Regulation of linoleic acid delta 6-desaturation by a cytosolic lipoprotein-like fraction in isolated rat liver microsomes

A peripheral component of the delta 6-fatty acid-desaturase system of rat liver microsomes has been isolated from the cytosol by ultracentrifugation at a saline density of 1.26 g/ml. It exhibited lipoprotein characteristics with an approximate protein/lipid ratio of 1.22 and free fatty acids and phosphatidylcholine as its main lipid components. Linoleic acid desaturation activity diminished in washed microsomes, since they lost the adsorbed cytosolic fraction. Addition of the factor reactivated the reaction and the recovery was dependent on the concentration of the factor in the medium. Linoleic acid and linoleyl-CoA were bound by the cytosolic fraction. However, the transport of substrate to the desaturase was not apparently a main function of the cytosolic fraction, since transport occurred equally in the absence of the factor. Moreover, the solubilization of linoleyl-CoA was not enhanced and the free monomeric concentration was not altered by the presence of the cytosolic fraction. In addition, the factor did not divert delta 6-desaturase substrate to or from other metabolic pathways such as esterification to phospholipids. gamma-Linolenic acid produced by delta 6-desaturation of linoleic acid in the microsomes inhibited the desaturase, but it was removed by the factor from the membrane towards the cytosol, preventing the inhibition. The anti-inhibitory effect of the cytosolic factor was blockaded by addition of columbinic acid or gamma-linolenic acid to the factor. Moreover, the inhibitory effect of arachidonic acid was not prevented by addition of the cytosolic fraction. These results suggest that the cytosolic fraction studied would optimize the delta 6-desaturation of linoleic acid in vitro in rat liver microsomes by removal of the product, gamma-linolenic acid, as it is formed.     

Modulation of rat liver lipid metabolism by prolactin

The effect of chronic hyperprolactinemia on the delta6- and delta5-desaturation activity, total lipid and fatty acid composition, as well as fluorescence anisotropy, was studied in liver microsomes from anterior pituitary-grafted rats. We observed a depression in delta6-desaturation activity but no changes in the delta5-desaturation activity in the grafted animals. The microsomal fraction from the hyperprolactinemic rats contained significantly less amount of linoleic acid and a higher content of 20:4 n-6, 22:5 n-6 and 22:6 n-3 acids. Lipid rotational mobility was increased in microsomes as well as in liposomes obtained from the microsomes of transplanted animals. The fluidifying effect induced by PRL was located in the deepest zone of the membrane. The results obtained indicate that high levels of prolactin induce changes in polyunsaturated fatty acid distribution in liver microsomes, which regulates the lipid rotational mobility and hence membrane fluidity.     

Comparative in vivo and in vitro study of the influence of experimental diabetes on rat liver linoleic acid delta 6- and delta 5-desaturation

Rats with experimental diabetes were administered in vivo a tracer dose of either [1-14C]-linoleic, [1-14C]-gamma-linolenic or [2-14C]-dihomo-gamma-linolenic acid and sacrificed 48 h later. With all three radioactive precursors, the radioactivity incorporated into arachidonic acid was lower in experimental diabetes, compared to nondiabetic rats similarly treated, while the weights of hepatic arachidonic acid were not significantly affected by the diabetic state. Streptozotocin-treated rats were administered moderate or excessive quantities of protamine-zinc-insulin. Streptozotocin diabetes inhibits rat liver homogenate [2-14C]-dihomo-gamma-linolenic acid delta 5-desaturation; only moderate injections of protamine-zinc-insulin restore the in vitro delta 5-desaturation. These results suggest that experimental diabetes, a reported inhibitor of delta 6-desaturation, also causes partial inhibition of delta 5-desaturation in rat liver; this suggests that dihomo-gamma-linolenic acid desaturation, a secondary regulatory step in linoleic acid metabolism, may be restored through an optimum insulin therapy.     

Two species of cytochrome P-450 involved in ergosterol biosynthesis of yeast

Discrimination of cytochrome P-450 involved in delta 22-desaturation of ergosta-5,7-dien-3 beta-o1 (P-450(22)-DS) from that involved in lanosterol 14 alpha-demethylation (P-450(14)-DM) in ergosterol biosynthesis was investigated with microsomes of several strains of Saccharomyces cerevisiae. In mutant N22 which is partially defective in the delta 22-desaturation, the 14 alpha-demethylation was not blocked. In contrast, mutant SG1 which is known to lack the 14 alpha-demethylation showed a significant activity of the delta 22-desaturation. The delta 22-desaturation activity was markedly increased upon aerobic adaptation of yeast cells but the 14 alpha-demethylation was not affected. Buthiobate, a specific inhibitor of P-450(14)-DM, and rabbit antibodies against P-450(14)-DM did not inhibit the delta 22-desaturation activity at all. It is evident from the obtained observations that these phenomena are not explainable in terms of NADPH-cytochrome P-450 reductase. These results indicate that P-450(22)-DS is different from P-450(14)-DM in molecular species.     

