Purification and some properties of ATP-sulfurylase from developing sea urchin embryos

ATP-sulfurylase (ATP:sulfate adenylyltransferase, EC 2.7.7.4), purified about 200-fold from sea urchin embryos, was free of ATPase and inorganic pyrophosphatase. The molecular weight of the enzyme was approx. 280 000 measured by gel filtration. The enzyme was activated by Mg2+, Ca2+ or Zn2+; EDTA and p-chloromercuriphenylsulfonate inhibited the enzyme activity. The inhibition was reversed by addition of Mg2+ and dithiothreitol, respectively. The enzyme activity increased continuously as the pH was raised from 5.6 to 10.6. The Km values for the enzyme were calculated to be 13 microM for adenosine 5'-phosphosulfate and 23 microM for pyrophosphate.     

Tritrichomonas foetus: purification and characterization of hydrogenosomal ATP:AMP phosphotransferase (adenylate kinase)

The hydrogenosomal enzyme ATP:AMP phosphotransferase (adenylate kinase) (EC 2.7.4.3) was purified to apparent homogeneity from the bovine parasite Tritrichomonas foetus. A fraction enriched for hydrogenosomes was obtained from cell homogenates which had been subjected to differential and isopycnic centrifugation. Adenylate kinase was solubilized in 50 mM Tris-HCl, pH 7.3, containing 0.8% Triton X-100, and purified by sequential Affi-Gel blue affinity chromatography and high-performance liquid chromatography gel filtration. The purified enzyme, a monomer of Mr 29,000, exhibited Km values of 100, 195, and 83 microM for ADP, ATP, and AMP, respectively. Substituting other mono-, di-, and trinucleotides for AMP, ADP, and ATP gave less than half the maximal activity. Full enzyme activity requires Mg2+, but Mn2+ and Co2+ yield half maximal activity. The enzyme has a broad optimal pH range between pH 6 and 9. The enzyme was competitively inhibited by P1,P5-di(adenosine-5')pentaphosphate, a specific adenylate kinase inhibitor: the Ki was 150 nM. The enzyme was also inhibited with 5,5'-dithiobis(2-nitrobenzoic acid), and this inhibition could be reversed by the addition of 2 mM dithiothreitol. T. foetus adenylate kinase has similar catalytic and physical properties to that of the biologically closely related human parasite Trichomonas vaginalis.     

Purification and characterization of phosphoenolpyruvate phosphomutase from Pseudomonas gladioli B-1.

Phosphoenolpyruvate phosphomutase (PEPPM) catalyzes C-P bond formation by intramolecular rearrangement of phosphoenolpyruvate to phosphonopyruvate (PnPy). We purified PEPPM from a gram-negative bacterium, Pseudomonas gladioli B-1 isolated as a C-P compound producer. The equilibrium of this reaction favors the formation of the phosphate ester by cleaving the C-P bond of PnPy, but the C-P bond-forming reaction is physiologically significant. The C-P bond-forming activity of PEPPM was confirmed with a purified protein. The molecular mass of the native enzyme was estimated to be 263 and 220 kDa by gel filtration and polyacrylamide gel electrophoresis, respectively. A subunit molecular mass of 61 kDa was determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that the native protein was a tetramer. The optimum pH and temperature were 7.5 to 8.0 and 40 degrees C, respectively. The Km value for PnPy was 19 +/- 3.5 microM, and the maximum initial velocity of the conversion of PnPy to phosphoenolpyruvate was 200 microM/s/mg. PEPPM was activated by the presence of the divalent metal ion, and the Km values were 3.5 +/- 1.4 microM for Mg2+, 16 +/- 5 nM for Mn2+, 3.0 +/- 1.5 microM for Zn2+, and 1.2 +/- 0.2 microM for Co2+.     

The assay of ATP-sulfurylase

An assay method for ATP-sulfurylase is described, based on the incorporation of 35SO4(2-) into adenosine-5'-phosphosulfate (and 3-phosphoadenosine-5'-phosphosulfate) and the separation of these compounds by HPLC. Since the enzyme is easily inactivated at temperatures above 20 degrees C during the assay, the reaction time should not exceed 10 min.     

