Purification and properties of rat kidney catechol-O-methyltransferase]

The S-adenosyl-methionine: catechol-O-methyltransferase (EC 2.1.1.6) from rat kidney was purified about 650 fold as compared with the homogenate and the result of disc electrophoresis presented. The purification involved extraction, precipitation at pH 5, ammonium sulfate fractionation, Chromatographies on Biogel 0.5 m, Ultrogel AcA 44 and DE Sephadex A 50. Affinity chromatography was tried but unsuccessful. The enzyme exhibited two pH optima at 7.9 and 9.6 with a minimum at about 8.9. The COMT had a temperature optimum of 50 degrees C, with activation energy of 23.1 Kcal/Mole between 25-35 degrees C, 18.9 Kcal/mole between 35-45 degrees C and the Q10 within the range of 25-35 degrees amounted to 3.5. The molecular weight was estimated to be 21500+/-1000 daltons from its behavior on Ultrogel AcA 44 and the pH1 determined by electrofocalisation was near 5.50. The time of half life of the best purified enzymatic extract was found to be 2 h 10 min. at -20 degrees C. At basic pH the instability of the enzyme was increased. Since O-methylation required the presence of divalent cations, our results show that apparent Michaelis constants for Mg++ and Mn++ were respectively 0.50 X 10(-3) M and 0.33 X 10(-5) M. The study of their Hill's number indicated that there was only one point of fixation on the enzyme. The Km value determined by Florini and Vestling's method were 2.5 X 10(-4) M and 11.9 X 10(-5) M for epinephrine and S-adenosyl-methionine respectively. All results were discussed with respect to other investigations.     

Studies of soluble rat liver mitochondrial acid ATPases. I. Purification and catalytic properties of ATPase 1.

A method for isolation of a soluble ATPase from rat liver mitochondria after freeze thaw cycling is described. Two enzymatically active fractions were separated by DEAE-cellulose chromatography (ATPase 1 and ATPase 2). ATPase 1 has been purified 300 fold. ATPase 1 was homogenous as judged by polyacrylamide gel electrophoresis. The optimum pH of the enzyme was 5.8-6.0 and the optimum temperature was 45 degrees C. The enzyme follows Michaelis-Menten kinetics: Km (9 X 10(-4) M), Vmax (23,6 mumoles Pi released X min -1 X mg protein -1). The enzyme hydrolysed nucleoside triphosphates, but was inactive upon nucleoside di and monophosphates, glucose 6-phosphate, phosphoserine, pyrophosphate and glycerol 2-phosphate. In contrast to membrane bound ATPase, cations have no effect on the enzyme activity. Nucleoside di and mono phosphates and glycerol 2 phosphate inhibited competitively the enzyme. The enzyme was not affected by oligomycin, but was stimulated by lactate, 2-mercaptoethanol and dithiothreitol.     

Acid phosphatase of the yeast Rhodotorula rubra. Purification and properties of the enzyme.

1. Acid phosphatase from the yeast Rhodotorula rubra was purified 44-fold. The purification procedure involved mechanical disruption of cells, precipitation with ethanol, chromatography on DEAE- and CM-cellulose. 2. The purified enzyme is homogeneous in polyacrylamide gels at pH 4.5, 9.5 and 8.4. Carbohydrate content accounts for 57% of the total weight. The optimum pH is at 4.0-4.6, and the enzyme is stable over pH range from 2.6 to 6.0. Full activity was retained on 60-min incubation at 50 degrees C, but it was reduced by half on 60-min incubation at 65 degrees C. 3. Specificity of the enzyme is fairly broad; monoesters of carbohydrates, and nucleosides and inorganic pyrophosphate can serve as substrates. Km was found to be 1 X 10(-4) M for p-nitrophenyl phosphate as a substrate. The enzyme is inhibited by molybdate, phosphate, arsenate and fluoride ions.     

