CURRENT TOPICS IN CELLULAR REGULATION, VOLUME 33
Association of Glycolytic Enzymes with the Cytoskeleton I
HARVEY R. KNULL
I Department of Biochemistry and Molecular Biology I I University of North Dakota School of I Medicine I Grand Forks, North Dakota 58202 I
JULIE L. WALSH
I Center for Biomédical Research I Lawrence, Kansas 66047
I. Introduction The cytoplasm of eukaryotic cells contains an extensive threedimensional array of proteins referred to as the cytoskeleton. The cy toskeleton is composed of three principal fibers: microtubules, interme diate filaments, and thin filaments. Cytosol was initially defined as that fraction of proteins appearing in the supernatant following a 100,000 g centrifugation (2); however, the term is sometimes expanded to refer to the portion of cytoplasm from which the cytosol is derived and as such is more loosely defined as the nonorganellar portion of the cytoplasm (2). The cytoskeleton, along with a host of proteins of the cytoplasm, many of which appear in the cytosolic fraction, makes up the cytomatrix. Many cytosolic proteins, specifically the glycolytic enzymes, have an affinity for cytoskeletal proteins. The premise of this article is that the dynamic interactions of the glycolytic enzymes with cytoskeletal proteins permit localized enrichment of these enzymes to provide a localized source of glycolytically derived energy for the energydependent and cytoskeletal affiliated intracellular functions. 15
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
16
HARVEY R. KNULL AND JULIE L. WALSH
II. Dynamic Involvement of the Cytoskeleton in Intracellular Structure and Organization, Transportation, and Cellular Contraction A. The Cytomatrix Influences Intracellular Structure and Organization A network involving the cytoskeleton and certain other cytoplasmic proteins that interacts with intracellular organelles such as lysosomes (3), mitochondria (4), and endoplasmic reticulum has been described (5, 6). Recent evidence implies not only that the endoplasmic reticulum is supported structurally by the cytoskeleton, but that construction of the endoplasmic reticular network (5) and its normal movement and rear rangement for maintenance (6) are dependent on the cytoskeleton, specifically microtubules. In addition to the macroscopic structural interactions with intracel lular organelles, the cytoskeletal framework is also involved at the microscopic level of cytoplasmic structure through orientation of ribosomes and the protein synthetic machinery. It has been shown that protein synthesis is dependent on mRNA binding to ribosomes attached to the cytoskeletal framework (7). Furthermore, synthesis of cytoskel etal proteins occurs at sites where these proteins are inserted into the cytoskeletal framework (8). This localized synthesis of cytoskeletal proteins occurs near the cell periphery and lamellipodia (9), where an increased concentration of a protein such as actin is necessary for the formation of new actin filaments. The dynamic nature of cytoskeletal filaments, with respect to an exchange between incorporated and unincorporated subunits, is sup ported by the work of Okabe and Hirokawa (10), who demonstrated that microtubules and actin filaments continuously disassemble/assemble along the length of the axon of cultured neurons. The continuous energy-dependent reincorporation of subunits and the energydependent processes physically associated with the cytoskeleton, such as protein synthesis, suggest that a localized energy source would opti mize cellular energy efficiency. B. Cytoskeletal Involvement with Intracellular Transport Intracellular transport includes processes such as the transport of endosomes and secretory vesicles and the aggregation and redispersal of pigment in specialized cells such as chromatophores. The intracellu lar transport of vesicles is commonly achieved by the movement of the vesicle along a microtubule via a motor protein. Three motor proteins, analogous to myosin, which contain microtubule-activated ATPases
ASSOCIATION OF GLYCOLYTIC ENZYMES WITH CYTOSKELETON
17
