Subcellular distribution of steryl ester biosynthesis in spinach leaves

Higher steryl ester biosynthetic activities were obtained with Triton X-100-phosphatidylcholine-cholesterol mixed micelles than with Tween 80-phosphatidylcholine-cholesterol mixed micelles when incubated with spinach leaf (Spinacia oleracea L.) acetone powder preparations. The best incorporation of [4-¹⁴C]cholesterol into [4-¹⁴C]cholesteryl ester was obtained with a Triton X-100-phosphatidylcholine-cholesterol (10:1:1, w/w) mixed micelle system. This mixed micelle system, however, required 1,2-dipalmitin and fatty acid-free bovine albumin for optimal activity. The reaction exhibited a diglyceride specificity since the dipalmitin requirement could be replaced with neither 1-monopalmitin nor tripalmitin. Significant amounts of steryl ester biosynthetic activity were detected in the chloroplast (1,000g pellet), mitochondrial (3,000g pellet), and microsomal (20,000g and 88,000g pellet) fractions. Little activity was detected in the water-soluble (88,000g supernatant) fraction. The highest specific activity occurred in the 88,000g pellet. The 88,000g supernatant contained a heatstable, water-soluble substance that was required for optimal steryl ester biosynthesis in all of the pellet fractions. This factor was not lost during extensive dialysis but was destroyed by ashing, indicating that it was large and organic. Silver nitrate thin layer chromatography indicated that 60% of the biosynthesized steryl esters contained saturated fatty acids in the absence of 1,2-dipalmitin and that 83% contained saturated fatty acids in the presence of 1,2-dipalmitin.     

Hydrolysis of neutral glycerides by lipases of rat brain microsomes.

The hydrolysis of monoacylglycerol and diacylglycerol by rat brain microsomes was followed by measuring the release of glycerol and monooleylglycerol from dispersions of water insoluble glyceryl esters of oleic acid. The microsomes showed three lipolytic activities. One activity, optimal at pH 4.8, catalyzed the hydrolysis of diacylglycerol but not monoacylglycerol. Two other lipolytic activities, optimal at pH 8.0-8.6, catalyzed the hydrolysis of both diacylglycerol and monoacylglycerol. The pH 8.0-8.6 activities were sensitive to heat and SH-reagents. Detergents were inhibitory in all cases. Extraction of the microsomes with KCl, KSCN, urea or Triton X-100 did not change the ratio of diacylglycerol hydrolysis at pH 4.8 and 8.0. The results of subcellular fractionation studies showed that there was no significant enrichment of the acid lipase in any fraction.     

Interfacial reaction dynamics and acyl-enzyme mechanism for lipoprotein lipase-catalyzed hydrolysis of lipid p-nitrophenyl esters

The fatty acyl (lipid) p-nitrophenyl esters p-nitrophenyl caprylate, p-nitrophenyl laurate and p-nitrophenyl palmitate that are incorporated at a few mol % into mixed micelles with Triton X-100 are substrates for bovine milk lipoprotein lipase. When the concentration of components of the mixed micelles is approximately equal to or greater than the critical micelle concentration, time courses for lipoprotein lipase-catalyzed hydrolysis of the esters are described by the integrated form of the Michaelis-Menten equation. Least square fitting to the integrated equation therefore allows calculation of the interfacial kinetic parameters Km and Vmax from single runs. The computational methodology used to determine the interfacial kinetic parameters is described in this paper and is used to determine the intrinsic substrate fatty acyl specificity of lipoprotein lipase catalysis, which is reflected in the magnitude of kcat/Km and kcat. The results for interfacial lipoprotein lipase catalysis, along with previously determined kinetic parameters for the water-soluble esters p-nitrophenyl acetate and p-nitrophenyl butyrate, indicate that lipoprotein lipase has highest specificity for the substrates that have fatty acyl chains of intermediate length (i.e. p-nitrophenyl butyrate and p-nitrophenyl caprylate). The fatty acid products do not cause product inhibition during lipoprotein lipase-catalyzed hydrolysis of lipid p-nitrophenyl esters that are contained in Triton X-100 micelles. The effects of the nucleophiles hydroxylamine, hydrazine, and ethylenediamine on Km and Vmax for lipoprotein lipase catalyzed hydrolysis of p-nitrophenyl laurate are consistent with trapping of a lauryl-lipoprotein lipase intermediate. This mechanism is confirmed by analysis of the product lauryl hydroxamate when hydroxylamine is the nucleophile. Hence, lipoprotein lipase-catalyzed hydrolysis of lipid p-nitrophenyl esters that are contained in Triton X-100 micelles occurs via an interfacial acyl-lipoprotein lipase mechanism that is rate-limited by hydrolysis of the acyl-enzyme intermediate.     

