Purification and properties of the S1 secondary alkylsulphohydrolase of the detergent-degrading micro-organism, Pseudomonas C12B

The S1 secondary alkylsulphohydrolase of the detergent-degrading micro-organism, Pseudomonas C12B, was separated from other alkylsulphohydrolases and purified to homogeneity. Under the experimental conditions used the enzyme completely hydrolysed d-octan-2-yl sulphate (d-1-methylheptyl sulphate), but showed no activity towards the corresponding l-isomer. Additional evidence has been obtained to indicate that it is probably optically stereospecific for d-secondary alkyl sulphate esters with the ester sulphate group at C-2 and with a chain length of at least seven carbon atoms. Enzyme activity towards racemic samples of heptan-2-yl sulphate (1-methylhexyl sulphate), octan-2-yl sulphate and decan-2-yl sulphate (1-methylnonyl sulphate) increased with increasing chain length. l-Octan-2-yl sulphate is a competitive inhibitor of the enzyme, as are certain primary alkyl sulphates and primary alkanesulphonates. Inhibition by each of the last two types of compounds is characteristic of the behaviour of an homologous series. Inhibition increases with increasing chain length and plots of log K(i) values against the number of carbon atoms in each alkyl chain show the expected linear relationship. A crude preparation of the S2 secondary alkylsulphohydrolase was used to show that this particular enzyme hydrolyses l-octan-2-yl sulphate, but is probably inactive towards the corresponding d-isomer. The similarity of the S1 and S2 enzymes to the CS2 and CS1 enzymes respectively of Comamonas terrigena was established, and some comments have been made on the possible roles of these and other alkylsulphohydrolases in the biodegradation of detergents.     

Purification and properties of the P2 primary alkylsulphohydrolase of the detergent-degrading bacterium pseudomonas C12B.

The P2 primary alkylsulphohydrolase of the soil bacterium Pseudomonas C12B was purified to homogeneity (200-250-fold) by column chromatography on DEAE-cellulose, Sephadex G-100 and butyl-agarose. The intact protein is a dimer with a mol. wt. of 160 000. Activity towards primary alkyl sulphate esters was maximal at pH 8.3, varied little in the range pH 7.8-8.7, but decreased sharply at higher pH. For a homologous series of primary alkyl sulphate substrates (C6-C12), logKm decreased linearly with increasing chain length, corresponding to a contribution to the free energy of association between enzyme and substrate of -2.5kJ/mol for each additional CH2 group in the alkyl chain. logKi for the competitive inhibition by secondary alkyl 2-sulphate esters followed a similar pattern (-2.4kJ/mol for each additional CH2 group) except that only n-1 carbon atoms effectively participate in hydrophobic bonding, implying that the C-1 methyl group is not involved. logKi values for inhibition primary alkanesulphonates also depended linearly on chain length but with a diminished gradient, indicating a free-energy increment of -1.2kJ/mol per additional CH2 group. The collective results showed the presence of a hydrophobic site on the enzyme capable of accomodating an alkyl chain of considerable length. Cationic structures (in the form of arginine, lysine or histidine), whose presence might be expected for binding the anionic sulphate group, were not detectable at the active site.     

Primary alkylsulphatase activities of the detergent-degrading bacterium Pseudomonas C12B. Purification and properties of the P1 enzyme.

The P1 primary alkylsulphatase of Pseudomonas C12B was purified 1500-fold to homogeneity by a combination of streptomycin sulphate precipitation of nucleic acids, (NH4)2SO4 fractionation and chromatography on columns of DEAE-cellulose, Sephacryl S-300 and butyl-agarose. The protein was tetrameric with an Mr of 181000-193000, and exhibited maximum activity at pH 6.1. Primary alkyl sulphates of carbon-chain length C1-C5 or above C14 were not substrates, but the intermediate homologues were shown to be substrates, either by direct assay (C6-C9 and C12) or by gel zymography (C10, C11, C13 and C14). Increasing the chain length from C6 to C12 led to diminishing Km. Values of delta G0' for binding substrates to enzyme were dependent linearly on chain length, indicating high dependence on hydrophobic interactions. Vmax./Km values increased with increasing chain length. Inhibition by alk-2-yl sulphates and alkane-sulphonates was competitive and showed a similar dependence on hydrophobic binding. The P1 enzyme was active towards several aryl sulphates, including o-, m- and p-chlorophenyl sulphates, 2,4-dichlorophenyl sulphate, o-, m- and p-methoxyphenyl sulphates, m- and p-hydroxyphenyl sulphates and p-nitrophenyl sulphate, but excluding bis-(p-nitrophenyl) sulphate and the O-sulphate esters of tyrosine, nitrocatechol and phenol. The arylsulphatase activity was weak compared with alkylsulphatase activity, and it was distinguishable from the de-repressible arylsulphatase activity of Pseudomonas C12B reported previously. Comparison of the P1 enzyme with the inducible P2 alkylsulphatase of this organism, and with the Crag herbicide sulphatase of Pseudomonas putida, showed that, although there are certain similarities between any two of the three enzymes, very few properties are common to all three.     