and have the ability either to move microtubules along a coverslip or to move latex beads along a microtubule have been identified. These proteins are dynein, kinesin, and dynamin (11). The involvement of motor proteins has not been investigated in all intracellular transport processes. However, the movement of pigment in chromatophores is ATP dependent (12), and the movement of lysosomes is microtubule dependent and has been hypothesized to occur via motor proteins (13), as has the microtuble- and ATPase-dependent movement of cytoplasmic granules in neutrophils (14). Neuronal cells present more complex problems of intracellular trans port owing to the presence of both anterograde and retrograde transport and the distance from the cell body to the nerve terminal. The transport of vesicles may proceed via the same mechanisms as discussed above for other cells utilizing the motor proteins and microtubules for cytoplasmic translocation of vesicles (11, 15). Because the axon itself is com posed of cytoplasmic elements which must play a role in facilitating transport, and because there are no ribosomes in the axon, the axoplasmic elements must be synthesized elsewhere and transported to the axoplasm. The cytoskeletal proteins themselves demonstrate move ment combined with polymerization/depolymerization of subunits along the axon (16,17). The polymerization/depolymerization reactions and the transport of vesicles along the axon are energy-dependent processes, showing the need for localized ATP synthesis. Although the glycolytic enzymes have been classically thought of as soluble proteins, the transport of nerve-specific enolase and the bulk of proteins pre sumed to make up the "metabolic machinery" with the slow component b complex has been demonstrated in neurons (18). This unique complex implies a structure in the axon involving enolase and suggests that glycolytic enzymes do not enter the axoplasm by random diffusion and that their intracellular location may be controlled. C. Cellular Contraction and the Cytoskeleton Myosin, the motor protein used as a prototype for the classification of other motor proteins, is most readily identified with muscle contraction. Muscle contracts as myosin, in the form of thick filaments, binds tran siently to actin, in the form of thin filaments, through the globular heads on the myosin which also contain actin-activated ATPase activ ity. Thus, through the cleavage of ATP coupled with orderly protein interactions, muscle contraction takes place. Myosin is also involved in other cytoskeletal movements such as the rounding up, constriction of cleavage furrows, capping of surface receptors, and establishment of cell polarity in Dictyostelium cells (19). Myosin, along with actin, is
18
HARVEY R. KNULL AND JULIE L. WALSH
involved in the physiological response of platelets, both in the charac teristic shape change from spheres to disks and in the formation of thrombosthenin, during clot formation (20,21). Thus, cellular contrac tion is mediated via energy-dependent processes directly linked to the cytoskeleton which could be efficiently supplied with ATP via localized glycolysis. III. Association of Glycolytic Enzymes with the Cytoskeleton A. Physical Interactions between the Cytoskeleton and Glycolytic Enzymes 1. GENERAL DEMONSTRATION OF LACK OF FREE SOLUBILITY OF GLYCOLYTIC ENZYMES IN THE CYTOPLASM
Interactions between glycolytic enzymes and the cytoskeleton have been demonstrated in several tissues including pig kidney cells and Swiss mouse fibroblast 3T3 cells, erythrocytes, brain, and skeletal mus cle. Two approaches have been used to study the mobility of the glyco lytic enzymes within the cytomatrix: first, extraction of the glycolytic enzymes from cells under various conditions and, second, use of fluo rescent probes to study intracellular movement of the glycolytic en zymes. The amount of enzyme extracted from mouse fibroblast 3T3 cells depends on the extraction conditions used. When compared to inositol, the extraction of the glycolytic enzymes, including glucose-phosphate isomerase, aldolase, GAPDH (glyceraldehyde-3-phosphate dehydrogenase), triose-phosphate isomerase, phosphoglycerate mutase, phosphoglycerate kinase, enolase, pyruvate kinase, and LDH (lactate dehydrogenase), was significantly reduced, with the glycolytic enzymes being retained by the cell due to associations with the cell substructure, presumably the cytoskeletal framework (22). Mouse L-929 cells permeabilized with dextran sulfate to the extent of showing lesions large enough for 400-kDa globular proteins to enter the cytoplasm demon strated very little release of cellular proteins, including the glycolytic enzymes, suggesting that these cytoplasmic proteins are not freely soluble, but interact with a substructure within the cytomatrix (23). Membraneless cells continued to carry out glycolysis (23) at rates simi lar to membrane-bound cells but unlike the rates of homogenates (24), implying metabolic control via the glycolytic enzyme associations within the cytomatrix. Pig kidney cells were similarly studied by ex traction under conditions of molecular crowding [i.e., bovine serum
