Production of Adenosine from Extracellular ATP at the Striatal Cholinergic Synapse

Abstract: The components of the ectonucleotidase pathway at the immunoaffinity-purified striatal cholinergic synapse have been studied. The ecto-ATPase (EC 3.6.1.15) had a K _m of 131 γ M , whereas the ecto-ADPase (EC 3.6.1.6) had a K _m of 58 γ M , was Ca²⁺-dependent, and was inhibited by the ATP analogue 5'-adenylylimidodiphosphate (AMPPNP). The ecto-5'-nucleotidase (EC 3.1.3.5) had a K _m of 21 γ M , was inhibited by AMPPNP and α,β-methylene ADP, and by a specific antiserum. The V _max values of the ATPase, ADPase, and 5'-nucleotidase enzymes present at this synapse were in a ratio of 30:14:1. Very little ecto-adenylate kinase activity was detected on these purified synapses. The intraterminal 5'-nucleotidase enzyme, which amounted to 40% of the total 5'-nucleotidase activity, was inhibited by AMPPNP, α,β-methylene ADP, and the antiserum, and also had the same kinetic properties as the ectoenzyme. The time course of ATP degradation to adenosine outside the nerve terminals showed a delay, followed by a period of sustained adenosine production. The delay in adenosine production was proportional to the initial ATP concentration, was a consequence of feedforward inhibition of the ADPase and 5'-nucleotidase, and was inversely proportional to the ecto-5'-nucleotidase activity. The function and characteristics of this pathway and the central role of 5'-nucleotidase in the regulation of extraterminal adenosine concentrations are discussed.     

An inhibitor of prostaglandin biosynthesis from human decidua: partial purification and properties

An inhibitor of prostaglandin synthetase which catalyzes the conversion of arachidonic acid into prostaglandin E2 was partially purified from the 105,000 x g supernatant fraction of the human decidual cell homogenate. By means of ammonium sulfate fractionation, Mono Q ion-exchange chromatography, and gel filtration chromatography, the inhibitor was purified about 15-fold, giving a preparation with a molecular weight of 55-60 KDa. The 50% inhibitory concentration of the purified substance was approximately 0.2 mg/ml. The inhibitor may play a role in suppression of prostaglandin production by decidua in early pregnancy.     

Salivary apyrase of Rhodnius prolixus. Kinetics and purification.

The salivary apyrase activity of the blood-sucking bug Rhodnius prolixus was found to reside in a true apyrase (ATP diphosphohydrolase, EC 3.6.1.5) enzyme. The crude saliva was devoid of 5'-nucleotidase, inorganic pyrophosphatase, phosphatase and adenylate kinase activities. ATP hydrolysis proceeded directly to AMP and Pi without significant accumulation of ADP. Km values for ATP and ADP hydrolysis were 229 and 291 microM respectively. Ki values for ATP and ADP inhibition of ADP and ATP hydrolysis were not different from the Km values, and these experiments indicated competitive inhibition. Activities were purified 126-fold by combined gel filtration and ion-exchange chromatography procedures with a yield of 63%. The purified enzyme displayed specific activities of 580 and 335 mumol of Pi released/min per mg of protein for ATP and ADP hydrolysis respectively. The action of the purified enzyme on several phosphate esters indicates that Rhodnius apyrase is a non-specific nucleosidetriphosphate diphosphohydrolase.     

Note: Purification of amylase secreted from Bifidobacterium adolescentis

Bifidobacterium adolescentis Int-57 isolated from human faeces produced extracellular amylase. The enzyme was purified from the culture supernatant fluids by ammonium sulphate precipitation, gel-filtration chromatography (Sephadex-G-75), ion-exchange chromatography (CM-cellulose) and FPLC. SDS-PAGE of the purified enzyme revealed a major band with an apparent molecular weight of 66 kDa. The pI was 5·2. Enzyme activity was optimal at 50°C, and at pH 5·5. The enzyme was stable at 20–40°C, and at pH 5–6 with a K _m value of 2·4 g l⁻¹ soluble starch. The activation energy was 42·3 kJ mol⁻¹. The enzyme was significantly inhibited by maltose (10%), glucose (10%), Cu²+ (5 mmol l⁻¹), Zn²+ (5 mmol l⁻¹), N- bromosuccinimide (5 mmol l⁻¹), EDTA (5 mmol l⁻¹), I₂ (1 mmol l⁻¹) and activated by β-mercaptoethanol (10 mmol l⁻¹).     

