Reaction of cytidine with semicarbazide in the presence of bisulfite. A rapid modification specific for single-stranded polynucleotide.

Semicarbazide reacted rapidly with 5,6-dihydrocytidine-6-sulfonate, which was formed from cytidine by addition of bisulfite across the 5,6-double bond. The transaminated product, 5,6-dihydro-4-semicarbazido-2-ketotopyrimidine-6-sulfonate ribofuranoside, was identified by comparison with that formed by treatment of 4-semicarbazido-2-ketopyrimidine ribofuranoside with bisulfite. The progress of the transamination was monitored spectrophotometrically by use of a strong absorbance of the product in alkali. The reaction between cytidine and the semicarbazide-bisulfite mixture was optimal at pH 4.5. Complete transformation of cytidine into the product required only 5 min with the use of 3M semicarbazide-1M sodium bisulfite, pH 5.0, at the reaction temperature 37 degrees C. The product was stable in unbuffered solution but in phosphate buffers it underwent elimination of bisulfite to give 4-semicarbazido-2-ketopyrimidine ribofuranoside. The rate of the elimination at pH 7.0 and 37 degrees C increased proportionally with the increase of the phosphate concentration. Complete elimination was obtained by treatment with 1 M sodium phosphate for 2 h. When heat-denatured calf-thymus DNA was treated with 3 M semicarbazide-1 M bisulfite at 37 degrees C and pH 5.0 the transamination of reactive cytosine residues was completed by 10 min of incubation. At 20 degrees C, it required 85 min of incubation. Cytosine residues in native DNA did not react at all even by prolonged incubations. The modified DNA samples were further treated with a phosphate buffer at pH 7, producing 4-semicarbazido-2-ketopyrimidine residues in the DNA. Analysis of the base compositions of these samples by perchloric acid hydrolysis showed that the modification was selective to cytosine, which had been expected from studies with monomers. It also showed that the reactive cytosine residues in the denatured DNA, constitute about 80% of the total cytosine, which was consistent with the view that heat-denatured DNA still contains a considerable amount of secondary structure. The semicarbazide-bisulfite modification is expected to be a sensitive method to locate cytosine residues in single-stranded regions of polynucleotides.     

A simple fluorimetric microassay for adenine compounds in platelets and plasma and its application to studies on the platelet release reaction

1. The formation of a stable fluorescent product between chloroacetaldehyde and adenine or its derivatives provides the basis of a rapid simple assay for total adenine compounds in blood platelets and in plasma. The assay will measure down to 200pmol of adenine nucleotides. An evaluation of the method established the optimum conditions for the production of maximum fluorescence. 2. Values obtained for total adenine compounds in platelets were 12.9nmol/10(8) cells in man and 7.8nmol/10(8) cells in rat. These closely agree with previous values for total platelet adenine nucleotides found by using a firefly luciferase assay, or a recycled NAD-linked photometric assay. This supports the concept that the chloroacetaldehyde reaction measures total adenine nucleotides in platelets. 3. Platelet aggregation induced by collagen was studied photometrically in 0.1ml volumes of citrated platelet-rich plasma, and total adenine nucleotides were assayed in platelets and plasma before and after aggregation. During aggregation 58% of adenine nucleotides were released from human platelets, and 36% from rat platelets. 4. The chloroacetaldehyde assay is no substitute for more sophisticated procedures, but is a simple sensitive means of monitoring the release of adenine nucleotides from blood platelets and is particularly valuable when small plasma samples must be used.     

Location and properties of glucose dehydrogenase in sporulating cells and spores of Bacillus subtilis.

Late during sporulation, Bacillus subtilis produces glucose dehydrogenase (GlcDH; EC 1.1.1.47), which can react with D-glucose or 2-deoxy-D-glucose and can use nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP) as a cofactor. This enzyme is found mainly in the forespore compartment and is present in spores; it is probably made exclusively in the forespore. The properties of GlcDH were determined both in crude cell extracts and after purification. The enzyme is stable at pH 6.5 but labile at pH 8 or higher; the pH optimum of enzyme activity is 8. After inactivation at pH 8, the activity can be recovered in crude extracts, but not in solutions of the purified enzyme, by incubation with 3 M KCl and 5 mM NAD or NADP. As determined by gel filtration, enzymatically active GlcDH has a molecular weight of about 115,000 (if the enzyme is assumed to be globular). GlcDH is distinct from a catabolite-repressible inositol dehydrogenase (EC 1.1.1.18), which can also react with D-glucose, requires specifically NAD as a cofactor, and has an electrophoretic mobility different from that of GlcDH.     