Adenylate kinase activity in Mycobacterium leprae

Adenylate kinase (ATP:AMP phosphotransferase, EC 2.7.4.3) was detected in partially purified preparations of cell-free extracts of Mycobacterium leprae. The apparent Km values of M. leprae adenylate kinase for ADP and Mg2+ were 1 X 10(-4) M, respectively. The enzyme was heat-labile: loss of activity by 80% at 45 degrees C and over 90% at 60 degrees C occurred within 5 min. M. leprae adenylate kinase was distinct from armadillo adenylate kinase in respect of affinity for substrate and heat-sensitivity.     

Purification and properties of GTP-cyclohydrolase from Bacillus subtilis]

Highly purified GTP-cyclohydrolase was obtained by fractionation of cell extracts with ammonium sulfate, ion-exchange and hydrophobic chromatography. The N-terminal amino acid sequence and amino acid composition of the protein were determined. According to SDS-PAGE data, the molecular weight of the enzyme is 45 kDa. The active enzyme has several isoforms separable by native electrophoresis. The maximal enzyme activity is determined at 1.5 mM Mn2+; 70% of enzymatic activity is detected with Mg2+. The enzyme is inhibited by heavy metal ions and chelators and is inactive in the absence of thiol-reducing agents. The enzyme activity is detected in a broad range of pH with a maximum at pH 8.2. The pyrimidine product of the GTP-cyclohydrolase reaction. 2.5-diamino-6-hydroxy-4-ribosylaminopyrimidine-5'-phosphate was purified and identified. Another product of this reaction is pyrophosphate.     

Purification and properties of the Pichia guilliermondii acid nucleotide pyrophosphatase hydrolyzing flavin adeninine dinucleotide

Acid nucleotide pyrophosphatase was isolated from the cell-free extracts of Pichia guilliermondii Wickerham ATCC 9058. The enzyme was 25-fold purified by saturation with ammonium sulphate, gel-filtration on Sephadex G-150 column and ion-exchange chromatography on DEAE-Sephadex A-50 column. The pH optimum was 5.9, temperature optimum--45 degrees C. The enzyme catalyzed the hydrolysis of FAD, NAD+ and NADH, displaying the highest activity with NAD+. The Km, values for FAD, NAD+ and NADH were 1.3 x 10(-5) and 2.9 x 10(-4) M, respectively. The hydrolysis of FAD was inhibited by AMP, ATP, GTP, NAD+ and NADP+. The K1 for AMP was 6.6 x 10(-5) M, for ATP--2.0 X 10(-5) M, for GTP--2.3 X 10(-6) M, for NAD+--1.7 X 10(-4) M. The molecular weight of the enzyme was 136 000 as estimated by gel-filtration on Sephadex G-150 and 142 000 as estimated by thin-layer gel-filtration chromatography on Sephadex G-200 (superfine). Protein-bound FAD of glucose oxidase was not hydrolyzed by acid nucleotide pyrophosphatase. The enzyme was stable at 2 degrees C in 0.05 M tris-maleate buffer, pH 6.2. Alkaline nucleotide pyrophosphatase hydrolyzing FAD was also detected in the cells of P. guilliermondii.     

Purification and characterization of heparinase from Flavobacterium heparinum

Heparinase (EC 4.2.2.7) isolated from Flavobacterium heparinum was purified to homogeneity by a combination of hydroxylapatite chromatography, repeated gel filtration chromatography, and chromatofocusing. Homogeneity was established by the presence of a single band on both sodium dodecyl sulfate and acid-urea gel electrophoretic systems. Amino acid analysis shows that the enzyme contains relatively high amounts of lysine residues (9%) consistent with its cationic nature (pI 8.5) but contains only 4 cysteine residues/polypeptide. The molecular weight of heparinase was estimated to be 42,900 +/- 1,000 daltons by gel filtration and 42,700 +/- 1,200 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is very specific, acting only on heparin and heparan monosulfate out of 12 similar polysaccharide substrates tested. It has an activity maximum at pH 6.5 and 0.1 M NaCl and a stability maximum at pH 7.0 and 0.15 M NaCl. The Arrhenius activation energy was found to be 6.3 kcal/mol. However, the enzyme is very sensitive to thermal denaturation and loses activity very rapidly at temperatures over 40 degrees C. Kinetic studies of the heparinase reaction at 37 degrees C gave a Km of 8.04 X 10(-6) M and a Vm of 9.85 X 10(-5) M/min at a protein concentration of 0.5 microgram/ml. By adapting batch procedures of hydroxylapatite and QAE (quaternary aminoethyl)-Sephadex chromatography, gram quantities of heparinase that is nearly free of catalytic enzyme contaminants can be purified in 4-5 h.     

