Rapid and convenient method for the assay of aminopropyltransferases.

A new method for the assay of aminopropyltransferase activity is described. The method measures the formation of [methyl-14C]methylthioadenosine from decarboxylated S-adenosyl[methyl-14C]methionine in the presence of an amine acceptor. When used with extracts from rat ventral prostate, kidney, liver or brain, and with putrescine or spermidine as amines, the method gave results in excellent agreement with those obtained by the much more time-consuming conventional method. It was found that 1,3-diamino-propane and 1,8-diamino-octane were not acceptors for the prostatic enzyme fraction, but 1,5-diaminopentane (cadaverine) was active and 1,9-diaminononane and 1,12-diaminododecane also lead to the production of [methyl-14C]methylthioadenosine.     

Intermediates in the recycling of 5-methylthioribose to methionine in fruits

The recycling of 5-methylthioribose (MTR) to methionine in avocado (Persea americana Mill, cv Hass) and tomato (Lycopersicum esculentum Mill, cv unknown) was examined. [¹⁴CH₃]MTR was not metabolized in cell free extract from avocado fruit. Either [¹⁴CH₃]MTR plus ATP or [¹⁴CH₃]5-methylthioribose-1-phosphate (MTR-1-P) alone, however, were metabolized to two new products by these extracts. MTR kinase activity has previously been detected in these fruit extracts. These data indicate that MTR must be converted to MTR-1-P by MTR kinase before further metabolism can occur. The products of MTR-1-P metabolism were tentatively identified as α-keto-γ-methylthiobutyric acid (α-KMB) and α-hydroxy-γ-methylthiobutyric acid (α-HMB) by chromatography in several solvent systems. [³⁵S]α-KMB was found to be further metabolized to methionine and α-HMB by these extracts, whereas α-HMB was not. However, α-HMB inhibited the conversion of α-KMB to methionine. Both [U-¹⁴C]α-KMB and [U-¹⁴C]methionine, but not [U-¹⁴C]α-HMB, were converted to ethylene in tomato pericarp tissue. In addition, aminoethoxyvinylglycine inhibited the conversion of α-KMB to ethylene. These data suggest that the recycling pathway leading to ethylene is MTR → MTR-1-P → α-KMB → methionine → S-adenosylmethionine → 1-aminocyclopropane-1-carboxylic acid → ethylene.     

Transmethylation, transsulfuration, and aminopropylation reactions of S-adenosyl-L-methionine in vivo

S-Adenosylmethionine (AdoMet) is metabolized through three main pathways, i.e. (a) transfer of its methyl group to a variety of methyl acceptors, (b) decarboxylation followed by aminopropylation leading to polyamine synthesis, and (c) cleavage of the bond between the sulfur atom and carbon 4 of the amino acid chain, resulting in formation of methylthioadenosine and homoserine thiolactone. In this study the metabolism of AdoMet through these pathways was studied after intravenous administration to rats of [1-14C]-, [3,4-14C]-, [methyl-14C]-, and [35S]AdoMet at various doses. The relative utilization of AdoMet and methionine was also investigated. The results show that intravenously administered AdoMet is efficiently metabolized in vivo up to the highest tested dose (250 mumol X kg-1 body weight), about two-thirds of the metabolized compound being utilized via transmethylation and cleavage to methylthioadenosine and one-third via decarboxylation. The efficient incorporation of the methyl group of AdoMet into muscle creatine indicates unambiguously that the compound is taken up and metabolized by the liver. Moreover, intravenously administered AdoMet is shown to be a better precursor than methionine both in creatine formation and in the utilization of the sulfur atom in transsulfuration reactions.     

Biosynthesis of the polyoxins, nucleoside peptide antibiotics: a new metabolic role for L-isoleucine as a precursor for 3-ethylidene-L-azetidine-2-carboxylic acid (polyoximic acid).