Lysophospholipase of Escherichia coli.

A lysophospholipase from Escherichia coli cells was purified about 1,500-fold to near homogeneity by extraction with Tris-HCl buffer, streptomycin treatment, (NH4)2SO4 fractionation, column chromatographies on Sephadex G-200, DEAE-cellulose and hydroxylapatite-cellulose, and polyacrylamide gel electrophoresis. The final preparation had a molecular weight of 39,500 plus or minus 500. The enzyme hydrolyzes 1-acylglycerylphosphorylethanolamine, 2-acylglycerylphosphorylethanoiamine, and 1-acylglycerylphosphorylglycerol, but does not attack diacylphospholipids with long chain fatty acids, such as phosphatidylethanolamine and phosphatidylglycerol. The enzyme does not show any esterase activity against p-nitrophenyl acetate or palmitate. Although it does not hydrolyze triacylglycerol or diacylglycerol, it hydrolyzes 1-acylglycerol at almost the same rate as 1-acyl-sn-glycerol-3-phosphorylethanolamine. Results indicated that the acyl-hydrolyzing activities toward monoacyl-glycerylphosphorylethanolamine and monoacylglycerol belong to the same enzyme. In general, acidic and nonionic detergents inhibited the reaction. This lysophospholipase preparation hydrolyzes the monomolecular and micellar forms of lysophospholipids as well as of monoacylglycerol. The monomolecular and micellar forms of Triton X-100 both inhibited the hydrolyses of lysophospholipids and monoacylglycerol.     

The enzymic deacylation of phospholipids and galactolipids in plants. Purification and properties of a lipolytic acyl-hydrolase from potato tubers

An enzyme preparation that catalyses the deacylation of mono- and di-acyl phospholipids, galactosyl diglycerides, mono- and di-glycerides has been partially purified from potato tubers. The preparation also hydrolyses methyl and p-nitrophenyl esters and acts preferentially on esters of long-chain fatty acids. Triglycerides, wax esters and sterol esters are not hydrolysed. The same enzyme preparation catalyses acyl transfer reactions in the presence of alcohols and also catalyses the synthesis of wax esters from long-chain alcohols and free fatty acids. Gel filtration, DEAE-cellulose chromatography and free-flow electrophoresis failed to achieve any separation of the acyl-hydrolase activities towards different classes of acyl lipids (phosphatidylcholine, monogalactosyl diglyceride, mono-olein, methyl palmitate and p-nitrophenyl palmitate) or any separation of these activities from a major protein component. For each class of lipid the acyl-hydrolase activity was subject to substrate inhibition, was inhibited by relatively high concentrations of di-isopropyl phosphorofluoridate and the pH responses were changed by Triton X-100. The hydrolysis of phosphatidylcholine was stimulated 30-40-fold by Triton X-100. The specific activities of the potato enzyme with galactolipids were at least 70 times higher than those reported for a homogeneous galactolipase enzyme purified from runner bean leaves. The possibility that a single lipolytic acyl-hydrolase enzyme is responsible for the deacylation of several classes of acyl lipid is discussed.     

Translocation of CTP: phosphocholine cytidylyltransferase from cytosol to membranes in HeLa cells: stimulation by fatty acid, fatty alcohol, mono- and diacylglycerol

Addition of oleate, oleyl alcohol, or palmitate to HeLa cell medium resulted in a rapid stimulation of PC synthesis and activation of CTP: phosphocholine cytidylyltransferase. Stimulation was optimal with 0.35 mM oleate, 0.3 mM oleyl alcohol and 5 mM palmitate, or 1 mM palmitate if EGTA were added to the medium. The cytidylyltransferase was activated by translocation of the inactive cytosolic form to membranes. In untreated cells approx. 30% of the total cytidylyltransferase was membrane bound, while in treated cells, 80-90% was membrane associated. Addition of bovine serum albumin (10 mg/ml) to cells previously treated with oleate (0.35 mM) rapidly removed cellular fatty acid, and the membrane-bound cytidylyltransferase activity returned to approx. 30%. Similar results were obtained by extraction of membranes with albumin in vitro. Although 95% of the free fatty acid was extracted, 30-40% of the membrane cytidylyltransferase remained bound. Translocation of cytidylyltransferase between isolated cytosol and microsomal fractions was promoted by addition of oleate, palmitate, oleyl alcohol, and monoolein. Addition of diacylglycerol, lysophosphatidylcholine, lysophosphatidylethanolamine, calcium palmitate, and detergents such as Triton X-100, cholate or Zwittergent did not stimulate translocation of the enzyme. Addition of oleoyl-CoA promoited translocation, however, 40% of it was hydrolyzed releasing free oleic acid. Cytosolic cytidylyltransferase bound to microsomes pre-treated with phospholipase C, which had 7-fold elevated diacylglycerol content. Fatty acid-promoted translocation was blocked by Triton X-100, but not by 1 M KCl. These results suggest that a variety of compounds with differing head group size and charge, and number of hydrocarbon chains can function as translocators, and that hydrophobic rather than ionic interactions mediate the binding of cytidylyltransferase to membranes.     