Novel bioluminescent assay of alkaline phosphatase using adenosine-3'-phosphate-5'-phosphosulfate as substrate and the luciferin-luciferase reaction and its application

This paper describes a novel bioluminescent assay of alkaline phosphatase (ALP) utilizing ATP-sulfurylase and the luciferin-luciferase reaction. The principle governing the assay is as follows. Adenosine-3'-phosphate-5'-phosphosulfate, which serves as the substrate for ALP, is hydrolyzed enzymatically to produce adenosine-5'-phosphosulfate (APS). APS is converted into ATP by ATP-sulfurylase in the presence of pyrophosphate. The ATP produced is detected by the luciferin-luciferase reaction. The measurable range was 1 zmol to 100 fmol/assay and the detection limit at blank+3 SD was 10 zmol/assay. The coefficient of variation (CV, n=5) was examined at each point of the standard curve; the mean CV percentage was 4.47% (n=6). This assay system was applied to enzyme immunoassay of human chorionic gonadotropin and allele-specific PCR enzyme-linked immunosorbent assay of verotoxin gene using ALP as the label enzyme; 10(-2) mIU/mL hCG in urine and 5 pg of Escherichia coli O157 DNA could be assayed directly and with high sensitivity by the proposed method.     

Specific magnesium-dependent diadenosine 5'',5''''''-P1,P3-triphosphate pyrophosphohydrolase in Escherichia coli.

A specific Mg2+-dependent bis(5'-adenosyl)-triphosphatase (EC 3.6.1.29) was purified 270-fold from Escherichia coli. The enzyme had a strict requirement for Mg2+. Other divalent cations, such as Mn2+, Ca2+, or Co2+, were not effective. The products of the reaction with bis(5'-adenosyl) triphosphate (Ap3A) as the substrate were ADP and AMP in stoichiometric amounts. The Km for Ap3A was 12 +/- 5 microM. Bis(5'-adenosyl) di-, tetra-, and pentaphosphates, NAD+, ATP, ADP, AMP, glucose 6-phosphate, p-nitrophenylphosphate, bis-p-nitrophenylphospate, and deoxyribosylthymine-5'-(4-nitrophenylphosphate) were not substrates of the reaction. The enzyme had a molecular mass of 36 kilodaltons (as determined both by gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis), an isoelectric point of 4.84 +/- 0.05, and a pH optimum of 8.2 to 8.5. Zn2+, a known potent inhibitor of rat liver bis(5'-adenosyl)-triphosphatase and bis(5'-guanosyl)-tetraphosphatase (EC 3.6 1.17), was without effect. The enzyme differs from the E. coli diadenosine 5',5'-P1, P4-tetraphosphate pyrophosphohydrolase which, in the presence of Mn2+, also hydrolyzes Ap3A.     

Partial purification and characterization of adenosine- and guanosine-3',5'-monophosphate phosphodiesterases from human lung tissue.

Crude extracts of human lung tissue were examined for cyclic adenosine- and guanosine-3',5'-monophosphate (cAMP and cGMP) phosphodiesterase activities. Nonlinear reciprocal plots were observed for each substrate. DEAE-Sephadex chromatography of the extracts revealed four main fractions of activity, which were further purified by Sephadex gel filtration. The phosphodiesterase activity of the resulting individual fractions was partially characterized with respect to substrate specificity, kinetic parameters, apparent molecular weight (gel filtration), thermal stability at 30 and 37 degrees C, effect of the cyclic nucleotide not utilized as substrate, and the possible influence of Ca2+-dependent protein activator. The results indicate that the tissue contains phosphodiesterases with strict specificity and a high apparent affinity for each of the two cyclic nucleotides (the Km values determined were approximately 0.3-0.4 muM). The high affinity cAMP phosphodiesterase activity was enriched in two of the purified fractions; both activities probably represent fragments of the native high affinity cAMP specific enzyme. A third purified phosphodiesterase showed mixed substrate specificity. The Km value recorded for hydrolysis of either substrate with this enzyme was approximately 25 muM. A fourth, irregularly occurring, phosphodiesterase activity also showed mixed substrate specificity. The Km value registered for hydrolysis of either substrate with this fraction was approximately 0.4 muM. There was no evidence for a Ca2+-dependent specific activation by a boiled lung tissue supernatant of any of the purified enzymes.     