Physical studies of chloroacetaldehyde labelled fluorescent DNA

The reaction of chloroacetaldehyde with denatured DNA produces a fluorescent DNA where both the adenine and cytosine bases are modified. The rate of modification of DNA by chloroacetaldehyde was measured using the absorption spectrum shift. The depolarization and quantum yield of native DNA and denatured DNA were investigated as a function of temperature.The melting points and the renaturation rates of a series of derivative DNA's were investigated. The melting point was decreased by 1.3°C for each base modified per 100 base pairs corresponding to a 2.8 Kcal destabilizing free energy per mismatched base pair. The renaturation rate of the derivative DNA is reduced by a factor 2 when the melting temperature is lowered by 13°C.     

Reconstitution of rabbit muscle aldolase after dissociation and denaturation at alkaline pH.

Tetrameric rabbit muscle aldolase is dissociated to the inactive monomer at strongly alkaline pH (pH greater than or equal to 12). As shown by sedimentation velocity, fluorescence emission, and specific activity, the final profiles of dissociation, denaturation, and deactivation run parallel. Increasing incubation time proves the enzyme to be metastable in the pH range of deactivation. At 10 less than pH less than 12 "hysteresis" of the deactivation-reactivation reaction is observed. Short incubation at pH greater than or equal to 12 leads to high yields of reactivation (greater than or equal to 60%), while irreversibly denatured enzyme protein is the final product after long incubation. The kinetics of reconstitution under essentially irreversible conditions (pH 7.6) can be described by a sequential uni-bimolecular mechanism, assuming partial activity of the isolated subunits. The kinetic constants correspond to those observed for the reactivation after denaturation at acid pH or in 6M guanidine. HCl. Obviously the pH-dependent deactivation and reactivation of aldolase at alkaline pH obeys the general transconformation/association model which has been previously reported to hold for the reconstitution of numerous oligomeric enzymes after denaturation in various denaturants.     

Formation of a Schiff base intermediate is not required for the adenine glycosylase activity of Escherichia coli MutY

The mutY gene product of Escherichia coli is a 39-kDa protein that catalyzes the removal of adenine bases mispaired with 2'-deoxyguanosine and 7,8-dihydro-8-oxo-2'-deoxyguanosine (OG) in DNA. Although adenine removal proceeds via monofunctional glycosylase activity, MutY is able to form covalent adducts with substrate DNA in the presence of borohydride, a trait otherwise known to be associated only with enzymes having bifunctional glycosylase/AP lyase activity. To help identify active site residues involved in the formation of MutY-DNA adducts in the presence of borohydride, a series of site-directed mutant forms of MutY were generated. Our data show that Lys 142 is the primary residue involved in cross-link formation. The absence of Lys 142 results in near elimination of the enzyme-DNA adducts formed relative to wild-type, suggesting that this residue is the primary one involved in forming covalent associations with DNA during MutY catalysis. Importantly, the enzymatic activity and DNA binding of the K142A enzyme is nearly identical to the WT enzyme. This shows that formation of the covalent intermediate is not required for adenine removal by MutY. Furthermore, this suggests that the covalent intermediate is formed by reaction of Lys 142 with the OG/G:(AP site) product, and this may be a consequence of MutY's unusually high affinity for the product of its glycosylase action.     

Adenylate kinase of plants. Properties of adenylate kinase of pea leaves]

The properties of adenylate kinase in 2 ADP in equilibrium ATP + AMP reaction have been studied. The dependence of the enzyme activity on medium pH, protein concentration, substrates, Mg++ ions, AMP, adenine and adenosine has been also investigated. pH optimum is found to be 8.5 for forward reaction and 8-9--for the reverse one. The Michaelis constants are as follows: for ADP--1.17-10(-4) M, for ATP--3.33-10(-4) M at 24 degrees C, in 50 mM tris-HCl pH 7.6. The optimal ratio, Mg++ ions/substrates (ADP, ATP + AMP), is 1:2. The chelates of adenine nucleotides with Mg++ ions are proved to be "true" reaction substrates. Unlike adenine and adenosine, the product of AMP reaction inhibits adenylate kinase activity. It is concluded that the properties of adenylate kinase in plants are similar to those of animals and humans (moikinase).     

