Effect of surface composition on triolein hydrolysis in phospholipid vesicles and microemulsions by a purified acid lipase

Sonicated dispersions of egg yolk phosphatidylcholine and triolein as vesicles and microemulsions have been used as substrates for the assay of a purified acid lipase. Previous studies have also shown that triolein localized in the surface phase of emulsions is the preferred substrate. In this study, we examined enzyme activity following several surface modifications using both vesicles and microemulsions. When the acidic phospholipids phosphatidylserine and phosphatidic acid were incorporated into both vesicles and microemulsions at up to 10 mol % of the total phospholipid, a dose-dependent reduction in the apparent Km was observed. Using the vesicles as substrate, a dose-dependent decrease in Vmax was also observed. Agarose gel electrophoresis was used to verify suspected changes in net particle charge. Analogous inclusion of phosphatidylethanolamine, sphingomyelin, or cholesterol did not affect kinetic parameters. Addition of oleic acid to sonication mixtures produced vesicles with a decreased apparent Km and Vmax, but triolein hydrolysis in microemulsions was not significantly altered. Triolein-containing vesicles prepared by using dimyristoyl- or dipalmitoylphosphatidylcholine were hydrolyzed maximally at the gel liquid-crystalline transition temperatures of the appropriate phospholipid. Differential scanning calorimetry was used to verify the temperatures of transition in these vesicles. The results indicate that acid lipase activity is influenced by the charge or physical state of the surface phase of model substrates and suggest that degradation of core components of naturally occurring substrates such as lipoprotein may be influenced by chemical changes on the surface of these particles.     

Conformational changes in human urokinase induced by a specific reduction of disulfide bond in Cys-194-Cys-222 associated with exhibition of enzymatic activity

The mechanism of action of hepatic triacylglycerol lipase (EC 3.1.1.3) was examined by comparing the hydrolysis of a water-soluble substrate, tributyrin, with that of triolein by hepatic triacylglycerol lipase purified from human post-heparin plasma. The hydrolyzing activities toward tributyrin and triolein were coeluted from heparin-Sepharose at an NaCl concentration of 0.7 M. The maximal velocity of hepatic triacylglycerol lipase (Vmax) for tributyrin was 17.9 mumol/mg protein per h and the Michaelis constant (Km) value was 0.12 mM, whereas the Vmax for triolein was 76 mumol/mg per h and the Km value was 2.5 mM. The hydrolyses of tributyrin and triolein by hepatic triacylglycerol lipase were inhibited to similar extends by procainamide, NaF, Zn2+, Cu2+, Mn2+, SDS and sodium deoxycholate. Triolein hydrolysis was inhibited by the addition of tributyrin. Triolein hydrolysis was also inhibited by the addition of dipalmitoylphosphaidylcholine vesicles. In contrast, the additions of triolein emulsified with Triton X-100 and dipalmitoylphosphatidylcholine vesicles enhanced the rate of tributyrin hydrolysis by hepatic triacylglycerol lipase. In the presence of dipalmitoylphosphatidylcholine, the Vmax and Km values of hepatic triacylglycerol lipase for tributyrin were 41 mumol/mg protein per h and 0.12 mM, respectively, indicating that the enhancement of hepatic triacylglycerol lipase activity for tributyrin by dipalmitoylphosphatidycholine vesicles was mainly due to increase in the Vmax. The enhancement of hepatic triacylglycerol lipase activity for tributyrin by phospholipid was not correlated with the amount of tributyrin associated with the phospholipid vesicles. On Bio-Gel A5m column chromatography, glycerol tri[1-14C]butyrate was not coeluted with triolein emulsion, and hepatic triacylglycerol lipase activity was associated with triolein emulsion even in the presence of 2 mM tributyrin. These results suggest that hepatic triacylglycerol lipase has a catalytic site for esterase activity and a separate site for lipid interface recognition, and that on binding to a lipid interface the conformation of the enzyme changes, resulting in enhancement of the esterase activity.     

Acyl chain unsaturation modulates distribution of lecithin molecular species between mixed micelles and vesicles in model bile. Implications for particle structure and metastable cholesterol solubilities.

