Phorbol ester stimulates PAF synthesisvia the activation of protein kinase C in rat leukocytes

When rat pleural mononuclear leukocytes were stimulated with 1 μM phorbol myristate acetate (PMA), platelet-activating factor (PAF)-like activity was detected in the supernatant and the cellular fractions of the incubation mixture, as measured by rabbit platelet aggregation. C₁₆PAF activity peaked at 30 min in both fractions. Acetyltransferase activity in the microsomal fraction of the stimulated cells also increased rapidly and showed a peak at 10 min. A protein kinase C inhibitor, staurosporine, and an inhibitor of phospholipase A₂,p-bromophenacylbromide, inhibited stimulated PAF formation in both fractions. Staurosporine also inhibited PMA induced acetyltransferase activity. The data suggest that PMA stimulates PAF synthesis by the remodeling pathway in rat pleural cells through activation of both phospholipase A₂ and acetyltransferase, and that the acetyltransferase, in turn, may be activated through activation of protein kinase C. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Purification and characterization of an extracellular lipase from Penicillium candidum

Penicillium candidum produces and secretes a single extracellular lipase with a monomer molecular weight of 29 kDa. However, this enzyme forms dimers and higher molecular weight aggregates under nondenaturing conditions. The lipase from P. candidum was purified 37-fold using Octyl-Sepharose CL-4B and DEAE-Sephadex columns. The optimal assay conditions for lipase activity were 35°C and pH 9. The lipase was stable in the pH range of 5–6 with a pl of 5.5, but rapid loss of the enzyme activity was observed above 25°C. Tributyrin was found to be the best substrate for the P. candidum lipase, among those tested. Metal ions such as Fe²⁺ and Cu²⁺ inhibited enzymatic activity and only Ca²⁺ was able to slightly enhance lipase activity. Ionic detergents inhibited the activity of the enzyme, whereas nonionic detergents stimulated lipase activity.     

蜡状芽抱杆菌壬基苯酚降解酶的分离纯化及其酶学性质(英文)

An extracellular NP-degrading enzyme secreted by Bacillus cereus.Frankland was purified to homogeneity by a combination of ammonium sulfate precipitation,Phenyl-Sepharose hydrophobic-interaction chromatography and DEAE anion-exchange chromatography.On SDS(sodium dodecyl sulfate)-polyacrylamide gel electrophoresis analysis,the purified enzyme showed a relative molecular mass of 58.3 kDa.The depolymerzation of subunits was accompanied with the loss of NP-degrading enzyme activity,and removing denaturing factors by dialysis could restore the dimer structure and enzymatic activity.The enzyme had an isoelectric point of 5.5 and an optimal temperature of 60℃,and was the most active at pH 6.0.The enzymatic activity was stable at pH 4-8 and inhibited by Cu2+.TenN-terminal amino acids were determined to be ASVNSIKIGY,demonstrating that the purified enzyme was a novel one.The hydrolysis pattern of the purified enzyme indicated that the NP-degrading enzyme was an endo NP-degrading enzyme.The extraordinary thermo-stability provided the enzyme with a good prospect to be used as a new tool for clean-production process for textile industry.     

Quantification of distinct molecular species of platelet activating factor in ulcerative colitis

The aim of this study was to characterize the synthesis and metabolism of platelet activing factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by colonic mucosa from patients with ulcerative colitis and healthy individuals. Tissue was obtained by endoscopic biopsy and by scraping the mucosa from surgical resections. Tissue was assayed for the various molecular species of PAF and its biologically inactive metabolite lyso-PAF using gas chromatography/mass spectrometry. Mucosa from surgical resections for ulcerative colitis contained C16:0 PAF (mean=156 ng/g of mucosa), but not C18:0 PAF. PAF was not identified in mucosa from normal surgical resections or in endoscopic biopsies from either patients with ulcerative colitis or normal individuals. Both C16:0 lyso-PAF and C18:0 lyso-PAF were found in mucosa from normal and ulcerative colitis surgical resections and in endoscopic biopsies from ulcerative colitis and normal tissue. Levels of lyso-PAF were similar in ulcerative colitis and normal mucosa. Incubation of mucosa from areas of active inflammation in ulcerative colitis with the calcium ionophore A23187 increased the levels of C16:0 PAF by 2–3 fold and also increased the levels of C16:0 lyso-PAF. Addition of³H-PAF to endoscopic biopsies from either normal individuals or patients with ulcerative colitis resulted in hydrolysis to³H-lyso-PAF. The data on colonic mucosal levels of PAF are consistent with the results of earlier studies measuring PAF in patients with ulcerative colitis by bioassay. This study examines the synthesis and metabolism of specific molecular species of PAF in ulcerative colitis for the first time.     

