Comparative study of the tRNA-methylases of normal and tumor tissues. II. Positional specificity of renal and carcinoma RA methylases

A comparative study of the position specificity of tRNA-methylases from normal and tumour tissues was performed on yeast tRNA1Val as the substrates using partially purified enzyme preparations from rat kidney and carcinoma RA. As in the case of rat liver and Novikoff hepatoma, two methylated compounds are formed in yeast tRNA1Val under the action of rat kidney and carcinoma enzyme preparations: m5C is formed in the sequence C49--C52 located in the extra loop and A59 in the Tpsi-loop is is converted into m1A. The activity of m5C-methylase [S-Ado-Met-tRNA-(cytosine-5)methyltransferase] (E. C. 2.1.1.29) is approximately equal in both tissues, whereas the activity of m1A-methylase [S-Ado-Met-tRNA-(adenine-1)methyltransferase] (E. C. 2.1.1.36) in carcinoma is twice as high as in the kidney. The two enzymes do not differ in their position specificity.     

On the mechanism of tRNA methylase-tRNA recognition.

In order to further elucidate the mechanism of tRNA methylase-tRNA intreaction the methylation of some individual tRNAs separately and by pairs was performed. In conditions of tRNA excess the methylation rates of positionally analogous nucleotides in tRNA molecules are not summed up when two substrates are simultaneously present in the reaction mixture. The inhibitory action of yeast tRNASer, possessing m5c in position 29, on the methylation of C29 in other individual tRNAs was shown. Yeast tRNAVal which possesses an A residue in position 27 was shown to inhibit the methylation of G27 in E. coli tRNAMet. The data obtained confirm the suggestion that tRNA methylases recognizes the tertiary structure of tRNAs. They show also that the recognition and the proper catalytic action are two autonomous processes and that the former at least in its first stage is rather unspecific.     

Participation of 5'-deoxyadenosyl-B12 in regulating methylation of tRNA]

The effects of different forms of cobalamines on the activities of tRNA-methylases of Zajdela ascite hepatoma were studied. Of six cobamides studied 5'-deoxyadenosyl-B12 and factor B containing as a ligand HSO3 in the concentrations of 2.4-10(-5) and 4.8-10(-5) M inhibited the tRNA-methylase activity by 21% and 15% correspondingly. The inhibitory effect of 5'-deoxyadenosyl-B12 is probably dependent on the adenosyl part of the molecule. 5'deoxyadenosyl-B12 exerted a selective effect of Zajdela ascite hepatoma tRNA-methylases, inhibiting largely the activity of 5-methyl cytosine methylase during the methylation of the E. coli K12W6 tRNA and yeast tRNA1 Val.     

Specificity of action of DNA methylases BcnI, CfrI and Cfr10I

The site specificity of three DNA methylases BcnI, CfrI and Cfr10I was determined to be 5'Cm4C(C/G)GG, 5'PyGGm5CCPu and 5'Pum5CCGGPy, respectively. Using the modification methylases under investigation with known restriction endonucleases, fourteen new DNA cleavage specificities can be created. Some aspects of the use of restriction endonucleases in DNA methylation analysis are discussed.     

Limited accessibility of DNA methylation sites for bacterial methylases M. Eco RII and M.Eco dam in chromatin at different levels of organization

The bacterial methylases M. Eco RII and M. Eco dam can methylate DNA in rat liver chromatin to form the 5-methylcytosine (m5C) and N6-methyladenine (m6A) residues, respectively. The CH3-accepting capacity of DNA in chromatin (mono- and dinucleosomes, mono- and dinucleomers) is 15 - 30 times less than that of free total DNA in rat liver. Such a low level of DNA methylation in chromatin in vitro suggests that the accessibility and recognition of methylation sites by DNA-methylases are decreased in comparison with free DNA both in the core-particle DNA and in the internucleosomal DNA. The degree of DNA methylation in chromatin particles depends on the ionic strength and Mg2+; when the former is decreased from 0.515 down to 0.176, the DNA methylation by both enzymes is increased 2-fold. An addition of Mg2+ (1 - 2 mM) decreases the CH3-accepting capacity of nucleomeric DNA, that of nucleosomal DNA remains unchanged. Thus, the accessibility of DNA for methylases is variable depending on the conformational changes of chromatin. The values of the m6A to m5C ratio for free and nucleosomal DNAs formed by methylation with a methylation of nucleomeric DNA, i. e. 1.01, 0.92 and 0.51, respectively. As Mg/4 concentration rises, the m6A/m5C ratio for nucleosomal and nucleomeric DNA is increased. It seems therefore that at different levels of organization and upon certain conformation changes the number and, probably, the nature of exposed DNA methylation sites in chromatin are different. Bacterial DNA-methylases can be used as an effective probe for a fine analysis of chromatin ultrastructure, in particular at its different functional states.     

