Enzymic mechanism of starch stynthesis in ripening rice grains. VII. Purification and enzymic properties of sucrose synthetase

Sucrose synthetase (UDP-glucose:d-fructose-2-glucosyltransferase, EC 2.4.1.13) from ripening rice seeds was purified by ammonium sulfate fractionation and column chromatography of microgranular DEAE-cellulose (DE-32) and Neusilin (MgO· Al₂O₃·2SiO₂). An enzyme preparation obtained was homogeneous as examined by polyacrylamide gel electrophoresis. The enzyme, having a molecular weight, 4.0 × 10⁵, consists of 4 identical subunits, each having a molecular weight, 1.0 × 10⁵.Examination of reaction kinetics of both sucrose synthesis and cleavage catalyzed by sucrose synthetase revealed that the rate of synthesis follows a Michaelis-Menten equation having the following parameters: K_m(fructose)_UDP-glucose, 6.9 mm; K_m(fructose)_ADP-glucose, 40 mm; K_m(UDP-glucose), 5.3 mm; and K_m(ADP-glucose), 3.8 mm. The cleavage reaction yielded the following values: K_m(UDP), 0.8 mm; K_m(ADP), 3.3 mm; and K_m(sucrose)_UDP, 290 mm. In the latter reaction the rate deviated from the Michaelis equation when ADP was used as the glucose acceptor, the n value being 1.6 by the Hill plot analysis and S_0.5(sucrose)_ADP, 400 mm. At high concentration of ADP the cleavage reaction was inhibited, while the synthesis reaction was inhibited with high concentrations of fructose.     

Isolation and characterization of an acyl-coenzyme A carboxylase from an erythromycin-producing Streptomyces erythreus

Streptomyces erythreus produces erythromycin presumably from methylmalonyl-coenzyme A, (CoA) which might be generated by carboxylation of propionyl-CoA. A biotin-containing enzyme which carboxylates acetyl-CoA, propionyl-CoA, and butyryl-CoA was purified to near homogeneity from S. erythreus using DEAE-cellulose, affinity chromatography on monomeric avidin-Sepharose, and blue Sepharose. The enzyme carboxylates propionyl-CoA (100%) with a K_m of 0.09 mm and V of 0.86μmol/mg/min, acetyl-CoA (16%) with a K_m of 0.17 mm and V of 0.08μmol/mg/min, and butyryl-CoA (7.7%) with a K_m of 0.67 mm and V of 0.044 μmol/mg/min. The native enzyme has a molecular weight of 537,000 and consists of two types of subunits with molecular weights of 67,000 and 61,000, respectively, indicating an octameric α₄β₄ type of structure. Biotin is associated with the large subunit (α). The enzyme has a pH optimum between 7.5 and 7.8. It is stimulated (three- to fourfold) by K⁺, Rb⁺ and Cs⁺ but not by Na⁺ or Li⁺ and is inhibited by high concentrations of NH₄⁺ and C1^?. Neither citrate nor free CoA stimulated the enzyme. The enzyme was shown to be stereospecific and generated onlyS-methylmalonyl-CoA from the carboxylation of propionyl-CoA. The present case appears to be the first enzyme possibly involved in erythromycin production to be isolated in homogeneous form.     

Purification and properties of two forms of hepatic phenylalanine:pyruvate transaminase from glucagon treated rats.

Two forms of phenylalanine:pyruvate transaminase (EC 2.6.1. aminotransferases, the exact EC number has not been assigned) termed A and B were obtained from the liver supernatant fraction of glucagon-treated rats by DEAE-Sephadex A-50 column chromatography. Each of the two forms was further purified by hydroxylapatite, Sephadex G-100 chromatography, and preparative gel electrophoresis. Both the A and B forms have been purified to homogeneity as judged by analytical and sodium dodecyl sulfate polyacrylamide gel electrophoresis. Moreover, histidine was found to be a competitive inhibitor of phenylalanine with both purified proteins. These findings conclusively support the view that phenylalanine:pyruvate transaminase and histidine:pyruvate transaminase reactions are catalyzed by the same protein. The overall purification was 710-fold for the A form and 1200-fold for the B form. The apparent molecular weight for both A and B are 74,000 ±6000 as determined by gel filtration. Sodium dodecyl sulfate gel electrophoresis revealed that the A form has two identical subunits of molecular weight 42,000, whereas the B form has two nonidentical subunits of molecular weight 42,000 and 44,000. The amino acid composition for the A and B forms of the enzyme are different. The major differences are in glycine, alanine and leucine. The isoelectric point for A was 7.8 and for B was 7.3. However, the A and B forms of the enzyme are of immunological identity. The substrate specificity determined for both the A and B form was phenylalanine >asparagine >alanine >leucine >histidine. The K_m for phenylalanine was 7.70 mm for the A form, 6.00 mm for the B form. For histidine, the K_m was 13.70 mm for the A form, 12.50 mm for the B form.     

