RESUMO
Palmitoylation is unique among lipid modifications in that it is reversible. In recent years, dynamic palmitoylation of G protein alpha subunits and of their cognate receptors has attracted considerable attention. However, very little is known concerning the acylation/deacylation cycle of the proteins in relation to their activity status. In particular, the relative contribution of the activation and desensitization of the signaling unit to the regulation of the receptors and G proteins palmitoylation state is unknown. To address this issue, we took advantage of the fact that a fusion protein composed of the stimulatory alpha subunit of trimeric G protein (Galpha(s)) covalently attached to the beta(2)-adrenergic receptor (beta(2)AR) as a carboxyl-terminal extension (beta(2)AR-Galpha(s)) can be stimulated by agonists but does not undergo rapid inactivation, desensitization, or internalization. When expressed in Sf9 cells, both the receptor and the Galpha(s) moieties of the fusion protein were found to be palmitoylated via thioester linkage. Stimulation with the beta-adrenergic agonist isoproterenol led to a rapid depalmitoylation of both the beta(2)AR and Galpha(s) and inhibited repalmitoylation. The extent of depalmitoylation induced by a series of agonists was correlated (0.99) with their intrinsic efficacy to stimulate the adenylyl cyclase activity. However, forskolin-stimulated cAMP production did not affect the palmitoylation state of beta(2)AR-Galpha(s), indicating that the agonist-promoted depalmitoylation is linked to conformational changes and not to second messenger generation. Given that, upon activation, the fusion protein mimics the activated receptor-G protein complex but cannot undergo desensitization, the data demonstrate that early steps in the activation process lead to the depalmitoylation of both receptor and G protein and that repalmitoylation requires later events that cannot be accommodated by the activated fusion protein.
Assuntos
Subunidades alfa Gs de Proteínas de Ligação ao GTP/metabolismo , Isoproterenol/farmacologia , Ácido Palmítico/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Animais , Linhagem Celular , Clonagem Molecular , Brometo de Cianogênio , Subunidades alfa Gs de Proteínas de Ligação ao GTP/química , Subunidades alfa Gs de Proteínas de Ligação ao GTP/isolamento & purificação , Humanos , Hidroxilamina/farmacologia , Cinética , Substâncias Macromoleculares , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/isolamento & purificação , Processamento de Proteína Pós-Traducional , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/isolamento & purificação , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Spodoptera , TransfecçãoRESUMO
Actin polymerization is essential for cell locomotion and is thought to generate the force responsible for cellular protrusions. The Arp2/3 complex is required to stimulate actin assembly at the leading edge in response to signalling. The bacteria Listeria and Shigella bypass the signalling pathway and harness the Arp2/3 complex to induce actin assembly and to propel themselves in living cells. However, the Arp2/3 complex alone is insufficient to promote movement. Here we have used pure components of the actin cytoskeleton to reconstitute sustained movement in Listeria and Shigella in vitro. Actin-based propulsion is driven by the free energy released by ATP hydrolysis linked to actin polymerization, and does not require myosin. In addition to actin and activated Arp2/3 complex, actin depolymerizing factor (ADF, or cofilin) and capping protein are also required for motility as they maintain a high steady-state level of G-actin, which controls the rate of unidirectional growth of actin filaments at the surface of the bacterium. The movement is more effective when profilin, alpha-actinin and VASP (for Listeria) are also included. These results have implications for our understanding of the mechanism of actin-based motility in cells.