Some features of cyclic adenosine monophosphate metabolism in mouse liver and hepatoma 22]

The levels of cyclic adenosine monophosphate (cAMP) and two forms of cAMP phosphodiesterase with low (PDE1) and high (PDE2) affinity for the substrate were determined in homogenates from mouse liver and transplanted hepatoma 22. The level of cAMP in the tumour is 3 times lower than that in liver. By te kinetic parameters (Vmax, Km, pH optimum) adenylate cyclase from tumour does not show any significant differences as compared to the liver enzyme; the enzyme from hepatoma is, however, more sensitive to activation by F- ions. The activities of adenylate cyclase in liver and tumour cells are the same. Phosphodiesterases of cAMP from tumour and liver cells are similar in their Km values (3,3-10(-4) M for PDE1 and 2-10(-6) M for PDE2); however, the maximal and real rates of cAMP hydrolysis in hepatoma are much higher than in liver. The fact that both cAMP phosphodiesterase activities have similar dependence on Mg2+ and Ca2+ concentrations, suggests that PDE1 is a latent form of PDE2. In tumour cells the equilibrium between these two forms is probably shifted towards the enzyme with high affinity for the substrate. The results suggest that a decreased cAMP level in hepatoma cells (as compared to the liver) is due to the activation of PDE2.     

DNA polymerase alpha from normal rat liver is different than DNA polymerases alpha from Morris hepatoma strains

To investigate whether DNA replication in rat hepatoma cells is altered compared with that in normal rat liver, the main replicative enzyme, i.e. the DNA polymerase alpha complex, was partially purified from a slow-growing (TC5123) and a fast-growing (MH3924) Morris hepatoma cell strain as well as from normal rat liver. The purified DNA polymerase alpha complexes contained RNA primase. DNA polymerase alpha activities of these complexes were characterized with regard to both their molecular properties and their dNTP and DNA binding sites. The latter were probed with competitive inhibitors of dNTP binding, resulting in Ki values, and with DNA templates, yielding Km values. The sedimentation coefficients of native DNA polymerases alpha from Morris hepatoma cells were found to be lower than that of polymerase alpha from normal rat liver. Consequently, when following the procedure of Siegel and Monty for determination of molecular mass considerably smaller molecular masses were calculated for polymerases of hepatoma strains (TC5123, 127 kDa; MH3924, 138 kDa; rat liver, 168 kDa). Similar differences were found when the dNTP binding site was probed with inhibitors. Ki values obtained with butylphenyl-dGTP were higher for polymerases of the hepatoma strains than for that of normal rat liver. However, Ki values measured with aphidicolin and butylanilino-dATP were lower for DNA polymerase alpha from the fast-growing hepatoma cell strain than for that from normal rat liver, indicating a reduced affinity of the dNTP binding sites for dATP and dCTP. This reduced affinity could be responsible for lowered specificity of nucleotide selection in the base-pairing process which in turn may cause an enhanced error rate in DNA replication in malignant cells. Furthermore, when the DNA binding site was characterized by Michaelis-Menten constants using gapped DNA as a template, Km values were similar for all three DNA polymerases. In contrast, the Km value measured with single-stranded DNA as a template was found to be lower for DNA polymerase alpha from the fast-growing hepatoma MH3924 than for that from normal rat liver. Thus, the DNA-polymerizing complex from MH3924 combines both higher binding strength to single-stranded DNA templates and decreased nucleotide selection, properties which may enhance replication velocity and may lower fidelity.     

