Reduction In Specific Activity Research Articles

Ethylene regulation of apoplastic invertase expression in autotrophic cells of Chenopodium rubrum

Physiological concentrations of ethylene in the growth medium of autotropic suspension culture Chenopodium rubrum L. cells reduced the activity of cell wall bound invertase by 25 – 47%, compared to controls. Northern blot analysis using homologous probe binding to total RNA preparations revealed that reduction in specific activity was paralleled by repression of the corresponding gene.

Functional Organization of Mammalian Hexokinases: Both N- and C-Terminal Halves of the Rat Type II Isozyme Possess Catalytic Sites

Previous work has shown that catalytic function is associated exclusively with the C-terminal half of the Type I isozyme of mammalian hexokinase. In contrast, we now demonstrate thatbothhalves of the Type II isozyme possess comparable catalytic activities. Mutation of a catalytically important Ser residue to Ala at analogous positions in either the N- or the C-terminal halves (S155A or S603A, respectively) of the rat Type II isozyme resulted in approximately 60% reduction in specific activity of the enzyme, with more than 90% reduction in the doubly mutated enzyme (S155A/S603A). Catalytic activity was retained in a chimeric hexokinase comprising the N-terminal half of Type II hexokinase and catalytically inactive (by site-directed mutation) C-terminal half of the Type I isozyme. The N- and C-terminal catalytic sites of Type II hexokinase are similar inVmaxandKm(≈130 μM) for glucose; however, the N-terminal site has a lower (0.45 vs 1.1 mM)Kmfor ATP, is slightly more sensitive to inhibition by the product analog 1,5-anhydroglucitol-6-P, and is much more sensitive to inhibition by Pi. It is suggested that the Type II isozyme most closely resembles the 100-kDa hexokinase which resulted from duplication and fusion of a gene encoding an ancestral 50-kDa hexokinase and which was the precursor for the contemporary Type I, Type II, and Type III mammalian isozymes. Subsequent evolutionary changes could then have led to functional differentiation of the N- and C-terminal halves, as seen with the Type I (and possibly the Type III) isozyme.

Characterization of the celB gene coding for beta-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus and its expression and site-directed mutation in Escherichia coli

The celB gene encoding the cellobiose-hydrolyzing enzyme beta-glucosidase from the hyperthermophilic archaeon Pyrococcus furiosus has been identified, cloned, and sequenced. The transcription and translation gene was overexpressed in Escherichia coli, resulting in high-level (up to 20% of total protein) production of beta-glucosidase that could be purified by a two-step purification procedure. The beta-glucosidase produced by E. coli had kinetic and stability properties similar to those of the beta-glucosidase purified from P. furiosus. The deduced amino acid sequence of CelB showed high similarity with those of beta-glycosidases that belong to glycosyl hydrolase family 1, implicating a conserved structure. Replacement of the conserved glutamate 372 in the P. furiosus beta-glucosidase by an aspartate or a glutamine led to a high reduction in specific activity (200- or 1,000-fold, respectively), indicating that this residue is the active site nucleophile involved in catalysis above 100 degrees C.

Local and Systemic Biosynthesis of Salicylic Acid in Infected Cucumber Plants.

Radiolabeling studies showed that salicylic acid (SA), an essential component in the signal transduction pathway leading to systemic acquired resistance, is synthesized from phenylalanine (Phe) and benzoic acid in cucumber (Cucumis sativus L.) plants inoculated with pathogens. Leaf discs from plants inoculated with either tobacco necrosis virus or Pseudomonas lachrymans incorporated more [14C]Phe into [14C]SA than mock-inoculated controls. The identity of SA was confirmed by gas chromatography-mass spectrometry. No reduction in specific activity of [14C]SA was observed for either free or bound SA between control and infected plants after feeding [14C]Phe. A specific inhibitor of Phe ammonia-lyase, 2-aminoindan-2-phosphonic acid, completely inhibited the incorporation of [14C]Phe into [14C]SA, although plants treated with 2-aminoindan-2-phosphonic acid could still produce [14C]SA from [14C]benzoic acid. Biosynthesis of SA in tissue inoculated with tobacco necrosis virus followed a transient pattern with the highest induction occurring 72 h postinoculation. Uninfected tissues from an infected plant synthesized de novo more SA than did controls. This suggests the involvement of a systemic signal triggering SA synthesis in tissue distant from the site of infection that display systemic acquired resistance.