Purification and molecular identification of two protein methylases I from calf brain. Myelin basic protein- and histone-specific enzyme

Two different molecular species of protein methylases I (S-adenosylmethionine:protein-arginine N-methyltransferase, EC 2.1.1.23), one specific for myelin basic protein (MBP) and the other for histone, have been purified from calf brain to near homogeneity, as discerned by nondenaturing polyacrylamide gel electrophoresis. Although both methylases share some common properties, such as utilization of S-adenosyl-L-methionine as the methyl donor and methylation of protein-bound arginine residues, they are distinctly different from each other in molecular weight and in catalytic, as well as the immunological, properties. The MBP-specific protein methylase I (approximately 500 kDa) methylates MBP preferentially (Km = 2 X 10(-7) M) and histone to a much lesser extent (Km = 1 X 10(-4) M), while the histone-specific methylase I (approximately 275 kDa) methylates histone only. Both methylases exhibit two major subunit bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis: 100 and 72 kDa for the MBP-specific and 110 and 75 kDa for the histone-specific. At 0.5 mM p-chloromercuribenzoate, about 50% of the MBP-specific enzyme remained as active, while most of the histone-specific enzyme activity was lost. In 2 mM guanidine HCl, approximately 90% of the former enzyme activity remained while nearly complete inactivation of the latter enzyme was observed. The enzymes also exhibited quite different inactivation profiles toward high temperature (45-65 degrees C); MBP-enzyme was stable up to 50 degrees C and was rapidly inactivated at higher temperatures with an inflection point at about 57 degrees C. However, under the identical conditions, histone-enzyme was inactivated progressively and linearly in the same temperature range. Finally, Western immunoblot analysis of polyclonal antibodies directed against either enzyme exhibited no cross-reactivity with the other.     

Characterization of DNA kinase from calf thymus

DNA kinase has been purified to homogeneity from calf thymus. The purified enzyme, with a specific activity of 16.7 units/mg protein at 25 degrees C, exhibited a sharp pH/activity curve with a pH optimum at 5.5 and low activity at alkaline pH. The molecular weight of the enzyme was estimated by dodecylsulfate/polyacrylamide gel electrophoresis to be 5.4 X 10(4). The enzyme has a sedimentation coefficient of 4.0 S. An apparent molecular weight of 5.6 X 10(4) and a Stokes' radius of 3.3 nm were estimated by gel-filtration on Sephadex G-100. The enzyme phosphorylates neither yeast RNA nor poly(A) instead of DNA. Compared with rat liver DNA kinase, calf thymus DNA kinase is relatively resistant to the inhibition by sulfate (Ki = 7 mM) and pyrophosphate (Ki = 5 mM). The enzyme activity is markedly stimulated by polyamines at the sub-optimal concentration of Mg2+ but not by monovalent cations.     

Mechanism of action of isopentenyl pyrophosphate isomerase: evidence for a carbonium ion intermediate

