Jerald L. Hoffman

The rate of transmethylation in mouse liver as measured by trapping S-adenosylhomocysteine

Abstract Periodate-oxidized adenosine has previously been shown to be a potent inhibitor in vitro of S -adenosylhomocysteine hydrolase (E.C. 3.3.1.1). This paper describes the inhibition of this enzyme in liver following injection of mice with periodate-oxidized adenosine. A maximally effective dose of 100 nmol/g of this compound causes liver S -adenosylhomocysteine to increase from 12 to 600 nmol/g within 30 min. This accumulation of S -adenosylhomocysteine provides an estimate of the rates of transmethylation, as well as adenosine and homocysteine production, as being at least 20 nmol/min/g liver. A doubling of S -adenosylmethionine in the liver of mice treated with periodate-oxidized adenosine suggests that the high levels of S -adenosylhomocysteine inhibit some transmethylation reactions.

A rapid liquid chromatographic determination of S-adenosylmethionine and S-adenosylhomocysteine in subgram amounts of tissue☆

Abstract A method is described for extraction and chromatographic determination of S -adenosylmethionine (SAM) and S -adenosylhomocysteine (SAH) from 1 g of tissue or less. Tissues are extracted by homogenization in 5% sulfosalicylic acid. Extracts are clarified by centrifugation and filtration and applied directly to a VYDAC cation-exchange column. Elution of the column with an ammonium formate gradient resolves SAM and SAH from other components. Livers of ethionine-treated mice are shown to accumulate large quantities of S -adenosylethionine while SAH decreases, indicative of reduced transalkylation.

Periodate-oxidized adenosine inhibits the formation of dimethylselenide and trimethylselenonium ion in mice treated with selenite.

The metabolic detoxification of selenite and many other selenium compounds involves a series of S-adenosylmethionine-dependent methylations yielding dimethylselenide (DMSe), which is exhaled, and trimethylselenonium ion (TMSe), which is excreted in the urine. This paper shows that periodate-oxidized adenosine (Adox) inhibits these methylation reactions in vivo and increases the toxicity of selenite. When Adox was injected in mice at 100 mumol/kg 30 min before injection of [75Se]selenite at 0.4 mg Se/kg the appearances of [75Se]DMSe in the breath and [75Se]TMSe in the liver were completely inhibited for 90 min. This was mediated by accumulation of S-adenosylhomocysteine, the methyltransferase inhibitor, in the livers of Adox-treated mice due to inhibition of its hydrolase enzyme. During 24 h, Adox-treated mice excreted no detectable urinary [75Se]TMSe and exhaled only 20% as much [75Se]DMSe as controls. The urine of Adox-treated mice also contained S-adenosylhomocysteine at a level (ca. 4 mM), 200 times that of untreated mice, which provided a convenient index of methylation potential in the intact animal. When three groups of three mice each were injected with 100 mumol Adox/kg, selenite at 4 mg Se/kg, or a combination of the two, the mice receiving the combination were dead within 2 days, while the mice in the other two groups all survived at least 4 days. These results verify the enzymatic nature of selenium methylation in vivo, support its importance in detoxification, and indicate the value of Adox in further studies of selenium metabolism.

Selenite toxicity, depletion of liver S-adenosylmethionine, and inactivation of methionine adenosyltransferase.

Abstract The possibility that dimethyl selenide production depletes liver S-adenosylmethionine was explored as a biochemical basis for selenite toxicity. Toxic doses of selenite (25 nmol/ g body weight) were found to rapidly decrease mouse liver S-adenosylmethionine and increase S-adenosylhomocysteine, indicative of an increased rate of transmethylation. However, S-adenosylmethionine levels remained depressed beyond the time when dimethyl selenide synthesis ceased, suggesting that selenite inactivated methionine adenosyltransferase. This was found to be the case in vivo by measuring the effect of graded doses of selenite on the conversion of the methionine analog, ethionine, to S-adenosylethionine. In vitro studies also indicated inactivation of this enzyme by selenite. Liver homogenates from mice injected with 25 nmol of selenite/g, as above, were found to have less than 50% of the methionine adenosyltransferase activity of saline-injected controls.

