Ca stimulated neutral lipase activity in castor bean lipid bodies

The membranes of lipid bodies from the endosperm of seeds of Ricinus communis have long been known to contain an acid lipase (triacylglycerol acyl hydrolase, EC 3.1.1.3). The means by which fat hydrolysis is initiated and controlled in the endosperm of the young seedling are not yet understood, although it is generally assumed that the acid lipase is the enzyme responsible for the conversion of stored triacylglycerols to fatty acids and glycerol. However, the enzyme from seeds is not an effective catalyst at cytoplasmic pH since it has a pH optimum at 4.5 and is virtually inactive above pH 6.0. The results described in this paper show that during early growth of castor seeds the lipid bodies acquire a lipase which is active at neutral pH values. The lipase is absent from dry seeds, appears at day 3, and increases rapidly in activity until day 5. The pattern of appearance of the lipase mirrors that of other enzymes involved in the conversion of fat to sugar. The lipase is stimulated 40-fold by 30 micromolar free Ca²⁺ and the activity at pH 7.0 to 7.5 adequately accounts for the known rate of triacylglycerol hydrolysis in vivo.     

Lipase in the Lipid Bodies of Corn Scutella during Seedling Growth

In the scutella of corn (Zea mays), lipase activity is absent in ungerminated seeds and increases during seedling growth. At the peak stage of lipolysis, about 50% of the lipase activity is recovered in the lipid body fraction after flotation centrifugation. The lipase is tightly bound to the lipid bodies, and resists solubilization by repeated washing with buffers or NaCl solutions. Isolated lipid bodies undergo autolysis of internal triacylglycerols, resulting in the release of fatty acids. After the triacylglycerols in isolated lipid bodies have been extracted with diethyl ether, the lipase is recovered in the membrane fraction. The lipase has an optimal activity at pH 7.5 in the autolysis of lipid bodies, or on trilinolein or N-methylindoxylmyristate. Of the various acylglycerols examined, the enzyme is active only on acylglycerols of linoleic and oleic acids which are the major fatty acid constituents of corn oil. The activity is not greatly affected by NaCl, CaCl₂, or pretreatment of the enzyme with p-chloromercuribenzoate or mersalyl, and detergents abolish the activity. The enzyme hydrolyzes trilinolein completely to fatty acids; during the course of reaction, there is little accumulation of di- or mono-linolein.     

Substrate specificity of potato lipoxygenase

The substrate specificity of potato lipoxygenase was examined using a partially purified enzyme preparation from tubers of a potato variety with low lipolytic acyl hydrolase activity. Potato lipoxygenase is fully active only on free linoleic acid or linolenic acid, and only acts directly on more complex glyceride moieties in the absence of any significant endogenous lipolytic acyl hydrolase activity.     

Involvement of glyoxysomal lipase in the hydrolysis of storage triacylglycerols in the cotyledons of soybean seedlings

The total cotyledon extract of soybean (Glycine max [L.] Merr. var. Coker 136) seedlings underwent lipolysis as measured by the release of fatty acids. The highest lipolytic activity occurred at pH 9. This lipolytic activity was absent in the dry seeds and increased after germination concomitant with the decrease in total lipids. Using spherosomes (lipid bodies) isolated from the cotyledons during the peak stage of lipolysis (5-7 days) as substrates, about 40% of the lipase activity was found in the glyoxysomes after organelle breakage had been accounted for; the remaining activity was distributed among other subcellular fractions but none was found in the spherosomal fraction. The glyoxysomal lipase had maximal activity at pH 9, and catalyzed the hydrolysis of tri-, di-, and monoacylglycerols of linoleic acid, the most abundant fatty acid in soybean. The spherosomes contained a neutral lipase that could hydrolyze monolinolein and N-methylindoxylmyristate, but not trilinolein. This spherosomal lipase activity dropped off rapidly during early seedling growth, preceding lipolysis. Spherosomes isolated from either dry or germinated seeds did not possess lipolytic activity, and spherosomes from germinated seeds but not from dry seeds could serve as substrates for the glyoxysomal lipase. It is concluded that the glyoxysomal lipase is the enzyme catalyzing the initial hydrolysis of storage triacylglycerols.     

