Muscle Aldolase Research Articles

Comparisons of Phosphofructokinases-1 from Rabbit, Chicken, and Fish

Background: Previous studies suggested that glycogen storage was facilitated by ascorbate inhibition of phosphofructokinase-1 (PFK-1) in resting mammalian muscle; these studies showed that purified PFK-1 from fish or chicken muscle has properties similar to PFK-1 from mammalian muscles. Materials and Methods: The enzymes utilized in the assay systems came from Sigma-Aldrich Co. Rabbit (Oryctolagus cuniculus) muscle G-actin (A 2522) was free of aldolase, LDH (EC 1.1.1.28) or AK (EC 2.7.4.3) activity and rabbit muscle aldolase (EC 4.1.2.13) was free of AK (EC 2.7.4.3) and LDH activity. Rabbit muscle PFK-1 (RPFK-1), chicken (Gallus gallus) muscle PFK- 1 (CPFK-1) and Pacific red snapper (Lutjanus peru) muscle PFK-1 (FPFK-1) used in these tests were prepared from frozen tissues with modifications of a method. AK, LDH, and aldolase activity were absent in purified FPFK- 1 and CPFK-1 preparations. Results: It can be shown that the following enzymes associated with glycolysis are not inhibited by 0.1 M ascorbate under our conditions: rabbit muscle aldolase (EC 4.1.2.13); rabbit muscle glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.12); rabbit muscle phosphoglucose isomerase (EC 5.3.1.9); rabbit muscle pyruvate kinase (EC 2.7.1.40); yeast hexokinase (EC 2,7.1.1); and yeast 3-phosphoglyceric phosphokinase (EC 2.7.2.3). Rabbit muscle enolase (EC4.2.1.11) is inhibited under our conditions. Conclusion: In summary, FPFK-1 and CPFK-1, possess characteristics and behaviors similar to RPFK-1, e.g., losses of activities due to dilutions and protections of some of these activity losses by rabbit muscle aldolase. These interactions of a mammalian aldolase with fish and a bird PFK-1’s suggests a conservative evolutionary relationship among aldolases and PFK-1’s.

Inhibition by Ascorbate among Phosphofructokinase-1, Aldolase, Enolase, and Lactate Dehydrogenase in Rabbit Muscle

Background: These studies examined mutual protective relationships among rabbit muscle aldolase, enolase, phosphofructokinase-1 (PFK-1) and LDH from inhibitions by ascorbate (AA). It was proposed earlier that specific inhibitions of PFK-1 and LDH by AA faciltated glycogen storage in resting muscle by inhibiting glycolysis. Materials and Methods: The L-ascorbate (AA), L-ascorbyl dibutyrate (AADB), L-ascorbyl dipalmitate (AADP), L-ascorbyl palmitate (AAP), and L- ascorbyl stearate (AS) are shown in Figure 1 and were obtained from TCI and Alfa Aesar. Unless otherwise stated, all enzymes come from rabbit and all experimental temperatures were 25°C, pH 8.0. Results: Rabbit muscle enolase was examined for its protective effect on other rabbit muscle glycolytic enzymes against inhibitions by ascorbate (AA) and some AA-faty acid derivatives. The IC50 values of enolase by ascorbate (AA) and IC50 values of AA-fatty acid derivatives were compared to estimate inhibition potency. For example, ascorbyl dipalmitate (AADP) was 156 times more inhibitory to enolase than AA. It was previously shown that rabbit muscle aldolase prevented LDH activity loses due to AA inhibition and prevented PFK-1 activity losses due both to dilution and AA inhibition; enolase was found to have the same effects as aldolase. Additionally, PFK-1 prevented enolase and LDH inhibitions by AA. LDH did not prevent enolase or PFK-1 from inhibition by AA. LDH did stimulate enolase activity but not PFK-1 activity. Conclusion: The results suggest that interactions among glycolytic enzyme serve to mutually protect one another from activity losses. The inhibition properties of the AA-fatty acid derivatives are discussed in relation to their possible roles in cancer and diabetes.