Effect of epinephrine on the oxidative desaturation of fatty acids in the rat.

The effect of epinephrine on the oxidative desaturation of fatty acids by liver microsomal preparations of rats has been studied. Administration of epinephrine (1 mg/kg body weight) produced a significant decrease in desaturation of [l-14C]=linoleic acid to gamma-linolenic acid and of [L-14C]alpha-linolenic acid to actadeca-6,9,12,15-tetraenoic acid 12 hr after the infection. Lower doses produced a lesser effect on the delta6-desaturation activity. Epinephrine administration modified the V max of linoleic acid desaturation but not the K m. There was also a slight increase in palmityl desaturation activity. The effect of epinephrine on delta6-desaturation activity was postulated to be mediated through an enhancement of the intracellular cyclic AMP levels that lead to an increase of a glucose metabolite. This metabolite would inhibit delta6-desaturation activity.     

Effect of dietary fats on arachidonic acid and eicosapentaenoic acid biosynthesis and conversion to C22 fatty acids in isolated rat liver cells

The desaturation, chain elongation and esterification of [1-14C]eicosapentaenoic acid, [1-14C]arachidonic acid, [1-14C]eicosatrienoic acid, [1-14C]linolenic acid and [1-14C]linoleic acid were studied in isolated liver cells. Rats fed diets with either 15% hydrogenated coconut oil or 15% partially hydrogenated marine oil, both deficient in essential fatty acids, 15% soybean oil or standard pellet diet with 6% fat, were used. The delta 4-desaturation of 22:5(n - 3) and 22:4(n - 6) as well as the delta 6-desaturase activity was distinctly higher in cells from animals fed coconut or marine oil than with soybean oil or standard pellet. The rate of delta 5-desaturation of 20:3(n - 6) and 20:4(n - 3) was nearly the same in cells from rats fed coconut, marine and soybean oils and higher than with standard pellet. The chain elongation of 20:5(n - 3) to 22:5(n - 3) was distinctly more pronounced than the elongation of 20:4(n - 6) with all four diets. 20:5(n - 3) was mainly esterified in the phospholipids with marine and coconut oils, and mainly in triacylglycerol with standard pellet and soybean oils. The proportion of [1-14C]20:4(n - 6) in the phospholipids to that in triacylglycerol decreased in the order marine oil greater than coconut oil greater than standard pellet greater than soybean oil. The different endogenous arachidonic acid content in the phospholipids induced by the different diets increased in the same order. 20:5(n - 3) was rapidly esterified in triacylglycerol and phospholipids, then liberated especially from the triacylglycerol fraction, chain elongated to 22:5(n - 3) and reesterified.     

Effects of dietary vitamin B-2 and vitamin E on the delta9-desaturase and catalase activities in the rat liver microsomes.

The effects of dietary vitamin B-2 and vitamin E on delta9-desaturation of stearoyl-CoA, catalase, glutathione peroxidase, superoxide dismutase and electron transport components in rat liver microsomes have been investigated. delta9-desaturase activities were decreased on diets deficient of vitamin B-2, E and supplemented with E. Among the peroxide-scavenging enzymes, only the catalase activity in microsomes correlates significantly with delta9-desaturase activity. In vitro addition of bovine catalase had no effect on microsomal delta9-desaturase activity on control diet. However, it enhanced the delta9-desaturation in microsomes on vitamin B-2-deficient diet which contained low catalase and high superoxide dismutase activities, compared to those in microsomes of control diet. It is suggested that the hydrogen peroxide-generating and -decomposing systems may play an important role on the delta9-desaturase activity in microsomes.     

