Effect of Cibacron Blue F3GA on phosphoglycerate kinase of Lactobacillus plantarum and phosphoglycerate mutase of Leuconostoc dextranicum

Blue dextran or Cibacron Blue F3GA has been shown to inhibit yeast phosphoglycerate kinase [EC 2.7.2.3] competitively with respect to ATP (Thompson et al. (1975) Proc. Natl. Acad. Sci. U.S. 72, 663--667; Beissner and Rudolph (1979) J. Biol. Chem. 254, 6273--6277). However, we have found that phosphoglycerate kinase of Lactobacillus plantarum was inhibited by Cibacron Blue F3GA, the blue chromophore of blue dextran, noncompetitively with respect to ATP, but competitively with respect to 3-phosphoglycerate. Further inhibition studies with Cibacron Blue F3GA suggest that one molecule of the dye was bound per molecule of phosphoglycerate kinase at a saturated level of either substrate, but two molecules of the dye were bound per molecule of the kinase with an unsaturated level of either substrate used as a fixed substrate. Furthermore, phosphoglycerate mutase [EC 2.7.5.3] of Leuconostoc dextranicum was also inhibited by Cibacron Blue F3GA competitively with respect to 3-phosphoglycerate and noncompetitively with respect to 2,3-bisphosphoglycerate. These results suggest that the 3-phosphoglycerate-binding site on both phosphoglycerate kinase and phosphoglycerate mutase can interact with Cibacron Blue F3GA.     

Bacteriophage T4 deoxynucleotide kinase: gene cloning and enzyme purification.

Gene 1 of bacteriophage T4 has been cloned into a lambda pL expression vector, resulting in the overproduction of deoxynucleotide kinase. A procedure that includes affinity chromatography on Cibacron Blue F3GA-agarose has been used to purify milligram quantities of enzymes from a 2-liter culture. The enzyme has been partially characterized in vitro and in vivo, and it appears to be identical to the deoxynucleotide kinase isolated from T4-infected Escherichia coli. These results prove the earlier contention that the phosphorylation of three dissimilar deoxynucleotides (5-hydroxymethyldeoxycytidylate, dTMP, and dGMP), to the exclusion of most others, is catalyzed by a single protein.     

Ferrocene-functionalized SWCNT for electrochemical detection of T4 polynucleotide kinase activity

A novel electrochemical strategy for monitoring the activity and inhibition of T4 polynucleotide kinase (PNK) is developed by use of titanium ion (Ti(4+)) mediated signal transition coupled with signal amplification of single wall carbon nanotubes (SWCNTs). In this method, a DNA containing 5'-hydroxyl group is self-assembled onto the gold electrode and used as substrate for PNK. The biofunctionalized SWCNTs with anchor DNA and ferrocene are chosen as the signal indicator by virtue of the intrinsic 5'-phosphate end of anchor DNA and the high loading of ferrocene for electrochemical signal generation and amplification. The 5'-hydroxyl group of the substrate DNA on the electrode is phosphorylated by T4 PNK in the presence of ATP, and the resulting 5'-phosphoryl end product can be linked with the signal indicator by Ti(4+). The redox ferrocene group on the SWCNTs is grafted to the electrode and generates the electrochemical signal, the intensity of which is proportional to the activity of T4 PNK. This assay can measure activity of T4 PNK down to 0.01 UmL(-1). The developed method is a potentially useful tool in researching the interactions between proteins and nucleic acids and provides a diversified platform for a kinase activity assay.     

Interaction of Cibacron Blue F3GA with Escherichia coli DNA polymerase I and with T4 DNA polymerase.

Individual rapid procedures for the enrichment of Escherichia coli DNA polymerase I and of bacteriophage T4 DNA polymerase free of endonuclease activity are described using Blue dextran-Sepharose chromatography. The blue dye of Blue dextran-Sepharose selectively binds to the deoxynucleoside triphosphate substrate site of the E. coli but not the T4 enzyme indicating that the catalytic sites of these two enzymes which catalyze the same polymerization reaction in vitro are quite distinct.     