We determined the distribution of lecithin molecular species between vesicles and mixed micelles in cholesterol super-saturated model biles (molar taurocholate-lecithin-cholesterol ratio 67:23:10, 3 g/dl, 0.15 M NaCl, pH approximately 6-7) that contained equimolar synthetic lecithin mixtures or egg yolk or soybean lecithins. After apparent equilibration (48 h), biles were fractionated by Superose 6 gel filtration chromatography at 20 degrees C, and lecithin molecular species in the vesicle and mixed micellar fractions were quantified as benzoyl diacylglycerides by high performance liquid chromatography. With binary lecithin mixtures, vesicles were enriched with lecithins containing the most saturated sn-1 or sn-2 chains by as much as 2.4-fold whereas mixed micelles were enriched in the more unsaturated lecithins. Vesicles isolated from model biles composed of egg yolk (primarily sn-1 16:0 and 18:0 acyl chains) or soy bean (mixed saturated and unsaturated sn-1 acyl chains) lecithins were selectively enriched (6.5-76%) in lecithins with saturated sn-1 acyl chains whereas mixed micelles were enriched with lecithins composed of either sn-1 18:1, 18:2, and 18:3 unsaturated or sn-2 20:4, 22:4, and 22:6 polyunsaturated chains. Gel filtration, lipid analysis, and quasielastic light scattering revealed that apparent micellar cholesterol solubilities and metastable vesicle cholesterol/lecithin molar ratios were as much as 60% and 100% higher, respectively, in biles composed of unsaturated lecithins. Acyl chain packing constraints imposed by distinctly different particle geometries most likely explain the asymmetric distribution of lecithin molecular species between vesicles and mixed micelles in model bile as well as the variations in apparent micellar cholesterol solubilities and vesicle cholesterol/lecithin molar ratios.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and characterization of lipoprotein lipase and hepatic triglyceride lipase from human postheparin plasma: production of monospecific antibody to the individual lipase

Lipoprotein lipase (LPL) and hepatic triglyceride lipase (HTGL) were purified to homogeneity from human postheparin plasma. Molecular, catalytic and immunological properties of the purified enzymes were investigated. The native molecular weights of LPL and HTGL were 67,200 and 65,500, respectively, by gel chromatography. The subunit molecular weights of LPL and HTGL were 60,600 and 64,600, respectively, suggesting that these enzymes are catalytically active in a monomeric form. In addition, the purified LPL and HTGL each gave a single protein band when they were detected as glycoproteins with a probe of concanavalin A. The purified enzyme preparations were free of detectable antithrombin III by Western blot analysis. Catalytic properties of the purified enzymes were examined using triolein-gum arabic emulsion and triolein particles stabilized with phospholipid monolayer as substrates. LPL catalyzed the complete hydrolysis of triolein to free oleate and monooleate in the presence of apolipoprotein C-II. Apparent Km values for triolein and apolipoprotein C-II were 1.0 mM and 0.6 microM, and Vmax was 40.7 mmol/h per mg. HTGL hydrolyzed triolein substrate at a rate much slower than LPL, and produced mainly free oleate with little monooleate. Apparent Km and Vmax values were 2.5 mM and 16.1 mmol/h per mg, respectively. Polyclonal antibodies were developed against the purified LPL and HTGL. The purity and specificity of these antisera were ascertained by immunotitration, Ouchterlony double diffusion and Western blot analyses. The anti-human LPL and anti-human HTGL antiserum specifically reacted with the corresponding either native or denaturated enzyme, indicating that two enzymes were immunologically distinct. We developed an assay system for LPL and HTGL in human PHP by selective immunoprecipitation of each enzyme with the corresponding antiserum.     

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.     

Isolation and characterization of a thermophilic lipase from Bacillus thermoleovorans ID-1

A thermophilic microorganism, Bacillus thermoleovorans ID-1, isolated from hot springs in Indonesia, showed extracellular lipase activity and high growth rates on lipid substrates at elevated temperatures. On olive oil (1.5%, w/v) as the sole carbon source, the isolate ID-1 grew very rapidly at 65 degrees C with its specific growth rate (2.50 h(-1)) and its lipase activity reached the maximum value of 520 U l(-1) during the late exponential phase and then decreased. In addition to this, isolate ID-1 could grow on a variety of lipid substrates such as oils (olive oil, soybean oil and mineral oil), triglycerides (triolein, tributyrin) and emulsifiers (Tween 20, 40). The excreted lipase of ID-1 was purified 223-fold to homogeneity by ammonium sulfate precipitation, DEAE-Sephacel ion-exchange chromatography and Sephacryl S-200 gel filtration chromatography. As a result, the relative molecular mass of the lipase was determined to be 34 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme showed optimal activity at 70-75 degrees C and pH 7.5 and exhibited 50% of its original activity after 1 h incubation at 60 degrees C and 30 min at 70 degrees C and its catalytic function was activated in the presence of Ca(2+) or Zn(2+).     