Transfer ribonucleic acid methylases of bone. Studies on vitamin A and D deficiency

Methods were devised for the assay of tRNA methylases of rat bone. The activities of bone tRNA methylases are similar to those from other mammalian tissues. However, unlike reports on liver methylases, no inhibitors were found in the supernatant fraction from pH5 precipitate of bone extracts. The effects of vitamins A and D on the methylation of tRNA by cell-free extracts of rat bone were studied. Deficiency of either vitamin resulted in a decrease in the rate and extent of tRNA methylation, whereas the administration of vitamin A to hypovitaminotic-A rats and vitamin D to hypovitaminotic-D rats increased the rate and extent of tRNA methylation. These effects appear to be apart from changes in ribonuclease activity or in concentrations of calcium or magnesium. No evidence of inhibitors of tRNA methylases was found in bone extracts from vitamin-deficient rats nor of activators in bone extracts from deficient rats given vitamin A or D. The pattern of tRNA methylation under conditions of vitamin A or D deficiency was not changed, suggesting a generalized cellular deficiency. It was of significance to find that the specificity for methylation of specific bases in tRNA was different after the administration of vitamin A as contrasted with the effects of vitamin D. The possible significance of tRNA methylation to the biochemical action of the vitamins on bone is discussed.     

Temperature dependency of the anomeric specificity of yeast and bovine hexokinases

The phosphorylation of alpha- and beta-D-glucose by either yeast hexokinase or beef heart hexokinase was measured at both 10 and 30 degrees C. At 30 degrees C, the anomeric specificity of yeast hexokinase represented a mirror image of that of bovine hexokinase, in terms of both maximal velocity and affinity. A decrease in temperature apparently accentuated the anomeric difference in both maximal velocity and affinity of bovine hexokinase. Such a difference consisted in a higher maximal velocity with beta- than alpha-D-glucose, but a greater affinity for the alpha- than beta-anomer. In yeast hexokinase, however, the decrease in temperature suppressed the anomeric difference in maximal velocity and inversed the anomeric difference in affinity. In the case of both enzymes, the fall in temperature decreased more the maximal velocity recorded with alpha-D-glucose than that measured with beta-D-glucose, and severely lowered the Km for alpha-D-glucose, whilst failing to affect significantly the Km for beta-D-glucose. These findings, which allow to reconcile prior apparently conflicting data, reveal that the anomeric behaviour of hexokinase is affected by the ambient temperature. Our data also support the view that hexokinase underwent a phylogenic evolution in terms of its anomeric specificity.     

Interaction of AluI, Cfr6I and PvuII restriction-modification enzymes with substrates containing either N4-methylcytosine or 5-methylcytosine

The cleavage specificity of R.Cfr6I, an isoschizomer of PvuII restriction endonuclease was determined to be 5'CAG decreases CTG and the methylation specificity of Cfr6I and PvuII methylases, 5'CAG4mCTG. Thus, M.Cfr6I and M.PvuII are new additions to the list of methylases with N4-methylcytosine specificity. Neither of the above RM enzymes acts on the substrates containing either N4-methylcytosine or 5-methylcytosine in a cognate methylation position.     

Model substrates of enzymes modifying ribonucleic acids. Synthesis of deca- and undecanucleotid-fragments of 21-member oligoribonucleotide simulating T psi C-branch of yeast valine tRNA

Decanucleotide (Ap)6GpTpUpC and undecanucleotide GpApUpCpCp (Up)5U have been synthesised. They constitute 5'- and 3'-parts of a 21-mer which imitates T psi C-arm of yeast tRNA(Val1) and is a potential substrate for m1A-methylases and pseudouridine synthetase. The oligonucleotide blocks, synthesised enzymatically by means of ribonucleases of various substrate specificity and polynucleotide phosphorylases (TpUpC, ApUpCpC, pGpTpUpC, GpApUpCpC) or obtained by hydrolysis of poly(U) and poly(A) with Serratia marcescens endonuclease (hexauridilate and hexaadenilate), were joined by T4 RNA ligase.     

Procaryotic and eucaryotic traits of DNA methylation in spiroplasmas (mycoplasmas).