Kinetic properties and inhibition of human T lymphoblast deoxycytidine kinase

The kinetic properties of 50,000-fold purified cultured human T lymphoblast (MOLT-4) deoxycytidine kinase were examined. The reaction velocity had an absolute requirement for magnesium. Maximal activity was observed at pH 6.5-7.0 with Mg:ATP for 1:1. High concentrations of free Mg2+ or free ATP were inhibitory. Double reciprocal plots of initial velocity studies yielded intersecting lines for both deoxycytidine and MgATP2-. dCMP was a competitive inhibitor with respect to deoxycytidine and ATP. ADP was a competitive inhibitor with respect to ATP and a mixed inhibitor with respect to deoxycytidine. dCTP, an important end product, is a very potent inhibitor and was a competitive inhibitor with respect to deoxycytidine and a non-competitive inhibitor with respect to ATP. TTP reversed dCTP inhibition. The data suggest that (a) MgATP2- is the true substrate of deoxycytidine kinase; (b) the kinetic mechanism of deoxycytidine kinase is consistent with rapid equilibrium random Bi Bi; (c) deoxycytidine kinase may be regulated by its product ADP and its end product dCTP as well as the availability of deoxycytidine. While many different nucleotides potently inhibit deoxycytidine kinase, their low intracellular concentrations make their regulatory role less important.     

Vita Blue: a new 633-nm excitable fluorescent dye for cell analysis

Several new derivatives of fluorescein were synthesized. The dyes were characterized by NMR; and the absorbance, excitation, and emission spectra were measured. The fluorescence quantum yields of the dyes were determined. The pKa3 values of the dyes were measured by fluorescence titration. The characteristics of the fluorescein and sulfonefluorescein derivatives were compared. The most promising dye for use in cell analysis appeared to be compound 9, which was given the name Vita Blue. The dibutyrate ester of Vita Blue was made and the compound was given the name Vita Blue dibutyrate (VBDB, 14). The Km of VBDB with pig liver esterase was measured and found to be 4 x 10(-5) M. The pKa3 of Vita Blue was 7.56 +/- 0.03; both acidic and basic forms were fluorescent (dual fluorescence). The use of VBDB as an intramolecular esterase substrate and its utility for the discrimination between live and dead cells by flow cytometry is described.     

Structure of a tRNA repair enzyme and molecular biology workhorse: T4 polynucleotide kinase

T4 phage polynucleotide kinase (PNK) was identified over 35 years ago and has become a staple reagent for molecular biologists. The enzyme displays 5'-hydroxyl kinase, 3'-phosphatase, and 2',3'-cyclic phosphodiesterase activities against a wide range of substrates. These activities modify the ends of nicked tRNA generated by a bacterial response to infection and facilitate repair by T4 RNA ligase. DNA repair enzymes that share conserved motifs with PNK have been identified in eukaryotes. PNK contains two functionally distinct structural domains and forms a homotetramer. The C-terminal phosphatase domain is homologous to the L-2-haloacid dehalogenase family and the N-terminal kinase domain is homologous to adenylate kinase. The active sites have been characterized through structural homology analyses and visualization of bound substrate.     

Studies on pig liver mevalonate-5-diphosphate decarboxylase

A procedure in which three sequential enzymes of cholesterol biosynthesis, mevalonate kinase (ATP: (R)-mevalonate 5-phosphotransferase, EC 2.7.1.36), phosphomevalonate kinase (ATP: (R)-5-phosphomevalonate phosphotransferase, EC 2.7.4.2) and mevalonate-5-diphosphate decarboxylase (ATP: (R)-5-diphosphomevalonate carboxy-lyase (dehydrating), EC 4.1.1.33), from pig liver, could be purified in the one operation is described. Mevalonate kinase and phosphomevalonate kinase were utilized for the enzymic synthesis of mevalonate 5-diphosphate (both 1-14C-labelled and unlabelled), the substrate for mevalonate-5-diphosphate decarboxylase, using excess free ATP4-. A radioactive assay for the enzyme, based on the release of 14CO2 from [1-14C]mevalonate-5-diphosphate, was developed. The assay allowed reassessment of the metal and nucleotide specificity of the decarboxylase. ATP could be partially replaced by GTP and ITP, but no activity was observed with CTP, UTP or TTP. Apparent activation of the enzyme by ATP4- was observed as found for mevalonate kinase (C.S. Lee and W.J. O'Sullivan (1983) Biochim. Biophys. Acta 747, 215-224) and phosphomevalonate kinase (C.S. Lee and W.J. O'Sullivan (1985) Biochim. Biophys. Acta 839, 83-89). The presence of 1 mM excess free ATP4-, above that complexed as the substrate MgATP2-, decreased the Km for MgATP2- from 0.45 mM to 0.15 mM. MgADP- was shown to act as a competitive inhibitor with respect to MgATP2-.     

Purification of orotate phosphoribosyltransferase and orotidylate decarboxylase by affinity chromatography on Sepharose dye derivatives.