ASSOCIATION OF GLYCOLYTIC ENZYMES WITH CYTOSKELETON
19
albumin (BSA) added to the extraction buffer], which resulted in re duced extraction, apparently because of interactions among the glycolytic enzymes and with structures within the cytomatrix (25). Studies using the injection of fluorescently tagged aldolase and enolase into intact cells and measuring their mobilities with fluorescence recovery after photobleaching {FRAP} lead to the conclusion that al dolase is transiently associated with stress fibers and more tightly associated with structures in the perinuclear region, whereas enolase seems to diffuse readily (26, 27). However, these data may be reinter preted in light of other relevant observations. As presented below, aldolase interacts not only with F-actin, but also with microtubules; therefore, the perinuclear structures to which aldolase is attached could be either microtubules or F-actin. As presented above, the detergent extraction studies indicate that enolase is associated within the cytomatrix (22,25), and its involvement in glycolysis in cells freed of their plasma membranes (23) implies affiliation with other glycolytic en zymes. Furthermore, enolase interacts with other glycolytic enzymes (28) although it does not appear to associate with microtubules or actin (29). These observations suggest that although enolase appeared freely diffusible in the FRAP study, this may have occurred because of indi rect and more transient interactions of this enzyme than aldolase. 2. GLYCOLYTIC ENZYME ASSOCIATIONS WITH THIN FILAMENT PROTEINS
The enrichment of glycolytic enzymes in the thin filament region of skeletal muscle has been investigated over the past three decades. Immunofluorescent (30), histochemical (31), and X-ray diffraction stud ies (32) show that most if not all of the glycolytic enzymes are compartmented in this region. This apparent compartmentation has lead to research to determine the specific interactions between the thin fila ment proteins and the glycolytic enzymes. Because the thin filaments are mainly composed of actin, the interactions between the glycolytic enzymes and actin filaments have been rigorously studied. Several techniques have demonstrated specific interactions between glycolytic enzymes and actin and include centrifugation (33-35), centrifugation with molecular crowding (36), moving boundary electrophoresis (37), countercurrent distribution (38), electron microscopy (39), and affinity chromatography (40). With this variety of techniques, interactions have been observed for glucose-phosphate isomerase, phosphofructokinase, aldolase, GAPDH, phosphoglycerate kinase, pyruvate kinase, and muscle type LDH with actin, as previously reviewed (29). Although nonmuscle actin shows considerable homology with muscle
20
HARVEY R. KNULL AND JULIE L. WALSH
actin, some of the properties such as critical concentration for polymer ization (41) differ; therefore, interactions between F-actin and glycolytic enzymes in nonmuscle cells cannot simply be assumed. There are also differences between nonmuscle and skeletal muscle cells in thin filament structure. Nonmuscle tropomyosin is shorter than muscle tropomyosin (42), and nonmuscle cells do not have troponin. Although aldolase binds to actin (34, 35, 43) and actin-tropomyosin complexes (40), troponin and tropomyosin both have been shown to influence aldolase association with thin filaments from skeletal muscle (39, 44). Nonetheless, interactions with F-actin in nonmuscle cells may be relevant. Glycolytic enzymes from crude brain homogenates showed increased pelleting when F-actin was added to the crude preparation (45). It was suggested that increased pelleting may have been due to an interaction with the added F-actin. In another study three brain enzymes, aldolase, GAPDH, and LDH, were found to adsorb to an affinity chromatography column containing muscle F-actin-tropomyosin complexes, indicating affinity of these enzymes for thin filaments (46). The occurrence of such interactions in tissue other than skeletal muscle has not been fully established; however, associations between nonmuscle thin filament proteins and the glycolytic enzymes are expected to be a general phe nomenon of cytomatrix association occurring in many cell types. 3. GLYCOLYTIC ENZYMES ASSOCIATE WITH TUBULIN
