Synthesis of an organoinsulin molecule that can be activated by antibody catalysis.
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We have developed a methodology of prodrug delivery by using a modified insulin species whose biological activity potentially can be regulated in vivo. Native insulin was derivatized with aldol-terminated chemical modifications that can be selectively removed by the catalytic aldolase antibody 38C2 under physiologic conditions. The derivatized organoinsulin (insulin(D)) was defective with respect to receptor binding and stimulation of glucose transport. The affinity of insulin(D) for the insulin receptor was reduced by 90% in binding studies using intact cells. The ability of insulin(D) to stimulate glucose transport was reduced by 96% in 3T3-L1 adipocytes and by 55% in conscious rats. Incubation of insulin(D) with the catalytic aldolase antibody 38C2 cleaved all of the aldol-terminated modifications, restoring native insulin. Treatment of insulin(D) with 38C2 also restored insulin(D)'s receptor binding and glucose transport-stimulating activities in vitro, as well as its ability to lower glucose levels in animals in vivo. We propose that these results are the foundation for an in vivo regulated system of insulin activation using the prohormone insulin(D) and catalytic antibody 38C2 with potential therapeutic application. (+info)
Renaturation of acid-denatured rabbit muscle aldolase. Existence and properties of a stable monomeric intermediate.
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The effects of temperature, pH and the substrate, fructose 1,6-bisphosphate, upon the kinetics and yield of renaturation of acid-denatured rabbit muscle aldolase have been investigated. The results are discussed in terms of a sequential set of events leading from the unfolded polypeptide chain to the renatured oligomeric enzyme. One of the intermediate molecular species in this sequence has been characterized as a folded monomer with a sedimentation coefficient of 3.1 S. This monomer is shown to be much more heat-labile than the tetramer under identical conditions, thus demonstrating stabilization of the tertiary structure of the polypeptide chain by the quaternary interactions between protomers. (+info)
PCR primers for an aldolase-B intron in acanthopterygian fishes.
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BACKGROUND: Nuclear DNA sequences provide genetic information that complements studies using mitochondrial DNA. Some 'universal' primer sets have been developed that target introns within protein-coding loci, but many simultaneously amplify introns from paralogous loci. Refining existing primer sets to target a single locus could circumvent this problem. RESULTS: Aldolase intron 'G' was amplified from four fish species using previously described primer sets that target several loci indiscriminately. Phylogenetic analyses were used to group these fragments and other full-length aldolase proteins from teleost fishes into orthologous clades and a primer set was designed to target specifically an intron within the aldolase-B locus in acanthopterygian fishes. DNA amplifications were tried in a variety of acanthopterygian fishes and amplification products, identifiable as aldolase-B intron 'G', were observed in all atherinomorph and percomorph taxa examined. Sequence variation within this locus was found within and among several species examined. CONCLUSIONS: Using 'universal' primer sets coupled with phylogenetic analyses it was possible to develop a genetic assay to target a specific locus in a variety of fish taxa. Sequence variation was observed within and among species suggesting that this targeted assay might facilitate interspecific and intraspecific comparisons. (+info)
The effects of aeration on glucose catabolism in Penicillium expansum.
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Polyacrylamide-disc gel electrophoresis and quantitative enzyme assays showed that the pathways of glucose catabolism and secondary metabolism in Penicillium expansum were dependent on the degree of aeration of the cultures. The isoenzyme patterns and specific activities of aldolase and succinate dehydrogenase indicated that glycolysis and the tricarboxylic acid cycle operated under conditions of both limited and efficient aeration (i.e. in cultures grown statically or on an orbital shaker). At high levels of aeration the growth rate was faster and synthesis of extracellular pectolytic enzymes was enhanced, whilst the activities of glucose 6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase showed that the pentose-phosphate shunt was important in glucose catabolism during the trophophase of growth. In contrast, under conditions of low aeration this latter pathway was virtually undetectable, growth was slower, pectolytic enzyme production low and large concentrations of secondary metabolites (6-methylsalicylic acid, patulin and citrinin) accumulated. (+info)
A conserved glutamate residue exhibits multifunctional catalytic roles in D-fructose-1,6-bisphosphate aldolases.