Isolation and characterization of acetylornithine delta-transaminase of wild-type Escherichia coli W. Comparison with arginine-inducible acetylornithine delta-transaminase.

The enzyme that catalyzes the reversible conversion of N-acetylglutamic γ-semialdehyde and l-glutamate to α-N-acetyl-l-ornithine and α-ketoglutarate, acetylornithine δ-transaminase, has been isolated in homogeneous form and crystallized from both the wild-type and the arginine-inducible strains of Escherichia coli W. The molecular weight of the wild-type transaminase is 119,000 while the molecular weight of the arginine-inducible enzyme is 61,000. However, the arginine-inducible acetylornithine δ-transaminase is not a breakdown product of the wild-type, arginine-repressible transaminase. Analysis of crude extracts of the wild-type and arginine-inducible strains by varying the acrylamide concentration in polyacrylamide disc gel electrophoresis showed that arginine-inducible and wild-type transaminases differed in ionic charge. Immunochemical analysis of the two transaminases showed that neither enzyme would cross-react with antibodies prepared against its counterpart. Treatment of the two enzymes with sodium dodecyl sulfate, followed by disc gel electrophoresis revealed that both transaminases were composed of 31,000-dalton subunits. Tryptic digestion of the two transaminases showed that nearly identical peptides were present. The overall data suggest that the wild-type and inducible transaminases were products of two different structural genes. The two transaminases have different molecular weights, ionic charges, and antigenic determinants, but both are composed of similar molecular weight subunits and show a high degree of similarity in amino acid content and peptide composition.     

Isolation of 3-methylcrotonyl-coenzyme A carboxylase from bovine kidney

3-Methylcrotonyl-CoA carboxylase (MCase), an enzyme of the leucine oxidation pathway, was highly purified from bovine kidney. The native enzyme has an approximate molecular weight of 835,000 as measured from exclusion limits by polyacrylamide gel electrophoresis at pH 7.3. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate demonstrated two subunits, identified as a biotin-free subunit (A subunit; M_r = 61,000) and a biotin-containing subunit (B subunit; M_r = 73,500). The biotin content of the enzyme was 1 mol/ 157,000 g protein, consistent with an AB protomeric structure for the enzyme. The isoelectric point of the enzyme was found to be 5.4. Maximal MCase activity was found at pH 8 and 38 °C in the presence of Mg²⁺ and an activating monovalent cation such as K⁺. Kinetic constants (K_m values) for the enzyme substrates were: 3-methylcrotonyl-CoA, 75 μm; ATP, 82 μm; HCO₃^?, 1.8 mm. Certain acyl-CoA derivatives, including crotonyl-CoA, (2Z)-3-ethylcrotonyl-CoA, and acetoacetyl-CoA, were also substrates for the enzyme. Some data on inhibition of the enzyme by acyl-CoA derivatives, and sulfhydryl- and arginyl-reagents, are presented.     

NADP-malic enzyme from maize leaf: purification and properties.