The biosynthetic origin of the carbon skeleton of 3-ethylidene-L-azetidine-2-carboxylic acid (polyoximic acid) is described. This unique cyclic amino acid is the C terminus of the nucleoside peptide antibiotics, the polyoxins, elaborated by Streptomyces cacaoi var, asoensis. In vivo experiments show that 14-C from [1-14-C]isoleucine, [U-14-C]isoleucine, [1-14-C]methionine, [U-14-C]methionine, [U-14-C]threonine, and [1-14-C]glutamate is incorporated into polyoximic acid; however, 14-C from [5-14-C]glutamate and [methyl-14-C]methionine is not incorporated. The distribution of 14-C in polyoximic acid clearly shows that the intact carbon skeleton of L-isoleucine is utilized directly. The incorporation of 14-C from [U-14-C]methionine, [U-14-C]threonine, and [1-14-CA1glutamate into polyoximic acid occurred only after their conversion to isoleucine via 2-ketobutyrate. A scheme is presented in which either of the two beta-unsaturated amino acids isolated from Bankera fuligineoalba, L-2-amino-3-hydroxymethyl-3-pentenoic acid or L-2-amino-3-formyl-3-penetenoic acid, is regarded as a possible intermediate amino acid between isoleucine and polyoximic acid.     

An assay for adenosine 5'-diphosphate (ADP)-glucose pyrophosphorylase that measures the synthesis of radioactive ADP-glucose with glycogen synthase

Adenosine 5'-diphosphate (ADP)-glucose pyrophosphorylase (ADP-Glc PPase) catalyzes the conversion of glucose 1-phosphate and adenosine 5'-triphosphate to ADP-glucose and pyrophosphate. We present a radioactive assay of this enzyme with a higher signal/noise ratio. After stopping the reaction that uses [14C]glucose 1-phosphate as a substrate, the ADP-[14C]glucose formed as a product is converted to [14C]glycogen by the addition of glycogen synthase and nonradioactive glycogen as primer. The final product is precipitated and washed, and the radioactivity is measured in a scintillation counter. The [14C]glucose 1-phosphate that did not react is easily eliminated during the washes. We have found that this assay produces much lower blanks than previously described radioactive methods based on binding of ADP-[14C]glucose to O-(diethylaminoethyl)-cellulose paper. In addition, we tested the kinetic parameters for the effectors of the Escherichia coli ADP-Glc PPase and both assays yielded identical results. The presented method is more suitable for Km or S(0.5) determinations of ADP-Glc PPases having high apparent affinity for glucose 1-phosphate. It is possible to use a higher specific radioactivity to increase the sensitivity at lower concentrations of [14C]glucose 1-phosphate without compromising the blanks obtained at higher concentrations.     

Production of [14C]fumonisin B1 by Fusarium moniliforme MRC 826 in corn cultures.

Kinetics of growth and fumonisin production by Fusarium moniliforme MRC 826 in corn "patty" cultures were investigated, and a technique was developed for the production of [14C]fumonisin B1 ([14C]FB1) by using L-[methyl-14C]methionine as the precursor. A significant (P < 0.01) correlation exists between fungal growth and FB1 (r = 0.89) and FB2 (r = 0.87) production in corn patties, beginning after 2 days and reaching the stationary phase after 14 days of incubation. [14C]FB1 was produced by adding L-[methyl-14C]methionine daily to cultures during the logarithmic phase of production. Incorporation of the isotope occurred at C-21 and C-22 of the fumonism molecule and was enhanced in the presence of unlabeled L-methionine. Although the concentration of exogenous unlabeled methionine is critical for incorporation of the 14C label, optimum incorporation was achieved by adding 50 mg of unlabeled L-methionine and 200 mu Ci of L-[methyl-14C]methionine to a corn patty (30 g) over a period of 9 days, yielding [14C]FB1 with a specific activity of 36 mu Ci/mmol.     

Non-enzymatic DNA methylation by S-adenosylmethionine results in the formation of minor thymine residues and 5-methylcytosine from cytosine

It was found that nonenzymatic DNA methylation proceeds in water solution in the presence of S-adenosylmethionine (AdoMet). The main reaction products are thymine and 5-methylcytosine residues. It was shown that labelled thymine residues are formed also upon DNA incubation in the presence of [methyl-14C]methionine as well as [methyl-14C]cobalamine. Only cytosine reacts with AdoMet resulting in thymine production. AdoMet may be a potential mutagen that induces GC----AT transitions during DNA replication in the cell.     