Calmodulin-dependent NAD kinase of human neutrophils

NAD kinase from human neutrophils has been partially purified by sequential application of Red Agarose, ion-exchange, and gel-filtration chromatography. The enzyme has a broad pH optimum, 7.0-9.5, is strictly dependent upon the presence of Mg2+, and in the absence of calcium exhibits Km values of 0.6 and 0.9 mM for NAD and ATP, respectively. NAD kinase activity is extremely sensitive to free calcium concentration, with half-maximal activity observed at free calcium concentrations of approximately 0.4 microM. In cellular extracts calcium-dependent activation of NAD kinase increases the maximum velocity of the reaction from 2- to 5-fold while not affecting Km values for NAD and ATP. The activity of the partially purified NAD kinase is stimulated 3.5-fold by the addition of calmodulin in the presence of calcium. This stimulation is inhibited by the addition of 20 microM trifluoperazine to the incubation. These data are interpreted as implicating calmodulin in NAD kinase regulation. The total concentration of NADP + NADPH in the human neutrophil used increased 2.2-fold in response to activation by phorbol myristic acetate. Finally, neutrophil NAD kinase has a Mr, based upon gel filtration, of 169,000.     

Purification and characterization of ATP:citrate lyase from Hydrogenobacter thermophilus TK-6.

ATP:citrate lyase [ATP citrate (pro-3S)-lyase; EC 4.1.3.8] was purified and characterized from the cells of Hydrogenobacter thermophilus, an aerobic, thermophilic, hydrogen-oxidizing bacterium which fixes carbon dioxide by a reductive carboxylic acid cycle. The enzyme was quite stable, even in the absence of sulfhydryl reagents. Optimum pH for reaction was 6.7 to 6.9, and optimum temperature was around 80 degrees C. The molecular weight of native enzyme was estimated to be 260,000 by gel filtration analysis, and that of a subunit was estimated to be 43,000 by sodium dodecyl sulfate-polyacrylamide gel analysis. Km values for reaction components were as follows: citrate, 6.25 mM; ATP, 650 microM; coenzyme A, 40.8 microM; and Mg2+, 8 mM. The enzyme showed citrate synthase activity in the presence of Mg2+, but the reaction rate was very low (less than 1/200 of the lyase activity).     

Purification and characterization of acetylcoenzyme A: deacetylvindoline 4-O-acetyltransferase from Catharanthus roseus

The enzyme acetylcoenzyme A:deacetylvindoline 4-O-acetyltransferase (EC 2.3.1.-) (DAT), which catalyzes the final step in vindoline biosynthesis in Catharanthus roseus, was purified 3300-fold using ammonium sulfate precipitation followed by gel filtration, anion exchange, hydroxyapatite, and affinity chromatographies. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) of the purified DAT showed the presence of two major proteins having Mr values of 33,000 and 21,000, whereas native PAGE showed three protein bands, and isoelectric focusing-PAGE one diffuse protein band (pI = 4.7-5.3) plus two minor protein bands (pI = 5.7 and 6.1). Purified DAT possessed Km values of 6.5 microM and 1.3 microM for acetylcoenzyme A and deacetylvindoline, respectively, and Vmax values of 12.6 pkat/microgram protein (acetylcoenzyme A) and 10.1 pkat/micrograms protein (deacetylvindoline). Inhibition of DAT by tabersonine, coenzyme A, and cations (K+, Mg2+, and Mn2+) was observed, while the pH optimum of this enzyme was determined to be 7.5 to 9.     

Purification and characterization of hyoscyamine 6 beta-hydroxylase from root cultures of Hyoscyamus niger L. Hydroxylase and epoxidase activities in the enzyme preparation

Hyoscyamine 6 beta-hydroxylase, a 2-oxoglutarate-dependent dioxygenase that catalyzes the hydroxylation of l-hyoscyamine to 6 beta-hydroxyhyoscyamine in the biosynthetic pathway leading to scopolamine [Hashimoto, T. & Yamada, Y. (1986) Plant Physiol. 81, 619-625] was purified 310-fold from root cultures of Hyoscyamus niger L. The enzyme has an average Mr of 41,000 as determined by gel filtration on Superose 12 and exhibited maximum activity at pH 7.8 l-Hyoscyamine and 2-oxoglutarate are required for the enzyme activity, with respective Km values of 35 microM and 43 microM. Fe2+, catalase and a reductant such as ascorbate significantly activated the enzyme. 2-Oxoglutarate was not replaced by any of ten other oxo acids tested, nor was Fe2+ by nine other divalent cations tested. The enzyme was inhibited moderately by EDTA, Tiron and various oxo acids and aliphatic dicarboxylic acids, and strongly by nitroblue tetrazolium and divalent cations Mn2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+ and Hg2+. Several pyridine dicarboxylates and o-dihydroxyphenyl derivatives inhibited the hydroxylase. Pyridine 2,4-dicarboxylate and 3,4-dihydroxybenzoate are competitive inhibitors with respect to 2-oxoglutarate with the respective Ki values of 9 microM and 90 microM. Several alkaloids with structures similar to l-hyoscyamine were hydroxylated by the enzyme at the C-6 position of the tropane moiety. The enzyme preparation also epoxidized 6,7-dehydrohyoscyamine, a hypothetical precursor of scopolamine, to scopolamine (Km 10 microM). This epoxidation reaction required the same co-factors as the hydroxylation reaction and the epoxidase activities were found in the same fractions with the hydroxylase activities during purification. Two possible pathways for scopolamine biosynthesis are discussed in the light of the hydroxylase and epoxidase activities found in the partially purified preparation of hyoscyamine 6 beta-hydroxylase.     

