Modulation by the ratio of cyclic AMP-dependent phosphorylation of the 50 kDa protein of rat liver phospholipid methyltransferase

The present results show that the catalytic subunit of cyclic AMP-dependent protein kinase phosphorylates the 50 kDa protein of rat liver phospholipid methyltransferase at one single site on a serine residue. Phosphorylation of this site is stimulated 2- to 3-fold by S-adenosylmethionine. S-adenosylmethionine-dependent protein phosphorylation is time- and dose-dependent and occurs at physiological concentrations. S-adenosylhomocysteine has no effect on protein phosphorylation but inhibits S-adenosylmethionine-dependent protein phosphorylation. ratios varying from 0 to 5 produce a dose-dependent stimulation of the phosphorylation of the 50 kDa protein. In conclusion, these results show, for the first time, that the ratio can modulate phosphorylation of a specific protein.     

Modulation by the ratio S-adenosylmethionineS-adenosylhomocysteine of cyclic AMP-dependent phosphorylation of the 50 kDa protein of rat liver phospholipid methyltransferase

The present results show that the catalytic subunit of cyclic AMP-dependent protein kinase phosphorylates the 50 kDa protein of rat liver phospholipid methyltransferase at one single site on a serine residue. Phosphorylation of this site is stimulated 2- to 3-fold by S-adenosylmethionine. S-adenosylmethionine-dependent protein phosphorylation is time- and dose-dependent and occurs at physiological concentrations. S-adenosylhomocysteine has no effect on protein phosphorylation but inhibits S-adenosylmethionine-dependent protein phosphorylation. $\text{S-}\text{Adenosylmethionine}\text{S-}\text{adenosylhomocysteine}$ ratios varying from 0 to 5 produce a dose-dependent stimulation of the phosphorylation of the 50 kDa protein. In conclusion, these results show, for the first time, that the ratio $\text{S-}\text{adenosylmethionine}\text{S-}\text{adenosylhomocysteine}$ can modulate phosphorylation of a specific protein.     

Suppression of methionine-induced hyperhomocysteinemia by dietary eritadenine in rats

The effect of dietary eritadenine on the plasma homocysteine concentration was investigated in methionine-induced hyperhomocysteinemic rats. The rats were fed on the control or eritadenine-supplemented (50 mg/kg) diet for 10 d. The animals were then injected with saline or methionine at a level of 100 or 300 mg/kg of body weight, and sacrificed 2 h or a more appropriate time after injection. The methionine injection increased the post-2 h concentration of plasma homocysteine in a dose-dependent manner in the control rats, this increase being significantly suppressed in the eritadenine-fed rats. This effect persisted up to 8 h after the methionine injection. The hepatic concentrations of S-adenosylmethionine and S-adenosylhomocysteine were increased by eritadenine, whereas the hepatic homocysteine concentration was inversely decreased. The cystathionine beta-synthase activity in the liver was increased by eritadenine. It is suggested from these results that eritadenine might suppress the methionine-induced increase in plasma homocysteine concentration by dual mechanisms: slowing the homocysteine production from S-adenosylhomocysteine and increasing the removal of homocysteine due to the enhanced activity of cystathionine beta-synthase.     

Basis for resistance to 3-deazaaristeromycin, an inhibitor of S-adenosylhomocysteine hydrolase, in human B-lymphoblasts

