Synthesis of biopterin from dihydroneopterin triphosphate by rat tissues

High performance liquid chromatography procedure for the analysis of pterins of biopterin synthesis from dihydroneopterin triphosphate via sepiapterin in rat tissues has been described. Sepiapterin-synthesizing enzyme 1, which catalyzes in the presence of Mg²⁺ the conversion of dihydroneopterin triphosphate to an intermediate designated compound X was assayed by determining pterin which is formed from compound X under acidic conditions. Sepiapterin- and biopterin-synthesizing activity were also assayed by determining sepiapterin and biopterin, respectively. Analytical results revealed the presence of these activities in most rat tissues examined and high levels were found in kidney, pineal gland and liver. Activities were also detectable in peripheral erythrocytes.     

Regulation of tyrosine hydroxylase from human pheochromocytoma, bovine adrenal and rat striatum.

Soluble tyrosine hydroxylase from human pheochromocytoma, bovine adrenal medulla and rat striatum can be activated by Mg²⁺, ATP and cyclic AMP. In pheochromocytoma, this activation is due to a decreased Km for the pterin cofactor, whereas in adrenal medulla, it is a result of an increase in the Vmax. Norepinephrine increases the Km for pterin cofactor for tyrosine hydroxylase from both of these tissues. The Ki for norepinephrine is not altered by the presence of Mg²⁺, ATP and cyclic AMP with enzyme from pheochromocytoma or adrenal medulla. On the other hand, striatal tyrosine hydroxylase shows a two-fold increase in the Ki for dopamine after exposure to Mg²⁺, ATP and cyclic AMP.     

Lithosperm inhibition of anterior pituitary hormones.

The enzyme system for the synthesis of the pteridine pigment, sepiapterin, from $\text{2-amino-4-hydroxy-6-(D-}\text{erythro}\text{-1',2',3'-trihydroxyprophyl)}$ triphosphate (dihydroneopterin triphosphate) has been found in extracts of $\text{Drosophila melanogaster}$. NADP⁺ or NADPH and Mg²⁺ are required for this enzymatic transformation. No sepiapterin is produced when dihydroneopterin is supplied as substrate in place of dihydroneopterin triphosphate.     

Some Properties of GTP Cyclohydrolase from Serratia indica and the Pterin Product by Its Enzymatic Reaction

GTP cyclohydrolase which catalyzes the formation of formic acid and a pterin compound from guanosine-5′-triphosphate (GTP) has been partially purified from extracts of Serratia indica IFO 3759. ¹⁴C-Formic acid eliminated from (8-¹⁴C)GTP is oxidized with mercury acetate to ¹⁴CO₂, which is trapped by β-phenylethylamine. The molecular weight of the enzyme is approximately 170,000 and the enzyme is relatively heat-stable. The enzyme activity is strongly inhibited by GDP and ATP, but not by other nucleotides. Inhibition by GDP is competitive with GTP. Metals, such as Fe²⁺, Co²⁺, Ni²⁺, Zn²⁺, Cd²⁺, Al³⁺, Hg²⁺ and p-chloromercuribenzoate strongly inhibit the enzyme activity. The activity is also inhibited by . The pterin product has been characterized as a derivative of neopterin triphosphate by enzymatic degradations, ultraviolet spectra, fluorescence and excitation spectra, thin-layer chromatography and thin-layer electrophoresis. The product is estimated to differ from d-erythro-neopterin triphosphate prepared from the enzyme system of Escherichia coli B, since (1) only one mole of phosphate can be liberated by alkaline phosphatase and two moles of phosphates by phosphodiesterase and alkaline phosphatase from the product, and (2) the retention time of the product on high-performance liquid chromatography is different from that of d-erythro-neopterin triphosphate.     

