Differential effects of detergents upon the soluble and particulate guanylate cyclases from rat lung.

The enzyme guanylate cyclase is present in both particulate and soluble form in rat lung homogenates. As previously reported, the soluble enzyme can be activated by preincubation in the presence of O₂. The inactive (nonactivated) soluble enzyme is also stimulated by nonionic detergents, in the order Tween 20 > Lubrol PX > Triton X-67 > Triton X-100. The activated enzyme, however, was inhibited by these detergents in the reverse order. Sodium deoxycholate and lysolecithin were potent inhibitors of both inactive and activated enzyme. The activity of the particulate enzyme was stimulated by Lubrol PX > Triton X-100 > Triton X-67 > Tween 20. At a low concentration of lysolecithin or deoxycholate the particulate activity was increased; however, when detergent/protein > 1, inhibition was seen. In the case of deoxycholate, the inhibition could be reversed if excess deoxycholate was removed either by chromatography or by forming mixed micelles with Lubrol PX; however, deoxycholate inhibition of the soluble enzyme was irreversible. The stimulation by detergents of the particulate enzyme was apparently the result of solubilization. The effects upon the activity of the soluble enzyme were interpreted in terms of a model which assumes two hydrophobic regions on the enzyme surface. The two regions differ in hydrophobicity with the more hydrophobic region only being exposed as a result of activation. Interaction of a nonionic detergent with the less hydrophobic region stimulates activity, while interaction with the more hydrophobic region results in inhibition.     

Proteolytic stimulation and solubilization of membrane-bound acetylcholinesterase from muscle sarcotubular system

Incubation of membranes derived from sarcotubular system of rabbit skeletal muscle with increasing concentrations of Triton X-100 produced both stimulation of the AChE activity and solubilization of this enzyme. Mild proteolytic treatment of microsomal membranes produced a several fold activation of the still membrane-bound acetylcholinesterase (AChE) activity. Attempts were made to solubilize AChE from microsomal membranes by proteolytic treatment. About 30–40% of the total enzyme activity could be solubilized by means of trypsin or papain. Short trypsin treatment of the microsomal membranes produced first an activation of the membrane-bound enzyme followed by solubilization. Incubation of muscle microsomes for a short time with papain yielded a significant portion of soluble enzyme. Membrane-bound enzyme activation was measured after a prolonged incubation period. These results are compared with those of solubilization obtained by treatment of membranes with progressive concentrations of Triton X-100. The occurrence of molecular forms in protease-solubilized AChE was investigated by means of centrifugation analysis and slab gel electrophoresis. Centrifugation on sucrose gradients revealed two main components of 4.4S and 10–11S in either trypsin or papain-solubilized AChE. These components behaved as hydrophilic species whereas the Triton solubilized AChE showed an amphipatic character. Application of slab gel electrophoresis showed the occurrence of forms with molecular weights of 350,000; 175,000; 165,000; 85,000 and 76,000. The stimulation of membrane-bound AChE by detergents or proteases would indicate that most of the enzyme molecules or their active sites are sequestered into the lipid bilayer through lipid-protein or protein-protein interactions and these are broken by proteolytic digestion of the muscle microsomes.     

THE DISSOCIATION OF RAT BRAIN MEMBRANES BEARING ACETYLCHOLINESTERASE BY THE NON-IONIC DETERGENT TRITON X-100 AND AN EXAMINATION OF THE PRODUCT

Abstract— The action of Triton X-100 on a membrane preparation from rat brain was studied with reference to the solubilization of acetylcholinesterase and the product was characterized by exclusion chromatography. The AChE and membrane protein were readily solubilized to form particles corresponding to a mol. wt. of about 5 × 10⁵. The solubility of these particles depended on the continued presence of the detergent. It was concluded that these soluble particles formed an intermediate stage in organization between membrane-bound AChE and the soluble protein enzyme, and perhaps represented preexisting lipoprotein subunits of the membranes.     

Activation parameters of the adenosine triphosphatase of Micrococcus lysodeikticus. A comparison of the soluble and membrane-bound forms of the enzyme.