Purification and properties of a second enzyme catalyzing the splitting of carbon-mercury linkages from mercury-resistant Pseudomonas K-62.

An enzyme (splitting enzyme 2) which catalyzes the splitting of carbon-mercury linkage of arylmercury compounds was found in extracts of mercury-resistant Pseudomonas K-62. This enzyme was purified about 725-fold by treatment with streptomycin, precipitation with ammonium sulfate, and successive chromatography on Sephadex G-75 and diethylaminoethyl-cellulose. A purified preparation of the enzyme showed a single band in electrophoresis either on polyacrylamide or sodium dodecyl sulfate-containing polyacrylamide gels. The molecular weight of the enzyme was estimated to be 20,000 (determined by Sephadex G-75 gel filtration) 17,000 (determined by sodium dodecyl sulfate-polyacrylamide disc gel electrophoresis). The enzyme showed a Km of 180 micron and a Vmax of 3.1 mumol/min per mg for p-chloromercuribenzoic acid and a Km of 250 micron and a Vmax of 20 mumol/min per mg for phenylmercuric acetate. The optimum temperature and pH for the reaction were 40 degrees C and 5.0, respectively.     

Characterization of a Ca2+-calmodulin-stimulated cyclic GMP phosphodiesterase from bovine brain

A calmodulin-stimulated form of cyclic nucleotide phosphodiesterase from bovine brain has been extensively purified (1000-fold). Its specific activity is approximately 4 mumol min-1 (mg of protein)-1 when 1 microM cGMP is used as the substrate. This form of calmodulin-sensitive phosphodiesterase activity differs from those purified previously by showing a very low maximum hydrolytic rate for cAMP vs. cGMP. The purification procedure utilizing ammonium sulfate precipitation, ion-exchange chromatography on DEAE-cellulose, gel filtration on Sephacryl S-300, isoelectric focusing, and affinity chromatography on calmodulin-Sepharose and Cibacron blue-agarose results in a protein with greater than 80% purity with 1% yield. Kinetics of cGMP and cAMP hydrolysis are linear with Km values of 5 and 15 microM, respectively. Addition of calcium and calmodulin reduces the apparent Km for cGMP to 2-3 microM and increases the Vmax by 10-fold. cAMP hydrolysis shows a similar increase in Vmax with an apparent doubling of Km. Both substrates show competitive inhibition with Ki's close to their relative Km values. Highly purified preparations of the enzyme contain a major protein band of Mr 74 000 that best correlates with enzyme activity. Proteins of Mr 59 000 and Mr 46 000 contaminate some preparations to varying degrees. An apparent molecular weight of 150 000 by gel filtration suggests that the enzyme exists as a dimer of Mr 74 000 subunits. Phosphorylation of the enzyme preparation by cAMP-dependent protein kinase did not alter the kinetic or calmodulin binding properties of the enzyme. Western immunoblot analysis indicated no cross-reactivity between the bovine brain calmodulin-stimulated gGMP phosphodiesterase and the Mr 60 000 high-affinity cAMP phosphodiesterase present in most mammalian tissues.     

Properties and Partial Purification of Choline Acetyltransferase from the Nematode Caenorhabditis elegans

We have stabilized and studied choline acetyltransferase from the nematode Caenorhabditis elegans. The enzyme is soluble, and two discrete forms were resolved by gel filtration. The larger of these two forms (MW approximately 154,000) was somewhat unstable and in the presence of 0.5 M NaI was converted to a form indistinguishable from the "native" small form (MW approximately 71,000). We have purified the small form of the enzyme greater than 3,300-fold by a combination of gel filtration, ion-exchange chromatography, and nucleotide affinity chromatography. The purified preparation has a measured specific activity of 3.74 mumol/min/mg protein, and is free of acetylcholinesterase and acetyl-CoA hydrolase activities. The Vmax of the purified enzyme is stimulated by NaCl, with half-maximal stimulation at 80 mM NaCl. The Km for each substrate is also affected by salt, but in different manners from each other and the Vmax; the kinetic parameter Vmax/Km thus changes significantly as a function of the salt concentration.     