Conversion of 5-S-methyl-5-thio-D-ribose to methionine in Klebsiella pneumoniae. Stable isotope incorporation studies of the terminal enzymatic reactions in the pathway

Extracts of Klebsiella pneumoniae convert 5-S-methyl-5-thio-D-ribose (methylthioribose) to methionine and formate. To probe the terminal steps of this biotransformation, [1-13C]methylthioribose has been synthesized and its metabolism examined. When supplemented with Mg2+, ATP, L-glutamine, and dioxygen, cell-free extracts of K. pneumoniae converted 50% of the [1-13C]methylthioribose to [13C]formate. The formation of [13C]formate was established by 13C and 1H NMR spectroscopy studies of the purified formate, and by 13C and 1H NMR spectroscopy and mass spectrometry studies of its p-phenylphenacyl derivative. By contrast, no incorporation of label from [1-13C]methylthioribose into the biosynthesized methionine was detected by either mass spectrometry or 13C and 1H NMR spectroscopy. The most reasonable interpretation of these results is that C-1 of methylthioribose is converted directly to formate concomitant with the conversion of carbon atoms 2-5 to methionine. The penultimate step in the conversion of methylthioribose to methionine and formate is an oxidative carbon-carbon bond cleavage reaction in which an equivalent of dioxygen is consumed. To investigate the fate of the dioxygen utilized in this reaction, the metabolism of [1-13C]methylthioribose in the presence of 18O2 was also examined. Mass spectrometry revealed the biosynthesis of substantial amounts of both [18O1]methionine and [13C, 18O1]formate under these conditions. These results suggest that the oxidative transformation in the conversion of methylthioribose to methionine and formate may be catalyzed by a novel intramolecular dioxygenase. A mechanism for this dioxygenase is proposed.     

Methionine metabolism in apple tissue: implication of s-adenosylmethionine as an intermediate in the conversion of methionine to ethylene

If S-adenosylmethionine (SAM) is the direct precursor of ethylene as previously proposed, it is expected that 5′-S-methyl-5′-thioadenosine (MTA) would be the fragment nucleoside. When [Me-¹⁴C] or [³⁵S]methionine was fed to climacteric apple (Malus sylvestris Mill) tissue, radioactive 5-S-methyl-5-thioribose (MTR) was identified as the predominant product and MTA as a minor one. When the conversion of methionine into ethylene was inhibited by _l-2-amino-4-(2′-aminoethoxy)-trans-3-butenoic acid, the conversion of [³⁵S] or [Me¹⁴C]methionine into MTR was similarly inhibited. Furthermore, the formation of MTA and MTR from [³⁵S]methionine was observed only in climacteric tissue which produced ethylene and actively converted methionine to ethylene but not in preclimacteric tissue which did not produce ethylene or convert methionine to ethylene. These observations suggest that the conversion of methionine into MTA and MTR is closely related to ethylene biosynthesis and provide indirect evidence that SAM may be an intermediate in the conversion of methionine to ethylene.     

Identification of a tyrosine residue in rat guanidinoacetate methyltransferase that is photolabeled with S-adenosyl-L-methionine.

