THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 250, No. 5, Issue of March 10, PP. 1892-1897, 1975

Printed in U.S.A.

Intracellular Localization of Fructose 1, GBisphosphate Aldolase*

(Received for publication, June 18, 1974)

RICHARD S. FOEMMEL,~ ROBERT H. GRAY, AND ISADORE A. BERNSTEIN

From the Department of Biological Chemistry and the Cellular Chemistry Laboratory of the Department of En- vironmental and Industrial Health, The University of Michigan, Ann Arbor, ikfichigan &?10&

SUMMARY

Submission of a rat liver homogenate made in 250 mM sucrose-l mM EDTA to centrifugation between 9,500 x g for 10 min and 105,000 X g for 60 min results in the sedimenta- tion of 60 to 70% of the total cellular fructose 1,6-bisphosphate aldolase (EC 4.1.2.13). Under these conditions only about one-quarter of the total triose phosphate dehydrogenase and phosphoglycerate kinase appears in the microsomal fraction. Ultrastructural immunologic localization techniques have demonstrated that the aldolase is associated with the endo- plasmic reticulum, in situ. The binding of this enzyme to the membrane is sensitive to changes in pH with an optimum at 6.0, and to increasing concentrations of NaCl and fructose 1,6-bisphosphate, being about loo-fold more sensitive to the ester than to the inorganic salt.

Traditionally, the glycolytic enzymes have been considered to be located in the soluble portion of the cell (cf. Ref. 1) although there are data which suggest that particular enzymes of this se- quence are associated with specific subcellular particulate ele- ments (cf. Ref. 2).

Green et al. (2) reported that when bovine erythrocytes were treated with 250 mM sucrose and then lysed in distilled water, most of the glycolytic enzymes remained at least partially associ- ated with t.he membranous fraction. Hexokinase, fructose- 1, 6-PZ1 aldolase, and triose phosphate isomerase were recovered in 507, or greater yield by sedimentation at 60,000 X g. How- ever, in the presence of 150 mM NaCl, these enzymes dissociated from the membranes which sedimented at 60,000 X g and re- mained in the supernatant solution. Green and colleagues suggested that all glycolytic enzymes were associated with mem-

* This work is supported by Research Grant ES-00339 from the National Institutes of Health, United States Public Health Serv- ice.

1 Predoctoral Research Trainee, Training Grant 5-TOl-GM- 00187.9 from the National Institutes of Health, United States Public Health Service, and Predoctoral Research Fellow, Institute for Environmental Quality, The University of Michigan. Sub- mitted in partial fulfillment of the requirements for the Ph.D. degree at the University of Michigan. Present address, Diagnos- t,ics Division, Abbott Laboratories, North Chicago, Illinois (50064.

1 The abbreviations used are : fructose-Pn, fructose bisphos- phate; fructose-l,S-Pz, fructose 1,6-bisphosphate; glucose-1,6- Pz, glucose 1,6-bisphosphate.

branes in the bovine erythrocyte. These workers obtained sup- porting data for this concept with Xaccharonayces cerevisiae.

Hernandez and Crane (3) reported the sedimentation of 80 to 90% of the hexokinasc activity when muscle from pig heart was homogenized in 300 mM sucrose and submitted to centrifuga- tion at 105,000 X g. Addition of 400 rnfix KC1 solubilized about one-third of the activity, while increasing the pH from 5 to 9 solubilized nearly all of the enzyme. The enzyme reassociated with the particulate fraction after removal of the salt by dialysis. The solubilization could also be effected by 0.5 mM glucose-6-I’, the enzyme’s product. The authors postulated an equilibrium dependent upon salt and pH between the soluble and particulate states of the enzyme with glucose-6-P as a modulator of this equilibrium.