The Arrhenius plots for the membrane-bound ATPase and its soluble form purified from Micrococcus lysodeikticus, presented discontinuities near 30 degrees C at pH 7.5. Glycerol-containing lipids were not responsible for these discontinuities. The values of the enthalpies of activation were 12 (soluble) and 22 (membrane-bound) kcal/mol (50.2 and 92.0 kJ/mol) above 30 degrees C and 42 (soluble) and 29 (membrane-bound) kcal/mol (175.7 and 121.3 kJ/mol) below that temperature. The results suggested that both molecular forms of the ATPase were able to adopt at least two different structures, above and below the critical temperature. Of the two, only the high-temperature structure seemed to be enzymically active. In the case of lipid-dependent ATPases, such as the Escherichia coli enzyme, the transition between both enzyme structures probably occurred with simultaneous "melting" of their lipid microenvironment.     

Kinetic characterization and partial purification of the membrane-bound inorganic pyrophosphatase from Rhodopseudomonas palustris

A membrane-bound inorganic pyrophosphatase from Rhodopseudomonas palustris has been studied by kinetic analysis. The enzymatic activity was stimulated by Mg2+, and the (Mg-PPi) complex is regarded to be the functional substrate. Free Mg2+ revealed a significant influence on the membrane-bound PPiase activity. Kinetic data were determined at various fixed concentrations of free Mg2+. Mg2+ is proposed to act as an activator in two ways. It may interact with the enzyme directly, and may combine with PPi to yield the functional substrate Mg-PPi. Ca2+ revealed a non-competitive type of inhibition on the Mg2+-activated enzyme. The membrane-bound PPiase activity was firmly attached to the chromatophore membrane. To achieve an almost entire solubilization, both, Triton X-100 and high concentrations of Mg2+, had to be applied. An enrichment method along with stepwise lowering the concentrations of Triton X-100 and Mg2+ after the solubilization has been established. The solubilized and partially purified enzyme was stimulated by phospholipids while the influence of free Mg2+ was lost. Three different energies of activation as a function of temperature were derived from Arrhenius plots for the membrane-bound as well as for the solubilized PPiase activity.     

Brain myelin-bound Zn2+-glycerophosphocholine cholinephosphodiesterase is a glycosylphosphatidylinositol-anchored enzyme of two different molecular forms

A Zn²⁺-GPC cholinephosphodiesterase activity, which is present more predominently in myelin than in microsome or cytosol, has been examined using -nitrophenylphosphocholine as a substrate. In the solubilization of enzyme activity from myelin membranes, lysolecithin was found to be more effective than Triton X-100 or deoxycholate. Especially, the myelin-bound phosphodiesterase was suggested to be a glycosylphosphatidyl-inositol-anchored protein, based on solubilization by B. cereus phospholipase C and Triton X-114 phase separation. Interestingly, it was found that while phospholipase C-solubilized enzyme, a hydrophilic protein, was associable with Concanavalin A column, detergent-solubilized amphiphilic form of enzyme was not. Either detergent extract or cytosol was observed to contain both amphiphilic form and hydrophilic one. In CM-sephadex chromatography, the soluble hydrophilic phosphodiesterase was observed to be separatable into two forms of enzyme. In comparative studies, both forms of phosphodiesterase showed much similarity in substrate specificity, optimum pH, Km value and Zn²⁺ requirement, although they differed in charge property and molecular weight.     

Resolution and reconstitution of Rhodospirillum rubrum pyridine dinucleotide transhydrogenase

The Rhodospirillum rubrum pyridine dinucleotide transhydrogenase system is comprised of a membrane-bound component and an easily dissociable soluble factor. Active transhydrogenase complex was solubilized by extraction of chromatophores with lysolecithin. The membrane component was also extracted from membranes depleted of soluble factor. The solubilized membrane component reconstituted transhydrogenase activity upon addition of soluble factor. Various other ionic and non-ionic detergents, including Triton X-100, Lubrol WX, deoxycholate, and digitonin, were ineffectual for solubilization and/or inhibited the enzyme at higher concentrations. The solubilized membrane component was significantly less thermal stable than the membrane-bound component. None of the pyridine dinucleotide substrate affected the thermostability of the solubilized membrane-bound component, whereas NADP⁺ and NADPH afforded protection to membrane-bound component. NADPH stimulated trypsin inactivation of membrane-bound component to a greater extent than NADP⁺, but inactivation of solubilized membrane component was stimulated to the same extent by both pyridine dinucleotides. The solubilized membrane component appears to have a slightly higher affinity for soluble factor than does the membrane-bound component.Abbreviations AcPyAD⁺ oxidized 3-acetylpyridine adenine dinucleotide - BChl bacteriochlorophyll - C_T-particles chromatophores depleted of soluble transhydrogenase factor and devoid of transhydrogenase activity This work was supported by Grant GM 22070 from the National Institutes of Health, United States Public Health Service. Paper I of this series is R. R. Fisher et al. (1975)     

