Structural characterization of the cytoplasmic pole of band 3 from bovine erythrocyte membranes

In an earlier study, we found that chymotryptic digestion of band 3 isolated from bovine erythrocyte membranes produces a 38,000-Da fragment in nonaethyleneglycol-n-dodecylether solution or a 50,000-Da fragment in deoxycholate solution as a primary fragment [Makino et al. (1984) J. Biochem. 95, 1019]. In the present study, these fragments were purified in an aqueous medium without detergent and their structural properties were examined. Several lines of evidence showed that the 50,000-Da fragment constitutes the entire cytoplasmic pole of bovine band 3 and that the 38,000-Da fragment is a subfragment of the 50,000-Da fragment. The large fragment was suggested to be divided into two distinct regions, the 12,000- and 38,000-Da portions, differing in their conformational thermal stability. However, attempts to identify the 12,000-Da portion as an isolable segment were without success. The cytoplasmic pole was characterized as a dimer which adopts an elongated gross conformation with helix of approximately 35%. Treatment of the fragments with dimethylmaleic anhydride dissociated the dimers into the monomers, accompanied by a significant conformational change of the 38,000-Da portion. Comparative studies suggested that the cytoplasmic domain of bovine band 3 has structurally different region(s) from that of human band 3, though their gross conformation shows extensive similarity.     

An ATP-modulated specific association of glyceraldehyde-3-phosphate dehydrogenase with human erythrocyte glucose transporter

Glyceraldehyde-3-phosphate dehydrogenase was found to bind in vitro to purified, human erythrocyte glucose transporter reconstituted into vesicles. Mild tryptic digestion of the glucose transporter totally inactivated the binding, suggesting that the cytoplasmic domain of the transporter is involved in the binding to glyceraldehyde-3-phosphate dehydrogenase. The binding was abolished in the presence of antisera raised against the purified glucose transporter, further supporting specificity of this interaction. The binding was reversible with a dissociation constant (Kd) of 3.3 x 10(-6) M and a total capacity (Bt) of approximately 30 nmol/mg of protein indicating a stoichiometry of one enzyme-tetramer per accessible transporter. The binding was sensitive to changes in pH showing an optimum at around pH 7.0. KCl and NaCl inhibited the binding in a simple dose-dependent manner with Ki of 40 and 20 mM, respectively. The binding was also inhibited by NAD+ with an estimated Ki of 3 mM. ATP, on the other hand, enhanced the binding by up to 3-fold in a dose-dependent manner with an apparent Ka of approximately 6 mM. The binding was not affected by D-glucose or cytochalasin B. The binding did not affect either the glucose or cytochalasin B in binding affinities or the transport activity of the transporter. However, the enzyme was inactivated totally upon binding to the transporter. Based on these findings, we suggest that a significant portion of glyceraldehyde-3-phosphate dehydrogenase in human erythrocytes exists as an inactive form via an ATP-dependent, reversible association with glucose transporter, and that this association may exert regulatory intervention on nucleotide metabolism in vitro.     

The complex of band 3 protein of the human erythrocyte membrane and glyceraldehyde-3-phosphate dehydrogenase: stoichiometry and competition by aldolase

The cytoplasmic domain of band 3, the main intrinsic protein of the erythrocyte membrane, possesses binding sites for a variety of other proteins of the membrane and the cytoplasm, including the glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and aldolase. We have studied the stoichiometry of the complexes of human band 3 protein and GAPDH and the competition by aldolase for the binding sites. In addition, we have tried to verify the existence of mixed band 3/GAPDH/aldolase complexes, which could represent the nucleus of a putative glycolytic multienzyme complex on the erythrocyte membrane. The technique applied was analytical ultracentrifugation, in particular sedimentation equilibrium analysis, on mixtures of detergent-solubilized band 3 and dye-labelled GAPDH, in part of the experiments supplemented by aldolase. The results obtained were analogous to those reported for the binding of hemoglobin, aldolase and band 4.1 to band 3: (1) the predominant or even sole band 3 oligomer forming the binding site is the tetramer. (2) The band 3 tetramer can bind up to four tetramers of GAPDH. (3) The band 3/GAPDH complexes are unstable. (4) Artificially stabilized band 3 dimers also represent GAPDH binding sites. In addition it was found that aldolase competes with GAPDH for binding to the band 3 tetramer, and that ternary complexes of band 3 tetramers, GAPDH and aldolase do exist.     