A case for glycerol as an acceptable additive for single-particle cryoEM samples.

Buffer-composition and sample-preparation guidelines for cryo-electron microscopy are geared towards maximizing imaging contrast and reducing electron-beam-induced motion. These pursuits often involve the minimization or the complete removal of additives that are commonly used to facilitate proper protein folding and minimize aggregation. Among these admonished additives is glycerol, a widely used osmolyte that aids protein stability. In this work, it is shown that the inclusion of glycerol does not preclude high-resolution structure determination by cryoEM, as demonstrated by an ∼2.3 Å resolution reconstruction of mouse apoferritin (∼500 kDa) and an ∼3.3 Å resolution reconstruction of rabbit muscle aldolase (∼160 kDa) in the presence of 20%(v/v) glycerol. While it was found that generating thin ice that is amenable to high-resolution imaging requires long blot times, the addition of glycerol did not result in increased beam-induced motion or an inability to pick particles. Overall, these findings indicate that glycerol should not be discounted as a cryoEM sample-buffer additive, particularly for large, fragile complexes that are prone to disassembly or aggregation upon its removal.

Benchmarking cryo-EM Single Particle Analysis Workflow.

Cryo electron microscopy facilities running multiple instruments and serving users with varying skill levels need a robust and reliable method for benchmarking both the hardware and software components of their single particle analysis workflow. The workflow is complex, with many bottlenecks existing at the specimen preparation, data collection and image analysis steps; the samples and grid preparation can be of unpredictable quality, there are many different protocols for microscope and camera settings, and there is a myriad of software programs for analysis that can depend on dozens of settings chosen by the user. For this reason, we believe it is important to benchmark the entire workflow, using a standard sample and standard operating procedures, on a regular basis. This provides confidence that all aspects of the pipeline are capable of producing maps to high resolution. Here we describe benchmarking procedures using a test sample, rabbit muscle aldolase.

ChemInform Abstract: A Chemoenzymatic Route to Prepare Acyclic Nucleoside Analogues

Abstractinvolving an aldol condensation of aldehydes of various nucleobases catalyzed by a dihydroxyacetone phosphate‐dependent aldolase (rabbit muscle aldolase)

A Chemoenzymatic Route To Prepare Acyclic Nucleoside Analogues

AbstractAcyclic nucleosides are potential antiviral and antitumor agents. In this work, their preparation through a novel chemoenzymatic procedure involving the N‐alkylation of a nucleobase and subsequent aldol condensation catalyzed by a dihydroxyacetone phosphate‐dependant aldolase (Rabbit Muscle Aldolase, RAMA) is described.

Prozessentwicklung zur enzymatischen Synthese eines biologisch abbaubaren Polymers

A single-pot enzymatic reaction sequence has been designed for the synthesis of a key intermediate metabolite called D-glyceraldehyde-3-phosphate (D-GAP). The reaction sequence consists of three enzymes and uses D-F16BP as a starting material (Fig.). The rabbit muscle aldolase (RAMA)catalyzed reaction step is in favor of D-F16BP formation with an equilibrium constant of 10M [1]. Therefore, the single-pot reaction sequence has been designed to shift the equilibrium and regenerate NADH. The capability of the reaction sequence was demonstrated by the enhanced equilibrium conversion from 1.4% to 96%. The reaction system was optimized in detail with regard to activity and stability of the enzymes and stability of the cofactors (NADH and NAD) and D-GAP. Reaction kinetics models for each of the enzymes were formulated. The effect of co-substances on the activity of the enzymes was evaluated. The results elucidated that D-F16BP and sn-G3P do not influence the activity of FDH and HCOO does not affect the activity of RAMA, whereas D-F16BP and HCOO suppress the activity of sn-G3PDH. From the kinetics perspective and due to the stability of the enzymes, a continuous operation based on an enzyme membrane reactor was preferred.