Interactions between tubulin and the glycolytic enzymes have not been studied as extensively as have the associations with actin. However, tubulin is a major component in the cytoskeletal network and may be involved in the general interactions with the cytomatrix as discussed previously. The first study to report an interaction of a glycolytic enzyme, GAPDH, with microtubules resulted from studies characterizing MAPs (microtubule-associated proteins). GAPDH was similar to MAPs in that it was retained with microtubules during tubulin purification and adsorbed to a tubulin affinity column (47). Follow-up studies indicated that GAPDH caused bundling of microtubules (48) and that the enzyme was inactivated on inclusion in polymerizing mixtures of tubulin (49). These initial observations of GAPDH-tubulin interactions lead to sev eral studies of the binding of glycolytic enzymes to tubulin and microtu bules. The techniques of coelectrophoresis (50), centrifugation with and without molecular crowding, and fluorescence anisotropy (51) have been used to investigate these interactions. Coelectrophoresis of the enzymes with tubulin in agarose gels indi-
ASSOCIATION OF GLYCOLYTIC ENZYMES WITH CYTOSKELETON
21
cated an affinity of aldolase, GAPDH, pyruvate kinase, and muscle type LDH for tubulin (50). The same four enzymes were located in the pellet fraction after centrifugation with microtubules. Additionally, under conditions of molecular crowding [addition of a solute to create a condi tion of volume exclusion (52) ], two more enzymes were found to copellet with microtubules: glucose-phosphate isomerase and phosphoglycerate kinase (36). The effect of molecular crowding on the association of
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Association of Glycolytic Enzymes with the Cytoskeleton I
HARVEY R. KNULL
I Department of Biochemistry and Molecular Biology I I University of North Dakota School of I Medicine I Grand Forks, North Dakota 58202 I
JULIE L. WALSH
I Center for Biomédical Research I Lawrence, Kansas 66047
I. Introduction The cytoplasm of eukaryotic cells contains an extensive threedimensional array of proteins referred to as the cytoskeleton. The cy toskeleton is composed of three principal fibers: microtubules, interme diate filaments, and thin filaments. Cytosol was initially defined as that fraction of proteins appearing in the supernatant following a 100,000 g centrifugation (2); however, the term is sometimes expanded to refer to the portion of cytoplasm from which the cytosol is derived and as such is more loosely defined as the nonorganellar portion of the cytoplasm (2). The cytoskeleton, along with a host of proteins of the cytoplasm, many of which appear in the cytosolic fraction, makes up the cytomatrix. Many cytosolic proteins, specifically the glycolytic enzymes, have an affinity for cytoskeletal proteins. The premise of this article is that the dynamic interactions of the glycolytic enzymes with cytoskeletal proteins permit localized enrichment of these enzymes to provide a localized source of glycolytically derived energy for the energydependent and cytoskeletal affiliated intracellular functions. 15
Copyright © 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
16
HARVEY R. KNULL AND JULIE L. WALSH
II. Dynamic Involvement of the Cytoskeleton in Intracellular Structure and Organization, Transportation, and Cellular Contraction A. The Cytomatrix Influences Intracellular Structure and Organization A network involving the cytoskeleton and certain other cytoplasmic proteins that interacts with intracellular organelles such as lysosomes (3), mitochondria (4), and endoplasmic reticulum has been described (5, 6). Recent evidence implies not only that the endoplasmic reticulum is supported structurally by the cytoskeleton, but that construction of the endoplasmic reticular network (5) and its normal movement and rear rangement for maintenance (6) are dependent on the cytoskeleton, specifically