Partial purification and properties of guanosine triphosphate cyclohydrolase from Drosophila melanogaster

The first enzyme (named GTP cyclohydrolase) in the pathway for the biosynthesis of pteridines has been partially purified from extracts of late pupae and young adults of Drosophila melanogaster. This enzyme catalyzes the hydrolytic removal from GTP of carbon 8 as formate and the synthesis of 2-amino-4-hydroxy-6-(d-erythro-1,2,3-trihydroxypropyl)-7,8-dihydropteridine triphosphate (dihydroneopterin triphosphate). Some of the properties of the enzyme are as follows: it functions optimally at pH 7.8 and at 42 C; activity is unaffected by KCl and NaCl, but divalent cations (Mg²⁺, Mn²⁺, Zn²⁺, and Ca²⁺) are inhibitory; the K _m for GTP is 22 m; and the molecular weight is estimated at 345,000 from gel filtration experiments. Of a number of nucleotides tested, only GDP and dGTP were used to any extent as substrate in place of GTP, and these respective compounds were used only 1.8% and 1.5% as well as GTP.This work was supported by research grants from the National Institutes of Health (AM03442) and the National Science Foundation (GB33929).     

Characteristics of the activation by dithiothreitol and Fe2+ of tryptophan hydroxylase from the rat brain

The preincubation of tryptophan hydroxylase extracted from various areas of the central nervous system of the rat with 30 mM dithiothreitol and 50 M ferrous ammonium sulfate under nitrogen atmosphere resulted in a persistent increase of its activity. Studies on the enzyme characteristics indicated that this activation was associated with a doubling in itsV _max and a shift (from 7.6 to 7.2) of the optimal pH for its activity. In contrast, the molecular weight and the apparent affinities of tryptophan hydroxylase for its pterin cofactor and for tryptophan were not significantly altered by the preincubation with dithiothreitol and ferrous ammonium sulfate. Since this treatment did not prevent the stimulatory effects of various compounds (phosphatidylserine, ATP and Mg²⁺, Ca²⁺) on tryptophan hydroxylase activity, this might be a good procedure to activate this enzyme with only minor changes in its regulatory properties.     

Regulation by ca of a cytosolic fructose-1,6-bisphosphatase from spinach leaves

Cytosolic fructose-1,6-bisphosphatase from spinach (Spinacia oleracea L.) leaves was purified over 1700-fold. The final preparation was specific for fructose-1,6-bisphosphate in the presence of either Mg²⁺ or Mn²⁺, and was free of interfering enzyme activities. Ca²⁺ was an effector of fructose-1,6-bisphosphatase activity, and showed different kinetics, depending on whether Mg²⁺ or Mn²⁺ was used as cofactor. In the presence of 5 millimolar Mg²⁺, Ca²⁺ appeared as activator or as inhibitor of the enzyme at low or high levels of substrate, respectively. In both cases, a rise in affinity for fructose-1,6-bisphosphate was observed. A model is proposed to describe the complex interaction of fructose-1,6-bisphosphatase with its substrate and Ca²⁺. However, with Mn²⁺ (60 micromolar) as cofactor, Ca²⁺ exhibited the Michaelis-Menten kinetics of a noncompetitive inhibitor. When assayed at constant substrate concentration, Ca²⁺ behaves as a competitive or noncompetitive inhibitor, depending on the use of Mg²⁺ or Mn²⁺ as cofactor, respectively, with a positive cooperativity in both cases. Fructose-2,6-bisphosphate showed a classic competitive allosteric inhibition in the presence of Mg²⁺ as cofactor, but this effect was low with Mn²⁺. From these results we suggest that Ca²⁺ plays a role in the in vivo regulation of cytosolic fructose-1,6-bisphosphatase.     

Properties of polyphosphatase ofAcinetobacter johnsonii 210A

Polyphosphatase, an enzyme which hydrolyses highly polymeric polyphosphates to P_i, was purified 77-fold fromAcinetobacter johnsonii 210A by Q-Sepharose, hydroxylapatite and Mono-Q column chromatography. The native molecular mass estimated by gel filtration and native gel electrophoresis was 55 kDa. SDS-polyacrylamide gel electrophoresis indicated that polyphosphatase ofAcinetobacter johnsonii 210A is a monomer. The enzyme was specific for highly polymeric polyphosphates and showed no activity towards pyrophosphate and organic phosphate esters. The enzyme was inhibited by iodoacetamide and in the presence of 10 mM Mg²⁺ by pyro- and triphosphate. The apparent K_m-value for polyphosphate with an average chain length of 64 residues was 5.9 µM and for tetraphosphate 1.2 mM. Polyphosphate chains were degraded to short chain polymers by a processive mechanism. Polyphosphatase activity was maximal in the presence of Mg²⁺ and K⁺.     