Synthesis of sugars catalysed by microgel conjugated rabbit muscle aldolase in aqueous and aqueous-organic solvents II

Kinetic runs of microgel-aldolase mediated reactions between dihydroxyacetone phosphate cyclohexylamine salt and D-glyceraldehydes were made in CH2CN, THF, DMF, DMSO and MeOH and aqueous-organic solution of these solvents at physiological temperature 37º. The same runs were effected between DHAP cyclohexylamine salt and non-natural aldehyde acceptors under similar conditions. Reaction was fastest in 1:3 DMSO/ H2O, faster than in aqueous medium the environment in which this enzyme normally operates. Preparation of a non-natural sugar using this system is related.Keywords: Microgel-aldolase, Kinetic physiological temperature, Natural acceptors, Non-natural acceptors.

Spinach aldolase interactions with rabbit, chicken, and fish muscle phosphofructokinase-1

Previous studies showed that rabbit muscle phosphofructokinase-1 (PFK-1) activity losses due to dilution, due to inhibition by ascorbate, and due to some lithium salts were prevented by rabbit muscle aldolase. Chicken PFK-1 and fish PFK-1 interacted with ascorbate and were inhibited, consistent with a previously proposed function that ascorbate facilitates glycogen in resting muscle by inhibiting glycolysis. This report shows that a plant enzyme, spinach aldolase, has the same ability to prevent rabbit muscle PFK-1 activity loses as rabbit muscle aldolase and in some instances it was a better protector from activity losses than rabbit aldolase. Spinach aldolase also protected chicken and fish PFK-1s from inhibitions by ascorbate and from activity losses due to dilution. Prevention of losses PFK-1 activities from animal species by a plant protein, spinach aldolase, suggests an evolutionary conservative relationship between PFK-1s and aldolases.

Synthesis of Carbohydrates in a Continuous Flow Reactor by Immobilized Phosphatase and Aldolase

Herein, we report a new flow process with immobilized enzymes to synthesize complex chiral carbohydrate analogues from achiral inexpensive building blocks in a three-step cascade reaction. The first reactor contained immobilized acid phosphatase, which phosphorylated dihydroxyacetone to dihydroxyacetone phosphate using pyrophosphate as the phosphate donor. The second flow reactor contained fructose-1,6-diphosphate aldolase (RAMA, rabbit muscle aldolase) or rhamnulose-1-phosphate aldolase (RhuA from Thermotoga maritima) and acid phosphatase. The immobilized aldolases coupled the formed dihydroxyacetone phosphate to aldehydes, resulting in phosphorylated carbohydrates. A final reactor containing acid phosphatase that dephosphorylated the phosphorylated product yielded the final product. Different aldehydes were used to synthesize carbohydrates on a gram scale. To demonstrate the feasibility of the flow systems, we synthesized 0.6 g of the D-fagomine precursor. By using immobilized aldolase RhuA we were also able to obtain other stereoisomers of the D-fagomine precursor.

Synthesis of sugars catalysed by microgel conjugated rabbit muscle aldolase in aqueous and aqueous-organic solvents I

Synthesis of sugars catalysed by microgel conjugated rabbit muscle aldolase in aqueous and aqueous-organic solvents I

Regulation of plant cytosolic aldolase functions by redox-modifications

From the five genes which code for cytosolic fructose 1,6-bisphosphate aldolases in Arabidopsis thaliana L., the cDNA clone of cAld2 (At2g36460), was heterologously expressed in E.coli and incubated under various oxidizing and reducing conditions. Covalent binding of a GSH moiety to the enzyme was shown by incorporation of biotinylated GSH (BioGEE) and by immunodetection with monoclonal anti-GSH serum. Nitrosylation after incubation with GSNO or SNP was demonstrated using the biotin-switch assay. Mass-spectrometry analysis showed glutathionylation and/or nitrosylation at two different cysteine residues: GSH was found to be attached to C68 and C173, while the nitroso-group was incorporated only into C173. Non-reducing SDS-PAGE conducted with purified wild-type and various Cys-mutant proteins revealed the presence of disulfide bridges in the oxidized enzyme, as described for rabbit muscle aldolase. Incubation of the purified enzyme with GSSG (up to 25mM) led to partial and reversible inactivation of enzyme activity; NADPH, in the presence of the components of the cytosolic NADP-dependent thioredoxin system, could reactivate the aldolase as did DTT. Total and irreversible inactivation occurred with low concentrations (0.1mM) of nitrosoglutathione (GSNO). Inactivation was prevented by co-incubation of cAld2 with fructose-1,6-bisphosphate (FBP). Nuclear localization of cAld2 and interaction with thioredoxins was shown by transient expression of fusion constructs with fluorescent proteins in isolated protoplasts.