Purification and characterization of bile acid-CoA:amino acid N-acyltransferase from human liver

The bile acid-conjugating enzyme, bile acid-CoA: amino acid N-acyltransferase, was purified 480-fold from the soluble fraction of homogenized frozen human liver. Purification was accomplished by a combination of anion exchange chromatography, chromatofocusing, glycocholate-AH-Sepharose affinity chromatography, and high performance liquid chromatography (HPLC) gel filtration. Following purification, the reduced, denatured enzyme migrated as a single 50-kDa protein band by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A similar molecular mass was obtained for the native enzyme by HPLC gel filtration. Elution from the chromatofocusing column suggested an apparent isoelectric point of 6.0 (+/- 0.2). Using a rabbit polyclonal antibody raised against the purified enzyme, Western blot analysis using 100,000 x g human liver supernatant confirmed that the affinity-purified polyclonal antibody was specific for human liver bile acid-CoA:amino acid N-acyltransferase. The purified enzyme utilized glycine, taurine, and 2-fluoro-beta-alanine (a 5-fluorouracil catabolite), but not beta-alanine, as substrates. Kinetic studies revealed apparent Km values for taurine, 2-fluoro-beta-alanine, and glycine of 1.1, 2.2, and 5.8 mM, respectively, with corresponding Vmax values of 0.33, 0.19, and 0.77 mumol/min/mg protein. These data demonstrate that a single monomeric enzyme is responsible for the conjugation of bile acids with glycine or taurine in human liver.     

Characterization of lysosomal acid lipase purified from rabbit liver

Lysosomal acid lipase from rabbit liver was solubilized with digitonin and purified 25,000-fold by Bio-Gel A-1.5 m, DEAE Bio-Gel A and phenyl Sepharose column chromatographies, preparative slab gel electrophoresis and finally Affi-Gel Blue affinity column chromatography. The purified enzyme gave a single protein band on polyacrylamide gel electrophoresis both in the presence and absence of sodium dodecyl sulfate. The molecular weight of the acid lipase was estimated to be 42,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis and 40,000 by gel filtration on Bio-Gel A-0.5 m. The enzyme was a hydrophobic glycoprotein with an isoelectric point of 5.15-5.90. The purified enzyme hydrolyzed tri-, di-, and monoolein and cholesterol oleate, with apparent Vmax values of 5.41, 56.1, 21.7, and 3.25 mumol/min/mg protein, and Km values of 50, 70, 200, and 40 microM, respectively. It hydrolyzed 4-methylumbelliferyl esters with fatty acids of different lengths in the order, medium length chains greater than long chains much greater than short chains. It did not hydrolyze dipalmitoylphosphatidylcholine. Its activity was inhibited by micromolar concentrations of p-chloromercuriphenyl sulfonic acid and p-bromophenacyl bromide and millimolar concentrations of Cu2+ and diethylpyrocarbonate. The activities of the enzyme towards the five substrates listed above showed almost identical thermal stabilities, mobilities on polyacrylamide gel electrophoresis and inhibition by several inhibitors. These findings support the idea that one enzyme is involved in the hydrolysis of both acylglycerols and cholesterol esters in lysosomes.     

Purification of 6-phosphogluconate dehydrogenase from chicken liver and investigation of some kinetic properties

6-phosphogluconate (6PG) dehydrogenase (EC 1.1.1.44; 6PGD) was purified from chicken liver; some kinetic and characteristic properties of the enzyme were investigated. The purification procedure consisted of four steps: preparation of the hemolysate, ammonium sulfate precipitation, 2',5'-ADP Sepharose 4B affinity chromatography, and Sephadex G-200 gel filtration chromatography. Thanks to the four consecutive procedures, product having a specific activity of 61 U (mg proteins)(-1), was purified 344-fold with a yield of 5.57%. Optimum pH, stable pH, optimum temperature, and KM and Vmax values for NADP+ and 6PG substrates were determined for the enzyme. Molecular weight of the enzyme was also determined by Sephadex G-200 gel filtration chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). In addition, Ki values and inhibition types were estimated by means of Lineweaver-Burk graphs obtained for NADPH and CO2 products.     