Preparation and characterization of substrates suitable for the study of stereospecific secondary alkylsulphohydrolases of detergent-degrading micro-organisms.

During the course of the purification of novel stereospecific secondary aklylsulphohydrolases present in certain detergent-degrading micro-organisms, it became apparent that substrates prepared by sulphating secondary alcohols with H2SO4 are heterogeneous. Apart from the racemization that occurs if resolved alcohols are sulphated, evidence is provided to show that other isomers are produced in which the position of the ester sulphate group on the alkyl chain has been altered. These changes can be avoided if pyridine/SO3 reagent (prepared with SO3) is substituted as sulphating agent. Experiments in which secondary alkyl sulphates prepared by both methods were tested as potential substrates for the two secondary alkylsulphohydrolase enzymes of Comamonas terrigena have provided initial information about the specificity of the enzymes.     

Purification, properties and cellular localization of the stereospecific CS2 secondary alkylsulphohydrolase of Comamonas terrigena.

The availability of homogeneous samples of the potassium salts of L- and D-octan-2-yl sulphate has enabled the separation of the optically stereospecific CS1 and CS2 secondary alkysulphohydrolases from extracts of cells of Comamonas terrigena. The CS2 enzyme was purified to homogeneity, and an initial study was made of its general properties, specificity, cellular localization and relationship to the CS1 enzyme. The CS2 enzyme has a molecular weight of approx. 250000 and a subunit size of approx. 58000, indicating that the molecule is a tetramer. Under the experimental conditions used the enzyme appears to be specific for (+)-secondary alkyl sulphate esters with the sulphate group at C-2 and with a chain length of at least six carbons. Enzyme activity towards racemic C-2 sulphates increases with increasing chain length up to C10, and there is some indirect evidence to suggest that activity declines when that chain length is exceeded. Other indirect evidence confirms that the CS1 enzyme exhibits similar specificity, except that only (-)-isomers can serve as substrates. Both enzymes are present in broth-grown stationary-phase cells of C. terrigena in approximately equal amounts.     

Chemically defined inducers of alkylsulphatases present in Pseudomonas C12B

When Pseudomonas C12B is grown on nutrient broth to the stationary phase, cell extracts contain two secondary alkylsulphatases (S1 and S2) active towards potassium decan-5-yl sulphate but not towards potassium pentan-3-yl sulphate and one primary alkylsulphatase (P1) active towards sodium dodecan-1-yl sulphate (sodium dodecyl sulphate). When 10mm-sodium hexan-1-yl sulphate is included in the nutrient broth an additional primary alkylsulphatase (P2) is produced. The S1, S2, P1 and P2 enzymes are also present in extracts of cells grown on broth containing the commercial detergent Oronite, together with an additional secondary alkylsulphatase (S3) active towards pentan-3-yl sulphate as well as decan-5-yl sulphate. The P2 primary alkylsulphatase can be induced by a number of primary and secondary alkyl sulphate esters but the induction of the S3 enzyme appears to be a more specific and complex process. Studies on the ability of different fractions separated from Oronite to act as inducers suggest that the combination of a long-chain secondary alkyl sulphate(s) and a long-chain secondary alcohol(s) is responsible for the appearance of the S3 enzyme. Potassium hexadecan-2-yl sulphate or potassium tetradecan-2-yl sulphate, in combination with either hexadecan-2-ol or tetradecan-2-ol, can serve as inducers for the enzyme. Some characteristics of these specific inducer systems have been elucidated.     

Substrate specificity and other properties of the inducible S3 secondary alkylsulphohydrolase purified from the detergent-degrading bacterium Pseudomonas C12B.