Blue Dextran-Sepharose and Cibacron Blue F3GA-Sepharose (Blue Sepharose) were found to act as affinity adsorbents for orotate phosphoribosyltransferase (PRTase) and orotidine 5′-monophosphate (OMP) decarboxylase from bakers' yeast. Experiments with columns of Blue Dextran-Sepharose and partially purified preparations of the PRTase and decarboxylase revealed that both enzymes were selectively eluted by a low concentration (0.1–2 mm) of their respective substrate or immediate product. On the other hand, a much higher concentration (50–400 mm) of NaCl was required to displace these two enzymes from the above columns. Larger scale experiments showed that OMP decarboxylase in crude extracts was purified about 5700- and 6600-fold on Blue Sepharose using 0.5 mm OMP and 2 mm uridine 5′-monophosphate (UMP) as the eluting ligand, respectively. In contrast, orotate PRTase did not bind to Blue Sepharose unless crude extracts were first subjected to gel filtration. The resulting preparation of orotate PRTase, purified about sixfold with respect to cell-free extracts, was purified an additional 200- and 40-fold when the enzyme was eluted from Blue Sepharose with 0.5 mm OMP and 1 mm 5-phosphoribosyl 1-pyrophosphate (PP-ribose-P), respectively. Blue Dextran-Sepharose, on the other hand, was found to provide a lower degree of enzyme purification and exhibited a lower sample-binding capacity. Samples of the PRTase and decarboxylase that had been purified about 200- and 6000-fold, respectively, on Blue Sepharose displayed a major protein band and one or more minor bands when subjected to polyacrylamide gel electrophoresis. Enzyme activity coincided with the major band in all cases.     

Dephosphorylation of phosphoproteins and synthetic phosphopeptides. Study of the specificity of the polycation-stimulated and MgATP-dependent phosphorylase phosphatases

The substrate specificity of different forms of polycation-stimulated (PCSH, PCSL, and PCSC) phosphorylase phosphatases and of the catalytic subunit of the MgATP-dependent protein phosphatase from rabbit skeletal muscle was investigated. This was done, with phosphorylase a as the reference substrate, using the synthetic phosphopeptides patterned after the phosphorylated sites of pyruvate kinase (type L) (Arg2-Ala-Ser(32P)-Val-Ala (S2), and its Thr(32P) substitute (T4)), inhibitor-1 (Arg4-Pro-Thr(32P)-Pro-Ala (T5), Arg2-Pro-Thr(32P)-Pro-Ala (T1), and its Ser(32P) substitute (S1)), and some modified phosphopeptides (Arg2-Ala-Thr(32P)-Pro-Ala (T2) and Arg2-Pro-Thr(32P)-Val-Ala (T3)), all phosphorylated by cyclic AMP-dependent protein kinase. In addition, casein(Thr-32P), phosphorylated by casein kinase-2, was also tested. The PCS phosphatases show a striking preference for the T4 configuration, PCSC being the least efficient. The catalytic subunit of the MgATP-dependent phosphatase was almost completely inactive toward all these substrates. As shown for the PCSH phosphatase, and comparing with T4, the two proline residues flanking the Thr(P) in T1 and T5, just as in inhibitor-1, drastically imparied the dephosphorylation by lowering the Vmax and not by affecting the apparent Km. The C-terminal proline (as in T2) by itself represents a highly unfavorable factor in the dephosphorylation. The critical effect of the sequence X-Thr(P)-Pro or Pro-Thr(P)-Pro (T1, T2, T5, and inhibitor-1) can be overcome by manganese ions. The additional finding that this is not the case with the Pro-Ser(P)-Pro sequence (S1) suggests that the effect of Mn2+ is highly substrate specific. These observations show the considerable importance of the primary structure of the substrate in determining the specificity of the protein phosphatases.     

Isolation and elucidation of some functional properties of the "mute" catalytic subunit of cAMP-dependent protein kinase

A mute isoenzyme of type II cAMP-dependent protein kinase from rat muscle has been reported that is released from the regulatory subunit by cAMP but remains inactive until combination with heat- and acid-stable modulator has occurred. This enzyme has now been obtained in isolation free of the normal catalytic subunit using affinity chromatography with both an ATP analog (Blue Dextran/Sepharose) and a protein substrate analog (Kemptide/CH-Sepharose). Separation can be effected in both cases before activation of the mute enzyme. Affinity of the mute enzyme for Blue Dextran--a ligand specific for the dinucleotide fold in this kinase--is somewhat higher than that of the normal enzyme. Conversely, before reaction with the modulatory protein the mute enzyme will not bind at all to Kemptide/CH-Sepharose, where the normal enzyme elutes at 50 mM KCl. When pretreated with the modulatory protein and so activated, mute enzyme binds to Kemptide with a very high affinity and can only be eluted using a natural substrate (phosphorylase kinase), up to 500 mM salt being ineffective. The modulator thus appears to act through alteration of the protein substrate binding site on the enzyme.     