Structural and Functional Roles of Cysteine 90 and Cysteine 240 in S-Adenosylmethionine Synthetase

Site-specific mutagenesis was performed on the structural gene for Escherichia coli S-adenosylmethionine (AdoMet) synthetase to introduce mutations at cysteines 90 and 240, residues previously implicated by chemical modification studies to be catalytically and/or structurally important. The AdoMet synthetase mutants (i.e. MetK/C90A, MetK/C90S, and MetK/C240A) retained up to approximately 10% of wild type activity, demonstrating that neither sulfhydryl is required for catalytic activity. Mutations at Cys-90 produced a mixture of noninterconverting dimeric and tetrameric proteins, suggesting a structural significance for Cys-90. Dimeric Cys-90 mutants retained approximately 1% of wild type activity, indicating a structural influence on enzyme activity. Both dimeric and tetrameric MetK/C90A had up to a approximately 70-fold increase in Km for ATP, while both dimeric and tetrameric MetK/C90S had Km values for ATP similar to the wild type enzyme, suggesting a linkage between Cys-90 and the ATP binding site. MetK/C240A was isolated solely as a tetramer and differed from wild type enzyme only in its 10-fold reduction in specific activity, suggesting that the mutation affects the rate-limiting step of the reaction, which for the wild type enzyme is the joining of ATP and L-methionine to yield AdoMet and tripolyphosphate. Remarkably all of the mutants are much more thermally stable than the wild type enzyme.

Physicochemical characterization of the cytoplasmic domain of the epidermal growth factor receptor and evidence for conformational changes associated with its activation by ammonium sulphate

The physiochemical properties of the purified cytoplasmic domain of the epidermal growth factor (EGF) receptor, its self-phosphorylation and peptide phosphorylation activities, and its activation by ammonium sulphate have been studied. Highly efficient purification procedures for the isolation of the recombinant cytoplasmic domain (Met644-Ala1186) of the EGF receptor, expressed in the baculovirus/insect cell system, are described. Physicochemical characterization of the protein included investigation of its isoelectric and hydrodynamic properties, stability, oligomeric status, and secondary structure using far-u.v. circular dichroism. The recombinant protein was not recognized by anti-phosphotyrosine antibodies, unless first self-phosphorylated in vitro. Tryptic phosphopeptide maps of self-phosphorylated recombinant cytoplasmic domain and the EGF-stimulated A431-membrane receptor were very similar, suggesting that the recombinant had similar self-phosphorylation capacity and specificity. The preparations were characterized by high specific activity towards peptide tyrosine phosphorylation. Although the cytoplasmic domain was isolated as a homogeneously monomeric protein, storage at 4 degrees C led to slow, spontaneous aggregation with reduction in specific activity. Both high activity and monomeric state were maintained by storage below 0 degree C. The dependence of the initial rate of self-phosphorylation on protein concentration was consistent with cross-phosphorylation but not with the known oligomerization-induced activation of holoreceptor. The peptide phosphorylation activity was stimulated by Mn2+, Mg2+ and (NH4)2SO4 at high concentrations. The substrate specificity of (NH4)2SO4 activation was studied using synthetic peptides. Self-phosphorylation was inhibited by (NH4)2SO4 in the range 0-0.25 M but activated at 1.0-1.5 M, possibly as a result of ionic and hydrophobic protein interactions respectively. Phosphopeptide maps of cytoplasmic domain phosphorylated in the presence of high (NH4)2SO4 showed that the protein was more extensively phosphorylated than in the absence of salt, or than the native receptor. Far-u.v. circular-dichroism spectra of the cytoplasmic domain changed dramatically at 1 M (NH4)2SO4, raising the possibility that (NH4)2SO4 activates the kinase catalytic domain by inducing conformational changes.

