1. Pantothenate kinase, which is present in cytosol, was studied in preparations from livers of rats fed normal or clofibrate-enriched diets. Effects of CoA, dephospho-CoA and different acyl-CoA derivatives on this enzyme activity were examined in vitro.2. With partially purified pantothenate kinase or crude particle-free supernatant from the liver of normal or clofibrate-treated rats, K, for pantothenic acid was 0.016 mmol/l at the pH optimum 6.1 3. Acetyl-CoA, propionyl-CoA, malonyl-CoA and other short-chain acyl-CoA derivatives were strong inhibitors of pantothenate kinase, with K i in the range 0.001 -0.003 mmol/l. The mechanism of inhibition appeared to be of an uncompetitive type.4. Free CoA has been held to be the main regulator of pantothenate kinase. We found, however, that free CoASH, dephospho-CoA and long-chain acyl-CoA (with Ki 0.003 -0.08 mmol/l) were less efficient inhibitors than acetyl-CoA.5. With pantothenate kinase from clofibrate-treated animals, all inhibitors were less potent. This was most pronounced when the enzyme was assayed in a crude supernatant fraction, possibly because the inhibitors were degraded and/or protein bound. Such a reduction of normal inhibition may contribute to the increased biosynthesis of CoA previously observed during clofibrate treatment.6. Fasting or diabetes leads to an increase of long-chain acyl-CoA and total CoA in the liver. The increase of CoA has been explained by increased acylation of CoA, and thereby reduced feed-back inhibition by free CoASH at the pantothenate kinase level. We propose another explanation. In these metabolic states, the cytosolic pool of acetyl-CoA is decreased. Since pantothenate kinase is present only in the cytosol, its activity will be released and the biosynthesis of CoA will increase.7. Acetyl-CoA is probably a more important physiological regulator of pantothenate kinase activity than is free CoASH.The total amount of CoA in the liver varies considerably in different physiological states [l -41. An increase is seen in fasting and diabetes, mainly due to accumulation of acyl-CoA [2-41. On feeding rats clofibrate, however, total CoA may increase by 2 -300 % because of augmentation of free CoASH Surprisingly few studies have appeared [8 -111 (cf. also [12]) on the mechanisms involved in the regulation of the biosynthesis of CoA in mammalian tissues. Pantothenate kinase is thought to be the most important regulatory enzyme, and CoASH and 4'-phosphopantetheine to be the most potent feed-back inhibitors at this initial step [8,9,11]. Only few potential inhibitors of pantothenate kinase have been studied, however, and the kinetics are incompletely characterized [I 11.We studied the pantothenate kinase activity in crude particle-free supernatant and in partially purified enzyme preparations from livers of normal and clofibrate-fed rats. A number of CoA-esters were studied for their effects on this reaction, and attempts were made to characterize the kinetics of inhibition. We conclude that acetyl-CoA, which acts by an uncompetitive type...
The amounts of CoA in rat liver increased about three fold after treatment with clofibrate. About 10 of total CoA were long‐chain acyl‐CoA in normal rats, and slightly less in clofibrate‐treated animals. The wet weight and protein content of the liver increased by about 25%. From livers of clofibrate‐treated rats, mitochondrial and lysosomal fractions were obtained in increased amounts by conventional tissue fractionation techniques. The supernatants from such livers had rather high activities of marker enzymes for lysosomes and peroxisomes. No changes of the specific activities of the two first enzymes in the degradation pathway for CoA, i.e. acid phosphatase and nucleotide pyrophosphatase, were found in the liver homogenate from clofibrate‐treated rats. In the mitochondrial fraction from such livers, both (contaminating) activities were decreased. The degradation of CoA was increased in the supernatant, however, probably because of an increased leakage of acid phosphatase from lysosomes during the fractionation procedure. The synthesis of CoA from pantothenic acid was estimated by two different methods in vitro: by quantitation of CoA formed and by estimation of the incorporation of [14C]pantothenic acid into CoA. By both methods, CoA synthesis was found to be increased 3–4‐fold in livers from clofibrate‐treated rats. Dephospho‐CoA kinase was found to have a dual localization, with a minor pool in the mitochondrial fraction. This enzyme was only slightly increased in the clofibrate‐treated animals. The activity of pantothenate kinase was more than doubled after clofibrate treatment. This is thought to be the most important factor for the increase of CoA. CoA exerted feed‐back inhibition at this step in liver supernatant from normal and clofibrate‐treated rats.
Pantetheinephosphate adenylyltransferase and dephospho‐CoA kinase, which are last in the sequence leading to the biosynthesis of CoA, are localized in mitochondria (about 30% of the activity in the cell) and cytosol. The first three enzymes in the pathway are only found in cytosol [Skrede, S. and Halvorsen, O. (1979) Biochem. Biophys. Res. Commun. 91, 1536–1542]. Both enzymes are present in the mitochondrial matrix, but dephospho‐CoA kinase also has a more ‘external’ pool and is less easily removed from the membrane fractions of the mitochondria. In accordance with the submitochondrial fractionation studies, pantetheinephosphate adenylyltransferase is to a larger extent activated by detergents or mechanical disruption of the mitochondria than is dephospho‐CoA kinase. In intact mitochondria, the biosynthesis of CoA from 4‐phosphopantetheine was stimulated by uncoupling agents, probably because of facilitated penetration of the substrate through the inner mitochondrial membrane and or of leakage of a regulator. Intact mitochondria could increase the synthesis of CoA 5–10 times, when conditions were chosen to promote biosynthesis from 4‐phosphopantetheine and to remove CoA by acylation and/or ‘leakage’ out of the mitochondria. The kinetic properties of both mitochondrial enzymes are quite similar to the cytosolic ones (but our reinvestigation of the latter enzymes only partially confirm previous studies). Km for 4‐phosphopantetheine and dephosplio‐CoA were 0.015 and 0.01 mmol/1, respectively, for the mitochondrial enzymes. Mitochondrial biosynthesis of CoA was strongly inhibited by dephospho‐CoA at the pantetheinephosphate adenylyltransferase level and more slightly by CoA at the dephospho‐CoA kinase level
Summary:The pathway for the olfactory response may be affected at an early stage of Alzheimer's disease. Measurement of the olfactory response in the elderly is therefore of particular interest. In this feasibility study, near-infrared spectroscopy was used to measure the olfactory response in 21 patients aged 56 to 79 years. Eight subjects had no memory complaints whereas 13 had subjective memory complaints, mild cognitive impairment, or very mild Alzheimer's disease. The optodes were placed over the temporal lobe, with the emitting optode over the pole and the receiving optode over the superior gyrus. The response to vanilla (1% in sterile H 2 O) in a test tube held immediately beneath the nostrils was compared to the response to sterile H 2 O only. Four control subjects had a clearly definable response with increased oxyhemoglobin and decreased deoxyhemoglobin bilaterally. The response was measured as the sum of the deviation of oxygenated and deoxygenated hemoglobin from baseline mean. With a cut-off determined after examination of responses to vanilla and sham stimulus, group difference was significant for response to vanilla ( 2 test, P ס 0.03). Response amplitudes to vanilla in the patient group were within the range of those to sham stimuli.
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