S-Adenosyl- L-Methionine And S-Adenosyl- L-Homocysteine: A Nuclear Magnetic Resonance Study And Purification Of S-Adenosyl- L-Methionine: L-Homocysteine S-Methyltransferase From Saccharomyces Cerevisiae

An analysis of the 360 MHz (\u271)H NMR spectra of the title compounds in (\u272)H(,2O) is presented. The (\u273)J values for the ribose vicinal protons of S-adenosyl-L-methionine are consistent with a predominantly C(,3\u27)-exo conformation and with one highly favored gauche-anti conformation about the C(,4\u27)-C(,5\u27) bond. The corresponding (\u273)J values for S-adenosyl-L-homocysteine imply a similar C(,3\u27)-exo ribose ring conformation, but the orientation about the C(,4\u27)-C(,5\u27) bond is distributed between two gauche-anti rotamers. The methionine side chain of S-adenosyl-L-methionine has approximately equal populations of rotational isomers about the C(,(alpha))-C(,(beta)) and C(,(beta))-C(,(gamma)) bonds, whereas the side chain of S-adenosyl-L-homocysteine exhibits a conformational preference for the gauche-anti conformations about the C(,(alpha))-C(,(beta)) bond. (\u271)H and (\u2713)C NMR spectra of commercially available samples of (-)S-adenosyl-L-methionine consistently available samples of (-)S-adenosyl-L-methionine consistently reveal the presence of a small amount of the (+) sulfonium diastereomer. This assignment was confirmed by the synthesis of both the (\u271)H and (\u2713)C methyl derivatives of S-adenosyl-L-homocysteine. Arguments are presented to explain the failure of previous workers to detect (+)S-adenosyl-L-methionine in biological preparations. The possible biological significance of this finding is discussed with reference to the enzyme S-adenosyl-L-methionine: L-homocysteine S-methyltransferase which employs both (-)S-adenosyl-L-methionine and (+)S-adenosyl-L-methionine as methyl donors. An improved purification of S-Adenosyl-L-methionine: L-homocysteine S-methyltransferse (EC. 2.1.1.10) from Saccharomyces cerevisiae is reported. The enzyme was purified approximately 1500-fold by toluene extraction, ammonium sulfate precipitation, Sephadex G25 gel exclusion chromatography, DEAE Sephadex ion-exchange chromatography and affinity chromatography on L-methionine AH-Sepharose 4B. The procedure affords a 12-fold increase in yield and a 3-fold increase in purification over the previous fractionation scheme. It is hoped that the availability of a highly purified preparation of the enzyme will provide the basis for an investigation of enzymatic transmethylation in which the fate of the sulfonium diastereomers of S-adenosyl-L-methionine is directly observable by NMR spectroscopy

Evaluation of integrin αvβ6 cystine knot PET tracers to detect cancer and idiopathic pulmonary fibrosis.

Advances in precision molecular imaging promise to transform our ability to detect, diagnose and treat disease. Here, we describe the engineering and validation of a new cystine knot peptide (knottin) that selectively recognizes human integrin αvβ6 with single-digit nanomolar affinity. We solve its 3D structure by NMR and x-ray crystallography and validate leads with 3 different radiolabels in pre-clinical models of cancer. We evaluate the lead tracer's safety, biodistribution and pharmacokinetics in healthy human volunteers, and show its ability to detect multiple cancers (pancreatic, cervical and lung) in patients at two study locations. Additionally, we demonstrate that the knottin PET tracers can also detect fibrotic lung disease in idiopathic pulmonary fibrosis patients. Our results indicate that these cystine knot PET tracers may have potential utility in multiple disease states that are associated with upregulation of integrin αvβ6

S-Adenosyl- L-Methionine And S-Adenosyl- L-Homocysteine: A Nuclear Magnetic Resonance Study And Purification Of S-Adenosyl- L-Methionine: L-Homocysteine S-Methyltransferase From Saccharomyces Cerevisiae

