Alpha-glycerophosphate dehydrogenase in Drosophila melanogaster: kinetic differences and developmental differentiation of the larval and adult isozymes

The isozymes of α-glycerophosphate dehydrogenase (α-GPDH) differ markedly with respect to their kinetic and stability parameters. Adult-limited GPDH-1 is stable at 50°C but decays at 57°C, while GPDH-3 is labile at 50°C under similar experimental conditions. By extrapolation of the thermal denaturation curves of crude adult extracts, we estimate GPDH-1 to constitute 76 per cent of the adult α-GPDH activity. Substrate kinetic studies revealed that, at pH 9·5, GPDH-3 exhibits an affinity for α-glycerophosphate which is twofold higher than that of GPDH-1, while K_m's for NAD⁺ are indistinguishable. The apparent K_m of GPDH-3 for dihydroxyacetone phosphate is consistently lower than that of GPDH-1 at pH 7·5, whereas at pH 6·7 the latter isozyme's apparent K_m approximates that of GPDH-3 at pH 7·5. Indistinguishable molecular weights of 66,000 were estimated by gel filtration for both GPDH-1 and 3. Gene dosage studies indicate that all three α-GPDH isozymes are simultaneously affected by dosage of the Gdh⁺ locus. These observations support a homomultimeric model of α-GPDH and the isozymes just discussed arise through epigenetic modification of the product of a single structural gene locus.     

Kinetic properties and regulation of glycerol-3-phosphate dehydrogenase from the overwintering, freezing-tolerant gall fly larva, Eurosta solidagenis

The kinetic properties of cytoplasmic glycerol-3-P dehydrogenase from the third instar larva of the gall fly, Eurosta solidaginis, were studied with emphasis on temperature effects on the enzyme and the regulation of enzyme activity during the synthesis of the cryoprotectant, glycerol. Isoelectrofocusing revealed one major and two minor forms of the enzyme with no alteration in the pI's or relative activities of the forms in larvae acclimated to 24 versus ?30 °C. Kinetic properties of the enzyme were also the same in larvae acclimated to high and low temperatures. Arrhenius plots were linear over a 30 to 0 °C range with an activation energy of 12,630 ± 185 cal/mol and a Q₁₀ of 2.16. The K_m for dihydroxyacetone-P was constant, at 50 μM, between 30 and 10 °C but increased by 75% at 0 °C; this increase may be a factor in the cessation of glycerol synthesis which occurs below 5 °C in this species. The K_m(NADH), by contrast, was higher (5–6 μM) at 30 °C but decreased (3 μM) at lower temperatures. In the reverse direction, K_m's were 340 μM for glycerol-3-P and 12 μM for NAD⁺. Effects of most inhibitors (of the forward reaction), glycerol-3-P (K_i = 2.4 mM), NAD⁺ (K_i = 0.2 mM), ATP, Mg·ATP, and P_i, were unaltered by assay temperature but ADP effects were potentiated by low temperature while citrate inhibition was greatest at high temperatures. Glycerol and sorbitol, which accumulate as cryoprotectants in E. solidaginis, had no significant effects on kinetic constants at any temperature but decreased the V_max activity of the enzyme. Thermal inactivation studies showed an increased thermal stability of the larval enzyme compared to the homologous enzyme from rabbit muscle while added polyols stabilized enzyme activity, decreasing the rate of enzyme inactivation at 50 °C.     

Molecular cloning and expression of a novel laccase showing thermo- and acid-stability from the endophytic fungus Phomopsis liquidambari and its potential for growth promotion of plants

A novel laccase (LACB3) from the endophytic fungus, Phomopsis liquidambari, was cloned and its potential to promote peanut growth was evaluated. The full-length cDNA is 1,731 bp, encoding a mature protein of 556 amino acids with a molecular mass of 60.1 kDa. Using 2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonate), LACB3 exhibited a K _m and k _cat of 85 μM and 92.7 s^?1, respectively. The enzyme had maximal activity at pH 2.5 and 50 °C and retained 50 % of its activity after 20 h at 50 °C. When LACB3 was applied to soil, the peanut biomass was increased by 12 %, and the content of vanillic acid, coumaric acid and 4-hydroxybenzoic acid in soil were decreased by 21, 27 and 40 %, respectively. These results suggest substantial potential for the use of P. liquidambari or its laccase in agriculture.     

