Activity In Cell-free Extracts Research Articles

Enzymological features of aromatic amino acid biosynthesis reflect the phylogeny of mycoplasmas.

Acholeplasma laidlawii possesses a biochemical pathway for tyrosine and phenylalanine biosynthesis, while Mycoplasma iowae and Mycoplasma gallinarum do not. The detection of 7-phospho-2-dehydro-3-deoxy-D-arabino-heptonate (DAHP) synthase (EC 4.1.2.15), dehydro-shikimate reductase (EC 1.1.1.25) and 3-enol-pyruvoylshikimate-5-phosphate synthase (EC 2.5.1.19) activities in cell-free extracts established the presence in A. laidlawii of a functional shikimate pathway. L-Phenylalanine synthesis occurs solely through the phenylpyruvate route via prephenate dehydratase (EC 4.2.1.51), no arogenate dehydratase activity being found. Although arogenate dehydrogenase was detected, L-tyrosine synthesis appears to occur mainly through the 4-hydroxyphenylpyruvate route, via prephenate dehydrogenase (EC 1.3.1.12), which utilized NAD+ as a preferred coenzyme substrate. L-Tyrosine was found to be the key regulatory molecule governing aromatic biosynthesis. DAHP synthase was feedback inhibited by L-tyrosine, but not by L-phenylalanine or L-tryptophan; L-tyrosine was a potent feedback inhibitor of prephenate dehydrogenase and an allosteric activator of prephenate dehydratase. Chorismate mutase (EC 5.4.99.5) was sensitive to product inhibition by prephenate. Prephenate dehydratase was feedback inhibited by L-phenylalanine. It was also activated by hydrophobic amino acids (L-valine, L-isoleucine and L-methionine), similar to results previously found in a number of other genera that share the Gram-positive line of phylogenetic descent. Aromatic-pathway-encoded cistrons present in saprophytic large-genome mycoplasmas may have been eliminated in the parasitic small-genome mycoplasmas.

Comparing Short-Term Effects of Ammonia and Methylamine on Nitrogenase Activity in Anabaena variabilis (ATCC 29413)

Cyanobacterium . Anabaena variabilis, Nitrogenase Regulation, Ammonia, Methylamine Using the heterocystous cyanobacterium A nabaena variabilis (ATCC 29413) in an alkaline environment its nitrogenase activity is rapidly inhibited by ammonia and methylamine. Nitro­genase inhibition by ammonia is probably caused by a mechanism comparable to the switch-off effect, which has been described for several species of the Rhodospirillaceae, whereas methyl-amine-induced inhibition is caused by an uncoupling effect only. Evidence for these different effects is obtained by comparing nitrogenase activity in cell-free extracts of filaments pretreated by ammonia or methylamine. In addition, ammonia-dependent nitrogenase inhibition is shown to be dependent on protein synthesis and on light intensity.

Purification and characterization of glutathione reductase encoded by a cloned and over-expressed gene in Escherichia coli.

An expression vector, pKGR, for the gor gene from Escherichia coli encoding glutathione reductase was constructed by subcloning of an AvaII fragment of the Clarke & Carbon bank plasmid pGR [Greer & Perham (1986) Biochemistry 25, 2736-2742] into the plasmid pKK223-3. The expression of glutathione reductase from the plasmid pKGR was found to have been successfully placed under the control of the tac promoter. Transformation of E. coli cells with this plasmid resulted in 100-200-fold increase in glutathione reductase activity in cell-free extracts. A rapid purification procedure for the enzyme, based on affinity chromatography on Procion Red HE-7B-CL-Sepharose 4B, was developed. The purified enzyme was homogeneous as judged by SDS/polyacrylamide-gel electrophoresis, and all its properties were consistent with the DNA sequence of the gene [Greer & Perham (1986) Biochemistry 25, 2736-2742] and with those previously reported for E. coli glutathione reductase [Mata, Pinto & Lopez-Barea (1984) Z. Naturforsch. C. Biosci. 39, 908-915]. These experiments have enabled an investigation of the protein chemical and mechanistic properties of the enzyme by site-directed mutagenesis.

Inorganic pyrophosphatase activity in cell-free extracts of Ureaplasma urealyticum.

