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Formyltetrahydrofolates Associated with Mitochondria Have Longer Polyglutamate Chains Than the Methyltetrahydrofolates Associated with Cytoplasm in Rat Brain^1,2,

G. Franklin Carl^*,,3, Farlyn Z. Hudson^* and Byron S. Mcguire,, Jr.^*

^* Medical Research, VA Medical Center, Augusta, GA 30904 Department of Neurology, Medical College of Georgia, Augusta, GA 30912

The subcellular distribution of folate coenzymes in the brain is unknown. Brain folate concentrations are low and hence require a sensitive assay to determine the subcellular distribution. Rat brain was fractionated by differential centrifugation into cytoplasmic, mitochondrial and crude synaptosomal fractions. The compositions of the folate pools in these subcellular fractions were determined by differential conversion of one-carbon forms enzymatically to 5,10-methylenetetrahydrofolate (5,10CH₂H₄PteGlu_n) followed by reaction of the 5,10CH₂H₄PteGlu_n with thymidylate synthetase and [³H]fluorodeoxyuridylate to form ternary complexes, which were then separated as a function of polyglutamate chain length by isoelectric focusing, visualized by fluorography and quantified by densitometry. The distribution of the pteridine derivatives in brain was very similar to the distribution of these derivatives in liver. Cytoplasm contained primarily 5-methyltetrahydropteroylpolyglutamates with smaller amounts of unsubstituted tetrahydropteroylpolyglutamates, whereas mitochondria contained approximately equal concentrations of unsubstituted and formyl-substituted tetrahydropteroylpolyglutamates. The subcellular distribution of polyglutamate derivatives in brain, however, was different from that in liver. In the brain, the mitochondrial folates exhibited longer polyglutamate chains than did the cytoplasmic folates, a pattern opposite to that in the liver. Whereas the brain cytoplasmic pteroylpolyglutamates were primarily penta and hexa glutamates, the brain mitochondrial pteroylpolyglutamates were primarily hexa and hepta glutamates. The brain also contained small but measurable levels of oxidized folates, which were seen in crude synaptosomal fractions but not in cytoplasmic or mitochondrial fractions.

KEY WORDS: • rats • brain • mitochondria • cytoplasm • folate

¹ The authors acknowledge the support of the Medical Research Service of the Department of Veterans Affairs.

² The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.

³ To whom correspondence should be addressed.

Manuscript received 21 June 1996. Revision accepted 4 September 1996.