Purification and molecular characterization of bovine pregastric lipase

A pregastric lipase was purified from calf pharyngeal tissues. The purification procedure was based on chromatographies on octyl-Sepharose and lentil-lectin-Sepharose followed by gel filtration. The final preparation, with an overall recovery of 26% of activity, gave a single protein band on dodecyl sulfate/polyacrylamide gel electrophoresis with a Mr of 55000. The Mr on gel filtration was 44-48000. The discrepancy may be due to the fact that pregastric lipase is a glycoprotein containing approximately 10% (w/w) of carbohydrate. The pI was around 7.0 and the enzyme protein is characterized by a high content of branched, aliphatic amino acid residues. The NH2-terminal amino acid sequence is: H2N-Phe-Leu/(Ile)-Gly-. Rabbit antibodies to the purified preparation detected only one component in the crude starting material in immuno-blotting experiments. Preincubation with antiserum resulted in loss of enzyme activity, showing that the antibodies were directed against the lipase.     

Sunflower seed lipase: extraction, purification, and characterization

A simple procedure for the extraction of the lipolytic activity from sunflower seed has been developed. Various conditions of extraction have been optimized in order to obtain maximum yield of lipase. A new lipase enzyme was purified by the fractional salt precipitation from the supernatant, dialysis on a cellulose membrane, and gel column chromatography on Sephadex G-75. The lipase was monomeric, with an apparent Mr of 22 kDa and a pI of 8, with the electrophoretic analysis. Kinetics of the enzyme activity versus substrate concentration showed typical lipase behavior, with Km and Vmax, values of 1.33 mM and 555 U/mg. All triglycerides were efficiently hydrolyzed by the enzyme, but this showed a preference towards triglycerides of natural mono unsaturated fatty acids. The optimum temperature, pH, and incubation time for lipolytic activity were 50 degrees C, 7.5, and 5 min, respectively. The stability of the sunflower lipase was investigated under operational and storage conditions. It was found that this enzyme preserved its lipolytic activity at temperatures between at 35-50 degrees C, alkaline pH, and for a period of about four months.     

Purification and characterization of lipoprotein lipase from pig myocardium.

1. Lipoprotein lipase was purified from pig myocardium by a two-step purification procedure involving (a) the formation of an enzyme-substrate complex and (b) affinity chromatography on Sepharose which contained covalently linked heparin. The purified enzyme gave in sodium dodecyl sulphate-polyacrylamide-gel electrophoresis one main band with an apparent molecular weight of 73 000. The enzyme, which was purified 70 000-fold, had a specific activity of 860 mumol of unesterified fatty acid liberated/h per mg of protein. 2. The purified enzyme hydrolysed [14C]triolein emulsions in the absence of added cofactors but its activity was increased fivefold by adding normal human serum. Of the low-density lipoprotein apoproteins only apolipoprotein CII could be substituted for serum in activating the enzyme. This lipase had maximum activity at 0.05-0.15 M-NaCl. Heparin increased the activity of the purified enzyme twofold at low concentrations, but high concentrations inhibited. The triglyceride lipase of pig myocardium thus resembles lipoprotein lipase purified from adipose tissue and from plasma, but is clearly different from pig hepatic triglyceride lipase.     

Purification and characterization of a lipase from Pichia burtonii

An extracellular lipase from Pichia burtonii was purified to homogeneity by a combination of DEAE-Sephadex A-50 ion-exchange chromatography, Sephadex G-100 gel filtration, and isoelectric focusing. The purified enzyme preparation showed a single protein band corresponding to a molecular mass of 51 kDa on sodium dodecyl sulphate/polyacrylamide gel electrophoresis. The molecular mass of the enzyme was estimated to be 47 kDa on Superdex 200 gel filtration, suggesting that the enzyme was a monomeric protein. The pI was about 5.8. The optimum pH and temperature for the hydrolysis of olive oil were about 6.5 and 45°C respectively. Rapid loss of the enzyme activity was observed above 30°C in the absence of olive oil, but the addition of olive oil or trimethylolpropane diallyl ether greatly stabilized the enzyme. At 30°C, the enzyme hydrolysed Spans and Tweens as well as simple triglycerides of short- and middle-chain fatty acids. Although the enzyme cleaved all the ester bonds of triolein, it showed some preference for the outer ester bonds.     