Skeletal Muscle Aldolase an Overexpression in Endotoxemic Rats and Inhibited by GSNO via Potential Role for S-nitrosylation In Vitro

Hepatic aldolase (ALD) A mRNA transcription and ALD B S-nitrosylation have been confirmed in endotoxemic rats and mice, respectively. In the present study we investigated whether the skeletal muscle ALD A shared potential for S-nitrosylation to act as a hypoxia-related signaling mechanism in lipopolysaccharide (LPS) challenged rats. Male Sprague Dawley rats were treated (i.p.) as follows, control group (n = 6) with 0.9% NaCl, tested group (n = 6) with a single dose of 2 mg/kg LPS. Protein S-nitrosylation was determined by biotin switch and dot blotting analysis. ALD A, hypoxia-inducible factor 1α and vascular endothelial growth factor were determined by western blotting. ALD A catalytic activity treated with S-nitrosoglutathione (GSNO), an exogenous NO-donor, was examined in vitro. There were several S-nitrosylated proteins under basal conditions. ALD A was over-expressed in a hypoxia-related way in the skeletal muscle of LPS challenged rats. Importantly, treatment of ALD A with GSNO at concentration 50 μmol/L ∼ 1000 μmol/L that inhibited catalytic activity, increased the number of S-nitrosylated bands and led to hyper-nitrosylation of basally S-nitrosylated proteins of ALD A. Quantization of enzyme S-nitrosothiol showed that a maximal of four cysteines per subunit was modified by S-nitrosylation in the presence of GSNO. These findings suggested that S-nitrosylation of ALD A might serve as a novel mechanism for controlling ALD A activity at the post-translational level in endotoxemic rats.

Gram scale synthesis of 3-fluoro-1-hydroxyacetone phosphate: a novel donor substrate in rabbit muscle aldolase-catalyzed aldol reactions

An efficient gram scale synthesis of 3-fluoro-1-hydroxyacetone phosphate (FHAP) has been developed. As a close analog to dihydroxyacetone phosphate, FHAP was used as a novel donor substrate for rabbit muscle aldolase catalyzed reactions. The different binding affinities of the gem-diol and keto form of FHAP were studied by (19)F-NMR.

ChemInform Abstract: A New Approach to Cyclitols Based on Rabbit Muscle Aldolase (RAMA).

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

ChemInform Abstract: Synthesis of Perfluoroalkylated Sugars Catalyzed by Rabbit Muscle Aldolase (RAMA).

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Dramatic improvement of crystal quality for low-temperature-grown rabbit muscle aldolase.

Rabbit muscle aldolase (RMA) was crystallized in complex with the low-complexity domain (LC4) of sorting nexin 9. Monoclinic crystals were obtained at room temperature that displayed large mosaicity and poor X-ray diffraction. However, orthorhombic RMA-LC4 crystals grown at 277 K under similar conditions exhibited low mosaicity, allowing data collection to 2.2 A Bragg spacing and structure determination. It was concluded that the improvement of crystal quality as indicated by the higher resolution of the new RMA-LC4 complex crystals was a consequence of the introduction of new lattice contacts at lower temperature. The lattice contacts corresponded to an increased number of interactions between high-entropy side chains that mitigate the lattice strain incurred upon cryocooling and accompanying mosaic spread increases. The thermodynamically unfavorable immobilization of high-entropy side chains used in lattice formation was compensated by an entropic increase in the bulk-solvent content owing to the greater solvent content of the crystal lattice.