Metabolism of cytokinin: Phosphoribosylation of cytokinin bases by adenine phosphoribosyltransferase from wheat germ

Adenine phosphoribosyltransferase (AMP:pyrophosphate phosphoribosyltransferase EC 2.4.2.8) which catalyzes the phosphoribosylation of cytokinin bases and adenine to form the corresponding nucleotides were partially purified from the cytosol of wheat (Triticum aestivum) germ. This enzyme (molecular weight, 23,000 ± 500) phosphoribosylates the bases at an optimum Mg²⁺ concentration of 5 mm and optimum pH of 7.5 (50 mm Tris-HCl buffer). K_m values for N⁶-(Δ²-isopentenyl)adenine, N⁶-furfuryladenine, N⁶-benzyladenine, and adenine are 130, 110, 154, and 74 μm, respectively, in 50 mm Tris-HCl buffer (pH 7.5) at 37 °C. Hypoxanthine and guanine are not substrates for the enzyme. In concerting with other cytokinin metabolic enzymes, this enzyme may play a significant role in maintaining the supply of adequate levels of “active cytokinin.”     

An alkali tolerant α-l-rhamnosidase from Fusarium moniliforme MTCC-2088 used in de-rhamnosylation of natural glycosides

Analkali tolerant α-l-rhamnosidase has been purified to homogeneity from the culture filtrate of a new fungal strain, Fusarium moniliforme MTCC-2088, using concentration by ultrafiltration and cation exchange chromatography on CM cellulose column. The molecular mass of the purified enzyme has been found to be 36.0 kDa using SDS-PAGE analysis. The K_m value using p-nitrophenyl-α-l-rhamnopyranoside as the variable substrate in 0.2 M sodium phosphate buffer pH10.5 at50 °C was 0.50 mM. The catalytic rate constant was15.6 s⁻¹giving the values of k_cat/K_m is 3.12 × 10⁴M⁻¹ s⁻¹. The pH and temperature optima of the enzyme were 10.5 and 50 °C, respectively. The purified enzyme had better stability at 10 °C in basic pH medium. The enzyme derhamnosylated natural glycosides like naringin to prunin, rutin to isoquercitrin and hesperidin to hesperetin glucoside. The purified α-l-rhamnosidase has potential for enhancement of wine aroma.     

Fractionation of Chromatin by thermal precipitation in phosphate buffer.

The stability of sonicated rat liver chromatin in sodium phosphate buffer, pH 6.8 was studied as a function of buffer concentration (0.012 to 0.16 m) and temperature (20 to 98 °C). It was found that as the temperature was increased a stepwise precipitation of chromatin took place which was revealed by the presence of three plateaux (20 to 50 °C, 70 to 75 °C and above 90 °C) and two transitional zones (55 to 70 °C and 75 to 90 °C) on the A₃₂₀ curves and on the percentage precipitated nucleoprotein versus temperature curves as well.This permitted the fractionation of chromatin in 0.08 m-phosphate buffer into three fractions by a stepwise heating at 50 °C (50 °C-pellet) and 98 °C (50–98 °C-pellet and post 98 °C-supernatant). DNA isolated from these fractions was characterized in respect to sedimentation velocity and hybridization with heterogeneous nuclear RNA. The hybridization studies showed a different ability of these three DNA preparations in binding nuclear heterogeneous RNA: 16%, 8% and 30% for DNA isolated from 50 °C-pellet, 50–98 °C-pellet and post 98 °C-supernatant, respectively. The results are discussed in terms of chromatin structure and function.     

Three-dimensional structure of an alkaline xylanase Xyn11A-LC from alkalophilic Bacillus sp. SN5 and improvement of its thermal performance by introducing arginines substitutions

The alkaline xylanase Xyn11A-LC from the alkalophilic Bacillus sp. SN5 was expressed in E. coli, purified and crystallized. The crystal structure was determined at a resolution of 1.49 Å. Xyn11A-LC has the β-jelly roll structure typical of family 11 xylanases. To improve its thermostability and thermophilicity, a mutant SB3 was constructed by introducing three arginines on the different sides of the protein surface. SB3 increased the optimum temperature by 5 °C. The wild type and SB3 had the half-lives of 22 and 68 min at 65 °C at pH 8.0 (Tris/HCl buffer), respectively. CD spectroscopy revealed that the melting temperature (T _m) of the wild type and SB3 were 55.3 and 66.9 °C, respectively. These results showed that the introduction of arginines enhance the thermophilicity and thermostability of Xyn11A-LC.     