Purification and characterization of cutinase from a fluorescent Pseudomonas putida bacterial strain isolated from phyllosphere

Cutinase, an extracellular enzyme, was induced by cutin in a fluorescent Pseudomonas putida strain that was found to be cohabiting with an apparently nitrogen-fixing Corynebacterium. This enzyme was purified from the culture fluid by acetone precipitation followed by chromatography on DEAE-cellulose, QAE-Sephadex, Sepharose 6B, and Sephadex G-100. The purified enzyme showed a single band when subjected to polyacrylamide electrophoresis and the enzymatic activity coincided with the protein band. Sodium dodecyl sulfate-polyacrylamide electrophoresis showed a single band at a molecular weight of 30,000 and gel filtration of the native enzyme through a calibrated Sephadex G-100 column indicated a molecular weight of 30,000, showing that the enzyme is a monomer. The amino acid composition of bacterial cutinase is distinctly different from that of fungal or plant cutinases. This bacterial cutinase showed a broad pH optimum between 8.5 and 10.5 with 3H-labeled apple cutin as the substrate. Linear rates of cutin hydrolysis were measured up to 20 min of incubation time and 4 mg/ml of cutin gave the maximum hydrolysis rate. This cutinase catalyzed hydrolysis of p-nitrophenyl esters of C4 to C16 fatty acids with decreasing V and increasing Km for the longer chain esters. It did not hydrolyze tripalmitoyl glycerol or trioleyl glycerol, indicating that this is not a general lipase. Active serine-directed reagents such as organophosphates and organoboronic acids severely inhibited the enzyme, suggesting that bacterial cutinase is an "active serine" enzyme. Neither thiol-directed reagents nor metal ion chelators had any effect on this enzyme. Antibody raised against purified enzyme gave a single precipitin line on Ouchterlony double diffusion analysis. Western blot analysis of the extracellular fluid of induced Ps. putida showed a single band at 30 kDa. No immunological cross-reactivity was detected between the present bacterial enzyme and the fungal enzyme from Fusarium solani pisi when rabbit antibodies against either enzyme was used.     

Purification and properties of intracellular clotting factor, factor B, from horseshoe crab (Tachypleus tridentatus) hemocytes

An intracellular clotting factor, factor B, which is closely associated with the hemolymph coagulation system of horseshoe crab (Tachypleus tridentatus), was purified and characterized. The purified preparation gave a single band (Mr = 64,000) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) in the absence of 2-mercaptoethanol, while three bands (Mr = 64,000, 40,000, and 25,000) were detected on SDS-PAGE after reduction. This preparation was converted by limulus clotting factor C to an activated form, factor B, with Mr = 56,000 consisting of a heavy chain (Mr = 32,000) and a light chain (Mr = 25,000) bridged by disulfide linkage(s). The factor B, which was produced separately by treating the partially purified factor B with factor C, was also purified. It gave a single band on unreduced SDS-PAGE and two bands on reduced SDS-PAGE. The purified factor B had Mr of 56,000 consisting of a heavy chain (Mr = 32,000) and a light chain (Mr = 25,000). These results indicated that the purified factor B zymogen is a mixture of single-chain and two-chain forms, both of which have the same molecular weight of 64,000, and that these two forms are converted to factor B by factor C. The diisopropyl phosphorofluoridate-sensitive site of factor B was found in the heavy chain. The reconstitution studies using purified factor C, factor B, proclotting enzyme and coagulogen in the presence of lipopolysaccharide indicated that factor B is an essential component to complete sequential activation of the limulus clotting system, and that it specifically activates proclotting enzyme to the active clotting enzyme.     

Iduronate sulfatase from human placenta

The major enzyme component of iduronate sulfatase from human placenta was purified 30 000-fold by a five-step procedure. Sucrose gradient centrifugation of the native enzyme gave a molecular weight estimate of 80 000 +/- 10 000. Electrophoresis in sodium dodecyl sulfate of the enzyme reduced with mercaptoethanol showed a protein band of Mr 82 000. We suggest that the enzyme is composed of a single polypeptide chain of Mr 80 000-90 000.     