Homoserine kinase from the phototrophic bacterium Rhodospirillum rubrum is not sensitive to feedback inhibition by l-threonine

Abstract Homoserine kinase (EC 2.7.1.39), one of the enzymes of L -threonine synthesis, was purified 200-fold from the phototrophic bacterium Rhodospirillum rubrum strain S1 by salt precipitation, hydrophobic interaction chromatography and gel filtration. The enzyme had a M _r of about 145000 and was active with L -homoserine ( K _m= 3 mM) and ATP ( K _m= 0.44 mM). In contrast to the kinase from the enteric bacterium, Escherichia coli , the R. rubrum enzyme was neither stabilized nor inhibited by L -threonine. Of 18 amino acids and metabolites tested (including L -allo-threonine, D -allo-threonine, DL -homocysteine, o -phosphoserine and L -norleucine), none was found to be inhibitory.     

Purification and Properties of Purified Hexokinase from the African Trypanosomes and Trypanosoma equiperdum

SYNOPSIS. Hexokinase was in both soluble and particulate fractions of extracts of Trypanosoma brucei, T. gambiense, T. rhodesiense and T. equiperdum. These enzymes have been purified some 350-fold from crude extracts by sonication, (NH₄)₂SO₄ fractionation, and DEAE Sephadex column chromatography. Purified hexokinases had: A) temperature optimum 45-50 C; B) pH optimum 6.5-7.0; C) Mg⁺⁺ and ATP requirements; D) inhibition by ADP, p -hydroxymercuribenzoate, glucose-6-phosphate, and competitive inhibition by mannose, glucosamine, N -acetyl-D-glucosamine and xylose; E) ability to phosphorylate glucose, fructose, mannose, 2-deoxy-D-glucose and glucosamine; F) a glucose requirement for stabilization. T. equiperdum hexokinase was inhibited 33% by ADP as compared to 17-20% with brucei group hexokinase, and showed 22% activity when ITP was substituted for ATP in contrast to 35-40% with brucei group hexokinase.     

Ornithine carbamoyltransferase of Streptomyces fradiae: purification and properties

Abstract The enzyme ornithine carbamoyltransferase was purified from Streptomyces fradiae . A 1200-fold increase in specific activity was achieved by ammonium sulphate precipitation, DEAE-cellulose and aminohexyl-agarose chromatography and gel filtration. The purified enzyme has a M _r of 87 000. Its isoelectric point is 5.3 as determined by isoelectric focusing. Apparent K _m values at pH 7.7 for ornithine and carbamoyl phosphate are 1.8 and 1.2 mM, respectively.     

Yeast mitochondria import ATP through the calcium-dependent ATP-Mg/Pi carrier Sal1p, and are ATP consumers during aerobic growth in glucose

Sal1p, a novel Ca²⁺-dependent ATP-Mg/Pi carrier, is essential in yeast lacking all adenine nucleotide translocases. By targeting luciferase to the mitochondrial matrix to monitor mitochondrial ATP levels, we show in isolated mitochondria that both ATP-Mg and free ADP are taken up by Sal1p with a K _m of 0.20 ± 0.03 mM and 0.28 ± 0.06 mM respectively. Nucleotide transport along Sal1p is strictly Ca²⁺ dependent. Ca²⁺ increases the V _max with a S _0.5 of 15 μM, and no changes in the K _m for ATP-Mg. Glucose sensing in yeast generates Ca²⁺ transients involving Ca²⁺ influx from the external medium. We find that carbon-deprived cells respond to glucose with an immediate increase in mitochondrial ATP levels which is not observed in the presence of EGTA or in Sal1p-deficient cells. Moreover, we now report that during normal aerobic growth on glucose, yeast mitochondria import ATP from the cytosol and hydrolyse it through H⁺-ATP synthase. We identify two pathways for ATP uptake in mitochondria, the ADP/ATP carriers and Sal1p. Thus, during exponential growth on glucose, mitochondria are ATP consumers, as those from cells growing in anaerobic conditions or deprived of mitochondrial DNA which depend on cytosolic ATP and mitochondrial ATPase working in reverse to generate a mitochondrial membrane potential. In conclusion, the results show that growth on glucose requires ATP hydrolysis in mitochondria and recruits Sal1p as a Ca²⁺-dependent mechanism to import ATP-Mg from the cytosol. Whether this mechanism is used under similar settings in higher eukaryotes is an open question.     