Assuntos
Actinas/fisiologia , Proteínas Contráteis , Proteínas do Citoesqueleto , Listeria monocytogenes/fisiologia , Shigella/fisiologia , Fatores de Despolimerização de Actina , Proteína 2 Relacionada a Actina , Proteína 3 Relacionada a Actina , Trifosfato de Adenosina/fisiologia , Animais , Proteínas de Bactérias , Bovinos , Moléculas de Adesão Celular/fisiologia , Proteínas de Ligação a DNA/fisiologia , Escherichia coli/fisiologia , Humanos , Proteínas dos Microfilamentos/fisiologia , Movimento , Proteínas do Tecido Nervoso/fisiologia , Fosfoproteínas/fisiologia , Profilinas , Coelhos , Proteínas Recombinantes , Fatores de Transcrição/fisiologia , Proteína Neuronal da Síndrome de Wiskott-AldrichRESUMO
To propel itself in infected cells, the pathogen Shigella flexneri subverts the Cdc42-controlled machinery responsible for actin assembly during filopodia formation. Using a combination of bacterial motility assays in platelet extracts with Escherichia coli expressing the Shigella IcsA protein and in vitro analysis of reconstituted systems from purified proteins, we show here that the bacterial protein IcsA binds N-WASP and activates it in a Cdc42-like fashion. Dramatic stimulation of actin assembly is linked to the formation of a ternary IcsA-N-WASP-Arp2/3 complex, which nucleates actin polymerization. The Arp2/3 complex is essential in initiation of actin assembly and Shigella movement, as previously observed for Listeria monocytogenes. Activation of N-WASP by IcsA unmasks two domains acting together in insertional actin polymerization. The isolated COOH-terminal domain of N-WASP containing a verprolin-homology region, a cofilin-homology sequence, and an acidic terminal segment (VCA) interacts with G-actin in a unique profilin-like functional fashion. Hence, when N-WASP is activated, its COOH-terminal domain feeds barbed end growth of filaments and lowers the critical concentration at the bacterial surface. On the other hand, the NH(2)-terminal domain of N-WASP interacts with F-actin, mediating the attachment of the actin tail to the bacterium surface. VASP is not involved in Shigella movement, and the function of profilin does not require its binding to proline-rich regions.
Assuntos
Actinas/metabolismo , Proteínas de Ciclo Celular/metabolismo , Proteínas Contráteis , Proteínas do Citoesqueleto , Proteínas de Ligação a DNA/metabolismo , Proteínas de Ligação ao GTP/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Fatores de Transcrição/metabolismo , Proteína 2 Relacionada a Actina , Proteína 3 Relacionada a Actina , Animais , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Plaquetas/citologia , Plaquetas/metabolismo , Encéfalo/citologia , Encéfalo/metabolismo , Bovinos , Moléculas de Adesão Celular/metabolismo , Proteínas de Ligação a DNA/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/fisiologia , Células HeLa , Humanos , Listeria/fisiologia , Proteínas dos Microfilamentos/genética , Proteínas dos Microfilamentos/metabolismo , Movimento , Mutação , Proteínas do Tecido Nervoso/química , Fosfoproteínas/metabolismo , Polímeros , Profilinas , Prolina/metabolismo , Shigella flexneri/genética , Shigella flexneri/fisiologia , Fatores de Transcrição/genética , Proteína Neuronal da Síndrome de Wiskott-Aldrich , Proteína cdc42 de Saccharomyces cerevisiae de Ligação ao GTPRESUMO
Intracellular propulsion of Listeria monocytogenes is the best understood form of motility dependent on actin polymerization. We have used in vitro motility assays of Listeria in platelet and brain extracts to elucidate the function of the focal adhesion proteins of the Ena (Drosophila Enabled)/VASP (vasodilator-stimulated phosphoprotein) family in actin-based motility. Immunodepletion of VASP from platelet extracts and of Evl (Ena/VASP-like protein) from brain extracts of Mena knockout (-/-) mice combined with add-back of recombinant (bacterial or eukaryotic) VASP and Evl show that VASP, Mena, and Evl play interchangeable roles and are required to transform actin polymerization into active movement and propulsive force. The EVH1 (Ena/VASP homology 1) domain of VASP is in slow association-dissociation equilibrium high-affinity binding to the zyxin-homologous, proline-rich region of ActA. VASP also interacts with F-actin via its COOH-terminal EVH2 domain. Hence VASP/ Ena/Evl link the bacterium to the actin tail, which is required for movement. The affinity of VASP for F-actin is controlled by phosphorylation of serine 157 by cAMP-dependent protein kinase. Phospho-VASP binds with high affinity (0.5 x 10(8) M-1); dephospho-VASP binds 40-fold less tightly. We propose a molecular ratchet model for insertional polymerization of actin, within which frequent attachment-detachment of VASP to F-actin allows its sliding along the growing filament.