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The aldolase catalytic cycle consists of a number of proton transfers that interconvert covalent enzyme intermediates. Glu-187 is a conserved amino acid that is located in the mammalian fructose-1,6-bisphosphate aldolase active site. Its central location, within hydrogen bonding distance of three other conserved active site residues: Lys-146, Glu-189, and Schiff base-forming Lys-229, makes it an ideal candidate for mediating proton transfers. Point mutations, Glu-187--> Gln, Ala, which would inhibit proton transfers significantly, compromise activity. Trapping of enzymatic intermediates in Glu-187 mutants defines a proton transfer role for Glu-187 in substrate cleavage and Schiff base formation. Structural data show that loss of Glu-187 negative charge results in hydrogen bond formation between Lys-146 and Lys-229 consistent with a basic pK(a) for Lys-229 in native enzyme and supporting nucleophilic activation of Lys-229 by Glu-187 during Schiff base formation. The crystal structures also substantiate Glu-187 and Glu-189 as present in ionized form in native enzyme, compatible with their role of catalyzing proton exchange with solvent as indicated from solvent isotope effects. The proton exchange mechanism ensures Glu-187 basicity throughout the catalytic cycle requisite for mediating proton transfer and electrostatic stabilization of ketamine intermediates. Glutamate general base catalysis is a recurrent evolutionary feature of Schiff base0forming aldolases. (+info)
Onchocerca volvulus glycolytic enzyme fructose-1,6-bisphosphate aldolase as a target for a protective immune response in humans.
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To identify potential vaccine candidates for the prevention of infection with the filarial nematode Onchocerca volvulus, we screened an O. volvulus L3 stage cDNA library with sera from putatively immune (PI) subjects, and a prominent immunogenic clone of 1,184 nucleotides was identified. It contained an open reading frame of 363 amino acids encoding the glycolytic enzyme fructose 1,6 bisphosphate aldolase (Ov-fba-1). Immunolocalization experiments demonstrated that the protein was most abundantly expressed in metabolically active tissues, including body wall muscle and the reproductive tract of adult female worms. Immunoelectron microscopy of L3 demonstrated binding in the region where the cuticle separates during molting, in the channels connecting the esophagus to the cuticle, and in the basal lamina surrounding the esophagus and the body cavity. Among subjects from areas where this organism is endemic specific humoral and cellular immune responses to recombinant protein were observed in both PI and infected subjects, whereas responses were not observed among subjects who had not been exposed to O. volvulus. Despite the absence of differential responsiveness in parasite-exposed human populations, when the recombinant was tested for protective efficacy in a mouse chamber model, a reduction in survival of larvae by ca. 50% was seen. This observation provides support for the further study of this parasite enzyme as a vaccine candidate in larger animal models. (+info)
The complex of band 3 protein of the human erythrocyte membrane and glyceraldehyde-3-phosphate dehydrogenase: stoichiometry and competition by aldolase.
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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. (+info)
A cofactor approach to copper-dependent catalytic antibodies.
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A strategy for the preparation of semisynthetic copper(II)-based catalytic metalloproteins is described in which a metal-binding bis-imidazole cofactor is incorporated into the combining site of the aldolase antibody 38C2. Antibody 38C2 features a large hydrophobic-combining site pocket with a highly nucleophilic lysine residue, Lys(H93), that can be covalently modified. A comparison of several lactone and anhydride reagents shows that the latter are the most effective and general derivatizing agents for the 38C2 Lys residue. A bis-imidazole anhydride (5) was efficiently prepared from N-methyl imidazole. The 38C2-5-Cu conjugate was prepared by either (i) initial derivatization of 38C2 with 5 followed by metallation with CuCl2, or (ii) precoordination of 5 with CuCl2 followed by conjugation with 38C2. The resulting 38C2-5-Cu conjugate was an active catalyst for the hydrolysis of the coordinating picolinate ester 11, following Michaelis-Menten kinetics [kcat(11) = 2.3 min(-1) and Km(11) 2.2 mM] with a rate enhancement [kcat(11)k(uncat)(11)] of 2.1 x 10(5). Comparison of the second-order rate constants of the modified 38C2 and the Cu(II)-bis-imidazolyl complex k(6-CuCl2) gives a rate enhancement of 3.5 x 10(4) in favor of the antibody complex with an effective molarity of 76.7 M, revealing a significant catalytic benefit to the binding of the bis-imidazolyl ligand into 38C2. (+info)