Alanine Aminotransferase in Bovine Brain: Purification and Properties

Abstract: Mitochondrial and cytosolic alanine aminotransferases (EC 2.6.1.2) were partially purified (140- and 180-fold, respectively) from bovine brain cortex by means of (NH₄)₂SO₄ precipitation, gel filtration on Sephadex G-150, and ion-exchange chromatography on DEAE A-50 and characterized. The enzymes exhibited identical molecular weights (110,000 ± 10,000) and pH optima (7.8), but were eluted from CM Sephadex C-50 at different ionic strengths. Isoelectric focusing of the enzymes indicated a pi value of 5.2 for the cytosolic enzyme and 7.2 for the mitochondrial enzyme. The K _m values of the mitochondrial enzyme were 5.1 m M , 6.6 m M , 0.7 m M , and 0.4 m M and of the cytosolic isozyme were 30.3 m M , 4.3 m M , 0.7 m M , and 0.5 m M for alanine, glutamate, 2-oxoglutarate, and pyruvate, respectively. The results indicated that two forms of alanine aminotransferase exist in nerve tissue, which suggests that they may play different roles in the cellular metabolism of nerve tissue.     

Isolation of pure anhydrotetracycline oxygenase from Streptomyces aureofaciens.

Anhydrotetracycline oxygenase was purified to homogeneity from Streptomyces aureofaciens, a producer of tetracycline. The enzyme was purified 60-fold in a 40% yield by a two-step procedure using a combination of hydrophobic chromatography and ion-exchange h.p.l.c. Purified anhydrotetracycline oxygenase was homogeneous according to SDS/polyacrylamide-gel electrophoresis, isoelectric focusing, ion-exchange h.p.l.c. on a Mono Q HR 5/5 column and size-exclusion h.p.l.c. on a TSK G 3000 SW column. The enzyme consists of two subunits of Mr 57,500, as determined by SDS/polyacrylamide-gel electrophoresis.     

A comparison of the properties of 5'-nucleotidase purified from the cytosolic and synaptic plasma membrane fractions of rat forebrain.

1. 5'-Nucleotidase was purified 1247-fold from the post-microsomal supernatant (I) and 3862-fold from the synaptic plasma membrane (II) of rat brain homogenates. 2. The apparent molecular masses of I and II were 131 and 72 kDa respectively by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate and 268 and 286 kDa respectively by Sephacryl S-300 chromatography. 3. The activities of both I and II were strongly inhibited by concanavalin A but were affected differently by digestion with glycosidases. for II, these were 0.083 and 0.056 mM respectively. 5. Activities of both I and II were strongly inhibited by ATP and ADP.     

N 5, N10-Methylenetetrahydromethanopterin reductase from Methanosarcina barkeri

N ⁵ N ¹⁰-Methylenetetrahydromethanopterin reductase was purified 13-fold to apparent homogeneity from methanol grown Methanosarcina barkeri . The colourless enzyme was found to be composed of four identical subunits of apparent molecular mass 36 kDa. It catalysed the reduction of methylenetetrahydromethanopterin ( K _m=15 μM) to methyltetrahydromethanopterin with reduced coenzyme F₄₂₀ ( K _m=12 μM) at a specific rate ( V _max) of 2200 μmol min⁻¹· mg protein⁻¹ ( K _cat=1320 s⁻¹). With respect to coenzyme specificity, molecular properties and catalytic mechanism the enzyme was found to be similar to CH₂=H₄MPT reductase of Methanobacterium thermoautotrophicum which phylogenetically is only distantly related to M. barkeri .     