Cell-free extracts of Ureaplasma urealyticum strains Pi and T960 (CX8) (serovars 6 and 8, respectively) metabolized inorganic pyrophosphate (PPi). The inorganic pyrophosphatase (PPase) activity was greatest with Mg2+ as cofactor, but Mn2+ acted as a poor substitute. The PPases of the two serovars differed electrophoretically. Although the highest PPase activity was obtained using PPi as substrate, the enzyme could also utilize to a lesser degree both tripolyphosphate and trimetaphosphate. No activity was observed against beta-glycerophosphate, naphthyl phosphates, glucose 6-phosphate, fructose 6-phosphate, fructose 1,6-bisphosphate, thiamin pyrophosphate, phosphoribosylpyrophosphate, ADP or ATP. Acid- and alkaline-phosphatase activities were observed with naphthyl phosphates as substrates, but they did not have the same electrophoretic mobility on gels as the PPase activity. U. urealyticum PPase was inhibited by oxidized glutathione, 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide, phenylglyoxal, p-chloromercuribenzoic acid, Mn2+, Zn2+ and Ca2+. Neither reduced glutathione, L-cysteine nor Co2+ enhanced activity. PPi can act as a substrate or regulator of certain metabolic reactions, and PPi metabolism can function in bacterial bioenergetics; its role in ureaplasmas is presently unclear.

Inhibition of membrane damage due to phospholipase activity during fractionation of wheat aleurone tissue

Fractionation of [Me- 14C]choline-labelled wheat aleurone tissue in the absence of phospholipase inhibitors resulted in a 60% loss of label from phosphatidylcholine and even larger losses of NADH-cytochrome c reductase activity from microsomal fractions. Several putative inhibitors of phospholipase D were tested for their ability to protect the membranes during fractionation. The addition of choline and O-phosphorylcholine, together with glycerol-1-phosphate to inhibit any phosphatidic acid phosphatase activity, proved to be the best protectants. In tissue from freshly imbibed seeds, however, the addition of p-chloromercuribenzoate to a cocktail of inhibitors was necessary for the best recovery of radiolabelled membranes. Effects of the inhibitors on phospholipase D activity in cell free extracts were studied in an attempt to confirm the enzyme as the cause of membrane damage.

Enzyme activities in cell-free extracts of shikonin-producing Lithospermum erythrorhizon cell suspension cultures

Lithospermum erythrorhizon cell cultures excrete large quantities of naphthoquinone pigments, viz. shikonin derivatives, which have made enzymatic studies impossible so far. This paper describes methods for the removal of shikonin derivatives from the cells with liquid paraffin during culture growth and for the preparation of active enzyme extracts. Chorismate mutase and shikimate dehydrogenase activities were used as indicators for the preparation of active extracts. The efficacy of the methods developed was proved by the demonstration of the enzymatic formation of m-geranyl- p-hydroxybcnzoic acid from geranylpyrophosphate and p-hydroxybenzoic acid in cell-free extracts of L. erythrorhizon cultures, the first detection of a key enzyme of shikonin biosynthesis.

Degradation of the nitrogenase proteins during encystment of Azotobacter vinelandii

Nitrogenase activity in cell-free extracts of Azotobacter vinelandii declines during encystment. Upon germination a rapid increase in activity is observed, which is suppressed by rifampicin, suggesting that de novo biosynthesis of the nitrogenase proteins is required. The decline of activity during encystment is accompanied by disappearance of both nitrogenase proteins from cell extracts, indicating irreversible proteolysis. Total proteinase activity does not change significantly during encystment.

Structural genes for phosphatases inAspergillus nidulans

SUMMARYAlthough the fungusAspergillus nidulanshas a multiplicity of phosphatases and of genes where mutations affect one or more phosphatases, we have succeeded in identifying structural genes for three phosphatases as well as one other gene which might encode a fourth. Using both conditional and non-conditional mutations,palD has been shown to be the structural gene for a phosphate-repressible alkaline phosphatase,palG to be the structural gene for a non-repressible alkaline phosphatase which apparently exists in two electrophoretically distinct forms (but whose rates of thermal inactivation are apparently very similar) andpacA to be the structural gene for both intracellular and secreted forms of a phosphate-repressible acid phosphatase. Colony staining techniques for the enzymes specified bypalD andpacA have been described previously but we have now shown that the enzyme specified bypalG can be detected by staining toluene-permeabilized colonies. Mutations inpacG lead to loss of non-repressible acid phosphatase as judged by colony staining and electrophoretic patterns but their effects on assays of activity in cell-free extracts are only marginal. Under phosphate-limited, but not phosphate-starved or phosphate-sufficient, conditions,pacG−mutations also affect the regulation of other, phosphate-repressible phosphatases. None of these phosphatases, alone or in combination, plays an essential role.

