Immobilization in the presence of Triton X-100: modifications in activity and thermostability of <Emphasis Type="Italic">Geobacillus thermoleovorans</Emphasis> CCR11 lipase

A partially purified lipase produced by the thermophile Geobacillus thermoleovorans CCR11 was immobilized by adsorption on porous polypropylene (Accurel EP-100) in the presence and absence of 0.1% Triton X-100. Lipase production was induced in a 2.5% high oleic safflower oil medium and the enzyme was partially purified by diafiltration (co. 500,000 Da). Immobilization conditions were established at 25 °C, pH 6, and a protein concentration of 0.9 mg/mL in the presence and absence of 0.1% Triton X-100. Immobilization increased enzyme thermostability but there was no change in neither the optimum pH nor in pH resistance irrelevant to the presence of the detergent during immobilization. Immobilization with or without Triton X-100 allowed the reuse of the lipase preparation for 11 and 8 cycles, respectively. There was a significant difference between residual activity of immobilized and soluble enzyme after 36 days of storage at 4 °C (P < 0.05). With respect to chain length specificity, the immobilized lipase showed less activity over short chain esters than the soluble lipase. The immobilized lipase showed good resistance to desorption with phosphate buffer and NaCl; minor loses with detergents were observed (less than 50% with Triton X-100 and Tween-80), but activity was completely lost with SDS. Immobilization of G. thermoleovorans CCR11 lipase in porous polypropylene is a simple and easy method to obtain a biocatalyst with increased stability, improved performance, with the possibility for re-use, and therefore an interesting potential use in commercial conditions.     

Partial Purification of an Ethylene-binding Component from Plant Tissue

An ethylene binding component(s) has been partially purified from mung bean sprouts. Tissue was homogenized in 0.3 molar sucrose and 0.2 molar potassium phosphate buffer (pH 7.0). The homogenate was centrifuged, and resuspended fractions were assayed by incorporating them onto cellulose fibers (0.7 grams per milliliter). These were exposed to [¹⁴C]ethylene (3.7 × 10⁻² microliters per liter of 120 millicurie per millimole) in the presence or absence of 1000 microliters per liter unlabeled ethylene. The cellulose was transferred to separate containers and the [¹⁴C]ethylene was absorbed in mercury perchlorate and counted. Distribution of ethylene binding to various fractions was: 0 to 3,000g, 3%; 3,000 to 12,000g; 4%; 12,000 to 100,000g, 69%; cellular debris, 24%; 100,000g supernatant, 0%. Adjustment of the pH to 4.0 precipitates the ethylene-binding component. Neutralization, addition of Triton X-100, and readjustment of the pH to 4.0 “solubilized” most of the binding component. Further purification was obtained by chromatography on CM-Sephadex in 10 millimolar potassium acetate buffer, (pH 5.0) containing 1% Triton X-100. Elution was with 200 millimolar potassium phosphate (pH 6.0) containing 1% Triton X-100. Upon treatment of the Triton “solubilized” component with cold acetone, over 90% of the binding capacity was lost. Extraction of the acetone-precipitated residue with 2% Triton X-100 restored some of the binding capacity which was found in the soluble fraction. The pH optimum for binding is 6.0. Passing the Triton X-100 extract of the acetone powder through Sepharose 6B provides considerable purification. The binding component moved ahead of most of the protein.     

Characteristics of lysosomes in the rat placental cells

Six acid hydrolases, cytochrome oxidase, and alkaline phosphatase were demonstrated in 0.25 m sucrose homogenates of rat chorioallantoic placenta. The acid hydrolases were: acid phosphatase, β-glucuronidase, N-acetyl-β-glucosaminidase, β-galactosidase, and acid deoxyribonuclease, showing optimum activity near pH 4.5; cathepsin, with optimum activity near pH 3.6. The free acid hydrolases present in cytoplasmic extracts expressed 20–40% of their total activity. “Total” activity was defined as the enzyme activity observed in the presence of Triton X-100, while “free” activity denoted enzyme activity measured under similar assay conditions except in the presence of sucrose and absence of Triton X-100. The decreased activity or latency in the assays for the free activity of acid phosphatase, acid deoxyribonuclease, and cathepsin persisted after incubation at pH 5 and 37 ° up to an hour. In contrast, this latency did not persist after incubation of the β-glycosidases. Additionally, the free activity of all the designated enzymes of the cytoplasmic extract was in excess of the nonsedimentable activity observed.     