Uptake of Lead and Cadmium by Maize Seedlings and the Effect of Heavy Metals on the Activity of Phosphoenolpyruvate Carboxylase Isolated from Maize

Maize seeds and five-day-old maize seedlings were incubated in media containing Pb²⁺ at concentrations of 50, 100, and 200 mg 1^-1 and Cd²⁺ at concentrations of 1, 5, 10 and 50 mg 1^-1. After five days of incubation, both heavy metals were determined by means of AAS following wet mineralisation of roots and shoots. The results obtained indicate that Pb²⁺ were transported to shoots from roots at a lower rate than Cd²⁺. Phosphoenolpyruvate carboxylase (PEPC) isolated from germinating maize seeds was inhibited to a comparable degree by solutions containing 0.001 mmol 1^-1 Pb²⁺, 0.01 mmol 1^-1 Cd²⁺, and 0.005 mmol 1^-1 Cu²⁺. The enzyme was protected against this inhibition by the addition of mercaptoethanol, the substrate (PEP), or the cofactor (Mg²⁺). The inhibition increased during a 20 min incubation of the enzyme with salts of the metals. Mn²⁺, Ni²⁺, and Co²⁺ ions could partially substitute for the metal cofactor Mg²⁺. K_m values for these metal ions were as follows: for Mg²⁺ 0.07 mmol 1^-1 in the range from 0 to 0.30 mmol 1^-1 Mg²⁺; 0.71 mmol 1^-1 for 0.30 to 2.50 mmol 1^-1 Mg²⁺; for Mn²⁺ 0.36 mmol 1^-1; for Ni²⁺ 0.34 mmol 1{-1}; and for Co²⁺ 0.20 mmol 1^-1. The activity of the enzyme reached with Mn²⁺ 85 %, with Ni²⁺ 65 %, and with Co²⁺ 55 % of the activity recorded with Mg²⁺.     

Production of 2-Methylisocitric Acid from n-Paraffins by Mutants of Candida lipolytica

Vitamin B₁₂-dependent ribonucleotide reductase purified from Rhizobium meliloti catalyzes the reduction of 5′-diphosphates of guanosine, adenosine, cytidine and uridine (GDP, ADP, CDP and UDP). The enzyme activities were regulated by Mg²⁺ and deoxyribonucleoside triphosphate effectors as follows: in the presence of Mg²⁺, allosteric effector deoxyguanosine triphosphate (dGTP) had the most stimulatory effect on reduction of ADP and UDP; deoxyadenosine triphosphate (dATP) on reduction of CDP; and thymidine triphosphate (dTTP) on reduction of GDP. These stimulatory effectors were active at a low concentration of 10 μm. Other deoxyribonucleotides may be negative or weakly positive effectors. Without effectors, the rate profile of ADP and GDP reduction showed a sigmoidal curve. In the absence of Mg²⁺, the activities of the reductase showed nearly maximal levels, and the addition of effectors rather decreased the activities, except in the case of UDP reduction which was most strongly stimulated by dGTP. The effect of Mg²⁺ can be replaced by Ca²⁺. Monovalent cations such as Na⁺ and K⁺ had a negligible effect on the activities of ribonucleotide reductase.     