Differences in the type of base methylated (cytosine or adenine) and in the extent of methylation were detected by high-pressure liquid chromatography in the DNAs of five spiroplasmas. Nearest neighbor analysis and digestion by restriction enzyme isoschizomers also revealed differences in methylation sequence specificity. Whereas in Spiroplasma floricola and Spiroplasma sp. strain PPS-1 5-methylcytosine was found on the 5' side of each of the four major bases, the cytosine in Spiroplasma apis DNA was methylated only when its 3' neighboring base was adenine or thymine. In Spiroplasma sp. strain MQ-1 over 95% of the methylated cytosine was in C-G sequences. Essentially all of the C-G sequences in the MQ-1 DNA were methylated. Partially purified extracts of S. apis and Spiroplasma sp. strain MQ-1 were used to study substrate and sequence specificity of the methylase activity. Methylation by the MQ-1 enzyme was exclusively at C-G sequences, resembling in this respect eucaryotic DNA methylases. However, the MQ-1 methylase differed from eucaryotic methylases by showing high activity on nonmethylated DNA duplexes, low activity with hemimethylated DNA duplexes, and no activity on single-stranded DNA.     

Two transfer RNA (1-methylguanine) methylases from yeast.

Two distinct tRNA (m-1G) methylases have been found in the yeast Saccharomyces cerevisiae. They differ in their chromatographic properties on hydroxyapatite, in their response to spermine, and in their site specificity. Only one of the methylases is active against normal tRNA from Escherichia coli.     

Further investigation of the increased transfer ribonucleic acid methylase activity in tumours of the mouse colon

1. Extracts prepared from tumours of the mouse colon induced by 1,2-dimethylhydrazine were considerably more active in catalysing the methylation of tRNA than were extracts from normal colon. The enhanced activity was observed when both unfractionated ;methyl-deficient' tRNA and purified tRNA preparations from yeast and bacteria were used as substrates for methylation. 2. The methylated bases produced in these reactions were identified. There were no differences between the products of the reaction catalysed by extracts of tumour and normal colon. 3. The increased activity of tRNA methylases was not due to the presence in the extracts of stimulatory or inhibitory molecules of low molecular weight such as polyamines or S-adenosylhomocysteine. 4. Other enzymes concerned with tRNA metabolism (RNA polymerase, ATP-tRNA adenylyltransferase, aminoacyl-tRNA ligases) were also increased in activity in the tumour tissue. 5. The extent of methylation of a limiting amount of tRNA was greater when tumour extracts were compared with controls, but in no case was it possible to achieve a stoicheiometric methylation of the purified tRNA preparations used as substrates, and the tumour extracts were not able to methylate tRNA obtained from normal mouse colon. We conclude that the tumours contained greater activities of tRNA methylases but that there was no evidence for changes in the specificity of these enzymes during neoplastic growth.     

Purification and characterization of two tRNA-(guanine)-methyltransferases from rat liver

tRNA(guanine-1-)-methyltransferase (EC 2.1.1.31) and tRNA(N2-guanine)-methyltransferase I (EC 2.1.1.32) were isolated from rat liver. The (guanine-1-)-methyltransferase preparation is 6800-fold purified and is free from contaminating methyltransferases or ribonuclease. The molecular weight of (guanine-1-)-methyltransferase is 83 000. Of seven purified Escherichia coli tRNAs examined, only tRNAMetf was utilized as substrate by (guanine-1-)-methyltransferase. The methylation of tRNAMetf is maximally stimulated by 40 mM putrescine with a pH optimum of 8.0. Using E. coli K-12 tRNA, the Km for S-adenosylmethionine is 3 micrometer and Ki for S-adenosylhomocysteine is 0.11 micrometer for (guanine-1-)-methyltransferase. (N2-Guanine-)-methyltransferase is 6200-fold purified and is also free of interfering enzymes. It has a molecular weight of 69 000. E. coli tRNAPhe, tRNAVal and tRNAArg are substrates for this enzyme which introduces a methyl at the 2-amino group of the guanine at position 10 from the 5'-terminus of these tRNAs. The methylation of tRNAPhe is maximally stimulated by 100 micrometer spermidine with a pH optimum of 8.0. (N2-Guanine-)-methyltransferase has a Km for S-adenosylmethionine of 2 micrometer and a Ki for S-adenosylhomocysteine of 23 micrometer with E. coli K-12 tRNA as methyl acceptor.     