The inducible S3 secondary alkylsulphohydrolase of the soil bacterium Pseudomonas C12B was purified to homogeneity (683-fold from cell-free extracts by a combination of column chromatography on DEAE-cellulose. Sephadex G-100 and Blue Sepharose CL-6B. The enzyme has a molecular weight in the region of 40000--46000, and is active over a broad range of pH from 5 to 9, with maximum activity at pH 8.2. The preferred substrates of the enzyme are the symmetrical secondary alkylsulphate esters such as heptan-4-yl sulphate and nonan-5-yl sulphate and the asymmetric secondary octyl and nonyl sulphate esters with the sulphate group attached to C-3 or C-4. However, for each asymmetric ester, the L-isomer is much more readily hydrolysed than the D-isomer. This specificity is interpreted in terms of a three-point attachment of the substrate to the enzyme's active site. The alkyl chains on either side of the esterified carbon atom are bound in two separate sites, one of which can only accommodate alkyl chains of limited size. The third site binds the sulphate group. Enzymic hydrolysis of this group is accompanied by complete inversion of configuration at the asymmetric carbon atom. The implied cleavage of the C--O bond of the C--O--S ester linkage was confirmed by 18O-incorporation studies.     

The primary alkylsulfatase of Pseudomonas aeruginosa: inducer specificity and induction kinetics

The ability of primary alkylsulfate esters and alkanesulfonates to induce alkylsulfatase formation in Pseudomonas aeruginosa was compared on the basis of maximum enzyme levels, induction rate, and levels of induction as a function of inducer concentration. Apparent K inducer values for these effectors were calculated from linear relationships between reciprocals of induction rate and inducer concentration. Maximum enzyme levels estimated from linear progress relationships for each effector indicated that little major distinction could be made between effectors. Excepting carbon chain lengths of C8 which induced about the same level of enzyme, sulfate esters were generally better inducers than sulfonates with little or no apparent induction occurring with effectors of chain length less than or equal to C6. These observations also held true when rates were compared, except that the rate for the C8 ester was approximately ninefold greater than that for the analogous sulfonate. Apparent K inducer constants decreased with increasing alkyl chain length for the esters (C6-C12) and the sulfonates (C8-C14). Values for the esters were approximately sixfold greater than those for sulfonates of equivalent chain length. Plots of log apparent K inducer values against carbon chain length for each series of esters and sulfonates yielded straight-line relationships characteristic of an homologous series in each instance.     

Protein--non-ionic detergent interaction. Interaction of bovine serum albumin with alkyl glucosides studied by equilibrium dialysis and infrared spectroscopy.

The binding isotherms of bovine serum albumin with octylglucoside and decyl glucoside were determined at 7 degrees C and 25 degrees C at pH 7.4 and ionic strength 0.1 M. The average number of detergent molecules bound was found to increase with increasing hydrocarbon chain length. Competitive binding indicates that alkylglycosides combine with the same sites as alkyl sulphates. Native bovine serum albumin has about 12 and 10 sites for non-ionic ligands at 7 degrees C and about 15 and 13 sites at 25 degrees C for octyl and decyl glucosides respectively. The values for standard free energy changes--delta G0, were calculated from the intrinsic association constants. Fourier-transformed infrared spectroscopy was used to study the effects of alkyl glucosides on the conformation of albumin. The results obtained indicate that there are no significant changes in protein structure.     

Activation of brain K+-phosphatase and Mg2+, Na+, K+-AtPase by sodium alkyl sulfates

K+-dependent phosphatase and Mg2+, Na+, K+-ATPase were studied under the activating effect of surfactant homologs of the alkyl sulphate series with the hydrocarbon radical long chain C4-C15. The homologs are shown to activate the enzymes when they are in the molecular-disperse but not in micellar state. A clear regularity is observed in the effect of these surfactants on K+-phosphatase depending on the length of the hydrocarbon radical chain: the degree of the activating effect rises with the chain lengthening, reaching the maximum value when the number of carbon atoms is 12. The lower and upper bounds of the alkyl sulphate hydrocarbon radical chain length necessary for manifestation of the activating effect shift somewhat for K+-dependent phosphatase as compared with Mg2+, Na+, K+-ATPase. The data obtained evidence for a stronger stability of the phosphatase to a destructive effect of the surfactants as compared with transport ATPase.     