Isopentenyl pyrophosphate isomerase catalyzes the interconversion of isopentenyl pyrophosphate and dimethylallyl pyrophosphate. The isomerase from yeast has been purified to near homogeneity (purity greater than 90%). The substrate analogue (Z)-3-(trifluoromethyl)-2-butenyl pyrophosphate reacts at less than 1.8 X 10(-6) times the rate of dimethylallyl pyrophosphate. The enzyme is irreversibly inactivated by 2-(dimethyl-amino)ethyl pyrophosphate (I). These observations are consistent with a carbonium ion mechanism for the isomerization. Compound I is an analogue of the intermediate carbonium ion and probably acts as a transition state analogue. For I, kon' = 2.1 X 10(6) M-1 min-1. No off-rate was detected and, therefore, Ki less than 1.4 X 10(-11) M. Upon denaturation of the inactivated enzyme, I is released unchanged. 2-(Trimethylammonio)ethyl pyrophosphate also inhibits with Ki' = 7 X 10(-7) M, kon' = 4.4 X 10(4) M-1 min-1, and koff = 0.03 min-1. Substrate analogues without a positively charged nitrogen were relatively poor inhibitors. The best inhibitor of these is ethyl pyrophosphate, Ki = 10(-4) M. The enzyme is inactivated by sulfhydryl-selective reagents. These reagents also prevent binding of I to the enzyme. The inactivation by iodoacetamide is dependent upon one ionizable group (pK = 9.3). The pH dependence of V and V/K for the isomerase-catalyzed reaction also depends upon a group with pK = 9.3.     

Purification and various properties of pantothenate kinase from the rat liver

Homogeneous (according to disc gel electrophoresis data) ATP: D-pantothenate-4'-phosphotransferase (pantothenate kinase, EC 2.7.1.33) was obtained from rat liver cytosol of heterogeneous stock rats. The enzyme was purified 199-fold with a 9.3% yield. The enzyme was relatively unstable but retained its activity in the presence of 10% glycerol containing 5.10(-4) M ATP over 10 days at 4 degrees C. The pH optimum was 6.5; the apparent Km values were equal to 1.2 X 10(-5) M and 1.4 X 10(-3) M for pantothenate and ATP, respectively, at the ATP/Mg2+ ratio of 1. Pantetheine produced a competitive inhibition of pantothenate kinase. Pantethine or pantetheine disulfide did not inhibit the enzyme.     

Purfication and properties of a specific isoflavone 7-O-glucoside-6'-malonate malonyestrase from roots of chickpea (Cicer arietinum L.)

Protein extracts from roots of chickpea (Cicer arietinum L.) plants contained high esterase activity hydrolyzing malonate hemiesters of isoflavone 7-O-glucosides. Using 5,7-dihydroxy-4'-methoxyisoflavone (biochanin A) 7-O-glucoside-6"-malonate as a substrate, a specific malonylesterase was purified about 700-fold to near homogeneity. The purified enzyme possesses an extremely low enzyme activity with synthetic esterase substrates. Various putative nonspecific esterases, as tested with alpha-naphthylacetate, were removed during enzyme purification. The malonylesterase demonstrated a very high molecular mass in gel chromatography and in sedimentation analyses with sucrose gradients (greater than or equal to 2 X 10(6)). Analytical sodium dodecyl sulfate-polyacrylamide gel electrophoresis pointed to a single subunit of 32,000. The catalyzed reaction showed a pH optimum at 7.5 and a temperature optimum between 30 and 35 degrees C. The apparent Km for biochanin A 7-O-glucoside-6"-malonate was (4.2 +/- 1.2) X 10(-4) M. The malonylesterase was insensitive to the esterase inhibitors eserine and neostigmine (10(-3) M) as well as phenylmethylsulfonyl fluoride, paraoxon, and diisopropylfluorophosphate (10(-4) M). On the other hand enzyme activity was totally inhibited by Hg2+ ions (10(-5) M) and p-hydroxymercuribenzoate (10(-4) M), whereas iodoacetamide (10(-6)-10(-4) M) inhibited only partially. Di- and tricarboxylic acids strongly stimulated enzyme activity at 10(-2) M. These properties indicate that the malonylesterase from chickpea roots greatly differs from other known esterases. The possible biological function of the specific malonylesterase is discussed in relation to isoflavone conjugate metabolism in chickpea.     

