LOCALIZATION OF LIPOGENIC ENZYMES IN MICROPEROXISOMES FROM 3T3-L1 ADIPOCYTES (SUBCELLULAR FRACTIONATION)
ROTHBLATT, JONATHAN A.
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At confluence, fibroblast-like 3T3-L1 cells undergo terminal differentiation acquiring the morphological and physiological characteristics of tissue adipocytes. Enzyme cytochemistry (Novikoff, et al., J. Cell Biol. 87:180, 1980) indicated that adipocyte conversion of 3T3-L1 cells is accompanied by an increase in the number of microperoxisomes, and that smooth endoplasmic reticulum (ER) and microperoxisomes appose the cytosolic triacylglycerol droplets. Assay of catalase activity, a biochemical marker for microperoxisomes, in homogenates of undifferentiated and differentiating 3T3-L1 cells confirms the induction of microperoxisomes with the onset of triacylglycerol accumulation. Two days after the stimulation of adipocyte conversion, catalase undergoes a very rapid 5- to 10-fold increase in specific activity which peaks at 6 days. Further, a cyanide-insensitive, carnitine-independent fatty acyl-CoA oxidizing activity increases in parallel with the catalase activity. Differentiation of 3T3-L1 cells in biotin-deficient growth medium results in morphological alterations at the light microscopic level characteristic of adipocyte conversion (e.g. rounding of cell shape), but significantly reduced accumulation of cytosolic triacylglycerol. Under these conditions, catalase activity still increases and with a time course identical to that observed in normal differentiation conditions, suggesting that the induction of microperoxisomes may reflect their participation in metabolic pathways regulated independently of the synthesis and turnover of triacylglycerol. To explore the role(s) of microperoxisomes in lipid biosynthesis and degradation we have developed a method for the analytical subcellular fractionation of differentiating 3T3-L1 cells. Fractions enriched in plasma membrane, ER and microperoxisomes were obtained, as assessed by biochemical and morphological criteria. The distribution and recovery of subcellular components were quantitatively analyzed by the measurement of enzymes indicative of particular cellular structures. This approach has permitted us to investigate the subcellular localization of glycerolipid-synthesizing enzymes. Glycerol-3-phosphate acyltransferase co-enriches with the ER marker, NADPH cytochrome c reductase. However, the enzymes of the dihydroxyacetone phosphate (DHAP) pathway of glycerolipid synthesis, namely, DHAP acyltransferase and alkyl-DHAP synthase, co-purify with catalase. Acyl/alkyl-DHAP oxidoreductase is present in both subcellular compartments. The activities of DHAP and G-3-P acyltransferases and acyl/alkyl-DHAP oxidoreductase, like catalase and fatty acyl-CoA oxidation, increase 5- to 10-fold during adipocyte conversion.
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