Effects of temperature and irradiance on quantum yield of PSII photochemistry and xanthophyll cycle in a tropical and a temperate species

The effect of a wide range of temperatures (?15 and 60°C) in darkness or under strong irradiation [1,600 μmol(photon) m^?2 s^?1] on quantum yield of photosystem II photochemistry and xanthophyll cycle pigments was investigated in a tropical fruit crop (Musa sp.) and a temperate spring flowering plant (Allium ursinum L.). In darkness within the nonlethal thermal window of A. ursinum (from ?6.7 to 47.7°C; 54.5 K) and of Musa sp. (from ?2.2°C to 49.5°C; 51.7 K) maximal quantum yield of PSII photochemistry (F_v/F_m) was fairly unaffected by temperature over more than 40 K. At low temperature F_v/F_m started to drop with ice nucleation but significantly only with initial frost injuries (temperature at 10% frost damage; LT₁₀). The critical high temperature threshold for PSII (T_c) was 43.8°C in A. ursinum and 44.7°C in Musa sp. Under strong irradiation, exposure to temperatures exceeding the growth ones but being still nonlethal caused photoinhibition in both species. Severity of photoinhibition increased with increasing distance to the growth temperature range. ΔF/F_m′ revealed distinctly different optimum temperature ranges: 27–36°C for Musa sp. and 18–27°C for A. ursinum exceeding maximum growth temperature by 2–7 K. In both species only at temperatures > 30°C zeaxanthin increased and violaxanthin decreased significantly. At nonlethal low temperature relative amounts of xanthophylls remained unchanged. At temperatures > 40°C β-carotene increased significantly in both species. In Musa sp. lutein and neoxanthin were significantly increased at 45°C, in A. ursinum lutein remained unchanged, neoxanthin levels decreased in the supraoptimal temperature range. In darkness, F_v/F_m was highly temperature-insensitive in both species. Under strong irradiation, whenever growth temperature was exceeded, photoinhibition occurred with xanthophylls being changed only under supraoptimal temperature conditions as an antiradical defence mechanism.     

Isolation and general characterization of a heat-stable proteinase from Pseudomonas fluorescens AFT 36

An extracellular proteinase from Pseudomonas fluorescens, strain AFT 36, was isolated to homogeneity by chromatography on DEAE-cellulose and Sephadex G-150; a 230-fold increase in specific activity with a recovery of 53% was obtained. The enzyme was optimally active at pH 6.5 and 45°C; activity declined rapidly at higher temperatures but significant activity persisted down to 4°C. Activity was strongly inhibited by 10^?3 M EDTA and was partially restored by addition of Zn²⁺, Ca²⁺ or Co²⁺. The K_m values on methylated casein and sodium caseinate were 18.2 and 7.1 mg/ml, respectively. The enzyme was very labile in phosphate buffer and in a milk salts buffer at 55°C but was very stable in the latter at more than 80°C.     

Purification and characterization of human lung calmodulin-independent cyclic AMP phosphodiesterase

A high-affinity calmodulin-independent cyclic AMP phosphodiesterase was purified to homogeneity from human lung tissue. This enzyme has a molecular weight of 60,000, a sedimentation coefficient of 3.2–3.4 S, and an isoelectric pH of 4.6–4.8. Neither Ca²⁺ nor calmodulin (in the presence or absence of added Ca²⁺) stimulates the enzymatic activity. This enzyme appears to be very similar to that described previously from dog kidney (W. J. Thompson, P. M. Epstein, and S. J. Strada, (1979) Biochemistry18, 5228–5237). Hydrolysis of cyclic AMP is greatly enhanced by Mg²⁺ (25–30× at 10 mm Mg²⁺) and Mn²⁺ (20× at 10 mm Mn²⁺). Zn²⁺, Cu²⁺, and Co²⁺ are ineffective at these concentrations. Cyclic AMP is the exclusive substrate with a K_m of 0.7–0.8 μm. The I₅₀ of cyclic GMP is 1 mm using 1 μm cyclic AMP as substrate. In contrast, aminophylline, MIX, and SQ 20009 have I₅₀s of 0.28, 0.021, and 0.001 mm, respectively). The purified enzyme is susceptible to temperature inactivation and protease degradation. Significant (10%) inhibition is seen at 37 °C for 20 min. Trypsin, at 0.1 μg/ml, destroys 50% of the activity in 30 min at 25 °C. Our observations concerning its lability to temperature and proteases coupled with its lack of response to calmodulin suggest this enzyme is a basic catalytic subunit of other cyclic AMP phosphodiesterases present within human lung tissue.     