Exposure of rat guanidinoacetate methyltransferase to ultraviolet light in the presence of S-adenosyl-L-[methyl-3H]methionine ([methyl-3H]AdoMet) results in covalent linking of radioactivity to the enzyme protein. The incorporation of radioactivity shows no lag and is linear with respect to time up to 1 h. The photolabeling is saturable with [methyl-3H]AdoMet, and the binding constant of the enzyme for AdoMet determined in this experiment is similar to that obtained by equilibrium dialysis. Low concentrations of competitive inhibitors S-adenosyl-L-homocysteine and sinefungin effectively prevent the photoinduced labeling by AdoMet. Although guanidinoacetate methyltransferase is irreversibly inactivated upon ultraviolet irradiation in the absence of AdoMet, the enzyme inactivated by 1-h exposure to ultraviolet irradiation has been shown to bind AdoMet with an affinity identical to that of the native enzyme. These results indicate that photolabeling occurs at the active site. Following proteolysis of the [methyl-3H]-AdoMet-labeled enzyme with chymotrypsin, a radioactive peptide is isolated having a sequence Asp-Thr-X-Pro-Leu-Ser-Glu-Glu-Thr-Trp. The peptide corresponds to residues 134-143, with X being modified Tyr-136. The same peptide is photolabeled when [carboxy-14C]AdoMet is used. High-performance liquid chromatography of this peptide after acid hydrolysis and phenyl isothiocyanate derivatization suggests that the entire molecule of AdoMet is attached to Tyr-136.     

Expression of human cathepsin D in Xenopus oocytes: phosphorylation and intracellular targeting

We have obtained expression of a cDNA clone for human cathepsin D in Xenopus laevis oocytes. Biosynthetic studies with [35S]methionine labeling demonstrated that most of the cathepsin D remained intracellular and underwent proteolytic cleavage, converting a precursor of Mr 47,000 D to a mature form of Mr 39,000 D with processing intermediates of Mr 43,000-41,000 D. greater than 90% of the cathepsin D synthesized by oocytes bound to a mannose 6-phosphate (Man-6-P) receptor affinity column, indicating the presence of phosphomannosyl residues. An analysis of [2-3H]mannose-labeled oligosaccharides directly demonstrated phosphomannosyl residues on cathepsin D. Sucrose-gradient fractionation, performed to define the membranous compartments that cathepsin D traversed during its biosynthesis, demonstrated that cathepsin D is targeted to a subpopulation of yolk platelets, the oocyte equivalent of a lysosome. Xenopus oocytes were able to endocytose lysosomal enzymes from the medium and this uptake was inhibited by Man-6-P, thus demonstrating the presence of Man-6-P receptors in these cells. Therefore, the entire Man-6-P dependent pathway for targeting of lysosomal enzymes is present in the oocytes. Xenopus oocytes should be a useful system for examining signals responsible for the specific targeting of lysosomal enzymes to lysosomes.     

Uridine phosphorylase from Acholeplasma laidlawii: purification and kinetic properties.

Uridine phosphorylase was purified 1,370-fold from sonicated extracts of Acholeplasma laidlawii by ammonium sulfate precipitation, DEAE-Sephadex column chromatography, hydroxylapatite chromatography, and Sephadex G-200 fractionation. The molecular weight of the enzyme as determined by gel filtration was approximately 65,000. [U-14C]ribose-1-phosphate (Rib-1-P), prepared enzymatically from [U-14C]inosine, was utilized in initial velocity studies of uridine synthesis, which indicated a sequential reaction with a KmUra of 110 microM and a KmRib-1-P of 17 microM. The kinetics of uridine cleavage were assessed at a saturating cosubstrate concentration, resulting in a KmUrd of 170 microM and a KmPi of 120 microM. These results indicate that an intracellular flux from uracil to uridine is kinetically feasible. However, such flux would be metabolically unproductive, since the low affinity of uridine kinase (KmUrd = 3.2 mM) precludes the operation of uridine phosphorylase and uridine kinase in tandem to convert uracil to UMP. We conclude that uridine phosphorylase performs only a catabolic function in A. laidlawii.     

Influenza viral mRNA contains internal N6-methyladenosine and 5''-terminal 7-methylguanosine in cap structures.

Influenza viral complementary RNA (cRNA), i.e., viral mRNA was radioactive when purified from the cytoplasmic fraction of cordycepin-treated canine kidney cells that were incubated with [methyl-3H]methionine during infection. Approximately 55 to 60% of the methyl-3H radioactivity was in internal N6-methyladenosine, a feature distinguishing this mRNA from those viral mRNA's that are known to be synthesized in the cytoplasm. The remaining methyl-3H radioactivity was in 5'-terminal cap structures that consisted of 7-methylguanosine in pyrophosphate linkage to 2'-o-methyladenosine, N6, 2'-O-dimethyladenosine, or 2'-O-methylguanosine. Methylated adenosine was the predominant penultimate nucleoside in caps, suggesting that cRNA synthesis in infected cells initiates preferentially with adenosine at the 5' end. In contrast to cRNA, influenza virion RNA segments extracted from purified virus contained mainly 5'-terminal ppA and no detectable cap structures.     