Purification and properties of the 5 alpha-dihydrotestosterone 3 alpha(beta)-hydroxysteroid dehydrogenase from human prostatic cytosol.

5 alpha-Dihydrotestosterone 3 alpha(beta)-hydroxysteroid dehydrogenase [3 alpha(beta)-HSDH] [EC 1.1.1.50/EC 1.1.1.51] which catalyses the conversion of 5 alpha-dihydrotestosterone (5 alpha-DHT) to both 5 alpha-androstane-3 alpha,17 beta-diol and 5 alpha-androstane-3 beta,17 beta-diol was purified to an apparent homogeneous state using cytosol of three human hyperplastic prostates by a 4-step purification procedure. After each purification step 3 alpha-HSDH activity was coincident with 3 beta-HSDH activity. On average, specific 3 alpha-HSDH activity was enriched 856-fold, specific 3 beta-HSDH activity 749-fold compared to human prostatic cytosol using anion exchange, hydrophobic interaction, gel filtration and affinity chromatography. Examination of the purified enzyme by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate (SDS) revealed a single protein band with silver staining. The molecular weight of the enzyme was estimated as 33 kDa by SDS-polyacrylamide gel electrophoresis and as 28 kDa by Sephacryl S-200 gel filtration indicating that the native 3 alpha(beta)-HSDH is a monomer. In the presence of the preferred co-factor, NADPH, the purified enzyme had a mean apparent Km for 5 alpha-DHT of 3.9 microM and a Vmax of 93.3 nmol (mg protein)-1 h-1 with regard to 3 alpha-HSDH activity, and a Km of 6.3 microM and a Vmax of 20.6 nmol (mg protein)-1 h-1 with regard to 3 beta-HSDH activity.     

Purification and characterization of 17 beta-hydroxysteroid dehydrogenase from Cylindrocarpon radicicola

An NAD+-linked 17 beta-hydroxysteroid dehydrogenase was purified to homogeneity from a fungus, Cylindrocarpon radicicola ATCC 11011 by ion exchange, gel filtration, and hydrophobic chromatographies. The purified preparation of the dehydrogenase showed an apparent molecular weight of 58,600 by gel filtration and polyacrylamide gel electrophoresis. SDS-gel electrophoresis gave Mr = 26,000 for the identical subunits of the protein. The amino-terminal residue of the enzyme protein was determined to be glycine. The enzyme catalyzed the oxidation of 17 beta-hydroxysteroids to the ketosteroids with the reduction of NAD+, which was a specific hydrogen acceptor, and also catalyzed the reduction of 17-ketosteroids with the consumption of NADH. The optimum pH of the dehydrogenase reaction was 10 and that of the reductase reaction was 7.0. The enzyme had a high specific activity for the oxidation of testosterone (Vmax = 85 mumol/min/mg; Km for the steroid = 9.5 microM; Km for NAD+ = 198 microM at pH 10.0) and for the reduction of androstenedione (Vmax = 1.8 mumol/min/mg; Km for the steroid = 24 microM; Km for NADH = 6.8 microM at pH 7.0). In the purified enzyme preparation, no activity of 3 alpha-hydroxysteroid dehydrogenase, 3 beta-hydroxysteroid dehydrogenase, delta 5-3-ketosteroid-4,5-isomerase, or steroid ring A-delta-dehydrogenase was detected. Among several steroids tested, only 17 beta-hydroxysteroids such as testosterone, estradiol-17 beta, and 11 beta-hydroxytestosterone, were oxidized, indicating that the enzyme has a high specificity for the substrate steroid. The stereospecificity of hydrogen transfer by the enzyme in dehydrogenation was examined with [17 alpha-3H]testosterone.     

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.     

