Citryl-CoA and the citrate condensing enzyme

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/32278/1/0000340.pd

The citrate cleavage enzyme. III. Citryl coenzyme a as a substrate and the stereospecificlty of the enzyme

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Getting a handle on lipid droplets: Insights into ER-lipid droplet tethering.

Lipid droplets (LDs) are hubs for lipid metabolism that form membrane contact sites with multiple organelles. In this issue, Hariri et al. (2019. J. Cell Biol. https://doi.org/10.1083/jcb.201808119) reveal the functions of Mdm1-mediated endoplasmic reticulum (ER)-LD tethering in yeast and Datta et al. (2019. J. Cell Biol. https://doi.org/10.1083/jcb.201808133) identify a role for the Mdm1 orthologue, Snx14, as an ER-LD tether that regulates lipid metabolism in human cells

Rapid Diffusion of Green Fluorescent Protein in the Mitochondrial Matrix

Abstract. It is thought that the high protein density in the mitochondrial matrix results in severely restricted solute diffusion and metabolite channeling from one enzyme to another without free aqueous-phase diffusion. To test this hypothesis, we measured the diffusion of green fluorescent protein (GFP) expressed in the mitochondrial matrix of fibroblast, liver, skeletal muscle, and epithelial cell lines. Spot photobleaching of GFP with a 100× objective (0.8-μm spot diam) gave half-times for fluorescence recovery of 15–19 ms with >90% of the GFP mobile. As predicted for aqueous-phase diffusion in a confined compartment, fluorescence recovery was slowed or abolished by increased laser spot size or bleach time, and by paraformaldehyde fixation. Quantitative analysis of bleach data using a mathematical model of matrix diffusion gave GFP diffusion coefficients of 2–3 × 10−7 cm2/s, only three to fourfold less than that for GFP diffusion in water. In contrast, little recovery was found for bleaching of GFP in fusion with subunits of the fatty acid β-oxidation multienzyme complex that are normally present in the matrix. Measurement of the rotation of unconjugated GFP by time-resolved anisotropy gave a rotational correlation time of 23.3 ± 1 ns, similar to that of 20 ns for GFP rotation in water. A rapid rotational correlation time of 325 ps was also found for a small fluorescent probe (BCECF, ∼0.5 kD) in the matrix of isolated liver mitochondria. The rapid and unrestricted diffusion of solutes in the mitochondrial matrix suggests that metabolite channeling may not be required to overcome diffusive barriers. We propose that the clustering of matrix enzymes in membrane-associated complexes might serve to establish a relatively uncrowded aqueous space in which solutes can freely diffuse

Renal cortical mitochondrial aconitase is regulated in hypo- and hypercitraturia

Renal cortical mitochondrial aconitase is regulated in hypo- and hypercitraturia.BackgroundChronic metabolic acidosis and K+ deficiency increase, while alkali feeding decreases proximal tubule citrate absorption and metabolism. The present studies examined the regulation of mitochondrial aconitase (m-aconitase), the first step in mitochondrial citrate metabolism, in these conditions.MethodsRats were fed appropriate diets, and m-aconitase activity and protein abundance measured.ResultsIn chronic metabolic acidosis and chronic K+ deficiency, renal cortical m-aconitase activity was increased 17% and 43%, respectively. This was associated with respective 90% and 221% increases in renal cortical m-aconitase protein abundance. With chronic alkali feeding, there was a 12% decrease in renal cortical m-aconitase activity, associated with a 35% decrease in m-aconitase protein abundance. Hepatic m-aconitase activity was not regulated in a similar manner. There was no regulation of citrate synthase, the enzyme responsible for mitochondrial citrate synthesis.ConclusionsThese studies demonstrate tissue specific chronic regulation of renal cortical m-aconitase activity and protein abundance, which likely contributes to the hypocitraturia and hypercitraturia seen in these conditions. As m-aconitase is the only step in citrate transport and metabolism found to be regulated in alkali feeding, its regulation likely plays a significant role in mediating the hypercitraturia seen in this condition

Starch-gel electrophoresis of citrate-condensing enzyme from pig heart

Citrate-condensing enzyme from pig heart can exist in vitro as two distinct species which are separable by starch-gel electrophoresis. Several mild types of treatment can interconvert these enzymes and suggest that the separate forms arise in the process of purification; the two enzymes may differ only in the state of reduction of their sulfhydryl groups.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/32276/1/0000338.pd