other branched polymer (Fig. 2C, upper, Highpreparations. The extent of branching might MW-rabbit; lower, High-MW-rat). This glyreflect the capability of the Type-X material cogen has been shown to readily bind to bind glycogen-metabolizing enzymes in soluble synthase (14). the liver extracts. In order to facilitate the isolation of It can be seen in Fig. 2 that the A,,, glycogen synthase from liver of newborn of Type-X (Fig. 2A, lower curve) and GE- rats, wherein glycogen contents are low complex (Fig. 2A, upper curve) is more (12), Type-X glycogen was added to an clearly defined and at a shorter wavelength 8000g (10 min) supernatant extract of liver than the A,,, of KOH-rabbit (Fig. 2B, homogenized in 5 vol (v/w) of Buffer A. upper curve) suggesting the high-molecularThe results of the experiment are shown weight glycogens are highly branched. in Table 2 where varying amounts of Type-X These results agree with observations by glycogen are added to the SOOOg extract, Krisman (17) wherein the A,,, of glycogen the preparation centrifuged (75,OOOg, 60 is less than the A,,, of amylose or amylomin), and the synthase activity determined
142
HAVERSTICK
AND GOLD
TABLE ISOLATION
GLYCOGEN
OF LIVER
Percentage glycogen”
2
SYNTHASEFROM
Control
NEWBORN
0.25
RATS
0.50
1.0
Step
Yield
Purity*
Yield
Purity
Yield
Purity
Yield
Purity
8,OOOg supernatantr 75,OOOg supernatant 75,OOOg microsomal layer 75,OOOg pellet
100 5 25 38
0.1 1.2 9
100 11 29 46
0.2 1.1 35
100 23 70
0.1 0.6 23
100 13 16 50
0.1 0.06 28
U Percentage glycogen indicates the final concentration (w/v) of glycogen Type-X added to an SOOOgsupernatant prepared from a homogenate of livers of 24- to 48-h-old rats (homogenized in 5 vol of 0.1 M glycylglycine, pH 7.4, 0.25 M sucrose, 0.1 M KF, and 0.02 M 2-mercaptoethanol, Buffer A). c Purity represents the ratio of the specific activity (units synthaseimg protein) to the specific activity of the SOOOgsupernatant. c The 8OOOg supernatant was centrifuged at 75,OOOg for 60 min and three fractions were isolated: supernatant: soft microsomai layer (resuspended in 50% of the 8OOOgsupernatant volume in 0.1 M glycylglycine, pH 7.4, 20% (v/v) glycerol, 0.1 M KF, and 0.02 M 2-mercaptoethanol, Buffer B): firmly packed glycogen pellet (resuspended in the same manner as the microsomal layer).
in the resulting supernatant, microsomal, and glycogen pellet fractions. It can be seen that increasing the concentration of Type-X glycogen results in an increase in the amount of synthase sedimenting in the glycogen pellet when compared to the preparation to which no Type-X glycogen was added. It is noted that there is some glycogen in the control preparation which sediments as a pellet containing about 38% of the synthase in the 80008 supernatant. However, this pellet contains more protein as reflected by the specific enzyme activity (units/mg protein). In this preparation it is about one-fourth the specific activities of the preparations to which Type-X glycogen was added. The results show also that the addition of 0.5% (w/v) of Type-X glycogen is optimal in terms of the yield of synthase obtained in the pellet although enzyme purity is somewhat less than with the other preparations. The glycogen pellet obtained as outlined in Table 2 can be taken through room temperature phosphorolysis in the presence of phosphate buffer to remove glycogen, followed by DEAE-cellulose (DE-52, Whatman) ion-exchange chromatography for sep-
aration of synthase and phosphorylase (6). The isolation of synthase in this experiment is shown in Table 3. For this experiment, the glycogen pellet of the 0.5% (w/v) addition of Type-X glycogen (Table 2) was centrifuged a second time at 75,OOOg (60 min). The glycogen pellet from the second centrifugation was then suspended in modified Buffer B (Buffer B at pH 6.9 containing 0.05 M KH,PO,) and dialyzed overnight at room temperature against this buffer. The phosphate was removed by a second dialysis, in the cold, against Buffer B. Comparative anthrone analysis shows that about 90% of the glycogen was removed by this method. This solution (cleared solution, Table 3) was then adsorbed to a DE-52 column (7 x 2 cm) equilibrated with Buffer B. After the “breakthrough” fraction (as monitored by 280-nm absorbance) washed from the column, phosphorylase was eluted with Buffer B supplemented with 0.1 M NaCl and synthase eluted in Buffer B containing 0.2 M NaCl. Sodium chloride was removed from the peak synthase fractions by dialysis against Buffer B; these fractions represent the “column eluate” of Table 3. It can be seen that synthase isolated by this
HIGH-MOLECULAR-WEIGHT
GLYCOGEN
TABLE ISOLATION
OF SYNTHASE
FROM
LIVERS
143
ISOLATION
3
OF NEWBORN
ANIMALS
USING
TYPE-X
GLYCOCEN
Fraction
Protein (total mg)
Units (synthase)
S.A.”