Clones resistant to 3-deazaaristeromycin, a potent inhibitor of S-adenosylhomocysteine hydrolase, were selected from a nucleoside kinase-deficient derivative of the WIL-2 human B-lymphoblastoid cell line. The resistant clones took up 3-deazaaristeromycin and showed no alteration in the level of S-adenosylhomocysteine hydrolase activity or in the sensitivity of the enzyme to inhibition by 3-deazaaristeromycin. However, they displayed markedly elevated S-adenosylmethionine content during growth in 3-deazaaristeromycin and, following prolonged selection, enhanced export of S-adenosylhomocysteine. As a result they maintained a high ratio of S-adenosylmethionine to S-adenosylhomocysteine and thus were resistant to the inhibition of S-adenosylmethionine turnover and transmethylation caused by 3-deazaaristeromycin. Expanded S-adenosylmethionine pools declined over several weeks of nonselective growth, suggesting a metabolic adaptation rather than a mutational mechanism. No alterations in S-adenosylmethionine synthetase activity were found in the 3-deazaaristeromycin-resistant clones. S-Adenosylhomocysteine export appeared to be carrier-mediated and largely unidirectional. The resistant clones showed a 5-fold increased rate of S-adenosylhomocysteine export compared with parental cells, but a similar Km for intracellular S-adenosylhomocysteine, estimated to be approximately 1 mM. Our results highlight the opposing effects of S-adenosylmethionine and S-adenosylhomocysteine on transmethylation and suggest that the ability to elevate S-adenosylmethionine pools and to export S-adenosylhomocysteine may provide for homeostatic control of transmethylation in lymphoid cells when S-adenosylhomocysteine hydrolase activity is limited.     

Effects of a phorbol ester and clomiphene on protein phosphorylation and insulin secretion in rat pancreatic islets.

The potentiation of glucose-stimulated insulin release induced by 100 nM-12-O-tetradecanoylphorbol 13-acetate (TPA) was inhibited by clomiphene, an inhibitor of protein kinase C (PK C), in a dose-dependent manner. Clomiphene at concentrations up to 50 microM had a modest inhibitory action (27%) on insulin release stimulated by 10 mM-glucose alone, but had no effect on the potentiation of insulin release induced by forskolin. Islet PK C activity, associated with a particulate fraction, was stimulated maximally by 100 nM-TPA. This stimulation was blocked by clomiphene in a dose-dependent manner, with 50% inhibition at 30 microM. Incubation of intact islets with TPA after preincubation with [32P]Pi and 10 mM-glucose to label intracellular ATP resulted primarily in enhanced phosphorylation of a 37 kDa protein (mean value, +/- S.E.M., 36,700 +/- 600 Da; n = 7). This increased phosphorylation was blocked by the simultaneous inclusion of clomiphene. Subcellular fractionation revealed the presence of the 37 kDa phosphoprotein in a 24,000 g particulate fraction of islet homogenates. Neither clomiphene nor TPA affected the rate of glucose oxidation by islets. These results show that the phosphorylation state of a 37 kDa membrane protein parallels the modulation of insulin release induced by TPA and clomiphene and support a role for PK C in the insulin-secretory mechanism.     

Selective augmentation by recombinant interferon-gamma of the intracellular content of S-adenosylmethionine in murine macrophages

Treatment of mouse peritoneal macrophages with IFN-gamma augmented the intracellular content of S-adenosylmethionine, as measured by quantitative high-performance liquid chromatography. Accumulation of S-adenosylhomocysteine, a competitive product of S-adenosylmethionine, was not detectable, either by direct measurement of absorbance or by radioisotopic techniques in IFN-gamma-treated macrophages. However, accumulation of S-adenosylhomocysteine was observed after treatment of macrophages with known inhibitors of S-adenosylhomocysteine catabolism. No inhibition of phospholipid methylation was observed upon IFN-gamma treatment, indicating that no reduction of the S-adenosylmethionine to S-adenosylhomocysteine ratio is induced by IFN-gamma in murine macrophages. The increased content of S-adenosylmethionine was associated with the acquisition of tumoricidal activity by macrophages upon IFN-gamma treatment. LPS also augmented the cellular content of S-adenosylmethionine and activated macrophages to become cytotoxic, suggesting a common mechanism of action for IFN-gamma and LPS in macrophage activation. Treatment of macrophages with cycloleucine, an agent that induces depletion of cellular S-adenosylmethionine, made the macrophages refractory to induction of cytolytic activity by IFN-gamma, suggesting a critical role for S-adenosylmethionine in macrophage activation.     