Multiple forms of acetylcholinesterase from pig brain

1. A number of methods of solubilization of pig brain acetylcholinesterase (EC 3.1.1.7) were studied. The multiple enzymic forms of the resultant preparations were examined by polyacrylamide-gel electrophoresis. 2. Butanol extraction, Nagarase treatment and ultrasonication proved unsuitable as preparatory methods, but detergent treatment (Triton X-100, Triton X-100-KCl and lysolecithin) gave good yields. 3. Separation of soluble enzyme in three systems of polyacrylamide-gel electrophoresis were compared and the relative advantages are discussed. 4. By using a 6% (w/v) gel and continuous buffer system two forms of acetylcholinesterase were detected in Triton X-100-solubilized enzyme, but the incorporation of a sample and spacer gel and a discontinuous buffer system resolved this into four components. The forms of the soluble enzyme extracted by different methods differed in mobility. 5. With gradient polyacrylamide-gel electrophoresis between two and six forms were detected, depending on the method used for extraction. The average molecular weights of the five forms most frequently found were 60000, 130000, 198000, 266000 and 350000. 6. Treatment of the Triton X-100-extracted enzyme with 2.5m-urea altered the pattern and evidence of dissociation was observed. 7. The results are discussed in the light of present theories on the molecular structure of acetylcholinesterase.     

Factors influencing β-glucosidase production, activity and stability inNectria catalinensis

The influence of different cultivation conditions on β-glucosidase production and of some parameters on the activity and stability of this enzyme were studied inNectria catalinensis. Maximal β-glucosidase production was achieved with ammonium nitrate (0.5 g N/L) as nitrogen source. Tween 80, Tween 20 and Triton X-100 increased β-glucosidase yields, Tween 80 was the most effective for enzyme release and growth at a concentration of 3.4 mmol/L. On the other hand, Tween 20 and Triton X-100 had an inhibitory effect onN. catalinensis growth. A temperature of 23°C and an initial pH of cultures of 6.5 were optimal for biomass and β-glucosidase production. Under optimal cultural conditions (ammonium nitrate, 0.5 g N/L; Tween 80, 3.4 mmol/L; 23°C; initial pH 6.5) the β-glucosidase yield was increased more than five fold respect to the initial state. Optimal temperature for β-glucosidase activity was 45°C, the initial activity dropped 60 % after 6 h of incubation at this temperature. Optimal pH for enzyme activity was 5.3. At this pH the β-glucosidase was completely stable after 3 d of incubation. TheV andK _m values calculated from Lineweaver-Burk and Eadie-Hofstee plots were 0.23 μmol 4-nitrophenol per min per mg of protein and 0.25 mmol 4-nitrophenol β-d-glucopyranoside per L, respectively. The activation energy according to Arrhenius plot was 49.6 KJ/mol.     

INFLUENCE OF COLD EXPOSURE ON THE ACTIVITIES OF SOLUBLE AND MEMBRANE-BOUND DOPAMINE β-HYDROXYLASE IN RAT HEART VESICLES

Abstract— A fraction containing noradrenaline storage vesicles of the sympathetic nerve terminals in the rat heart was obtained by differential centrifugation. In this preparation, 17% of the dopamine β-hydroxylase was present in a soluble form. Cold exposure (3°C) for periods from 5 to 30 min led to an increase in the activity of soluble dopamine β-hydroxylase by about 50%, while the activity of membrane-bound dopamine β-hydroxylase was simultaneously decreased by approx 30%. The nor-adrenaline content of the vesicles rose concomitantly with the increase in the activity of soluble dopamine β-hydroxylase. This rise in noradrenaline content was caused by an enhanced synthesis and not by an alteration in the subcellular distribution. The results are discussed with respect to the fate of dopamine β-hydroxylase during enhanced sympathetic nerve activity.     

Characterization of the interactions between lysophosphatides, Triton X-100, and sarcoplasmic reticulum.