Antigenic determinants of the cytoplasmic domain of band 3 from bovine erythrocyte membrane

Rabbit antibodies were prepared against the cytoplasmic 38K-Da fragment of bovine band 3 and the immunological cross-reactivity with human, murine, rat, and chicken band 3 was examined. The antibodies cross-reacted with human and rodent band 3, indicating that there is an antigenic determinant(s) common to primate and nonprimate species. However, the antibodies did not recognize chicken band 3. Antigenic sites on the 38K-Da fragment were determined via amino acid sequence and immunoblotting analyses of proteolytic peptides of the fragment. Positions of antigenic determinants which were assumed to be common to primate and nonprimate species were mapped to the areas of residues 127-160 and of residues 259-304 in the primary structure of human band 3. Another epitope(s), which is absent in human band 3, existed in a region having a bovine-specific amino acid sequence. In addition, comparison of sequence data from different species showed that a proposed hinge region and a tryptophan-rich region on the cytoplasmic domain of band 3 [P. S. Low et al. (1984) J. Biol. Chem. 259, 13,070-13,076; R. R. Kopito and H. F. Lodish (1985) Nature (London) 316, 234-238] are also conserved in the bovine case.     

Mapping of the interaction site of CP12 with glyceraldehyde-3-phosphate dehydrogenase from Chlamydomonas reinhardtii. Functional consequences for glyceraldehyde-3-phosphate dehydrogenase

The 8.5 kDa chloroplast protein CP12 is essential for assembly of the phosphoribulokinase/glyceraldehyde-3-phosphate dehydrogenase (GAPDH) complex from Chlamydomonas reinhardtii. After reduction of this complex with thioredoxin, phosphoribulokinase is released but CP12 remains tightly associated with GAPDH and downregulates its NADPH-dependent activity. We show that only incubation with reduced thioredoxin and the GAPDH substrate 1,3-bisphosphoglycerate leads to dissociation of the GAPDH/CP12 complex. Consequently, a significant twofold increase in the NADPH-dependent activity of GAPDH was observed. 1,3-Bisphosphoglycerate or reduced thioredoxin alone weaken the association, causing a smaller increase in GAPDH activity. CP12 thus behaves as a negative regulator of GAPDH activity. A mutant lacking the C-terminal disulfide bridge is unable to interact with GAPDH, whereas absence of the N-terminal disulfide bridge does not prevent the association with GAPDH. Trypsin-protection experiments indicated that GAPDH may be also bound to the central alpha-helix of CP12 which includes residues at position 36 (D) and 39 (E). Mutants of CP12 (D36A, E39A and E39K) but not D36K, reconstituted the GAPDH/CP12 complex. Although the dissociation constants measured by surface plasmon resonance were 2.5-75-fold higher with these mutants than with wild-type CP12 and GAPDH, they remained low. For the D36K mutation, we calculated a 7 kcal.mol(-1) destabilizing effect, which may correspond to loss of the stabilizing effect of an ionic bond for the interaction between GAPDH and CP12. It thus suggests that electrostatic forces are responsible for the interaction between GAPDH and CP12.     

Interaction of phosphate analogues with glyceraldehyde-3-phosphate dehydrogenase.

The glycolytic enzyme glyceraldehyde-3-phosphate dehydrogenase catalyzes the oxidative phosphorylation of D-glyceraldehyde 3-phosphate. A variety of phosphonates have been shown to substitute for phosphate in this reaction [Gardner, J. H., & Byers, L. D., (1977) J. Biol. Chem. 252, 5925--5927]. The dependence of the logarithm of the equilibrium constant for the reaction on the pKa2 value of the phosphonate is characterized by a Br?nsted coefficient, betaeq, of approximately 1. This represents the sensitivity of the transfer of the phosphoglyceroyl group between the active-site sulfhydryl residue (in the acyl-enzyme intermediate) and the acyl acceptor on the basicity of the acyl acceptor. Molybdate (MoO42-) can also serve as an acyl acceptor in the glyceraldehyde-3-phosphate dehydrogenase catalyzed reaction. The second-order rate constant for the reaction with molybdate is only approximately 12 times lower than the reaction with phosphate even though the pKa2 of molybdate is 3.1 units lower than the pKa2 of phosphate. The immediate product of the molybdate reaction is the acyl molybdate, 1-molybdo-3-phosphoglycerate. The acyl molybdate, like the acyl arsenate (the immediate product of the reaction when arsenate is the acyl acceptor), is kinetically unstable. At pH 7.3 (25 degrees C), the half-life for hydrolysis of the acyl molybdate, or the acyl arsenate, is less than 2.5 s. Thus, hydrolysis of 1-molybdo- and 1-arseno-3-phosphoglycerate is at least 2000 times faster than hydrolysis of 1,3-diphosphoglycerate under the same conditions. Glyceraldehyde-3-phosphate dehydrogenase has a fairly broad specificity for acyl acceptors. Most tetrahedral oxy anions tested are substrates for the enzyme (except SO4(2-) and SeO4(2-)). Tetrahedral monoanions such as ReO4- and GeO(OH)3- are not substrates but do bind to the enzyme. These results suggest the requirement of at least one anionic site on the acyl acceptor required for binding and another anionic group on the acyl receptor required for nucleophilic attack on the acyl enzyme.     