Preferential transcription of HTLV-I LTR in cell-free extracts of human T cells producing HTLV-I viral proteins.

The promoters of the adenovirus 2 major late gene, the mouse beta-globin gene, the mouse immunoglobulin VH gene and the LTR of the human T-lymphotropic retrovirus type I were tested for their transcription activities in cell-free extracts of four cell lines; HeLa, CESS (Epstein-Barr virus-transformed human B cell line), MT-1 (HTLV-I-infected human T cell line without viral protein synthesis), and MT-2 (HTLV-I-infected human T cell line producing viral proteins). LTR was preferentially transcribed in the extracts of MT-2 although the other three genes were transcribed with relatively constant efficiencies in different extracts. The results agree well with the previous in vivo studies on the promoter activity of HTLV-I LTR. Mixing of HeLa and MT-2 extracts revealed the presence of a LTR-specific stimulating activity in MT-2 extracts.

Conversion of leukotrienes A 4 to C 4 in cell-free systems

A procedure for assaying leukotriene C 4 synthase activity in cell-free extracts has been presented. Leukotriene A 4 methyl ester was as active a substrate as leukotriene A 4 (Na salt) for the synthesis. The methyl ester is the substrate of choice, because (1) it is more stable than the sodium salt, (2) it is not a substrate of epoxide hydrolase for leukotriene B 4 synthesis, and (3) it gives a lower blank than an equimolar concentration of leukotriene A 4. The enzyme activity in rat liver, guinea pig and human lungs, and human nasal polyp was chiefly membrane-bound, although the cytosol contained some activity.

Regulation of glutamine synthetase, aspartokinase, and total protein turnover in Klebsiella aerogenes

When suspensions of Klebsiella aerogenes are incubated in a nitrogen-free medium there is a gradual decrease in the levels of acid-precipitable protein and of aspartokinase III (lysine-sensitive) and aspartokinase I (threonine-sensitive) activities. In contrast, the level of glutamine synthetase increases slightly and then remains constant. Under these conditions, the glutamine synthetase and other proteins continue to be synthesized as judged (a) by the incorporation of [ 14C]leucine into the acid-precipitable protein fraction and into protein precipitated by anti-glutamine synthetase antibodies, (b) by the fact that growth-inhibiting concentrations of chloramphenicol also inhibit the incroporation of [ 14C]leucine into protein and into protein precipitated by anti-glutamine synthetase antibody, and (c) by the fact that chloramphenicol leads to acceleration in the loss of aspartokinases I and III and promotes a net decrease in the level of glutamine synthetase and its cross-reactive protein. The loss of aspartokinases I and III in cell suspensions is stimulated by glucose and is inhibited by 2,4-dinitrophenol. Glucose also stimulates the loss of aspartokinases and glutamine synthetase in the presence of chloramphenicol. Cell-free extracts of K. aerogenes catalyze rapid inactivation of endogenous glutamine synthetase as well as exogeneously added pure glutamine synthetase. This loss of glutamine synthetase is not associated with a loss of protein that cross-reacts with anti-glutamine synthetase antibodies. The inactivation of glutamine synthetase in extracts is not due to adenylylation. It is partially prevented by sulfhydryl reagents, Mn 2+, antimycin A, 2,4-dinitrophenol, EDTA, anaerobiosis and by dialysis. Following 18 h dialysis, the capacity of extracts to catalyze inactivation of glutamine synthetase is lost but can be restored by the addition of Fe 2+ (or Ni 2+ together with ATP (or other nucleoside di- and triphosphates. After 40–60 h dialysis Fe 3+ together with NADH (but not ATP) are required for glutamine synthetase inactivation. The results suggest that accelerated protein degradation in cells exposed to nitrogen-limited conditions reflects the differential destruction of some proteins, including aspartokinases I and III, in order to sustain the biosynthesis of others such as glutamine synthetase. The loss of glutamine synthetase activity in cell-free extracts is likely mediated in part by mixed-function oxidation systems and could represent a ‘marking’ step in protein turnover.