Isolation of rat liver microsomal short-chain beta-ketoacyl-coenzyme A reductase and trans-2-enoyl-coenzyme A hydratase: evidence for more than one hydratase

An enzyme preparation (IIIB) isolated from liver microsomes of untreated male rats was found to contain two activities--short-chain trans-2-enoyl-CoA hydratase and beta-ketoacyl-CoA reductase. The hydratase was purified more than 1000-fold, while the reductase activity was purified over 600-fold. Employing sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, a single band with a molecular weight of 76,000 was observed. Although attempts to separate these two activities have failed, it remains to be established whether the final preparation contains a single enzyme with two activities or two separate enzymes. The hydratase was most active toward crotonyl-CoA, followed by trans-2-hexenoyl-CoA (6:1) and -octenoyl-CoA (8:1); the enzyme was essentially inactive toward substrates containing more than eight carbon atoms. The Vmax for crotonyl-CoA was 2117 mumol/min/mg protein, while the Km was 59 microM. Using acetoacetyl-CoA as substrate, the Vmax for the beta-ketoacyl-CoA reductase was over 60 mumol/min/mg protein and the Km was 37 microM; the Vmax for beta-ketopalmitoyl-CoA was only 15% of that observed with acetoacetyl-CoA, although the Km was 6 microM. During the course of purification, a second short-chain hydratase was discovered (fraction IVA); unlike IIIB, this fraction catalyzed the hydration of 4:1, 6:1, and 8:1 at similar rates. The partially purified preparation yielded maximal activity with 8:1 CoA (apparent Vmax 35 mumol/min/mg), followed by 6:1 CoA, 4:1 CoA, and 10:1 CoA; longer chain CoA's were relatively poor substrates, with trans-2-hexadecenoyl CoA about 0.1 as active as 8:1 CoA. On SDS-gels, fraction IVA contained four bands, all of which were below 60,000 Mr. Proteases, such as trypsin, chymotrypsin, and subtilisin, were found to completely inactivate both enzyme fractions.     

Hydrolysis of lipid monolayers and the substrate specificity of hepatic lipase

The substrate specificities of the phospholipase and triglyceridase activities of purified rat liver hepatic lipase were compared using lipid monolayers so that the substrates were presented to the enzyme in a controlled physical state. The rate of hydrolysis of 14C-labeled lipid at constant surface pressure in the presence of hepatic lipase and fatty acid-free bovine serum albumin at 33 degrees C was determined by monitoring the decrease of surface radioactivity. In monolayers of sphingomyelin/cholesterol (2:1, mol/mol) containing either 1 mol% triacylglycerol, 1 mol% phosphatidylethanolamine, or 10 and 20 mol% phosphatidylcholine, hepatic lipase clearly showed a preference for unsaturated over saturated lipids. In addition, with a sphingomyelin/cholesterol (2:1) monolayer containing 1 mol% of lipid substrate, hepatic lipase showed the following preference: triolein = dioleoylphosphatidylethanolamine much greater than dioleoylphosphatidylcholine; the respective rates of hydrolysis were 15.3 +/- 1.2, 14.9 +/- 0.8, and 0.5 +/- 0.1 mumol fatty acid produced/h per mg hepatic lipase. Overall, it appears that when comparing rates of hydrolysis of molecules within a given lipid class, hydrocarbon chain interactions are important. However, when comparing different lipid classes such as phosphatidylcholines and phosphatidylethanolamines, it is apparent that the polar group has a significant influence on the rate of hydrolysis. The rate of [14C]triolein hydrolysis, when mixed at surface concentrations of up to 2 mol% in a sphingomyelin/cholesterol (2:1) monolayer, was significantly faster than when triolein was present in a 1-oleyl-2-palmitylphosphatidylcholine monolayer; the rates of hydrolysis were 47.7 +/- 5.4 and 8.9 +/- 0.8 mumol fatty acid produced/h per mg hepatic lipase, respectively. The monolayer physical state and the miscibility of the substrate in the inert matrix influence the presentation of the substrate to the enzyme, thereby affecting the hydrolysis rate.     

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.     