Molecular properties of cytochrome P-45011 beta from adrenal cortex mitochondria.

Cytochrome P-45011 beta has been solubilized and partially purified from bovine adrenal cortex mitochondria using chromatography on Octyl-Sepharose CL-4B or DEAE-Sepharose CL-6B. The partially purified P-450 preparations were about 90% pure as judged by SDS-polyacrylamide gel electrophoresis. In the presence of purified preparations of adrenodoxin reductase and adrenodoxin, the partially purified P-450 preparations catalyzed NADPH-supported 11 beta-hydroxylation of unconjugated and sulphoconjugated deoxycorticosterone. In presence of Triton X-100 the partially purified cytochrome P-45011 beta had a Stoke's radius of 4.5 nm, a sedimentation coefficient of 3.1 S and a partial specific volume of about 0.85 cm3/g. These results indicate that the cytochrome P-45011 beta-Triton X-100 complex has a molecular weight of about 100 000 and that P-45011 beta bound about 1.1 g of Triton X-100 per g of protein. The P-45011 beta-Triton X-100 complex was catalytically active in hydroxylation reactions supported by NADPH or the hydroxylating agent ortho-nitroiodosobenzene, suggesting that the monomer of cytochrome P-45011 beta is an active form of the protein.     

Solubilization of proteins from bovine brain coated vesicles by protein perturbants and Triton X-100

To identify integral and peripheral membrane proteins, highly purified coated vesicles from bovine brain were exposed to solutions of various pH, ionic strength, and concentrations of the nonionic detergent Triton X-100. At pH 10.0 or above most major proteins were liberated, but four minor polypeptides sedimented with the vesicles. From quantitative analysis of phospholipids in the pellet and extract, we determined that at a pH of up to 12 all phospholipids could be recovered in the pellet. Electron microscopic examination of coated vesicles at pH 12.0 showed all vesicles devoid of coat structures. Treatment with high ionic strength solutions (0-1.0 M KCl) at pH 6.5-8.5 also liberated all major proteins, except tubulin, which remained sedimentable. The addition of Triton X-100 to coated vesicles or to stripped vesicles from which 90% of the clathrin had been removed resulted in the release of four distinct polypeptides of approximate Mr 38,000, 29,000, 24,000 and 10,000. The 38,000-D polypeptide (pK approximately 5.0), which represents approximately 50% of the protein liberated by Triton X-100, appears to be a glycoprotein on the basis of its reaction with periodic acid-Schiff reagent. Extraction of 90% of the clathrin followed by extraction of 90% of the phospholipids with Triton X-100 produced a protein residue that remained sedimentable and consisted of structures that appeared to be shrunken stripped vesicles. Together our data indicate that most of the major polypeptides of brain coated vesicles behave as peripheral membrane proteins and at least four polypeptides behave as integral membrane proteins. By use of a monoclonal antibody, we have identified one of these polypeptides (38,000 mol wt) as a marker for a subpopulation of calf brain coated vesicles.     

Temperature-dependent oligomerization of hsp85 in vitro

The failure of conventional subcellular fractionation methods to identify interactions between the bulk of hsp85 and other cellular structures suggested that critical stress protein interactions might be detectable only at elevated temperatures. This was confirmed by showing that incorporation of hsp85 and grp95 into sedimentable complexes in Triton X-100 extracts of L929 cells increased progressively over the 30 degrees C-43 degrees C temperature range. Whereas several other proteins, including hsp110 and hsp69, became sedimentable under these conditions, this effect required temperatures of approximately 43 degrees C and was only partially detergent-dependent. In contrast, hsp85 became sedimentable at temperatures as low as 33 degrees C, and this effect was highly detergent-dependent. Temperature-dependent conversion of purified hsp85 to a sedimentable form was shown to result from limited oligomerization of the protein, which occurred in the presence of detergent. Since the detergent requirement could be met by a variety of compounds, including sphingosine, these findings suggest that hsp85 oligomerization may occur when intact cells are exposed to elevated temperature.     