Expression, purification and characterization of soluble human thrombopoietin receptor from Escherichia coli

The extracellular domain (edMpl) of human thrombopoietin (TPO) receptor, c-Mpl was expressed in Escherichia coli by changing some nucleotides before and after the translation initiation codon. The mutations increased the expression by approx. 15-fold. The inclusion bodies were solubilized in 8 M guanidine-HCl under reducing conditions and refolded using a glutathione-redox system. The monomeric form of edMpl was purified to near homogeneity by two successive steps of ion-exchange chromatography using DEAE-Sephacel and Mono Q columns. The purified monomeric edMpl inhibited the TPO-dependent cell proliferation, suggesting that it was binding to TPO. Also, antisera raised against the edMpl bound specifically to the soluble receptor secreted by mammalian cells.     

Inhibition of Aldehyde Dehydrogenases in Rat Brain and Liver by Disulfiram and Coprine

Abstract: Rats were treated with either coprine or disulfiram and the inhibition of aldehyde dehydrogenase (ALDH) in liver and brain mitochondria was measured with acetaldehyde, 3,4-dihydroxyphenylacetaldehyde (DOPAL), and succinate semialdehyde at different concentrations. The inhibition pattern was similar for both inhibitors, but the degree of inhibition was lower with disulfiram. The ALDH activity both in the liver and the brain was inhibited at low concentrations of acetaldehyde and DOPAL, but not with succinate semialdehyde. The high- K _m enzyme activities with acetaldehyde were not inhibited in liver and brain. The activity at high concentration of DOPAL was inhibited in the liver, but only slightly affected in the brain, suggesting the presence of a brain enzyme with an intermediate K _m value for DOPAL. In contrast with the results observed in viva, it was found that the high- K _m activities with acetaldehyde and DOPAL in brain mitochondrial preparations were more sensitive to the inhibitors in vitro than the low- K _m activities. Kinetic studies on ALDH preparations from brain and liver mitochondria suggested that acetaldehyde and DOPAL are metabolized by the same low- K _m ALDH.     

The ATP-dependent synthesis of factor 390 by cell-free extracts of Methanobacterium thermoautotrophicum (strain ΔH)

Abstract Cell-free extracts of Methanobacterium thermoautotrophicum (strain ΔH) converted the 8-OH-5-deazaflavin coenzyme F₄₂₀ to factor 390, a 8-adenylyl derivative (F₄₂₀-AMP). Activity was only observed upon exposure of the crude cell-free extract to oxygen. The ability to synthesize F₃₉₀ was lost when crude cell-free extract was subsequently brought to an anaerobic reducing environment. The enzymatic reaction used ATP and oxidized coenzyme F₄₂₀ as substrates and inorganic pyrophosphate was formed next to F₃₉₀. GTP could be used instead of ATP resulting in a guanylylated derivative. The crude cell-free extract showed K _m values of 154 μM for coenzyme F₄₂₀ and 2.4 mM for ATP. A partially purified enzyme preparation exhibited a K _eq of 0.32. In accordance, coenzyme F₄₂₀ and ATP could be synthesized from F₃₉₀ and PPi by the reverse reaction.     

Some kinetic properties of a purified glutamine synthetase from bacteroids of Glycine max

Abstract A purified glutamine synthetase was prepared from bacteroids of Rhizobium japonicum from nodules of Glycine max . For the biosynthetic assay the K _m values (mM) were l -glutamate 12.9, NH₄Cl 8.9 and ATP 14.3. When the enzyme was assayed by the γ-glutamyltransferase reaction the K _m values (mM) were l -glutamine 11.1 and hydroxylamine 3.3 compared with 7.7 and 1.2, respectively, for the purified enzyme from Rhizobium japonicum grown in culture. The enzyme prepared from bacteroids of Glycine max was 80% adenylylated.     

An Endogenous Aminoenkephalinase Inhibitor: Purification and Characterization of Arg0-Met5-Enkephalin from Bovine Striatum

