Further evidence for changes in the level of palmitoyl-CoA desaturase during thermal adaptation in Tetrahymena pyriformis.

Palmitoyl-CoA desaturase activity in microsomes was increased up to about 4-fold within 2 h after temperature shift from 39.5 to 15 degrees C. Compared with control cells, cycloheximide-treated cells indicated no induction of palmitoyl-CoA desaturase by a decrease in temperature. The results suggest that temperature acclimation requires an increase in the level of the desaturase enzyme content.     

Mechanism of thermal adaptation of membrane lipids in Tetrahymena pyriformis NT-1. Possible evidence for temperature-mediated induction of palmitoyl-CoA desaturase.

The regulatory mechanism of a key enzyme, palmitoyl-CoA desaturase, involved in the adaptation to temperature shift was investigated by labeling Tetrahymena pyriformis cells with [14C]palmitic acid. The rate of conversion of [14C]palmitate to [14C]palmitoleate was shown to be dependent on incubation temperature and also to be maximal at 2 h after the shift 39.5 to 15 degrees C. Addition of cycloheximide before the temperature shift produced no increase in desaturation of [14C]palmitate after the shift. These data would provide evidence for temperature-triggered increase of palmitoyl-CoA desaturase level and are also discussed in relation to membrane fluidity.     

Further evidence for changes in the level of palmitoyl-CoA desaturase during thermal adaptation in Tetrahymena pyriformis

Palmitoyl-CoA desaturase activity in microsomes was increased up to about 4-fold within 2 h after temperature shift from 39.5 to 15°C. Compared with control cells, cycloheximide-treated cells indicated no induction of palmitoyl-CoA desaturase by a decrease in temperature. The results suggest that temperature acclimation requires an increase in the level of the desaturase enzyme content.     

Thermoadaptive regulation of microsomal desaturase and electron-transport enzyme activities in lipid-manipulated Tetrahymena cells. Extent of unsaturated fatty acid production is dependent on membrane fluidity before temperature down-shift

Exposure of Tetrahymena pyriformis NT-1 to chimyl alcohol (1-O-hexadecyl glycerol) produced a reproducible enhancement in unsaturated fatty acids and a great decrease in order parameter (S), which result from the 2-fold increases of stearoyl-CoA and oleoyl-CoA desaturase activities in microsomes. When the chimyl alcohol-fed cells were shifted from 34 to 15 degrees C (down-shift), unlike the drastic increases in palmitoyl-CoA, stearoyl-CoA and oleoyl-CoA desaturase activities in the native cells, there was only a slight increase in palmitoyl-CoA desaturase activity with a parallel rise in the activity of the terminal component (cyanide-sensitive factor; CSF) of the desaturase system. During cold acclimation, the decrease of order parameter in chimyl alcohol-fed cells was smaller than that in native cells, since the order parameter had already been decreased by the addition of chimyl alcohol before the shift. These results suggest that chimyl alcohol-fed cells are easily able to accomplish temperature acclimation without requiring great modification of fatty acid composition and membrane fluidity, while the non-fed control cells have difficulty doing so.     

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.     

Properties of rat liver microsomal stearoyl-coenzyme A desaturase.

1. Rat liver microsomal stearoyl-CoA desaturase activity was shown to be stimulated by both bovine serum albumin and a basic cytoplasmic protein from rat liver. 2. Partially purified desaturase is unaffected by either of these two proteins. 3. Bovine serum albumin appears to exert its effect on the crude system by protecting the desaturase substrate, stearoly-CoA, from the action of endogenous thiolesterases. 4. By using partially purified enzyme preparations, it was possible to establish the substate specificity of the delta9-fatty acyl-CoA desaturase with the C14, C15, C16, C17, C18 and C19 fatty acyl-CoA substrates. Maximum enzyme activity was shown with stearoyl-CoA decreasing with both palmitoyl-CoA and nonadecanoyl-CoA, as reported previously for free fatty acids. 5. Both cytochrome b5 and NADH-cytochrome b5 reductase (EC 1.6.2.2) are required for these studies and a method is described for the purification of homogeneous preparations of detergent-isolated cytochrome b5 from rat liver. 6. From amino acid analyses, a comparison was made of the hydrophobicity of the membrane portion of cytochrome b5 with the hydrophobicity reported for stearoyl-CoA desaturase. The close resemblance of the two values suggested that unlike cytochrome b5 and its reductase, the stearoyl-CoA desaturase may be largely buried in the endoplasmic reticulum.     