Ca2+-dependent in vitro phosphorylation of soluble proteins from germinating wheat (Triticum turgidum) endosperm

A 40000 g supernatant fraction from extracts of germinating wheat ( Triticum turgidum Desf. cv. Edmore) endosperm contains protein kinase activity that phosphorylates several endogenous proteins. In vitro incorporation of radiolabel from [³²P]-ATP into phosphoproteins was maximal in the presence of 1 m M CaCl₂ and 5 m M MgCl₂Ca²⁺ at micromolar concentrations greatly stimulated the phosphorylation of 49 and 47 kDa polypeptides and also inhibited the phosphorylation of a few specific polypeptides. The phosphorylation of the 49 and 47 kDa polypeptides was present at 2 days after seed germination and was maximal at 8 days. Quantitative protein changes were also detected during the seed germination, but differences could not be correlated with changes in protein phosphorylation. Phosphoamino acid analysis by two dimensional thin-layer electrophoresis showed that the Ca²⁺-dependent protein kinase phosphorylates a serine residue of the 47 kDa polypeptide. Ca²⁺-dependent protein kinase phosphorylates a serine residue of the 47 KDa polypeptide. Ca²⁺ dependent protein phosphorylktion was inhibited by phenothiazine-derived drugs. Addition of S-adenosylmethionine to the in vitro phosphorylation reaction specifically inhibited the Ca²⁺-dependent protein phosphorylation.     

A new method for the assay of tissue. S-adenosylhomocysteine and S-adenosylmethione. Effect of pyridoxine deficiency on the metabolism of S-adenosylhomocysteine, S-adenosylmethionine and polyamines in rat liver.

The hepatic synthesis and accumulation of S-adenosylhomocysteine, S-adenosylmethionine and polyamines were studied in normal and vitamin B-6-deficient male albino rats. A method involving a single chromatography on a phosphocellulose column was developed for the determination of S-adenosylhomocysteine and S-adenosylmethionine from tissue samples. Feeding the rat with pyridoxine-deficient diet for 3 or 6 weeks resulted in a four- to five-fold increase in the concentration of S-adenosylhomocysteine, whereas that of S-adenosylmethionine was only slighly elevated. The concentration of putrescine was decreased to half, that of spermidine was somewhat decreased and that of spermine remained fairly constant. The activities of L-ornithine decarboxylase, S-adenosyl-L-methionine decarboxylase, L-methionine adenosyltransferase and S-adenosyl-L-homocysteine hydrolase were moderately increased. S-Adenosylmethionine decarboxylase showed no requirement for pyridoxal 5'-phosphate. The major effect of pyridoxine deficiency of S-adenosylmethionine metabolism seems to be a block in the utilization of S-adenosylhomocysteine, resulting in the accumulation of this metabolite to a concentration that may inhibit biological methylation reactions.     

Photoaffinity labeling of catechol O-methyltransferase with 8-azido-S-adenosylmethionine

An in vitro system using an enzyme extract containing ATP:L-methionine S-adenosyltransferase from Escherichia coli MRE 600 cells was used to synthesize 8-azido-S-adenosyl-L-methionine from methionine and 8-azidoadenosine 5'-triphosphate. In the absence of ultraviolet light and analog can serve as a methyl donor for porcine catechol O-methyltransferase. Photolysis of 8-azido-S-adenosyl[35S]methionine in the presence of catechol O-methyltransferase results in covalent incorporation. Addition of either authentic S-adenosylmethionine or S-adenosylhomocysteine, but not adenosine 5'-monophosphate, to the photolysis reaction mixture eliminates the photoincorporation. These results indicate that the incorporation is occurring at the S-adenosylmethionine binding site in the catechol O-methyltransferase.     