Formaldehyde dehydrogenase from Pseudomonas putida. Purification and some properties

Formaldehyde dehydrogenase was isolated and purified in an overall yield of 12% from cell-free extract of Pseudomonas putida C-83 by chromatographies on columns of DEAE-cellulose, DEAE-Sephadex A-50, and hydroxyapatite. The purified enzyme was homogeneous as judged by disc gel electrophoresis and was most active at pH 7.8 using formaldehyde as a substrate. The enzyme was also active toward acetaldehyde, propionaldehyde, glyoxal, and pyruvaldehyde, though the reaction rates were low. The enzyme was NAD+-linked but did not require the external addition of glutathione, in contrast with the usual formaldehyde dehydrogenase from liver mitochondria, baker's yeast, and some bacteria. The enzyme was markedly inhibited by Ni2+, Pd2+, Hg2+, p-chloromercuribenzoate, and phenylmethanesulfonyl fluoride. The molecular weight of the enzyme was estimated to be 150,000 by the gel filtration method, and analysis by SDS-polyacrylamide gel electrophoresis indicated that the enzyme was composed of two subunit monomers. Kinetic analysis gave Km values of 67 microM for formaldehyde and 56 microM for NAD+, and suggested that the reaction proceeds by a "Ping-pong" mechanism. The enzyme catalyzed the oxidation of formaldehyde accompanied by the stoichiometric reduction of NAD+, but no reverse reaction was observed.     

Isolation and characterization of tropomyosin kinase from chicken embryo

Tropomyosin kinase is partially purified from 14-day-old chicken embryos using DEAE-cellulose, cellulose phosphate and gel filtration chromatography. The purest enzyme preparation consists of two major bands of Mr = 76,000 and 43,000 on SDS-polyacrylamide gel electrophoresis. The molecular weight of the enzyme is 250,000 determined by gel filtration chromatography. It phosphorylates casein and skeletal tropomyosin equally well but histone and phosvitin at a much slower rate. Smooth muscle myosin light chain, tropomyosin from platelet, erythrocyte and smooth muscle are not phosphorylated. The apparent Km for skeletal alpha-tropomyosin and ATP is 50 microM and 200 microM, respectively. Vmax varies between 100-300 nmol/min per mg depending on the purity of the preparation. Mg2+ and dithiothreitol are essential for activity but Ca+, calmodulin and cAMP are not required. The optimum temperature is 37 degrees C and optimum pH is about 7.5. Heparin, a potent inhibitor of casein kinase II, has no inhibitory effect on the enzyme. Similar tropomyosin kinase activity is not detected in skeletal muscle in adult rabbit and chicken. The tropomyosin kinase described here represents a hitherto uncharacterized kinase responsible for phosphorylation of tropomyosin in the chicken embryo.     

Isolation and characterization of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from bovine liver

6-Phosphofructo-2-kinase and fructose-2,6-bisphosphatase activities were copurified to homogeneity from bovine liver. The purification scheme consisted of polyethylene glycol precipitation, anion-exchange and Blue-Sepharose chromatography, substrate elution from phosphocellulose, and gel filtration. The bifunctional enzyme had an apparent molecular weight of 102,000 and consisted of two subunits (Mr 49,000). The kinase had a Km for ATP of 12 microM and a S0.5 for fructose 6-phosphate of 150 microM while the bisphosphatase had a Km for fructose 2,6-bisphosphate of 7 microM. Both activities were subject to modulation by various effectors. Inorganic phosphate stimulated both activities, while alpha-glycerolphosphate inhibited the kinase and stimulated the bisphosphatase. The pH optimum for the 6-phosphofructo-2-kinase activity was 8.5, while the fructose-2,6-bisphosphatase reaction was maximal at pH 6.5. Incubation of the purified enzyme with [gamma-32P]ATP and the catalytic subunit of the cAMP-dependent protein kinase resulted in 32P incorporation to the extent of 0.7 mol/mol enzyme subunit with concomitant inhibition of the kinase activity and activation of the bisphosphatase activity. The mediation of the bisphosphatase reaction by a phosphoenzyme intermediate was suggested by the isolation of a stable labeled phosphoenzyme when the enzyme was incubated with fructose 2,6-[2-32P]bisphosphate. The pH dependence of hydrolysis of the phospho group suggested that it was linked to the N3 of a histidyl residue. The 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase from bovine liver has properties essentially identical to those of the rat liver enzyme, suggesting that hepatic fructose 2,6-bisphosphate metabolism is under the same control in both species.     