Partial purification of a triacylglycerol lipase isolated from steelhead trout (Salmo gairdneri) adipose tissue

A triacylglycerol (TG) lipase (EC 3.1.1.2), assayed by monitoring [1-¹⁴C]-oleic acid release from [carboxyl-¹⁴C]-triolein after liquid-liquid partition of the fatty acid from the unhydrolyzed triacylglycerol substrate, was isolated and partially purified from steelhead trout adipose tissue. The TG lipase was resolved from contaminating lipoprotein lipase by heparin-sepharose affinity chromatography and purified ca. 71-fold over the original fraction. Optimal enzyme activity occurred at pH 7.5. The purified enzyme migrated on SDS-polyacrylamide gels with an apparent molecular weight of 48,000.     

Toxic inhibition of some dehydrogenases by methyl sterculate--a natural occurring substance in cotton seeds

According to the present study, crude preparations of rainbow trout liver and rabbit muscle lactate dehydrogenase (LDH,EC 1.1.1.27) were not inhibited by methyl sterculate and oleate, while trout liver glucose-6-phosphate dehydrogenase (G6PDH,EC 1.1.1.49) was activated by these esters. Methyl sterculate inhibited purified preparations of trout liver, rabbit muscle, and bovine heart LDH in contrast to methyl oleate which inhibited only a portion of the activity of purified rabbit muscle LDH and had no appreciable effect on the activities of the other purified LDH preparations. Trout liver LDH preparations were not inhibited by p-Chloromercuribenzoate (pCMB), while rabbit muscle and bovine heart LDH were sensitive to the presence of this inhibitor. Trout liver G6PDH was activated at the lower concentrations of pCMB. These data suggest that the reduction of the activities of liver dehydrogenases in the rainbow trout fed a diet containing methyl sterculate was not due to inhibition of these dehydrogenases by this cyclic fatty acid.     

Anaerobic lipoxygenase activity from Chlorella pyrenoidosa responsible for the cleavage of the 13‐hydroperoxides of linoleic and linolenic acids

An enzyme from the alga Chlorella pyrenoidosa, previously identified as a hydroperoxide lyase (HPLS), cleaves the 13‐hydroperoxide derivatives of linoleic and linolenic acids into a volatile C5 fragment and a C13 oxo‐product, 13‐oxo‐9(Z),11(E)tridecadienoic acid (13‐OTA). Gas chromatography/mass spectrometry (GC/MS) headspace analysis of the volatile products indicated the formation of pentane when the substrate was the 13‐hydroperoxide derivative of linoleic acid, whereas a more complex mixture of hydrocarbons was formed when the 13‐hydroperoxide derivative of linolenic acid was the substrate. Analysis of the nonvolatile products by GC/MS and liquid chromatography/mass spectrometry (LC/MS) indicated the formation of 13‐OTA along with the 13‐ketone derivative. This enzymatic activity was inhibited by oxygen but was restored with nitrogen. The enzymatic cleavage activity was coincidental in purified fractions with lipoxygenase activity that produced the 13‐ and 9‐hydroperoxide derivatives of linolenic acid. The results suggest that the enzymatic cleavage activity in Chlorella pyrenoidosa was not a consequence of hydroperoxide lyase activity as previously thought, but was due to anaerobic lipoxygenase activity. This enzyme fraction was purified by (NH₄)₂ SO₄ precipitation, gel filtration, and hydrophobic interaction chromatography. The purified enzyme has an approximate MW of 120 KDa and maximum activity at pH 8.0.     