Assuntos
Actinas/fisiologia , Moléculas de Adesão Celular/fisiologia , Proteínas Contráteis , Proteínas do Citoesqueleto , Proteínas de Ligação a DNA/fisiologia , Listeria monocytogenes/fisiologia , Fosfoproteínas/fisiologia , Actinas/química , Actinas/ultraestrutura , Animais , Sequência de Bases , Sítios de Ligação , Plaquetas/metabolismo , Encéfalo/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/fisiologia , Moléculas de Adesão Celular/genética , Primers do DNA/genética , Proteínas de Ligação a DNA/genética , Listeria monocytogenes/genética , Camundongos , Camundongos Knockout , Proteínas dos Microfilamentos/fisiologia , Microscopia Eletrônica , Modelos Biológicos , Movimento/fisiologia , Fosfoproteínas/genética , Profilinas , Ligação Proteica , Proteínas/genética , Proteínas/fisiologiaRESUMO
We have recently demonstrated that wild-type beta2-adrenergic receptors (beta2AR) form homodimers and that disruption of receptor dimerization inhibits signalling via Gs [Hebert, Moffett, Morello, Loisel, Bichet, Barret and Bouvier (1996) J. Biol. Chem. 271, 16384-16392]. Here taking advantage of the altered functional properties of a non-palmitoylated, constitutively desensitized mutant beta2AR (C341Gbeta2AR), we sought to study whether physical interactions between mutant and wild-type beta2AR expressed in Sf9 cells could occur and have functional consequences. Using metabolic labelling with [3H]palmitate and co-immunoprecipitation we demonstrated the existence of heterodimerization between wild-type and C341Gbeta2AR. Furthermore, we show that, in co-expression experiments, wild-type receptors have a dominant positive effect resulting in the functional complementation of C341Gbeta2AR. Indeed, when expressed alone, the mutant C341G receptor displays altered functional characteristics in that (1) the response of the receptor to agonist is reduced as compared to the wild-type receptor and (2) the desensitization of the receptor in response to prolonged exposure to agonist is minimal. In contrast, when C341G and the wild-type beta2AR were expressed together, both the response to agonist and subsequent desensitization (at a constant level of total receptor) were equivalent to the wild-type beta2AR expressed alone. This dominant positive effect was also seen when C341G was co-expressed with a second receptor mutant in which the two protein kinase A phosphorylation sites (S261, 262, 345, 346A beta2AR) were mutated. Taken together these data suggest that intermolecular interactions between receptors may have both functional and structural implications for G-protein-mediated signalling.
Assuntos
Receptores Adrenérgicos beta 2/química , Adenilil Ciclases/metabolismo , Animais , Dimerização , Mutação Puntual , Ligação Proteica , Agregação de Receptores , Proteínas Recombinantes , Transdução de Sinais , Spodoptera , Relação Estrutura-AtividadeRESUMO
Expression in baculovirus-infected insect cells allows sufficient production of G-protein coupled receptor for structural studies. An important drawback of this expression system comes from the presence of unprocessed and biologically inactive receptors that have to be eliminated during receptor purification steps. We show that viral particles released from Sf9 cells infected with a recombinant baculovirus coding for the human beta 2-adrenergic receptor (beta 2AR) cDNA contain glycosylated and biologically active beta 2AR. In addition, post-translational modifications known to modulate receptor activity were found to occur in these particles.