The active form of cytochrome P-45011beta from adrenal cortex mitochondria.

Cytochrome P-45011beta has been solubilized and partially purified from bovine adrenal cortex mitochondria by means of chromatography on Octyl-Sepharose CL-4B or DEAE-Sepharose CL-6B. The partially purified P-450 preparations were about 90% pure as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but had a low specific content of P-450 (between 1 and 2 nmol of P-450 per mg of protein). In the presence of purified preparations of adrenodoxin reductase and adrenodoxin, the partially purified P-450 preparations catalyzed NADPH-supported 11beta-hydroxylation of unconjugated and sulfoconjugated deoxycorticosterone. In the reconstituted system the hydroxylation of deoxycorticosterone sulfate proceeded at a much higher rate than in intact mitochondria, indicating that in the former case interactions between the hydrophilic substrate and P-450 were facilitated. In the presence of Triton X-100 the partially purified cytochrome P-45011beta had a Stokes radius of 4.5 nm, a sedimentation coefficient of 3.1 S, and a partial specific volume of about 0.85 cm3/g. These results indicate that the cytochrome P-45011beta . Triton X-100 complex had a molecular weight of about 100,000 and that P-45011beta bound about 1.1 g of Triton X-100 per g of protein. The P-45011beta . Triton X-100 complex was catalytically active in hydroxylation reactions supported by NADPH or the hydroxylating agent ortho-nitroiodosobenzene, suggesting that the monomer of cytochrome P-45011beta is the active form of the protein.     

Solubilization of thyroid peroxidase by nonionic detergents.

We have examined the ability of nonionic detergents to solubilize thyroid peroxidase from a porcine thyroid particulate fraction, as measured by the release of peroxidase activity into the supernatant fraction after centrifugation at 105,000 X g for 1 hour and the retardation of the supernatant peroxidase of Sepharose 6B. The parameters of peroxidase solubilization by Triton X-100 have been investigated in detail. Under optimum conditions, 60 to 95% of the thryoid peroxidase and about 50% of the total protein is released into the 105,000 X g, 1-hour supernatant. Under the optimum conditions established with Triton X-100, a series of Brij detergents of different chemical structure were equally effective in releasing peroxidase and protein. The protein patterns of the supernatants obtained with these detergents were similar on sodium dodecyl sulfate-polyacrylamide electrophoresis gels, suggesting that the detergents studied release similar membrane proteins. The Triton X-100 and Brij 58 supernatants were chromatographed separately on Sepharose 6B equilibrated with 0.1% Triton X-100 or Brij 58, respectively. In both cases, 75 to 80% of the peroxidase activity was retarded, thereby indicating that the nonionic detergents effect solubilization of the peroxidase rather than dispersal of nonsedimentable membrane fragments. These studies report the first successful solubilization of thyroid peroxidase by nonionic detergents. Together with previous evidence from our laboratory, these experiments indicate that thyroid peroxidase is an integral membrane protein.     

A highly thermostable,alkaline CMCase produced by a newly isolated Bacillus sp. VG1

An extracellular, highly thermostable and alkaline CMCase was purified from Bacillus sp. VG1 using ion exchange and gel filtration chromatography. Enzyme was optimally produced in a medium containing 1.0% CMC and 0.5% tryptone. The purified CMCase had a pH optimum of 9–10 and a half life of 12 min even at 100 °C. The enzyme activity was reduced by Hg²⁺ and stimulated by Co²⁺, Na⁺ and K⁺. Various detergents and proteinases moderately inhibited the CMCase activity. The molecular weight studies showed a single band on SDS–PAGE.     