Isolation of a stearoyl CoA Desaturase form Tetrahymena thermophila

Cell free preparations of Tetrahymena thermophila contain an enzyme that catalyzes the direct desaturation of stearoyl CoA to octadecenoic acid. The enzyme is associated with the microsomal fraction of the ciliate. Substrate of the enzyme consists of either free stearic acid or stearoyl CoA. Both ATP and CoA are required when free stearate is the substrate and are also highly stimulatory when stearoyl CoA is the substrate. With stearoyl CoA as the substrate, either NADH or NADPH are required for desaturase activity. In presence of ATP and CoA, either NAD or NADP can replace NADH and NADPH. Desaturase activity is optimal when the enzyme is incubated at pH of 7.2 and a temperature of 30-35 degrees C. Highest levels of the stearoyl CoA desaturase are found in stationary phase ciliates grown at 35 degrees C.     

Lack of stearoyl-CoA desaturase-1 function induces a palmitoyl-CoA Delta6 desaturase and represses the stearoyl-CoA desaturase-3 gene in the preputial glands of the mouse

The mouse preputial gland (PG), a specialized sebaceous structure, is rich in wax esters, triglycerides, and alkyl-2,3-diacylglycerol. We have found that the mouse PG expresses the three gene isoforms (SCD1, SCD2, and SCD3) of the Delta9 stearoyl-CoA desaturase enzyme that catalyzes the biosynthesis of monounsaturated fatty acids mainly, C16:1n-7 and C18:1n-9. However, mice with a targeted disruption in the SCD1 isoform (SCD1(-/-)) have undetectable SCD3 mRNA expression in the PG while the expression of SCD2 isoform was not altered. The levels of C16:1n-7 were reduced by greater than 70% while that of C18:1n-9 were reduced by 28%. The content of the C16:1n-10 (Delta6 hexadecenoic acid) isomer and a major fatty acid of the PG was increased by greater than 2-fold, mainly in the wax ester fraction of the SCD1(-/-) mouse. We demonstrate that the increase in C16:1n-10 is due to induction of a specific palmitoyl-CoA Delta6 desaturase activity. Testosterone administration to the SCD1(-/-) mouse induced SCD3 mRNA expression and resulted in an increase in the Delta9 desaturation of 16:0-CoA, but not of 18:0-CoA. These observations demonstrate that loss of SCD1 function alters the expression of SCD3 and reveal for the first time the presence and regulation of a palmitoyl-CoA Delta6 desaturase enzyme in mammals.     

Characterization of sn-glycerol 3-phosphate acyltransferase from guinea pig harderian gland microsomes

Microsomal sn-glycerol 3-phosphate acyltransferase from the guinea pig Harderian gland was studied. Its specific activity (1.0 nmol/min X mg, with palmitoyl-CoA as a substrate) was almost the same as that of the rat liver microsomal enzyme. The enzyme acted on various types of acyl-CoA, the relative reaction rates being as follows: palmitoyl-CoA, 100(%); stearoyl-CoA, 30; oleoyl-CoA, 50; linoleoyl-CoA, 40; and arachidonoyl-CoA, 20. When assayed in the presence of 1 mM 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), the activity on palmitoyl-CoA was inhibited by only 20-30%, whereas those for other acyl-CoAs were completely abolished. The DTNB-resistant activity was inhibited by 0.1 mM dihydroxyacetonephosphate and 0.5 mM dithiothreitol, whereas the DTNB-sensitive activity was not affected. Furthermore, heat treatment at 50 degrees C for 15 min abolished most of the DTNB-sensitive activity, but not the DTNB-resistant activity. These results, taken together, suggested that the microsomal fraction of the guinea pig Harderian gland contained at least two types of sn-glycerol 3-phosphate acyltransferase, and that, in contrast to in the case of rat liver microsomes, a DTNB-resistant enzyme that utilized exclusively palmitoyl-CoA was predominant.     