Differential responses to fasting and subsequent feeding by microsomal systems of rat liver: 6- and 9-desaturation of fatty acids

Hepatic microsomal preparations from nonfasted, fasted, and fasted-fed rats were employed, together with cofactors, in studies of 9-desaturation of stearate-1-(14)C and 6-desaturation of linolenate-1-(14)C. Prior fasting sharply reduced 9-desaturation but did not affect 6-desaturation; feeding restored 9-desaturation. Position of desaturation was determined by permanganate-periodate oxidation and separation of the dicarboxylic acids. Feeding after fasting stimulated both desaturase systems but either dl-ethionine or actinomycin D prevented this. Dietary carbohydrate or saturated fat increased 9-desaturation and dietary protein increased 6-desaturation. Insulin treatment of nonfasted rats increased 9-desaturation but not 6-desaturation. High dietary unsaturated fat (20% safflower oil) stimulated 6-desaturation but inhibited the 9-desaturation response to feeding. The results indicate that the two desaturases are distinct and are inducible in response to different substances.     

In vitro study of delta-6- and delta-5-desaturation of linoleic and dihomo-gamma-linolenic acids in WKY rats on a high-sodium diet

Delta 6- and delta 5-desaturation activities of linoleic [1(4)C] and dihomo-gamma-linolenic [2(14)C] acids are increased, in vitro, in liver microsomes of normotensive rats WKY, which receive an hypernatriuric diet (NaCl 3%). These results lead to a best known of delta 6- and delta 5-desaturases, which are involved in fundamental steps of linoleic acid metabolites biosynthesis, implicated in blood pressure regulation.     

Changes of fatty acid composition of phospholipids and lipid structural order in rat liver mitochondrial membrane subsequent to galactosamine intoxication. Effect of clofibrate

Since it has been earlier reported that D-galactosamine induces an inhibition of palmitoylcarnitine transferase I and a depletion of mitochondrial phospholipids which were both prevented by clofibrate, an evaluation of the effects of these drugs on mitochondrial fatty acid composition was made. Galactosamine does not alter the fatty acid pattern of these fatty acids whereas clofibrate induces a 2-fold increase in monounsaturated/saturated fatty acids ratio and a 10-fold decrease of the 20:4 (n - 6)/20:3 (n - 6) ratio in phosphatidylcholine. These alterations suggest an increase of delta 9-desaturation and a decrease of delta 5-desaturation. To determine whether the drug-induced changes in mitochondrial phospholipids has an effect on the physical properties of the membrane, the lipid structural order of mitochondrial preparations was studied using the lipophilic probes DPH and TMA-DPH. Mitochondrial isolated either from galactosamine- or clofibrate-treated rats showed a decrease in fluorescence polarization, indicating an overall decrease in lipid structural order. This alteration is more drastic when both drugs are administered. This phenomenon suggests drastic changes in the bulk phase of inner mitochondrial membrane lipids after treatments and could explain the altered kinetic properties of palmitoylcarnitine transferase I.     

Human placental 3beta-hydroxysteroid dehydrogenase: delta5-isomerase. Demonstration of an intermediate in the conversion of 3beta-hydroxypregn-5-en-20-one to pregn-4-ene-3,20-dione.

3beta-Hydroxypregn-5-en-20-one (pregnenolone) and NAD+ were incubated with a solubilized preparation of the coupled enzyme 3beta-hydroxysteroid:NAD(P) oxidoreductase-3-ketosteroid delta4,delta5-isomerase (3beta-hydroxysteroid dehydrogenase: delta5-isomerase) from the mitochondrial fraction of human placenta. Unconverted pregnenolone, pregn-4-ene-3,20-dione (rogesterone), and a small but detectable amount of pregn-5-ene-3,20-dione were isolated from the medium by Sephadex LH-20 chromomatography. The identification of pregn-5-ene-3,20-dione, confirmed by mass fragmentography, has provided the first direct evidence for the formation of the hypothetical delta5,3-ketone intermediate in the conversion of pregnenolone to progesterone. When tritium-labeled pregnenolone and [4-14C]pregnenolone were incubated simultaneously the 3H:14C ratio in isolated pregn-5-ene-3,20-dione was 4.6 times greater than in isolated progesterone and pregnenolone, indicating a kinetic isotope effect in the enzymatic isomerization of tritium-labeled pregn-5-ene-3,20-dione. Exposure of the enzyme to two steroids which inhibit the overall enzyme reaction, 2alpha-cyano-17beta-hydroxy-4,4,17alpha-trimethylandrost-5-en-3-one (cyanoketone) and 3-hydroxyestra-1,3,5(10),6,8-pentaen-17-one (equilenin), increased the relative yield of labeled pregn-5-ene-3,20-dione as well as the recovery of radioactivity remaining as unconverted pregnenolone, suggesting that both the dehydrogenase and isomerase activities were inhibited. Exposure of the enzyme to equilenin increased the ratio of isolated pregn-5-ene-3,20-dione radioactivity to progesterone radioactivity as progesterone synthesis was inhibited. Equilenin also diminished the tritium isotope effect on the isomerase reaction. Both findings suggest that it is possible to inhibit the isomerase to a greater extent than the dehydrogenase. In order to measure the rate of progesterone produced by the coupled enzymes, we have modified a radiochemical method which involves precipitation of pregnenolone by digitonin. Digitonin precipitation proved to be effective in separating unconverted pregnenolone from the steroid products of both enzyme reactions, progesterone and pregn-5-ene-3,20-dione. Neither the steroidal inhibitors nor the kinetic isotope effect altered the accuracy of the method for routine measurement of the overall rate of conversion of delta5,3beta-hydroxysteroid to delta4,3-ketosteroid.     

