Labeled Substrates. I. The Synthesis of DL-2-Deuteriolactic Acid 1

The pathways of peroxisomal and mitochondrial fatty acid oxidation were monitored with the use of substrates which produce NAD3H. I used as marker substrates: D-[3-3H]3-hydroxybutyrate for mitochondrial NAD3H production, [2-3H]glycerol for cytosolic NAD3H production, and [2-3H]acetate to measure carbon-bound 3H which was also generated by the metabolism of the commercial 9,10-3H-labelled fatty acids. The assumption that peroxisomal NAD3H can be considered to be equivalent to cytosolic NAD3H was supported using a specific inhibitor of mitochondrial fatty acid oxidation. The approach involves determination of the specific yields, and the relative distribution on carbons 4 and 6, of 3H in glucose from the marker substrates and the labelled fatty acids. In hepatocytes from clofibrate-treated rats, the amount of palmitate or oleate oxidation which starts in the peroxisomes is comparable with that which starts in the mitochondria.

Section: Resultsmentioning

confidence: 99%

Estimation of peroxisomal and mitochondrial fatty acid oxidation in rat hepatocytes using tritiated substrates

Rognstad

1991

“…The consequences of malate exchange between compartments include effects onI the specific radioactivity ofthe NAD3H produced from tritiated substrates that form NAD3H in either the cytosol or the mitochondria. Hoberman & D'Adamo (1960) were the first to use L[2_2H]and L-[2_3H] lactate as tracers for reductive hydrogen in gluconeogenesis. In gluconeogenesis from L-[2-3H]lactate, the molar 3H specific radioactivity of the glucose formed, in the absence of isotope discrimination or exchange reactions with water, should be about 1.0.…”

Section: Resultsmentioning

confidence: 99%

Gluconeogenesis in the kidney cortex. Flow of malate between compartments

Rognstad

1970

1. Kidney-cortex slices from starved rats were incubated with l-[U-(14)C]lactate or l-[U-(14)C]malate plus unlabelled acetate and the specific radioactivity of the glucose formed was determined. In parallel experiments the specific radioactivity of the glucose formed from [1-(14)C]acetate plus unlabelled l-lactate and l-malate was determined. 2. By analytical methods the major products formed from the substrates were measured. The glucose formed was purified by paper chromatography for determination of specific radioactivity. 3. The specific radioactivity of the glucose formed from l-[U-(14)C]lactate agrees with predictions of a model based on interaction of the gluconeogenic and the oxidative pathways. 4. The specific radioactivity of the glucose formed from l-[U-(14)C]malate agrees with the predicted value if rapid malate exchange between the cytosol and mitochondria is assumed. 5. The rate of malate exchange between compartments was estimated to be rapid and at least several times the rate of glucose formation. 6. The specific radioactivity of the glucose formed from [1-(14)C]acetate plus unlabelled l-lactate or l-malate agrees with the predictions from the model, again assuming rapid malate exchange between compartments. 7. Malate exchange between compartments together with reversible malate dehydrogenase activity in the mitochondria and cytosol also tends to equilibrate isotopically the NADH pool in these compartments. (3)H from compounds such as l-[2-(3)H]lactate, which form NAD(3)H in the cytosol, appears in part in water; and (3)H from dl-beta-hydroxy[3-(3)H]butyrate, which forms NAD(3)H in the mitochondria, appears in part in glucose, largely on C-4.

“…A rational approach to the study of regulation of metabolic pathways has been described by Newsholme & Gevers (1967), and this approach was followed in the present investigation. Glycerol kinase (EC 2.7.1.30) has been shown to catalyse a non-equilibrium reaction in liver (see the Results section); and, as it is the first enzyme in the metabolism of glycerol in this tissue (see Bublitz & Kennedy, 1954b;Hoberman & D'Adamo, 1960), its catalytic activity must regulate glycerol utilization. Therefore factors that could modify the catalytic activity of this enzyme could be important in the regulation of glycerol utilization.…”

mentioning

confidence: 99%

Some properties of hepatic glycerol kinase and their relation to the control of glycerol utilization

Robinson

Newsholme

1969

1. Glycerol kinase (EC 2.7.1.30) is shown to catalyse a non-equilibrium reaction in rat liver; and, as it is the first enzyme in the pathway metabolizing glycerol, its properties may be pertinent to the metabolic regulation of glycerol uptake and utilization by this tissue. 2. The properties of hepatic glycerol kinase were studied by using a radiochemical technique to measure the enzyme activity. When the concentration of ATP is low the activity of glycerol kinase is inhibited by high concentrations of glycerol; but when the concentration of ATP is high there is no inhibition and the double-reciprocal plot is linear, providing a K(m) for glycerol of 3.16x10(-6)m. Glycerol kinase is activated by high ATP concentrations provided that the concentration of the second substrate (glycerol) is high; at low concentrations of glycerol ATP does not activate the enzyme so that the double-reciprocal plot is linear, providing a K(m) for ATP of 5.8x10(-5)m. It is suggested that these kinetics may be explained by a model similar to that described by Ferdinand (1966) for phosphofructokinase. 3. Hepatic glycerol kinase is inhibited by ADP and AMP, and raising the Mg(2+) concentration increases the inhibition by these two compounds; this suggests that ADP-Mg(2+) and AMP-Mg(2+) complexes are the inhibitory species. The physiological significance of these inhibitions may be to prevent phosphorylation of glycerol when the hepatic ATP concentration is low. It is suggested that this inhibition may provide an approach to the problem of measurement of rates of lipolysis by glycerol release in tissues that contain glycerol kinase (e.g. liver, kidney, muscle, adipose tissue). 4. Hepatic glycerol kinase is inhibited by l-3-glycerophosphate competitively with respect to glycerol. The physiological significance of this inhibition may be that factors that change the intracellular concentration of l-3-glycerophosphate could change glycerol uptake by the tissue. Thus it is suggested that thyroxine treatment or feeding rats on a diet high in glycerol, which increase the activity of glycerophosphate oxidase in liver and kidney cortex respectively, lead to an increased glycerol uptake through a decrease in the concentration of glycerophosphate in these tissues. It is known that ethanol administration decreases glycerol uptake by liver, and this can be explained by the increased concentration of l-3-glycerophosphate causing inhibition of glycerol kinase.