NADP-malic enzyme (EC 1.1.1.40), which is involved in the photosynthetic C⁴ pathway, was isolated from maize leaf and purified to apparent homogeneity as judged by polyacrylamide gel electrophoresis. At the final step, chromatography on Blue-Sepharose, the enzyme had been purified approximately 80-fold from the initial crude extract and its specific activity was 101 μmol malate decarboxylated/mg protein/min at pH 8.4. The enzyme protein had a sedimentation coefficient (s_20,w) of 9.7 and molecular weight of 2.27 × 10⁵ in sucrose density gradient centrifugation, and molecular weight of 2.26 × 10⁵ calculated from sedimentation equilibrium analysis. The molecular weight of the monomeric form was determined to be 6.3 × 10⁴ by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In the pyruvate carboxylation reaction, HCO₃^? proved to be the active molecular species involved. With all other substrates at saturating concentration, the following kinetic constants were obtained: K_m (malate), 0.4 mm; K_m (NADP), 17.6 μm; K_m (Mg²⁺), 0.11 mm. The maize leaf malic enzyme was absolutely specific for NADP. The Arrhenius plot obtained from enzyme activity measurements was linear in a temperature range of 13 to 48 °C, and the activation energy was calculated to be 9500 cal/mol.     

Purification of NAD malic enzyme from potato and investigation of some physical and kinetic properties

A procedure is described for purification of NAD malic enzyme (EC 1.1.1.39) to near homogeneity from potato tuber mitochondria. The purified enzyme is active with either NAD or NADP, and functions with either Mg²⁺ or Mn²⁺. V_app is greatest when the enzyme is assayed with Mg²⁺ and NAD. When Mn²⁺ replaces Mg²⁺ the V_app of the NAD-linked reaction decreases but the K_m values for all substrates drop substantially. When NADP is used in place of NAD, the V_app of the Mg²⁺-linked reaction decreases and the K_m values for most substrates increase. The pH optimum of the enzyme depends on the metal ion and cofactor used and varies between 6.4 and 6.8. At pH 6.8, with saturating levels of Mg²⁺ and NAD, the turnover number of the enzyme is 37,000 min^?1. The shape of the pH profile indicates the involvement of two to three protons in the activation of the enzyme, whereas only one proton is involved in the inactivation process. The molecular weight of the enzyme in the presence of 5 mm dithiothreitol and 2 mm MgCl₂ is 490,000 as determined by gel filtration. A lower molecular weight form of the enzyme predominates in gel filtration at lower levels of dithiothreitol and in native gel electrophoresis. Sodium dodecyl sulfate gel electrophoresis of the enzyme reveals two main bands with molecular weights of 61,000 and 58,000, suggesting that the subunit stoichiometry of the high-molecular-weight form may be α₄β₄. However, given the possibility that the smaller subunit may be a proteolytic artifact, the enzyme may prove to be an octamer of identical subunits.     

Isolation and properties of a Mn2+-activated phosphohistone phosphatase from canine heart.

A Mn²⁺-activated phosphohistone phosphatase has been isolated from canine heart. The s_{20, w} for the enzyme is 3.8. Using this value and the value for Stokes radius (39 Å), the molecular weight for the enzyme was calculated to be 61,000. The enzyme is inactive in the absence of divalent cations, among which Mn²⁺ is the most effective activator. Co²⁺ and Mg²⁺ are less effective than is Mn²⁺. Zn²⁺, Fe²⁺, and Cu²⁺ are inhibitory. The enzyme has a pH optimum between 7 and 7.5 and has an apparent K_m for phosphohistone and Mn²⁺ of about 17 μm and 0.5 mm, respectively. The enzyme is inhibited by nucleoside triphosphate, ADP, AMP, phosphate, and pyrophosphate, but is not affected by cyclic AMP or cyclic GMP. The dephosphorylation of phosphohistone is stimulated by salts. Kinetic studies reveal that KCl and other salts greatly affect both the rate of hydrolysis and the K_m for either Mn²⁺ or phosphohistone by interacting with the substrate. The data suggest that modification at substrate level is an important regulatory mechanism for the enzyme. The enzyme preparation also dephosphorylates phosphorylase a and phosphocasein. Evidence suggests that one enzyme possesses both phosphohistone and phosphorylase phosphatase activities and that a different enzyme catalyzes the Mg²⁺- and Mn²⁺-activated dephosphorylation of phosphocasein.     