Age-dependent modifications in membrane lipids: lipid composition, fluidity and palmitoyl-CoA desaturase in Tetrahymena membranes

The membrane lipid composition of Tetrahymena pyriformis NT-I was observed to change in a manner markedly dependent on the progress of culture age. The pellicular, mitochondrial and microsomal membranes were isolated from cell harvested at various growth phases (I, early exponential; II, mid-exponential; III, late exponential; IV, early stationary; V, late stationary) and their lipid composition was analyzed by thin-layer and gas-liquid chromatography. Although the phospholipid composition varied somewhat among membrane fractions, the most general age-dependent alteration was a considerable decrease in the content of phosphatidylethanolamine accompanied by a small increase in phosphatidylcholine. The 2-aminoethylphosphonolipid, enriched in the surface membrane pellicle, did not undergo a consistent change. As for fatty acid composition the most notable variation occurred in unsaturated fatty acids; a great increase in oleic and linoleic acids and a compensatory decrease in palmitoleic acid. This resulted in an augmented unsaturation of the overall phospholipid fatty acid profile of the aged membranes. The age-associated drastic decline in the palmitoleic acid content in membrane phospholipids could be accounted for by the markedly lowered activity of palmitoyl-CoA desaturase. The microsomes from the early exponential phase cells possess a 4-fold higher activity of the desaturase as compared to that of the late stationary phase microsomes. The decreased desaturase activity associated with the culture age was also reflected in the corresponding decrease in the conversion rate of [14C]palmitate to [14C]palmitoleate in cells labelled in vivo. The ESR spectra of the spin-labeled phospholipids extracted from the pellicular and microsomal membranes have led to the suggestion that these types of membrane would become more fluid with the age of growth.     

Studies in Tetrahymena membranes substrates for desaturation of fatty acyl chains in Tetrahymena pyriformis microsomes

[1-14-C]Palmitoyl-Co A was incubated with Tetrahymena microsomes containing the complete enzyme system for desaturation during various time periods. The level of [1-14C]palmitoleoyl-CoA increased to a maximum during the 1--3 min incubation time, while [1-14C]palmitoleic acid in the phospholipid reached a maximum level during 6--7 min incubation time. The radioactivity of [1-14C]palmitoleic acid in free fatty acid and the triglyceride fraction was not significantly observed upon 3 min incubation. Incubation of [1-14C]palmitoyl-CoA with microsomes in the absence of NADH produced [1-14C]palmitoyl lipid without desaturation. Radioactive palmitic acids in the microsomal lipids were not converted to palmitoleic acids after addition of NADH by the complete enzyme system. When microsomes prepared from cells labeled with [1-14C]palmitic acid or [1-14C]stearic acid were incubated alone in the presence of O2 and NADH, no significant increase in [1-14C]palmitoleic acid in the phospholipid was observed, wherease an increase in [1-14C]linoleic acid and gamma-[1-14C]linolenic acid did occur at the expense of [1-14C]oleic acid in the phospholipid. From these results it can be concluded that the enzyme involving desaturation of palmitic acid to palmitoleic acid requires palmitoyl-CoA as the substrate. However, the possibility of oleoyl and linoleoyl phospholipids being substrates in the desaturation of Tetrahymena microsomes was suggested.     

Isolation of a Stearoyl CoA Desaturase from Tetrahymena thermophila

Cell free preparations of Tetrahymena thermophila contain an enzyme that catalyzes the direct desaturation of stearoyl CoA to octadecenoic acid. The enzyme is associated with the microsomal fraction of the ciliate. Substrate for the enzyme consists of either free stearic acid or stearoyl CoA. Both ATP and CoA are required when free stearate is the substrate and are also highly stimulatory when stearoyl CoA is the substrate. With stearoyl CoA as the substrate, either NADH or NADPH are required for desaturase activity. In the presence of ATP and CoA, either NAD or NADP can replace NADH and NADPH. Desaturase activity is optimal when the enzyme is incubated at a pH of 7.2 and a temperature of 30–35°C. Highest levels of the stearoyl CoA desaturase are found in stationary phase ciliates grown at 35°C.     