Characterization of wax-ester hydrolase from roots of white mustard (Sinapis alba L.) seedlings

The activity of wax-ester hydrolase in roots of white mustard (Sinapis alba L.) seedlings is located in a membranous fraction sedimenting at 15000 g. The enzyme which shows a high degree of hydrophobicity was solubilized with a synthetic detergent Triton X-100 and purified about 70-fold by acetone precipitation and gel permeation chromatography on Sepharose 6B. The purified enzyme preparation was active within a broad pH range of 5.8-8.5. Hydrolase activity with hexadecanyl palmitate as the substrate was stimulated by Triton X-100 and dithioerythritol. Of wax esters containing saturated fatty acids C2-C22 and saturated, primary alcohols C2-C24 the highest rate of hydrolysis was found with the esters containing palmitic acid (C16) and tetradecanol (C14). Data presented suggest that wax esters and steryl esters are either hydrolyzed by different specific enzymes or that two enzymes are present of different specificity towards the two substrates.     

Diacylglycerol activation of protein kinase C is modulated by long-chain acyl-CoA

The activity of rat brain protein kinase C, measured in the presence of diacylglycerol, phosphatidylserine and Ca+2, was found to be greatly increased by micromolar amounts of long chain acyl-CoAs, using two different assay systems (lipids added as sonicated dispersion or as mixed micelles with Triton X-100). The potentiation phenomenon required the presence of both diacylglycerol and phosphatidylserine; it was observed at low and saturating concentrations of these effectors, and it was inhibited at high, non physiological Ca+2 concentrations. Under similar conditions, fatty acids alone or coenzyme A were ineffective. The data strongly suggest that acyl-CoAs at the intracellular concentration levels, are important in the modulation of protein kinase C, after activation of the enzyme by the phospholipase C/phosphatidylinositol pathway.     

Endurance training increases FFA oxidation and reduces triacylglycerol utilization in contracting rat soleus

We examined the effects of 8 wk of intense endurance training on free fatty acid (FFA) transporters and metabolism in resting and contracting soleus muscle using pulse-chase procedures. Endurance training increased maximal citrate synthase activity in red muscles (+54 to +91%; P 相似文献   

Phosphatidylglycerol synthesis in spinach chloroplasts: characterization of the newly synthesized molecule

Intact chloroplasts from spinach (Spinacia oleracea L., hybrid 424) readily incorporate [¹⁴C]glycerol-3-phosphate and [¹⁴C]acetate into diacylglycerol, monoacylglycerol, diacylglycrol, free fatty acids (only when acetate is the precursor), phosphatidic acid, phosphatidylcholine, and most notably phosphatidylglycerol. The fraction of phosphatidylglycerol synthesized is greatly increased by the presence of manganese chloride in the reaction mixture. Glycerol-3-phosphate-labeled phosphatidylglycerol is equally labeled in the two glycerol moieties of the molecule. Acetate-labeled phosphatidylglycerol is equally labeled in both acyl groups. Position one contains primarily oleate, linoleate and small amounts of palmitate. Position two contains primarily palmitate. No radioactive trans-Δ³-hexadecenoate was detected. The labeling patterns indicate that the radioactive phosphatidylglycerol is the product of de novo chloroplast lipid biosynthesis and furthermore, phosphatidylglycerol may be a substrate for fatty acid desaturation.     