Glucose 6-phosphate dehydrogenase isozymes of maize leaves : some comparative properties

Two different forms of glucose 6-phosphate dehydrogenase (EC 1.1.1.49) have been purified from etiolated and green leaves, respectively, of 6-day maize (Zea mays L. cv Fronica) seedlings. The procedure includes an ammonium sulfate step, an ion exchange chromatography, and a second gel filtration in Sephadex G-200 in the presence of NADP⁺ to take advantage of the corresponding molecular weight increase of the enzyme. The isozyme from etiolated leaves is more stable and has been purified up to 200-fold. Subunit molecular weight, measured by sodium dodecyl sulfate-gel electrophoresis, is 54,000. The active protein, under most conditions, has a molecular weight 114,000, which doubles to molecular weight 209,000 in the presence of NADP⁺. The association behavior of enzyme from green leaves is similar, and the molecular weight of the catalytically active protein is also similar to the form of etiolated leaves.

Glucose 6-phosphate dehydrogenase of dark-grown maize leaves isoelectric point (pI) 4.3 is replaced by a form with pI 4.9 during greening. The isozymes show some differences in their kinetic properties, K_m of NADP⁺ being 2.5-fold higher for pI 4.3 form. Free ATP (K_m = 0.64 millimolar) and ADP (K_m = 1.13 millimolar) act as competitive inhibitors with respect to NADP⁺ in pI 4.3 isozyme, and both behave as less effective inhibitors with pI 4.9 isozyme. Magnesium ions abolish the inhibition.

     

Microbial oxidation of methanol: Purification and properties of formaldehyde dehydrogenase from a Pichia sp. NRRL-Y-11328

An NAD⁺-linked, reduced glutathione-dependent formaldehyde dehydrogenase was purified to homogeneity from soluble extracts of methanol-grown yeast, Pichia sp. Formaldehyde and methylglyoxal are oxidized in the presence of NAD⁺ as an electron acceptor. NADP⁺ could not replace NAD⁺. Other straight chain aldehydes (C₂–C₆ tested), branched-chain aldehydes (e.g., isobutyaldehyde), aromatic aldehydes (e.g., salicylal-dehyde, benzaldehyde), glutyraldehyde, glyceraldehyde, glycoaldehyde, and glyoxal-dehyde tested were not oxidized by the purified formaldehyde dehydrogenase. The product of formaldehyde oxidation by purified enzyme was demonstrated to be S-for-mylglutathione by measuring the absorption at 240 nm due to the formation of thioester of formaldehyde and reduced glutathione. The K_m values for NAD⁺, formaldehyde, and reduced glutathione were 0.12, 0.31, and 0.16 mm, respectively, for the forward reaction at pH 8.0. The purified formaldehyde dehydrogenase also catalyzed the reduction of S-formylglutathione in the presence of NADH. Formate was not reduced by the purified enzyme. The K_m values for S-formylglutathione and NADH were 0.60 and 0.25 mm, respectively, for the reverse reaction at pH 6.0. Formaldehyde dehydrogenase has a molecular weight of 84,000 as determined by gel filtration and subunit molecular weight of 41,000 as determined by sodium dodecyl sulfate-gel electrophoresis. S-Formylglutathione, a product of formaldehyde oxidation, was oxidized by the partially purified formate dehydrogenase from Pichia sp. Formate dehydrogenase has a higher affinity toward S-formylglutathione (K_m value 1.8 mm) than toward formate (K_m value 25 mm). Antiserum prepared against the purified formaldehyde dehydrogenase from Pichia sp. NRRL-Y-11328 forms strong precipitin bands with isofunctional enzymes from methanol-grown Pichia pastoris NRRL-Y-7556 and Torulopsis candida Y-11419 and weak precipitin bands with Hansenula polymorpha NRRL-Y-2214. No cross-reaction was observed with isofunctional enzyme derived from methanol-grown Kloeckera sp.     