Studies on the selectivity of enzymes involved in platelet-activating factor formation in stimulated cells

The present studies were undertaken to obtain further insight into the selectivities of the enzymes, i.e., phospholipase A₂ and acetyltransferase, involved in platelet-activating factor (PAF) production upon stimulation of human polymorphonuclear leukocytes (PMN) and platelets. After appropriate stimulation of the cells in the presence of [³H]acetate the total PAF and analogs, i.e., 1-alkyl-2-acetyl-, 1-alkenyl-2-acetyl-, and 1-acyl-2-acetyl-glycero-3-phosphocholine were isolated by high performance liquid chromatography. The isolated mixture was subjected to treatment with phospholipase A₁ to differentiate acetate incorporation into 1-ether linked and 1-ester linked species. The ratio of acetate incorporation into 1-ether linked vs 1-ester linked PAF analogs amounted to 13.8±1.0 and 1.3±0.1 for PMN and platelets, respectively. When compared to the ratio of 1-ether linked and 1-ester linked species in the diradylglycerophosphocholine precursors in each cell type, i.e., 1.13 for PMN and 0.22 for platelets, these data suggested a pronounced selectivity for the phospholipase A₂ and/or acetyltransferase in the process of PAF production. When the experiments were repeated with cells that had been pretreated with phenylmethanesulfonylfluoride (PMSF) to block the acetylhydrolase, the most dramatic effects were observed on acetate incorporation into 1-acyl-2-acetyl-glycero-3-phosphocholine, which increased much more than that into 1-alk(en)yl-2-acetyl-glycero-3-phosphocholine. Under these conditions, the ratio of acetate incorporation into 1-ether linked vs 1-ester linked PAF analogs became 1.4±0.2 and 0.17±0.02 for PMN and platelets, respectively. These values are very close to the 1-ether linked vs 1-ester linked species in the diradylglycerophosphocholine precursors for PAF in the respective cell type. These data suggested that the selectivities of phospholipase A₂ and/or acetyl transferase for etherlinked species, as observed in non-PMSF treated cells, are only apparent and caused by rapid degradation of the 1-acyl analog either before or after acetylation. In line with this interpretation, we demonstrated that 1-acyl-2-acetyl-GPC can be deacylated to water-soluble acetyl-GPC and GPC by sonicated PMN and platelets and that this deacylation is completely blocked in sonicates from PMSF-pretreated cells. In addition, evidence is presented which indicates that the enzyme responsible for deacylation may be a lysophospholipase. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.     

Platelet‐activating factor detection,metabolism, and inhibitors in the ethanologenic bacterium Zymomonas mobilis

Platelet‐activating factor (PAF) is a signaling phospholipid with a significant physiological role in multicellular and unicellular organisms, including fermentative organisms such as yeast. Zymomonas mobilis is an ethanologenic α‐proteobacterium currently studied for bioethanol production. In order to examine the presence of PAF and/or PAF inhibitors in Z. mobilis, a new one‐step high performance liquid chromatography (HPLC) separation procedure of total lipids was performed, using a C8 reversed‐phase semi‐preparative column. According to this method and to bioassays based on washed rabbit platelet aggregation, two lipid molecules with PAF‐like activity and same retention times as those of standard PAF were detected; electron‐spray ionization MS and MS/MS analysis revealed that they share similar structure with 16:0 and 18:0 PAF. Furthermore, other lipids extracted from Z. mobilis were found to exhibit a potent anti‐PAF activity. Enzyme activities indicative of key PAF biosynthetic enzymes, such as dithiothreitol‐insensitive cholinephosphotransferase (PAF‐CPT) and lyso‐PAF acetyltransferase were detected in Z. mobilis homogenates. As for PAF degradation, activity similar to that of PAF acetylhydrolase was also discovered. Overall, the presence of PAF, PAF‐specific inhibitors, and enzyme activities relating to PAF metabolism, suggests that PAF may play an intrinsic role in this biotechnological organism. Practical applications: Z. mobilis is a platform microorganism for bioethanol production and a potential source of high‐value chemicals of interest to the food and healthcare industries. Further investigation of PAF's role is bound to affect applications involving this and other biotechnological organisms. The finding that Z. mobilis lipids exhibit potent anti‐PAF activity opens up prospects for their identification, overproduction and pharmaceutical use. The presented HPLC method for lipid fractionation accomplishes a one‐step separation of lipids from dense samples, which may be successfully employed to other lipid‐rich sources such as blood.     