1,2-Cyclohexanedione modification of arginine residues in egg-white riboflavin-binding protein

1. Reaction of 1,2-cyclohexanedione with arginine residues of egg white riboflavin-binding protein results in a loss of the binding activity. 2. In borate buffer pH 8.0, with 0.15 M cyclohexanedione, the inactivation proceeds with a pseudo-first-order rate constant 0.084 hr.-1. 3. At least 65% of lost riboflavin binding capacity can be recovered on 12 hr incubation in 0.5 M hydroxylamine pH 7.0. 4. All 5 arginine residues are modified, 2-3 of them seem to react much easier than others. 5. The correlation between modification of arginines and protein inactivation, as analyzed by kinetic and statistical methods, suggests that one of low-reactivity residues is "essential" for riboflavin binding. 6. In the holoprotein, one arginine residue is almost completely protected from 1,2-cyclohexanedione modification. 7. Riboflavin does not dissociate from holoprotein, even on prolongated incubation with the reagent. 8. The protected arginine residue seems to be located in the riboflavin binding pocket of protein macromolecule.     

The reaction of 14C-labelled platinum ethylenediamine dichloride with adenine compounds and DNA

Various derivatives of adenine have been studied with regard to their rate of reaction with ¹⁴C-labelled platinum ethylenediamine dichloride, Pt(¹⁴C-en)Cl₂. The reactivities have been calculated from the “rate of disappearance” of Pt(¹⁴C-en)Cl₂ using chromatographic separation of reactants and products.Adenine and adenosine react very slowly at 37° whereas other adenine derivatives react much more readily in the order: poly A > AMP > ApA > poly d(AT). From the numerical values of the rate constants it is concluded that the presence of a phosphate group increases the reaction rate considerably. This is partly the explanation of the rapid reaction of poly A which possesses terminal phosphate groups. However adjacent adenine moieties such as those in polyadenylic acid (poly A) and adenosyl-3′5′-adenosine (ApA) may also react by another mechanism which involves the 6-NH₂ groups.The energies of activation of the second order reaction with platinum ethylenediamine dichloride (PtenCl₂) are 12.9, 18.8, 19.0 kcal/mole for poly A, AMP and ApA respectively.In DNA, no free phosphate groups are present, and the occurrence of adjacent adenines will be low. The reaction of PtenCl₂ with DNA seems to involve a rapid attack on deoxyguanosine (GdR) and a slow reaction with deoxyadenosine (AdR) and deoxycytidine (CdR).     

Adenine specific DNA chemical sequencing reaction.

Reaction of DNA with K2PdCl4 at pH 2.0 followed by a piperidine workup produces specific cleavage at adenine (A) residues. Product analysis revealed the K2PdCl4 reaction involves selective depurination at adenine, affording an excision reaction analogous to the other chemical DNA sequencing reactions. Adenine residues methylated at the exocyclic amine (N6) react with lower efficiency than unmethylated adenine in an identical sequence. This simple protocol specific for A may be a useful addition to current chemical sequencing reactions.     

Stopped-flow chemical modification with N-bromosuccinimide: a good probe for changes in the microenvironment of the Trp 62 residue of chicken egg white lysozyme

The stopped-flow chemical modification with N-bromosuccinimide (NBS) of Trp 62 of hen (chicken) egg white lysozyme (EC 3.2.1.17) was found to depend greatly on pH: it was not observed at pH's above 7, but it was observed at pH's lower than 6. In addition, at pH's between 6 and 7 the NBS modification showed a delta epsilon pH profile similar to a "titration curve," giving a pK (congruent to 6.5) nearly equal to the pK (congruent to 6.2) of a catalytic residue, Glu 35. The stopped-flow chemical (NBS) modification of N-acetyl-L-tryptophan ethyl ester, a model compound of Trp 62, does not depend on pH at the pH's examined, approximately 3.5-8.5. These experimental results suggest that a change in the state of Trp 62 at Subsite C is induced by protonation-deprotonation of an ionizable residue, which could be Glu 35 (catalytic site), indicating that stopped-flow NBS modification is a good probe for detection of changes in the micorenvironment around the tryptophan residue(s) of enzymes.     