Inhibition of Neutral Lipase from Castor Bean Lipid Bodies by Coenzyme A (CoA) and Oleoyl-CoA

The neutral lipase (EC 3.1.1.3) in lipid body membranes isolated from the endosperm of 4 day old castor (Ricinus communis L.) seedlings catalyzes the hydrolysis of [¹⁴C]trioleoylglycerol, releasing [¹⁴C]oleic acid for up to 4 hours. However, the addition of Mg-ATP and coenzyme A (CoA), which are present in the cytoplasm of plant cells, caused a progressive inhibition of the neutral lipase such that after 15 minutes, release of [¹⁴C]oleic acid was almost undetectable. A fatty acyl CoA synthetase was found in the lipid body membrane which converts [¹⁴C]oleic acid produced from the lipase reaction to [¹⁴C]oleoyl-CoA under these conditions. The concentration of free oleoyl-CoA in the reaction mixture when the lipase was inhibited by 50% was calculated to be about 21 micromolar. It was found that a mixture of exogenously added oleoyl-CoA and CoA was most effective in causing lipase inhibition. Little inhibition of lipase was detected in the presence of CoA alone. It is possible that this effect is important In vivo in coordinating lipase activity with fatty acid oxidation.     

Does Gibberellic Acid Induce the Transfer of Lipase from Protein Bodies to Lipid Bodies in Barley Aleurone Cells?

We have examined the effect of gibberellic acid (GA₃) on the distribution of the enzyme responsible for mobilizing storage triacylglycerol in aleurone cells of Hordeum vulgare L. cv Himalaya. Using cellular fractionation techniques, we find that, in cells that have not been exposed to hormone, neutral lipase activity is principally associated with a pellet containing the membranes of protein bodies. If the cells are exposed to GA₃ for at least 1 hour, the majority of the lipase activity becomes associated with the lipid body fraction. The nature of the in vivo association between lipid bodies and protein bodies was examined using ultrarapid freezing followed by freeze-fracture electron microscopy. Our analysis indicates that the phospholipid monolayer surrounding the lipid body is directly continuous with the outer leaflet of the bilayer surrounding the protein body. Based on our data, we propose that lipase can be transferred from protein bodies (storage form) to lipid bodies (active form) by lateral diffusion within the plane of the fused phospholipid monolayer, and that the transfer can be controlled by gibberellic acid by an unknown mechanism.     

Studies on catalytic properties of purified high molecular weight pancreatic lipase.

The properties of the 500-fold purified high-molecular-weight lipase have been studied. The rate of hydrolysis of the triglycerides decreases with increasing fatty acid chain length. The lipolytic activity also increases with increase in unsaturation in the fatty acyl moiety. Diglycerides are hydrolyzed at more than twice the rate for triglycerides while monoglycerides are not hydrolyzed. Methyl esters are generally hydrolyzed at a higher rate which increases with increasing chain length of the fatty acid but the enzyme does not act on phospholipids. Emulsifying agents such as Tween 20, gum arabic, and albumin increase the rate of hydrolysis. Metal ions such as Hg2+, Zn2+, Cu2+, and Fe2+ strongly inhibit the lipolytic activity of the high-molecular-weight lipase while Ca2+ or Mg2+ by themselves show no stimulating effect. Treatment of the high-molecular-weight lipase with P-chloromercurybenzoate inhibits hydrolytic activity by 70% while iodoacetic acid has no effect.     

Rice bran lipase: extraction, activity, and stability

A simple procedure for the extraction of the lipolytic activity from rice bran has been developed. Various conditions of extraction have been optimized so as to obtain maximum yield of the lipase. It was found that high enzyme activity could be obtained by first defatting the rice bran to remove the lipid component. This was followed by five cycles of aqueous extraction (potassium phosphate buffer, 50 mM and pH 7, containing 0.5 mM of CaCl(2)). The stability of the rice bran lipase under storage and operative conditions was investigated. Further, the influence of glycerol as a stabilizer has been assessed. It was found that further purification using micro- and ultrafiltration yielded an enzyme preparation with higher activity and specific activity and better stability.     