Purification and characterization of an organic solvent and detergent‐tolerant novel protease produced by Bacillus sp. RKY3

BACKGROUND: Purification and characterization of a novel protease produced by Bacillus sp. RKY3, has been investigated, with special emphasis on the stability of the enzyme in the presence of different oxidizing and reducing agents as well as organic solvents. The enzyme was purified in two steps through concentration of the crude enzyme by ammonium sulfate precipitation, followed by anion exchange chromatography. RESULTS: The purified protease had a molecular mass of approximately 38 kDa, which was highly active over a broad range of pH between 7.0 and 9.0 and was also stable over a wide pH range from 5.0 to 11.0. Although the optimum temperature for enzyme activity was found to be 60 °C, it was rapidly deactivated at temperatures above 60 °C. It also showed good stability at 50 °C, with a 70 min half‐life. Ca²⁺ ions did not greatly enhance the activity or the stability of the enzyme. PMSF (1 mmol L^?1) completely inhibited the protease activity, and thus the purified protease was considered to be serine protease. The purified protease was stable with oxidants (H₂O₂, 2%), reducing agents (sodium dodecyl sulfate, 2%), and organic solvents (25%) such as benzene, hexane, and toluene. CONCLUSION: The purified enzyme, protease, seems to possess potential applications in protease‐based detergent and bleaching industries. The enzymatic activity against a wide variety of substrates suggests that the purified enzyme should be investigated for a range of commercial applications, especially for soy protein and gelatin hydrolysis in the food processing industry. Copyright © 2008 Society of Chemical Industry     

Purification and characterization of a cytochrome P450 isozyme catalyzing lanosterol 14α-demethylation (P45014DM) in hamster liver

To characterize cholesterol synthesis in Syrian golden hamster, an isozyme of cytochrome P450, lanosterol 14α-demethylase (P45014DM), which catalyzes the initial step in the biosynthesis of cholesterol from lanosterol, was purified and its mode of induction by microsomal enzyme inducers was characterized. P450450DM was purified from hamster livers by chromatography using aminooctyl-Sepharose CL-4B, hydroxylapatite, DEAE-5PW, and CM-Sephrose CL-6B columns, to a specific content of 12.8 nmol/mg-protein. The purified protein displayed a single band on SDS-polyacrylamide gel electrophoresis with an apparent molecular weight of 52,000. The absorption spectra of the oxidized form of the purified protein howed a Soret peak at 417 nm in a low-spin state and a Soret peak of reduced CO-binding complex at 448 nm. In a reconstituted system, the purified protein catalyzed 14α-demethylation of 24,25-dihydrolanosterol (1.58 nmol/min/nmol-P450), although it did not show any activities toward testosterone and 7-ethoxyresorufin, marker substrates of other P450 families. Immunoblot analysis using an antibody against porcine P45014DM, which inhibited the activity of lanosterol 14α-demethylation in the hamster liver microsomes, demonstrated that the level of this isozyme protein was markedly decreased in dexamethasone-treated hamster livers. This was accompanied by a decrease in the enzyme activity. In contrast, the levels and the activity in the phenobarbital-and 3-methylcholanthrene-treated hamsters were almost equal to that in the untreated animals.     