Chemical mapping of cytosines enzymatically flipped out of the DNA helix

Haloacetaldehydes can be employed for probing unpaired DNA structures involving cytosine and adenine residues. Using an enzyme that was structurally proven to flip its target cytosine out of the DNA helix, the HhaI DNA methyltransferase (M.HhaI), we demonstrate the suitability of the chloroacetaldehyde modification for mapping extrahelical (flipped-out) cytosine bases in protein-DNA complexes. The generality of this method was verified with two other DNA cytosine-5 methyltransferases, M.AluI and M.SssI, as well as with two restriction endonucleases, R.Ecl18kI and R.PspGI, which represent a novel class of base-flipping enzymes. Our results thus offer a simple and convenient laboratory tool for detection and mapping of flipped-out cytosines in protein-DNA complexes.     

The reactions of thiouridines and thiouracils with chloroacetaldehyde; mechanistic considerations.

2-Thiouridine, 4-thiouridine and the corresponding thiouracils were quantitatively modified with aqueous chloroacetaldehyde /37 degrees C, pH 3.0--6.5/. The rate-pH dependence found for the disappearance of the substrates suggested initial S-alkylation. The unstable S-acetaldehydyl intermediates were not detected due to their further rapid transformations. The following possibilities of such transformations are discussed: 1. intramolecular addition of the endocyclic nitrogen atom to the aldehyde carbonyl group to form the "hydroxyethano" bridged compounds, 2. hydrolysis to the corresponding "oxo" analogues of the substrates, 3. hydrolysis of the N-glycoside bond. The structures of new compounds formed in these reactions were assigned on the basis of their FD-MS, UV, IR and PMR spectra. The reaction rates were similar to those found for modification of adenosine and cytidine with chloroacetaldehyde.     

Properties of a DNA-adenylate complex formed in the reaction between mammalian DNA ligase I and DNA containing single-strand breaks.

The major DNA ligase from calf thymus (mammalian DNA ligase I) forms a covalent enzyme-AMP complex on incubation with ATP [S?derh?ll & Lindahl, J. Biol. Chem. 248, 672-675, (1973)]. The reaction of this complex with DNA has now been studied. When the ligase-adenylate complex is incubated at 0 degrees C for short time periods with DNA containing single-strand breaks, a DNA-AMP complex can be isolated from the reaction mixture by isopycnic centrifugation in CsCl. Incubation at pH 6.5 increased the amount of DNA-AMP complex that could be isolated 10-20-fold relative to that obtained at pH 7.4. Under the same conditions, incubation of the ligase-AMP complex with DNA free from single-strand breaks did not lead to detectable DNA-AMP formation. The DNA-AMP complex was resistant to treatment with dilute acid and alkali indicating the presence of a covalent linkage. Further, this complex was sensitive to DNase but resistant to pronase and RNase. Free AMP was released on further incubation of the isolated DNA-AMP complex with thymus DNA ligase I and Mg2+, suggesting that the complex is a reaction intermediate. Degradation of the DNA-AMP complex with several reagent enzymes indicated that the AMP residues were bound at the 5' ends of the single-strand breaks in DNA by pyrophosphate bonds.     

Intracellular distinction between peroxidase and catalase in exocrine cells of rat lacrimal gland: a biochemical and cytochemical study.