Wax ester formation catalysed by isoenzymes of lipolytic acyl hydrolase

Wax ester formation by esterification of a long chain fatty acid (palmitic acid) with a long chain fatty alcohol (octadecanol) was enzymically catalysed by acetone dried powder preparations of potato tubers. The enzyme responsible for wax ester formation had multiple isoenzymic forms and was identical with lipolytic acyl hydrolase, a lipid deacylating enzyme. Tubers from different varietiees of potato (Solanum tuberosum) demonstrated markedly different levels of activity and electrophoretic patterns for both wax ester formation and lipid deacylation.     

New thermophilic and thermostable esterase with sequence similarity to the hormone-sensitive lipase family, cloned from a metagenomic library

A gene coding for a thermostable esterase was isolated by functional screening of Escherichia coli cells that had been transformed with fosmid environmental DNA libraries constructed with metagenomes from thermal environmental samples. The gene conferring esterase activity on E. coli grown on tributyrin agar was composed of 936 bp, corresponding to 311 amino acid residues with a molecular mass of 34 kDa. The enzyme showed significant amino acid similarity (64%) to the enzyme from a hyperthermophilic archaeon, Pyrobaculum calidifontis. An amino acid sequence comparison with other esterases and lipases revealed that the enzyme should be classified as a new member of the hormone-sensitive lipase family. The recombinant esterase that was overexpressed and purified from E. coli was active above 30 degrees C up to 95 degrees C and had a high thermal stability. It displayed a high degree of activity in a pH range of 5.5 to 7.5, with an optimal pH of approximately 6.0. The best substrate for the enzyme among the p-nitrophenyl esters (C(4) to C(16)) examined was p-nitrophenyl caproate (C(6)), and no lipolytic activity was observed with esters containing an acyl chain length of longer than 10 carbon atoms, indicating that the enzyme is an esterase and not a lipase.     

Identification of a putative triacylglycerol lipase from papaya latex by functional proteomics

Latex from Caricaceae has been known since 1925 to contain strong lipase activity. However, attempts to purify and identify the enzyme were not successful, mainly because of the lack of solubility of the enzyme. Here, we describe the characterization of lipase activity of the latex of Vasconcellea heilbornii and the identification of a putative homologous lipase from Carica papaya. Triacylglycerol lipase activity was enriched 74-fold from crude latex of Vasconcellea heilbornii to a specific activity (SA) of 57 μmol·min(-1)·mg(-1) on long-chain triacylglycerol (olive oil). The extract was also active on trioctanoin (SA = 655 μmol·min(-1)·mg(-1) ), tributyrin (SA = 1107 μmol·min(-1)·mg(-1) ) and phosphatidylcholine (SA = 923 μmol·min(-1)·mg(-1) ). The optimum pH ranged from 8.0 to 9.0. The protein content of the insoluble fraction of latex was analyzed by electrophoresis followed by mass spectrometry, and 28 different proteins were identified. The protein fraction was incubated with the lipase inhibitor [(14) C]tetrahydrolipstatin, and a 45 kDa protein radiolabeled by the inhibitor was identified as being a putative lipase. A C. papaya cDNA encoding a 55 kDa protein was further cloned, and its deduced sequence had 83.7% similarity with peptides from the 45 kDa protein, with a coverage of 25.6%. The protein encoded by this cDNA had 35% sequence identity and 51% similarity to castor bean acid lipase, suggesting that it is the lipase responsible for the important lipolytic activities detected in papaya latex.     

Purification and characterization of a cold-active lipase from Pichia lynferdii Y-7723: pH-dependant activity deviation

Lipases with abnormal functionalities such as high thermostability and optimal activity at extreme conditions gain special attentions because of their applicability in the restricted reaction conditions. In particular, coldactive lipases have gained special attentions in various industrial fields such as washer detergent, pharmaceutical catalyst, and production of structured lipid. However, production of cold-active lipase is mostly found from psychrophilic microorganisms. Recently we found a novel cold-active lipase from Pichia lynferdii Y-7723 which is mesophilic yeast strain. In this study, we purified the cold active lipase and the enzyme was further characterized in several parameters. The enzyme was purified with 33 purification fold using chromatographic techniques and the purified lipase represented maximum lipolytic activity at 15°C and the maximum activity was highly dependent on pH.     