Abstract: Arg⁰-Met⁵-enkephalin (Arg⁰-MEK) was isolated from bovine striatum and purified to homogeneity. The peptide was extracted with trichloroacetic acid, followed by column chromatography successively on Bio-Sil C₈, semipreparative HPLC Radial-Pak C₁₈, fast protein liquid chromatography (FPLC) Mono S, HPLC Ultrasphere-ODS, Supelco C₁₈, Lichromsorb C₁₈, and μBondapak C₁₈. The peptide content was followed by radioimmunoassay with an antibody against synthetic Met-enkephalin. For each of the six HPLC and FPLC systems, the elution time of the immunoreactive fractions coincided exactly with that of synthetic Arg⁰-MEK. The purified peptide showed a highly homogeneous profile in three different analytical HPLC systems. Its retention time and three-dimensional UV spectrum were identical to those of the synthetic Arg⁰-MEK. The structure of the purified material was identified by microsequencing as the hexapeptide Arg-Tyr-Gly-Gly-Phe-Met. Ninety percent of the purified peptide was in oxidized form containing equimolar amounts of Met-( R )- and Met-( S )-sulfoxide. The reduced Arg⁰-MEK inhibited aminoenkephalinase with a K _i of 2.2 µ M , and its sulfoxide analogue inhibited it with a K _i of 8.9 µ M . The reduced or oxidized peptide suppressed the electrically induced contraction of rat vas deferens with an ED₅₀ of 5 µ M , and the effect could be reversed by equimolar naloxone. Our data indicate that Arg⁰-MEK is an immediate Met-enkephalin precursor and an endogenous inhibitor.     

Soluble 5''-Nucleotidase Activities in Rat Brain

5'-Nucleotidase activity was assayed in 105,000-g supernatants from rat brain by following conversion of [3H]AMP into adenosine. The effect of ATP on this process was complex and suggested the presence of at least two soluble 5'-nucleotidase activities: one inhibited by ATP and another activated by ATP. The relative proportions of these activities differed considerably among brain regions. Activity changes induced by hypothyroidism also suggested that these activities may be regulated independently. These findings may have consequences for the regional regulation of adenosine formation in the brain.     

Purification and Characterization of a Ca2+- and Calmodulin-Dependent Protein Kinase from Rat Brain

Abstract: A Ca²⁺- and calmodulin-dependent protein kinase was purified from rat brain cytosol fraction to apparent homogeneity at approximately 800-fold and with a 5% yield. The purified enzyme had a molecular weight of 640,000 as determined by gel filtration analysis on Sephacryl S-300 and a sedimentation coefficient of 15.3 S by sucrose density gradient centrifugation, and resulted in a single protein band of MW 49,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These results suggest that the native enzyme has a large molecular weight and consists of 11 to 14 identical subunits. The purified enzyme exhibited K _m values of 109 and 30 μM for ATP and chicken gizzard myosin light chain, respectively, and K _a values of 12 n M and 1.9 μM for brain calmodulin and Ca²⁺, respectively. In addition to myosin light chain, myelin basic protein, casein, arginine-rich histone, microtubule protein, and synaptosomal proteins were phosphorylated by the enzyme in a Ca²⁺- and calmodulin-dependent manner. The purified enzyme was phosphorylated without the addition of the catalytic subunit of cyclic AMP-dependent protein kinase. Our findings indicate that there is a multifunctional Ca²⁺- and calmodulin-dependent protein kinase in the brain and that this enzyme may regulate the reactions of various endogenous proteins.     

Soils contain two different activities for oxidation of hydrogen

Abstract Hydrogen oxidation rates were measured in a neutral compost soil and an acidic sandy loam at H₂ mixing ratios of 0.01 to 5000 ppmv. The kinetics were biphasic showing two different K _m values for H₂, one at about 10–40 nM dissolved H₂, the other at about 1.2–1.4 μM H₂. The low- K _m activity was less sensitive to chloroform fumigation than the high- K _m activity. If sterile soil was amended with Paracoccus denitrificans or a H₂-oxidizing strain isolated from compost soil, it exhibited only a high- K _m (0.7–0.9 μM) activity. It also failed to utilize H₂ mixing ratios below a threshold of 1.6–3.0 ppmv H₂ (160–300 mPa). A similar result was obtained when fresh soil samples were suspended in water, and H₂ oxidation was determined from the decrease of dissolved H₂. However, H₂ was again utilized to mixing ratios lower than 0.05 ppmv, if the supernatant of the soil suspension or the settled soil particles were dried onto sterile soil or purified quarz sand. Obviously, soils contain two different activities for oxidation of H₂: (1) a high- K _m, high-threshold activity which apparently is due to aerobic H₂-oxidizing bacteria, and (2) a low- K _m, low-threshold activity whose origin is unknown but presumably is due to soil enzymes.