Chromatography of Nucleic Acids on Cross-Linked Cyciodextrin Gels Having Inclusion-Forming Capacity

Abstract The characterization by UV spectral methods of the interaction between cyclodextrins and nucleic acid components yields the following information. Adenine nucleotides are found to interact most strongly with cycloheptaamylose, presumably by inclusion of the base within the cyciodextrin cavity. Factors influencing the interaction include pH, the position of phosphorylation of nucleotides, and the degree of polymerization of oligonucleotides. When epichloro-hydrin cross-linked cycloheptaamylose gel is used as a stationary phase for nucleic acid chromatography, adenine-containing compounds are retained most strongly. The factors affecting complex formation with free cycloheptaamylose have the same effects on gel chromatography of nucleic acids. It appears that cyciodextrin stationary phases are useful for fractionation of nucleic acids and of other classes of compounds with functional groups which can interact with cyciodextrin cavities.

Differential activation of rat liver methionine adenosyltransferase isozymes by dimethylsulfoxide

Summary Dimethylsulfoxide at a concentration of 10% stimulates one isozyme of rat liver methionine adenosyltransferase 50–70 fold when assayed at low (25 μM) methionine. A second liver isozyme is stimulated only 1.5 fold. The dimethylsulfoxide-stimulated liver isozyme elutes from DEAE-cellulose and Sephadex G-150 later than the less sensitive form. Methionine adenosyltransferases from rat brain, heart, and kidney are not affected, or perhaps slightly inhibited by dimethylsulfoxide. Dimethylsulfoxide activation thus provides a simple test for the relative distribution of liver methionine adenosyltransferase isozymes.

Separation of rat liver methionine adenosyltransferase isozymes by hydrophobic chromatography.

Abstract Rat liver methionine adenosyltransferase was fractionated into two activity peaks by chromatography on a hydrophobic resin, phenyl-Sepharose. The first peak of enzyme activity (methionine adenosyltransferase I), eluted in low-ionic strength buffer, was only slightly activated by 10% dimethylsulfoxide (twofold) and possessed a low K m (Met) of 14 μ m which was unaffected by Me 2 SO. A second peak of enzyme activity (methionine adenosyltransferase II) was more tightly bound to the hydrophobic resin but could be eluted with 40% dimethylsulfoxide. Upon removal of the eluting dimethylsulfoxide from methionine adenosyltransferase II on a DEAE-Sephacel column, this methionine adenosyltransferase isozyme was strongly activated by 10% dimethylsulfoxide. This unique liver isozyme possessed a high apparent K m (Met) of 1.3 m m with upward curvature of the double-reciprocal plot. In the presence of 10% dimethylsulfoxide the kinetics became hyperbolic with the K m lowering to 33 μ m . Dimethylsulfoxide did not significantly alter the V of methionine adenosyltransferase II.

Chromatography of nucleic acid components on cyclodextrin gels

Abstract The application of gels consisting of cross-linked cycloheptaamylose to column chromatographic separations of nucleic acid derivatives has been described. Separations of various nucleotide and nucleoside mixtures were achieved, most notably those containing adenine. Separation is believed to rely on principles of inclusion complex formation similar to those for cyclodextrin molecules in solution.

Ion chromatographic analysis of the purity and synthesis of sulfonium and selenonium ions

The use of single-column ion chromatography with conductometric detection was shown to be useful for the analysis of sulfonium and selenonium ions. A Hamilton PRP X-200 cation column was eluted with either solvent A (5 mM nitric acid in 30% methanol) or solvent B (4 mM nitric acid). With solvent B, trimethylsulfonium ion was separated from trimethylselenonium ion. With solvent A, amounts of trimethylsulfonium ion from 2 to 250 nmol were detected with a linear response. The retention times and response factors for a series of sulfonium ions with various organic groups were determined. In general the ions with more hydrophobic groups eluted later, but all had similar response factors. The method was shown to be useful for optimizing conditions for the synthesis of methylsulfonium ions, specifically the reaction of methyl iodide with diallyl sulfide.

Effect of γ Subunit Carboxyl Methylation on the Interaction of G Protein α Subunits with βγ Subunits of Defined Composition

Abstract A baculovirus expression system was used to determine the contribution of carboxyl methylation of specific G protein γ subunits to the interaction between α and βγ subunits. βγ subunits were carboxyl methylated by a membrane bound methyltransferase in Sf9 cells, and periodate-oxidized adenosine inhibited this methylation by 90%. Carboxyl methylation of β1γ2, β2γ3, and β2γ7 enhanced pertussis toxin-catalyzed ADP-ribosylation of αi2 and αi3 by about 2-fold. On the other hand, methylation did not enhance membrane attachment of βγ subunits. These results suggest that methylation of isoprenylated γ subunits is required for optimal G protein-mediated signal transduction, but not membrane attachment.