Rose and Warms (4) localized hexokinase in the mitochondria of cells in an ascitcs tumor. Solubilization was accomplished by exposure to 1 M NaCl or 0.1 m&f glucose-6-P. Again reassoci- ation could be achieved after dialysis. Li and Ch’ien (5) sug- gested that this equilibrium might play a regulatory role since the K, for ATP was about 3 times greater for the soluble than for the bound enzyme.

Roodyn (6, 7) found that when nuclei from rat hepatocytes were ultrasonicated in 250 mM sucrose-O.018 mM CaC12, only 3% of the total fructose-P2 aldolase was released into solution. How- ever, addition of 150 mM NaCl to the sucrose-CaClz mixture caused the solubilization of more than 90% of the enzyme with- out ultrasonication. Clarke et al. (8) found that 20 to 30% of the total fructose-P2 aldolase sedimented when a homogenate of rat brain or muscle in 250 mM sucrose-10 mM Tris-HCl, pH 7.4, was centrifuged at 100,000 x g. A reduction of the pH from 7.4 to 5.5 with lactic acid, but not with acetic acid, resulted in sedimentation of 60% of the brain enzyme but only 10% of the muscle enzyme. These workers proposed that this effect was a function of the differential effect of lactic acid on the binding of the two isozymes of this aldolase in these tissues.

Arnold and Pette (9, 10) found that fructose-P2 aldolase and triose phosphate dehydrogenase from rabbit muscle could be reversibly bound in vitro to F-actin, one of the structural proteins of the muscle contractile apparatus. The binding of the aldolase was sensitive to alkaline pH, to ionic strengths great.er t’han 100 mM, and to the presence of metabolites such as fructose-l, 6-Pz, glucose-l, 6-l’*, dihydroxyacetone-I’, and 2,3-Pz-glycerate. The K, of the bound enzyme was nearly 10 times greater than the K, for the soluble enzyme while the Vmax was twice as great. Hist.o- chemical staining confirmed the relevance of this observation, in situ. Staining for fructose-Pz aldolase at the light microscopic

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level indicated that in sections of rabbit muscle, the enzyme was

located at the sites of the actin filaments.

Data in the present report show that in rat liver, fructose-P’s

aldolase is associated with the endoplasmic reticulum. By dif-

ferential centrifugation of a homogenate of this tissue made in

250 m&f sucrose-l mM EDTA, 60 to 70% of the total cellular

aldolase sedimented in the microsomal fraction. Ultrastructural

immunologic localization techniques showed specific labeling for

fructose-P2 aldolase on and around the endoplasmic reticulum.

The binding is sensitive to changes in pH, and to variations in the

concentration and identity of salts and metabolic intermediates.

IIIETHODS AND MATERIALS

All work was done with adult male rats of the CFN strain (Cars- worth Farms) from a randomly inbred colony maintained in this laboratory.

Subfractionation of 1Zat Liver Homogenate-Microsomal frac- tions were prepared from a 1O70 (w/v) homogenate in 250 mM sucrose-l mM EDTA as described by Dallner (11). Rat liver ho- mogenates were subfractionated into nuclear, mitochondrial, mi- crosomal, and soluble fractions by a slight modification of the method of Schachter (12).

Ellzymatic Assays-The conversion of fructose-1,6-P* to 1,3- Pz-glycerate was measured by the conversion in 10 min at 37” of 32Pi to organically bound [32P]phosphate in the presence of 1 mM ADP, 1 rnM ATP, 1 rn~ NAD+, 1.7 mM MgC12, 1 mM fructose-1,6-Pz, 33 mM Tris-HCl, pH 7.6, 1.8 mM sodium 32P;, and homogenate or extract (0.1 to 1.0 mg of protein) in a final volume of 3.0 ml. After addition of 1 ml of 10% NH40H containing 300 mM Pi, all radio- active P; was precipitated at 0” by the addition of 1.0 ml of mag- nesia mixture (cf. Ref. 13) leaving organically bound 32P, primarily ATP, in solution. Radioactivity was measured by monitoring Cerenkov radiation (14) at the normal setting for tritium in a Packard Tri-Carb 3375 liquid scintillation counter, corrected for quench by the channel’s ratio method (15).