Role of ribosomal RNA methylases in the regulation of ribosome production in mammalian cells.

The activity of rRNA methylases was stimulated by high-energy precursors of RNA (ribonucleoside triphosphates) and inhibited by degradation products of RNA (ribonucleotides and oligoribonucleotides). The response of methylases from rat Novikoff ascites tumor and liver to these metabolites was strikingly different. The highly active tumor enzymes responded preferentially to inhibition by catabolic metabolites, whereas the less active liver enzymes responded exclusively to stimulation by anabolic metabolites. When the activity of rRNA methylases was assayed in response to increasing concentration of S-adenosylmethionine, the tumor enzymes responded with a hyperbolic substrate dependence curve and the liver enzymes with a sigmoidal curve. In the presence of an inhibitory dinucleotide, ApA, the tumor enzymes responded with a sigmoidal curve; in the presence of a stimulator, adenosine 5'-triphosphate, the liver enzymes responded with a hyperbolic substrate concentration curve. When normal rats were subject to a series of treatments by thioacetamide, a hepatocarcinogen, the liver nucleolar rRNA methylases became responsive to inhibition by ApA and relatively unresponsive to stimulation by adenosine 5'-triphosphate. When tumor-bearing rats were treated with polyinosinate:polycytidylate, an antitumor agent, the tumor nucleolar rRNA methylases became unresponsive to inhibition by ApA and more responsive to stimulation by adenosine 5'-triphosphate. A correlation was noted between increased methylation efficiency in vivo and increased stability of nucleolar RNA during incubation in vitro, or vice versa. These results are interpreted to indicate that rRNAmethylases are regulated by cellular metabolites during the nucleolar biosynthesis of ribosomes and that rRNA methylases may provide a favorable site for selective action by cancer chemotherapeutic agents.     

Steady state kinetics and binding of eukaryotic cytochromes c with yeast cytochrome c peroxidase.

1. The steady state kinetics for the oxidation of ferrocytochrome c by yeast cytochrome c peroxidase are biphasic under most conditions. The same biphasic kinetics were observed for yeast iso-1, yeast iso-2, horse, tuna, and cicada cytochromes c. On changing ionic strength, buffer anions, and pH, the apparent Km values for the initial phase (Km1) varied relatively little while the corresponding apparent maximal velocities varied over a much larger range. 2. The highest apparent Vmax1 for horse cytochrome c is attained at relatively low pH (congruent to 6.0) and low ionic strength (congruent to 0.05), while maximal activity for the yeast protein is at higher pH (congruent to 7.0) and higher ionic strength (congruent to 0.2), with some variations depending on the nature of the buffering ions. 3. Direct binding studies showed that cytochrome c binds to two sites on the peroxidase, under conditions that give biphasic kinetics. Under those ionic conditions that yield monophasic kinetics, binding occurred at only one site. At the optimal buffer concentrations for both yeast and horse cytochromes c, the KD1 and KD2 values approximate the Km1 and Km2 values. At ionic strengths below optimal, binding becomes too strong and above optimal, too weak. 4. Under ionic conditions that are optimal and give monophasic kinetics with horse cytochrome c but are suboptimal for the yeast protein, yeast cytochrome c strongly inhibits the reaction of horse cytochrome c with peroxidase, uncompetitively at one site and competitively at a second site. The appearance of the second site under monophasic conditions is interpreted as an allosteric effect of the inhibitor binding to the first site. 5. The simplest model accounting for these observations postulates two kinetically active sites on each molecule of peroxidase, a high affinity and a low affinity site, that may correspond to the free radical and the heme iron (IV) of the oxidized enzyme, respectively. Both oxidizing equivalents may be discharged at either site. Furthermore, the enzyme appears to exist as an equilibrium mixture of a high ionic strength form, EH and a low ionic strength form, EL, the former reacting optimally with yeast cytochrome c, and the latter with horse cytochrome c.