Kinetics of acylglycerol sequential hydrolysis by human milk bile salt activated lipase and effect of taurocholate as fatty acid acceptor

The simplest reaction scheme for the conversion of trioleoylglycerol to glycerol catalyzed by human milk bile salt activated lipase can be described by consecutive first-order reactions: triacylglycerol k1----diacylglycerol k2----monoacylglycerol k3----glycerol. In these equations, k1, k2, and k3 represent the pseudo-first-order rate constants for the indicated reactions. The results from this study show that although the relative ratio of k2/k1 or k3/k1 may change somewhat, depending on the reaction conditions, the enzyme has a reactivity with the order of dioleoylglycerol greater than trioleoylglycerol greater than monooleoylglycerol. The incomplete equilibration of the intermediary diacylglycerol and monoacylglycerol with the bulk of the substrate during sequential lipolysis of triacylglycerol provides a means for their efficient lipolysis and minimizes the effect of partial acylglycerol as competitive substrates for intact triacylglycerol lipolysis. Taurocholate functions both as an activator of the enzyme and also as fatty acid acceptor to relieve product inhibition. In the presence of sufficient taurocholate, bovine serum albumin is no longer required as a fatty acid acceptor for the in vitro lipolysis.     

Effects of ethanol alone or after pretreatment with 20% ethanol on phospholipid metabolism in rat gastric mucosa

Changes in phospholipid metabolism in gastric mucosa caused by instillation of absolute ethanol (a cell-damaging agent) into the stomach of rats and the effects of pretreatment with 20% ethanol (a mild irritant) were investigated by using radioisotope-labeled fatty acids and glycerol. The labeled precursors were incorporated mainly into phosphatidylcholine and triacylglycerol, and also to lesser extents into phosphatidylethanolamine and phosphatidylinositol + phosphatidylserine. The instillation of absolute ethanol reduced the incorporation of fatty acids and glycerol into phospholipids within 15 min, indicating the inhibition by ethanol of de novo synthesis of phospholipids. Pretreatment with 20% ethanol caused the incorporation of fatty acids into phospholipids to be maintained after absolute ethanol instillation. These results suggest that the pretreatment with 20% ethanol may protect the cellular synthetic activity of phospholipids against damage by absolute ethanol. The incorporation of fatty acids into the free fatty acid fraction, monoacylglycerol and diacylglycerol was increased by absolute ethanol instillation, suggesting damage to the blood vessels of the gastric mucosa, and these changes were inhibited to some extent by the pretreatment with 20% ethanol.     

Triacylglycerol synthesis in cultured rat hepatocytes. The rate-limiting role of diacylglycerol acyltransferase

The limiting role of diacylglycerol acyltransferase with respect to triacylglycerol synthesis in cultured rat hepatocytes was evaluated by following the inhibition of the overall synthetic flux by 2-bromooctanoate acting as an inhibitor of the diacylglycerol acyltransferase step. The flux-control coefficient of diacylglycerol acyltransferase in intact cultured hepatocytes amounted to 0.76 in the presence of saturating glycerol and either palmitate or oleate as the fatty acyl substrates. The flux-control coefficient of diacylglycerol acyltransferase in lysolecithin-permeabilized cultured hepatocytes amounted to 0.80 and 0.99 in the presence of saturating glycerol 3-phosphate and either palmitate or oleate as the fatty acyl substrate, respectively. Hence, triacylglycerol synthesis in liver cells under the experimental conditions employed is rate-limited by the diacylglycerol acyltransferase.     

Synthesis of novel lipids in Saccharomyces cerevisiae by heterologous expression of an unspecific bacterial acyltransferase

The bifunctional wax ester synthase/acyl-coenzyme A:diacylglycerol acyltransferase (WS/DGAT) is the key enzyme in storage lipid accumulation in the gram-negative bacterium Acinetobacter calcoaceticus ADP1, mediating wax ester, and to a lesser extent, triacylglycerol (TAG) biosynthesis. Saccharomyces cerevisiae accumulates TAGs and steryl esters as storage lipids. Four genes encoding a DGAT (Dga1p), a phospholipid:diacylglycerol acyltransferase (Lro1p) and two acyl-coenzyme A:sterol acyltransferases (ASATs) (Are1p and Are2p) are involved in the final esterification steps in TAG and steryl ester biosynthesis in this yeast. In the quadruple mutant strain S. cerevisiae H1246, the disruption of DGA1, LRO1, ARE1, and ARE2 leads to an inability to synthesize storage lipids. Heterologous expression of WS/DGAT from A. calcoaceticus ADP1 in S. cerevisiae H1246 restored TAG but not steryl ester biosynthesis, although high levels of ASAT activity could be demonstrated for WS/DGAT expressed in Escherichia coli XL1-Blue in radiometric in vitro assays with cholesterol and ergosterol as substrates. In addition to TAG synthesis, heterologous expression of WS/DGAT in S. cerevisiae H1246 resulted also in the accumulation of fatty acid ethyl esters as well as fatty acid isoamyl esters. In vitro studies confirmed that WS/DGAT is capable of utilizing a broad range of alcohols as substrates comprising long-chain fatty alcohols like hexadecanol as well as short-chain alcohols like ethanol or isoamyl alcohol. This study demonstrated the highly unspecific acyltransferase activity of WS/DGAT from A. calcoaceticus ADP1, indicating the broad biocatalytic potential of this enzyme for biotechnological production of a large variety of lipids in vivo in prokaryotic as well as eukaryotic expression hosts.     

