Abstract
Many murine cells express two mRNAs with markedly different sizes (2.0 and 3.1 kilobases (kb)) that hybridize with cDNA probes for cytosolic malic enzyme ((S)-malate NADP+ oxidoreductase (oxaloacetate-decarboxylating, EC 1.1.1.40). A series of overlapping cDNA clones corresponding to 3129 nucleotides of malic enzyme mRNA was isolated and sequenced to determine the relationship between the two mRNAs and establish the primary structure of mouse malic enzyme. The larger mRNA has an open reading frame of 1716 nucleotides followed by a 3' untranslated region of 1348 nucleotides. The sequence of an exceptionally G/C-rich (88%) portion (65 nucleotides) of the 5' noncoding region was also established. An uncommon poly A addition signal (AUUAAA) is used during the processing of the 3.1-kb mRNA. The 2.0-kb mRNA results from the utilization of another poly A addition signal that truncates the 3' noncoding sequence by approximately 1 kb. The mRNA coding sequence indicates that the malic enzyme subunit contains 572 amino acid residues and has a Mr of 64,000. Two putative components of an NADP-binding domain are located between residues 100 and 165. During the differentiation of 3T3-L1 preadipocytes into adipocytes both the rate of synthesis and relative mRNA concentration for malic enzyme and another lipogenic enzyme, ATP-citrate lyase, are coordinately increased 5-7-fold. However, as preadipocytes approach confluence, the mRNA levels for both lipogenic enzymes transiently increase 3-4-fold, whereas the rates of synthesis of the two proteins are only slightly elevated. Thus, lipogenic enzyme expression is controlled at a pretranslational level during adipogenesis, but the accumulation of the same enzymes may also be subject to translational control in the fibroblast-like preadipocytes. In contrast, mRNA coding for a third enzyme required for lipogenesis, glycerol-3-phosphate dehydrogenase, is not detected in 3T3-L1 preadipocytes, but rapidly accumulates during adipocyte development.
Highlights
Edly different sizes (2.0 and 3.1 kilobases) that that catalyzes the oxidative decarboxylationof malate: malate hybridize with cDNA probes for cytosolic malic en- + NADP+ +pyruvate + COz + NADPH + H’ [1].NADPH zyme ((s-malate NADP+ oxidoreductase(oxaloace- generated by malic enzyme is a major source of the reducing tate-decarboxylating, EC 1.1.1.40)
During the differentia- cultured in the absence of insulin ( 6 ),suggesting that the tion of 3T3-Ll preadipocytes into adipocytes both the polypeptide hormone is a synergistic modulator in the longrate of synthesis and relativme RNA concentration for term regulation of malicenzyme content.Hormonesthat ctHmimwnioRatcorlwrNaiecetAapeevsnreeozllreyyt3,veam-aie4snsele-ssfa,opfrlroeaaderrn,oabedndwoliayctpnhhoosoeolclritirypghedtoaheeigsntrsleayanttplehiipelpceylroieeongnvarcezaacrnytteheioemaccdfsseoe.esnsnydzTftnyrlhtuamhuneees5nss,i-cAie7selnT,ioftptPoflhoy-ltedgh.eeniciedrnenicgeTzrutyehalmaraeysteoesrrcaytaatherreabesnooionfdhftisytrneydahnncriatebhtcleielotu.solirasdmroinCaf lamAitceeMalnlyiPzcryecemgonuenzlacytemeindnetdraaunbtcdyitoinohonotahrrmeberyoclionpTueo3sngteaaennnrid-dc enzyme expression is controlled a t a pretranslational dietarymanipulations [2, 10, 11]
We report the characterization of the primary structure of murine malic enzyme mRNA and its derived amino acid sequence, an assessment of the relationships between the two malic enzyme mRNAs; temporal aspects of the regulation of malic enzyme mRNA during adipocyte differentiation; acomparison of the coordinate regulation of malic enzyme and ATP-citratelyase with the regulation of glycerol-3-phosphate dehydrogenase; and evidence that malic enzyme is controlled at a pretranslationallevel during differentiation, but is subject to translationalcontrol in preadipocytes
Summary
Edly different sizes (2.0 and 3.1 kilobases (kb)) that that catalyzes the oxidative decarboxylationof malate: malate hybridize with cDNA probes for cytosolic malic en- + NADP+ +pyruvate + COz + NADPH + H’ [1].NADPH zyme ((s-malate NADP+ oxidoreductase(oxaloace- generated by malic enzyme is a major source of the reducing tate-decarboxylating, EC 1.1.1.40). The amino acid coding sequence accounts for 55% of the nucleotides in the larger malic enzyme mRNA and more than 85% of the primary structure of the 2-kbmRNA.
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