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J I Pedersen

Publications and source records attributed to J I Pedersen.

At least 109 records · Page 6Linked to original sources
PubMed1979

Assay of 1,25-dihydroxy vitamin D3 by isotope dilution--mass fragmentography.

We describe a specific assay of 1,25-dihydroxy vitamin D3 in human serum, based on isotope dilution-mass fragmentography. We added [26-2H3]-1,25-dihydroxy vitamin D3 to a fixed amount of serum. The steroids were extracted with chloroform/methanol and purified by liquid chromatography. The purified materal was converted into the trimethylsilyl ether and analyzed. Unlabeled 1,25-dihydroxy vitamin D3 was quantitated from the ratio between the tracings at m/e 452 and 455. The two ions used correspond to loss of two trimethylsilyloxo functions from the molecular ion. Essentially the same results, with some problems of interference, were obtained when we used the more intense ions at m/e 131 and 134 (corresponding to cleavage between C-24 and C-25). The detection limit was about 5 ng/L of serum; the coefficient of variation was about 6%. The accuracy of the method was assessed by recovery experiments. 1,25-Dihydroxy vitamin D3 in sera from 15 healthy subjects was found to average 55 +/- 10 ng/L(+/- SD). We believe this represents the first determination of 1,25-dihydroxy vitamin D3 in serum by use of a method not based on radioimmunoassay or receptor assay. Slightly lower values have been reported by those latter techniques.

Chromatography, High Pressure Liquid↗
PubMed1978

Purification of NADPH-ferredoxin reductase from rat liver mitochondria.

NADPH-ferredoxin reductase (NADPH:ferredoxin oxidoreductase, EC 1.6.7.1) has been identified in rat liver mitochondria and purified to homogeneity as judged by sodium dodecyl sulfate (SDS) gel electrophoresis. The protein was detected by its ability to reconstitute NADPH-cytochrome c reductase in the presence of adrenal ferredoxin. The purified protein had properties very similar to adrenal NADPH-ferredoxin reductase. The molecular weight was 52 000, as estimated by gel filtration. On SDS-polyacrylamide gels, mobility was identical to that of adrenal NADPH-ferredoxin reductase (Mr = 52 000). The enzyme exhibited a typical oxidized flavoprotein absorbance spectrum with maxima at 269, 377 and 450 nm and gave an absorbance ratio A450nm/A269nm of 0.138. The fluorescence excitation spectrum was identical to that of FAD. In the presence of NADPH and a ferredoxin, the reductase was found to be active in a reconstituted cytochrome P-450-dependent steroid 26-hydroxylase, which was recently isolated from rat liver mitochondria (Pedersen, J.I. (1978) FEBS Lett. 85, 35-39).

Adrenodoxin↗
PubMed1978

Soluble 25-hydroxyvitamin D3-1 alpha-hydroxylase from kidney mitochondria of rachitic pigs.

1. Mitochondria isolated from the kidneys of rachitic pigs have been shown to contain an active 25-hydroxyvitamin D3-1 alpha-hydroxylase. From these mitochondria a cytochrome P-450 has been solubilized with a specific content of 0.02-0.04 nmol/mg protein. 2. In the presence of a bovine adrenal NADPH-ferredoxin reductase, bovine adrenal ferredoxin and NADPH, the cytochrome P-450 supported the formation of 1,25-dihydroxyvitamin D3 from 25-hydroxyvitamin D3. 3. The hydroxylation reaction was linear with time up to 40 min, and with the amount of enzyme up to 0.03 nmol cytochrome P-450. The pH optimum for the reaction was 7.4, and the apparent Km was 3 x 10(-10) mol/mg protein. 4. The results show that 25-hydroxyvitamin D3 is metabolized in mammals by the same enzyme system as has been demonstrated in birds.

Animals↗
PubMed1976

Isolation of chick renal mitochondrial ferredoxin active in the 25-hydroxyvitamin D3-1alpha-hydroxylase system.

An iron-sulfur protein has been isolated from chick kidney mitochondria and purified (200-fold as determined enzymatically by its NADPH-cytochrome c reductase activity in the presence of adrenodoxin reductase) on DEAE-cellulose and gel filtration on Sephadex G-100. The purified protein showed an absorption peak at 411 nm with a shoulder at 460 nm. The electron paramagnetic resonance spectrum was typical of a ferredoxin-type iron-sulfur protein with g values: gx=gy-1.94 and gz=2.02. The molecular weight was estimated by gel filtration to be 12,500. When tested against anti-adrenodoxin gamma-globulin, the protein showed a precipitin line that fused completely with that of adrenodoxin. Based on these findings it is concluded that this protein is an iron-sulfur protein quite similar to adrenal ferredoxin. In the presence of adrenoxodin reductase, NADPH, and carbon monoxide, the purified renal ferredoxin was found to be active in the reduction of cytochrome P-450 solubilized from chick kidney mitochondria. It was also effective in the reconstituted 25-hydroxyvitamin D3-1alpha-hydroxylase composed of the cytochrome P-450 from rachitic chick kidneys and adrenodoxin reductase. A ferredoxin reductase isolated from chick kidney mitochondria could replace adrenodoxin reductase in the reconstituted system. These results strongly support a previous conclusion that the kidney mitochondrial 25-hydroxyvitamin D3-1alpha-hydroxylation system consists of a renal ferredoxin reductase (presumably a flavoprotein), renal ferredoxin, and cytochrome P-450.

25-Hydroxyvitamin D3 1-alpha-Hydroxylase↗
PubMed1975

The intracellular localization of long-chain acyl-CoA synthetase in brown adipose tissue.

1. The acyl-CoA synthetase activity in brown adipose tissue of cold-exposed guinea pig has been studied by measuring the rate of palmitoylcarnitine formation in the presence of excess carnitine palmitoyltransferase. 2. The rate of palmitoylcarnitine formation in the mitochondria was found to be 161 plus or minus 64 nmol.mg-minus-1. min-minus-1 (n=9). 3. In the absence of added palmitate and bovine serum albumin a total of 35 plus or minus 1 nmol endogenous fatty acids.mg-minus-1 were activated with three different mitochondrial preparations. 4. Three different experimental approaches have been used to study the subcellular localization of the enzyme: (a) conventional differential centrifugation (De Duve, C., Pressman, B.C., Gianetto, R., Wattiaux, R. and Appelmans, F. (1955) Biochem. J. 60, 604-617) (B) the determination of the sediterm of different marker enzymes (Slinde, E. and Flatmark. T. (1973) Anal. Biochem. 56, 324-340) and (c) the determination of the stoichiometry between the activities of these enzymes sedimented at higher centrifugal effects. 5. Throughout all fractionation procedures, the long-chain acyl-CoA synthetase follows strictly the amine oxidase generally considered to be exclusively located on the mitochondrial outer membrane.

Adipose Tissue, Brown↗