Effects of detergents on the lecithin:cholesterol acyltransferase reaction

This paper describes the effect of an ionic (sodium dodecyl sulfate; SDS) and a nonionic detergent (Triton X-100) on the substrate and enzyme components of the lecithin: cholesterol acyltransferase (LCAT) reaction. When the enzyme sources (purified or partially purified) or the respective substrates [high-density lipoproteins (HDL) or proteoliposomes] were preincubated with detergents, a consistent trend in LCAT activity was only seen when partially purified LCAT was used as the enzyme source. This trend indicated an approximately 25% increase in enzyme activity over the control when 10(-4) M SDS and 2 X 10(-3)% Triton X-100 were present in the preincubation mixtures, respectively. Those observations suggested that, during the preincubations and subsequent assays, the enzyme (in the presence of detergents) was allowed to dissociate from the endogenous substrate and subsequently interact with the exogenous substrate molecules. Additional experiments utilizing molecular-sieve chromatography with whole plasma and partially purified enzyme also showed that dissociation of LCAT/lipoprotein complexes occurred in the presence of detergent. SDS was also shown to enhance the reaction of LCAT in whole plasma with anti-LCAT antibody in an enzyme-linked immunoassay system, indicating that the detergent treatment facilitated the exposure of additional antigenic sites, perhaps via dissociation of the enzyme from plasma lipoproteins.     

Purification and preliminary characterization of mitochondrial complex I (NADH: ubiquinone reductase) from broad bean (Vicia faba L.).

NADH:ubiquinone reductase (EC 1.6.19.3), or complex I, was isolated from broad bean (Vicia faba L.) mitochondria. Osmotic shock and sequential treatment with 0.2% (v/v) Triton X-100 and 0.5% (w/v) [3-cholamidopropyl)dimethylammonio]-1-propanesulfate (CHAPS) removed all other NADH dehydrogenase activities. Complex I was solubilized in the presence of 4% Triton X-100 and then purified by sucrose-gradient centrifugation in the presence of the same detergent. The second purification step was hydroxylapatite chromatography. Substitution of CHAPS for Triton X-100 helped remove contaminants such as ATPase. The high molecular mass complex is composed of at least 26 subunits with molecular masses ranging from 6000 to 75,000 kD. The purified complex I reduced ferricyanide and ubiquinone analogs but not cytochrome c. NADPH could not substitute for NADH as an electron donor. The KM for NADH was 20 microM at the optimum pH of 8.0. The NH2-terminal sequence of several subunits was determined, revealing the ambiguous nature of the 42-kD subunit.     

Characterization and localization of neutral sphingomyelinase in bovine adrenal medulla

Homogenates of bovine adrenal medullae hydrolyzed exogenous sphingomyelin at 4.3 +/- 1.6 nmol X mg-1 X min-1 and 97% of this sphingomyelinase activity was sedimentable at 110,000 g. The sphingomyelinase had a broad pH optimum centered at pH 7. Enzymatic activity was maximal with 80 microM added Mn2+; Mg2+ supported less than half maximal activity and both Ca2+ and EDTA inhibited activity. No activity was detected in the absence of Triton X-100. Response to detergent was biphasic with dose-dependent stimulation from 0.02% to 0.05% Triton X-100 followed by inhibition with increasing concentrations of detergent. Activity in response to detergent was also modulated by protein concentration. Sphingomyelinase activity was associated with a plasma membrane-microsomal fraction. Phosphatidylcholine was not hydrolyzed under optimal conditions for sphingomyelin hydrolysis and a variety of other conditions. Neutral-active sphingomyelinase activity in adrenal medulla was similar in magnitude to that observed in other non-neural bovine tissues. This study demonstrates the presence of a potent neutral-active sphingomyelinase in a plasma membrane-microsomal fraction of bovine adrenal medulla. This enzyme may be involved in membrane fusion and lysis during catecholamine secretion through its ability to alter membrane composition.     