De novo biosynthesis of biodiesel by Escherichia coli in optimized fed-batch cultivation

Biodiesel is a renewable alternative to petroleum diesel fuel that can contribute to carbon dioxide emission reduction and energy supply. Biodiesel is composed of fatty acid alkyl esters, including fatty acid methyl esters (FAMEs) and fatty acid ethyl esters (FAEEs), and is currently produced through the transesterification reaction of methanol (or ethanol) and triacylglycerols (TAGs). TAGs are mainly obtained from oilseed plants and microalgae. A sustainable supply of TAGs is a major bottleneck for current biodiesel production. Here we report the de novo biosynthesis of FAEEs from glucose, which can be derived from lignocellulosic biomass, in genetically engineered Escherichia coli by introduction of the ethanol-producing pathway from Zymomonas mobilis, genetic manipulation to increase the pool of fatty acyl-CoA, and heterologous expression of acyl-coenzyme A: diacylglycerol acyltransferase from Acinetobacter baylyi. An optimized fed-batch microbial fermentation of the modified E. coli strain yielded a titer of 922 mg L(-1) FAEEs that consisted primarily of ethyl palmitate, -oleate, -myristate and -palmitoleate.     

UDP-Glucose: Sterol Glucosyltransferase and a Steryl Glucoside Acyltransferase Activity in Amyloplast Membranes from Potato Tubers

Envelope membranes were isolated from potato tuber amyloplastby a discontinuous sucrose density gradient and high speed centrifugation.These membranes catalyzed the transfer of [¹⁴C]glucose fromUDP-[¹⁴C]glucose to endogenous sterol acceptors and, in turn,catalyzed the esterification of steryl glucosides with fattyacids from an endogenous acyl donor. The synthesis of sterylglucosides was stimulated in the presence of Triton. X-100,while formation of acyl steryl glucosides was inhibited by thedetergent. However, in the presence of an added sterol acceptorand Triton X-100, the inhibition of acyl steryl glucoside synthesiswas overcome by the addition of phosphatidylethanolamine. Theenzyme involved in steryl glucoside formation was solubilizedby treatment of the envelope membranes with 0.3% Triton X-100.The solubilized enzyme had an almost absolute requirement forsterol acceptors. Key words: Solanum tuberosum, Sterol glucosylation, Steryl glucoside acylation, Amyloplast membrane     

Glycosyl-phosphatidylinositol anchored acetylcholinesterase as substrate for phosphatidylinositol-specific phospholipase C from Bacillus cereus.

We investigated the enzymatic properties of phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus towards glycosyl-phosphatidylinositol anchored acetylcholinesterase (AChE) from bovine erythrocytes and Torpedo electric organ as substrate. The conversion of membrane from AChE to soluble AChE by PI-PLC depended on the presence of a detergent and of phosphatidylcholine. In presence of mixed micelles containing Triton X-100 (0.05%) and phosphatidylcholine (0.5 mg/ml) the rate of AChE conversion was about 3 times higher than in presence of Triton X-100 alone. Furthermore, inhibition of PI-PLC occurring at Triton X-100 concentrations higher than 0.01% could be prevented by addition of phosphatidylcholine. Ca2+, Mg2+ and sodium chloride had no effect on PI-PLC activity in presence of phosphatidylcholine and Triton X-100, whereas in presence of Triton X-100 alone sodium chloride largely increased the rate of AChE conversion. Determination of kinetic parameters with three different substrates gave Km-values of 7 microM, 17 microM and 2 mM and Vmax-values of 0.095 microM.min-1, 0.325 microM.min-1 and 56 microM.min-1 for Torpedo AChE, bovine erythrocyte AChE and phosphatidylinositol, respectively. The low Km-values for both forms of AChE indicated that PI-PLC not only recognized the phosphatidylinositol moiety of the anchor but also other components thereof.     