Monoclonal antibodies to bovine milk lipoprotein lipase. Evidence for proteolytic degradation of the native enzyme

Lipoprotein lipase was purified from bovine skim milk by chromatography on heparin-Sepharose. Polyacrylamide gel electrophoresis showed a single protein with an apparent molecular weight of 55,000 in the trailing edge of the elution profile; fractions in the leading edge contained additional proteins with molecular weights of 36,000 and 18,000-22,000. Nine monoclonal antibodies were prepared against the 55,000-dalton protein. By immunoblotting, we show that the Mr = 18,000-22,000 components share common antigen determinants with the 55,000-dalton protein, suggesting that they represent proteolytic degradation products. Incubation of partially purified lipoprotein lipase for 24 h at 37 degrees C results in breakdown of the 55,000-dalton protein with concomitant enrichment in lower Mr components; the proteolytic activity is prevented by incubating the milk with phenylmethane, sulfonyl fluoride prior to chromatography on heparin-Sepharose. This study shows the presence of milk proteases which co-purify and degrade lipoprotein lipase. We suggest that this degradation could account for part of the known instability of the enzyme.     

Purification to homogeneity of human placental acid sphingomyelinase

Acid sphingomyelinase was purified to homogeneity from human placenta in the presence of a dialyzable detergent, n-octyl-beta-D-glucopyranoside. The major steps in the procedure included column chromatographies with Con A-Sepharose, sphingosylphosphorylcholine-Sepharose 4B, hexyl-agarose, and Mono P. The purified enzyme with pI 7.4 had a specific activity of approx 170,000 units/mg protein with a yield of 3.6%. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a single protein band of Mr 62,000. Gel filtration with a Superose 12 column gave a single peak, and the enzyme in the presence 50 mM n-octyl-beta-D-glucopyranoside was of Mr 123,000, indicating that the native enzyme occurs in a dimeric form. The optimal pH was 5.5 with both sphingomyelin and an artificial substrate, 2-N-hexadecanoylamino-4-nitrophenylphosphorylcholine. The Km values were 55 microM with sphingomyelin and 340 microM with the artificial substrate. The enzyme activity was not affected by Mg2+ (1-5 mM), confirming that the enzyme is acid sphingomyelinase. The enzyme was stable at -80 degrees C for more than 4 months. In addition to the enzyme with pI 7.4, the Mono P chromatofocusing gave two peaks (pI 7.0 and 6.7) possessing the enzymatic activity.     

Penicillium chrysogenum extracellular acid phosphatase: purification and biochemical characterization

An extracellular acid phosphatase (EC 3.1.3.2) from crude culture filtrate of Penicillium chrysogenum was purified to homogeneity using high-performance ion-exchange chromatography and size-exclusion chromatography. SDS-PAGE of the purified enzyme exhibited a single stained band at an Mr of approx. 57,000. The mobility of the native enzyme indicated the Mr to be 50,000, implying that the active form is a monomer. The isoelectric point of the enzyme was estimated to be 6.2 by isoelectric focusing. Like acid phosphatases from several yeasts and fungi the Penicillium enzyme was a glycoprotein. Removal of carbohydrate resulted in a protein band with an Mr of 50,000 as estimated by SDS-PAGE, suggesting that 12% of the mass of the enzyme was carbohydrate. The enzyme was catalytically active at temperatures ranging from 20 degrees C to 65 degrees C with a maximum activity at 60 degrees C and the pH optimum was at 5.5. The Michaelis constant of the enzyme for p-nitrophenyl phosphate was 0.11 mM and it was inhibited competitively by inorganic phosphate (ki = 0.42 mM).     

Purification and characterization of an extracellular lipase from a thermophilic Rhizopus oryzae strain isolated from palm fruit