Assuntos
Baculoviridae/genética , Receptores Adrenérgicos beta 2/fisiologia , Adenilil Ciclases/metabolismo , Animais , Linhagem Celular , Humanos , Microscopia Imunoeletrônica , Ligação Proteica , Processamento de Proteína Pós-Traducional , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Spodoptera , Vírion/genética , Vírion/ultraestruturaRESUMO
We have characterized the dynamic nature of beta 2-adrenergic receptor palmitoylation in Sf9 cells. Under basal conditions, the turnover of receptor-bound palmitate is rapid (half-life = 9.8 +/- 1.8 min) compared to the turnover rate of the receptor protein itself (half-life = 109 +/- 10 min). This suggests that an equilibrium between the palmitoylated and nonpalmitoylated forms of the receptor exists at resting state. Stimulation of the receptor by the agonist isoproterenol reduces the half-life of the beta 2-adrenergic receptor-bound palmitate by 1.8 fold without affecting the turnover rate of the receptor itself. Upon sustained stimulation, this increased palmitate turnover rate shifted the equilibrium toward the nonpalmitoylated form of the receptor, suggesting that prolonged activation either increases the rate of depalmitoylation or prevents receptor palmitoylation. Consistent with the latter possibility, pretreatment of cells with agonist, prior to metabolic labeling, reduced the incorporation of [3H]palmitate into the beta 2-adrenergic receptor by more than 80%. This suggests a link between receptor desensitization occurring upon sustained agonist stimulation and the decrease in receptor palmitoylation. Supporting this hypothesis, mutation of PKA phosphorylation sites known to be involved in receptor desensitization abolished the agonist-promoted reduction in palmitate incorporation. We have previously reported that palmitoylation of the beta 2-adrenergic receptor is important in controlling receptor phosphorylation by PKA [Moffett, S., et al. (1993) EMBO J. 12, 349-356; Moffett, S., et al. (1996) J. Biol. Chem. 271, 21490-21497]. The present study now demonstrates that the receptor palmitoylation state is regulated by agonist stimulation and suggests the existence of concerted reciprocal regulatory interactions between palmitoylation and phosphorylation upon sustained receptor stimulation.
Assuntos
Agonistas de Receptores Adrenérgicos beta 2 , Ácido Palmítico/metabolismo , Sequência de Aminoácidos , Animais , Baculoviridae/genética , Sítios de Ligação , Células Cultivadas , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Eletroforese em Gel de Poliacrilamida , Humanos , Isoproterenol/farmacologia , Cinética , Dados de Sequência Molecular , Mutação/genética , Ligação Proteica/efeitos dos fármacos , Receptores Adrenérgicos beta 2/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Spodoptera/metabolismoRESUMO
We previously showed that substitution of a glycine residue for the palmitoylated cysteine 341 of the human beta2-adrenergic receptor (Gly341beta2AR), increases the basal level of the receptor phosphorylation and reduces its ability to functionally interact with Gs. In the present study, we show that additional mutation of serines 345 and 346 (Ala345,346Gly341beta2AR) restored normal phosphorylation and receptor-Gs coupling, thus suggesting that the increased phosphorylation of this site, rather than the lack of palmitoylation per se, is responsible for the poor coupling of the unpalmitoylated receptor. This is supported by the observation that chemical depalmitoylation of purified beta2AR did not affect the ability of the receptor to stimulate adenylyl cyclase in reconstitution assays. Furthermore, mutation of Ser345,346 in a wild type receptor background (Ala345,346beta2AR) significantly decreased the rate of agonist-promoted desensitization of the receptor-stimulated adenylyl cyclase activity, supporting a role for this phosphorylation site in regulating the functional coupling of the receptor. Since serines 345 and 346 are located in a putative cyclic AMP-dependent protein kinase (PKA) phosphorylation site immediately downstream of the palmitoylated cysteine 341, the hypothesis that the accessibility of this site may be regulated by the receptor palmitoylation state was further assessed in vitro. In membrane phosphorylation assays, Gly341beta2AR was found to be a better substrate for PKA than the wild type receptor, thus supporting the notion that palmitoylation restrains access of the phosphorylation site to the enzyme. Taken together, the data demonstrate that palmitoylation of cysteine 341 controls the phosphorylation state of the PKA site located in the carboxyl tail of the beta2AR and by doing so modulates the responsiveness of the receptor.