microtubules. In addition to the macroscopic structural interactions with intracel lular organelles, the cytoskeletal framework is also involved at the microscopic level of cytoplasmic structure through orientation of ribosomes and the protein synthetic machinery. It has been shown that protein synthesis is dependent on mRNA binding to ribosomes attached to the cytoskeletal framework (7). Furthermore, synthesis of cytoskel etal proteins occurs at sites where these proteins are inserted into the cytoskeletal framework (8). This localized synthesis of cytoskeletal proteins occurs near the cell periphery and lamellipodia (9), where an increased concentration of a protein such as actin is necessary for the formation of new actin filaments. The dynamic nature of cytoskeletal filaments, with respect to an exchange between incorporated and unincorporated subunits, is sup ported by the work of Okabe and Hirokawa (10), who demonstrated that microtubules and actin filaments continuously disassemble/assemble along the length of the axon of cultured neurons. The continuous energy-dependent reincorporation of subunits and the energydependent processes physically associated with the cytoskeleton, such as protein synthesis, suggest that a localized energy source would opti mize cellular energy efficiency. B. Cytoskeletal Involvement with Intracellular Transport Intracellular transport includes processes such as the transport of endosomes and secretory vesicles and the aggregation and redispersal of pigment in specialized cells such as chromatophores. The intracellu lar transport of vesicles is commonly achieved by the movement of the vesicle along a microtubule via a motor protein. Three motor proteins, analogous to myosin, which contain microtubule-activated ATPases
ASSOCIATION OF GLYCOLYTIC ENZYMES WITH CYTOSKELETON
17
and have the ability either to move microtubules along a coverslip or to move latex beads along a microtubule have been identified. These proteins are dynein, kinesin, and dynamin (11). The involvement of motor proteins has not been investigated in all intracellular transport processes. However, the movement of pigment in chromatophores is ATP dependent (12), and the movement of lysosomes is microtubule dependent and has been hypothesized to occur via motor proteins (13), as has the microtuble- and ATPase-dependent movement of cytoplasmic granules in neutrophils (14). Neuronal cells present more complex problems of intracellular trans port owing to the presence of both anterograde and retrograde transport and the distance from the cell body to the nerve terminal. The transport of vesicles may proceed via the same mechanisms as discussed above for other cells utilizing the motor proteins and microtubules for cytoplasmic translocation of vesicles (11, 15). Because the axon itself is com posed of cytoplasmic elements which must play a role in facilitating transport, and because there are no ribosomes in the axon, the axoplasmic elements must be synthesized elsewhere and transported to the axoplasm. The cytoskeletal proteins themselves demonstrate move ment combined with polymerization/depolymerization of subunits along the axon (16,17). The polymerization/depolymerization reactions and the transport of vesicles along the axon are energy-dependent processes, showing the need for localized ATP synthesis. Although the glycolytic enzymes have been classically thought of as soluble proteins, the transport of nerve-specific enolase and the bulk of proteins pre sumed to make up the "metabolic machinery" with the slow component b complex has been demonstrated in neurons (18). This unique complex implies a structure in the axon involving enolase and suggests that glycolytic enzymes do not enter the axoplasm by random diffusion and that their intracellular location may be controlled. C. Cellular Contraction and the Cytoskeleton Myosin, the motor protein used as a prototype for the classification of other motor proteins, is most readily identified with muscle contraction. Muscle contracts as myosin, in the form of thick filaments, binds tran siently to actin, in the form of thin filaments, through the globular heads on the myosin which also contain actin-activated ATPase activ ity. Thus, through the cleavage of ATP coupled with orderly protein interactions, muscle contraction takes place. Myosin is also involved in other cytoskeletal movements such as the rounding up, constriction of cleavage furrows, capping of surface receptors, and establishment of cell polarity in Dictyostelium cells (19). Myosin, along with actin, is