We have isolated a lipolytic strain from palm fruit that was identified as a Rhizopus oryzae. Culture conditions were optimized and highest lipase production amounting to 120 U/ml was achieved after 4 days of cultivation. The extracellular lipase was purified 1200-fold by ammonium sulfate precipitation, sulphopropyl-Sepharose chromatography, Sephadex G 75 gel filtration and a second sulphopropyl-Sepharose chromatography. The specific activity of the purified enzyme was 8800 U/mg. The lipolytic enzyme has a molecular mass of 32 kDa by SDS-polyacrylamide gel electrophoresis and gel filtration. The enzyme exhibited a single band in active polyacrylamide gel electrophoresis and its isoelectric point was 7.6. Analysis of Rhizopus oryzae lipase by RP-HPLC confirmed the homogeneity of the enzyme preparation. Determination of the N-terminal sequence over 19 amino acid residues showed a high homology with lipases of the same genus. The optimum pH for enzyme activity was 7.5. Lipase was stable in the pH range from 4.5 to 7.5. The optimum temperature for lipase activity was 35 degrees C and about 65% of its activity was retained after incubation at 45 degrees C for 30 min. The lipolytic enzyme was inhibited by Triton X100, SDS, and metal ions such as Fe(3+), Cu(2+), Hg(2+) and Fe(2+). Lipase activity against triolein was enhanced by sodium cholate or taurocholate. The purified lipase had a preference for the hydrolysis of saturated fatty acid chains (C(8)-C(18)) and a 1, 3-position specificity. It showed a good stability in organic solvents and especially in long chain-fatty alcohol. The enzyme poorly hydrolyzed triacylglycerols containing n-3 polyunsaturated fatty acids, and appeared as a suitable biocatalyst for selective esterification of sardine free fatty acids with hexanol as substrate. About 76% of sardine free fatty acids were esterified after 30 h reaction whereas 90% of docosahexaenoic acid (DHA) was recovered in the unesterified fatty acids.     

Characterization of tyrosine hydroxylase from bovine adrenal medulla

Tyrosine hydroxylase purified to apparent homogeneity from the soluble fraction of bovine adrenal medulla had an apparent Mr of about 280,000 by Bio-Gel A-1.5m chromatography, and gave a single band with a Mr of 60,000 by sodium dodesyl sulfate polyacrylamide gel electrophoresis. The enzyme is considered to be composed of four identical subunits. Isoelectric point of purified enzyme was pH 6.0. The amino acid composition of the enzyme was characterized by fairly high contents of glutamic acid and alanine residues. The N-terminal amino acid was determined to be glutamic acid.     

Extracellular lipase of Pseudomonas sp. strain ATCC 21808: purification, characterization, crystallization, and preliminary X-ray diffraction data.

A procedure for the purification of a very hydrophobic lipase from Pseudomonas sp. strain ATCC 21808 was elaborated by avoiding the use of long-chain detergents in view of subsequent crystallization of the enzyme. The purification procedure included chromatography on Q-Sepharose in the presence of n-octyl-beta-D-glucopyranoside, Ca2+ precipitation of fatty acids, and Octyl-Sepharose chromatography. The enzyme was purified 260-fold to a yield of 35% and a specific activity of 3,300 U/mg. The molecular weight was determined as 35,000; a polyacrylamide gel under nondenaturing conditions revealed a band at 110,000, and the isoelectric point proved to be at 4.5 to 4.6. The lipase crystallized with different salts and ethylene glycol polymers in the presence of n-octyl-beta-D-glucopyranoside and one alkyloligooxyethylene compound (CxEy) in the range from C5E2 to C8E4. The crystals diffract to a resolution of about 0.25 nm. Precession photographs revealed that they belong to space group C2 with lattice constants of a = 9.27 nm, b = 4.74 nm, c = 8.65 nm, and beta = 122.3 degrees, indicating a cell content of one molecule per asymmetric unit of the crystal. In hydrolysis of triglycerides, the lipase showed substrate specificity for saturated fatty acids from C6 to C12 and unsaturated long-chain fatty acids. Monoglycerides were hydrolyzed very slowly. The N-terminal sequence is identical to that of the lipase from Pseudomonas cepacia. Treatment with diethyl-p-nitrophenylphosphate affected the activities toward triolein and p-nitrophenylacetate to the same extent and with the same velocity.     

Detergent compatible alkaline lipase produced by marine Bacillus smithii BTMS 11

Bacillus smithii BTMS 11, isolated from marine sediment, produced alkaline and thermostable lipase. The enzyme was purified to homogeneity by ammonium sulfate precipitation and ion exchange chromatography which resulted in 0.51 % final yield and a 4.33 fold of purification. The purified enzyme was found to have a specific activity of 360 IU/mg protein. SDS-PAGE analyses, under non-reducing and reducing conditions, yielded a single band of 45 kDa indicating the single polypeptide nature of the enzyme and zymogram analysis using methylumbelliferyl butyrate as substrate confirmed the lipolytic activity of the protein band. The enzyme was found to have 50 °C and pH 8.0 as optimum conditions for maximal activity. However, the enzyme was active over wide range of temperatures (30–80 °C) and pH (7.0–10.0). Effect of a number of metal salts, solvents, surfactants, and other typical enzyme inhibitors on lipase activity was studied to determine the novel characteristics of the enzyme. More than 90 % of the enzyme activity was observed even after 3 h of incubation in the presence of commercial detergents Surf, Sunlight, Ariel, Henko, Tide and Ujala indicating the detergent compatibility of B. smithii lipase. The enzyme was also found to be efficient in stain removal from cotton cloths. Further it was observed that the enzyme could catalyse ester synthesis between fatty acids of varying carbon chain lengths and methanol with high preference for medium to long chain fatty acids showing 70 % of esterification. Results of the study indicated scope for application of this marine bacterial lipase in various industries.     