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Cisteína/metabolismo , Ácidos Palmíticos/metabolismo , Receptores Adrenérgicos beta 2/metabolismo , Agonistas Adrenérgicos beta/farmacologia , Sequência de Aminoácidos , Animais , Linhagem Celular , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Ácido Palmítico , Fosforilação , Receptores Adrenérgicos beta 2/efeitos dos fármacos , Receptores Adrenérgicos beta 2/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , SpodopteraRESUMO
One of the assumptions of the mobile receptor hypothesis as it relates to G protein-coupled receptors is that the stoichiometry of receptor, G protein, and effector is 1:1:1 (Bourne, H. R., Sanders, D. A., and McCormick, F.(1990) Nature 348, 125-132). Many studies on the cooperativity of agonist binding are incompatible with this notion and have suggested that both G proteins and their associated receptors can be oligomeric. However, a clear physical demonstration that G protein-coupled receptors can indeed interact as dimers and that such interactions may have functional consequences was lacking. Here, using differential epitope tagging we demonstrate that beta2-adrenergic receptors do form SDS-resistant homodimers and that transmembrane domain VI of the receptor may represent part of an interface for receptor dimerization. The functional importance of dimerization is supported by the observation that a peptide derived from this domain that inhibits dimerization also inhibits beta-adrenergic agonist-promoted stimulation of adenylyl cyclase activity. Moreover, agonist stimulation was found to stabilize the dimeric state of the receptor, while inverse agonists favored the monomeric species, which suggests that interconversion between monomeric and dimeric forms may be important for biological activity.
Assuntos
Fragmentos de Peptídeos/farmacologia , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/fisiologia , Adenilil Ciclases/metabolismo , Sequência de Aminoácidos , Animais , Baculoviridae , Linhagem Celular , Chlorocebus aethiops , Cromatografia de Afinidade , Cricetinae , Cricetulus , Humanos , Isoproterenol/farmacologia , Substâncias Macromoleculares , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas c-myc/biossíntese , Receptor Muscarínico M2 , Receptores Adrenérgicos beta 2/isolamento & purificação , Receptores de Dopamina D2/química , Receptores de Dopamina D2/fisiologia , Receptores Muscarínicos/biossíntese , Receptores de Vasopressinas/biossíntese , Receptores de Vasopressinas/química , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Sitios de Sequências Rotuladas , Spodoptera , TransfecçãoRESUMO
The ability of insulin to promote phosphorylation of the human beta 2-adrenergic receptor (beta 2AR) was assessed in Chinese hamster fibroblasts transfected with beta 2AR cDNA. Phosphotyrosine residues were detected in purified beta 2AR using a polyclonal anti-phosphotyrosine antibody and by phosphoamino acid analysis following metabolic labelling with inorganic 32P. Treatment of the cells with insulin induced a 2.4-fold increase in the phosphotyrosine content of the receptor. The insulin-promoted phosphorylation of the beta 2AR was accompanied by an increase in the beta-adrenergic-stimulated adenyl cyclase activity. Substitution of a phenylalanine residue for tyrosine-141 completely prevented both the increased tyrosine phosphorylation and the enhanced responsiveness of the beta 2AR promoted by insulin treatment. Mutation of three other tyrosines located in the cytoplasmic domain of the receptor, tyrosine-366, tyrosine-350 and tyrosine-354, did not abolish the insulin-promoted tyrosine phosphorylation. Taken together, these results suggest that insulin promotes phosphorylation of the beta 2AR on tyrosine-141 and that such phosphorylation leads to a supersensitization of the receptor.