Effect of di-n-octyl phthalate on fatty acid composition of phosphatidylcholine in Tetrahymena

We examined the effect of di-n-octyl phthalate (DOP) on fatty acid composition of phosphatidylcholine (PC) in Tetrahymena pyriformis NT-1. When Tetrahymena cells were grown in DOP-containing proteose peptone medium, the cell growth was repressed. This repression was attended by decreases in the PC content of the cells and decreases in oleic (18:1), linoleic (18:2) and linolenic (18:3) acids of PC and an increase in palmitoleic acid (16:1). The ratio of 18:1/stearic acid (18:0) of PC in cells grown in DOP-containing medium was lower than that of control cells, while the ratio of 16:1/palmitic acid (16:0) was higher than that of control. On the other hand, no changes in the ratios of 18:2/18:1 and 18:3/18:2 were observed. The activity of microsomal stearoyl-CoA desaturase from cells grown with DOP (0.63 mumol/ml medium) decreased to 27% of that from control cells, while the microsomal palmitoyl-CoA desaturase activity increased to 210% of the control value. By the addition of dioleoyl glyceride to the DOP-containing medium, the effects of DOP on Tetrahymena cells were completely blocked. These results suggest that the changes in fatty acid composition of PC may be due to the alteration of the substrate specificity of microsomal delta 9-desaturase, and the decrease in stearoyl-CoA desaturase activity may be a cause for the cell growth repression.     

Lipid hydrogenation induces elevated 18:1-CoA desaturase activity in Candida lipolytica microsomes.

Microsomal membranes prepared from the mesophilic yeast Candida lipolytica grown at 10 degrees C were hydrogenated by the homogeneous Pd-catalyst, palladium di (sodium alizarine sulfonate) (Pd(QS)2). After hydrogenation to various levels, the microsomes were washed free of the Pd-complex and transferred to a reaction mixture (containing NADH, MgCl2, ATP, CoA and [14C]18:1-CoA) for assay of 18:1-CoA desaturase activity. Microviscosity alterations were also followed by measuring changes in DPH fluorescence polarization. Rapid catalytic hydrogenation of unsaturated fatty acids of the lipids occurred within 20-120 s, resulting in large increases in 16:0, 18:0 and 18:1 acids and decreases in 18:2 acid. In the range 7-20% 18:0 content, a pronounced increase in desaturase activity was observed, with a maximum of greater than 2-fold at a 18:0 content of 12%, followed by a decrease to the initial activity at 33% 18:0 content. These changes were well-correlated with changes in microviscosity, maximal desaturase activity occurring in the DPH fluorescence anisotropy range of 0.23-0.24; above and below this range, desaturase activities were close to the initial control values. It is suggested that the hydrogenation-induced increase in the formation of 18:2 from 18:1-CoA (proceeding partly through direct desaturation of PC) may be due to changes in conformation of the membrane-bound desaturase enzyme complex as a result of controlled rigidification of the surrounding lipids. The operation of such a self-regulating control mechanism would be consistent with a previously proposed model for microsomal desaturase action.     

Sterol manipulation that modulates the alteration in membrane fluidity of Tetrahymena pyriformis during temperature acclimation

Our previous results [Umeki and Nozawa (1983) Biochem. Biophys. Res. Commun. 113, 96-101] suggested that ergosterol-replaced Tetrahymena cells (ergosterol-cells) accomplish an adaptive modification of fatty-acid composition by a preferential increase in palmitoyl-CoA desaturase activity, which is principally due to the increased content of the terminal component (cyanide-sensitive factor) of the desaturase system. The present study was designed to obtain information as to how the membrane fluidity of ergosterol-cells is changed during cold temperature acclimation. The order parameter (S) of liposomes prepared from ergosterol-cell lipids was reduced more rapidly after a temperature shift-down than that of control liposomes prepared from native cells containing tetrahymanol. These results indicate that, unlike native cells containing tetrahymanol, ergosterol-cells strive to accomplish cold temperature acclimation by undergoing a great modification of membrane fluidity because of the altered microsomal desaturase activity.     