Isolation and characterization of glyceraldehyde-3-phosphate dehydrogenase from human erythrocyte membranes.

A rapid and convenient procedure for isolating human glyceraldehyde-3-phosphate dehydrogenase from erythrocytes has been developed and yields enzyme with a specific activity of 33–52. The physical and catalytic properties of the enzyme are similar to those of rabbit muscle enzyme. Reassociation of freshly isolated human glyceraldehyde-3-phosphate dehydrogenase with washed erythrocyte membranes increases the specific activity and stability of the enzyme suggesting that enzyme-membrane interactions may have an important effect on the conformation and catalytic activity. That the human enzyme behaves as a dimer of dimers, similar to the behavior or rabbit muscle glyceraldehyde-3-phosphate dehydrogenase, is suggested by its half-of-the-sites reactivity toward 4-iodoacetamido-1-naphthol. The human enzyme binds nicotinamide hypoxanthine dinucleotide, a structural analog of NAD⁺, with negative cooperativity, further indicating its similarity to rabbit muscle enzyme.     

Fluorescence studies on glyceraldehyde-3-phosphate dehydrogenase from bovine heart muscle

Glyceraldehyde-3-phosphate dehydrogenase is a glycolytic enzyme that catalyses conversion of glyceraldehyde-3-phosphate to 1,3-diphosphoglycerate. ATP has been found to have an inhibitory effect on this enzyme. To establish the interaction between the enzyme and ATP, a fluorescence technique was used. Fluorescence quenching in the presence of ATP suggests cooperative binding of ATP to the enzyme (the Hill obtained coefficient equals 2.78). The interaction between glyceraldehyde-3-phosphate dehydrogenase and ATP may control not only glycolysis but other activities of this enzyme, such as binding to the cytoskeleton.     

Mechanism of glyceraldehyde-3-phosphate transfer from aldolase to glyceraldehyde-3-phosphate dehydrogenase

The catalytic interaction of glyceraldehyde-3-phosphate dehydrogenase with glyceraldehyde 3-phosphate has been examined by transient-state kinetic methods. The results confirm previous reports that the apparent Km for oxidative phosphorylation of glyceraldehyde 3-phosphate decreases at least 50-fold when the substrate is generated in a coupled reaction system through the action of aldolase on fructose 1,6-bisphosphate, but lend no support to the proposal that glyceraldehyde 3-phosphate is directly transferred between the two enzymes without prior release to the reaction medium. A theoretical analysis is presented which shows that the kinetic behaviour of the coupled two-enzyme system is compatible in all respects tested with a free-diffusion mechanism for the transfer of glyceraldehyde 3-phosphate from the producing enzyme to the consuming one.     

Interaction between mammalian glyceraldehyde-3-phosphate dehydrogenase and L-lactate dehydrogenase from heart and muscle

The exceptionally high protein concentration in living cells can favor functional protein-protein interactions that can be difficult to detect with purified proteins. In this study we describe specific interactions between mammalian D-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and L-lactate dehydrogenase (LDH) isozymes from heart and muscle. We use poly(ethylene-glycol) (PEG)-induced coprecipitation and native agarose electrophoresis as two independent methods uniquely suited to mimic some of the conditions that can favor protein-protein interaction in living cells. We found that GAPDH interacts with heart or muscle isozymes of LDH with approximately one-to-one stoichiometry. The interaction is specific; GAPDH shows interaction with two LDH isozymes that have very different net charge and solubility in PEG solution, while no interaction is observed with GAPDH from other species, other NAD(H) dehydrogenases, or other proteins that have very similar net charge and molecular mass. Analytical ultracentrifugation showed that the LDH and GAPDH complex is insoluble in PEG solution. The interaction is abolished by saturation with NADH, but not by saturation with NAD(+) in correlation with GAPDH solubility in PEG solution. The crystal structures show that GAPDH and LDH isozymes share complementary size, shape, and electric potential surrounding the active sites. The presented results suggest that GAPDH and LDH have a functional interaction that can affect NAD(+)/NADH metabolism and glycolysis in living cells.     