Synthesis of platelet activating factor by cholinephospho-transferase in developing fetal rabbit lung

Developing fetal lung is a possible source of the platelet activating factor (PAF, 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) present in amniotic fluid of women in labor. We have assayed the microsomal activities of a specific enzyme for the de novo synthesis of PAF in developing fetal and neonatal rabbit lung, 1-alkyl-2-acetyl-glycerol-dependent dithiothreitol-insensitive cholinephosphotransferase. The specific activity of this enzyme increased from 0.92 to 3.60 nmol×min⁻¹×mg⁻¹ protein between day 21 and day 31 of gestation. In contrast, during this same period the activity of the PAF-biosynthetic cholinephos-photransferase in developing rabbit kidney did not change significantly. The specific activity of the diacyl-glycerol-dependent, dithiothreitol-sensitive cholinephos-photransferase that catalyzes the final step in phosphatidylcholine biosynthesis was not altered during late gestation in either fetal lung or kidney. Previously, increased amounts of pulmonary PAF had been found during the latter stages of gestation (Hoffman, Truong and Johnston (1986)Biochim. Biophys. Acta 879, 88–96) and may be attributed to increased activity of the PAF biosynthetic enzymes found in this investigation. This elevated level of PAF in fetal lung may serve to facilitate breakdown of glycogen that provides, in part, the carbon and energy source for surfactant biosynthesis. In addition, PAF may be secreted in association with surfactant into amniotic fluid in which it may interact with amnion tissue and subsequently participate in the events associated with the initiation of parturition.     

Purification and characterization of an extracellular lipase from <Emphasis Type="Italic">Geotrichum marinum</Emphasis>

An extracellular lipase (EC 3.1.1.3) from Geotrichum marimum was purified 76-fold with 46% recovery using Octyl Sepharose 4 Fast Flow and Bio-Gel A 1.5 m chromatography. The purified enzyme showed a prominent band on SDS-PAGE and a single band on native PAGE based on the activity staining. The molecular mass of the lipase was estimated to be 62 kDa using SDSPAGE and Bio-Gel A chromatography, indicating that the lipase likely functions as a monomer. The pl of the lipase was determined to be 4.54. The apparent V _max and K _m were 1000 μmol/min/mg protein and 11.5 mM, respectively, using olive oil emulsified with taurocholic acid as substrate. The lipase demonstrated a pH optimum at pH 8.0 and a temperature optimum at 40°C. At 6 mM, Na⁺, K⁺, Ca²⁺, and Mg²⁺ stimulated activity, but Na⁺, and K⁺ at 500 mM and Fe²⁺ and Mn²⁺ at 6 mM reduced lipase activity. The anionic surfactant, taurocholic acid, and the zwitterionic surfactant, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, enhanced the activity at 0.1 mM. Other anionic surfactants such as SDS and sodium dioctyl sulfosuccinate, the cationic surfactants methylbenzethonium bromide and cetyltriethylammonium bromide, and the nonionic surfactants Tween-20 and Triton X-100 inhibited the lipase activity to different extents. The lipase was found to have a preference for TG containing cis double bonds in their FA side chains, and the reaction rate increased with an increasing number of double bonds in the side chain. The lipase had a preference for ester bonds at the sn-1 and sn-3 positions over the ester bond at the sn-2 position.     

Purification and characterization of an extracellular lipase from Geotrichum marinum

An extracellular lipase (EC 3.1.1.3) from Geotrichum marinum was purified 76-fold with 46% recovery using Octyl Sepharose 4 Fast Flow and Bio-Gel A 1.5 m chromatography. The purified enzyme showed a prominent band on SDS-PAGE and a single band on native PAGE based on the activity staining. The molecular mass of the lipase was estimated to be 62 kDa using SDS-PAGE and Bio-Gel A chromatography, indicating that the lipase likely functions as a monomer. The pl of the lipase was determined to be 4.54. The apparent V(max) and Km were 1000 micromol/min/mg protein and 11.5 mM, respectively, using olive oil emulsified with taurocholic acid as substrate. The lipase demonstrated a pH optimum at pH 8.0 and a temperature optimum at 40 degrees C. At 6 mM, Na+, K+, Ca2+, and Mg2+ stimulated activity, but Na+ and K+ at 500 mM and Fe2+ and Mn2+ at 6 mM reduced lipase activity. The anionic surfactant, taurocholic acid, and the zwitterionic surfactant, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate, enhanced the activity at 0.1 mM. Other anionic surfactants such as SDS and sodium dioctyl sulfosuccinate, the cationic surfactants methylbenzethonium bromide and cetyltriethylammonium bromide, and the nonionic surfactants Tween-20 and Triton X-100 inhibited the lipase activity to different extents. The lipase was found to have a preference for TG containing cis double bonds in their FA side chains, and the reaction rate increased with an increasing number of double bonds in the side chain. The lipase had a preference for ester bonds at the sn-1 and sn-3 positions over the ester bond at the sn-2 position.     