18
HARVEY R. KNULL AND JULIE L. WALSH
involved in the physiological response of platelets, both in the charac teristic shape change from spheres to disks and in the formation of thrombosthenin, during clot formation (20,21). Thus, cellular contrac tion is mediated via energy-dependent processes directly linked to the cytoskeleton which could be efficiently supplied with ATP via localized glycolysis. III. Association of Glycolytic Enzymes with the Cytoskeleton A. Physical Interactions between the Cytoskeleton and Glycolytic Enzymes 1. GENERAL DEMONSTRATION OF LACK OF FREE SOLUBILITY OF GLYCOLYTIC ENZYMES IN THE CYTOPLASM
Interactions between glycolytic enzymes and the cytoskeleton have been demonstrated in several tissues including pig kidney cells and Swiss mouse fibroblast 3T3 cells, erythrocytes, brain, and skeletal mus cle. Two approaches have been used to study the mobility of the glyco lytic enzymes within the cytomatrix: first, extraction of the glycolytic enzymes from cells under various conditions and, second, use of fluo rescent probes to study intracellular movement of the glycolytic en zymes. The amount of enzyme extracted from mouse fibroblast 3T3 cells depends on the extraction conditions used. When compared to inositol, the extraction of the glycolytic enzymes, including glucose-phosphate isomerase, aldolase, GAPDH (glyceraldehyde-3-phosphate dehydrogenase), triose-phosphate isomerase, phosphoglycerate mutase, phosphoglycerate kinase, enolase, pyruvate kinase, and LDH (lactate dehydrogenase), was significantly reduced, with the glycolytic enzymes being retained by the cell due to associations with the cell substructure, presumably the cytoskeletal framework (22). Mouse L-929 cells permeabilized with dextran sulfate to the extent of showing lesions large enough for 400-kDa globular proteins to enter the cytoplasm demon strated very little release of cellular proteins, including the glycolytic enzymes, suggesting that these cytoplasmic proteins are not freely soluble, but interact with a substructure within the cytomatrix (23). Membraneless cells continued to carry out glycolysis (23) at rates simi lar to membrane-bound cells but unlike the rates of homogenates (24), implying metabolic control via the glycolytic enzyme associations within the cytomatrix. Pig kidney cells were similarly studied by ex traction under conditions of molecular crowding [i.e., bovine serum
ASSOCIATION OF GLYCOLYTIC ENZYMES WITH CYTOSKELETON
19
albumin (BSA) added to the extraction buffer], which resulted in re duced extraction, apparently because of interactions among the glycolytic enzymes and with structures within the cytomatrix (25). Studies using the injection of fluorescently tagged aldolase and enolase into intact cells and measuring their mobilities with fluorescence recovery after photobleaching {FRAP} lead to the conclusion that al dolase is transiently associated with stress fibers and more tightly associated with structures in the perinuclear region, whereas enolase seems to diffuse readily (26, 27). However, these data may be reinter preted in light of other relevant observations. As presented below, aldolase interacts not only with F-actin, but also with microtubules; therefore, the perinuclear structures to which aldolase is attached could be either microtubules or F-actin. As presented above, the detergent extraction studies indicate that enolase is associated within the cytomatrix (22,25), and its involvement in glycolysis in cells freed of their plasma membranes (23) implies affiliation with other glycolytic en zymes. Furthermore, enolase interacts with other glycolytic enzymes (28) although it does not appear to associate with microtubules or actin (29). These observations suggest that although enolase appeared freely diffusible in the FRAP study, this may have occurred because of indi rect and more transient interactions of this enzyme than aldolase. 2. GLYCOLYTIC ENZYME ASSOCIATIONS WITH THIN FILAMENT PROTEINS