Identification and functional characterization of the moss Physcomitrella patens delta5-desaturase gene involved in arachidonic and eicosapentaenoic acid biosynthesis

The moss Physcomitrella patens contains high levels of arachidonic acid and lesser amounts of eicosapentaenoic acid. Here we report the identification and characterization of a delta5-desaturase from P. patens that is associated with the synthesis of these fatty acids. A full-length cDNA for this desaturase was identified by data base searches based on homology to sequences of known delta5-desaturase cDNAs from fungal and algal species. The resulting P. patens cDNA encodes a 480-amino acid polypeptide that contains a predicted N-terminal cytochrome b5-like domain as well as three histidine-rich domains. Expression of the enzyme in Saccharomyces cerevisiae resulted in the production of the delta5-containing fatty acid arachidonic acid in cells that were provided di-homo-gamma-linolenic acid. In addition, the expressed enzyme generated delta5-desaturation products with the C20 substrates omega-6 eicosadienoic and omega-3 eicosatrienoic acids, but no products were detected with the C18 fatty acid linoleic and alpha-linolenic acids or with the C22 fatty acid adrenic and docosapentaenoic acids. When the corresponding P. patens genomic sequence was disrupted by replacement through homologous recombination, a dramatic alteration in the fatty acid composition was observed, i.e. an increase in di-homo-gamma-linolenic and eicosatetraenoic acids accompanied by a concomitant disappearance of the delta5-fatty acid arachidonic and eicosapentaenoic acids. In addition, overexpression of the P. patens cDNA in protoplasts isolated from a disrupted line resulted in the restoration of arachidonic acid synthesis.     

Characterization of nystatin-resistant mutants of Saccharomyces cerevisiae and preparation of sterol intermediates using the mutants

Analysis of sterols of Saccharomyces cerevisiae mutants N3, N15, N26, and N3H, defective in sterol biosynthesis, was performed. Strains N3, N15, and N26 were isolated from their mother strain, M10, by screening with nystatin (Nagai et al. (1980) Mie Med. J. 30, 215-224), and strain N3H was isolated from N3 as a doubly-mutated strain. The main sterols of N3, N15, N26, and N3H were ergosta-7,22-dienol, ergost-8-enol, cholesta-5,7,24-trienol, and ergosta-7,22,24(28)-trienol, respectively. The former three strains were characterized as defective in delta 5-desaturation, delta 8--delta 7 isomerization, and C-24 transmethylation. Strain N3H was found to be defective in delta 5-desaturation as well as in delta 24(28)-reduction. However, the defect of N26 and N3H was suggested to be leaky, since small amounts of ergosterol and ergosta-7,22-dienol were found in these mutants, respectively. In N15, an accumulation (2% in total sterols) of the compound likely to be hydroxylated sterol was found. By aerobic adaptation of these strains, the accumulation of these strains, the accumulations of ergosta-7,22-dienol (22 mg/g dry cells), ergosta-7,22,24(28)-trienol (24 mg), ergosta-8,24(28)-dienol (18 mg), and cholesta-8,24-dienol (22 mg) reached a maximum in N3, N3H, N15, and N26 after 20, 20, 30, and 30 h, respectively. These strains appear to be useful for making 14C-labeled and non-labeled preparations of the above sterols.     

Gamma-linolenic acid dietary supplementation can reverse the aging influence on rat liver microsome delta 6-desaturase activity.