Isolation and characterization of human liver guanine deaminase

Guanine deaminase (EC 3.5.4.3, guanine aminohydrolase [GAH]) was purified 3248-fold from human liver to homogeneity with a specific activity of 21.5. A combination of ammonium sulfate fractionation, and DEAE-cellulose, hydroxylapatite, and affinity chromatography with guanine triphosphate ligand were used to purify the enzyme. The enzyme was a dimer protein of a molecular weight of 120,000 with each subunit of 59,000 as determined by gel filtration and sodium dodecyl sulfate-gel electrophoresis. Isoelectric focusing gave a pI of 4.76. It was found to be an acidic protein, as evidenced by the amino acid analysis, enriched with glutamate, aspartate, alanine and glycine. It showed a sharp pH optimum of 8.0. The apparent Km for guanine was determined to be 1.53 X 10(-5) M at pH 6.0 and 2 X 10(-4) M for 8-azaguanine as a substrate at pH 6.0. The enzyme was found to be sensitive to p-hydroxymercuribenzoate inhibition with a Ki of 1.53 X 10(-5) M and a Ki of 5 X 10(-5) M with 5-aminoimidazole-4-carboxamide as an inhibitor. The inhibition with iodoacetic acid showed only a 7% loss in the activity at 1 X 10(-4) M and a 24% loss at 1 X 10(-3) M after 30 min of incubation, whereas p-hydroxymercuribenzoate incubation for 30 min resulted in a 91% loss of activity at a concentration of 1 X 10(-4) M. Guanine was the substrate for all of the inhibition studies. The enzyme was observed to be stable up to 40 degrees C, with a loss of almost all activity at 65 degrees C with 30 min incubation. Two pKa values were obtained at 5.85 and 8.0. Analysis of the N-terminal amino acid proved to be valine while the C-terminal residue was identified as alanine.     

Purification and properties of beta-mannosyltransferase that synthesizes Man-beta-GlcNAc-GlcNAc-pyrophosphoryl-dolichol

The beta-mannosyltransferase that adds mannose, from GDP-mannose, to GlcNAc-GlcNAc-pyrophosphoryl-dolichol, to form Man-beta-GlcNAc-GlcNAc-pyrophosphoryl-dolichol was solubilized from pig aorta microsomal preparations, using 0.5% NP-40, and was purified about 116-fold using conventional methods. The purified enzyme was mostly free of alpha 1,3- or alpha 1,6-mannosyltransferase activities, since Man beta-GlcNAc-GlcNAc-PP-dolichol (PP = pyrophosphoryl) accounted for more than 95% of the product when enzyme was incubated with GDP-[14C]mannose and GlcNAc-GlcNAc-PP-dolichol. Very little Man-beta-GlcNAc-GlcNAc-PP-dolichol was formed when GDP-[14C]mannose was replaced by dolichol-phosphoryl-[14C]mannose, indicating that GDP-mannose was the mannosyl donor. The oligosaccharide portion of this lipid was released by mild acid hydrolysis and was characterized by gel filtration as well as by susceptibility to beta-mannosidase and resistance to alpha-mannosidase. The partially purified enzyme could be stabilized by the addition of 20% glycerol and 0.5 mM dithiothreitol to the buffer, and could be kept in this solution for 5 or 6 days in ice. The enzyme was greatly stimulated by the addition of detergent (NP-40) with optimum activity being observed at 0.1%. However, no stimulation was seen with any phospholipid. The partially purified enzyme had a pH optimum of about 7.0, and showed an almost absolute requirement for Mg2+ with optimal activity occurring at about 5 mM Mg2+. Mn2+ and Ca2+ were only slightly active. The Km for GDP-mannose was about 5 X 10(-7) M and that for GlcNAc-GlcNAc-PP-dolichol about 1 X 10(-6) M. Beta-Mannosyltransferase activity was inhibited competitively by a variety of guanosine nucleotides with GDP and GDP-glucose being most active, but GTP, GMP, guanosine, and periodate-oxidized guanosine were also effective. The enzyme was strongly inhibited by p-chloromercuribenzenesulfonic acid and this inhibition was partially prevented by the addition of dithiothreitol.     