S-Adenosylmethionine metabolism and its relation to polyamine synthesis in rat liver. Effect of nutritional state, adrenal function, some drugs and partial hepatectomy

S-Adenosylmethionine metabolism and its relation to the synthesis and accumulation of polyamines was studied in rat liver under various nutritional conditions, in adrenalectomized or partially hepatectomized animals and after treatment with cortisol, thioacetamide or methylglyoxal bis(guanylhydrazone) {1,1'-[(methylethanediylidine)dinitrilo]diguanidine}. Starvation for 2 days only slightly affected S-adenosylmethionine metabolism. The ratio of spermidine/spermine decreased markedly, but the concentration of total polyamines did not change significantly. The activity of S-adenosylmethionine decarboxylase initially decreased and then increased during prolonged starvation. This increase was dependent on intact adrenals. Re-feeding of starved animals caused a rapid but transient stimulation of polyamine synthesis and also increased the concentrations of S-adenosylmethionine and S-adenosylhomocysteine. Similarly, cortisol treatment enhanced the synthesis of polyamines, S-adenosylmethionine and S-adenosylhomocysteine. Feeding with a methionine-deficient diet for 7-14 days profoundly increased the concentration of spermidine, whereas the concentrations of total polyamines and of S-adenosylmethionine showed no significant changes. The results show that nutritional state and adrenal function play a significant role in the regulation of hepatic metabolism of S-adenosylmethionine and polyamines. They further indicate that under a variety of physiological and experimental conditions the concentrations of S-adenosylmethionine and of total polyamines remain fairly constant and that changes in polyamine metabolism are not primarily connected with changes in the accumulation of S-adenosylmethionine or S-adenosylhomocysteine.     

Serine phosphorylation of protein tyrosine phosphatase (PTP1B) in HeLa cells in response to analogues of cAMP or diacylglycerol plus okadaic acid

The major intracellular protein tyrosine phosphatase (PTP1B) is a 50kDa protein, localized to the endoplasmic reticulum. This PTP is recovered in the particulate fraction of mamalian cells and can be solubilized as a complex of 150 kDa by extraction with non-ionic detergents. Previous work from this laboratory implicated phosphorylation of serine/threonine residues in the regulation of this PTP. Activity was several-fold higher in cells treated with activators of cAMP-dependent or Ca²⁺/phospholipid-dependent protein kinases or inhibitors of protein phosphatase 2A. Here we show that these treatments result in more than an 8-fold increase in the phosphorylation of the 50kDa PTP catalytic subunit within the 150kDa form of the phosphatase in HeLa cells. The phosphorylation occurred exclusively on serine residues, and the same tryptic and cyanogen bromide,³²P-phosphopeptides were recovered in the PTP from control and stimulated cells. Either multiple kinases phosphorylate a common site in the PTP1B, or a single kinase is activated downstream of cAMP- and Ca²⁺/phospholipid-dependent kinases. The results indicate that phosphorylation of a serine residue in the segment 283–364, probably serine 352 in the sequence Lys-Gly-Ser-Pro-Leu, occurs in response to cell stimulation. Phosphorylation in this region of PTP1B, between the N-terminal catalytic domain and the C-terminal membrane localization segment, is proposed to regulate phosphatase activity.     

Phosphorylation reactions in bovine rod outer segments studied by 32P-labelling of intact retina

The protein phosphorylation pattern in the intact bovine retina has been investigated by labelling with 32P-phosphate under incubation conditions that preserve the electrical photoresponse of the photoreceptor cells. The phosphorylation of rod outer segment proteins was analysed after isolation of outer segments from the labelled retina. The global influence of light, Ca2+ and the phosphodiesterase inhibitor, isobutylmethylxanthine, on protein phosphorylation in rod outer segments was analysed. A 12 kDa protein is the most prominent phosphorylated species in the intact bovine retina. Its phosphorylation is increased by light and/or Ca2+. Evidence is presented that this strongly phosphorylated protein is not located in the outer segment, and we suggest that it may be a synaptic protein. Retinal rod outer segment membrane proteins with apparent molecular weights of 245, 226, 125, 110, 50, 46, 38 and 20 all show light-stimulated phosphorylation. Lowering the extracellular Ca2+ levels results in a decrease of the phosphorylation level of some of these proteins, viz. at 125, 50, 38 and probably at 20 kDa. Such proteins, whose phosphorylation level is influenced both by light and by elevated Ca2+, are candidates for mediators of phototransduction. The phosphorylated species at 245, 226, 110, 50 and 20 kDa are enriched in rod outer segment plasma membrane preparations. These protein species could participate in the light-regulated modulation of the Na+-conductance of the plasma membrane.     