Purification and characterization of a third glucosyltransferase from Streptococcus mutans serotype g

Streptococcus mutans strain AHT (serotype g) secretes at least two glucosyltransferases with different pI values. A novel glucosyltransferase with a pI of 5.8 was purified 244-fold from the ammonium sulphate fraction by DEAE-cellulose chromatography, FPLC (Mono Q column, Pharmacia) and hydrophobic chromatography. The enzyme preparation gave a single protein band on analysis by both PAGE and SDS-PAGE, and did not form multiple protein bands detectable by IEF. The Mr was estimated to be about 130,000 by SDS-PAGE and about 135,000 by ultracentrifugal analysis. The apparent Km value and pH optimum of the enzyme were 3.9 +/- 0.2 mM (mean +/- SD) and about 4.7, respectively. The enzyme synthesized water-soluble glucan from sucrose, and the glucan consisted of over 90 mol% 1,6-alpha-D-glucosidic linkages. The enzyme activity was not stimulated by primer dextran. Anti-enzyme serum produced a single precipitin band with the purified enzyme preparation, whereas it did not react with either of the other two known glucosyltransferases.     

Purification and characterization of a novel thermostable lipase from Bacillus sp

A thermostable lipase from Bacillus sp. has been purified to homogeneity as judged by disc-PAGE, SDS-PAGE, and isoelectric focusing. The purification included ammonium sulfate fractionation, treatment with acrinol, and sequential column chromatographies on DEAE-Sephadex A-50, Toyopearl HW-55F, and Butyl Toyopearl 650M. The purified enzyme was found to be a monomeric protein with Mr of 22,000, and pI of 5.1. The optimal pH at 30 degrees C, and optimal temperature at pH 5.6 were 5.5-7.2, and 60 degrees C, respectively, when olive oil was used as the substrate. The substrate specificity towards simple triglycerides was broad and 1- and 3-positioned ester bonds were hydrolyzed in preference to a 2-positioned ester bond. The addition of acetone to the assay mixture in the range of 0-60% (v/v) stimulated the enzyme remarkably, whereas n-hexane had an inhibitory effect.     

N5-(L-1-carboxyethyl)-L-ornithine:NADP+ oxidoreductase from Streptococcus lactis. Purification and partial characterization

N5-(L-1-Carboxyethyl)-L-ornithine:NADP+ oxidoreductase (EC 1.5.1.-) from Streptococcus lactis K1 has been purified 8,000-fold to homogeneity. The NADPH-dependent enzyme mediates the reductive condensation between pyruvic acid and the delta- or epsilon-amino groups of L-ornithine and L-lysine to form N5-(L-1-carboxyethyl)-L-ornithine and N6-(L-1-carboxyethyl)-L-lysine, respectively. The five-step purification procedure involves ion-exchange (DE52 and phosphocellulose P-11), gel filtration (Ultrogel AcA 44), and affinity chromatography (2',5'-ADP-Sepharose 4B). Approximately 100-200 micrograms of purified enzyme of specific activity 40 units/mg were obtained from 60 g of cells, wet weight. Anionic polyacrylamide gel electrophoresis revealed a single enzymatically active protein band, whereas three species (pI 4.8-5.1) were detected by analytical electrofocusing. The purified enzyme is active over a broad pH range of 6.5-9.0 and is stable to heating at 50 degrees C for 10 min. Substrate Km values were determined to be: NADPH, 6.6 microM; pyruvate, 150 microM; ornithine, 3.3 mM; and lysine, 18.2 mM. The oxidoreductase has a relative molecular mass (Mr = 150,000) as estimated by high pressure liquid chromatography exclusion chromatography and by polyacrylamide gradient gel electrophoresis. Conventional gel filtration indicated an Mr = 78,000, and a single protein band of Mr = 38,000 was revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is composed of identical subunits of Mr = 38,000, which may associate to yield both dimeric and tetrameric forms. Polyclonal antibody to the purified protein inhibited enzyme activity. The amino acid composition of the enzyme is reported, and the sequence of the first 37 amino acids from the NH2 terminus has been determined by stepwise Edman degradation.     