Radiometric measurement of phosphoribosylpyrophosphate and ribose 5-phosphate by enzymatic procedures.

Methods for the measurement of phosphoribosylpyrophosphate (PRPP) and ribose 5-phosphate (R-5-P) in tissues have been developed. The lability of these compounds during tissue extraction and the recovery of standards from tissue preparations have been examined. Enzymatic conversion of phosphoribosylpyrophosphate to [14C]AMP in the presence of labeled adenine or formation of [14C]GMP ([14C]IMP) in the presence of labeled guanine or hypoxanthine was accomplished in the first step. In the second step, the labeled product was separated from the substrate. For the measurement of R-5-P, the first step included phosphoribosylpyrophosphate synthetase, as well as the appropriate substrate and effector (ATP and Pi), in combination with adenine phosphoribosyl transferase. The product [14C]AMP was measured in three ways: (1) HPLC separation with an on-line radioisotope detector; (2) butanol extraction of the labeled base, and measurement of an aliquot of the aqueous phase in a scintillation counter; (3) filtration of the incubation mixture with chromatographic filter paper disks, which were then counted in a scintillation counter. When [14C]guanine was the substrate, HPLC separation was used because the butanol or paper separation was not adequate. Measurement of 5-125 pmol of PRPP or R-5-P gave a linear response.     

NADPH-dependent generation of a cytosolic dithiol which activates hepatic iodothyronine 5''-deiodinase. Demonstration by alkylation with iodoacetamide.

We have assessed a previously proposed mechanism mediating 5'-deiodinase activation involving enzymic reduction of disulphides to thiols in non-glutathione cytosolic components of Mr approx. 13,000 (Fraction B) catalysed by NADPH in the presence of other cytosolic components of Mr greater than 60,000 (Fraction A). The extent of Fraction B reduction under various experimental conditions was monitored by determining the amount of 14C incorporated into chromatographically isolated Fractions B and A after their alkylation with iodo[14C]acetamide. Incorporation of 14C into B was found to require the simultaneous presence of NADPH and A, to be directly proportional to the concentration of NADPH added, and to be unaffected by either propylthiouracil or iopanoate. Activation of 5'-deiodinase attainable using B after its partial reduction by various concentrations of NADPH and subsequent alkylation with non-radioactive iodoacetamide was inversely proportional to the previously added concentration of NADPH. Fraction B was stable at 100 degrees C for 5 min, while similar heat treatment of Fraction A or omission of NADPH resulted in a complete loss of 14C incorporation. A greater than 90% reduction in iodo[14C]acetamide incorporation was revealed when 0.2 mM-sodium arsenite was added after enzymic reduction of B, as well as when NADPH was replaced by NADH. Fraction B could be labelled more extensively after reduction non-specifically, with dithiothreitol or NaBH4, but not by GSH. These observations provide strong evidence for the presence in vivo of a cytosolic disulphide (DFBS2) in Fraction B which can be reduced enzymically to a dithiol [DFB(SH)2] by NADPH and cytosolic components in Fraction A. The degree of activation of hepatic 5'-deiodinase correlated with the amount of available (unalkylated) Fraction B.     