Use of sodium dodecyl sulphate for stimulation of biodegradation of n-alkanes without residual contamination by the surfactant

The capacity of a range of aliphatic alkanes (C₆–C₁₆), intermediates of n-decane oxidation and sodium dodecyl sulphate (SDS) to induce decane-mineralization activity in the cells of Pseudomonas C12B was compared with that for n-decane. The comparison on quantitative basis had two serious limitations: low solubility of tested inducers in aqueous solutions and their toxicity to bacterial cells. Carbon chain length and the presence of hydroxyl group were the important factors for induction activity. However, presence of hydroxyl groups at both ends of alkyl chain prevented the induction of decane-mineralization activity. Good induction activity by SDS was caused either by the presence of free end of alkyl chain, or by bacterial hydrolysis of sulphate group to yield alcohol, which in turn served as true inducer. The presence of SDS in the culture medium with n-decane as main source of carbon and energy accelerated the growth of Pseudomonas C12B. SDS disappeared from the culture medium in early stages of cultivation suggesting preferential degradation by the bacterium, while the consumption of n-decane was accelerated. This may be associated with the capacity of SDS to induce decane-mineralization system in Pseudomonas C12B and/or with the ability of SDS to stimulate the surface attachment of competent bacteria resulting in the close proximity of the cells with alkane droplets, and thus, enhanced breakdown of the hydrocarbon pollutant.     

The structure of keratan sulphates from various sources.

Quantitative structural comparisons were made between keratan sulphates isolated from various sources, namely pig nucleus pulposus, bovine cornea, and the costal cartilages of children, a young adult with Marfan syndrome and of old human autopsies. In human costal cartilage the amount of keratan sulphate increases markedly with age, although total mucopolysaccharide decreases to some extent, concomitant with a decrease in chondroitin 4-sulphate and an increase in chondroitin 6-sulphate. Comparison of molecular weights estimated by gel chromatography with those calculated from the molar ratio of galactose to mannose indicates that keratan sulphates of human costal cartilages of children and of a young adult with Marfan syndrome, and of pig nucleus pulposus, contain one mannose residue per chain, whereas keratan sulphates of old human costal cartilage and of bovine cornea contain one to two, and two, per chain respectively. After mild acid-catalysed desulphation of pig nucleus pulposus keratan sulphate, approx. 12% of the mucopolysaccharide aggregates irreversibly once the water is removed from the polysaccharide. The following conclusions have been drawn from a methylation analysis of keratan sulphates of various sources, aided by g.l.c.-mass spectrometry. (1) Fucose and N-acetylneuraminic acid are non-reducing terminal residues and the sialic acid is linked to the 3-position of galactose residues. (2) Pig nucleus pulposus keratan sulphate has approximately 4 non-reducing terminal groups per molecule and appears to be slightly less branched than the costal-cartilage keratan sulphate of children. The branching in human costal-cartilage keratan sulphates decreases with age. Bovine corneal keratan sulphate appears to be unbranched. (3) Mannose residues are linked by 3 different substituents in human costal-cartilage and bovine corneal keratan sulphates, and by two different substituents in pig nucleus pulposus keratan sulphate. (4) The sulphate ester groups are all on the 6-position of N-acetyl-glucosamine and galactose residues. The degree of sulphation increases with age in costal keratan sulphates with the increase mainly of the galactose 6-sulphate residues.     

Synergistic effects of anacardic acids and methicillin against methicillin resistant Staphylococcus aureus

The synergistic effects of 6-alk(en)ylsalcylic acids, also known as anacardic acids, in combination with methicillin against Staphylococcus aureus ATCC 33591 (MRSA) was investigated. The double bond in C15-anacardic acids is not essential in eliciting the antibacterial activity but is associated with increasing the activity. The synergistic effects decreased with increasing the number of double bonds in the alkyl chain. On the other hand, the antibacterial activity of anacardic acids possessing different alkyl chain lengths against the same MRSA strain was found to be a parabolic function of their lipophilicity and maximized with the alkyl chain length of C10 and C12. Notably, the synergistic effects were noted to increase with increasing the alkyl chain length.     

Constant and variable domains of different disaccharide structure in corneal keratan sulphate chains.