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 characterization of N-acetylglutamate 5-phosphotransferase from pea (Pisum sativum) cotyledons.

N-Acetylglutamate 5-phosphotransferase (acetylglutamate kinase, EC 2.7.2.8) has been isolated from pea (Pisum sativum) cotyledons and purified 312-fold by using heat treatment, (NH4)2SO4 fractionation, affinity chromatography on ATP--Sepharose and ion-exchange chromatography on DEAE-cellulose. This preparation was shown on polyacrylamide-gel electrophoresis to yield one band staining with Coomassie Blue. The enzyme was shown by a variety of techniques to be composed of two different kinds of subunits, of mol.wts. 43000 and 53000 respectively. These subunits are arranged to give either a dimeric or tetrameric enzyme composed of equal numbers of each type of subunit. The dimeric and tetrameric enzyme forms are thought to be interconvertible, the equilibrium between these forms being influenced by the type of ligand bound to the subunits. Kinetic studies performed on the purified enzyme, indicated a random Bi Bi type of mechanism. The enzyme displayed apparent negative co-operativity with respect to one of its substrates, N-acetylglutamate; as a result, two Km values were found for this substrate, one at 1.9 X 10(-3) M and the other at 6.2 X 10(-3) M. A single Km value for ATP was found to be 1.7 X 10(-3) M. Allosteric regulation by arginine was also shown. A model, based on the Koshland, Némethy & Filmer [(1966) Biochemistry 5, 365-385] Sequential model, which adequately describes the kinetic and structural properties of N-acetylglutamate 5-phosphotransferase, is presented.     

Protein kinase of bacteriophage T7. 2. Properties, enzyme synthesis in vitro and regulation of enzyme synthesis and activity in vivo.

Protein kinase, which was isolated from cells infected with T7, is indeed a viral gene product. This is shown by DNA-dependent synthesis in vitro. The protein kinase transfers phosphate from ATP to seryl or threonyl residues in protein. The enzyme has only a relative requirement for magnesium ions, but is only active at low ionic strength. The best substrate is lysozyme. T7 protein kinase activity is not stimulated by cyclic 3':5'-AMP and/or cyclic 3':5'-GMP. The T7 protein kinase carries -- SH groups essential for activity. There is indication that the enzyme phosphorylates itself and causes self inactivation, which may explain the fast disappearance of enzyme activity in vivo. Bacteriophage T3 also induces a protein kinase which is similar to the T7-induced enzyme in all respects tested.     

Thyroid hormones and the reactivities of genetic variants of human erythrocytic glucose-6-phosphate dehydrogenase

Thyroid hormones, throxine (T4) and triiodothyronine (T3) which are known to activate glucose-6-phosphate dehydrogenase (G6PD) activity in vivo act as substrate inhibitors of G6PD in vitro. T4 competitively inhibits NADP in human erythrocyte G6PD variants G6PDA, G6PDB and G6PDA- with inhibition constants of 2.40 +/- 0.90 X 10(-6), 3.44 +/- 0.63 X 10(-6) and 6.53 +/- 0.60 X 10(-6) mol/l, respectively. The inhibition is, however, noncompetitive with respect to G6P in the three variants. T3 also has similar inhibition pattern to T4 with inhibition constants for NADP of 1.9 +/- 0.08 X 10(-5) and 1.28 +/- 0.17 X 10(-5) mol/l for G6PDB and G6PDA-, respectively. cAMP on the other hand inhibits G6P competitively with inhibition constants 1.50 +/- 0.22 X 10(-4), 1.06 +/- 0.24 X 10(-4) and 1.76 +/- 0.14 X 10(-4) mol/l for G6PDB, G6PDA and G6PDA-, respectively. There are significant differences in the inhibition effects of T4 and cAMP with respect to NADP as substrates for the normal enzyme G6PDA or G6PDB and the deficient enzyme G6PDA- when NADP is the substrate, the latter being much more inhibited. The activation effect of thyroid hormones in vivo may therefore not be a direct result of thyroid hormone binding to the G6PD enzyme nor mediated through the action of cAMP but plausibly be through complexation of inhibitory trace metal ions by the thyroid hormones T4 and T3.     