Mechanism of Aldolase Control of Sorting Nexin 9 Function in Endocytosis

Sorting nexin 9 (SNX9) functions in a complex with the GTPase dynamin-2 at clathrin-coated pits, where it provokes fission of vesicles to complete endocytosis. Here the SNX9.dynamin-2 complex binds to clathrin and adapter protein complex 2 (AP-2) that line these pits, and this occurs through interactions of the low complexity domain (LC4) of SNX9 with AP-2. Intriguingly, localization of the SNX9.dynamin-2 complex to clathrin-coated pits is blocked by interactions with the abundant glycolytic enzyme aldolase, which also binds to the LC4 domain of SNX9. The crystal structure of the LC4 motif of human SNX9 in complex with aldolase explains the biochemistry and biology of this interaction, where SNX9 binds near the active site of aldolase via residues 165-171 that are also required for the interactions of SNX9 with AP-2. Accordingly, SNX9 binding to aldolase is structurally precluded by the binding of substrate to the active site. Interactions of SNX9 with aldolase are far more extensive and differ from those of the actin-nucleating factor WASP with aldolase, indicating considerable plasticity in mechanisms that direct the functions of the aldolase as a scaffold protein.

Fructose‐1,6‐Bisphosphate Aldolase‐Mediated Synthesis of Aminocyclitols (Analogues of Valiolamine) and their Evaluation as Glycosidase Inhibitors

AbstractA highly stereoselective method for the preparation of nitro‐ and aminocyclitols, using fructose‐1,6‐bisphosphate aldolase, which catalyzes the aldol reaction of dihydroxyacetone phosphate (DHAP) on hydroxynitrobutanals, is reported. The key part of the synthesis is based on a one‐pot /two‐enzyme process whereby three reactions take place; rabbit muscle aldolase (RAMA) catalyzed aldolization, phytase‐catalyzed phosphate hydrolysis, and intramolecular spontaneous nitroaldolization. Two families of nitrocyclitols were obtained depending on the carbon configuration in the β position to the nitro group. Reduction of the latter afforded the aminocyclitols. Evaluation of the inhibition properties of the amines towards five commercially available glycosidases has shown selectivity for β‐glucosidase and β‐galactosidase.

Charge Stabilization and Entropy Reduction of Central Lysine Residues in Fructose-Bisphosphate Aldolase

Fructose-1,6-bisphosphate muscle aldolase is an essential glycolytic enzyme that catalyzes reversible carbon-carbon bond formation by cleaving fructose 1,6-bisphosphate to yield dihydroxyacetone phosphate (DHAP) and d-glyceraldehyde phosphate. To elucidate the mechanistic role of conserved amino acid Asp-33, Asn-33 and Ser-33 mutants were examined by kinetic and structural analyses. The mutations significantly compromised enzymatic activity and carbanion oxidation in presence of DHAP. Detailed structural analysis demonstrated that, like native crystals, Asp-33 mutant crystals, soaked in DHAP solutions, trapped Schiff base-derived intermediates covalently attached to Lys-229. The mutant structures, however, exhibited an abridged conformational change with the helical region (34-65) flanking the active site as well as pK(a) reductions and increased side chain disorder by central lysine residues, Lys-107 and Lys-146. These changes directly affect their interaction with the C-terminal Tyr-363, consistent with the absence of active site binding by the C-terminal region in the presence of phosphate. Lys-146 pK(a) reduction and side chain disorder would further compromise charge stabilization during C-C bond cleavage and proton transfer during enamine formation. These mechanistic impediments explain diminished catalytic activity and a reduced level of carbanion oxidation and are consistent with rate-determining proton transfer observed in the Asn-33 mutant. Asp-33 reduces the entropic cost and augments the enthalpic gain during catalysis by rigidifying Lys-107 and Lys-146, stabilizing their protonated forms, and promoting a conformational change triggered by substrate or obligate product binding, which lower kinetic barriers in C-C bond cleavage and Schiff base-enamine interconversion.