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Subunit ratios of separated hybrid hexamers of Neurospora NADP-specific glutamate dehydrogenase containing complementing mutationally modified monomers.

ABSTRACT: The am1 and am3 mutational variants of the Neurospora crassa NADP-specific glutamate dehydrogenase show complementation activity in hybrid hexamers. A freeze-thaw hybridization method was used to construct hybrids from purified enzymes and the products were separated into species of different monomer ratio by affinity chromatography. Hexamers with am1:am3 ratios of 1:5, 2:4, 3:3, 4:2 and 5:1 were all recovered as resolved or partially resolved peaks in quantities approximating to a binomial distribution. Reassociation of monomers during the hybridization process was random, except for some differential loss of am3 protein by precipitation and an apparent absence of reassociated am1 homohexamers. Complementation activity was shown by hybrids of all five monomer ratios, owing to activation of am3 monomers by conformational constraints arising from the intrinsically inactive am1 monomers. The activating effect of such constraints was greatest in hexamers containing only a single am1 monomer and least in the 5 am1:1am3 species. When fully activated by L-glutamate all am3 monomers were equivalent in intrinsic catalytic activity, irrespective of the number of am1 monomers per hexamer.

SUBMITTER: Watson DH

PROVIDER: S-EPMC1186176 | biostudies-other | 1978 Dec

REPOSITORIES: biostudies-other

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Project description:The synthesis of an affinity adsorbent, 8-(6-aminohexyl)aminoadenosine 2'-phosphate-Sepharose 4B, is described. The assembly of the 2'-AMP ligand and the hexanediamide spacer arm was synthesized in free solution before its attachment to the Sepharose matrix. This adsorbent retarded the hexameric NADP-specific glutamate dehydrogenase of Neurospora crassa, showing a capacity for this enzyme similar to that of comparable coenzyme-analogue adsorbents for other dehydrogenases. The enzyme was eluted either at pH 6.8 in a concentration gradient of NADP+, or at pH 8.5 in the presence of NADP+ in concentration gradients of either dicarboxylates or NaCl. Anomalous effects of dicarboxylates in facilitating elution are discussed. 2'-AMP and its derivatives, 8-bromoadenosine 2'-phosphate and 8-(l-aminohexyl)aminoadenosine 2'-phosphate, which were used in the synthesis of the adsorbent, all acted as enzyme inhibitors competitive with NADP+. The chromatographic properties of the wild-type enzyme were compared with those of mutationally modified variants containing defined amino acid substitutions. This approach was used to assess the biospecificity of adsorption and elution and the contribution of non-specific binding. The adsorbent showed a low capacity for the enzyme from mutant am1 (Ser-336 replaced by Phe), a variant that has a localized defect in NADP binding, but an otherwise almost normal conformation, suggesting that non-specific interactions are at most weak. The enzyme from mutant am3, a variant modified in a conformational equilibrium, was fully retarded by the adsorbent, but showed a significantly earlier elution position than the wild-type enzyme. This is consistent with measurements in free solution that showed the am3 enzyme to have a higher Ki for 2'-AMP than the wild-type enzyme. The enzyme from mutant am19 was eluted as two distinct peaks at both pH 6.8 and 8.5. The adsorbent was used to separate hybrid hexamers constructed in vitro by a freeze-thaw procedure from pairs of purified variants. Several chromatographically distinct peaks of differing enzymological properties were purified from each hybridization mixture in quantities of up to a few milligrams, and represented distinct species of hybrid hexamers differing in subunit ratio.

Project description:ObjectivesThe susceptibility of methacrylates to hydrolytic and enzymatic degradation may be a contributing factor limiting the clinical lifespan of resin composite restorations. The elimination of labile ester bonds is a potential advantage of methacrylamides, which have been shown to produce more stable restorative interfaces. The rationale of this study is to design hydrolytically and enzymatically stable adhesive monomers, with the added benefit of being able to form crosslinked networks. The objective of this study was to synthesize difunctional, hybrid methacrylate-methacrylamide monomers, and evaluate them as potential monomers for dental adhesives.Materials and methodsHEMA, TEGDMA (controls) or secondary methacrylamides (HEMAM - commercially available, 2EM and 2dMM - newly synthesized) either bearing a hydroxyl group or a methacrylate functionality (Hybrids-Hy), were added at 40mass% to bisGMA. The photoinitiator system consisted of 2-dimethoxyphenyl acetophenone (DMPA) and diphenyl iodonium hexafluorophosphate (DPI-PF6) at 0.2 and 0.4mass%, respectively. Polymerization kinetics were followed in real-time by near-IR spectroscopy during light activation at 630mW/cm2 for 300s. Water sorption and solubility (WS, SL) were measured according to ISO 4049. Storage modulus in shear (G') for 300s was obtained by oscillatory rheometry. For the microtensile bond strength (μTBS), fully formulated adhesives containing 40vol% ethanol were used to restore caries-free human third molars. Bonded specimens with 1mm2 cross-sectional area were tested after 48h and 6 months storage in water at 37°C. Single bond (SB) was tested as a commercial control. Data were analysed with one-way ANOVA and Tukey's test and Student's t-test (α=0.05).ResultsIn general, hybrid versions showed lower polymerization rate and degree of conversion, whereas the methacrylate controls, HEMA and TEGDMA, showed the highest values. The hybrid versions showed lower values of WS and SL than their monofunctional versions. HEMAM Hy showed the highest values of G' and TEGDMA, 2EM, and 2dMM-Hy the lowest. The μTBS values between 48h and 6 months were statistically reduced only for the HEMA and both 2dMM materials. The formulation containing the monofunctional methacrylamide (HEMAM) showed only 9% reduction in μTBS after 6 months of aging, while the other groups showed a decrease ranging between 18% and 33%.ConclusionOverall, hybrid monomers showed lower reactivity than their analogous monofunctional versions, but had markedly lower water sorption. Shear storage modulus was affected differently by the addition of the second functionality. HEMAM-containing systems were able to maintain stable long-term dentin bond strength, which demonstrates that bonding stability is a result of the complex interplay among the factors studied.Clinical significanceThe novel monomers showed here are potential alternatives to the current methacrylate adhesives, with selected formulations presenting greater bond stability.

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Subunit ratios of separated hybrid hexamers of Neurospora NADP-specific glutamate dehydrogenase containing complementing mutationally modified monomers.

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