Purification of Phosphatidylinositol Synthetase from Rat Brain by CDP-Diacylglycerol Affinity Chromatography and Properties of the Purified Enzyme

A purification procedure for rat brain phosphatidylinositol synthetase (PI synthetase; CDP-1,2-diacyl-sn-glycerol:myo-inositol 3-phosphatidyltransferase; EC 2.7.8.11) is described. The enzyme was purified 200-250-fold from the homogenate by solubilization with Triton X-100 from microsomal membranes and affinity chromatography on CDP-diacylglycerol-Sepharose. Elution of enzyme activity required the presence of Triton X-100, CDP-diacylglycerol, and either phosphatidylcholine or asolectin. The product that was obtained in 5-10% yield from whole brain and in 70% yield from the microsomal fraction contained three protein bands as determined by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The final preparation contained levels of CDP-diacylglycerol hydrolase and CDP-diacylglycerol: sn-glycero-3-phosphate 3-phosphatidyltransferase activities that were less than 1% of PI synthetase activity. The purified enzyme displayed a pH optimum of 8.5-9.0, required either Mg2+ or Mn2+ and exhibited a Km of 4.6 mM for myo-inositol.     

Solubilization and properties of UDP-D-glucose:N-acetylglucosaminyl pyrophosphorylundecaprenol glucosyltransferase from Bacillus coagulans AHU 1366 membranes

The glucosyltransferase which catalyzes the conversion of GlcNAc-PP-undecaprenol into Glc(beta 1----4)GlcNAc-PP-undecaprenol in the presence of UDP-glucose was solubilized from Bacillus coagulans AHU 1366 membranes by treatment with 0.1% Triton X-100 and partially purified by means of column chromatography on Sephacryl S-300 and DEAE-Sephacel. The final preparation was virtually free from other enzymes involved in the de novo synthesis of teichoic acid. The enzyme had a pH optimum of 6.6-8.0 and a Km value for UDP-glucose of 21 microM. The enzyme required 40 mM MgCl2, 0.6 M KCl, and 0.1% Nonidet P-40 for full activity.     

Biochemical and Electron Microscopic Studies of the Streptomyces reticuli Cellulase (Avicelase) in Its Mycelium-Associated and Extracellular Forms

Streptomyces reticuli is able to grow efficiently with crystalline cellulose (Avicel) as the sole carbon source. Cultivation in the presence of the nonionic detergent Tween 80 at a concentration of 0.1% led to a 10-fold increase in extracellular cellulolytic activity. Under these conditions, one single 82-kDa cellulase (Avicelase) capable of degrading crystalline and soluble cellulose as well as cellodextrins and p-nitrophenylcellobioside was purified to apparent homogeneity by a procedure which consisted of two consecutive anion-exchange chromatographies followed by chromatofocusing. Aggregation, which was a major problem during protein purification, could be avoided by including Triton X-100 at a concentration of 0.1% in every chromatographic step. The Avicelase was identified in extracellular and mycelium-associated forms, the latter of which could be released efficiently by nonionic detergents. In addition, a 42-kDa truncated form retaining cellulolytic activity was identified which had been generated from the 82-kDa enzyme by a protease. Antibodies raised against the mycelium-associated Avicelase reacted with the 42-kDa derivative and the extracellular form. The mycelial association of the enzyme was confirmed by immunofluorescence and immunoelectron microscopies.     

Purification and characterization of membrane-bound phosphatidylinositol kinase from rat brain

A membrane-bound phosphatidylinositol (PI) kinase was purified from rat brain. The enzyme was solubilized with Triton X-100 from salt-washed membrane and purified 11,183-fold, with a final specific activity of 150 nmol/min/mg of protein. Purification steps included several chromatography using Q-Sepharose Fast Flow, cellulose phosphate, Toyopearl HW 55 and Affi-Gel Blue. The purified PI kinase had an estimated molecular weight of 80,000 by gel filtration and 76,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified kinase phosphorylated only PI and did not phosphorylate phosphatidylinositol 4-phosphate or diacylglycerol. Km values for PI and ATP were found to be 115 and 150 microM, respectively. The enzyme required Mg2+ (5-20 mM) or Mn2+ (1-2 mM) for activity, was stimulated by 0.1-1.0% (w/v) Triton X-100, and completely inhibited by 0.05% sodium dodecyl sulfate. The enzyme activity showed a broad pH optimum at around 7.4. The enzyme utilized ATP and not GTP as phosphate donor. Nucleoside triphosphates other than ATP and diphosphates significantly inhibited the kinase activity. However, inhibitory effects of adenosine, cAMP, and quercetin were weak.     

Purification and chemical modification of a phosphatidylinositol kinase from sheep brain.