The lacrimal gland (Glandula orbitalis externa) of rat contains both peroxidase and catalase and was used as a model for biochemical and cytochemical distinction between peroxidase and catalase. Both enzymes were isolated by ammonium sulfate precipitation from tissue homogenates, and the effects of fixation with glutaraldehyde and various conditions of incubation were investigated colorimetrically using DAB as hydrogen donor. The lacrimal gland peroxidase is strongly inhibited by glutaraldehyde treatment. In contrast, for catalase the fixation with glutaraldehyde is the prerequistie for demonstration of its peroxidatic activity. The maximal peroxidatic activity was obtained after treatment of catalase with 3% glutaraldehyde, higher concentrations being inhibitory. For lacrimal gland peroxidase, the maximal rate of oxidation of DAB is at pH 6.5, whereas for catalase it is at pH 10.5. The optimal concentration of H2O2 for lacrimal gland peroxidase is at 10(-3)M and for peroxidatic activity of catalase at 10(-1)M. These optimal conditions obtained biochemically were applied to tissue sections of rat lacrimal gland. After the fixation of tissue with a low concentration of glutaraldehyde and incubation in the DAB medium at neutral pH containing 10(-3)M H2O2 (Peroxidase medium), the reaction product was localized in the cisternae of the rough endoplasmic reticulum, in elements of the Golgi apparatus, and in secretory granules. After the fixation of tissue with 3% glutaraldehyde and incubation in the DAB-medium containing 10(-1)M H2O2 and at pH 10.5 (catalase medium), the staining in the endoplasmic reticulum, the Golgi-apparatus and in secretory granules was completely inhibited and reaction product was localized exclusively in small (0.2-0.5 mu) particles similar to small peroxisomes described in various other cell-types.     

Purine metabolism in microplasmodia of Physarum polycephalum.

The uptake and utilization of purine nucleosides and purines in microplasmodia of Physarum polycephalum were investigated. The results revealed a unique pattern, namely that exogenous purine nucleosides are readily taken up and metabolised, while free purine bases are hardly taken up. The pathways of incorporation have been elucidated in studies with whole cells and with cell-free extracts. The ribonucleosides (adenosine, inosine and guanosine) can be converted into ribonucleotides in two ways; either directly catalysed by a kinase or by a phosphorolytic cleavage to the free base (adenine, hypoxanthine and guanine respectively) which can then be activated by a purine phosphoribosyltransferase. Apparently the purine phosphoribosyltransferases do not react with exogenous purine bases. The deoxyribonucleosides (deoxyadenosine, deoxyinosine and deoxyguanosine) are also phosphorolysed by purine nucleoside phosphorylase to adenine, hypoxanthine and guanine respectively. A portion of deoxyadenosine is directly phosphorylated to dAMP. It appears that only a minor part of the soluble nucleotide pool can be synthesised from exogenous supplied nucleosides and that none of the deoxyribonucleosides specifically label DNA. There is no catabolism of the purine moiety. In agreement with the above findings, we have found that analoguees of purine nucleosides are more toxic than their corresponding purine base analogues.     

Azoindoxyl methods for the histochemical investigation of hydrolases. I. Lactase (lactase-beta-glucosidase complex) (author's transl)]

An azoindozyl method for the histochemical demonstration of lactase (lactase-beta-glucosidase complex) is described. The incubation medium consists of 5 mg 5-Br-4-Cl-3-indolyl-beta-D-fucoside (dissolved in 0.5 ml N,N-dimethylformamide) and 0.02 ml hexazotized prosaniline in 10 ml 0.1 M citric acid phosphate buffer, pH 6-6.5. By means of this method lactase can be exactly localized in the brush border of the enterozytes in the jejunum of suckling rats. Compared to the corresponding indigogenic method the azoindoxyl reaction proceeds faster and the reaction product is often precipitated more precisely.     

Microphotometric determination of enzymes in brain sections. V. Glycerophosphate dehydrogenases