Purification and enzymatic properties of the extracellular and constitutive chitosanase produced by Bacillus subtilis RKY3

BACKGROUND: Purification and enzymatic properties of a chitosanase from Bacillus subtilis RKY3 have been investigated to produce a chitooligosaccharide. The enzyme reported was extracellular and constitutive, which was purified by two sequential steps including ammonium sulfate precipitation and ion exchange chromatography. RESULTS: Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of the purified chitosanase revealed one single band corresponding to a molecular weight of around 24 kDa. The highest chitosanase activity was found to be at pH 6.0 and at 60 °C. Although the mercaptide forming agents such as Hg²⁺ (10 mmol L^?1) and p‐hydroxymercuribenzoic acid (1 mmol L^?1, 10 mmol L^?1) significantly or totally inhibited the enzyme activity, its activity was enhanced by the presence of 10 mmol L^?1 Mn²⁺. The enzyme showed activity for hydrolysis of soluble chitosan and glycol chitosan, but colloidal chitin, carboxymethyl cellulose, crystalline cellulose, and soluble starch were not hydrolyzed. The analysis of chitosan hydrolysis by thin‐layer chromatography and viscosity variation revealed that the purified enzyme should be endosplitting‐type chitosanase. CONCLUSION: The chitosanase produced by Bacillus subtilis RKY3 was a novel chitosanlytic enzyme with relatively low molecular weight, which is a versatile enzyme for chitosan hydrolysis because it could hydrolyze soluble chitosan into a biofunctional oligosaccharide at a high level. Copyright © 2011 Society of Chemical Industry     

Purification and properties of aortic cholesteryl ester hydrolase

The enzyme(s) present in acetonedried powder of rat and rabbit aortas, which catalyzes the synthesis and hydrolysis of cholesteryl ester, was purified partially by acid precipitation, acetone fractionation, 0-(diethylaminoethyl) cellulose chromatography, and Sephadex G-100 filtration. The synthetic activity was purified by 120-fold (rat) and 140-fold (rabbit). Purification of hydrolytic activity was 90-fold (rat) and 103-fold (rabbit). Cholesteryl ester hydrolase activity was separated from nonspecific, esterase by column chromatography. Both synthetic and the hydrolytic activities are apprently the functions of one enzyme. The mol wt of the enzyme was estimated to be 140,000 dalton as determined by Sephadex G-200 gel filtration. The extracts of the acetone-dired powders of aortas of both species contained an inhibitor of synthetic activity. The inhibitor was nondialyzable and was precipitated at pH 5.7 Both activities were found to be fairly nonspecific with regard to sterol and fatty acids. With oleic acid, the relative rates of sterol ester synthesis were: cholesterol, 100; cholestanol, 94; desmosterol, 35; corprostanol, 24; ergosterol, 20; and β-sitosterol, 19. Epicholesterol was not esterified. Oleic acid was most active in cholesteryl ester synthesis, the relative rates being: oleic>linoleic>arachidonic>palmitic>stearic>butyric. The rate of hydrolysis was maximum with cholesteryl linoleate followed by oleate, linolenate, palmitate, stearate and laurate in decreasing order.     

Activation of the phospholipase A1 activity of lipoprotein lipase by apoprotein C-II

The effect of apo very low density lipoprotein (apo VLDL) and apoprotein C-II on the phospholipase A₁ activity associated with lipoprotein lipase (E.C.3.1.1.3) was studied using purified bovine milk lipoprotein lipase. The enzyme degraded¹⁴C phosphatidylcholine (PC) to¹⁴C 2-acyl lysophosphatidylcholine at a rate of 0.28±0.01 nmol/min/ml and triolein at a rate of 20.3±0.4 nmol/min/ml in mixed emulsions of PC and triolein. The phospholipase activity and triacylglycerol lipase activity were both increased by the addition of apo VLDL and apoprotein C-II. After maximal activation, the rate of PC degradation was 1.19±0.02 nmol/min/ml and triolein degradation 64.4±0.4 nmol/min/ml. Activation of phospholipase A₁ activity and triacylglycerol lipase activity occurred in parallel.     