Sucrose synthase of soybean nodules

Sucrose synthase (UDPglucose: d-fructose 2-α-d-glucosyl transferase, EC 2.4.1.13) has been purified from the plant cytosolic fraction of soybean (Glycine max L. Merr cv Williams) nodules. The native enzyme had a molecular weight of 400,000. The subunit molecular weight was 90,000 and a tetrameric structure is proposed for soybean nodule sucrose synthase. Optimum activity in the sucrose cleavage and synthesis directions was at pH 6 and pH 9.5 respectively, and the enzyme displayed typical Michaelis-Menten kinetics. Soybean nodule sucrose synthase had a high affinity for UDP (K_m, 5 micromolar) and a relatively low affinity for ADP (apparent K_m, 0.13 millimolar) and CDP (apparent K_m, 1.1 millimolar). The K_m for sucrose was 31 millimolar. In the synthesis direction, UDPglucose (K_m, 0.012 millimolar) was a more effective glucosyl donor than ADPglucose (K_m, 1.6 millimolar) and the K_m for fructose was 3.7 millimolar. Divalent cations stimulated activity in both the cleavage and synthesis directions and the enzyme was very sensitive to inhibition by heavy metals.     

Products of rat liver mitochondrial protein synthesis: electrophoretic analysis of the number and size of these proteins and their solubility in chloroform: methanol

Rat liver mitochondria were incubated in vitro with radioactive leucine, and submitochondrial particles prepared by several methods. Analysis of the labeled mitochondrial membrane fractions by sodium dodecylsulfate gel electrophoresis revealed three labeled bands of molecular weights corresponding to 40,000; 27,000; and 20,000 daltons. Electrophoresis for longer times at higher concentrations of acrylamide revealed eight labeled bands, ranging in molecular weights from 48,000 to 12,000.Mitochondria were incubated for 5 min with [³H]leucine followed by a chase of unlabeled leucine. Gel electrophoresis of the membranes obtained after labeling for 5 min indicated significant synthesis of polypeptides in the 40,000 M_r, range and very little labeling of low molecular-weight polypeptides. After addition of the chase, increased synthesis of the high molecular-weight polypeptides was observed; however, no significant increase or decrease of radioactivity in the bands of low molecular-weight was observed, suggesting that rat liver mitochondria have the ability to synthesize complete proteins in the M_r 27,000–40,000 range.Approximately 16% of the total leucine incorporated into protein by isolated rat liver mitochondria in vitro could be extracted by chloroform: methanol. Gel electrophoresis of the chloroform: methanol extract revealed several bands containing radioactivity with the majority of counts in a band of 40,000 molecular weight. Gel electrophoresis of the chloroform: methanol extract of lyophilized submitochondrial particles indicated label in two broad bands in the low molecular-weight region of 14,000-10,000 with insignificant counts in the higher molecular-weight regions of the gel.Yeast cells were pulse labeled in vivo with [³H]leucine in the presence of cycloheximide and the submitochondrial particles extracted with chloroform:methanol. The extract separated after gel electrophoresis into four labeled bands ranging in molecular weight from 52,000 to 10,000. Preincubation of the yeast cells with chloramphenicol prior to the pulse labeling caused a 6-fold stimulation of labeling into the band of lowest molecular weight of the chloroform: methanol extract. These results suggest that the accumulation of mitochondrial proteins synthesized in the cytoplasm, when chloramphenicol is present in the medium, may stimulate the synthesis of certain specific mitochondrial proteins which are soluble in chloroform: methanol.     

Localization and characteristics of rat liver mitochondrial aldehyde dehydrogenases.

Two NAD-dependent aldehyde dehydrogenase enzymes from rat liver mitochondria have been partially purified and characterized. One enzyme (enzyme I) has molecular weight of 320,000 and has a broad substrate specificity which includes formaldehyde; NADP is not a cofactor for this enzyme. This enzyme has K_m values for most aldehydes in the micromolar range. The isoelectric point was found to be 6.06. A second enzyme (enzyme II) has a molecular weight of 67,000, a K_m value for most aldehydes in the millimolar range but no activity toward formaldehyde. NADP does serve as a coenzyme, however. The isoelectric point is 6.64 for this enzyme. By utilization of the different substrate properties of these two enzymes it was possible to demonstrate a time-dependent release from digitonin-treated liver mitochondria. The high K_m, low molecular weight enzyme (enzyme II) is apparently in the intermembrane space while the low K_m, high molecular weight enzyme (enzyme I) is in the mitochondrial matrix and is most likely responsible for oxidation of acetaldehyde formed from ethanol.     