An incubation medium was adapted for the microphotometric determination (kinetic and end-point measurements) of the activities of mitochondrial alpha-glycerophosphate dehydrogenase (GPDH) in the rat hippocampus. For comparison, the activities of the cytoplasmic NAD-linked alpha-glycerophosphate dehydrogenase were also measured. The study showed that in the demonstration of both enzymes the use of an exogenous electron carrier is necessary. Both enzymes react to phenazine methosulfate (PMS) which transfers reduction equivalents to the electron acceptor nitroblue tetrazolium chloride (NBT), thus causing a coreaction of GPDH in the demonstration of NAD-GPDH. Therefore, only the NAD-independent GPDH which is stimulated by menadione, can be selectively demonstrated in the histochemical procedure applied. The final incubation medium of GPDH consisted of 15 mM L-glycerol 3-phosphate, 5 mM NBT, 0.4 mM menadione, 7.5% polyvinyl alcohol in 0.5 M Hepes buffer, pH 8; the final pH of the incubation medium was 7.5. A linear response of the reaction lasted about 5 min. There was a linear relationship between section thickness and the formation of reaction product up to a section thickness of 14 microns. The apparent Km value at 25 degrees C was 0.6 mM. It is concluded that using menadione histochemical methods are suited to determine the mitochondrial GPDH activities in brain sections whereas using PMS a coreaction of GPDH takes place in the demonstration of NAD-GPDH, so that a histochemical quantification of NAD-GPDH cannot be recommended.     

Formation of ribonucleotides in DNA modified by oxidative damage in vitro and in vivo. Characterization by 32P-postlabeling.

Oxygen free radicals generated by the interaction of Fe2+ and H2O2 (Fenton reaction) are capable of reacting with DNA bases, which may induce premutagenic and precarcinogenic lesions. Products formed in DNA by such reactions have been characterized as hydroxylated derivatives of cytosine, thymine, adenine, and guanine and imidazole ring-opened derivatives of adenine and guanine. As shown here by 32P-postlabeling, incubation of DNA under Fenton reaction conditions gave rise to additional oxidation products in DNA that were characterized as putative ribonucleosides by enzymatic hydrolysis of the oxidized DNA, 32P-postlabeling, and co-chromatography in multiple systems with authentic markers. Formation of these products in DNA was enhanced by the presence of L-ascorbic acid in the reaction mixtures and their total amounts were similar to those of the major DNA oxidation product, 8-hydroxy-2'-deoxyguanosine. The ribonucleoside guanosine was also formed in kidney DNA of male rats treated with ferric nitrilotriacetate, a renal carcinogen. It is postulated that ribonucleotides alter conformation and function of DNA and thus their presence in DNA may lead to adverse health effects.     

Analysis of bacteriophage phi X174 gene A protein-mediated termination and reinitiation of phi X DNA synthesis. II. Structural characterization of the covalent phi X A protein-DNA complex

In the preceeding paper (Brown, D. R., Roth, M. J., Reinberg, D., and Hurwitz, J. (1984) J. Biol. Chem. 259, 10545-10555), it was shown that following bacteriophage phi X174 (phi X) DNA synthesis in vitro using purified proteins, the phi X A protein could be detected covalently linked to nascent 32P-labeled DNA. This phi X A protein-[32P]DNA complex was the product of the reinitiation reaction. The phi X A protein-[32P]DNA complex could be trapped as a protein-32P-oligonucleotide complex by the inclusion of ddGTP in reaction mixtures. In this report, the structure of the phi X A protein-32P-oligonucleotide complex has been analyzed. The DNA sequence of the oligonucleotide bound to the phi X A protein has been determined and shown to be homologous to the phi X (+) strand sequence immediately adjacent (3') to the replication origin. The phi X A protein was directly linked to the 5' position of a dAMP residue of the oligonucleotide; this residue corresponded to position 4306 of the phi X DNA sequence. The phi X A protein-32P-oligonucleotide complex was exhaustively digested with either trypsin or proteinase K and the 32P-labeled proteolytic fragments were analyzed. Each protease yielded two different 32P-labeled peptides in approximately equimolar ratios. The two 32P-labeled peptides formed after digestion with trypsin (designated T1 and T2) and with proteinase K (designated PK1 and PK2) were isolated and characterized. Digestion of peptide T1 with proteinase K yielded a product which co-migrated with peptide PK2. In contrast, peptide T2 was unaffected by digestion with proteinase K. These results suggest that the phi X A protein contains two active sites that are each capable of binding covalently to DNA. The peptide-mononucleotide complexes T1-[32P]pdA and T2-[32P]pdA were isolated and subjected to acid hydrolysis in 6.0 N HCl. In each case, the major 32P-labeled products were identified as [32P] phosphotyrosine and [32P]Pi. This indicates that each active site of the phi X A protein participates in a phosphodiester linkage between a tyrosyl moiety of the protein and the 5' position of dAMP.