Characterization of rat brain microsomal acyl-coenzyme A ligases: different enzymes for the synthesis of palmitoyl-coenzyme A and lignoceroyl-coenzyme A

Palmitic acid solubilized with Triton WR-1339 was converted to palmitoyl-CoA by microsomal membranes but lignoceric acid solubilized with Triton WR-1339 was not an effective substrate even though the detergent dispersed the same amount of these fatty acids and was also not inhibitory to the enzyme [I. Singh, R. P. Singh, A. Bhushan, and A. K. Singh (1985) Arch. Biochem. Biophys. 236, 418-426]. This observation suggested that palmitoyl-CoA and lignoceroyl-CoA may be synthesized by two different enzymes. We have solubilized the acyl-CoA ligase activities for palmitic and lignoceric acid of rat brain microsomal membranes with Triton X-100 and resolved them into three separate peaks (fractions) by hydroxylapatite chromatography. Fraction A (palmitoyl-CoA ligase) had high specific activity for palmitic acid and Fraction C (lignoceroyl-CoA ligase) for lignoceric acid. Specific activity of palmitoyl-CoA ligase for palmitic acid was six times higher than in Fraction C and specific activity of lignoceroyl-CoA ligase for lignoceric acid was four times higher than in Fraction A. At higher concentrations of Triton X-100 (0.5%), lignoceroyl-CoA ligase loses activity whereas palmitoyl-CoA ligase does not. Lignoceroyl-CoA ligase lost 60% of activity at 0.6% Triton X-100. Palmitoyl-CoA ligase (T1/2 of 4.5 min) is more stable at 40 degrees C than lignoceroyl-CoA ligase (T1/2 of 1.5 min). The pH optimum of palmitoyl-CoA ligase was 7.7 and that of lignoceroyl-CoA ligase was 8.4. Similar to our results with intact membranes, palmitic acid solubilized with Triton WR-1339 was converted to palmitoyl-CoA by palmitoyl-CoA ligase whereas lignoceric acid when solubilized with Triton WR-1339 was not able to act as substrate for lignoceroyl-CoA ligase. Since solubilized enzyme activities for synthesis of palmitoyl-CoA and lignoceroyl-CoA from microsomal membranes can be resolved into different fractions by column chromatography and demonstrate different properties, we suggest that in microsomal membranes palmitoyl-CoA and lignoceroyl-CoA are synthesized by two different enzymes.     

Growth temperature-dependent stearoyl coenzyme A desaturase activity of Fusarium oxysporum microsomes.

The characteristics of the microsomal stearoyl CoA desaturase (EC 1.14.99.5) of vegetative Fusarium oxysporum cells grown at different temperatures were studied. The enzyme had an unusual preference for NADPH (Km = 38 micrometers) over NADH (Km = 89 micrometers) as electron donor, and a relatively high optimum pH of 8.3. Enzyme activity was highest in microsomes from cells grown at 37 degrees C and lowest in cells grown at 15 degrees C. This result correlated well with the observed changes in oleic acid content of the microsomal lipids. Both NADPH-linked reductase activities and hemoprotein content were lowest in cells grown at 37 degrees C. Spectrophotometric analysis of the microsomal hemoproteins indicated the absence of cytochrome b5 and the presence of a b-type heme with a pyridine hemochrome alpha band absorption maximum at 565 nm. Labile sulfide analysis and inhibitor studies with thenoyltrifluoroacetone suggested a role for an iron-sulfur protein in the electron transfer system associated with the desaturase.     

Antarctic marine bacterium Pseudoalteromonas sp. 22b as a source of cold-adapted beta-galactosidase