Preparation and properties of proteases immobilized on anion exchange resin with glutaraldehyde

High activity alkaline protease was obtained when the enzyme was immobilized on Dowex MWA-1 (mesh 20–50) with 10% glutaraldehyde in chilled phosphate buffer (M/15, pH 6.5). Activity yields of the protease and rennet were 27 and 29, respectively. The highest activities appeared at 60°C, pH 10 for alkaline protease and 50°C, pH 4.0 for rennet. The properties of both proteases were not essentially changed by the immobilization except that the K_m values of both enzymes were increased about tenfold as a result of immobilization. Both proteases in the immobilized state were more stable than those in the free state at 60°C. Other peptide hydrolases, β-galactosidase, invertase, and glucoamylase, were successfully immobilized with high activities, but lipase, hexokinase, glucose-6-phosphate dehydrogenase, and xanthine oxidase became inactive.     

Enhancement of the thermostability of a recombinant β-agarase, AgaB, from Zobellia galactanivorans by random mutagenesis

Random mutagenesis was performed on β-agarase, AgaB, from Zobellia galactanivorans to improve its catalytic activity and thermostability. The activities of three mutants E99K, T307I and E99K–T307I were approx. 140, 190 and 200%, respectively, of wild type β-agarase (661 U/mg) at 40°C. All three mutant enzymes were stable up to 50°C and E99K–T307I had the highest thermostability. The melting temperature (T _m) of E99K–T307I, determined by CD spectra, was increased by 5.2°C over that of the wild-type enzyme (54.6°C). Activities of both the wild-type and E99K–T307I enzymes, as well as their overall thermostabilities, increased in 1 mM CaCl₂. The E99K–T307I enzyme was stable at 55°C with 1 mM CaCl₂, reaching 260% of the activity the wild-type enzyme held at 40°C without CaCl₂.     

In vivo temperature limitations of photosynthesis in Phaseolus vulgaris L

The purpose of this study was to evaluate the temperature response of photosynthesis in two common bean genotypes differing in crop yield when grown under warm conditions. The cultivar Nobre is sensitive to high temperatures, whereas Diplomata shows better crop yield under high temperatures. Plants were grown in a greenhouse prior to transferring to a controlled environment cabinet for the temperature treatments. In a first experiment, 30 days-old plants were subjected to a short exposure (1 day) at temperatures that varied from 9 °C to 39 °C. Diplomata had lower net CO₂ assimilation rate (A) at 15 °C and 21 °C, but higher from 27 °C to 39 °C. Photosynthetic parameters calculated from modeling the response of A to the intercellular CO₂ concentration suggested that the different temperature responses of the two genotypes are caused by different rates of diffusion of CO₂ to the assimilation site, not by differences in biochemical limitations of photosynthesis. While stomatal conductance (g_s) did not differ between the genotypes, mesophyll conductance (g_m) was slightly greater for Nobre at 15 °C, but much higher in Diplomata from 21 °C to 39 °C. In a second experiment, no difference was observed in biomass accumulation between the two genotypes after growth for 24 days under a 35/20 °C (day/night) regime. Hence, the differences in photosynthesis did not cause variation in plant growth at the vegetative stage. The differential genotypic response of g_m to temperature suggests that g_m might be an important limitation to photosynthesis in Nobre, the common bean genotype sensitive to elevated temperature. However, more studies are needed employing other methods for g_m evaluation to validate these results.     