Anaerobic and Aerobic Metabolism of Sorbitol in Streptococcus sanguis and Streptococcus mitior

Sorbitol-fermenting strains of Streptococcus sanguis and Streptococcus mitior were grown both anaerobically and in the presence of oxygen in a sorbitol-containing complex medium. Washed-cell suspensions were incubated with an excess of sorbitol, and the production of lactate, formate, ethanol, and acetate was analyzed. Moreover, we determined the lactate dehydrogenase and pyruvate formate-lyase activities in cell-free extracts of anaerobically grown cells. The anaerobically grown cells produced lactate, formate, ethanol, and acetate under anaerobic conditions. When these cells were exposed to air, the amounts of formate, ethanol, and acetate were reduced in comparison with those of the strictly anaerobic cells. Cells grown in the presence of oxygen only produced detectable levels of lactate and acetate. Anaerobically grown cells possessed lactate dehydrogenase and pyruvate formate-lyase activities under strictly anaerobic conditions. The level of pyruvate formate-lyase was dramatically reduced when cells were exposed to air, while the level of lactate dehydrogenase was about the same as that under strictly anaerobic conditions. Thus, the results indicate that S. sanguis and S. mitior both metabolize sorbitol differently under anaerobic and aerobic conditions. This difference may depend on the oxygen-sensitivity of the pyruvate formatelyase of these micro-organisms.

Fermentation of acetylene by an obligate anaerobe,Pelobacter acetylenicus sp. nov.

Four strains of strictly anaerobic Gram-negative rod-shaped non-sporeforming bacteria were enriched and isolated from marine and freshwater sediments with acetylene (ethine) as sole source of carbon and energy. Acetylene, acetoin, ethanolamine, choline, 1,2-propanediol, and glycerol were the only substrates utilized for growth, the latter two only in the presence of small amounts of acetate. Substrates were fermented by disproportionation to acetate and ethanol or the respective higher acids and alcohols. No cytochromes were detectable; the guanine plus cytosine content of the DNA was 57.1±0.2 mol%. Alcohol dehydrogenase, aldehyde dehydrogenase, phosphate acetyltransferase, and acetate kinase were found in high activities in cell-free extracts of acetylene-grown cells indicating that acetylene was metabolized via hydration to acetaldehyde. Ethanol was oxidized to acetate in syntrophic coculture with hydrogen-scavenging anaerobes. The new isolates are described as a new species in the genusPelobacter, P. acetylenicus.

A sodium ion gradient as energy source for Peptostreptococcus asaccharolyticus.

The determination of enzymatic activities in cell-free extracts of Acidaminococcus fermentans and Peptostreptococcus asaccharolyticus led to a refined scheme for the pathway of glutamate fermentation via (R)-2-hydroxyglutarate to acetate and butyrate. From the ratio of these products the amount of ATP generated by substrate level phosphorylation was calculated. Growth experiments with the organisms including Clostridium symbiosum and Clostridium tetanomorphum indicated that a sodium gradient contributed additional energy for growth. The high growth yields found in organisms containing the biotin dependent sodium pump glutaconyl-CoA decarboxylase could be reduced by the sodium ionophor monensin. In P. asaccharolyticus energy equivalent up to 0.6 mol ATP per mol of glutaconyl-CoA decarboxylated was conserved via the Na+ gradient. The data may explain the growth promoting effects of monensin in cattle.

Regulation of methylotrophic and heterotrophic metabolism in Arthrobacter P1. Growth on mixtures of methylamine and acetate in batch and continuous cultures

Incubations of Arthrobacter P1 in batch culture in media with mixtures of acetate and methylamine resulted in sequential utilization of the two carbon substrates, but not in diauxic growth. Irrespective of the way cells were pregrown, acetate was the preferred substrate and subsequent studies showed that this is due to the fact that acetate is a strong inhibitor of the methylamine transport system and amine oxidase in Arthrobacter P1. An analysis of enzyme activities in cell-free extracts showed that synthesis of amine oxidase occurred already in the first growth phase with acetate, whereas rapid synthesis of hexulose phosphate synthase was only observed once methylamine utilization started. It is therefore concluded that in Arthrobacter P1 the synthesis of the enzymes specific for methylamine oxidation is not regulated co-ordinately with those involved in formaldehyde fixation, but induced sequentially by methylamine and formaldehyde, respectively.