Diacylglycerol kinase from pig brain. Purification and phospholipid dependencies

Diacylglycerol kinase (EC 2.7.1.-) was purified 1,650-fold from pig brain cytosol. The purified enzyme showed a single protein band on polyacrylamide gel electrophoresis in the presence and absence of sodium dodecyl sulfate. The molecular weight of the kinase was estimated to be 78,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A similar value (76,000) was obtained by Sephadex G-150 gel filtration. The activity of the purified enzyme was markedly enhanced by either deoxycholate or phospholipids. The extent of activation by phospholipids was in the order of phosphatidylcholine greater than lysophosphatidylcholine greater than phosphatidylethanolamine approximately equal to phosphatidylserine greater than sphingomyelin. Other phospholipids and unsaturated fatty acids were ineffective. Phosphatidylcholines from egg yolk and pig brain, and dioleoyl phosphatidylcholine were similarly effective. Saturated phosphatidylcholines with acyl chain lengths shorter than palmitate also gave a considerable activation. The activity with phosphatidylcholine was from 1.5- to 2.5-fold higher than that measured with deoxycholate. A very small amount of phosphatidylinositol or phosphatidylglycerol potently inhibited the phosphatidylcholine-dependent (but not deoxycholate-dependent) kinase activity. The inhibition by phosphatidylinositol was varied according to its molar ratio to phosphatidylcholine. As little as about 2.5 mol per cent of phosphatidylinositol resulted in 50% inhibition of the phosphatidylcholine-dependent kinase activity. The deoxycholate- and phosphatidylcholine-dependent kinase activities showed almost the same Km values for the substrates. In both cases, the apparent Km values for ATP and diacylglycerol were 300 microM and about 60 microM, respectively. The kinase required Mg2+ for its activity. When compared to deoxycholate, phosphatidylcholine was more effective at higher Mg2+ concentrations. The deoxycholate-dependent activity showed a broad pH optimum at around 8.0, whereas the phosphatidylcholine-dependent activity formed a clear peak at pH 7.4.     

Adenosine-5'-phosphosulfate kinase from Penicillium chrysogenum: ligand binding properties and the mechanism of substrate inhibition.

[35S]Adenosine-5'-phosphosulfate (APS) binding to Penicillium chrysogenum APS kinase was measured by centrifugal ultrafiltration. APS did not bind to the free enzyme with a measurable affinity even at low ionic strength where substrate inhibition by APS is quite marked. However, APS bound with an apparent Kd of 0.54 microM in the presence of 5 mM MgADP. In the presence of 0.1 M (NH4)2SO4, Kd,app was increased to 2.1 +/- 0.7 microM. Bound [35S]APS was displaced by low concentrations of 3'-phosphoadenosine-5'-phosphosulfate (PAPS), or iso-(2') PAPS, or (less efficiently) by adenosine-3,5'-diphosphate (PAP) or adenosine-5'-monosulfate (AMS). The results support our conclusion that substrate inhibition of the fungal enzyme by APS results from the formation of a dead end E. MgADP.APS complex. That is, APS binds to the subsite vacated by PAPS in the compulsory (or predominately) ordered product release sequence (PAPS before MgADP). Radioligand displacement was used to verify the Kd for APS dissociation from E.MgADP.APS and to determine the Kd values for the dissociation of iso-PAPS (13 +/- 5 microM), PAP (4.8 mM), or AMS (5.2 mM) from their respective ternary enzyme.MgADP.ligand complexes. Incubation of the fungal enzyme with [gamma-32P]MgATP did not yield a phosphoenzyme that survives gel filtration or gel electrophoresis.     

Purification and characterization of aquacobalamin reductase (NADPH) from Euglena gracilis

Euglena aquacobalamin reductase (NADPH: EC 1.6.99.-) was purified, and its subcellular distribution was studied to elucidate the mechanism of the conversion of hydroxocobalamin to 5'-deoxyadenosylcobalamin. The enzyme was found in the mitochondria. It was purified about 150-fold over the Euglena mitochondrial extract in a yield of 38%. The purified enzyme was homogeneous in polyacrylamide gel electrophoresis. Spectra of the purified enzyme showed that it was a flavoprotein. The molecular weight of the enzyme was calculated to be 66,000 by Sephadex G-100 gel filtration and 65,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was specific to NADPH with an apparent Km of 43 microM and to hydroxocobalamin with an apparent Km of 55 microM. The enzyme did not require FAD or FMN as a cofactor. The optimum pH and temperature were 7.0 and 40 degrees C, respectively.