The interaction of lysophosphatidylcholines and lysophosphatidylethanolamines with lobster abdominal muscle sarcoplasmic reticulum was studied. Only lysophosphatidylcholines with 16 and 18 carbon acyl chains were effective solubilizing agents. The rate of membrane solubilization was most rapid with the palmitoyl and oleoyl derivatives. All lysophosphatides partially inhibited calcium-dependent ATPase activity between 0.0 and 2.0 μmol of lysophosphatide mg^?1 of membrane protein. Lysophosphatides that were active in solubilizing membranes exhibited a reactivating effect on ATPase activity between 2.0 and 6.0 μmol of lysophosphatide mg^?1 of membrane protein. Arrhenius plots of temperature-dependent ATPase activity showed high activation energies and loss of discontinuities in Arrhenius plots when inhibiting concentrations of the lysophosphatides were present. These results suggest that the inhibiting effect of lysophosphatides on membrane enzyme activity is due to intrusion of the lysophosphatide into the membrane, which results in a less fluid lipid environment around the enzyme. Subsequent membrane solubilization at higher lysophosphatide concentrations may release the enzyme from the inhibiting effects of the lysophosphatide by increasing lipid fluidity neighboring the enzyme. The effects of lysophosphatides on a membrane enzyme system were also examined in the presence of 10 mm Triton X-100. Under these conditions, little effect on membrane enzyme activity was generated by increasing concentrations of the lysophosphatidylcholines (lauryl, palmitoyl, and steroyl), while the unsaturated lysophosphatidylcholine and the lysophosphatidylethanolamines caused a two- to threefold increase in enzyme activity. Temperature-dependent enzyme activity studies showed that discontinuities in the Arrhenius plots of enzyme activity occurred at varying temperatures, depending on the lysophosphatide used. Lowest transition temperatures occurred for lysophosphatidylcholine (oleoyl) and the lysophosphatidylethanolamines. These results suggest that, in the presence of 10 mm Triton X-100, lipid exchange occurs around the sarcoplasmic reticulum ATPase enzyme and the fluidity of this lipid-protein complex is increased by lysophosphatides with unsaturated acyl chains or ethanolamine head groups.     

Phosphatidylinositol biosynthesis in Saccharomyces cerevisiae: purification and properties of microsome-associated phosphatidylinositol synthase

The membrane-associated phospholipid biosynthetic enzyme phosphatidylinositol synthase (cytidine 5'-diphospho-1,2-diacyl-sn-glycerol:myo-inositol 3-phosphatidyltransferase, EC 2.7.8.11) was purified 1,000-fold from the microsomal fraction of Saccharomyces cerevisiae. The purification procedure included Triton X-100 solubilization of the microsomal membranes, CDPdiacylglycerol-Sepharose (Larson et al., Biochemistry 15:974-979, 1976) affinity chromatography, and chromatofocusing. The procedure resulted in the isolation of a nearly homogeneous protein preparation with an apparent minimum subunit molecular weight of 34,000, as determined by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Phosphatidylinositol synthase was dependent on manganese and Triton X-100 for maximum activity. The pH optimum was 8.0. Thioreactive agents inhibited enzyme activity. The energy of activation was found to be 35 kcal/mol (146,540 J/mol). The enzyme was reasonably stable at temperatures of up to 60 degrees C.     

Effects of reconstitution of membrane-bound lipoprotein lipase and dispersity of substrate on its reaction characteristics

Reaction characteristics of a membrane-bound lipoprotein lipase acting on a hydrophobic substrate were investigated in aggregated structures—lipid bilayers of liposomes and mixed micelles of Triton X-100. The enzyme activity was enhanced with increases in Triton X-100 and phospholipid concentrations in micellar and liposomal structures. This higher activity was found to be due to both the solubilization state of the hydrophobic substrate and the hydrophobic interactions of the enzyme with either phospholipid or Triton X-100 molecules as a result of its incorporation into the aggregated systems. The enzyme reconstituted into lipid bilayers of liposomes prepared from 15 mM DMPC in the presence of 0.05% Triton X-100 showed a further 1.5-fold higher activity in comparison with the activity without reconstitution in micelles of 1.0% Triton X-100. These results indicate the necessity of the bilayer structure to retain the membrane-bound enzyme in an active conformation.     

Kinetic properties of membrane-bound and Triton X-100-solubilized human brain monoamine oxidase

The kinetic properties of membrane-bound and Triton X-100-solubilized human brain mitochondrial type A and B monoamine oxidase were examined. These studies reveal that the K_m values for phenylethylamine and benzylamine, type B monoamine oxidase substrates, were only slightly increased by the solubilization procedure. The K_m value for 5-hydroxytryptamine, a type A monoamine oxidase substrate, was similarly increased by treatment with Triton X-100. The K_m values for oxygen with all three amine substrates were unaffected by solubilization of the oxidase. Similarly, the optimum pH for deamination of substrates for the B isoenzyme was essentially unaltered in the solubilized preparation as compared to the membrane-bound enzyme whereas that for 5-hydroxytryptamine metabolism was decreased from pH 8.5 to approximately 7.75 on solubilization. The energy of activation with all three substrates was altered on solubilization of the oxidases with Triton X-100. The energy of activation for the B monoamine oxidase substrates increased whereas that for 5-hydroxytryptamine decreased. These data support the contention that the lipid environment surrounding the two forms of monoamine oxidase controls, in part, the activity and kinetic properties of the enzymes.     