Macromolecular Synthesis in Leishmania donovani Amastigotes

Amastigotes (non-flagellated tissue forms) of Leishmania sp. reside and multiply within the phagolysosomes in tissue macrophages of vertebrate hosts. They are true obligate, intracellular parasites and do not persist as viable entities outside of host cells. This absolute dependence on host cells raises interesting questions about the metabolic capabilities of the amastigote stage. It is assumed that in general, intracellular stages of parasitic protozoa, exhibit reduced metabolic capabilities when compared with insect or culture forms. This hypothesis is largely untested in Leishmania sp. although there is evidence to indicate that several enzymes in amastigotes are less active than their counterparts in promastigotes (Coombs et al., 1982, Molecular and Biochemical Parasitology 5: 199-211; Meade et al., 1984, Journal of Protozoology 31: 156-161). Both forms also differ in their ability to utilize various substrates (Hart et al., 1981, Molecular and Biochemical Parasitology 4: 39-51; Hart and Coombs, 1982, Experimental Parasitology 54: 397-409). The presence of several enzymes of intermediary metabolism in amastigotes of L. donovani (Looker et al., 1983, Molecular and Biochemical Parasitology 9: 15-28; Meade et al., loc. cit.) and L. mexicana (Coombs et al., 1982, loc. cit.) suggests that they are indeed capable of carrying out independent metabolic activities. Since in vitro demonstration of enzyme activity in cell free extracts in and of itself does not ensure functional activity in vivo, the capacity of L. donovani amastigotes to catabolize and incorporate substrates or precursors into macromolecules was tested. Amastigotes of L. donovani (Sudan strain 1S) were isolated and purified from infected hamster spleens as described before (Meade et al., 1984, loc. cit.). Parasites were washed twice and resuspended in basal salts solution (Mukkada et al., 1974, Journal of Protozoology 21: 393-397), pH 5.5 to a density of 0.35 mg protein/ml. Incorporation of glucose and proline into macromolecules was determined by measuring the incorporation of label into cold trichloroacetic acid (TCA) insoluble materials (Mukkada and Simon, 1977, Experimental Parasitology 42: 87-96). Amastigote suspensions (4.9 ml) were incubated on a shaking waterbath at 30 C for 20 min for temperature equilibration. [U-_4C]D-glucose and [U-_4C]-L-proline were added to a final concentration of 0.1 mM and a specific radioactivity of 0.4 A,Ci/umole. Samples of 0.5 ml were withdrawn at various intervals and immediately added to 0.5 ml cold 10% TCA. After 15 min of extraction, 0.5 ml of the suspension was filtered through a millipore filter (0.8 ,tm porosity), washed with 3 ml basal salts and the filter pads with the cells thereon were dropped into scintillation vials containing 10 ml Bray's solution (Bray, 1960, Analytical Biochemistry 1: 279-285); radioactivity was determined in a Packard scintillation spectrometer Model 3003. Radioactivity in amastigotes after extraction with cold TCA represents incorporation of label into macromolecules. Synthesis of nucleic acids was followed by determining the incorporation of [3H] thymidine and [3H] uridine into TCA insoluble materials. Amastigote suspensions were incubated with [3H] thymidine and [3H] uridine at a final concentration of 0.4 mM and specific radioactivity of 0.6 ACi/,umole. Samples of 0.5 ml were withdrawn at intervals, filtered rapidly through glass microfiber filters (1.2 ,um porosity; Whatman GF/C) and immediately washed with 3 ml cold 10% TCA. They were then rinsed with 1 ml ethanol (95%) and transferred to vials containing 10 ml Biofluor scintillation fluid (NEN) and the radioactivity determined. Protein was determined by the method of Oyama and Eagle (1956, Proceedings of the Society of Experimental Biology and Medicine 91: 305-307). Since amastigotes were found to carry out a variety of metabolic activities optimally at pH 5.0-5.5 (data not shown) these studies were conducted at pH 5.5. Label from glucose and proline were readily incorporated into TCA insoluble materials in a time dependent fashion (Fig. 1). Though details are not shown, trapping in methyl benzethonium hydroxide (Weiss et al., 1967, Nature 213: 1020-1022) showed the evolution of significant amounts of labelled CO2 from both substrates which clearly indicate that amasti-

Effect of the dialdehyde derivative of 5′-deoxyinosine on pyrimidine deoxyribonucleoside metabolism in L1210 cells