Hexose kinases from Rhodotorula glutinis. Identification and properties of an hexokinase and a glucokinase.

An hexokinase (EC 2.7.1.1) and a glucokinase (EC 2.7.1.2) from the red yeast Rhodotorula glutinis are described. Both enzymes have been separated and some of their properties studied. The two enzymes share many properties, the K_mfor glucose is 0.1 mm for both enzymes and the K_m values for ATP are 0.5 mm and 0.6 mm respectively for hexokinase and glucokinase. The hexokinase shows a K_m of 2 mm for fructose and 0.1 mm for mannose; the glucokinase has a K_m for mannose of 0.2 mm. Both enzymes are constitutive, show competitive inhibition by N-acetylglucosamine and xylose, have weak affinity for glucosamine and exhibit a broad pH optimum. The molecular weights determined by gel filtration are 110,000 for glucokinase and 96,000 for hexokinase. The maximal activity of both hexose kinases nearly accounts for glucose utilization by Rh. glutinis.     

Binding of heparin to antithrombin III: The use of dansyl and rhodamine labels

Low molecular weight heparin of low-anticoagulant activity and high molecular weight heparin of correspondingly high activity were prepared by chromatography on protamine-Sepharose; preparations subjected to limited N-desulfation (5–10% free amino groups) by solvolysis were labeled with 5-dimethylaminonaphthalene-1-sulfonyl chloride (dansyl chloride) or rhodamine B isothiocyanate (RITC). The fluorescent heparins retained approximately 50% of the original anticoagulant activities. Dansyl-heparin on binding to antithrombin III (ATIII) exhibited a 2.5-fold enhancement of dansyl fluorescence intensity. This effect could be prevented by excess unlabeled heparin. A 7900 molecular weight dansyl-heparin preparation bound to ATIII with a stoichiometry of close to 2:1 and with an apparent association constant for binding (K_a) of 4.9 × 10⁵, m^?1, whereas a 21,600 molecular weight fraction bound at 0.7:1 with the protein and with an apparent K_a = 7.9 × 10⁵, m^?1. When ATIII reacted with a mixture of low molecular weight dansyl-heparin and low molecular weight RITC-heparin, there was enhancement of RITC fluorescence emission when excited at the dansyl excitation maximum; this effect was not observed when either of the labeled heparin species was prepared from high molecular weight material. The results are consistent with the proposal that a single molecule of high molecular weight, high-activity heparin occupies two sites when it binds to ATIII, whereas low molecular weight, low-activity heparin binds to the two sites separately.     

Purification and Properties of Oxalate Oxidase from NaCl Stressed Grain Sorghum Seedlings

The effect of NaCl stress on molecular and biochemical properties of oxalate oxidase (OXO) was studied in leaves of grain sorghum hybrid (var CSH-14) seedlings. There was no effect on molecular weight and number of subunits of the enzyme but it showed some important changes in its kinetic parameters such as K_m for oxalate and V_max. Optimum pH (5.8), activation energy (5.084 kcal mole^?1), time of incubation (6 min) and K_m for oxalate (1.21×10^t-4M) were increased, while V_max (0.182 mmole min^?1) decreased and no change in optimum temperature was observed. This showed that substrate affinity and maximum activity of the enzyme was adversely affected. The specific activity of oxalate oxidase was increased in seedlings grown in a NaCl containing medium compared to normal, which reveals the increased de novo synthesis of the enzyme to sustain oxalate degradation.     