A Carboxyl Terminal Leucine Zipper Is Required for Tyrosine Hydroxylase Tetramer Formation

Tyrosine hydroxylase catalyzes the rate-limiting reaction in the biosynthesis of the catecholamine neurotransmitters and hormones (dopamine, norepinephrine, and epinephrine). Rat tyrosine hydroxylase exists, in its native form, as a tetramer composed of identical 498 amino acid subunits. There is currently no information describing the molecular interactions by which the four monomeric tyrosine hydroxylase subunits assemble into an active tetramer. Mutational analysis was performed on bacterially expressed enzyme to assess the role of a putative C-terminal leucine zipper in the assembly of subunits into the tetrameric holoenzyme. Deletion of the C-terminal 19 amino acids, or mutation of a leucine residue (to an alanine), converts the enzyme from a tetrameric to a dimeric form that exhibits greater structural heterogeneity. This change in macromolecular form is accompanied by a 75% (deletion mutation) to 20% (Leu-->Ala mutation) reduction in specific activity of the enzyme. This represents the first report of the functional involvement of a region containing a leucine zipper motif in the assembly and activity of a neuronal enzyme.

Evaluation of catalytic activity and synergism between two xylanase isoenzymes in enzymic hydrolysis of two separate xylans in different states of solubility

Synergism of two xylanase (1,4) β- d-xylan xylanohydrolase, EC 3.2.1.8) isoenzymes from Streptomyces A451 was observed in enzymic hydrolysis of water-soluble and -insoluble fractions of two commercial xylan preparations. The reaction modes were determined qualitatively by TLC and quantitatively by GLC. With oat-spelt xylan as substrate, the total amount of xylose and xylobiose produced by using xylanase I and xylanase II in sequence was significantly higher than that by xylanase I or xylanase II individually, while the amounts of the relatively higher-molecular-weight oligosaccharides xylotriose to xylopentaose obtained by using isoxylanases individually were more than those obtained by using isoxylanases in sequence. A time-course study on the release of hydrolysis products over a 24-h period revealed that higher oligosaccharides were released initially followed by release of simpler sugars. A study of the hydrolysis of model oligosaccharides by crude enzyme preparation revealed a preference for higher oligosaccharides and demonstrated that xylopentaose was hydrolyzed to xylose, xylobiose, and xylotriose. A comparison was made of the hydrolysis of two different xylans, birchwood (hardwood) and oat-spelt (softwood), by crude xylanase. Product profiles showed that there were significant differences between the range and amounts of products released from the two different xylans. Studies on relative specific activities showed that xylanase II had overall lower specific activity toward various xylans than did xylanase I, and xylanase I, xylanase II, and crude xylanase showed lower specific activity toward insoluble xylan fractions than toward soluble xylan fractions. In addition, the reduction in specific activity was greater for insoluble birchwood than for insoluble oat-spelt xylan.

High-level expression and deletion mutagenesis of human tryptophan hydroxylase.

Human tryptophan hydroxylase has been expressed as a soluble and active form in Escherichia coli by fusion with an affinity tag, maltose-binding protein. The fusion protein has been purified to near homogeneity by affinity chromatography on crosslinked amylose resin. The purified fusion protein has a specific activity of 86 nmol of 5-hydroxytryptophan per min per mg of fusion protein. A series of truncation mutants have also been made to explore the domain organization of tryptophan hydroxylase. All deletion mutants were subject to affinity purification and kinetic characterization. While removal of the N-terminal 164 amino acids completely inactivates the enzyme, deletion of the first 91 residues results in a 7-fold reduction in specific activity. From the C terminus, deletion of 36, 55, or 112 amino acids abolishes the activity, whereas deletion of 19 residues decreases the specific activity by approximately 11-fold. These results are consistent with a model for tryptophan hydroxylase in which the enzyme consists of an N-terminal regulatory domain, a catalytic core, and a small C-terminal region of uncertain but important function.