Thermal stability and chain conformational studies of xanthan at different ionic strengths

The aim of the present study was to determine the influence of the ionic strength on the thermal stability of xanthan, i.e. xanthan resistance to chain breaking at high temperatures. Xanthan solutions of various ionic strengths were kept at 80, 90 and 95°C for periods up to 95 h. The thermal stability was determined by measuring the intrinsic viscosity after the heating periods. The experiments showed a critical ionic strength for the thermal stability of xanthan between 10 and 100 mm NaCl or KCl in this temperature range. Below the critical ionic strength the intrinsic viscosity was rapidly reduced, whereas above the critical ionic strength the intrinsic viscosity was virtually unaffected by heating.We then looked for a possible correlation between thermal stability and secondary structure of xanthan. The transition ionic strength (I_m) of xanthan solutions, i.e. where xanthan is midway between an ordered and a disordered structure, was determined by NMR at constant temperatures. I_m was found to be in the range of 24 mm at 80°C to 60 mm NaCl at 95°C, thus lying in the range of the critical ionic strength of the thermal stability. This suggests a close relationship between thermal stability and secondary structure of xanthan, indicated by the enhanced thermal stability in the ordered state. We believe this enhanced thermostability arises from a double-stranded conformation in the ordered state, as in DNA. The presence of double-stranded xanthan is also indicated by electron micrographs taken at both high and low ionic strengths.The transition temperature (T_m) of xanthan was determined by NMR and optical rotation measurements. At the ionic strength of 7·5 mm the two methods resulted in T_m values of 67 and 52°C respectively. This difference in T_m can possibly be due to the fact that the observed NMR and optical rotation (OR) effects are caused by different molecular phenomena.     

Agro-climatic zoning of Jatropha curcas as a subside for crop planning and implementation in Brazil

As jatropha (Jatropha curcas L.) is a recent crop in Brazil, the studies for defining its suitability for different regions are not yet available, even considering the promises about this plant as of high potential for marginal zones where poor soils and dry climate occur. Based on that, the present study had as objective to characterize the climatic conditions of jatropha’s center of origin in Central America for establishing its climatic requirements and to develop the agro-climatic zoning for this crop for some Brazilian regions where, according to the literature, it would be suitable. For classifying the climatic conditions of the jatropha’s center of origin, climate data from 123 weather stations located in Mexico (93) and in Guatemala (30) were used. These data were input for Thornthwaite and Mather’s climatological water balance for determining the annual water deficiency (WD) and water surplus (WS) of each location, considering a soil water-holding capacity (SWHC) of 100 mm. Mean annual temperature (T _m), WD, and WS data were organized in histograms for defining the limits of suitability for jatropha cultivation. The results showed that the suitable range of T _m for jatropha cultivation is between 23 and 27 °C. T _m between 15 and 22.9 °C and between 27.1 and 28 °C were classified as marginal by thermal deficiency and excess, respectively. T _m below 15 °C and above 28 °C were considered as unsuitable for jatropha cultivation, respectively, by risk of frosts and physiological disturbs. For WD, suitability for rain-fed jatropha cultivation was considered when its value is below 360 mm, while between 361 and 720 mm is considered as marginal and over 720 mm unsuitable. The same order of suitability was also defined for WS, with the following limits: suitable for WS up to 1,200 mm; marginal for WS between 1,201 and 2,400 mm, and unsuitable for WS above 2,400 mm. For the crop zoning, the criteria previously defined were applied to 1,814 climate stations in the following Brazilian regions: Northeast (NE) region and the states of Goiás (GO), Tocantins (TO), and Minas Gerais (MG). The suitability maps were generated by crossing the crop climate requirements with the interpolated climate conditions of the selected regions. The maps showed that only 22.65 % of the areas in the NE region are suitable for jatropha as a rain-fed crop. The other areas of the region are classified as marginal (62.61 %) and unsuitable (14.74 %). In the states of GO and TO, the majority of the areas (47.78 %) is classified as suitable, and in the state of MG, 33.92 % of the territory has suitability for the crop. These results prove that jatropha cannot be cultivated everywhere and will require, as any other crop, minimum climatic conditions to have sustainable performance and high yields.     

Dicarboxylate transport in maize mesophyll chloroplasts

Evidence is presented for high rates of carrier-mediated dicarboxylate anion transport in maize mesophyll chloroplasts. Radioactively labeled malate is transported across the chloroplast envelope leading to accumulation in the stroma. Malate in the stroma will exchange for external malate, oxaloacetate, glutamate, aspartate, and oxoglutarate. At 4 °C the V of malate uptake is 50 μmol·h^?1·mg Chl^?1 and the K_m for malate is 0.5 mm. Oxaloacetate competitively inhibits malate uptake with a K_i estimated to be 0.3 mm. The temperature dependence of malate uptake indicates an activation energy of 12 kcal/mol, and extrapolation using this value gives a rate of transport at 30 °C of approximately 300 μmol·h^?1·mg Chl^?1. This rate approximates the rates of photosynthetic malate production by these chloroplasts.     