Effects of lipid removal on the molecular size and kinetic properties of bovine plasma arylesterase

A purified arylesterase preparation from bovine plasma was characterized to the extent that it has a partial specific volume of 0.91ml/g and an apparent z-average molecular weight of 440000. The relatively large magnitude of the former reflects the presence of phospholipids, cholesterol, triglycerides and β-carotene, the last-named being responsible for the pronounced yellow colour of the preparation. Removal of the lipid material is accompanied by a decrease in the apparent z-average molecular weight to 120000, the size of the smallest species detected by high-speed sedimentation equilibrium being in the vicinity of 70000 daltons: denaturation of the lipid-free preparation with 6m-guanidine hydrochloride caused essentially complete breakdown into subunits of this size. In kinetic studies on the enzyme the maximal velocity for the hydrolysis of phenyl acetate was found to increase by 60% on addition of 1 mm-Ca²⁺, with the K_m showing a concomitant decrease from 6.6 to 2.1 mm. Removal of lipid had no detectable effect on V_max. or K_m in either the presence or the absence of Ca²⁺. It is concluded that the bovine plasma arylesterase preparation is either a lipoprotein or an enzyme–lipoprotein complex with properties very similar to those of the α₁-lipoprotein or high-density lipoprotein (HDL₂) fraction of serum.     

Purification and properties of a D-fructose 1,6-bisphosphatase from Saccharomyces cerevisiae.

Fructose 1,6-bisphosphatase (EC 3.1.3.11) from Saccharomyces cerevisiae has been purified to homogeneity. A molecular weight of 115,000 has been obtained by gel filtration. The enzyme appears to be a dimer with identical subunits. The apparent K_m for fructose bisphosphatase varies with the Mg²⁺ concentration of the enzyme, being 1 × 10^?6m at 10 mm Mg²⁺ and 1 × 10^?5m at 2 mm Mg²⁺. Other phosphorylated compounds are not significantly hydrolyzed by the enzyme. An optimum pH of 8.0 is exhibited by the enzyme. This optimum is not changed by addition of EDTA. AMP inhibits the enzyme with a K_i of 8.0 × 10^?5m at 25 °C. The inhibition is temperature dependent, the value of K_i increasing with raising temperature. 2-Deoxy-AMP is also inhibitory with a K_i value at 25 °C of 1.6 × 10^?4m. An ordered uni-bi mechanism has been deduced for the reaction with phosphate leaving the enzyme as the first product and the fructose 6-phosphate as the second one.     

Human kidney alpha-L-iduronidase: purification and characterization.

α-l-Iduronidase has been purified 25,000-fold from the soluble proteins of human kidney by chromatography on heparin-Sepharose, hydroxylapatite, and Bio-Gel P-100. The α-l-iduronidase activity is associated with 80% of the protein in the most purified preparation. It has a molecular weight of 60,000 ± 6500, determined by sedimentation equilibrium, and can be dissociated by reduction into subunits of molecular weight 31,000 ± 6500 determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate in the presence of dithiothreitol. It contains glucosamine and binds to concanavalin A. The pH optimum, K_m and V_max for two substrates, phenyl iduronide and [³H]anhydromannitol iduronide, were found to be 4.0, 1.05 mm, 16 μmol/mg protein/min, and 4·5, 9 mm and 270 μmol/mg protein/min, respectively. The enzyme is of the low uptake, noncorrective form with respect to fibroblasts cultured from the skin of patients with Hurler syndrome. It is inhibited by 10⁶ m p-chloromercuribenzoate and 10^?3 m Cu²⁺, but is not significantly affected by other divalent cations, EDTA, or sulfhydryl compounds. Antibodies to α-l-iduronidase have been raised in goats.     

Monomeric structure of glutamyl-tRNA synthetase in Escherichia coli.

An investigation of the subunit structure of glutamyl-tRNA synthetase (EC 6.1.1.17) from Escherichia coli indicates that this enzyme is a monomer. The enzyme purified to apparent homogeneity is a single polypeptide chain with a molecular weight of 62,000 ± 3,000 and K^Glu_m ? 50 μM in the aminoacylation reaction. Analytical gel electrophoretic procedures were used to determine the molecular weight of species exhibiting glutamyl-tRNA synthetase activity in freshly prepared extracts of several strains of E. coli, which had been grown under various nutritional conditions and harvested at different stages of growth. In all cases, glutamyl-tRNA synthetase activity was associated with a protein having about the same molecular weight and K^Glu_m as the purified enzyme. Thus, no evidence of an oligomeric form of glutamyl-tRNA synthetase with a greater affinity for l-glutamate was obtained, in contrast to a previous report of J. Lapointe and D. Söll (J. Biol. Chem.247, 4966–4974, 1972).