The effects of the dialdehyde derivatives of inosine (Inox) and 5′-deoxyinosine (5′-dInox) on L1210 cells were compared. The growth of L1210 cells was inhibited to a greater extent by 5′-dInox than by Inox. The increased inhibition of L1210 cell growth by 5′-dInox was also reflected by the increased inhibition of the incorporation of precursors into RNA, DNA and proteins. Even though 5′-dInox was a more potent inhibitor, Inox accumulated in the L1210 cells to levels 4- to 5-fold greater than 5′-dInox. The metabolism of [5- 3H]deoxycytidine and [5- 3H]deoxyuridine by L1210 cells in culture, in the presence of Inox or 5′-dInox, indicated that dCMP deaminase was an intracellular site of action for 5′-dInox. The dCMP deaminase activity in cell-free extracts prepared from 5′-dInox-treated cells was reduced markedly. This decrease in activity was not reversed by increased substrate concentrations nor was the activity subject to allosteric activation by dCTP. Deoxyuridine and deoxycytidine were able to reverse the effects of 5′-dInox on the inhibition of L1210 cell growth.

A Direct Spectrophotometric Assay for Pyruvic Oxime Hydrolase

Abstract A sensitive spectrophotometric method, which directly measures the formation of pyruvic oxime from pyruvate and NH2OH in the UV, is described for the detection of pyruvic oxime hydrolase activity in biochemical systems. The method was used in an attempt to detect pyruvic oxime hydrolase activity in cell-free extracts from an Alcaligenes sp. which can grow on pyruvic oxime. Although substantial pyruvic oxime + nitrite oxidative activity was detected in cells and cell-free extract of this bacterium, catalysis of pyruvic oxime formation was not observed within the error of the method (∼20% of the uncatalyzed rate constant of 1.9x10−3 s−1 at pH 7, 22°C). The rate of pyruvic oxime oxidation by cells and cell-free extract was at least 105−106 times greater than the rate of its hydrolysis, thus implying that oxidation of pyruvic oxime need not require prior hydrolysis. The method would appear to be applicable to hydrolases directed toward a variety of oximes.

Metronidazole-inactivating activity of cell-free extracts of Streptococcus faecalis

Streptococcus faecalisの菌体を超音波処理して無細胞抽出物とし,この抽出物のMetronidazole不活化活性および不活化因子とみなされるnitroreductase活性について検討した。MetronidazoleはS. faecalisの抽出物によつて液体培地中で不活化され,GLCおよびbioassayで検出されなくなる。この不活化因子の活性は培地の種類によつて低いことがあり,培養〓液中にも認められないため,見逃がされることもあるが,培養時の酸素の有無にかかわらず産生され,Metronidazoleによつて誘導されず,同剤を含まない培地でも産生される構成的な不活化因子である。Metronidazole不活化活性は生菌の場合と同様にEscherichia coliの抽出物にも認められ,Streptococcus agalactiae, Staphylococcus aureus, Serratia marcescensの抽出物には認められない。nitroreductase活性はMetronidazole不活化活性を示す菌の抽出物に認められ,同剤を不活化しない菌の抽出物には認められない。したがつてS. faecalisの抽出物によるMetronidazoleの不活化は,抽出物のnitroreductaseがMetronidazoleのnitro基を還元するためであると考える。

T-DNA of Agrobacterium tumefaciens encodes an enzyme of cytokinin biosynthesis.

Phytohormone overproduction in crown gall tumors is due to the expression of several T-DNA genes. The data strongly suggest that the tmr gene (transcript 4) is responsible for cytokinin overproduction by encoding dimethylallyl-pyrophosphate:AMP dimethylallyltransferase (DMA transferase), an enzyme directly involved in cytokinin biosynthesis. Cell-free extracts of Escherichia coli strains containing the tmr gene from pTiA6NC had DMA transferase activity. No activity was present in the control strain containing only the plasmid vector. The cytokinins synthesized were isopentenyladenine, isopentenyladenosine, and isopentenyladenosine 5'-monophosphate. DMA transferase activity was also detected in cloned crown gall tumors incited by Agrobacterium tumefaciens wild-type A6NC and a tms mutant. Enzymatic activity in cell-free extracts of E. coli and tumors could be abolished by transposon insertion within the tmr gene.