S-adenosyl-L-methionine:thioether S-methyltransferase, a new enzyme in sulfur and selenium metabolism

The final urinary excretion product of selenium detoxification is trimethylselenonium ion. An assay has been developed for the enzyme, S-adenosylmethionine:thioether S-methyltransferase, responsible for this final methylation reaction. This assay employed high pressure liquid chromatography separation and quantitation of the trimethylselenonium ion produced by thioether methyltransferase acting on S-adenosylmethionine and dimethyl selenide. The enzyme was shown to reside primarily in the cytosol of mouse lung (30 pmol/mg protein/min) and liver (7 pmol/mg protein/min). Purification from mouse lung to a preparation that exhibited a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was achieved by DEAE, gel filtration, and chromatofocusing chromatographies. Thioether methyltransferase is monomeric with a molecular weight of 28,000 and has a pI of 5.3. The pH optimum was 6.3, and Km values for dimethyl selenide and S-adenosylmethionine were 0.4 and 1.0 microM, respectively. The enzyme was inhibited 50% by 25 microM sinefungin, an analog of S-adenosylmethionine, or 40 microM S-adenosylhomocysteine, the reaction product. Pure thioether methyltransferase methylated selenium in dimethyl selenide, tellurium in dimethyl telluride, and S in dimethyl sulfide and many other thioethers. These data suggest a general role for this novel enzyme in the synthesis of onium compounds with increased aqueous solubility helpful in their excretion.     

The kinetic properties and reaction mechanism of histamine methyltransferase from human skin.

The substrate kinetic properties of histamine methyltransferase from human skin were studied at limiting concentrations of both histamine and S-adenosylmethionine. Substrate inhibition by histamine was observed at concentrations above 10 microM. Primary plots showed evidence of a sequential reaction mechanism. The Michaelis constants were derived from secondary plots of slopes from the primary plots ([S]/v versus [S]) versus reciprocal of the second substrate concentration. The mean Km values for histamine and S-adenosylmethionine were 4.2 and 1.8 microM respectively. Histamine in concentrations of 25-100 microM inhibited enzyme activity uncompetitively with respect to S-adenosylmethionine. No substrate inhibition was observed with S-adenosylmethionine. To elucidate the reaction mechanism further, inhibition by the two products, S-adenosylhomocysteine and 1-methylhistamine, was studied. S-Adenosylhomocysteine inhibited non-competitively with respect to histamine and competitively with respect to S-adenosylmethionine. 1-Methylhistamine inhibited non-competitively with respect to histamine and to S-adenosylmethionine. These results are interpreted as providing evidence for an ordered sequential Bi Bi reaction mechanism, with the methyl-group donor S-adenosylmethionine as the first substrate that adds to the enzyme and histamine as the second substrate. 1-Methylhistamine is the first product to leave the enzyme and S-adenosylhomocysteine is the second. The results are discussed in terms of the possible role that this enzyme could play in the modulation of histamine-mediated reactions in skin.     

Role of calcium on protein phosphorylation in collagen-stimulated platelets. Effect of a new dihydropyridine derivative

We investigated the effect of extracellular calcium on protein phosphorylation stimulated by collagen in rabbit platelets. We found that collagen-induced increase in 40 kDa protein phosphorylation was maximum at 2 mM Ca2+, and was evident in buffer with zero Ca2+ but not in the presence of EGTA. We also studied the effects of a new dihydropyridine derivative, which has antithrombotic properties, on protein phosphorylation induced by collagen. This compound inhibited the phosphorylation of 40 kDa and 20 kDa protein independently of the extra-cellular Ca2+. The inhibitory effect was dose-dependent but not time-dependent and was more evident when the drug was added before or simultaneously with collagen.     

Protein kinase C-alpha produces reciprocal effects on the phorbol ester stimulated tyrosine phosphorylation of a 50 kDa kinase in Jurkat cells.