Identification of yin-yang regulators and a phosphorylation consensus for male germ cell-associated kinase (MAK)-related kinase

MAK (male germ cell-associated protein kinase) and MRK/ICK (MAK-related kinase/intestinal cell kinase) are human homologs of Ime2p in Saccharomyces cerevisiae and of Mde3 and Pit1 in Schizosaccharomyces pombe and are similar to human cyclin-dependent kinase 2 (CDK2) and extracellular signal-regulated kinase 2 (ERK2). MAK and MRK require dual phosphorylation in a TDY motif catalyzed by an unidentified human threonine kinase and tyrosine autophosphorylation. Herein, we establish that human CDK-related kinase CCRK (cell cycle-related kinase) is an activating T157 kinase for MRK, whereas active CDK7/cyclin H/MAT1 complexes phosphorylate CDK2 but not MRK. Protein phosphatase 5 (PP5) interacts with MRK in a complex and dephosphorylates MRK at T157 in vitro and in situ. Thus, CCRK and PP5 are yin-yang regulators of T157 phosphorylation. To determine a substrate consensus, we screened a combinatorial peptide library with active MRK. MRK preferentially phosphorylates R-P-X-S/T-P sites, with the preference for arginine at position -3 (P-3) being more stringent than for prolines at P-2 and P+1. Using the consensus, we identified a putative phosphorylation site (RPLT(1080)S) for MRK in human Scythe, an antiapoptotic protein that interacts with MRK. MRK phosphorylates Scythe at T1080 in vitro as determined by site-directed mutagenesis and mass spectrometry, supporting the consensus and suggesting Scythe as a physiological substrate for MRK.     

Conformational restraint is a critical determinant of unnatural nucleotide recognition by protein kinases

This report describes the synthesis of N(4)-(benzyl) AICAR triphosphate, a conformationally restrained analogue of N(4)-(benzyl) ribavirin triphosphate. Both of these nucleotides were evaluated as phosphodonors for wild-type p38MAP kinase and T106G p38MAP kinase, a designed mutant with expanded nucleotide specificity. The conformationally restrained nucleotide, N(4)-(benzyl) AICAR triphosphate, is orthogonal to (not accepted as a substrate by) wild-type p38MAP kinase, in contrast to N(4)-(benzyl) ribavirin triphosphate. Furthermore, N(4)-(benzyl) AICAR triphosphate, is accepted as a substrate by T106G p38MAP kinase, in contrast to N(4)-(benzyl) ribavirin triphosphate. We hypothesize that the presence of an internal hydrogen bond in N(4)-(benzyl) AICAR and its absence in N(4)-(benzyl) ribavirin triphosphate is the main determinant for their differing structure-activity relationships.     

Mutagenicity of selected sulfonated azo dyes in the Salmonella/microsome assay: use of aerobic and anaerobic activation procedures

A selection of 16 sulfonated azo dyes of both the monoazo type and diazo dyes based on benzidine, o-tolidine and o-dianisidine were assayed for mutagenicity in Salmonella typhimurium strains TA98 and TA100 employing both aerobic and anaerobic preincubation procedures. 3 food dyes, FD & C Red No. 40 and Yellows No. 5 and No. 6 were non-mutagenic in all tests. 5 dyes were mutagenic with aerobic treatment (trypan blue, Pontacyl Sky Blue 4BX, Congo Red, Eriochrome Blue Black B, dimethylaminoazobenzene) and 6 were mutagenic aerobically with riboflavin and cofactors (Deltapurpurin, trypan blue, Pontacyl Sky Blue 4BX, Congo Red, methyl orange, Ponceau 3R). Anaerobic preincubation involving enzymatic reduction of the dyes led to a different pattern of mutagenicity, with trypan blue giving much enhanced mutagenicity; Eriochrome Blue Black B, Pontacyl Sky Blue 4BX, Deltapurpurin and Congo Red exhibiting similar activity to aerobic preincubation; and methyl orange and Ponceau 3R yielding no mutagenicity. The results are interpreted with respect to an hypothesis involving partial reduction of the azo bond under differing degrees of aerobiosis via azo-anion radicals and hydrazo intermediates.     

Design, synthesis, and characterization of an ATP-peptide conjugate inhibitor of protein kinase A

An ATP-peptide conjugate was synthesized as a bisubstrate analogue inhibitor of the serine/threonine kinase protein kinase A. The compound was found to be a linear, competitive inhibitor with respect to ATP substrate, exhibiting a Ki of 3.8 microM. The compound was noncompetitive with respect to peptide substrate. The inhibitor was shown to be selective for protein kinase A versus the closely related protein kinase C as well as tyrosine kinase Csk. This analysis provides new evidence for the dissociative transition state of protein serine/threonine kinases and illustrates a simple method to convert a low affinity peptide substrate to a selective and moderately potent inhibitor for these enzymes.