We have recently demonstrated that in rats the process of delta 6-desaturation of linoleic and alpha-linolenic acids slows with aging. One method of counteracting the effect of slowed desaturation of linoleic acid would be to provide the 6-desaturated metabolite, gamma-linolenic acid (18:3(n-6) GLA) directly. We have here investigated the 6-desaturation of both linoleic and alpha-linolenic acids in liver microsomes of young and old rats given GLA in the form of evening primrose oil (EPO) (B diet) in comparison to animals given soy bean oil alone (A diet), monitoring also the fatty acid composition of liver microsomes and relating this to the microviscosity of the membranes. In young rats the different experimental diets did not produce any difference in delta 6-desaturase (D6D) activity on either substrate suggesting that, when D6D activity is at or near its peak, the variations in diet tested are unable to influence it. In the old animals the rate of 6-desaturation of linoleic and particularly of alpha-linolenic acid was significantly greater in the B diet fed animals than in the A diet fed. The effects of the diets on the fatty acid composition of liver microsomes were consistent with the findings with regard to 6-desaturation. Administration of GLA partially corrected the abnormalities of n-6 essential fatty acid (EFA) metabolism by raising the concentration of 20:4(n-6) and other 6-desaturated EFAs. Furthermore, the GLA rich diet also increased the levels of dihomo-gamma-linolenic acid and of 6-desaturated n-3 EFAs in the liver microsomes. The microviscosity of microsomal membranes as indicated by DPH polarization was correlated with the unsaturation index of the same membranes. There was a very strong correlation between the two. In both young and old rats the B diet reduced the microviscosity and increased the unsaturation index. However, the effect was much greater in the old animals.     

Accumulation of 14 alpha-methylergosta-8,24(28)-dien-3 beta,6 alpha-diol in 14 alpha-demethylation mutants of Candida albicans: genetic evidence for the involvement of 5-desaturase

14 alpha-Demethylation mutants of the fungus Candida albicans have been shown to accumulate 14 alpha-methylergosta-8,24(28)-dien-3 beta,6 alpha-diol. A derivative from one of these mutants (KD4900) that does not form this 6 alpha-hydroxylated sterol but is still defective in 14 alpha-demethylation (KD4950) was obtained. Mutational restitution of 14 alpha-demethylation capacity to this derivative resulted in the formation of the 5,6-saturated sterol ergosta-7,22-dien-3 beta-ol as the major product, clearly indicating that 5-desaturase deficiency exists in this demethylation-proficient revertant (KD4952). This implies that its parent, KD4950, which has lost the ability to form the hydroxylated sterol, also is deficient in 5-desaturation. We infer from the results that 5-desaturase is responsible for the formation of the hydroxylated sterol. However, it is unclear whether the hydroxylation represents a genuine step of the normal 5-desaturation reaction.     

Preliminary characterization of the delta-9 desaturase of Tetrahymena pyriformis W.

1. In vitro assay conditions have been defined for measurement of delta 9 desaturase activity in Tetrahymena pyriformis W. 2. The reaction depends on the presence of oxygen and a reduced pyridine nucleotide cofactor. FAD supports a low level of enzymatic activity. 3. Both stearyl-CoA and palmityl-CoA are acceptable substrates. Oleate formation is maximal at 30 degrees C. 4. Delta-9 desaturase activity appears to be localized in the microsomal fraction. Delta-6 and/or delta 12 desaturase activities have also been observed. 5. When the specificity of the delta 9 desaturase towards stearyl-CoA and palmityl-CoA was observed at 30 and 16 degrees C it was found that lowering the assay temperature did not affect specificity. Stearyl-CoA was more readily desaturated at both temperatures. 6. Exogenous oleyl-CoA and diisopropylfluorophosphate had little effect on delta 9 desaturase activity. However, cyanide strongly inhibited desaturation and a sensitivity to sulfhydryl-binding reagents has also been demonstrated.     

PGJ2 and delta 12PGJ2 inhibit cell growth accompanied with inhibition of phosphoinositide turnover in human astrocytoma cells