Fructose-P% aldolase was assayed either spectrophotometrically (16) or calorimetrically (17) ; horseradish peroxidase was assayed as described by Maehly and Chance (18), and glucose B-phospha- tase was assayed by the method of Swanson (19) as modified by Hubscher and West (20).

Chemical Analysis-Protein was determined by the method of Lowry (21). DNAwasextracted from cellularsubfractions by the method of Schneider (22), and analyzed by the diphenylamine method of Dische (23). Calf thymus DNA was used as a standard.

Purijkation of Fructose-f ,6-Pp Aldolase (EC 4.1.2.13) from Rat Liver Microsomes for Preparation of Anti-Aldolase-A microsomal fraction was obtained from a homogenate of rat liver, made in 250 rnM sucrose-l mM EDTA, by sedimentation between 7,500 X g for 10 min at 4” (the post-mitochondrial fraction) and 70,000 X g for 105 min at 4”. After rinsing in the sucrose-EDTA solution, the pellet was rehomogenized in one-half the original volume of 250 mM sucrose-l mM EDTA-150 mM NaCl to liberate the bound aldolase which appeared in the supernatant solution after centri- fugation at 70,000 X g for 105 min at 4’. Fructose-Pz aldolase was then precipitated by dialysis against saturated (NHI),SO~. The precipitate was resuspended in 1 mM EDTA-20 mM Tris-IICl, pH 7.6, to a final concentration of 20 mg per ml, dialyzed against this buffer to remove all (NHa)#Oa, and fractionated two successive times on columns of phosphocellulose essentially as described by Gracy et al. (24). Elution of the second column with fructose- 1,6-Pn showed one peak with coincidence of protein and aldolase activity which exhibited only one band of protein by disc gel elec- trophoresis (25) on 77, polyacrylamide in 380 mM Tris-glycine at pH 8.3. The final recovery of enzyme, based upon the level of ac- tivity observed in the homogenate, was 10% at a purification of 62-fold with a specific activity of 6.03 units per mg of protein as- sayed (16) at 37”. These characteristics agreed well with values obtained for a crystalline preparation of this enzyme by Matsu- shima ei al. (26).

Preparation of Rabbit IgG Anti-Aldolase-Antiserum to purified fructose-P2 aldolase was obtained from New Zealand white rab- bits which had received, at IO-day intervals, three series of injec- tions (0.1 ml in each foot pad and 0.1 ml intradermally) of an emul- sified equal volume mixture of the aldolase in physiological saline

(0.9% NaCl solution) (2 mg per ml) and Freund’s complete ad- juvant. Beginning 2 weeks after the last injection, bleedings from the ear vein were done once each 2 weeks for 2 months. The IgG fraction was isolated from antiserum by precipitation with NazSOr and purified on DEAE-cellulose (27). The purity and specificity of the antibody was examined against the supernatant fraction of a homogenate (57, w/v) of rat liver in 250 mM sucrose-l mM EDTA- 150 mM NaCl centrifuged at 100,000 X g for 60 min. When the antiserum and anti-aldolase IgG were tested by immuno-diffusion, a single precipitin band with no spurring was observed. Control rabbit serum and IgG obtained from the same rabbits prior to im- munization showed no precipitin bands. Immunoelectrophoresis (28) indicated a single band when tested against the rat liver ex- tract. Anti-aldolase IgG precipitated 95% of the aldolase from the extract when incubated at 4” for 4 days and centrifuged at 10,000 X g for 20 min at 4”. Control rabbit serum had no such ef- fect.