Brain lysosomal hydrolases: I. Solubilization and electrophoretic behavior of acid hydrolases in nerve-ending and mitochondrial-lysosomal fractions from rat brain. Effects of autolysis,neuraminidase, and storage

In solubility studies of 7 acid hydrolases, the extent of solubilization by sonic disruption varied with the enzyme species and increased with increasing pH and Triton X-100 concentration of the suspension medium. Hydrolases in the nerve-ending (NE) fraction were more resistant to solubilization than those in the mitochondrial-lysosomal (M-L) fraction, but nearly quantitative solubilization was attained by sonication in an alkaline buffer containing 0,5% Triton X-100. Polyacrylamide gel electrophoresis of extracts revealed multiple components of acid phosphatase, acid esterase, arylsulfatase,-glucuronidase, and-N-acetyl-hexosaminidase. The enzyme patterns varied with the subcellular fraction and the composition of the medium. In general, the acidic (anodic) forms of these hydrolases were more readily solubilized by sonication in acidic buffer, alkaline pH and Triton X-100 being required to solubilize the basic (cationic) components. The acidic forms of these enzymes were converted to less anodic or cathodic forms, or both, during autolysis at pH 6 at 0 and 37°C, and during storage at –20°C.     

Binding, activation, and solubilization of the Ca2+-ATPase from sarcoplasmic reticulum by nonionic detergents

Interactions between delipidated Ca2+-ATPase from sarcoplasmic reticulum and four nonionic detergents--dodecyl octaoxyethyleneglycol monoether (C12E8), Triton X-100, Brij 58, and Brij 35--were characterized with respect to activation of ATPase activity, binding, and solubilization. C12E8 and Triton X-100 activated the delipidated ATPase to at least 80% of the original activity at the critical micelle concentrations (CMCs), whereas Brij 58 and Brij 35 activated no more than 10% of the original activity. The inability of Brij 58 and Brij 35 to activate the delipidated enzyme was probably a result of reduced binding of these detergents below the CMCs; both detergents exhibited a sixteenfold reduction in binding at the CMC compared with C12E8. The two Brij detergents were also unable to solubilize the delipidated enzyme and form monomers, as determined by sedimentation experiments. Thus the reduced binding levels of these detergents may result from an inability to overcome protein/protein interactions in the delipidated preparation. However, the Brij detergents were capable of solubilizing active enzyme from membrane vesicles, although with lower efficiency than C12E8 and Triton X-100. These results suggest that Brij 58 and 35 may be useful for solubilization of membrane proteins without disrupting protein/protein interactions, while Triton X-100 and C12E8 are more useful when bulk solubilization is the goal.     

Methemoglobin reductase activity in fish erythrocytes

NADH-methemoglobin reductase activity of erythrocytes from the coho salmon, Oncorhynchus kisutch, sockeye salmon, Oncorhynchus nerka, and the rainbow trout, Salmo gairdneri exhibited a major band of activity that resembled the human enzyme in electrophoretic mobility. No polymorphism was found in 35 samples from rainbow trout, 4 samples from Dolly Varden, 29 samples from sockeye salmon, and 24 samples from coho salmon. All samples differed from the human enzyme in that they appeared to be membrane-bound and required the presence of a detergent, Triton X-100, for solubilization. Rainbow trout and coho salmon enzymatic activity is greater than the human enzyme activity at 15 degrees C.     