Escherichia coli alpha-ketoglutarate dehydrogenase complex

The alpha-ketoglutarate dehydrogenase complex from Escherichia coli catalyzes the hydrolysis of S-succinyl-CoA to succinate and CoASH. The reaction rate is dependent upon the presence of thiamin pyrophosphate and NADH, as well as the functional integrity of the alpha-lipoyl groups associated with the enzyme. The Km value for S-succinyl-CoA is 9.3 X 10(-5) M, and the maximum velocity is 0.02 mumol X min-1 X mg of protein-1 at pH 7 and 25 degrees C. This hydrolysis can be rationalized on the basis that succinyl thiamin pyrophosphate is generated under reductive succinylation conditions. Occasional diversion of succinyl thiamin pyrophosphate to hydrolysis produces succinate.     

Thermococcus profundus 2-ketoisovalerate ferredoxin oxidoreductase, a key enzyme in the archaeal energy-producing amino acid metabolic pathway

2-Ketoisovalerate ferredoxin oxidoreductase (VOR) is a key enzyme in hyperthermophiles catalyzing the coenzyme A-dependent oxidative decarboxylation of mainly aliphatic amino acid-derived 2-keto acids. The very oxygen-labile enzyme purified under anaerobic conditions from a hyperthermophilic archaeon, Thermococcus profundus, is a hetero-octamer (alphabetagammadelta)(2) consisting of four different subunits, alpha = 45,000, beta = 31,000, gamma = 22,000 and delta = 13,000, respectively. Electron paramagnetic resonance and resonance Raman spectra of the purified enzyme indicate the presence of approximately three [4Fe-4S] clusters per alphabetagammadelta-protomer, although one of the clusters has a tendency to be converted to a [3Fe-4S] form during purification. The optimal temperature for the enzyme activity is 93 +/- 2 degrees C and the cognate [4Fe-4S] ferredoxin serves as an electron acceptor of the enzyme. The purified enzyme is highly oxygen-labile (t(1/2), approximately 5 min at 25 degrees C), and is partly protected in the presence of magnesium ions, thiamine pyrophosphate and sodium chloride (t(1/2), approximately 25 min at 25 degrees C).     

Properties and function of malate enzyme from Pseudomonas putida

Malate enzyme (L-malate: NADP+ oxidoreductase (oxalacetate-decarboxylating, EC 1.1.1.40)) has been purified from Pseudomonas putida to 99 per cent homogeneity by heat, ammonium suphate fractionation, gel filtration and anion exchange chromatography. Sodium dodecylsulphate-(SDS)-polyacrylamide disc gel electrophoresis analysis showed an approximate tetrameric subunit with a molecular weight of 52,000. The purified enzyme showed a pH optimum between 8.0 and 8.5 (for Tris-HCl buffer) and required bivalent cations for catalysis; monovalent ions like K+ and NH4+ acted as very effective activators. The temperature-activity relationship for the malate enzyme from 35-80 degrees C showed broken Arrhenius plots with an inflexion at 65 degrees C. The enzyme halflife was 30s at 85 degrees C. The enzyme showed hyperbolic kinetics for both substrates with apparent Km values of 4.0 X 10(-4) M and 2.3 X 10(-5) M for L-malate and NADP+ respectively. From the study of the effects of some compounds on the enzyme, the physiological significance of those produced by fumarate, succinate and oxalacetate can be emphasized.     

Free energy of hydrolysis of tyrosyl adenylate and its binding to wild-type and engineered mutant tyrosyl-tRNA synthetases

The equilibrium constant for the formation of tyrosyl adenylate and pyrophosphate from ATP and tyrosine in solution has been measured by applying the Haldane relationship to wild-type and three mutant tyrosyl-tRNA synthetases from Bacillus stearothermophilus. The formation constant (=[Tyr-AMP] [PPi]/[ATP] [Tyr]) at pH 7.78, 25 degrees C, and 10 mM MgCl2 is (3.5 +/- 0.5) X 10(-7). This corresponds to a free energy of hydrolysis of tyrosyl adenylate at pH 7.0 and 25 degrees C of -16.7 kcal mol-1. All necessary rate constants had been determined previously for the calculations apart from the dissociation constant of tyrosyl adenylate from its enzyme-bound complex. This was measured by taking advantage of the 100-fold difference in hydrolysis rates of the tyrosyl adenylate when sequestered by the enzyme and when free in solution. These are technically difficult measurements because the dissociation constants are so low and the complexes unstable. The task was simplified by using mutants prepared by site-directed mutagenesis. These were designed to have different rate and equilibrium constants for dissociation of tyrosyl adenylate from the enzyme-bound complexes. The dissociation constants were in the range (3.5-38) X 10(-12) M, with that for wild type at 13 X 10(-12) M. The four enzymes all gave consistent data for the formation constant of tyrosyl adenylate in solution. This not only improves the reliability of the measurement but also provides confirmation of the reliability of the measured kinetic constants for the series of enzymes.     