Four peptidokeratan sulphate fractions of different Mr and degree of sulphation were cut from the pig corneal keratan sulphate distribution spectrum. After exhaustive digestion with keratanase, the fragments were separated on DEAE-Sephacel and Bio-Gel P-10 and analysed for their Mr, degree of sulphation and amino sugar and neutral sugar content. It was found that every glycosaminoglycan chain is constructed of a constant domain of non-sulphated and monosulphated disaccharide units and a variable domain of disulphated disaccharide units. Total neuraminic acid of the four peptidokeratan sulphates was recovered from their isolated linkage-region oligosaccharides. In kinetic studies, the four peptidokeratan sulphates were investigated for Mr distribution after various incubation times with keratanase. There was a continuous shift towards lower Mr and no appearance of a distinct intermediate-sized product at any degradation time. The linkage-region oligosaccharide was already being liberated after a very short incubation period. From the results of these kinetic investigations in connection with the results of neuraminic acid analyses it is suggested that there exists only one disaccharide chain per peptidokeratan sulphate molecule. A model of corneal keratan sulphate is postulated. One of the alpha-mannose residues in the linkage region is bound to an oligosaccharide consisting of a lactosamine and a terminal sialic acid. The other alpha-mannose residue is attached to the disaccharide chain. This chain contains one or two non-sulphated disaccharide units at the reducing end, followed by 10-12 monosulphated disaccharide units. The disulphated disaccharide moiety of variable length is positioned at the non-reducing end of the chain.     

Microorganisms that break down alkyl sulfates

The following bacteria assimilating alkyl sulphates as a sole source of carbon and energy were isolated from various substrates: Pseudomonas, Achromobacter, Flavobacterium, Citrobacter, Enterobacter. The bacteria decomposed sodium dodecyl sulphate at a high rate, and some of them, industrial preparations of alkyl sulphates. The ability to assimilate alkyl sulphates was tested in 257 collection cultures belonging to different taxonomic groups. Alkyl sulphates were found to be decomposed by heterotrophous gram-negative rod-like bacteria belonging to different families and genera. The frequency of bacteria destroying alkyl sulphates at a high rate was found most often in the Pseudomonas genus.     

Immobilization of lipases on alkyl silane modified magnetic nanoparticles: effect of alkyl chain length on enzyme activity

Background

Biocatalytic processes often require a full recycling of biocatalysts to optimize economic benefits and minimize waste disposal. Immobilization of biocatalysts onto particulate carriers has been widely explored as an option to meet these requirements. However, surface properties often affect the amount of biocatalysts immobilized, their bioactivity and stability, hampering their wide applications. The aim of this work is to explore how immobilization of lipases onto magnetite nanoparticles affects their biocatalytic performance under carefully controlled surface modification.

Methodology/Principal Findings

Magnetite nanoparticles, prepared through a co-precipitation method, were coated with alkyl silanes of different alkyl chain lengths to modulate their surface hydrophobicity. Candida rugosa lipase was then directly immobilized onto the modified nanoparticles through hydrophobic interaction. Enzyme activity was assessed by catalytic hydrolysis of p-nitrophenyl acetate. The activity of immobilized lipases was found to increase with increasing chain length of the alkyl silane. Furthermore, the catalytic activities of lipases immobilized on trimethoxyl octadecyl silane (C18) modified Fe₃O₄ were a factor of 2 or more than the values reported from other surface immobilized systems. After 7 recycles, the activities of the lipases immobilized on C18 modified nanoparticles retained 65%, indicating significant enhancement of stability as well through hydrophobic interaction. Lipase immobilized magnetic nanoparticles facilitated easy separation and recycling with high activity retaining.

Conclusions/Significance

The activity of immobilized lipases increased with increasing alkyl chain length of the alkyl trimethoxy silanes used in the surface modification of magnetite nanoparticles. Lipase stability was also improved through hydrophobic interaction. Alkyl silane modified magnetite nanoparticles are thus highly attractive carriers for enzyme immobilization enabling efficient enzyme recovery and recycling.     

Gentisides C–K: Nine new neuritogenic compounds from the traditional Chinese medicine Gentiana rigescens Franch

Nine new alkyl 2,3-dihydroxybenzoates, gentisides C–K, were isolated from the traditional Chinese medicine Gentiana rigescens Franch. Their structures and stereochemistry were elucidated by spectroscopic methods, and comparison of the specific rotation with that of the gentiside B. These metabolites are additional members of the gentisides which belong to a novel class of neuritogenic compounds. They are structurally different from one another because they possess varying alkyl chain lengths, with or without an isobutyl or isopropyl group at the end of the alkyl chain. These compounds are potent inducers of neurite outgrowth on PC12 cells. The gentiside C possessing the shortest alkyl chain length exhibited the highest neuritogenic activity among all of the gentisides. Gentiside C showed a significant neuritogenic activity at 1 μM against PC12 cells comparable to that seen for the best nerve growth factor (NGF) concentration of 40 ng/mL. In addition, evident neuritogenic activity was observed in the cells when treated with gentiside C at a concentration as low as 0.03 μM. The structure–activity relationships within the gentisides A–K revealed that alkyl chain length is important for the activity, but structure diversity at the end of the alkyl chain is not.     