An analysis of the 360 MHz (\u271)H NMR spectra of the title compounds in (\u272)H(,2O) is presented. The (\u273)J values for the ribose vicinal protons of S-adenosyl-L-methionine are consistent with a predominantly C(,3\u27)-exo conformation and with one highly favored gauche-anti conformation about the C(,4\u27)-C(,5\u27) bond. The corresponding (\u273)J values for S-adenosyl-L-homocysteine imply a similar C(,3\u27)-exo ribose ring conformation, but the orientation about the C(,4\u27)-C(,5\u27) bond is distributed between two gauche-anti rotamers. The methionine side chain of S-adenosyl-L-methionine has approximately equal populations of rotational isomers about the C(,(alpha))-C(,(beta)) and C(,(beta))-C(,(gamma)) bonds, whereas the side chain of S-adenosyl-L-homocysteine exhibits a conformational preference for the gauche-anti conformations about the C(,(alpha))-C(,(beta)) bond. (\u271)H and (\u2713)C NMR spectra of commercially available samples of (-)S-adenosyl-L-methionine consistently available samples of (-)S-adenosyl-L-methionine consistently reveal the presence of a small amount of the (+) sulfonium diastereomer. This assignment was confirmed by the synthesis of both the (\u271)H and (\u2713)C methyl derivatives of S-adenosyl-L-homocysteine. Arguments are presented to explain the failure of previous workers to detect (+)S-adenosyl-L-methionine in biological preparations. The possible biological significance of this finding is discussed with reference to the enzyme S-adenosyl-L-methionine: L-homocysteine S-methyltransferase which employs both (-)S-adenosyl-L-methionine and (+)S-adenosyl-L-methionine as methyl donors. An improved purification of S-Adenosyl-L-methionine: L-homocysteine S-methyltransferse (EC. 2.1.1.10) from Saccharomyces cerevisiae is reported. The enzyme was purified approximately 1500-fold by toluene extraction, ammonium sulfate precipitation, Sephadex G25 gel exclusion chromatography, DEAE Sephadex ion-exchange chromatography and affinity chromatography on L-methionine AH-Sepharose 4B. The procedure affords a 12-fold increase in yield and a 3-fold increase in purification over the previous fractionation scheme. It is hoped that the availability of a highly purified preparation of the enzyme will provide the basis for an investigation of enzymatic transmethylation in which the fate of the sulfonium diastereomers of S-adenosyl-L-methionine is directly observable by NMR spectroscopy

A fourier transform proton magnetic resonance study of the molecular conformation of S-adenosyl-L-methionine

Contrary to a previous report, S-adenosyl-L-methionine (SAM) affords stable solutions in D2O and the 1H NMR spectrum can be determined. Comparison with the spectra of the model compounds adenosine, L-methionine and L-methionine-S-methyl sulfonium iodide allows complete assignment of the proton resonances. Coupling constants were determined by homonuclear decoupling and graphical analysis and were confined by computer simulation. Details of the molecular conformation were determined by application of the Karplus equation and calculation of relative rotational isomer populations. Evidence indicates that the ribose ring is puckered preferentially in the C3\u27-exo conformation and that the C4\u27-C5\u27 bond is constrained to a rotamer in which the sulfonium center is gauche to H4\u27. No conformational constraints were detected for the Cα-Cβ and Cβ-Cα bonds of the methionine side chain. The purine ring was shown to be oriented preferentially anti by intermolecular association studies with adenosine 5\u27-phosphate in the presence of MN(II). Spectra of samples of (-)S-adenosyl-L-methionine of biological origin, differing in activity, counter ion and commercial source, have consistently revealed the presence of a small amount of the (+) sulfonium diastereomer. Arguments are presented to explain the failure of previous workers to detect (+)S-adenosyl-L-methionine in biological preparations

Evaluation of integrin αvβ6 cystine knot PET tracers to detect cancer and idiopathic pulmonary fibrosis.

Advances in precision molecular imaging promise to transform our ability to detect, diagnose and treat disease. Here, we describe the engineering and validation of a new cystine knot peptide (knottin) that selectively recognizes human integrin αvβ6 with single-digit nanomolar affinity. We solve its 3D structure by NMR and x-ray crystallography and validate leads with 3 different radiolabels in pre-clinical models of cancer. We evaluate the lead tracer's safety, biodistribution and pharmacokinetics in healthy human volunteers, and show its ability to detect multiple cancers (pancreatic, cervical and lung) in patients at two study locations. Additionally, we demonstrate that the knottin PET tracers can also detect fibrotic lung disease in idiopathic pulmonary fibrosis patients. Our results indicate that these cystine knot PET tracers may have potential utility in multiple disease states that are associated with upregulation of integrin αvβ6