A detergent extract isolated from the enriched fraction of integral membrane proteins of Jurkat cells showed an enhanced tyrosine phosphate level when phosphorylated in the presence of phorbol 12-myristate 13-acetate (TPA) and phorbol 12,13-dibutyrate (PDBu). The enhanced tyrosine phosphorylation was observed when the reaction time exceeded 6 min; at shorter incubation times, however, TPA inhibited tyrosine phosphorylation. When the reaction proceeded for a constant time period longer than 6 min and phorbol esters were added at different times after the start of the reaction, two phases of an enhanced tyrosine phosphorylation of a 50 kDa protein were observed. An increased phosphorylation of the 50 kDa protein was correlated with an enhanced phosphorylation of poly(Glu4,Tyr1). The two phases of enhanced phosphorylation differed in their TPA and PDBu requirement and in the proteins that were tyrosine phosphorylated. Studies with protein kinase C (PKC) inhibitors showed a negatively correlated effect on the enhanced tyrosine phosphorylation in phase I; tyrosine phosphorylation was further augmented. In phase II the regulation of tyrosine phosphorylation correlated with the efficiency of the PKC inhibitors on the alpha-isoform of PKC which was found in the cell extract. Separation of the proteins present in the investigated cell extract by gel filtration revealed a co-migration of the alpha-PKC and the 50 kDa protein. The metabolic labeling of intact Jurkat cells with 32Pi indicated that phorbol esters are also able to induce tyrosine phosphorylation of the 50 kDa protein underin vivo conditions. These data suggest an activation of two different tyrosine phosphorylation pathways by phorbol esters involving tyrosine phosphorylation/autophosphorylation of a 50 kDa kinase, as confirmed by 5'-p-fluorosulfonylbenzoyladenosine (FSBA) labeling, that are accurately regulated by alpha-PKC.     

Crystal structure of SAM-dependent O-methyltransferase from pathogenic bacterium Leptospira interrogans

The S-adenosylmethionine (SAM)-dependent O-methyltransferase from Leptospira interrogans (LiOMT) expressed by gene LA0415 belongs to the Methyltransf_3 family (Pfam PF01596). In this family all of the five bacterial homologues with known function are reported as SAM-dependent O-methylstransferases involved in antibiotic production. The crystal structure of LiOMT in complex with S-adenosylhomocysteine reported here is the first bacterial protein structure in this family. The LiOMT structure shows a conserved SAM-binding region and a probable metal-dependent catalytic site. The molecules of LiOMT generate homodimers by N-terminal swapping, which assists the pre-organization of the substrate-binding site. Based on the sequence and structural analysis, it is implied by the catalytic and substrate-binding site that the substrate of LiOMT is a phenolic derivative, which probably has a large ring-shaped moiety.     

Different effects of two proteinase inhibitors on insulin-induced cellular responses in rat adipocytes

Among various proteinase inhibitors, N-acetyl-L-tyrosine ethyl ester (ATEE), a chymotrypsin substrate analog, and N alpha-p-tosyl-L-lysine chloromethyl ketone (TLCK), a trypsin inhibitor, showed significant inhibitory effects on insulin stimulated glucose transport in rat adipocytes. ATEE did not affect insulin binding, but inhibited insulin internalization. In intact adipocytes, ATEE inhibited tyrosine phosphorylation of the beta-subunit of the insulin receptor, a 170 kDa protein and a 60 kDa protein at almost the same concentration (ID50 = 0.24 +/- 0.05 mM, n = 4, mean +/- S.E.), but in a plasma membrane fraction, ATEE did not appreciably inhibit the tyrosine phosphorylation of the beta-subunit of the insulin receptor, TLCK did not inhibit insulin binding. At 0.25 mM, TLCK did not inhibit insulin internalization, but inhibited 70% of the insulin-stimulated glucose transport (ID50 = 0.19 +/- 0.02 mM, n = 7). TLCK inhibited insulin internalization at more than 0.25 mM. TLCK did not inhibit the tyrosine phosphorylation of the beta-subunit of the insulin receptor in intact cells or in the plasma membrane fraction. In intact cells, TLCK inhibited the phosphorylation of the 60 kDa protein and simultaneously it stimulated the phosphorylation of the 170 kDa protein more than 3-fold. These results indicate that there are at least two sites in the insulin-induced signal transduction pathway where proteinase inhibitors act to suppress the insulin signal transduction. A major ATEE site is very close to phosphorylation of the beta-subunit of the insulin receptor. On the other hand, TLCK inhibits a step(s) in the signal transduction pathway after the insulin receptor but before the glucose transporter.     