Phospholipase,galactolipase and acyl transferase activities of a lipolytic enzyme from potato

Characterization of reaction products showed that an enzyme (lipolytic acyl hydrolase) isolated from potato tubers could act on endogenous substrates as a galactolipase (E.C. 3.1.1.26), lysophospholipase (E.C. 3.1.1.5) or a ‘phospholipase B’ but not as a lipase (E.C. 3.1.1.3). The affinity of the enzyme for methanol as acyl acceptor (acyl transferase activity) was higher than its affinity for water (acyl hydrolase activity). The nomenclature of acyl hydrolases in plants is discussed.     

Lysosomal triacylglycerol lipase activity in L6 myoblasts and its changes on differentiation.

L6 myoblasts, before fusion, accumulate large stores of neutral lipid when cultured in medium supplemented with fatty acid. Upon fusion to terminally differentiated myotubes, a noticeable decrease in these neutral-lipid stores was observed. Triacylglycerol lipase activity was examined in L6 myoblasts at various stages of cell differentiation to assess a possible role for this enzyme in the above phenomenon. In this first study to demonstrate lipolytic activity in cultured muscle cells, the activity was found to be totally dependent on the presence of a detergent, either Cutscum or Triton X-100, during homogenization. The inhibition by many thiol-specific reagents [N-ethylmaleimide, p-chloromercuribenzoate, iodoacetate, 5,5'-dithiobis-(2-nitrobenzoic acid)] suggest that a thiol group is at or near the active site. The observed acidic pH optimum (5.5-6.0), the acute inhibition by chlorpromazine (a lysosomal lipase inhibitor) and the distribution of lipolytic activity upon cell fractionation (which co-sediments with acid phosphatase, a lysosomal marker enzyme) suggest that the lipase may be of lysosomal origin. Under the optimal conditions described, the triacylglycerol lipase activity of L6 myoblasts was determined to be 2.9 +/- 0.4 nmol of oleic acid released/min per mg of DNA. This activity increased 3-fold, to 9.0 +/- 1.6 nmol/min per mg, in the myotube phase. This increase in lipolytic activity may be responsible for the observed decrease in neutral-lipid stores of differentiating myoblasts.     

Gluconeogenesis from storage wax in the cotyledons of jojoba seedlings

The cotyledons of jojoba (Simmondsia chinensis) seeds contained 50 to 60% of their weight as intracellular wax esters. During germination there was a gradual decrease in the wax content with a concomitant rise in soluble carbohydrates, suggesting that the wax played the role of a food reserve. Thin layer chromatography revealed that both the fatty alcohol and fatty acid were metabolized. The disappearance of wax was matched with an increase of catalase, a marker enzyme of the gluconeogenic process in other fatty seedlings. Subcellular organelles were isolated by sucrose gradient centrifugation from the cotyledons at the peak stage of germination. The enzymes of the β oxidation of fatty acid and of the glyoxylate cycle were localized in the glyoxysomes but not in the mitochondria. The glyoxysomes had specific activities of individual enzymes similar to those of the castor bean glyoxysomes. An active alkaline lipase was detected in the wax bodies at the peak stage of germination but not in the ungerminated seeds. No lipase was detected in glyoxysomes or mitochondria. After the wax in the wax bodies had been extracted with diethyl ether, the organelle membrane was isolated and it still retained the alkaline lipase. The gluconeogenesis from wax in the jojoba seedling appears to be similar, but with modification, to that from triglyceride in other fatty seedlings.     

Purification and properties of glyoxysomal lipase from castor bean

The alkaline lipase in the glyoxysomes from the endosperm of young castor bean seedlings, an integral membrane component, was solubilized in deoxycholate:KCl and purified to apparent homogeneity. The molecular weight on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was 62,000 daltons. The enzyme reaction was markedly stimulated by salts and inhibited by detergents. Triricinolein, the endogenous storage lipid, was hydrolyzed by the purified enzyme which is therefore a true lipase. Treatment of intact glyoxysomes with trypsin strongly diminished the lipase activity but did not affect matrix enzymes. An antibody preparation raised in a rabbit against the purified enzyme inhibited the purified enzyme and that in glyoxysomal membranes.     