Alanine scanning mutagenesis of human erythropoietin identifies four amino acids which are critical for biological activity.

Erythropoietin regulates the growth and proliferation of red blood cell progenitors. We demonstrated previously the important structural and functional roles of the amino acid region 99-110 in the biological activity of the hormone. [Chern, Y., Chung, T. & Sytkowski, A. J. (1991) Eur. J. Biochem. 202, 225-229]. We have now performed alanine scanning mutagenesis to identify which specific residues in this region are essential for function. Four substitutions reduced the biological activity of erythropoietin. Alanine substitution of Ser104, Leu105 and Leu108 reduced activity to 16, 44, and 37% that of the wild-type protein, respectively. Most significantly, substitution of Arg103 with Ala reduced activity to undetectable levels, indicating a > 230-fold reduction in specific activity. Immunochemical analyses using anti-peptide monoclonal antibodies demonstrated that this substitution does not cause major changes in the conformation of the protein or large, localized distortions within the amino acid region 99-129 of the molecule. Heat-denaturation kinetics for the arginine mutant and wild-type erythropoietin are virtually identical, further indicating the structural similarity between these two molecules. Based upon these findings, we propose that Arg103 plays an essential role in the biological activity of erythropoietin, presumably by interacting directly with the erythropoietin receptor.

A tyrosine residue is involved in the allosteric binding of tryptophan to yeast chorismate mutase

Comparative 1H-NMR studies have been carried out on wild-type chorismate mutase, which is activated by tryptophan and inhibited by tyrosine and phenylalanine, and mutant yeast chorismate mutase, which has a single point mutation (T226I) and is not allosterically regulated but ‘locked’ in the activated state. Double quantum-filtered COSY spectra show cross-peaks which have been assigned to a tyrosine that are absent in the mutant enzyme and in the wild-type enzyme plus tryptophan when compared with the wild-type enzyme alone. These observations indicate the involvement of a tyrosine at or near the allosteric binding site. The involvement of tyrosine in tryptophan binding was tested by modification of tyrosine in yeast chorismate mutase by nitration with tetranitromethane. All forms of the enzyme exhibited an pprox. 50% reduction in specific activity, but it was found that preincubation of the wild-type with the allosteric activator, tryptophan, lead to partial protection against loss in specific activity. Only one tyrosine residue was nitrated in the wild-type enzyme and this tyrosine was identified by tryptic digestion and sequencing, and found to be very close to the site of the single point mutation in the mutant enzyme. It is proposed that Tyr-234 is located at or near the allosteric activation site.

The nitrogenase proteins of Rhizobium meliloti: purification and properties of the MoFe and Fe components.

The alfalfa-Rhizobium meliloti symbiosis contributes a major portion of biologically fixed nitrogen to temperate zone forage crop production. Highly-purified molybdenum-iron (MoFe) and iron (Fe) nitrogenase components were obtained for the first time from extracts of R. meliloti bacteroids. Intact bacteroid cells were isolated anaerobically from 100 g quantities of alfalfa nodules following storage in liquid nitrogen. Centrifuged bacteroid extracts showed a marked reduction in specific activity when assayed at protein concentrations less than 1 mg/ml. Both nitrogenase proteins were resolved and purified to homogeneity as determined spectroscopically and by SDS-PAGE. The purified MoFe protein differed in several respects from previously characterized nitrogenase proteins. Saturation of the acetylene-reducing and proton-reducing activities of the R. meliloti MoFe protein required higher relative concentrations of Fe protein than nitrogenase proteins purified from free living diazotrophs. Electron allocation to dinitrogen reduction was sustained at component ratios similar to those present in bacteroid extracts, suggesting that while the observed saturation effects were not detrimental to physiological function in the symbiotic system, overall activity could be enhanced by higher levels of iron protein. Analyses of the MoFe protein gave 22 Fe, 22 labile sulfide and 1.7 Mo atoms per molecular unit of 215 kDa. Dithionite-reduced MoFe protein contained a spin 3/2 iron centre but had a lower visible absorbance at 360 nm than the equivalent Azotobacter chroococcum component. Amino-acid composition indicated a notably lesser tryptophan content, and cysteine content greater than that of the equivalent tetrameric protein of free living diazotrophs. Ratios of acidic and basic residues were similar to other MoFe proteins. Calculation of hydrophobicity and discriminant parameters gave values midway between those expected for soluble cytoplasmic proteins and peripheral membrane associated proteins. ADP was tightly bound by the dithionite-free MoFe protein containing reduced iron-molybdenum cofactor. The R. meliloti iron protein was found to be a 64 kDa homodimer containing a single 4Fe-4S metal centre.