GH52 xylosidase from Geobacillus stearothermophilus: characterization and introduction of xylanase activity by site-directed mutagenesis of Tyr509

A xylosidase gene, gsxyn, was cloned from the deep-sea thermophilic Geobacillus stearothermophilus, which consisted of 2,118 bp and encoded a protein of 705 amino acids with a calculated molecular mass of 79.8 kDa. The GSxyn of glycoside hydrolase family 52 (GH52) displayed its maximum activity at 70 °C and pH 5.5. The K _m and k _cat values of GSxyn for ρNPX were 0.48 mM and 36.64 s^?1, respectively. Interestingly, a new exo-xylanase activity was introduced into GSxyn by mutating the tyrosine509 into glutamic acid, whereas the resultant enzyme variant, Y509E, retained the xylosidase activity. The optimum xylanase activity of theY509E mutant displayed at pH 6.5 and 50 °C, and retained approximately 45 % of its maximal activity at 55 °C, pH 6.5 for 60 min. The K _m and k _cat values of the xylanase activity of Y509E mutant for beechwood xylan were 5.10 mg/ml and 22.53 s^?1, respectively. The optimum xylosidase activity of theY509E mutant displayed at pH 5.5 and 60 °C. The K _m and k _cat values of the xylosidase activity of Y509E mutant for ρNPX were 0.51 mM and 22.53 s^?1, respectively. This report demonstrated that GH52 xylosidase has provided a platform for generating bifunctional enzymes for industrially significant and complex substrates, such as plant cell wall.     

The effect of temperature on the activity of acetylcholinesterase preparations from rat brain

Homogenization of rat brain with dilute buffer shows that about 15% of the acetylcholinesterase is soluble while the remaining 85% is present in a membrane-bound form which can be brought into solution by extraction with Triton X-100. The effect of temperature on the values of V_max and K_m of the buffer-soluble, the membrane-bound and the Triton-soluble forms of acetylcholinesterase have been compared and the results discussed in terms of possible changes in the conformation, dissociation or aggregation of the enzyme molecule.Gradient-gel electrophoresis of the soluble preparations carried out at 4°C or 37°C suggest that the normal tetrameric structure present at 4°C dissociates into monomers and forms some higher molecular weight species at 37°C.The effect of prior storage of the brains in toluene on these properties is also considered.     

Effect of temperature on germination,growth, and infectivity of the mosquito pathogen Tolypocladium cylindrosporum (deuteromycotina; hyphomycetes)

The mosquito pathogen Tolypocladium cylindrosporum was examined with regard to its response to temperature. Similar temperature ranges were found for growth, germination, and infectivity of blastospores and conidia. Germination occurred at 8° and 33°C but not at 6° and 35°C. Optimal germination and growth was noted between 24° and 27°C for both spore types. Infectivity of blastospores and conidia at different temperatures was examined by exposing L₂Aedes sierrensis larvae to concentrations of 5 × 10⁵ blastospores/ml or 5 × 10⁶ conidia/ml. Larvae were incubated at 12°, 15°, 25°, and 30°C. Infection occurred at all temperatures tested with LT₅₀ values ranging from 22.7 days (12°C) to 5.6 (25°C) days for conidia and 4.7 days (12°C) to 0.6 day (25°C) for blastospores. These results confirmed earlier findings that blastospores infected and killed host larvae more rapidly than conidia and suggested that this difference is largely due to the more rapid germination rate of blastospores. These experiments demonstrated that T. cylindrosporum can be active against mosquito larvae over a broad range of temperatures encompassing both the cold-water habitat of certain temperate mosquito species as well as the habitat of tropical vector species.     

Prostaglandin E2-9-ketoreductase of rat testis.

A divalent metal dependent gluconolactonase has been isolated from porcine liver and purified to apparent homogeneity. Its molecular weight is estimated at 223,000 and that of the subunits is 37,200 as determined by gel electrophoresis. A K_m value of 6.2 mM was obtained at 27° in 50 mM tris HCl buffer. Gluconolactonase is specific for gluconolactone, and manganese is preferred over magnesium for maximum activity. The hepatic concentration of gluconolactonase is estimated to be 7.2 μmol of enzyme per kg of porcine liver, and a subcellular fractionation study indicates that this enzyme is located primarily within the cytosol.