The decarboxylation of S-adenosylmethionine by detergent-treated extracts of rat liver

1. The production of (14)CO(2) from S-adenosyl[carboxyl-(14)C]methionine by rat liver extracts was investigated. It was found that, in addition to the well-known cytosolic putrescine-activated S-adenosylmethionine decarboxylase, an activity carrying out the production of (14)CO(2) could be extracted from a latent, particulate or membrane-bound form by treatment with buffer containing 1% (v/v) Triton X-100 [confirming the report of Sturman (1976) Biochim. Biophys. Acta428, 56-69]. 2. The formation of (14)CO(2) by such detergent-solubilized extracts differed from that by cytosolic S-adenosylmethionine decarboxylase in a number of ways. The reaction by the solubilized extracts did not require putrescine and was not directly proportional to time of incubation or the amount of protein added. Instead, activity a showed a distinct lag period and was much greater when high concentrations of the extracts were used. The cytosolic S-adenosylmethionine decarboxylase was activated by putrescine, showed strict proportionality to protein added and the reaction proceeded at a constant rate. Cytosolic activity was not inhibited by homoserine or by S-adenosylhomocysteine, whereas the Triton-solubilized activity was strongly inhibited. 3. By using an acetone precipitate of Triton-treated homogenates as a source of the activity, it was found that decarboxylated S-adenosylmethionine was not present among the products of the reaction, although 5'-methylthioadenosine and 5-methylthioribose were found. Such extracts were able to produce (14)CO(2) when incubated with [U-(14)C]-homoserine, and (14)CO(2) production was greater when S-adenosyl[carboxyl-(14)C]methionine that had been degraded by heating at pH6 at 100 degrees C for 30min (a procedure known to produce mainly 5'-methylthioadenosine and homoserine lactone) was used as a substrate than when S-adenosyl[carboxyl-(14)C]methionine was used. 4. These results indicate that the Triton-solubilized activity is not a real S-adenosylmethionine decarboxylase, but that (14)CO(2) is produced via a series of reactions involving degradation of the S-adenosyl-[carboxyl-(14)C]methionine. It is probable that this degradation can occur via several pathways. Our results would suggest that part of the reaction occurs via the production of S-adenosylhomocysteine, which can then be converted into 2-oxobutyrate via the transsulphuration pathway, and that part occurs via the production of homoserine by an enzyme converting S-adenosylmethionine into 5'-methylthioadenosine and homoserine lactone.     

Purification and characterization of methyl-accepting chemotaxis protein methyltransferase I in Bacillus subtilis.

A methyltransferase that methylates one of the proteins involved in chemotactic adaptation to sensory stimuli in Bacillus subtilis was purified to homogeneity. The enzyme utilizes S-adenosylmethionine as donor for a methyl group that is transferred to a glutamate residue in a 69 000-mol.wt. membrane protein and also to a protein of 19 000 mol.wt. The molecular weights of the denatured enzyme by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and of the native enzyme by gel-filtration chromatography both show the protein to be a 44 000-mol.wt. monomer. Isoelectric focusing of the purified methyltransferase showed the protein to be a single species with isoelectric point pI 5.4. On the basis of a molecular weight of 44 000, the molar absorption coefficient at 262 nm of the enzyme is 10.9 x 10(4) M-1 . cm-1. The Km of the enzyme for S-adenosylmethionine is about 2 microM. The Ki for S-adenosylhomocysteine is about 0.2 microM. Ca2+ is a competitive inhibitor of methylation, with a Ki of 0.065 microM. The enzyme methylates membranes from the wild-type more efficiently than membranes isolated from a mutant strain defective in chemotaxis. The enzyme is unable to methylate Escherichia coli membranes.