Lipase in lipid bodies of cotyledons of rape and mustard seedlings

Lipolytic activity was absent in the crude cotyledon extract of ungerminated rapeseed (Brassica napm L. var. Dwarf Essex), and increased to a peak at day 4 in seedling growth, concomitant with the decrease in total lipids. About 50% of the lipase activity was recovered in the lipid bodies isolated from the cotyledon extract by flotation centrifugation. Isolated lipid bodies underwent autolysis of internal triacylglycerols resulting in the release of fatty acids. After the triacylglycerols in isolated lipid bodies had been extracted with diethyl ether, the lipase was recovered in the remaining membrane fraction. The lipase had a maximal activity at pH 6.5 on trierucin, trilinolein, or endogenous triacylglycerols, and at pH 8.0 on N-methylindoxylmyristate. The lipase was most active on trierucin and trilinolein, and hydrolyzed the related di- and monoacylglycerols at lower rates. There was little enhancement of the lipase activity in the presence of NaCl, CaCl₂, or detergents, and detergents in general reduced the activity. The hydrolysis of trierucin was linear until about 50% of the trierucin had been converted to erucic acid, and there was little accumulation of dierucin and monoerucin. Lipase extracted from lipid bodies isolated from germinated rapeseed of the variety Tower, which contains little or no erucic acids in the storage triacylglycerols, also had the highest activities on trierucin and trilinolein. A comparative study on mustard seed (Brassica juncea) revealed that the mustard lipase possessed characteristics very similar to those of the rapeseed lipase.     

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.     

Lipase Activities in Castor Bean Endosperm during Germination

Two lipases were found in extracts from castor bean (Ricinus communis L.) endosperm. One, with optimal activity at pH 5.0 (acid lipase), was present in dry seeds and displayed high activity during the first 2 days of germination. The second, with an alkaline pH optimum (alkaline lipase), was particularly active during days 3 to 5. When total homogenates of endosperm were fractionated into fat layer, supernatant, and particulate fractions, the acid lipase was recovered in the fat layer, and the alkaline lipase was located primarily in the particulate fraction. Sucrose density gradient centrifugation showed that the alkaline lipase was located mainly in glyoxysomes, with some 30% of the activity in the endoplasmic reticulum. When glyoxysomes were broken by osmotic shock and exposed to KCl, which solubilizes most of the enzymes, the alkaline lipase remained particulate and was recovered with the glyoxysomal “ghosts” at equilibrium density 1.21 g/cm³ on the sucrose gradient. Association of the lipase with the gly-oxysomal membrane was supported by the responses to detergents and to butanol. The alkaline lipase hydrolyzed only monosubstituted glycerols. The roles of the two lipases in lipid utilization during germination of castor bean are discussed.     

Post-heparin triacylglycerol lipases in ovine plasma

Lipoprotein lipase and hepatic lipase have been shown to be present in the post-heparin plasma of sheep. Intravenous injection of heparin into sheep produced a rapid increase in the free fatty acid concentration and lipolytic enzyme activity of the plasma, both peaking within 5-15 min and then falling to pre-heparin levels within 30-60 min. Lipolytic activity was not detected in plasma before heparin treatment. Two distinct lipolytic activities were separated from the plasma by chromatography on heparin-Sepharose 6B. Lipoprotein lipase was identified on the basis that the lipolytic activity was dependent upon the addition of plasma, inhibited by 1M NaCl, and inhibited by a specific antiserum against lipoprotein lipase. The second lipolytic activity of plasma was identified as hepatic lipase, as it was not dependent upon plasma for activity, nor was it inhibited by 1M NaCl or antiserum against lipoprotein lipase. Its properties were identical to the lipase extracted from the liver of sheep. Lipoprotein-lipase activity, but not hepatic-lipase activity, was dependent upon the nutritional state of the sheep at the time of heparin injection. However, hepatic lipase comprised a significant proportion of the total lipolytic activity.