Specificity and kinetics defining the interaction between a murine monoclonal autoantibody and DNA

Interactions between a murine monoclonal anti-DNA autoantibody (BV17-45) and DNA were examined by direct binding and competitive radioimmunoassays. Binding isotherms constructed by titration of purified BV17-45 with a series of distinct 32P-labeled double-stranded DNA ([32P]dsDNA) fragments were super-impossible, suggesting: 1) BV17-45/[32P]dsDNA binding is independent of dsDNA size using fragments greater than or equal to 192 base pairs in length, and 2) BV17-45 does not exhibit stringent sequence specificity. Single-stranded DNA-specific monoclonal antibody BV04-01 did not react with [32P]dsDNA, confirming its duplex character. In competition experiments, BV17-45 cross-reacted with phage (phi X174, M13) RF AND VIRION DNAS AT PICOMOLAR concentrations. Selectivity for B-form DNA was suggested by the ability of poly(dA) . poly(dT), but not other helical duplex forms, to block BV17-45/[32P] dsDNA binding. Among the four deoxyribohomopolymers, only deoxyadenylic acid polymers completely inhibited BV17-45/[32P]dsDNA complex formation. [32P]dsDNA binding was relatively insensitive to ionic strength, suggesting minimal contribution of electrostatic forces to the binding free energy. Measured BV17-45/[32P]dsDNA association and dissociation rate constants (4 degrees C) were 7.4 X 10(6) M-1 s-1 and 9.2 X 10(-5) s-1, respectively, yielding a functional affinity of 8 X 10(10) M-1. Results are discussed in terms of the relative contribution of B-DNA structural and substructural determinants to the mechanism of BV17-45 recognition.     

Anacystis nidulans mutants resistant to aromatic amino acid analogues.

Three classes of mutants of Anacystis nidulans were selected on the basis of resistance to fluorophenylalanine and 2-amino-3-phenylbutanoic acid. The most frequent type exhibited DAHP synthetase (7-phospho-2-keto-3-deoxy-D-arabino-heptonate-D-erythrose-4-phosphate-lyase [pyruvate phosphorylating], EC 4.1.2.15) activity identical to that of the parental strain. The second type was characterized by extremely low levels of the activity. The third type had a DAHP synthetase showing decreased sensitivity to inhibition by L-tyrosine. The enzyme was purified 140-fold from wild-type and feedback-insensitive strains, and the kinetics of the reaction was examined. The activity of the wild-type enzyme was inhibited 75% in the presence of 2.0 X 10-3 M tyrosine, and the altered enzyme was inhibited 10%. The following apparent constants were obtained from kinetic studies with partially purified wild-type enzyme: S0.5 for D-erythrose-4-phophate equal to 7.1 X 10-4 M; S0.5 for phosphoenolpyruvate equal to 1.4 X 10-4 M. Inhibition by tyrosine was mixed with respect to binding of both D-erythrose-4-phosphate and phosphoenolpyruvate. In addition, tyrosine promoted cooperative interactions in the binding of phosphoenolpyruvate. For the altered enzyme the following apparent constants were obtained: S0.5 for D-erythrose-4-phosphate equal to 7.1 X 10-4 M; S0.5 for phosphoenolpyruvate equal to 2.9 X 10-4 M. Inhibition by tyrosine was mixed with respect to D-erythrose-4-phosphate and competitive with respect to phosphoenolpyruvate. Tyrosine did not promote cooperative effects in the binding of phosphoenolpyruvate to the altered enzyme.