A membrane-bound phosphatidylinositol (PtdIns) kinase has been purified approximately 9500-fold to apparent homogeneity from sheep brains. The purification procedure involves: solubilisation of the membrane fraction with Triton X-100, ammonium sulphate fractionation and a number of ion-exchange and gel-filtration chromatography steps. The purified enzyme exhibited a final specific activity of 1149 nmol.min-1.mg-1. The molecular mass of the enzyme was estimated to be 55 kDa by SDS/PAGE and 150 +/- 10 kDa by HPLC gel filtration in the presence of Triton X-100. Kinetic measurements have shown that the apparent Km value of PtdIns kinase for the utilisation of PtdIns is 22 microM and for ATP 67 microM. Mg2+ was the most effective divalent cation activator of PtdIns kinase, with maximal enzymatic activity reached at a concentration of 10 mM Mg2+. In addition to adenosine and ADP, the 2'(3')-O-(2,4,6-trinitrophenyl) derivative of ATP was found to be a strong competitive inhibitor of the enzyme, with a Ki of 32 microM. Enzymatic activity was found to be stimulated by Triton X-100 but inhibited by deoxycholate.     

Calcium-Stimulated Proteolysis in Myelin: Evidence for a Ca2+-Activated Neutral Proteinase Associated with Purified Myelin of Rat CNS

Incubation of myelin purified from rat spinal cord with CaCl2 (1-5 mM) in 10-50 mM Tris-HCl buffer at pH 7.6 containing 2 mM dithiothreitol resulted in the loss of both the large and small myelin basic proteins (MBPs), whereas incubation of myelin with Triton X-100 (0.25-0.5%) and 5 mM EGTA in the absence of calcium produced preferential extensive loss of proteolipid protein (PLP) relative to MBP. Inclusion of CaCl2 but not EGTA in the medium containing Triton X-100 enhanced degradation of both PLP and MBPs. The Ca2+-activated neutral proteinase (CANP) activity is inhibited by EGTA (5 mM) and partially inhibited by leupeptin and/or E-64c. CANP is active at pH 5.5-9.0, with the optimum at 7-8. The threshold of Ca2+ activation is approximately 100 microM. The 150K neurofilament protein (NFP) was progressively degraded when incubated with purified myelin in the presence of Ca2+. These results indicate that purified myelin is associated with and/or contains a CANP whose substrates include MBP, PLP, and 150K NFP. The degradation of PLP (trypsin-resistant) in the presence of detergent suggests either release of enzyme from membrane and/or structural alteration in the protein molecule rendering it accessible to proteolysis. The myelin-associated CANP may be important not only in the turnover of myelin proteins but also in myelin breakdown in brain diseases.     

Extraction and detergent/lipid activation of dolichol kinase

The CTP-dependent dolichol kinase from bovine liver microsomes was optimally extracted using either 0.5% sodium deoxycholate or 0.5% Triton X-100 containing 0.5 M NH4Cl. All activity was found in the supernatant fraction following high-speed centrifugation. This fraction was depleted of phospholipid (phospholipid remaining, less than 5% of total) by gel chromatography of the 0.5% deoxycholate extract. This partially purified enzyme was maximally activated 9- or 53-fold over controls in the presence of 0.1% deoxycholate or 0.1% Triton X-100, respectively. Stimulation of the kinase was also observed with mixtures of dimyristoylphosphatidylcholine and deoxycholate. The level of stimulation by these mixtures was up to 20-fold higher than that observed in controls having deoxycholate alone. Dimyristoylphosphatidylcholine alone was not stimulatory. A 1:1 molar ratio of Triton X-100 or deoxycholate to dimyristoylphosphatidylcholine was optimal for enzyme activation. The half-maximum velocity of the dephospholipidated enzyme at 1:1 molar ratio of detergent to dimyristoylphosphatidylcholine was obtained at 150 or 550 microM CTP in the presence of deoxycholate or Triton X-100, respectively. It has been observed, therefore, that dolichol kinase may be extracted from liver microsomes, depleted of endogenous phospholipids and activated by specific molar ratios of detergent to phospholipid.