LOCALIZATION OF EXTRANEURONAL DOPAMINE-β-HYDROXYLASE IN RAT SALIVARY GLAND BY IMMUNOFLUORESCENCE HISTOCHEMISTRY

Abstract— Superior cervical ganglionectomy results in a complete noradrenergic neuronal denervation of the rat sublingual-submaxillary salivary gland. Dopamine-β-hydroxylase activity in the serous submaxillary gland falls approximately 90% after noradrenergic denervation; but in the mucinproducing sublingual gland dopamine-β-hydroxylase activity is reduced by only 33%. Dopamine-β-hydroxylase immunofluorescence in the submaxillary gland is distributed with noradrenergic neurons and is eliminated by superior cervical ganglionectomy. In the sublingual gland dopamine-β-hydroxylase immunofluorescence is localized within mucinous acini and small ducts, and the disposition and intensity of staining materials is not affected by noradrenergic denervation for up to 30 days. DBH protein in the sublingual gland had little physiologic activity in vivo. Low levels of authentic dopamine-β-hydroxylase activity were detected in saliva. Thus, dopamine-β-hydroxylase protein is present in the sublingual gland in an extraneuronal location and appears to be a secretory product of the gland.     

Lipid ordering and enzymic activities in chromaffin granule membranes.

1. The interaction of a variety of fluorescent probes with the membranes of adrenal medullary chromaffin granules is described. 2. Changes in the motional properties of the bound probes with temperature were investigated and evidence is presented which indicates that ordering of the membrane lipids occurs below 33 degrees C. 3. The ordering is characteristics of the membrane lipids and is retained by sonicated aqueous dispersions of the total lipid extracted from chromaffin granule membranes. 4. The ATPase and NADH:acceptor oxidoreductase activities of the chromaffin granule membrane have discontinuous Arrhenius temperature versus activity relationships with 'transitions' at 33 degrees C. 5. The ATPase has a second transition at 36.5 degrees C. 6. The 33 degrees C 'transition' for the NADH:acceptor oxidoreductase is removed by treatment with the detergent Triton X-100. 7. The correlation between the onset of lipid ordering and the change in activation energy of the membrane-bound enzyme activities is discussed in terms of the co-operative interactions of the different membrane components. The possible role of lipid ordering in exocytosis is discussed.     

Long-chain acyl-coenzyme A synthetase from rat brain microsomes. Kinetic studies using [1-14C]docosahexaenoic acid substrate

The activation of docosahexaenoic acid by rat brain microsomes was studied using an assay method based on the extraction of unreacted [1-14C]docosahexaenoic acid and the insolubility of [1-14C]docosahexaenoyl-CoA in heptane. This reaction showed a requirement for ATP, CoA, and MgCl2 and exhibited optimal activity at pH 8.0 in the presence of dithiothreitol and when incubated at 45 degrees C. The apparent Km values for ATP (185 microM), CoA (4.88 microM), MgCl2 (555 microM) and [1-14C]docosahexaenoic acid (26 microM) were determined. The presence of bovine serum albumin or Triton X-100 in the incubation medium caused a significant decrease in the Km and Vm values for [1-14C]docosahexaenoic acid. The enzyme was labile at 45 degrees C (t1/2:3.3 min) and 37 degrees C (t1/2:26.5 min) and lost 36% of its activity after freezing and thawing. The transition temperature (Tc) obtained from Arrhenius plot was 27 degrees C with the activation energies of 74 kJ/mol between 0 degrees C and 27 degrees C and 30 kJ/mol between 27 degrees C and 45 degrees C. [1-14C]Palmitic acid activation in rat brain and liver microsomes showed apparent Km values of 25 microM and 29 microM respectively, with V values of 13 and 46 nmol X min-1 X mg protein-1. The presence of Triton X-100 (0.05%) in the incubation medium enhanced the V value of the liver enzyme fourfold without affecting the Km value. Brain palmitoyl-CoA synthetase, on the other hand, showed a decreased Km value in the presence of Triton X-100 with unchanged V. The Tc obtained were 25 degrees C and 28 degrees C for brain and liver enzyme with an apparent activation energy of 109 and 24 kJ/mol below and above Tc for brain enzyme and 86 and 3.3 kJ/mol for liver enzyme. The similar results obtained for the activation of docosahexaenoate and palmitate in brain microsomes suggest the possible existence of a single long-chain acyl-CoA synthetase. The differences observed in the activation of palmitate between brain and liver microsomes may be due to organ differences. Fatty acid competition studies showed a greater inhibition of labeled docosahexaenoic and palmitic acid activation in the presence of unlabeled unsaturated fatty acids. The Ki values for unlabeled docosahexaenoate and arachidonate were 38 microM and 19 microM respectively for the activation of [1-14C]docosahexaenoate. In contrast, the competition of unlabeled saturated fatty acids for activation of labeled docosahexaenoate is much less than that for activation of labeled palmitate.(ABSTRACT TRUNCATED AT 400 WORDS)