The marine, psychrotolerant, rod-shaped and Gram-negative bacterium 22b (the best of 41 beta-galactosidase producers out of 107 Antarctic strains subjected to screening), classified as Pseudoalteromonas sp. based on 16S rRNA gene sequence, isolated from the alimentary tract of Antarctic krill Thyssanoessa macrura, synthesizes an intracellular cold-adapted beta-galactosidase, which efficiently hydrolyzes lactose at 0-20 degrees C, as indicated by its specific activity of 21-67 U mg(-1) of protein (11-35% of maximum activity) in this temperature range, as well as k(cat) of 157 s(-1), and k(cat)/K(m) of 47.5 mM(-1) s(-1) at 20 degrees C. The maximum enzyme synthesis (lactose as a sufficient inducer) was observed at 6 degrees C, thus below the optimum growth temperature of the bacterium (15 degrees C). The enzyme extracted from cells was purified to homogeneity (25% recovery) by using the fast, three-step procedure, including affinity chromatography on PABTG-Sepharose. The enzyme is a tetramer composed of roughly 115 kDa subunits. It is maximally active at 40 degrees C (190 U mg(-1) of protein) and pH 6.0-8.0. PNPG is its preferred substrate (50% higher activity than against ONPG). The Pseudoalteromonas sp. 22b beta-galactosidase is activated by thiol compounds (70% rise in activity in the presence of 10 mM dithiotreitol), some metal ions (K(+), Na(+), Mn(2+)-40% increase, Mg(2+)-15% enhancement), and markedly inactivated by pCMB and heavy metal ions, particularly Cu(2+). Noteworthy, Ca(2+) ions do not affect the enzyme activity, and the homogeneous protein is stable at 4 degrees C for at least 30 days without any stabilizers.     

Differential characteristics of purified hepatic triglyceride lipase and lipoprotein lipase from human postheparin plasma.

Evidence is presented that hepatic triglyceride lipase (H-TGL) and lipoprotein lipase (LPL), purified from human postheparin plasma, can each hydrolyze both glyceryl trioleate and palmitoyl-CoA. The average ratio of glyceryl trioleate/palmitoyl-CoA hydrolase activities, obtained with enzyme preparations from 15 human postheparin plasma samples was 1.30 (1.18-1.52) for H-TGL and 8.75 (7.45-10.25) for LPL. Albumin was identified as the serum cofactor required for the hydrolysis of palmitoyl-CoA by H-TGL. It protected this enzyme from inactivation by this substrate. In contrast, palmitoyl-CoA activated and protected LPL from denaturation by dilution and incubation at 25 degrees C. The effects of other detergents were investigated on glyceryl trioleate hydrolase activities of both enzymes. Sodium dodecyl sulfate (0.4 mM) and Trisoleate (0.4 mM), which also effectively activated and protected LPL against inactivation, had only moderate protective effect on H-TGL. Sodium dodecyl sulfate at a higher concentration (1 mM) produced little or no inhibition of LPL, while completely inactivating H-TGL. Conversely, sodium taurodeoxycholate (0.4 mM) protected and activated H-TGL, but had only moderate protective effect on LPL. Triton X-100 (0.1-0.8 mM) and egg lysolecithin (0.05-2 mM) also protected H-TGL, but not LPL. The very dissimilar effects of detergents on preparations on H-TGL and LPL may form the basis for the direct assay of each enzyme in the presence of the other.     

Mutants of Bacillus species isolated on the basis of protonophore resistance are deficient in fatty acid desaturase activity.

The fatty acid desaturase activity in cell extracts of Bacillus subtilis was characterized and found to be O2 dependent, NADH dependent, and cyanide sensitive. In cell fractionation studies, only 10% of the desaturase activity was recovered in the membrane fraction; the addition of cytosolic factors, which by themselves were devoid of activity, restored membrane activity to the level found in the unfractionated cell extracts. NADH was preferred over NADPH as an electron donor, and palmitoyl-coenzyme A was used preferentially over stearoyl-coenzyme A as the straight-chain fatty acid substrate. An increase in desaturase activity was observed when either the growth or the assay temperature was lowered from 37 to 20 degrees C, although the assay temperature appeared to be the more important parameter. Three protonophore-resistant mutants of B. subtilis and a comparable mutant of Bacillus megaterium had been found to possess reduced levels of unsaturated fatty acids in their membrane phospholipids; their protonophore resistance was abolished when grown in the presence of an unsaturated fatty acid supplement. All of these strains were found to be either significantly deficient in or totally lacking desaturase activity in comparison with their wild-type parent strains. Full, protonophore-sensitive revertants of the mutants had levels of desaturase activity comparable to those of the wild-type. Temperature-sensitive revertants of two of the mutants, which grew at 32 degrees C but not at 26 degrees C in the presence of protonophore, exhibited desaturase activity comparable to that of the wild-type at 26 degrees C but lacked activity at 32 degrees C. These results indicate that the biochemical basis for protonophore resistance in these Bacillus mutants is a fatty acid desaturase deficiency.