Purijkation of Peroxidase-labeled Goat IgG (Anti-Rabbit-ZgG)- Peroxidase was conjugated to goat (anti-rabbit-IgG) IgG using p,p’-difluoro-m,m’-dinitrodiphenylsulfone as the coupling reagent according to the procedure described by Nakane and Pierce (29). The conjugate was further purified by adsorption on activated charcoal and rat liver acetone powder.2 Ten milligrams of rat liver acetone powder, prepared according to Coons (30), were added to 0.1 ml of stock conjugate previously diluted to 1.0 ml with 50 mM phosphate-0.9oy0 NaCl, pH 7.2, and were shaken for 1 hour at room temperature. The mixture was then submitted to centrifugation at about 18,000 X g for 15 min at 4”. The superna- tant was exposed to the “acetone powder” procedure twice more with the addition each time of 10 mg of activated charcoal. The light yellow supernatant solution (about 0.7 ml) which finally re- sulted was dialyzed overnight against 100 mM Tris-HCl, pH 7.4 and used immediately.

Tissue Fixation-Liver was excised, diced into pieces of about 2 mm3 while in 2.77, formaldehyde-0.2yn picric acid-250 mM su- crose-20 mM cacodylate-HCl, pH 7.2, at 4”, and incubated in the ice-cold solution for 4 hours. After several rinses in 200 mM caco- dylate-HCl, pH 7.2, and washing overnight at 4” in the buffer with constant mixing, the fixed tissue was embedded in 7.5% agar. Sections were cut at 60 to 100 pm using a Sorvall TC-2 tissue sec- tioner and were washed overnight in 200 mM Tris-HCl, pH 7.4, to remove the last traces of fixative.

Cytochemical Procedure-Ultrastructural immunologic localiza- tion of fructose-P2 aldolase was carried out by first attaching rab- bit anti-aldolase IgG to the aldolase in the fixed rat hepatocytic tissue then exposing the combined aldolase-IgG to goat (anti- rabbit IgG) IgG conjugated to horseradish peroxidase. The entire complex was localized by incubating the tissue in 3,3’-di- aminobenzidine, and H,Os. After preparation of tissues for elec- tron microscopy, the peroxidase reaction product was electron opaque and visible as a dark precipitate in the tissue.

The immunochemical techniques which were used, were adapted from methods described by Kraehenbuhl et al. (31). A tissue sec- tion was exposed to 0.25 ml of rabbit IgG (either control or anti- aldolase) for 5 to 6 hours at room temperature, rinsed, and washed overnight in 200 mM Tris-HCl, pH 7.4, at 4” with constant agita- tion. The specimens were then incubated with 0.25 ml of goat IgG-peroxidase conjugate in 100 mM Tris-HCl, pH 7.4, for 5 to 6 hours at room temperature and again rinsed and washed overnight in Tris-HCl as before. After fixation for 3 hours in 37, glutaralde- hyde-100 mM cacodylate-HCl, pH 7.4, at 4”, and rinsing for 4 hours in 100 mM Tris-HCl, pH 7.4, the sections were stained for peroxi- dase by incubation in 0.05% 3,3’-diaminobenzidine for 1 hour at room temperature and then for 10 min more at room tempera- ture in 0.05y0 3,3’-diaminobenzidine-O.OvO H202 in 200 mM Tris- HCl, pH 7.4. The stained sections were finally rinsed in the Tris-HCl buffer, washed overnight with constant agitation in 200 mM cacodylate-HCl, pH 7.4, at 4”, and prepared for electron microscopy.

Preparation of Tissues for Electron l%!ficroscopy-Tissues were postfixed in 2% OsOa-200 mM cacodylate-HCl, pH 7.4, at 4” for 60 min, rinsed three times in buffer, dehydrated in alcohol, washed two times in propylene oxide, and embedded in Epon. Sections were cut at 60 to 100 nm and mounted on grids coated with Form-

2 B. Baker, Department of Anatomy, The University of Michi- gan, personal communication.

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var and carbon. The mounted sections were poststained in a drop of cacodylate-buffered 0~04 for 10 min at room temperature and washed with distilled water. The specimens were examined by an AEI Corinth 275 electron microscope.