The influence of tRNA located at the P-site on the turnover of EF-Tu·GTP on ribosomes

The turnover of EF-Tu·GTP on poly-U programmed ribosomes was measured both in the presence and in the absence of N-acetylated Phe-tRNA Phe at the P-site. The reaction was uncoupled fromrotein synthesis by omitting Phe-tRNA Phe at the A-site. In this reaction, the ribosome can be considered as an enzyme catalysing the transition of EF-Tu·GTP to EF-Tu·GDP. A constant EF-Tu·GTP concentration is maintained by regenerating GDP to GTP at the expense of phosphoenolpyruvate by pyruvate kinase. The rate constants are determined using a procedure which corrects for the reduction in specific activity of GTP due to regeneration of the nucleotide. Ribosomes with an occupied P-site are more efficient in stimulating the GTPase of EF-Tu·GTP than ribosomes with an empty P-site. The data suggest that this is mainly caused by an increased affinity of EF-Tu·GTP for ribosomes with a filled P-site rather than by an enhanced reactivity of the GTPase centre.

Identification and mechanism of action of phospholipids capable of modulating rat testicular microsomal 3 beta-hydroxysteroid dehydrogenase-isomerase activity in vitro.

Rat testicular 3 beta-hydroxysteroid dehydrogenase-isomerase (3 beta-HSD-Isomerase) converts pregnenolone to progesterone. The enzyme is localized to the microsomal membranes of testicular homogenates, and treatment of the microsomes with phospholipases causes a reduction in 3 beta-HSD-Isomerase activity. The relationship between the membrane microenvironment and 3 beta-HSD-Isomerase activity was investigated by adding phospholipids of known structure to microsomal incubations and determining the effects on the conversion of pregnenolone to progesterone. Phosphatidylcholines with saturated acyl chains of 8, 10, 12, and 14 carbon atoms, or unsaturated chains, were extremely inhibitory to 3 beta-HSD-Isomerase, causing reductions in specific activity to 10-40% of the control value. Furthermore, the inhibition appeared to be caused primarily by a reduction in the active enzyme concentration (Vmaxapp). Phosphatidylcholines with longer saturated acyl chains were without effect. Phosphatidylserine and phosphatidic acid were activators of 3 beta-HSD-Isomerase. These phospholipids decreased the Kmapp value (to 21% and 43% of control values, respectively), suggesting that the enhancement of 3 beta-HSD-Isomerase activity was through an active-site-oriented effect. Furthermore, for phosphatidic acid to activate 3 beta-HSD-Isomerase, saturated acyl chains of 16 carbon atoms were necessary, other configurations being slightly inhibitory. The remarkable specificity for certain phospholipid configurations and the different effects of these membrane components suggest that androgen biosynthesis may be regulated by changes in the phospholipid microenvironment of 3 beta-HSD-Isomerase.

Mutations in acute intermittent porphyria detected by ELISA measurement of porphobilinogen deaminase.