Redox regulation of methylthioadenosine phosphorylase in liver cells: molecular mechanism and functional implications

MTAP (5'-methylthioadenosine phosphorylase) catalyses the reversible phosphorolytic cleavage of methylthioadenosine leading to the production of methylthioribose-1-phosphate and adenine. Deficient MTAP activity has been correlated with human diseases including cirrhosis and hepatocellular carcinoma. In the present study we have investigated the regulation of MTAP by ROS (reactive oxygen species). The results of the present study support the inactivation of MTAP in the liver of bacterial LPS (lipopolysaccharide)-challenged mice as well as in HepG2 cells after exposure to t-butyl hydroperoxide. Reversible inactivation of purified MTAP by hydrogen peroxide results from a reduction of V(max) and involves the specific oxidation of Cys(136) and Cys(223) thiols to sulfenic acid that may be further stabilized to sulfenyl amide intermediates. Additionally, we found that Cys(145) and Cys(211) were disulfide bonded upon hydrogen peroxide exposure. However, this modification is not relevant to the mediation of the loss of MTAP activity as assessed by site-directed mutagenesis. Regulation of MTAP by ROS might participate in the redox regulation of the methionine catabolic pathway in the liver. Reduced MTA (5'-deoxy-5'-methylthioadenosine)-degrading activity may compensate for the deficient production of the precursor S-adenosylmethionine, allowing maintenance of intracellular MTA levels that may be critical to ensure cellular adaptation to physiopathological conditions such as inflammation.     

Sphingomyelin synthesis in Fasciola hepatica

Whole worms and/or homogenates of F. hepatica incorporate label from cytidine-5-diphospho[methyl-14C]choline,[1-14C]palmitoylCoA,[U- 14C]serine,[2-14C]methionine, [U-14C]glycine, [U-14C]threonine and [U-14C]aspartate into the various intermediates of sphingomyelin synthesis (ketosphinganine, sphinganine, sphingosine, ceramide and sphingomyelin). This suggests that sphingomyelin synthesis in F. hepatica occurs by a pathway similar to that found in mammals. However, there is some evidence that in F. hepatica 3-ketosphinganine may be N-acylated prior to reduction and dehydrogenation.     

The radiochemical assay for juvenile hormone biosynthesis in insects: problems and solutions

Measurement of juvenile hormone (JH) production using the radiochemical assay (RCA) for JH biosynthesis and release is usually a reliable and precise technique. However problems with radiolabeled precursors and misunderstanding of the data, the techniques and the calculations have contributed towards uncertainty with respect to published experimental results. Problems with the purity of [methyl-3H]-methionine or determination of its specific radioactivity have had detrimental effects on the reliability of results using the RCA. Proper control procedures and the use of 14C/3H-double-label RCA can be useful in detecting irregularities in the experimental results, and in determining contributing factors to any problems. The use of [methyl-14C]-methionine and an awareness of normally expected RCA values can also assist the researcher in checking the validity of results. The radiolabeled methyl moiety of methionine is incorporated into JH without discrimination relative to unlabeled methyl methionine, by the o-methyl transferase. However unexpected preferential incorporation of the [methyl-14C]- vs. [methyl-3H]-moiety into JH occurs, but is only evident at concentrations of radiolabeled methionine outside the normal range of the RCA. Changes in radioactive precursor formulation have no effect on the RCA.     

Impaired formylation and uptake of tetrahydrofolate by rat small gut following cobalamin inactivation

The effect of inactivation of cobalamin by N2O on the intestinal absorption of folate was studied using rat everted gut sacs. Further, in view of uncertainties about the presence of methionine synthetase in gut [1], this enzyme was measured. Everted gut sacs were incubated with [2-14C]tetrahydrofolate, and the subsequent appearance of labelled formyl- and methyl [14C] tetrahydrofolate in everted segments of small intestine of rats was studied. Considerable methionine synthetase activity was present in washed everted gut sacs but not in gut segments in the absence of such treatment. Methionine synthetase activity declined after exposure to N2O, which oxidizes and inactivates cob(I)alamin. Folate uptake by gut sacs was not affected by 24 h exposure of the animals to N2O but fell significantly after 7 days exposure. There was a significant fall in the amount of formyltetrahydrofolate formed after cobalamin inactivation and this was reversed by supplying either methionine, methylthioadenosine or sodium formate. Serine had no effect. The data support the hypothesis that methionine and methylthioadenosine act by supplying single carbon units at the formate level of oxidation.