Expression, purification and characterization of recombinant (E)-beta-farnesene synthase from Artemisia annua

A cDNA clone (GenBank Accession No. AY835398) encoding a sesquiterpene synthase, (E)-β-farnesene synthase, has been isolated from Artemisia annua L. It contains a 1746-bp open reading frame coding for 574 amino acids (66.9 kDa) with a calculated pI = 5.03. The deduced amino acid sequence is 30-50% identical with sequences of other sesquiterpene synthases from angiosperms. The recombinant enzyme, produced in Escherichia coli, catalyzed the formation of a single product, β-farnesene, from farnesyl diphosphate. The pH optimum for the recombinant enzyme is around 6.5 and the K_m- and k_cat-values for farnesyl diphosphate, is 2.1 μM and 9.5 × 10⁻³ s⁻¹, respectively resulting in the efficiency 4.5 × 10⁻³ M⁻¹ s⁻¹. The enzyme exhibits substantial activity in the presence of Mg²⁺, Mn²⁺ or Co²⁺ but essentially no activity when Zn²⁺, Ni²⁺ or Cu²⁺ is used as cofactor. The concentration required for maximum activity are estimated to 5 mM, 0.5 mM and <10 μM for Mg²⁺, Co²⁺ or Mn²⁺, respectively. Geranyl diphosphate is not a substrate for the recombinant enzyme.     

Purification and properties of the enzymes from Drosophila melanogaster that catalyze the synthesis of sepiapterin from Dihydroneopterin triphosphate

Sepiapterin synthase, the enzyme system responsible for the synthesis of sepiapterin from dihydroneopterin triphosphate, has been partially purified from extracts of the heads of young adult fruit flies (Drosophila melanogaster). The sepiapterin synthase system consists of two components, termed enzyme A (MW 82,000) and enzyme B (MW 36,000). Some of the properties of the enzyme system are as follows: NADPH and a divalent cation, supplied most effectively as MgCl₂, are required for activity; optimal activity occurs at pH 7.4 and 30 C; the K _m for dihydroneopterin triphosphate is 10 µm; and a number of unconjugated pterins, including biopterin and sepiapterin, are inhibitory. Dihydroneopterin cannot be used as substrate in place of dihydroneopterin triphosphate. Evidence is presented in support of a proposed reaction mechanism for the enzymatic conversion of dihydroneopterin triphosphate to sepiapterin in which enzyme A catalyzes the production of a labile intermediate by nonhydrolytic elimination of the phosphates of dihydroneopterin triphosphate, and enzyme B catalyzes the conversion of this intermediate, in the presence of NADPH, to sepiapterin. An analysis of the activity of sepiapterin synthase during development in Drosophila revealed the presence of a small amount of activity in eggs and young larvae and a much larger amount in late pupae and young adults. Sepiapterin synthase activity during development corresponds with the appearance of sepiapterin in the flies. Of a variety of eye color mutants of Drosophila melanogaster tested for sepiapterin synthase activity, only purple (pr) flies contained activity that was significantly lower than that found in the wild-type flies (22% of the wild-type activity). Further studies indicated that the amount of enzyme A activity is low in purple flies, whereas the amount of enzyme B activity is equal to that present in wild-type flies.This work was supported by research grants from the National Institutes of Health (AM03442) and the National Science Foundation (PCM75-19513 A02). G. G. K. was supported as a predoctoral trainee by National Institutes of Health Training Grant GM00515.     

Catalytic role of subunit A in ribulose-1,5-diphosphate carboxylase from Chromatium strain D

The oligomeric form of the larger subunit designated as A_m produced by alkali treatment of ribulose-1,5-diphosphate carboxylase from the purple sulfur bacterium, Chromatium strain D, retained partial enzymic activity in the absence of the small subunit (B). Supporting evidence was obtained by polyacrylamide gel electrophoresis at pH 8.9 and Sephadex G-200 gel filtration equilibrated with alkaline buffer at pH 9.2. The specific enzyme activity of A_m (45 nmoles CO₂ fixed/mg protein/min) was approximately 15% of the native intact enzyme molecule. By sodium dodecyl sulfatepolyacrylamide gel electrophoresis, the A_m preparation was proved to be free from contamination of subunit B. With reservation of the sensitivity limit of this particular technique we concur that the larger subunit is the catalytic entity of the carboxylase reaction. The optimum pH of the ribulose-1,5-diphosphate carboxylase reaction catalyzed by isolated A_m lies on the alkaline side at about pH 8.3 with or without Mg²⁺. The undissociated native enzyme possesses an optimum pH on the alkaline side in the absence of Mg²⁺, which shifts to the acidic side in the presence of Mg²⁺. From this behavior it is inferred that the association of the smaller subunit with the larger subunit causes conformational stabilization of the enzyme molecule with an accompanying change in the pH optimum due to Mg²⁺.     