Partial purification and characterization of acylester hydrolase from Lupinus mutabilis

An alkaline acylester hydrolase was partially purified from germinated seeds of Lupinus mutabilis. Hydrolytic activity was absent in the crude extract of ungerminated lupine seed, but it increased and peaked at the fourth day in the germinating seedling. The purification scheme involved homogenization, centrifugation, acetone precipitation, anion exchange chromatography, pH precipitation, and hydrophobic interaction chromatography. The acylester hydrolase was purified 126-fold, and the overall activity yield was 10%. The molecular weight estimated by sodium dodecylsulfate-polyacrylamide gel electrophoresis was 60 kDa. The enzyme had an isoelectric point of 6.2 and showed maximal activity at pH 8.0. The enzyme showed good stability between pH 5.0 and 9.0. In the pH range 7.0–7.5, enzyme precipitation was observed. The enzyme was stable from 0 to 25°C for 5 h and at 45°C lost 50% of its activity in the same period of time. At higher temperatures, the enzyme showed low thermal stability. However, the highest initial activity was found to be at 45°C. Nonionic surfactants and cholic acid enhanced the activity of the enzyme. The activity was reduced by the addition of toluene and isooctane and increased by the addition of diethyl ether, acetonitrile, methanol, and pyridine. The activity was reduced by 37% in the presence of 1 mM Cu²⁺ ions. The enzyme-hydrolyzed triolein showing no positional specificity.     

Purification and properties of a milk-clotting enzyme produced by Bacillus amyloliquefaciens D4

The milk-clotting enzyme from Bacillus amyloliquefaciens D4 was purified to 17.2-fold with 20% recovery by precipitation in ammonium sulfate and ion-exchange chromatography. The molecular mass of the enzyme was 58.2 kDa as determined by SDS-PAGE, and it was proved to be a metalloprotease by EDTA inhibition. The enzyme was active in the pH range 5.5–7.0 and was inactivated completely by heating at 55 °C for 20 min. The highest level of enzyme activity was obtained at 65 °C, pH 5.5, in the presence of 25mM CaCl₂. The milk-clotting activity was inhibited only slightly by Na⁺ and K⁺ and significantly by Cu²⁺, Zn²⁺ and Sn²⁺. The K _m value of this enzyme was 0.471 mg/mL. The high level of milk-clotting activity coupled with a low level of thermal stability suggested that the milk-clotting enzyme from B. amyloliquefaciens D4 should be considered as a potential substitute for calf rennet.     

Specific binding of antibodies to platelet-activating factor (PAF) as demonstrated by thin-layer chromatography/Immunostaining

The specificity of rabbit antibodies produced by injection of 1-O-(15'-carboxypentadecyl)-2-N,N-dimethylcar-bamoyl-sn-glycero-3-phosphocholine bovine serum albumin (BSA) conjugates was examined by a thin-layer chromatography (TLC)/immunostaining method. Phosphatidylcholine (PC), lysophosphatidylcholine (lysoPC), lyso platelet-activating factor (lysoPAF), phosphatidylethanolamine (PE), phosphatidylglycerol (PG), phosphatidylserine (PS), sphingomyelin (SM), phosphatidylinositol (PI), phosphatidic acid (PA) and cardiolipin (CL) were not immunostained. Among several synthetic PAF-related compounds, the antibodies only bound to PAF agonists which have the activity to induce washed rabbit platelet aggregation. The results suggest that the binding sites of the antibodies on the PAF molecule are the acetyl group at thesn-2 position and the choline moiety at thesn-3 position of glycerol, both of which are essential for exerting the biological function of PAF and for binding to the PAF receptors located on cellular membranes. Based on a paper presented at the Third International Conference on Platelet-Activating Factor and Structurally Related Alkyl Ether Lipids, Tokyo, Japan, May 1989.