Assuntos
Insulina/farmacologia , Receptores Adrenérgicos beta 2/metabolismo , Tirosina/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Sítios de Ligação , Células Cultivadas , Cricetinae , Primers do DNA , Fibroblastos/citologia , Humanos , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Fosforilação , Fosfotirosina/metabolismo , Conformação Proteica , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/genética , Relação Estrutura-Atividade , Transfecção , Tirosina/genéticaAssuntos
Proteínas de Ligação ao GTP/metabolismo , Ácidos Palmíticos/metabolismo , Receptores de Superfície Celular/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Ligação ao GTP/química , Proteínas de Ligação ao GTP/genética , Humanos , Dados de Sequência Molecular , Estrutura Molecular , Ácidos Palmíticos/química , Processamento de Proteína Pós-Traducional , Receptores Adrenérgicos beta 2/química , Receptores Adrenérgicos beta 2/genética , Receptores Adrenérgicos beta 2/metabolismo , Receptores de Superfície Celular/química , Receptores de Superfície Celular/genética , Transdução de SinaisAssuntos
Proteínas de Ligação ao GTP/metabolismo , Ácidos Palmíticos/metabolismo , Conformação Proteica , Receptores de Superfície Celular , Receptores Acoplados a Proteínas G , Receptores de Neurotransmissores/química , Receptores de Neurotransmissores/metabolismo , Sequência de Aminoácidos , Animais , Bovinos , Humanos , Dados de Sequência Molecular , Ácido Palmítico , Glicoproteínas da Membrana de Plaquetas/química , Glicoproteínas da Membrana de Plaquetas/metabolismo , Ratos , Receptores Adrenérgicos/química , Receptores Adrenérgicos/metabolismo , Receptores Muscarínicos/química , Receptores Muscarínicos/metabolismo , Receptores da Neurocinina-2/química , Receptores da Neurocinina-2/metabolismo , Receptores de Serotonina/química , Receptores de Serotonina/metabolismo , Rodopsina/química , Rodopsina/metabolismo , Homologia de Sequência de AminoácidosAssuntos
Proteínas de Ligação ao GTP/metabolismo , Ácidos Palmíticos/metabolismo , Processamento de Proteína Pós-Traducional , Receptores Adrenérgicos beta 2/metabolismo , Receptores de Superfície Celular/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Células Eucarióticas/metabolismo , Proteínas de Ligação ao GTP/biossíntese , Genes myc , Humanos , Radioisótopos do Iodo , Iodocianopindolol , Isoproterenol/farmacologia , Cinética , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Ácido Palmítico , Pindolol/análogos & derivados , Pindolol/metabolismo , Proteínas Proto-Oncogênicas c-myc/biossíntese , Técnica de Diluição de Radioisótopos , Ensaio Radioligante/métodos , Receptores Adrenérgicos beta 2/biossíntese , Receptores de Superfície Celular/biossíntese , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismo , Spodoptera , Transfecção/métodos , TrítioRESUMO
Site-directed mutagenesis was utilized to study the functional role of the COOH-terminal region in recombinant maize aldolase. A single mutation was created in each of the last nine amino acids of the COOH terminus and characterized kinetically. Point mutations in the COOH-terminal region were found to influence both the rate of fructose 1,6-bisphosphate and fructose 1-phosphate cleavage. Catalytic efficiency, kcat/Km, was not affected by the mutations within experimental error consistent with this region of the COOH terminus modulating product release. Concentrations of the carbanion-enamine enzyme intermediate complex produced upon substrate cleavage increased with the severity of the point mutation. A condensation assay was developed to directly measure fructose 1,6-bisphosphate synthesized by aldolases in the presence of high triose phosphate concentrations. The maximal rate of aldol condensation of triose phosphates, D-glyceralehyde-3-P and dihydroxyacetone-P, was affected by the point mutations to the same extent as the maximal rate of substrate cleavage. Interpretation of the data is consistent with point mutations in the COOH terminus predominantly affecting the proton exchange with the dihydroxyacetone-P enzymatic complex at the carbanion-enamine step and that this step is probably rate-limiting in the catalytic mechanism of recombinant maize aldolase. The role of the COOH-terminal region in aldolases is thus consistent with a sequence dependent modulation of catalytic activity.