Mobile loop mutations in an archaeal inositol monophosphatase: Modulating three‐metal ion assisted catalysis and lithium inhibition

The inositol monophosphatase (IMPase) enzyme from the hyperthermophilic archaeon Methanocaldococcus jannaschii requires Mg²⁺ for activity and binds three to four ions tightly in the absence of ligands: K_D = 0.8 μM for one ion with a K_D of 38 μM for the other Mg²⁺ ions. However, the enzyme requires 5–10 mM Mg²⁺ for optimum catalysis, suggesting substrate alters the metal ion affinity. In crystal structures of this archaeal IMPase with products, one of the three metal ions is coordinated by only one protein contact, Asp38. The importance of this and three other acidic residues in a mobile loop that approaches the active site was probed with mutational studies. Only D38A exhibited an increased kinetic K_D for Mg²⁺; D26A, E39A, and E41A showed no significant change in the Mg²⁺ requirement for optimal activity. D38A also showed an increased K_m, but little effect on k_cat. This behavior is consistent with this side chain coordinating the third metal ion in the substrate complex, but with sufficient flexibility in the loop such that other acidic residues could position the Mg²⁺ in the active site in the absence of Asp38. While lithium ion inhibition of the archaeal IMPase is very poor (IC₅₀～250 mM), the D38A enzyme has a dramatically enhanced sensitivity to Li⁺ with an IC₅₀ of 12 mM. These results constitute additional evidence for three metal ion assisted catalysis with substrate and product binding reducing affinity of the third necessary metal ion. They also suggest a specific mode of action for lithium inhibition in the IMPase superfamily.     

Purification and characterization of pyruvate dehydrogenase complex from borccoli floral buds.

The pyruvate dehydrogenase complex (PDC) was purified from Brassica oleracea var. italica floral buds to a specific activity of approximately 6 μmol of NADH formed/min/ mg of protein. The PDC had cofactor requirements for NAD⁺, thiamine pyrophosphate, coenzyme A, and a divalent cation (Mg²⁺, Ca²⁺, or Mn²⁺). The enzyme catalyzed the oxidative decarboxylation of pyruvate at a rate threefold faster than 2-oxobutyrate but was inactive toward 2-oxoglutarate. The PDC was competively inhibited by acetyl-CoA against CoA and NADH against NAD⁺. The enzyme was shown to be more sensitive to regulation by NADH than acetyl-CoA.     

Novel Microbial Inhibitors of Angiotensin-converting Enzyme,Aspergillomarasmines A and B

Brush border membrane vesicles (BBMV) from the midgut epithelial cells of silkworm larvae were prepared. ATP hydrolyzing activity (ATPase activity) was associated with the BBMV. ATPase activity without Mg^{2 +} was not observed at pH 7 but substantial ATP hydrolyzing activity was observed at pH 7 with Mg^{2 +}. The enzyme required Mn^{2 +}, Mg^{2 +}, or Ca²⁺ ions. The enzyme also hydrolyzed ITP and GTP but not p-NPP, ADP, or AMP. KNO₃ and NEM strongly inhibited the ATPase activity. Behaviours of the ATPase against inhibitors suggested that it resembled vacuolar type ATPase.     