Purity” fold
Yield (%)
8OOOgsupernatant First 75,OOOg pellet Second 75,OOOgpellet Cleared solution” Column eluate
1075.0’ 35.0 12.8 4.8 0.6
115 80 55 75 46
0.11 2.3 4.3 15.6 76.7
1.0 21 39 142 697
100 70 48 65 40
’ Specific activity, defined as units synthaseimg protein. B Purity is defined as in Table 2. V Equivalent to 19 g (wet wt) of liver from pooled liters of 24- to 4%h-old rats. ” Cleared solution refers to the second 75,OOOg pellet after endogenous phosphorolysis the text.
procedure is 700-fold purified relative to the 80008 extract and is about eight times more pure than when a DE-52 ion-exchange separation is used to isolate the synthase from an (NH&SO, (45% saturation) fraction as described previously by this laboratory (7). Table 3 is representative of the majority of preparations by this method; however, on two occasions enzyme purifications of 900-fold relative to the 8000g supernatant have been obtained. Fasting of adult rats for 16-24 h depletes liver glycogen to less than 1% of tissue wet weight resulting in the partial solubilization of glycogen synthase and phosphorylase. The 8000g supernatant from the fasted rats was used as synthase and phosphorylase source material to compare the efficiency of enzyme binding to Type-X and High-MWrabbit glycogens, the latter of which binds solubilized synthase quite readily (14). For this experiment, 1% (w/v) of the glycogen was added to the supernatant, the solution was mixed and centrifuged at 75,OOOg (60 mitt) to separate supernatant, microsomal layer, and glycogen pellet. A control was identically treated except that exogenous glycogen was not added to the 8000g supernatant. The choice of 1% (w/v) glycogen is based on a comparative study with adult liver preparations at varying concentrations of glycogen as done with the liver extracts
as described in
of young rats shown in Table 2 (data for the adult is not shown). The glycogen pellets were centrifuged a second time (75,OOOg; 60 min) after suspension in Buffer B. The washed glycogen pellets were resuspended in the phosphate-containing pH 6.9 buffer, as described for Table 3, for the endogenous phosphorolysis of the added glycogens. Phosphate was removed by a second dialysis against Buffer B and each fraction of the entire preparation was assayed for protein and glycogen contents as well as for synthase and phosphorylase activities. Table 4 shows the units of synthase and phosporylase activity per milligram of protein and the percentage recovery of the enzymes relative to the 8000g starting material for each fraction of the control and glycogen-supplemented preparations. The results show that after the first 75,000g centrifugation, the control fraction had the majority of synthase and phosphorylase activities in the supernatant whereas there was no synthase and very little phosphorylase in the supernatant of either the High-MW-rabbit or Type-X glycogen-supplemented extracts. With the Type-X glycogen, most of the synthase and phosphorylase recovered (65% of synthase, 59% of phosphorylase) sediments with the glycogen pellet. However, with the High-MW-rabbit glycogen-treated preparation, the synthase is equally distributed
144
HAVERSTICK
AND
TABLE ISOLATION
OF SYNTHASE
AND
4
PHOSPHORYLASE
FROM
Control Phosphorylase Yield
8,OoOg 75,000~ 75,OOOg 75,OOOg Second Cleared
Supernatant Supernatant Microsomal layer Pellet 75.OOOg pellet solution
” Definition
for S.A.,
S.A.”
(%)
S.A.
0.20 0.14 0.08 0.06
loo 50 8 4
5.0 5.0 1.0 1.0
-
-
and cleared
solution
LIVERS
High
Synthase
Fraction
GOLD
100 61 5 I -
DISCUSSION
This investigation describes a method for preparing a high-molecular-weight polydisperse glycogen from rat liver. The procedure is relatively simple and mild in that
RATS Type-X
Phosphorylase
Yield
Phosphorylase
Synthase Yield
S.A.
(%I
S.A.
(%)
S.A.
0.2 0.0 0.4 3.3 19.0 4.8
100 0 56 46 23 10
5.2 0.7 5.7 23.3 182.0 86.0
100 10 34 16 9 8
0.2 0.0 0.3 2.0 9.1 6.8
are the same as for Table
between microsomal and glycogen pellet fractions and the majority of total phosphorylase recovered (57%) is in the microsomes. Of significance as well is the observation that there is a threefold greater recovery of synthase and phosphorylase in the cleared solution of the Type-X glycogen preparation than of the High-MW-rabbit glycogen-treated preparation even though both glycogens were initially at the same concentration. These results suggest the polydisperse nature of the Type-X glycogen affords protection for synthase and phosphorylase activities. In addition, the Type-X glycogen possibly contains components which more readily bind these enzymes than does the cold water-ethanol extracted High-MW-rabbit glycogen. Analyses before and after phoshorolysis show that both glycogen-supplemented preparations lost about 96% of anthrone-positive material suggesting that the glycogens are equally efficient substrates for phosphorylase and other degradative enzymes which might be present.