Diacylglycerol metabolism in neonatal rat liver: characterization of cytosolic diacylglycerol lipase activity and its activation by monoalkylglycerols.

Diacylglycerol lipase (glycerol ester hydrolase, EC 3.1.1.3) activities were investigated in subcellular fractions from neonatal and adult rat liver in order to determine whether one or more different lipases might provide the substrate for the developmentally expressed, activity monoacylglycerol acyltransferase. The assay for diacylglycerol lipase examined the hydrolysis of sn-1-stearoyl,2- [14C]oleoylglycerol to labeled monoacylglycerol and fatty acid. Highest specific activities were found in lysosomes (pH 4.8) and cytosol and microsomes (pH 8). The specific activity from plasma membrane from adult liver was 5.8-fold higher than the corresponding activity in the neonate. In other fractions, however, no developmental differences were observed in activity or distribution. In both lysosomes and cytosol, 75 to 90% of the labeled product was monoacylglycerol, suggesting that these fractions contained relatively little monoacylglycerol lipase activity. In contrast, 80% of the labeled product from microsomes was fatty acid, suggesting the presence of monoacylglycerol lipase in this fraction. Analysis of the reaction products strongly suggested that the lysosomal and cytosolic diacylglycerol lipase activities hydrolyzed the acyl-group at the sn-1 position. The effects of serum and NaCl on diacylglycerol lipase from each of the subcellular fractions differed from those effects routinely observed on lipoprotein lipase and hepatic lipase, suggesting that the hepatic diacylglycerol lipase activities were not second functions of these triacylglycerol lipases. Cytosolic diacylglycerol lipase activity from neonatal liver and adult liver was characterized. The apparent Km for 1-stearoyl,2-oleoylglycerol was 115 microM. There was no preference for a diacylglycerol with arachidonate in the sn-2 position. Bovine serum albumin stimulated the activity, whereas dithiothreitol, N-ethylmaleimide, and ATP inhibited the activity. Both sn-1(3)- and 2-monooleylglycerol ethers stimulated cytosolic diacylglycerol lipase activity 2-3-fold. The corresponding amide analogs stimulated 28 to 85%, monooleoylglycerol itself had little effect, and 1-alkyl- or 1-acyl-lysophosphatidylcholine inhibited the activity. These data provide the first characterization of hepatic subcellular lipase activities from neonatal and adult rat liver and suggest that independent diacylglycerol and monoacylglycerol lipase activities are present in microsomal membranes and that the microsomal and cytosolic diacylglycerol lipase activities may describe an ambipathic enzyme. The data also suggest possible cellular regulation by monoalkylglycerols.     

Diacylglycerol kinase from suspension cultured plant cells : purification and properties

Diacylglycerol kinase (ATP:1,2-diacylglycerol 3-phosphotransferase, EC 2.7.1.107) from suspension-cultured Catharanthus roseus cells was extracted from a membrane fraction with 0.6% Triton X-100 and 150 millimolar NaCl and was purified about 900-fold by DEAE-cellulose, blue Sepharose, gel permeation, and phenyl-Sepharose chromatography. The enzyme is obviously membrane bound as activity in the cytosol could not be detected. In the presence of detergents such as Triton X-100 (3-[3-cholamidopropyl]dimethylamino)-1-propanesulfonate (Chaps), or deoxycholate, a molecular weight of about 250,000 was determined by gel filtration. In glycerol density gradients, the enzyme sedimented slightly more slowly than bovine serum albumin, indicating a molecular weight of less than 68,000. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis enzyme activity could be assigned to a protein of 51,000 daltons. As found previously for bacterial and animal diacylglycerol kinases, the purified enzyme was completely devoid of activity without the addition of phospholipids or deoxycholate. Cardiolipin was found to be most effective, whereas higher amounts of detergent were inhibitory. The enzyme needs divalent cations for activity, with Mg²⁺ ions being the most effective. Apparent K_m values for ATP and diacylglycerol were determined as 100 and 250 micromolar, respectively.     

How to prevent losses of protein by adsorption to glass and plastic

An improved procedure for reducing the loss of protein by adsorption to glass or plastic surfaces is reported. For working with proteins at the microgram level, the solvent is modified by adding glycerol (50% final concentration) or Triton X-100 (0.2 mM final concentration). Coating the plastic or glass surfaces with proteins such as bovine serum albumin or other materials is not as effective; adding proteins such as bovine serum albumin to the solvent is counterproductive.