Reagents-Reagents were obtained from the following sources. Fructose-l ,6-P2 heptahydrate was from Wessex Biochemicals, adenine nucleotides and 3,3’-diaminobenzidine from Sigma Chem- ical Co., NADH from Boehringer Mannheim Corp., ultrapure (NH~)~SOI from Schwarz-Mann, phosphocellulose from Mann Bio- chemicals, horseradish peroxidase (type II) from Sigma Chemical Co., goat (anti-rabbit-IgG) IgG from Pentex, Freund’s complete adjuvant from Difco Laboratories, agar and p,p’-difluoro-m,m’- dinitrodiphenylsulfone from General Biochemicals, activated charcoal from Atlas Power Co., 0~04 from Merck and Co., p-for- maldehyde from Mallinckrodt Chemical Works, and glutaralde- hyde (as 70yo solution in sealed ampules) was from Ladd Research Industries.

RESULTS

In a preliminary effort to delineate the extent to which certain of the glycolytic enzymes (fructose-Pa aldolase, triose phosphate dehydrogenase, and phosphoglycerate kinase) are present in the microsomal fraction from rat liver, the conversion of a2Pi to or- ganic “P, dependent upon added fructose-l, 6-Pa, was measured. When assaying for the level of each of the three enzymes, excesses of the other two enzymes, as commercial preparations, were added. The microsomal fraction contained 65% of the aldolase, but only 3% of the dehydrogenase, and 10% of the kinase which were present in the post-mitochondrial supernatant solution.

TABLE I

Subcellular localization of fructose-l ,6-PZ aldolase activity in nuclear, mitochondrial, microsomal, and soluble fractions

For procedural details, see “Methods and Materials” in text.

Fraction Aldolase Protein

%

Crude homogenate 100 100 Nuclear.. . . 19 9 Mitochondrial.. 2 29 Microsomal. 58 25 Soluble. 13 30

Relative specific activity

DNA

1.0 2.1 0.1 2.3 0.4

% 100 100

2 91 13 4 70 Cl 4 <l

Total Recovered Activity

FIG. 1. Effect of NaCl on the subcellular localization of fruc- tose-Pz aldolase activity. Subfractions of rat liver homogenates were prepared as described under “Methods and Materials” ex- cept that NaCl was added to the crude homogenate before sub- fractionation to make the final concentrations indicated. All buffers used in further subfractionation contained these NaCl concentrations.

Rat liver homogenates were further subfractionated into nu- clear, mitochondrial, microsomal, and soluble fractions, and fruc- tose-Pz aldolase was assayed spectrophotometrically (Table I). The nuclear fraction (including unbroken cells) contained 19% of the total aldolase activity of the crude homogenate while 80 y0 of the remaining enzyme appeared in the microsomal fraction (which had 80% of the total recovered cellular glucose-6-phos- phatase). The addition of 150 mM NaCl to the homogenate prior to fractionation resulted in 98 y0 of the total aldolase appear- ing in the soluble fraction. Upon dialysis, almost half of the

[FRUCTOSE -1,6-P2] rnE (0)

0 0 I 0 2 0.3 04 0.5 0.6 0.7 0.0 0.9 I.0 I 1 I I I 1 I I 1 I

0 IO

4o So

FIG. 2. Effect of added NaCl and fructose-1,6-Pt on the appar- ent subcellular localization of fructose-P8 aldolase. A 10% (w/v) liver homogenate was prepared in 250 mM sucrose-l mM EDTA and centrifuged at 7,500 X g for 10 min at 4O. The NaCl or fructose-Pz concentration of a IO-ml aliquot of the post-mitochondrial frac- tion was adjusted to the desired concentrations by addition of the salt and centrifuged at 100,000 X g for 60 min at 4”. The super- natant solution was dialyzed overnight against 250 mM sucrose-l mM EDTA at 4” and assayed for aldolase activity. To determine the total microsomal aldolase, an aliquot of the post-mitochon- drial fraction was centrifuged at 100,009 X g for 60 min at 4”, in 250 mM sucrose-l mM EDTA-150 mM NaCl, and the supernatant solution was dialyzed as above and assayed as described.