Novel redox surfactants and their interactions with glucose oxidase of Aspergillus niger

A number of novel redox surfactants (based on mixed bipyridine/dipyridylamine complexes of osmium (II) where the dipyridylamine ligands bears a saturated C(8), C(10), C(12), C(14), or C(16) alkyl chain) were synthesized and characterized electrochemically and biochemically as mediators for glucose oxidase (EC 1.1.3.4, GOD) of Aspergillus niger. These compounds exhibited critical micelle concentrations (CMCs) in phosphate-buffered saline solution (pH 7.4) in the range 10(-4) 10 10(-3) M, the value decreasing with increasing chain length. Dependence of a number of properties (speed of mediation, redox potential, denaturing action on the enzyme, adsorption on an electrode surface) on the length of the mediator alkyl chain was observed. The presence of an alkyl chain decreased the rate of mediation relative to otherwise similar nonsurfactant mediators, and the longer alkyl chain, the slower the rate of mediation. For each compound, mediation above the CMC was about tenfold slower than that observed below the CMC. However, for the cases of mediator absorbed on an electrode surface with GOD, longer chains give increased physisorption of mixed micelles of enzyme and mediator. The compounds were incidentally found to inhibit the glucose oxidase activity of GOD in a complex manner; inhibition increased with increasing chain length and the deactivation, for any given compound, was more pronounced below the CMC than above. Glucose oxidase activity assays and study of the action of surfactants and mediators on the fluorescent properties of carboxy-fluorescein-labeled GOD led to the consideration of a model for redox surfactant-GOD interaction where three mechanisms may operate: first, a selective interaction of mediators with the GOD active site; second, a nondenaturing association of short-chain (相似文献   

Requirement for omega and (omega;-1)-hydroxylations of fatty acids by human cytochromes P450 2E1 and 4A11.

Human liver microsomes and recombinant human P450 have been used as enzyme source in order to better understand the requirement for the optimal rate of omega and (omega;-1)-hydroxylations of fatty acids by cytochromes P450 2E1 and 4A. Three parameters were studied: alkyl chain length, presence and configuration of double bond(s) in the alkyl chain, and involvement of carboxylic function in the fatty acid binding inside the access channel of P450 active site. The total rate of metabolite formation decreased when increasing the alkyl chain length of saturated fatty acids (from C12 to C16), while no hydroxylated metabolite was detected when liver microsomes were incubated with stearic acid. However, unsaturated fatty acids, such as oleic, elaidic and linoleic acids, were omega and (omega;-1)-hydroxylated with an efficiency at least similar to palmitic acid. The (omega;-1)/omega ratio decreased from 2.8 to 1 with lauric, myristic and palmitic acids as substrates, while the reverse was observed for unsaturated C18 fatty acids which are mainly omega-hydroxylated, except for elaidic acid showing a metabolic profile quite similar to those of saturated fatty acids. The double bond configuration did not significantly modify the ability of hydroxylation of fatty acid, while the negatively charged carboxylic group allowed a configuration energetically favourable for omega and (omega;-1)-hydroxylation inside the access channel of active site.     

Variable product specificity of microsomal dehydrodolichyl diphosphate synthase from rat liver

Several detergents activated microsomal dehydrodolichyl diphosphate synthase of rat liver, but the chain length of products shifted downward from C90 and C95 with increasing concentration of the detergents. Maximum activation was observed at the concentration of 2% Triton X-100, 30 mM octyl glucoside, 30 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, and 10 mM deoxycholate with the product chain length being C80-C85, C65-C75, C70-C75, and C55-C65, respectively. The activity of Triton X-100 solubilized enzyme was decreased by asolectin, phosphatidylethanolamine, and phosphatidylcholine. The chain lengths of products formed in the presence of these phospholipids were C85 and C90. In the presence of both phosphatidylcholine and Mg2+ the solubilized enzyme was able to produce C90 and C95 dehydrodolichyl diphosphates like native microsomal enzyme. Microsomal enzyme preparations from rat liver, brain, and testis catalyzed the formation of dehydrodolichyl diphosphates with the same chain lengths as those of the natural dolichols occurring in individual tissues. The chain length distribution of dehydrodolichyl products by (rat liver) microsomes also depended on the concentration of substrates. Not only did increasing the concentration of isopentenyl diphosphate lead to longer chain product, but decreasing that of farnesyl diphosphate increased product chain length.