ADULT
MW-rabbit
Synthase
Yield (%)
OF FASTED
Yield (%) loo 0 33 60 40 30
Yield S.A.
cm
4.2 0.2 4.0 25.0 105.0 146.0
100 3 25 40 22 27
3.
the glycogen pellets obtained by high-speed centrifugation and complexed with synthase and phosphorylase can be used for starting material to isolate either enzymes or the polysaccharide (3,4,6,8). The method of glycogen preparation is mild in that a heat step in the absence of strong alkali appears sufficient to denature proteins of the enzymeglycogen complex. In addition, ethanol precipitation of glycogen is avoided as is the use of other organic solvents, as described by Bueding and Orrell (13). The polydisperse nature of the Type-X glycogen provides a structure to which synthase can bind for extraction from tissue homogenates which have low endogenous glycogen. Enzyme isolation procedures developed for normal animals can then be followed (6-8). The binding of phosphorylase as well as synthase to the high-molecular-weight glycogen allows for endogenous phosphorolysis of the added glycogen to remove the polysaccharide prior to enzyme isolation and does not necessitate the addition of amylase for hydrolysis of the polysaccharide (4,5), or removal of glycogen by chemical dissociation (3). The Type-X glycogen seems to protect both synthase and phosphorylase as there is no loss of either activity during the phosphorolysis at room temperature. This Type-X glycogen should prove suitable for isolation of synthase and phosphorylase
HIGH-MOLECULAR-WEIGHT
from livers of glycogenic hormone-deficient animals where glycogen levels are low (e.g., adrenalectomized, diabetic). It is interesting to note as well that the concentration of Type-X glycogen added to the 8OOOg supernatant is, when calculated on the amount added per gram liver wet weight, within the physiological range of 3-6%. Since High-MW-rabbit glycogen was added at the same concentration as Type-X, the greater fraction of synthase and phosphorylase bound to Type-X glycogen suggests that the Type-X material contains components that bind these enzymes more efficiently than the High-MW-rabbit glycogen. This observation suggests that Type-X glycogen might be used in further studies to describe the nature of the interaction between enzymes of glycogen metabolism and the polysaccharide (14).
GLYCOGEN
145
ISOLATION
5. Jett, M. F., and Soderling, Chem. 254,6739-6745.
T. R. (1979)
J. Biol.
6. Gold, A. H., Dickemper, D., and Haverstick, D. M. (1979) Mol. Cell. Biochem. 25, 47-59. 7. Haverstick, D. M., and Gold, A. H. (1980)1. Viol.
Chem. 255, 1351- 1357. 8. Lin, D. C., and Segal. H. L. (1973)5. Biol. Chem. 248, 7007-7011. 9. Mersmann, H. J., and Segal, H. L. (1969) J. Biol. Chem. 244, 1701-1704. 10. Gold, A. H. (1970) J. Biol. Chem. 245, 903-906. 11. Haverstick, D. M., Dickemper, D., and Gold, A. H. (1979) Biochem. Biophys. Res. Commun. 87, 177- 183. 12. Gold. A. H., and Haverstick, D. M. (1977) Arch. Biochem. Biophys. 184, 441-452. 13. Bueding, E., and Orrell, S. A. (1964)5. Biol. Chem. 239, 4018-4020. 14. Ernest, M. J., and Kim, K.-H. (1974) J. Biol. Chem. 249, 5011-5018. 15. Hassid, W. Z.. and Abraham, S. (1957)in Methods in Enzymology (Colowick, S. P., and Kaplan, N. O., eds.), Vol. 3, pp. 34-50, Academic Press, New York. 16. Martin, R. G., and Ames, B. N. (1961) J. Biol.
Chem. 236, 1372- 1379.
REFERENCES 1. Sanada, Y., and Segal, H. L. (1971) Biochem. Biophys. Res. Commun. 45, 1159-1168. 2. Hussein, A. I., and Mendicino, J. (1973) J. Bio/. Chem. 248,685-696. 3. McVerry, P. H., and Kim, K.-H. (1974) Biochemistry 13,3505-3511. 4. Killilea, S. D., and Whelan, W. J. (1976) Biochemistry 15, 1349- 1356.
17. Krisman, C. R. (1962) Anal. B&hem. 4, 17-23. 18. Bradford, M. M. (1976) Anal. Biochem. 72, 248254. 19. Lowry, 0. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951) J. Biol. Chem. 193, 265-275. 20. Thomas, J. A., Schlender, K. K., and Larner, J. (1968) Anal. Biochem. 25, 486-499. 21. Orrell, S. A., and Bueding, E. (1964)J. Biol. Chem. 239, 4021-4026.