The enrichment of glycolytic enzymes in the thin filament region of skeletal muscle has been investigated over the past three decades. Immunofluorescent (30), histochemical (31), and X-ray diffraction stud ies (32) show that most if not all of the glycolytic enzymes are compartmented in this region. This apparent compartmentation has lead to research to determine the specific interactions between the thin fila ment proteins and the glycolytic enzymes. Because the thin filaments are mainly composed of actin, the interactions between the glycolytic enzymes and actin filaments have been rigorously studied. Several techniques have demonstrated specific interactions between glycolytic enzymes and actin and include centrifugation (33-35), centrifugation with molecular crowding (36), moving boundary electrophoresis (37), countercurrent distribution (38), electron microscopy (39), and affinity chromatography (40). With this variety of techniques, interactions have been observed for glucose-phosphate isomerase, phosphofructokinase, aldolase, GAPDH, phosphoglycerate kinase, pyruvate kinase, and muscle type LDH with actin, as previously reviewed (29). Although nonmuscle actin shows considerable homology with muscle
20
HARVEY R. KNULL AND JULIE L. WALSH
actin, some of the properties such as critical concentration for polymer ization (41) differ; therefore, interactions between F-actin and glycolytic enzymes in nonmuscle cells cannot simply be assumed. There are also differences between nonmuscle and skeletal muscle cells in thin filament structure. Nonmuscle tropomyosin is shorter than muscle tropomyosin (42), and nonmuscle cells do not have troponin. Although aldolase binds to actin (34, 35, 43) and actin-tropomyosin complexes (40), troponin and tropomyosin both have been shown to influence aldolase association with thin filaments from skeletal muscle (39, 44). Nonetheless, interactions with F-actin in nonmuscle cells may be relevant. Glycolytic enzymes from crude brain homogenates showed increased pelleting when F-actin was added to the crude preparation (45). It was suggested that increased pelleting may have been due to an interaction with the added F-actin. In another study three brain enzymes, aldolase, GAPDH, and LDH, were found to adsorb to an affinity chromatography column containing muscle F-actin-tropomyosin complexes, indicating affinity of these enzymes for thin filaments (46). The occurrence of such interactions in tissue other than skeletal muscle has not been fully established; however, associations between nonmuscle thin filament proteins and the glycolytic enzymes are expected to be a general phe nomenon of cytomatrix association occurring in many cell types. 3. GLYCOLYTIC ENZYMES ASSOCIATE WITH TUBULIN
Interactions between tubulin and the glycolytic enzymes have not been studied as extensively as have the associations with actin. However, tubulin is a major component in the cytoskeletal network and may be involved in the general interactions with the cytomatrix as discussed previously. The first study to report an interaction of a glycolytic enzyme, GAPDH, with microtubules resulted from studies characterizing MAPs (microtubule-associated proteins). GAPDH was similar to MAPs in that it was retained with microtubules during tubulin purification and adsorbed to a tubulin affinity column (47). Follow-up studies indicated that GAPDH caused bundling of microtubules (48) and that the enzyme was inactivated on inclusion in polymerizing mixtures of tubulin (49). These initial observations of GAPDH-tubulin interactions lead to sev eral studies of the binding of glycolytic enzymes to tubulin and microtu bules. The techniques of coelectrophoresis (50), centrifugation with and without molecular crowding, and fluorescence anisotropy (51) have been used to investigate these interactions. Coelectrophoresis of the enzymes with tubulin in agarose gels indi-
ASSOCIATION OF GLYCOLYTIC ENZYMES WITH CYTOSKELETON
21
cated an affinity of aldolase, GAPDH, pyruvate kinase, and muscle type LDH for tubulin (50). The same four enzymes were located in the pellet fraction after centrifugation with microtubules. Additionally, under conditions of molecular crowding [addition of a solute to create a condi tion of volume exclusion (52) ], two more enzymes were found to copellet with microtubules: glucose-phosphate isomerase and phosphoglycerate kinase (36). The effect of molecular crowding on the association of
PK
GAPDH 1001
c
o GO
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+PEG
CD
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