Purification of a membrane-bound neutral endopeptidase from rat kidney and its activation by polyamines

An endopeptidase which cleaves succinyl trialanine p-nitroanilide (Suc(Ala)3-pNA) into succinyl dialanine and alanine p-nitroanilide (Ala-pNA) was solubilized from a microsomal membrane fraction of rat kidney with Nonidet P-40 following treatment with 1 M KCl and Brij 35. The solubilized enzyme was purified to homogeneity by DEAE-Sephadex chromatography, Sepharose CL-6B gel filtration and sucrose gradient centrifugation. The final enzyme preparation had a specific activity of 1.69 mumol/min/mg protein, representing about 140-fold purification over the starting membrane. The enzyme hydrolyzes Suc(Ala)3-pNA with a Km value of 0.28 mM and a Vmax value of 1.3 mumol/min. The molecular weight of the undenatured enzyme was estimated to be 360,000 by gel filtration on a Sepharose CL-6B column and that of the denatured enzyme to be 92,000 by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, revealing the presence of a single polypeptide chain. The enzyme was markedly activated by polyamines, producing increases in the values of both Km and Vmax. Comparatively less activation was found in the presence of some monovalent cations and Ca2+. The activation by polyamines was inversely proportional to the concentration of monovalent cations, but Ca2+ and polyamines seemed to stimulate additively.     

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.     

Purification and characterization of 17 beta-hydroxysteroid dehydrogenase from Cylindrocarpon radicicola

An NAD+-linked 17 beta-hydroxysteroid dehydrogenase was purified to homogeneity from a fungus, Cylindrocarpon radicicola ATCC 11011 by ion exchange, gel filtration, and hydrophobic chromatographies. The purified preparation of the dehydrogenase showed an apparent molecular weight of 58,600 by gel filtration and polyacrylamide gel electrophoresis. SDS-gel electrophoresis gave Mr = 26,000 for the identical subunits of the protein. The amino-terminal residue of the enzyme protein was determined to be glycine. The enzyme catalyzed the oxidation of 17 beta-hydroxysteroids to the ketosteroids with the reduction of NAD+, which was a specific hydrogen acceptor, and also catalyzed the reduction of 17-ketosteroids with the consumption of NADH. The optimum pH of the dehydrogenase reaction was 10 and that of the reductase reaction was 7.0. The enzyme had a high specific activity for the oxidation of testosterone (Vmax = 85 mumol/min/mg; Km for the steroid = 9.5 microM; Km for NAD+ = 198 microM at pH 10.0) and for the reduction of androstenedione (Vmax = 1.8 mumol/min/mg; Km for the steroid = 24 microM; Km for NADH = 6.8 microM at pH 7.0). In the purified enzyme preparation, no activity of 3 alpha-hydroxysteroid dehydrogenase, 3 beta-hydroxysteroid dehydrogenase, delta 5-3-ketosteroid-4,5-isomerase, or steroid ring A-delta-dehydrogenase was detected. Among several steroids tested, only 17 beta-hydroxysteroids such as testosterone, estradiol-17 beta, and 11 beta-hydroxytestosterone, were oxidized, indicating that the enzyme has a high specificity for the substrate steroid. The stereospecificity of hydrogen transfer by the enzyme in dehydrogenation was examined with [17 alpha-3H]testosterone.     

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 some properties of phospholipase C (alpha-toxin) of Clostridium perfringens.

1. Phospholipase C[EC 3.1.4.3] was purified from the culture filtrate of Clostridium perfringens by successive chromatographies on CM-Sephadex, DEAE-Sephadex, and Sephadex G-100. During the purification it was noted that, beside the monomer form of the enzyme which was purified, a part of the enzyme existed in active polymerized forms. 2. The purified preparation gave a single band on polyacrylamide gel electrophoresis and gave a single precipitin line in immunodiffusion with the National Standard gas gangrene (C. perfringens) antitoxin, indicating the homogeneity of the preparation. 3. The specific lecithin-hydrolyzing activity of the purified preparation was comparable to that of a preparation obtained by affinity chromatography, which had the highest specific activity previously reported. 4. The molecular weight of the purified enzyme was estimated to be 43,000 by SDS-polyacryl-amide gel electrophoresis, although the same preparation gave a molecular weight of 31,000 as determined by gel filtration on Sephadex G-150. From this and the above finding that a part of the enzyme exists in active polymerized forms, the discrepancy among reported values for the molecular weight of C. perfringens phospholipase C can be accounted for. 5. For maximum hydrolytic activity toward lecithin, the enzyme required sodium deoxycholate (SDC) and Ca2+ ions. In the presence of 6 mM Ca2+, the optimal molar ratio of SDC to lecithin for maximal hydrolytic activity was about 0.5 for dipalmitoyl lecithin and about 1.0 for egg lecithin. The effects of various divalent cations on the enzymatic hydrolysis were also investigated. 6. The effects of sodium deoxycholate and Ca2+ ions on the enzymatic hydrolysis are discussed, based on their possible roles in mixed micelle formation.