PGJ2 and delta 12PGJ2 (1 microM to 30 microM) inhibited the growth of human astrocytoma cells (1321N1) in a time-dependent manner within 48 hrs, determined by [3H]thymidine incorporation into acid-insoluble fraction or amounts of protein. The EC50 values for PGJ2 and delta 12PGJ2 were approximately 8 microM and 6 microM, respectively. [3H]Thymidine incorporation to acid insoluble fraction was inhibited by these PGs within 1 hr, indicating that these PGs rapidly affect cell functions. Although it has been reported that an increase in cyclic AMP inhibits cell growth, PGJ2 and delta 12PGJ2, but not PGE1, reduced isoproterenol (10 microM)-induced accumulation of cyclic AMP, suggesting that PGJ2 and delta 12PGJ2 may disturb adenylate cyclase system, which might be independent on cell growth. On the other hand, these PGs inhibited the incorporation of [3H]inositol into phospholipid fraction within 6 hrs. Furthermore, PGJ2 and delta 12PGJ2 inhibited carbachol- and/or histamine-induced accumulation of inositol phosphates with a similar dose-dependency to their inhibitions of cell growth. In membrane preparations, however, PGJ2 and delta 12PGJ2 failed to inhibit GTP gamma S (10 microM)- nor Ca2+ (1 mM)-induced accumulation of inositol phosphates. The site of PGJ2 or delta 12PGJ2 in inhibition of inositol phosphate accumulation would not be phospholipase C nor a putative GTP binding protein involved in activation of phospholipase C. The present results indicate that PGJ2 and delta 12PGJ2 inhibit cell growth in human astrocytoma cells and the inhibition of phosphoinositide turnover by these PGs might be involved in the inhibition of cell growth.     

Oxidative desaturation of eicosa-8,11-dienoic acid to eicosa-5,8,11-trienoic acid: comparison of different diets on oxidative desaturation at the 5,6 and 6,7 positions

The oxidative desaturation of [1-(14)C]eicosa-8,11-dienoic acid to eicosa-5,8,11-trienoic acid by rat liver microsomes was studied, and the kinetic conditions appropriate to measure the specific activity of the enzyme were determined. A comparative study of the effects of a balanced diet and essential fatty acid-free diets on the oxidative desaturation of oleic and linoleic acids at the 6,7 position and the oxidative desaturation of eicosadienoic acid at the 5,6 position were made. Eicosadienoic acid showed a higher conversion than oleic acid for all the diets. The conversion of oleic and linoleic acids to Delta6 acids was equally increased by fat-free diets with or without added methyl palmitate, whereas the oxidative 5-desaturation of eicosadienoic acid at the 5,6 position was not changed. The effect was apparently independent of the amount of endogenous free fatty acids. The results suggest that the rate-limiting and principal regulatory step in the biosynthesis of eicosa-5,8,11-trienoic acid is the 6-desaturation of oleic acid. The 5-desaturation of eicosadienoic acid was increased by a protein diet and decreased by alloxan diabetes to a lesser extent than the 6-desaturation of linoleic acid. The 5-desaturation of eicosadienoic acid would constitute a secondary regulatory step.     

Sex-related differences in desaturation and chain elongation of essential fatty acids studied in isolated rat hepatocytes

When [14C]linoleic acid (18:2(n-6)) or [14C]dihomogammalinolenic acid (20:3(n-6)) was incubated with isolated liver cells from rats fed an essential fatty acid deficient diet, delta 6- and delta 5-desaturation, chain elongation and synthesis of 14C-labelled C14-C18 fatty acids (from [14C]acetate) were enhanced in female cells compared with male ones. No sex difference in total secretion of very low density lipoproteins (VLDL) was observed. However, VLDL secreted from female cells contained significantly more C16-C18 fatty acids than male cells. It is suggested that the observed sex differences, at least in part, may be related to the different content of fatty acid binding proteins in female cells compared with males.     

Characteristics of 16-dehydroprogesterone reductase in cell extracts of the intestinal anaerobe, Eubacterium sp. strain 144

16-Dehydroprogesterone reductase (16-DHPR) activity was present in cell extracts of Eubacterium sp. strain 144 only when the organism was grown in the presence of steroids containing a delta 16-17 double bond and C-20-ketone. Cells grown with 16-dehydropregnenolone contained 16-DHPR activity but lacked delta 4-5-3-keto steroid reductase activity. Pyruvate or sodium dithionite served as electron donors for 16-DHPR and both reactions required methyl viologen as an electron carrier. Neither NADH nor NADPH, with or without flavin nucleotides, were used by 16-DHPR. Enzyme activity was detected in the cytoplasmic fraction (40%) and membrane fraction (20%) of crude cell extracts, but 40% of the activity was unaccounted for following ultracentrifugation. 16-DHPR activity was unaffected by pH in potassium phosphate buffer over the range 5.0 to 8.5, but was inhibited by Tris-HCl above pH 7.0. 16-DHPR activity was inhibited by sulfhydryl reagents, but inhibitors of electron transport reactions or metal chelators did not affect the enzyme.