1001

w 40-

2

g 30-

d 20-

5

z IO- tK

kf 0 5 6 7 8

PH

FIG. 3. Effect of pH on the binding of fructose-P2 aldolase to the microsomal fraction. Technical details were as described for Fig. 2 except that the pH of lo-ml aliquots of the post-mitochon- drial fraction was adjusted by addition of 100 rnrvr HCI or NaOH to the desired values (volume addition less than 0.02 ml).

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FIG. 4. Electron microscopic demonstration of fructose-Pz aldolase specific staining in the endoplasmic reticulum of fixed liver tis- sue. A and C, anti-aldolase IgG; B, control IgG. Post-stained in 2% 0~0~. Magnification: A and B, X 30,000; C, X 12,000.

total cellular aldolase reattached to the microsomes. The data in Fig. 1 indicate that not only was the enzyme almost completely solubilized from the particulate fractions by addition of 40 mM

NaCl but that the total recovered activity exceeded 100% of that present in the homogenate in the absence of salt. While it took 5 mM NaCl to solubilize 50% of the microsomal aldolase, as little as 0.05 mM fructose-1,6-I’2 produced the same effect (Fig. 2). The binding of aldolase to the microsomes was also found to be sensitive to changes in pH being maximal at pH 6.0 and 20% at pH 8.0 (Fig. 3).

Since the enzyme can be reversibly solubilized by changing the

pH or the concentrations of various salts, the binding might be an artifact and not exist in wivo. The best available proof for the physiological relevance of the binding would be the immuno- logical ultrastructural localization of the enzyme in tissue sec- tions. Fig. 4A demonstrates by this technique that the enzyme is associated with the endoplasmic reticulum. Control IgG produced no labeling (Fig. 4B). Although by biochemical frac- tionation, the nucleus appeared to contain aldolase, no nuclear labeling was seen (Fig. 4C) by the immunological technique. Fig. 4C shows that the enzyme is associated with both the smooth and rough endoplasmic reticular membranes. When the im-

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FIG. 5. Specific staining of microsomal membranes for fructose-P2 aldolase. Anti-aldolase IgG is used with no post-staining in osoa. Magnification: X 30,000. A, microsomes isolated in 250 mM sucrose-l mM EDTA; B, microsomes isolated in 250 mM sucrose- 1 mM EDTA-150 mM N&l.

muno-electron microscopic technique was applied to a micro- somal fraction made in sucrose-EDTA, labeling was seen (Fig. 5A), whereas addition of 150 mM NaCl resulted in a preparation in which no labeling is observed (Fig. 5B).

DISCUSSION

Although there are published data, obtained in vitro, which sug- gest that certain of the glycolytic enzymes are localized in an organelle or on a cellular structural element, most of these data could also be interpreted as representing an artifactual binding which occurred during the experimental procedure employed. The most compelling data favoring particulate localizations of these enzymes, in situ, would make use of ultrastructural im- munological localization techniques. The present study utilized such techniques to demonstrate the association of fructose-Pz aldolase with the endoplasmic reticulum, in situ. Rabbit anti- fructose-P2 aldolase IgG was shown by electron microscopy to localize on or. in close apposition to these membranes in tissue sections of rat liver. The specificity of the anti-aldolase IgG was shown by its ability to precipitate all of the aldolase from a crude extract of rat liver which had been made in the presence of 150 mM NaCl; the purity was shown by the formation of only one precipitin band when tested by immunoelectrophoresis against the extract. Although it is not presently possible to quantitate cytoimmunologically the percentage of the total aldolase which is present on the endoplasmic reticulum, biochemical fractiona- tion showed that at least 607, of the enzyme is associated with these membranes.