Catalytic properties of d-xylose isomerase from Streptomyces violaceoruber

The catalytic properties and stability of d-xylose isomerase from Streptomyces violaceoruber have been studied. The enzyme was activated by Mg²⁺, Co²⁺ and Mn²⁺ but Ni²⁺, Ca²⁺, Zn²⁺, Cu²⁺ and Hg were ineffective. Optimum catalytic conditions were obtained at 80°C in the pH range 7.5–9.5 and in the presence of 10 mm Mg²⁺. The specific activity of the enzyme increased after treatment with 10 mm EDTA (factor 2.4). A further increase of activity (factor 2.0–2.8) was observed after preincubation of the enzyme with Mg²⁺ or Co²⁺, the preincubation time depending on the incubation temperature. The thermal stability of the enzyme is very high. At 60°C the enzyme retained optimum activity following 30 days of storage in the presence of 1 mm Co²⁺ or 10 mm Mg²⁺. At 80°C, Co²⁺ is superior as a protector against thermal denaturation. At saturating concentrations of Mg²⁺ (35°C) the K_m-values of the EDTA-treated enzyme with respect to d-xylose and d-glucose were 2.8 and 149 mm and the dissociation constants of the enzyme-Mg²⁺ complex for xylitol and d-sorbitol were 0.455 and 4.47 mm, respectively.     

Properties of membrane-bound ATPase of some acidophilic heterotrophic bacteria

Membrane-bound ATPase (EC 3.6.1.4) of acidophilic heterotrophic bacteria from mine environment was isolated and characterized. The enzyme preparations fromAcidiphilium symbioticum KM2 and the strains GS18h and GS19h have a pH optimum of 7.7, 8.2 and 7.7, respectively, in the presence of Mg²⁺ which is required for activity. In an assay system containing Mn²⁺ or Ca²⁺ only, some activity was also evident. These enzymes hydrolyzed inorganic diphosphate (PPi), guanosine triphosphate (GTP) and inosine triphosphate (ITP) as the better substrate than ATP and theK _m values of the enzymes with respect to ATP were determined to be 238, 157 and 228 μmol/L forA. symbioticum KM2 and the strains GS18h and GS19h, respectively. The activity was stimulated by sulfite while Zn²⁺, Hg²⁺, 4-chloromercuribenzoic acid (p-CMB) and the specific inhibitors of F₀F₁ type ATPase,viz. N,N′-dicyclohexylcarbodiimide (DCCD), oligomycin and azide reduced the activity of the enzyme preparations.     

Purification and properties of fructose 1,6-bisphosphatase from turkey liver.

Fructose 1,6-bisphosphatase (EC 3.1.3.11) has been purified 360-fold from turkey liver. The purified enzyme appears to be homogeneous by disc gel electrophoresis and has a pH profile indistinguishable from that of the enzyme in crude extracts. Mn²⁺ is significantly more effective than Mg²⁺ as the essential metal cofactor of this enzyme. The maximal effect of histidine is equivalent to that of EDTA except that EDTA is more efficient at lower concentrations. The histidine effect is decreased with an increase in pH or if substrate is first bound to the enzyme. The enzyme activity is activated equally by d- and l-forms of histidine. Enzyme affinity for the substrate decreases with an increase in pH. The inhibition by high substrate concentrations observed at pH 7.5 is markedly reduced in the absence of chelating activator or when Mg² is replaced by Mn²⁺ as the metal cofactor. Turkeys liver fructose 1,6-bisphosphatase resembles the enzyme from mammalian sources in that the sensitivity to AMP inhibition is decreased with the increase in pH, temperature, and Mg² concentration.     

Characterization of Mg2+-ATPase from sheep kidney medulla: purification.

Magnesium-dependent adenosine triphosphatase has been purified from sheep kidney medulla plasma membranes. The purification, which is based on treatment of a kidney plasma membrane fraction with 0.5% digitonin in 3 mm MgCl₂, effectively separates the Mg²⁺-ATPase from (Na⁺ + K⁺)-ATPase present in the same tissue and yields the Mg²⁺-ATPase in soluble form. The purified enzyme is activated by a variety of divalent cations and trivalent cations, including Mg²⁺, Mn²⁺, Ca²⁺, Co²⁺, Fe²⁺, Zn²⁺, Eu³⁺, Gd³⁺, and VO²⁺. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme shows two bands with R_f values corresponding to molecular weights of 150,000 and 77,000. The larger peptide is phosphorylated by [γ-³²P]ATP, suggesting that this peptide may contain the active site of the Mg²⁺-ATPase. The Mg²⁺-ATPase activity is unaffected by the specific (Na⁺ + K⁺)-ATPase inhibitor ouabain.