Physiological regulation and optimization of lipase activity in Pseudomonas aeruginosa EF2.

Physiological regulation of extracellular lipase activity by a newly-isolated, thermotolerant strain of Pseudomonas aeruginosa (strain EF2) was investigated by growing the organism under various conditions in batch, fed-batch and continuous culture. Lipase activity, measured as the rate of olive oil (predominantly triolein) hydrolysis, was weakly induced by general carbon and/or energy limitation, strongly induced by a wide range of fatty acyl esters including triglycerides, Spans and Tweens, and repressed by long-chain fatty acids including oleic acid. The highest lipase activities were observed during the stationary phase of batch cultures grown on Tween 80, and with Tween 80-limited fed-batch and continuous cultures grown at low specific growth rates. The lipase activity of Tween 80-limited continuous cultures was optimized with respect to pH and temperature using response surface analysis; maximum activity occurred during growth at pH 6.5, 35.5 degrees C, at a dilution rate of 0.04 h-1. Under these conditions the culture exhibited a lipase activity of 39 LU (mg cells)-1 and a specific rate of lipase production (qLipase) of 1.56 LU (mg cells)-1 h-1 (1 LU equalled 1 mumol fatty acid released min-1). Esterase activity, measured with p-nitrophenyl acetate as substrate, varied approximately in parallel with lipase activity under all growth conditions, suggesting that a single enzyme may catalyse both activities.     

Hepatic lipase. Purification and characterization

Hepatic lipase has been purified to homogeneity from rat liver homogenates. The purified enzyme exhibits a single band on SDS-polyacrylamide gel electrophoresis. The molecular size of the native hepatic lipase is 200 000, while on SDS-polyacrylamide gel electrophoresis the apparent minimum molecular weight of the enzyme is 53 000, suggesting that the active enzyme is composed of four subunits. The relationship between triacylglycerol, monoacylglycerol and phospholipid hydrolyzing activities of the purified rat liver enzyme was studied. All three activities had a pH optimum of 8.5. The maximal reaction rates obtained with triolein, monoolein and dipalmitoylphosphatidylcholine were 55 000, 66 000 and 2600 mumol fatty acid/mg per h with apparent Michaelis constant (Km) values of 0.4, 0.25 and 1.0 mM, respectively. Hydrolysis of triolein and monoolein probably takes place at the same site on the enzyme molecule, since competitive inhibition between these two substrates was observed, and a similar loss of hydrolytic activity occurred in the presence of diisopropylfluorophosphate. Addition of apolipoproteins C-II and C-I had no effect on the hydrolytic activity of the enzyme with the three substrates tested. However, the triacylglycerol hydrolyzing activity was inhibited by the addition of apolipoprotein C-III. Monospecific antiserum to the pure hepatic lipase has been raised in a rabbit.     

The purification and characterization of CTP:phosphorylcholine cytidylyltransferase from rat liver

We have purified CTP:phosphorylcholine cytidylyltransferase from rat liver cytosol 2180-fold to a specific activity of 12,250 nmol/min/mg of protein. The purified enzyme was stable at -70 degrees C in the presence of Triton X-100 and 0.2 M phosphate. The purified enzyme gave a single protein and activity band on nondenaturing polyacrylamide electrophoresis. Separation by sodium dodecyl sulfate-polyacrylamide electrophoresis indicated that the purified enzyme contained subunits with Mr of 39,000 and 48,000. Gel filtration analysis indicated that the native enzyme was a tetramer containing two 39,000 and two 48,000 subunits. The purified enzyme appeared to bind to Triton X-100 micelles, one molecule of tetramer/micelle. Maximal activity was obtained with 100 microM phosphatidylcholine-oleic acid vesicles (8-10-fold stimulation). Phosphatidylglycerol produced a 4-5-fold increase in activity at 10 microM. The pH optimum and true Km values for CTP and phosphorylcholine were similar to those reported previously for crude preparations of cytidylyltransferase. The overall behavior of cytidylyltransferase during purification and subsequent analysis suggested that it has hydrophobic properties similar to those exhibited by membrane proteins.