Interaction of cytoplasmic L-glycerol-3-phosphate dehydrogenase with liposomes

NAD-linked L-glycerol-3-phosphate dehydrogenase binds to phosphatidylcholine liposomes as shown by the changes in the properties of both the enzyme and the membrane. The surface potential and the fluidity of the liposome membrane (monitored at the 5th C atom depth) change due to the presence of the enzyme, whereas the enzyme is activated by the liposomes. These findings suggest the occurrence of peripheral protein-lipid interactions.     

Lack of binding of glyceraldehyde-3-phosphate dehydrogenase to erythrocyte membranes under in vivo conditions

A filtration method is described for separating membrane-free cytoplasm from concentrated erythrocyte haemolysates. The method has been used to assess glyceraldehyde-3-phosphate dehydrogenase binding to erythrocyte membranes. The relative amounts of glyceraldehyde-3-phosphate dehydrogenase and lactate dehydrogenase in the cytoplasm (either oxygenated or deoxygenated) indicate there is no detectable binding of glyceraldehyde-3-phosphate dehydrogenase to the membranes under physiological conditions.     

Role of lysine residues in the binding of glyceraldehyde-3-phosphate dehydrogenase to human erythrocyte membranes

Glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.12) binds reversibly to human erythrocyte membranes. Several specific amino acid residues involved in the enzyme-membrane contact region have already been identified. These include tyrosine 46 and threonine 150. Covalent modification of lysines 212 and 191 with pyridoxal phosphate results in a decreased affinity of the enzyme for erythrocyte membranes if the enzyme-linked pyridoxal phosphate is not reduced prior to binding. Reduction of the pyridoxal phosphate-lysine complex completely inhibits the binding of the enzyme to erythrocyte membranes. These results suggest a role for lysines 212 and 191 in the interaction of glyceraldehyde-3-phosphate with human erythrocyte membranes.     

Effect of stilbenedisulfonate binding on the state of association of the membrane-spanning domain of band 3 from bovine erythrocyte membrane

The membrane-spanning domain of bovine band 3, the anion transport protein of erythrocyte membrane, was purified in the presence of nonaethyleneglycol lauryl ether (C12E9) and the effect of a covalent attachment of 4,4'-diisothiocyanostilbene-2,2'-disulfonate (DIDS), a potent transport inhibitor, on the state of association of the domain isolated (the 58 kDa fragment) was studied via gel filtration, gel electrophoresis and sedimentation velocity experiments. It was indicated that the DIDS-unlabeled fragment in C12E9 solution forms heterogeneous aggregates which are larger in size than the dimer. This contrasted with the behavior that bovine band 3 is present as dimers or tetramers in the same medium (Nakashima and Makino (1980) J. Biochem. 88, 933-947). When DIDS was covalently attached, the fragment was present as a single molecular species which was indicated to be a dimer by molecular weight determination. The secondary structure of the fragment was not affected by DIDS. The change in the state of association caused by the DIDS-binding was also found in the presence of sucrose monolaurate (SE12), which was a more potent detergent for extraction of the 58 kDa fragment from membranes than C12E9. However, the complex with SE12 was extremely unstable.     

Thermal destabilization of non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase from Streptococcus mutans upon phosphate binding in the active site

Catalysis by the NADP-dependent non-phosphorylating glyceraldehyde-3-phosphate dehydrogenase (GAPN) from Streptococcus mutans, a member of the aldehyde dehydrogenase (ALDH) family, relies on a local conformational reorganization of the active site. This rearrangement is promoted by the binding of NADP and is strongly kinetically favored by the formation of the ternary complex enzyme.NADP.substrate. Adiabatic differential scanning calorimetry was used to investigate the effect of ligands on the irreversible thermal denaturation of GAPN. We showed that phosphate binds to GAPN, resulting in the formation of a GAPN.phosphate binary complex characterized by a strongly decreased thermal stability, with a difference of at least 15 degrees C between the maximum temperatures of the thermal transition peaks. The kinetics of phosphate association and dissociation are slow, allowing both free and GAPN.phosphate complexes to be observed by differential scanning calorimetry and to be separated by native polyacrylamide electrophoresis run in phosphate buffer. Analysis of a set of mutants of GAPN strongly suggests that phosphate is bound to the substrate C-3 subsite. In addition, the substrate analog glycerol-3-phosphate has similar effects as does phosphate on the thermal behavior of GAPN. Based on the current knowledge on the catalytic mechanism of GAPN and other ALDHs, we propose that ligand-induced thermal destabilization is a mechanism that provides to ALDHs the required flexibility for an efficient catalysis.