To study the existence of different mutations in acute intermittent porphyria, erythrocyte porphobilinogen deaminase activity and enzyme protein concentration were investigated in 125 porphyria gene carriers from 31 families, and in 121 apparently healthy controls. Porphobilinogen deaminase concentration (micrograms/gHb) was quantified using a recently developed double-sandwich ELISA. The ratio of enzyme catalytic activity to the concentration of enzyme protein was expressed as the porphobilinogen specific activity (nkat/g). The controls had a mean porphobilinogen deaminase concentration of 160 +/- 35 micrograms/gHb and a specific activity of 762 +/- 127 nkat/g. Two different types of mutation causing acute intermittent porphyria were detected. The majority (91%) of gene carriers, from 25 families, had a diminished porphobilinogen deaminase concentration of 102 +/- 18 micrograms/gHb, with a slightly lowered specific activity of 634 +/- 105 nkat/g. In 9% of the gene carriers, representing six different families, an increase in porphobilinogen deaminase concentration to 269 +/- 46 micrograms/gHb, and a highly significant reduction in specific activity to 234 +/- 48 nkat/g, were found, which indicates the presence of a different mutation.

Protein S deficiency occurs in the nephrotic syndrome.

Protein S activity may be compromised in patients with the nephrotic syndrome and contribute to a thrombotic diathesis. Protein S is found in two forms in plasma as free and functionally active protein S, and complexed to C4b-binding protein. When compared with controls, patients with nephrotic syndrome had reduced functional levels of protein S (69% +/- 27% [SD], p less than 0.001) despite having elevated levels of total protein S antigen (139% +/- 42%, p less than 0.001). Decreased protein S activity was caused by significant reductions in free (active) protein S levels (90% +/- 38%, p less than 0.05) due to the selective urinary loss of free protein S and elevation of C4b-binding protein levels (170% +/- 52%, p less than 0.001) that favors complex formation; and in the specific activity of the circulating free protein S (0.76; p less than 0.001). Along with this reduction in specific activity, we noted the abnormal electrophoretic mobility of the protein S in the presence of calcium ions. We conclude that acquired protein S deficiency occurs in the nephrotic syndrome and may be a risk factor for the development of the thromboembolic complications.

Early phenobarbital-induced alterations in hippocampal acetylcholinesterase activity and behavior

Early exposure to phenobarbital (PhB) causes marked destruction of large neurons which are then forming both in the hippocampus and in the cerebellum. Such exposure to PhB also reduces the achievements of mice in hippocampus-related behaviors such as radial 8-arm maze performance. Experimental evidence suggests that these behaviors are partially mediated by cholinergic transmission. We studied the performance of mice, exposed to PhB prenatally or neonatally, in radial 8-arm maze. Both treatments caused significant impairments in the animals' performance in the maze. Acetylcholinesterase (AChE) and pseudocholinesterase (pChE) activities were studied in the hippocampus and cerebellum of mice who were exposed to PhB prenatally or neonatally. These enzymes are involved both in cholinergic transmission and in neuronal development. A significant decrease (13-16%, P less than 0.01) in hippocampal AChE specific activity was found between days 15 and 22 in animals exposed to PhB neonatally. The total hippocampal activity of AChE was also greatly reduced (25-39%, P less than 0.01) during that period as a result of both the reduction in specific activity and a reduction in hippocampal weight of the treated animals. These alterations were transient and were not detected in adulthood. No changes in hippocampal AChE or pChE activities were found in animals treated prenatally. Cerebellar AChE and pChE activities were not altered after prenatal nor after neonatal exposure to PhB. It is possible that the short-term effect of neonatal treatment on AChE specific activity might mediate the long-term impairments in hippocampus-related behaviors.

Trypsin-modified alkaline phosphatase. Formation of apoenzyme monomer and hybrid dimer.