The increase in total cellular enzymatic activity seen when all of the fructose-Pz aldolase was solubilized (Fig. 1) could be ex-. plained by an increase in the Vm,, of the enzyme as was found for fructose-P2 aldolase in rabbit muscle (10). Further investiga- tion of this point is in progress.

Recent studies in this laboratory3 have demonstrated an in- crease of at least 3507” in the total activity of microsomal fruc- tose-1’2 aldolase when 80 mM NaCl was added to a preparation of microsomes which had been isolated by centrifugation from a rat liver homogenate and resuspended in 250 mM sucrose-l mM EDTA. Simply adding 80 mM NaCl to a soluble dialyzed prepa- ration of the aldolase does not result in an increase of activity. These observations and the finding that the association of fruc- tose-Pz aldolase with the endoplasmic reticular membranes is sensitive to relatively small changes in the substrate suggest that reversible association of the enzyme with the membranes may be a mechanism for controlling the rate of glycolysis in rat liver. Similar results have been reported in rat brain where the addition of 2 mM fructose-l , 6-PZ completely solubilizes fructose-1’2 aldol- ase from microsomes isolated in sucrose-EDTA, whereas about 150 mM NaCl is necessary to accomplish the same effect (32). Arnold and Pette (10) found that the binding of aldolase to F- actin was sensitive to low concentrations of fructose-l ,6-Pz, dihydroxyacetone-I’, 2,3-Pz-glycerate, and glucose-l, 6-PZ but much less sensitive or insensitive to other glycolytic intermedi- ates.

Support for the proposal that modulation of the activity of fructose-Pz aldolase by interaction with the endoplasmic reticu- lum represents a regulatory mechanism for the glycolytic path- way would include evidence that (a) the enzyme can reversibly bind to the membranes, (b) the percentage of the enzyme bound to the membranes or free in the cytosol is affected by biochemical or physiological parameters; (c) the effective activity of soluble aldolase is different from bound aldolase; and (d) physiologically, this enzyme limits the rate of glycolysis in either direction by

3 ‘I’. Weiss, Department of Environmental and Industrial Health, The University of Michigan, personal communication.

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virtue of its transit between the particulate and soluble phases of the cell.

The reversible association of fructose-P2 aldolasc with the membranous cellular fraction has now been documented for cells in the brain and muscle (8) as well as in the liver of the rat; in the muscle of the rabbit (9, lo), in the bovine erythrocyte (a), and in yeast (a), in vitro, and for muscle actin in the rabbit (10) and the cndoplasmic reticulum in the hepatocytc of the rat, in

situ. In rat brain, OIE of two isozymcs of aldolasc is prefercn- tially bound to the microsomcs while the other is preferentially located in the cytosol (8). This binding has been showrl to be sensitive to lactic acid in the casr of the brain of the rat (8), to some glycolytic intcrmcdi~tes in the muscle of the rabbit (lo), and to fructose-l, 6-l’y in the liver of the rat. Insrcascs in total cellular activity of the enzyme up011 solubilization have bee11 demonstrated in the lattcxr two systems. That the rcvcrsibk dis- sociation of fructosc-l’2 aldolasc from the membranes can bc nrl

allostcric control rnc&~~lisrn is suggcstcd by the obscxrvation tllat upon adsorption 011 pl~os~~l~occllulosc, the enzyme (xhibits the sigmoidal kinetics of allostcric enzymes whw enzymatic activity is plotted against the concentration of fructose-l , G-l’, (33). Ilowcvcr, tlic crucial demonstration that the incrcasc~ in activity, resulting from solubilizatioll, is associated with an alteration of the physiological rate of glycolysis, has not ~1s yet been achieved.

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R S Foemmel, R H Gray and I A BernsteinIntracellular localization of fructose 1,6-bisphosphate aldolase.

1975, 250:1892-1897.J. Biol. Chem. 

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