Abstract
Fatty acid methyl esters are the major radioactive lipid products obtained after incubation of rat lung membranes with [methyl-3H or 14C]S-adenosylmethionine. Evidence which suggests an enzymatic transmethylation includes: time and protein dependence, lack of reaction at 0 degrees C or with heat-denatured membranes, an apparent affinity for S-adenosylmethionine of about 1 microM, inhibition by S-adenosylhomocysteine, and lack of inhibition by 0.1% methanol. Activity was highest in microsomes but present in other membranous fractions. Endogenous activity was highest in membranes from parotid, lung, and pancreas. Products were analyzed by organic solvent extraction, thin layer chromatography, column chromatography, high performance liquid chromatography, and gas chromatography. Identification of methylpalmitate, methylstearate, methyloleate, and methyllinoleate was confirmed by mass spectrometry. Presence of the radioactive methyl group was demonstrated by the variation of isotopic ratios with specific activity. Addition of oleate to incubation mixture increased the rate of product formation and preincubation experiments suggested the absence of long lived intermediates. The data suggest an enzymatic transfer of methyl groups from S-adenosylmethionine to free fatty acids.
Highlights
From the Gaboratoryof Clinical Science, National Institute ofMental HealthB, ethesda, Maryland 20205 and $Laboratory of Vision Research, National Eye Institute, Bethesda,Maryland 20205
Fatty acid methyl esters are the major radioactive lipidproductsobtainedafterincubation of ratlung Materials-S-Adeno~yl[methyZ-~H~methionin(1e2to 15Ci/mmol) membranes with [methyC'H or 14C]S-adenosylmethio- and S-aden~syl[methyl-'~C]methionine(58 to 60 mCi/mmol) were nine.Evidencewhich suggests an enzymatic trans- purchased from New England Nuclear Corp. or Amersham-Searle, methylationincludes:timeandproteindependence, Inc
Were analyzed by organic solvent extraction, thin lpaeyIIeerts obtained after 1O,OO X g for 10 min and 12,000 X g for 20 min chromatography,columnchromatography,highper- were discarded and thepellet obtained after 100O, OO X g for 60 min formance liquid chromatography, andgas chromatog- was used
Summary
Lipidproductsobtainedafterincubation of ratlung Materials-S-Adeno~yl[methyZ-~H~methionin(1e2to 15Ci/mmol) membranes with [methyC'H or 14C]S-adenosylmethio- and S-aden~syl[methyl-'~C]methionine(58 to 60 mCi/mmol) were nine.Evidencewhich suggests an enzymatic trans- purchased from New England Nuclear Corp. or Amersham-Searle, methylationincludes:timeandproteindependence, Inc. Formation a n d Assay of Nonpolar Methylated Lipids-In small scale experiments, lungs from two or three rats were removed into NaP04 buffer ( 5 0 mM, pH 7.2 to 7.4) containing 5 mMMgC12. Pellets were rehomogenizedin NaP04 buffer (50 to 66 mM, pH 7.2 to 7.4) containing 5 mM MgCL and 0.1% Triton X-100 Incubations contained these membranes (about 200 Mg of protein) and 10 pCi ofI3H]AdoMet We have identified and characterized these nonpolar lipid methylations in several systems.' In rat lung membranes,the major lipidproductsof transmethylation are fatty acid methyl esters. Rats were removed into buffer as above, disrupted by Waring blendor and Polytron, homogenized, and centrifuged to prepare high speed membranes. The costs of publication of this article were defrayed in High Performance Liquid Chromatography-Heptane extracts part by the payment of page charges. Typical conditions were gc interface temperature 250 "C, oxidation tube at 750 "C, and counter plateau 2400 V
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