The cleavage of an amino-terminal decapeptide from Escherichia coli alkaline phosphatase has been previously described (Roberts, C. H., and Chlebowski, J. F. (1984) J. Biol. Chem. 259, 729-733) by this laboratory. The modest reduction in specific activity of the modified enzyme is paralleled by an apparent alteration in the Zn(II) affinity at one of the three active center metal ion binding sites. In contrast to the behavior of the native enzyme, formation of the metal-free apoprotein results in an irreversible loss of catalytic activity; phosphohydrolase activity is not restored on addition of Zn(II) and Mg(II). Differential scanning calorimetry and velocity sedimentation data indicate that the apo form of the modified enzyme exists as a monomer form which, while capable of binding Zn(II) does not readily reassociate to active dimer. Processive cleavage of the amino termini of the dimer by trypsin results in the transient formation of a hybrid dimer consisting of cleaved and uncleaved subunits. This species can be directly observed and isolated by taking advantage of the differential chromatographic mobility of the native "isozymes" and the resulting products. Coupled with improved procedures for the preparation of the modified protein, these data indicate that the amino-terminal modification results in alterations in the subunit interface domain and provides a species (the hybrid dimer) for the investigation of the propagation of these effects.

The effects of pyrimidine nucleotides on alkylating agent induced cytotoxicity and spontaneous and induced mutation to purine analog resistance in V79 cells.

Exposure of three V79 cell lines to dT after treatment with monofunctional alkylating agents resulted in potentiation of alkylation induced cytotoxicity. The degree of potentiation achieved was dependent on the concentration and duration of exposure to dT and was reversed by equimolar concentrations of dCyd. Exposure to dT after UV or X-irradiation or treatment with HN2 or MMC did not affect the cytotoxic response. dT exposure at non-cytotoxic concentrations did not affect DNA synthesis as measured by [3H]-dT incorporation when allowance was made for reductions in specific activity of labelled thymidine. However, dT post treatment reversed the alkylation induced inhibition of DNA synthesis. Toxic concentrations of dT caused an increase in frequency of TGR colonies but this increase was shown to be due to effects of dT on cell growth rate, and differential sensitivity ot HGPRT- and HGPRT+ cells. The frequency of spontaneous and alkylation induced AZR and to a lesser extent TGR colonies was also increased by non-toxic dT concentrations. Evidence was obtained which suggests that this increase is more likely to be due to alterations in the selective efficiency of the purine analogs than alterations in coding fidelity due to altered dNTP pools.

Inhibition of (Na,K)-ATPase by tetravalent vanadium.

Vanadyl, the tetravalent state of vanadium and a divalent cation, VO2+, was a relatively powerful inhibitor of highly purified membrane-bound sodium and potassium ion transport adenosine triphosphatase. The sensitivity of the ATPase activity to vanadyl characteristically correlated positively with the specific activity of the enzyme preparation. Inhibition ranged from nearly complete inhibition at less than 5 microM vanadyl for some of the purest fractions (specific activity approximately 45 mumol/min/mg of protein) to no observable inhibition at 300 microM vanadyl in one crude preparation of the enzyme with a specific activity of 10 mumol/min/mg of protein. The level of free vanadyl was reduced by incubation with these membranes, but this reduction was not sufficient to account for the low sensitivity to vanadyl observed in crude preparations. A reduction in specific activity by partial inactivation of a sensitive preparation by treatment with FeCl3 and ascorbate reduced its sensitivity to vanadyl. Anionic ligands of the enzyme, vanadate or ATP, increased the rate of recovery from inhibition after chelation of free vanadyl. At pH 6.1, the inhibition was characteristically fully reversible (t1/2 approximately 10 min), whereas at pH 8.1 it was stable for hours. The degree and stability of enzyme inhibition by vanadyl increased for several hours during incubation of the vanadyl-enzyme mixture, and at pH 6.1 the properties of the inhibitor itself also changed with time. Preincubation of the ion at that pH for 5 h before addition of the enzyme produced a more stable inhibition. The time- and pH-dependent changes in the degree and stability of enzyme inhibition probably relate to the complex chemistry of the vanadyl ion in solution.